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Team:UNAM Genomics Mexico/Notebook/ANDSugar
2012-10-27T03:52:19Z
<p>Abieltega: </p>
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<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Arabinose/Xylose AND Gate Notebook'''</h1></center><br />
<br /><br />
<br /><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="20"><br />
<tr><br />
<td id="leftcolumn2"><center>2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JUNE | '''JUNE''']] </center> <br /><br />
<br /><br />
2.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F07.2F12 |06/07/12]] <br /><br />
2.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F12.2F12 |06/12/12]] <br /><br />
2.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F13.2F12 |06/13/12]] <br /><br />
2.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F14.2F12 |06/14/12]] <br /><br />
2.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F15.2F12 |06/15/12]] <br /><br />
2.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F18.2F12 |06/18/12]] <br /><br />
2.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F19.2F12 |06/19/12]] <br /><br />
2.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F22.2F12 |06/22/12]] <br /><br />
2.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F25.2F12 |06/25/12]] <br /><br />
2.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F26.2F12 |06/26/12]] <br /><br />
2.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F27.2F12 |06/27/12]] <br /><br />
2.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F29.2F12 |06/29/12]] <br /><br />
</td><br />
<br />
<td id="contentcolumn2"><center>3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JULY | '''JULY''']] </center> <br /><br />
<br /><br />
3.1[[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F03.2F12 | 07/03/12]] <br /><br />
3.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F04.2F12 | 07/04/12]] <br /><br />
3.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F06.2F12 | 07/06/12]] <br /><br />
3.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F07.2F12 | 07/07/12]] <br /><br />
3.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F08.2F12 | 07/08/12]] <br /><br />
3.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F09.2F12 | 07/09/12]] <br /><br />
3.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F10.2F12 | 07/10/12]] <br /><br />
3.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F11.2F12 | 07/11/12]] <br /><br />
3.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F12.2F12 | 07/12/12]] <br /><br />
3.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F13.2F12 | 07/13/12]] <br /><br />
3.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F14.2F12 | 07/14/12]] <br /><br />
3.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F16.2F12 | 07/16/12]] <br /><br />
3.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F17.2F12 | 07/17/12]] <br /><br />
3.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F18.2F12 | 07/18/12]] <br /><br />
3.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F19.2F12 | 07/19/12]] <br /><br />
3.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F20.2F12 | 07/20/12]] <br /><br />
3.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F23.2F12 | 07/23/12]] <br /><br />
3.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F24.2F12 | 07/24/12]] <br /><br />
3.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F25.2F12 | 07/25/12]] <br /><br />
3.20 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F26.2F12 | 07/26/12]] <br /><br />
3.21 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F27.2F12 | 07/27/12]] <br /><br />
3.22 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F30.2F12 | 07/30/12]] <br /><br />
3.23 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F31.2F12 | 07/31/12]] <br /><br />
</td><br />
<br />
<td id="rightcolumn2"><center>4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#AUGUST | '''AUGUST''']] </center> <br /><br />
<br /><br />
4.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F01.2F12 | 08/01/12]] <br /><br />
4.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F02.2F12 | 08/02/12]] <br /><br />
4.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F03.2F12 | 08/03/12]] <br /><br />
4.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F06.2F12 | 08/06/12]] <br /><br />
4.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F08.2F12 | 08/08/12]] <br /><br />
4.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F09.2F12 | 08/09/12]] <br /><br />
4.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F10.2F12 | 08/10/12]] <br /><br />
4.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F13.2F12 | 08/13/12]] <br /><br />
4.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F16.2F12 | 08/16/12]] <br /><br />
4.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F20.2F12 | 08/20/12]] <br /><br />
4.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F21.2F12 | 08/21/12]] <br /><br />
4.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F22.2F12 | 08/22/12]] <br /><br />
4.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F23.2F12 | 08/23/12]] <br /><br />
4.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F24.2F12 | 08/24/12]] <br /><br />
4.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F25.2F12 | 08/25/12]] <br /><br />
4.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F28.2F12 | 08/28/12]] <br /><br />
4.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F29.2F12 | 08/29/12]] <br /><br />
4.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F30.2F12 | 08/30/12]] <br /><br />
4.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F31.2F12 | 08/31/12]] <br /><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2"><center>5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#SEPTEMBER | '''SEPTEMBER''']] </center> <br /><br />
<br /><br />
5.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F01.2F12 | 09/01/12]] <br /><br />
5.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F02.2F12 | 09/03/12]] <br /><br />
5.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F04.2F12 | 09/04/12]] <br /><br />
5.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F05.2F12 | 09/05/12]] <br /><br />
5.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F06.2F12 | 09/06/12]] <br /><br />
5.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F14.2F12 | 09/14/12]] <br /><br />
5.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F16.2F12 | 09/16/12]]<br /><br />
5.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F17.2F12 | 09/17/12]]<br /><br />
5.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F18.2F12 | 09/18/12]]<br /><br />
5.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F19.2F12 | 09/19/12]]<br /><br />
5.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F19.2F12 | 09/19/12]]<br /><br />
5.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F20.2F12 | 09/20/12]]<br /><br />
5.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F21.2F12 | 09/21/12]]<br /><br />
5.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F22.2F12 | 09/22/12]]<br /><br />
5.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F23.2F12 | 09/23/12]]<br /><br />
5.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F24.2F12 | 09/24/12]]<br /><br />
5.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F26.2F12 | 09/26/12]]<br /><br />
5.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F27.2F12 | 09/27/12]]<br /><br />
<br />
<br />
<br />
</td><br />
<br />
<td id="contentcolumn2"><br />
'''OCTOBER'''<br /><br />
5.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F09.2F12 | 10/09/12]]<br /><br />
5.20 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F10.2F12 | 10/10/12]]<br /><br />
5.21 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F11.2F12 | 10/11/12]]<br /><br />
5.22 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F12.2F12 | 10/12/12]]<br /><br />
5.23 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F13.2F12 | 10/13/12]]<br /><br />
</td><br />
<br />
<td id="rightcolumn2"><center>[[File:UnmagenomicsBacteria sugar.png | 190px]]</center><p> '''On hover the images to see descriptions'''</p><td/><br />
</tr><br />
</table><br />
<br />
<br />
=JUNE=<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
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<br /><br /><br />
</html> <br />
<h2>06/07/12</h2><br /><br />
PY BROTH 10g salt/L<br /><br />
PSB2K3 kanamicin<br /><br />
We transformed RFP E1010. <br /><br />
plate 1 18F<br /><br />
plate 2 17E<br /><br />
Stock 50 mg/mL<br /><br />
''E.coli'' 1/1000<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
We made liquid cultures with colonies [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
We extracted plasmids [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PLASMID_EXTRACTION_2 | PLASMID EXTRACTION PROTOCOL]]. <br /><br />
We ran a gel to check the extraction [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<h2>06/12/12</h2><br /><br />
We made glycerols of the bacteria <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed plasmid pHp45 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/38/UnamgenomicsandsugarBitacora_2.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>06/13/12</h2><br /><br />
We ran gel and extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] and lysed. <br /><br />
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<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2d/UnamgenomicsandsugarBitacora_3.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
We extracted from gel<br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/14/12</h2><br /><br />
We extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made glycerols of pHp45 Ω [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450<br />
PSB4A5 Am (ampicillin) 1I BBa_J04450<br />
AraC BBa_C0080 <br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
2011 14L plate 1 pSB2K3 Km+<br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
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<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/5/56/UnamgenomicsandsugarBitacora_5.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
pHp45 Ω with E/P<br /><br />
We extracted from gel<br /><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/15/12</h2><br /><br />
> Digested pHp45 Ω with E/P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
> We extracted plasmid with kit. <br /><br />
> Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (5) <br /><br />
>Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br /><br />
ARAC<br /><br />
>From the overnight plate we prepared liquid culture [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
>We left them incubating overnight. <br /><br />
>We left a plate (LB Km DH5α C0080 and a control). <br /><br />
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<h2>06/18/12</h2><br /><br />
>Plasmid extraction AraC with kit. <br /><br />
>C0080-psB2K3 915 bp<br /><br />
>Streaked 4 LB Km 30 plates DH5α psB2K3. <br /><br />
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red. <br /><br />
>Ran gel with Spr/Strr (eppendorf -20ºC) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
>Made glycerols from one tube of DH5α C0080 Km 50 -> 04. <br /><br />
>Made glycerols from one tube of DH5α PSB4A5 Amp 100 -> 05 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]]. <br /><br />
>Digested C0080 X,S. <br /><br />
>Digested B0014 E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/5/52/UnamgenomicsandsugarBitacora_6.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
Digested B0014 with E, P and with E,X<br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<br /><br />
<br />
<h2>06/19/12</h2><br /><br />
>Digested B0014 with E, P and with E,X. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
>Ran gels with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation. <br /><br />
C0080 is in the first lane. <br /><br />
>Extracted from gel C0080 X,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Made glycerols from 4 plates LB Km DH5α pSB2K3 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
>Take out AmyE 5’ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
AmyE 5’ 18K plate 3 2010, 2011, 2012<br /><br />
AmyE 3’ 18M plate 3 2010, 2011, 2012<br /><br />
>Digested B0014 with E,X and E,P again [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>06/22/12</h2><br /><br />
<br />
>Ran gels with digestions B0014 with E,X and E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
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>Dephosphated B0014 E,X and B0014 E,P. <br /><br />
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<h2>06/25/12</h2><br /><br />
>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
<br />
<h2>06/26/12</h2><br /><br />
>Ran gel with psB2K3 and psB4A5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
<h2>06/27/12</h2><br /><br />
>Transformed with plasmid B0079 1576 bp psB1A2 12A [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011<br /><br />
AmyE 5’ BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011<br /><br />
AmyE 3’ BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011<br /><br />
AmyE 5’ grew 2 colonies. <br /><br />
<br />
<h2>06/29/12</h2><br /><br />
>We did a DH5α K143001 Km30 Amp 100 glycerol [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] 07. <br /><br />
>We plated twice DH5α K143002 Km 30 Amp 100 + control and twice DH5α B0079 Amp 100 + control<br /><br />
These were both plated in 2 plates each. <br /><br />
They were left overnight since they had not grown by 7:00 pm so the next day glycerols were made. <br /><br />
>Liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
2 tubes DH5α K143001 Km30 Amp 100 <br /><br />
2 tubes DH5α K143002 Km30 Amp 100 <br /><br />
2 tubes DH5α B0079 Amp 100 <br /><br />
1 tube LB Km 30 Amp 100 control<br /><br />
1 tube LB Amp 100 control<br /><br />
>From the 6 tubes we extracted plasmid from kit. <br /><br />
<br /><br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=JULY=<br />
<br />
07/02/12<br /><br />
>Digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /><br />
>PCR’s<br /><br />
•AraC<br /><br />
•Cassete ΩSpr/Strr <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PCR_Protocol_.2850.C2.B5l.29 | PCR PROTOCOL]] <br /><br />
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<div class='captionmoradoclaro'><br />
<p class='captionInside'>After Cassete ΩSpr/Strr PCR we ran a gel <br />
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<div class='captionmorado'><br />
<p class='captionInside'>B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /> <br />
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<h2>07/03/12</h2><br /><br />
>After Cassete ΩSpr/Strr PCR we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (8) <br /><br />
>Ran gel with digestions from yesterday. (9) <br /><br />
>Did band extractions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Stored at -20ºC. <br /><br />
>Did PCR to Cassete ΩSpr/Strr to add RBS and prefix/suffix. <br /><br />
>Left digesting with E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='captionazul'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
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</li><br />
</ul><br />
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<h2>07/04/12</h2><br /><br />
>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1). <br /><br />
>Ligated Cassete ΩSpr/Strr +K143002 dephosphated(digested with E,X and E,S). <br /><br />
>Transformed ligation and left overnight plated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs. <br /><br />
>Digested with PstI [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>07/06/12</h2><br /><br />
4 LB Km30 Spec60 DH5α ΩSpr/Strr +K143002 still have not grown. <br /><br />
Ran a gel with yesterday’s digestions to chek if they were done properly (10) <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
Ran another gel with the rest of the samples. <br /><br />
Extracted GusA fragment <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
Repeated ligation ΩSpr/Strr +K143002 E,X dephosphated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Repeated ΩSpr/Strr PCR. <br /><br />
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<br /><br />
<h2>07/07/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002 . <br /><br />
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<h2>07/08/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002. <br /><br />
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<div class='captiongray'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
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</ul><br />
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<div class='captionnaranja'><br />
<p class='captionInside'>1 PCR omega P<br /><br />
2 PCR omega I<br /><br />
3 PCR AraC P<br /><br />
4 PCR AraC I<br /><br />
5 ladder 1 Kb<br /><br />
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</li><br />
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<h2>07/09/12</h2><br /><br />
From yesterday’s transformations only one colony grew. <br /><br />
From the previous transformation only 2 colonies grew. <br /><br />
These 3 were streaked in 3 plates: <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km 30 Sp 100 control. <br /><br />
Did liquid cultures in 3 tubes: <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km30 Sp 100 control. <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
Ran a gel with: (11) <br /><br />
GusA P<br /><br />
PBBR1 GusA<br /><br />
Ω E PCR P<br /><br />
Ω PCR P<br /><br />
Ω PCR I<br /><br />
Ω E<br /><br />
Ω PCR I<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] <br /><br />
Did GusA primers dissolution for PCR. <br /><br />
GusA PCR <br /><br />
Gel Extraction by kit of lanes 3 and 5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
'''PCR omega P, PCR omega I, PCR AraC P, PCR AraC I'''<br /><br />
Add 10 μl of each primer (LW and UP). <br /><br />
Add 3 μl of plasmid (P). <br /><br />
Add 30.4 μl buffer. <br /><br />
Add 5 μl Mg. <br /><br />
Add 8 μl DNTp’s. <br /><br />
Add 42.6μl H2O miliQ. <br /> <br />
Add 1 μl RTTG polymerase. <br /><br />
Centrifuge (spin) 8 secs. <br /><br />
Add vegetable oil till the eppendorf is full. <br /><br />
Place eppendorf 1 mL in thermocycler. <br /><br />
Run PCR with program “BERNA”. <br /><br /><br />
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<div class='captionverde'><br />
<p class='captionInside'>GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S <br /><br />
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</li><br />
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<h2>07/10/12</h2><br /><br />
•GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S (2 times)[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTIONS]]. (11.2) <br /><br />
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<h2>07/11/12</h2><br /><br />
•GusA PCR digestions with E,S; Ω+AmyE 3’ with E,X [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•T.V. AraC PCR+ Ω+AmyE 3’ E,X dephosphated ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<div class='captionrojo'><br />
<p class='captionInside'>1. ladder. <br /><br />
2. GusA PCR E,S. <br /><br />
3. Ω+AmyE 3’ with E,X. <br /><br />
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</li><br />
</ul><br />
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<br />
<h2>07/12/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: (12) <br /><br />
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[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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•Transformed AraC PCR+ Ω+AmyE 3’ ligation in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
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•From pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid. <br /><br />
•Made glycerols from 2 LB Km 30 Sp100 DH5α Ω+AmyE 3’ plates and from 1 LB Amp100 pfrc54 (A3) plates with control[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
•Ligated GusA PCR with B0014 E,X desphophorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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•Digested Pfrc54 (A3) with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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•Desphophorylated Ω+AmyE 3’ E,X. <br /><br />
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<div class='captionrojo'><br />
<p class='captionInside'>•Ran gel with pfrc54 S,P<br /><br />
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<h2>07/13/12</h2><br /><br /><br />
•Ran gel with pfrc54 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (13) <br /><br />
•Transformation of GusA PCR + B0014 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Transformed with GFP E0040 psBIA2. <br /><br />
•Liquid Cultures with ligation: AraC+ Ω+AmyE 3’ for plasmid extraction at night [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•''B. Subtitils'' competent cells. <br /><br />
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<h2>07/14/12</h2><br /><br /><br />
•From 24 AraC+ Ω+AmyE 3’ LB Km 30 Sp 100 DH5α tubes: <br /><br />
•Extracted pellets<br /><br />
•Extracted plasmids. <br /><br />
•Ran gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br /><br />
•From transformed DH5α km 30: <br /><br />
GusA+B0014 DH5α Km50 24 pellets <br /><br />
E0040 DH5α LB Amp100 <br /><br />
From these two we: <br /><br />
•Did liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Extracted plasmid. <br /><br />
•From the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ran gel with this transformation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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<h2>07/16/12</h2><br /><br /><br />
•Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
•Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='captionrosa'><br />
<p class='captionInside'>Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
Ω+AmyE 3’ E,P <br /><br />
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</li><br />
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<h2>07/17/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (14) <br /><br />
•The gel we ran didn’t work, probably because the agarose was not prepared correctly. <br /><br />
•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/d/db/Bitacora_15.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S<br /><br />
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<h2>07/18/12</h2><br /><br /><br />
•From yesterday’s digestions we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (15) <br /><br /><br />
•We made liquid cultures of AraC+ Ω+AmyE 3’ 12, 15, 21 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br /><br />
•We ligated AraC PCR E,S dephosphated with + Ω+AmyE 3’ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
•We digested 1 + Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<h2>07/19/12</h2><br /><br /><br />
•We ran We digested 1 + Ω+AmyE 3’ E,P with Miguel’s enzymes and with Abiel’s enzymes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
•We transformed with ligations AraC+ Ω+AmyE 3’ E and + Ω+AmyE 3’ E [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br /><br />
•Extracted plasmids from AraC+ Ω+AmyE 3’ 12,15,21. <br /><br /><br />
•Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P <br /><br />
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<h2>07/20/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (16) <br /><br />
•Transformed GusA+B0014 in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Made liquid cultures from AraC+ Ω+AmyE 3’s transformation LB Km30 Sp 100 and control LB Km 30 Sp100 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<h2>07/23/12</h2><br /><br /><br />
•From 24 GusA+B0014 tubes (-) we didn’t do anything. <br /><br />
•From 4 AraC+ Ω+AmyE 3’ we extracted plasmid. <br /><br />
•Digested from the 4 AraC+ Ω+AmyE 3’ E,X; X; P and GusA PCR with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<h2>07/24/12</h2><br /><br /><br />
•Digested B0014 E with X<br />
B0014 X with E<br /><br />
•Digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>07/25/12</h2><br /><br /><br />
•Ran gel of digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Joined B0014 E with X B0014 X with E digestions. <br /><br />
•Made liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. Transformed [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•From LasR DH5α make liquid cultures and plate [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Digested Ω+AmyE 3’ with X,P; E,S; C0080 X,P; E,S; S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Ligated Gus PCR X,P with B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<div class='captionazul'><br />
<p class='captionInside'>1.Ω+AmyE 3’ X,P<br /><br />
2. Ω+AmyE 3’ E,S<br /><br />
3.C0080 X,P<br /><br />
4.C0080 E,S<br /><br />
5.C0080 S,P<br /><br />
6.ladder<br /><br />
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<h2>07/26/12</h2><br /><br />
•From C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5α C0179 LB Cb 100 (two cultures) and a control without bacteria [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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•We extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguel’s transformation), Puc, B.S. control and PSB4AE. <br /><br />
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•Due to problems with the way we did the transformations of ligations we repeated them: <br /><br />
GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
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GusAPCR X,P+ B0079 S,P dephosphorylated (-)<br /><br />
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GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
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GusAPCR X,P+ A3 S,P dephosphorylated (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•AraC+ Ω+AmyE 3’ S,P 1,2,3,4<br /><br />
•K143002 X,P<br /><br />
•AraC+ Ω S,P<br /><br />
•C0179 X,S<br /><br />
<br />
<br />
<h2>07/27/12</h2><br /><br /><br />
Ligated AraC+ Ω S,P dephosphorylated + K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<h2>07/30/12</h2><br /><br /><br />
•Transformed with ligations: <br /><br />
•AraC+ Ω dephosphprylated +K143002 X,P<br /><br />
•GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
•GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
•Transformed the following sythesis: <br /><br />
•91996 Pveg 140 bp<br /><br />
•91997 ArsR-CzrA_promoter 1 194 bp<br /><br />
•91998 ArsR-CzrA_promoter 2 221 bp<br /><br />
•91999 ArsR-CzrA_promoter 3 213 bp<br /><br />
•92000 pBad-pXyl 387 bp<br /><br />
•92001 XylR 1117pb<br /><br />
•92002 CI_pro_(NAND_INHIBITOR) 774 <br /><br />
•These synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked.<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Make liquid cultures of the following transformations for tomorrow [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]: <br /><br />
•AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+B0079<br /><br />
•Synthesis<br /><br />
<br />
<h2>07/31/12</h2><br /><br /><br />
Extracted plasmids from yesterday’s cultures and from K143002 cultures that were in the other lab. <br /><br />
<br /><br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=AUGUST=<br />
<br />
<h2>08/01/12</h2><br /><br /><br />
Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b1/UnamgenomicsandsugarBitacora_17.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1 PCR GusA I<br /><br />
2 PCR GusA I<br /><br />
3 PCR GusA P<br /><br />
4 PCR GusA P<br /><br />
5 ladder 1 Kb<br /></p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/02/12</h2><br /><br /><br />
•Extracted plasmids from liquid cultures: <br /><br />
AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+BBR1<br /><br />
<br />
PCR GusA, GusA I, PCR GusA P, PCR GusA I<br /><br />
<br />
•Add 10 μl of each primer (LW and UP). <br /><br />
•Add 3 μl of plasmid (P). <br /><br />
•Add 30.4 μl buffer. <br /><br />
•Add 5 μl Mg. <br /><br />
•Add 8 μl DNTp’s. <br /><br />
•Add 42.6μl H2O miliQ. <br /><br />
•Add 1 μl RTTG polymerase. <br /><br />
•Centrifuge (spin) 8 secs. <br /><br />
•Add mineral oil till the eppendorf is full. <br /><br />
•Place eppendorf 1 mL in thermocycler. <br /><br />
•Run PCR with program “BERNA”. <br /><br />
<br /><br />
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<br /><br />
<br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/82/UnamgenomicsandsugarBitacora_18.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.A3+GusA 1<br /><br />
4.A3+GusA 2<br /><br />
5.A3+GusA 3<br /><br />
6.P GusA<br /><br />
7.P GusA 2<br /><br />
8.Ladder<br /><br />
9.01<br /><br />
10.06<br /><br />
11.96<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<br /><br />
<br />
<h2>08/03/12</h2><br /><br /><br />
• Ran a gel with: (18) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Ran another gel to extract with: <br /><br />
1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.00<br /><br />
4.01<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]: <br /> <br />
<br />
• Ω+K143002 E,X; A3+GusA E,S; GusAPCR X,P <br /><br />
<br />
•Extracted GusAPCR X,P digestion [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Left the following ligation: AraC+ Ω+K143002 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/17/UnamgenomicsandsugarBitacora_19.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3+GusA 2<br /><br />
2.A3+GusA 3<br /><br />
3. Ω+AmyE 3’ 2<br /><br />
4. Ω+AmyE 3’ 3<br /><br />
5.XylR X,P<br /><br />
6.pBad-pXyl X,P<br /><br />
7.GusA PCR X,P<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/06/12</h2><br /><br /><br />
•Made liquid cultures of Ω+K143002 (2 cultures+1 control) and AraC+ Ω+K143002 (2 cultures+1 control) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ligated GusAPCR X,P+B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
•Ran a Gel with: (19) <br /><br />
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<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
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<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/8b/UnamgenomicsandsugarBitacora_19.1.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/08/12</h2><br /><br />
BBa_B0040 6I psB1A2 Amp+ plate 1<br /><br />
Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (19.1) <br /><br />
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<br />
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<br />
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<br /><br />
<br />
<br /><br />
<br />
<h2>08/09/12</h2><br /><br />
•From yesterday’s PCR’s : <br /><br />
• E0040 plasmid 1<br /><br />
E0040 plasmid 2<br /><br />
E0040 digested 1<br /><br />
E0040 digested 2<br /><br />
We purified with PCR kit<br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Made 14 PCR’s from AraC+ Ω+AmyE 3’. <br /><br />
•From B0079+GusA ligation and B0040 transformation:<br />
•Grew colonies. <br /><br />
•Made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Streaked these in a new plate. <br /><br />
•Diluted plasmid E0080 2 1/50. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<h2>08/10/12</h2><br /><br />
•Yesterday’s gel did not come out as expected so we repeated the PCR. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/e/ef/UnamgenomicsandsugarBitacora_20.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.AraC+ Ω+AmyE 3’ <br /><br />
2-14.AraC+ Ω+AmyE 3’ <br /><br />
15.AraC+ Ω 1<br /><br />
16.AraC+ Ω 1<br /><br />
17.AraC+ Ω 1<br /><br />
18.PCR 1 GFP<br /><br />
19.PCR GFP<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<h2>08/13/12</h2><br /><br />
•After running a gel with 14 PCR’s AraC+ Ω+AmyE 3’ and it did not come out as expected, we ran a gel with the ligation AraC+ Ω+AmyE 3’ to select candidate (that weight the same as the ligation) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (20) <br /><br />
<br />
•Extracted pasmids form liquid cultures: B0049, B0079+GusA. <br /><br />
<br />
•AraC+ Ω+AmyE 3’ undigested didn’t run in the gel as expected, it ran similarly to AraC+ Ω<br /><br />
<br />
• We repeated the ligation AraC+ Ω S,P dephosphorylated+K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
• GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.<br />
<br />
• We ran a gel to extract. <br /><br />
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<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f1/UnamgenomicsandsugarBitacora_22.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>1.6 AraC+ Ω+AmyE 3’ E with P. <br /><br /><br />
2.8 AraC+ Ω+AmyE 3’ E with P. <br /><br />
3.11 AraC+ Ω+AmyE 3’ E with P. <br /><br />
4.12 AraC+ Ω+AmyE 3’ E with P. <br /><br />
5.6 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
6.8 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
7.11 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
8.12 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
9.00 pBad/pXyl with S,P. <br /><br />
10.Ladder. <br /><br />
11.E0040 PCR E,S to extract the correct band. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/16/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: <br /><br />
<br />
•Digested: <br /><br />
ArSR-CzrA 97 with S,P<br /><br />
ArSR-CzrA 98 with S,P<br /><br />
ArSR-CzrA 99 with S,P<br /><br />
E0040 PCR with E,S<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
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<br /><br />
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<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br />
<h2>08/20/12</h2><br /><br />
•Ligated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/21/12</h2><br /><br />
•Joined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated. <br /><br />
•Extracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR. <br /><br />
•Digested Ω+AmyE 3’ with E,X and + Ω+AmyE 3’ II with E,X (obtained these two from glycerols stored at -80ºC [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/29/UnamgenomicsandsugarBitacora_22.1.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested K143001+PBad, pXyl with E,S <br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/1c/UnamgenomicsandsugarBitacora_23.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>1.K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
4.A3 PCR<br /><br />
5.A3 PCR<br /><br />
6.E0040 PCR E,S<br /><br />
7.B0014 E,X dephosphorylated<br /><br />
8. Ω+AmyE 3’ E,X <br /><br />
9. Ω+AmyE 3’ E,X II<br /><br />
10.Ladder<br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/22/12</h2><br /><br />
•We did 2 PCR’s for A3. <br /><br />
<br />
•Digested K143001+PBad, pXyl with E,S <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (22.1) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did band extraction of: <br /><br />
1. K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
•Made liquid cultures from E1010+Boo14 that the other lab gave us and did 2 plates for glycerol Km+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Did 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ Ω+AmyE 3’ 6,8,11,12 Km+ and Spectinomycin+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Transformed ligations: <br /><br />
B0079+GusA Amp+<br /><br />
Pveg+XylR Chloramphenicol+<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]].. <br /><br />
<br /><br />
<br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/78/UnamgenomicsandsugarBitacora_23.01.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1. Ω+AmyE 3’ E,X 6.2<br /><br />
2.Ω+AmyE 3’ II E,X<br /><br />
3.97 ArsR-CzrA<br /><br />
4.98 ArsR-CzrA<br /><br />
5.99 ArsR-CzrA<br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/23/12</h2><br /><br />
•The negative controls were contaminated. We didn’t manipulate the ones that had grown because of this. <br /><br />
•Ran gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
<br />
<br />
<h2>08/24/12</h2><br /><br />
From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ Ω+AmyE 3’ 6 and 8 we streaked 2 plates for glycerol of each ligation. <br />Repeated ligation B0079+GusA. <br /><br />
<br />
Ligated E0040 PCR E,S+B0014 E,X dephospohylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
Made 2 new plates from Pveg+XylR ligation and liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>08/25/12</h2><br /><br />
Extracted pellet from 16 tubes of pVeg+XylR that were left overnight. <br /><br />
<br />
Ligated B0079+GusA and B0040+B0014 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/28/12</h2><br /><br />
<br />
Made liquid cultures from transformations tht were left overnight <br />
-R0079+GusA<br /><br />
-R0079+GusA (-)<br /><br />
-E0040+B0014<br /><br />
-E0040+B0014 (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/25/UnamgenomicsandsugarBitacora_23.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. A3 PCR 1. <br /><br />
2. A3 PCR 2. <br /><br />
3. Ω+AmyE 3’ S,P *.<br /><br />
4. Ω+AmyE 3’ S,P . <br /><br />
5. Pveg+XylR E,S 2. <br /><br />
6. Pveg+XylR E,S 3. <br /><br />
7. Pveg+XylR E,S 4. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/29/12</h2><br /><br />
Digested Pveg+XylR 2,3,4 with E,S. <br /><br />
<br />
Digested Ω+AmyE 3’ with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTION]]. <br /><br />
<br />
Ran a PCR with A3 PCR (2 .6 ml tubes). <br /><br />
<br />
Ran a gel with <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2e/UnamgenomicsandsugarBitacoraBitacora_24.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1-23. E0040 + B0014 colonies<br /><br />
24-29. B0079+GusA 1<br /><br />
30. B0079<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/30/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (24) <br /><br />
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<br />
•Digested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='captionnaranja'><br />
<p class='captionInside'>1-3B0079+GusA E,S 3. <br /><br />
4-9. PVeg+XylR E,S 1. <br /><br />
10-17. E0040+B0014 X,P 3. <br /><br />
18. 1 kb ladder. <br /><br />
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</li><br />
</ul><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/d2/UnamgenomicsandsugarBitacora_26.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3 PCR. <br /><br />
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<div class='captionverde'><br />
<p class='captionInside'>E0040+B0014 X,P 4 to extract<br /><br />
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</li><br />
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<h2>08/31/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (25) <br /><br />
<br />
<br />
•Dephosphorylated: <br /><br />
Ω+AmyE 3’ S,P <br /><br />
Ω+AmyE 3’ II S,P <br /><br />
6 AraC+ Ω+AmyE 3’ E,X<br /><br />
8 AraC+ Ω+AmyE 3’ E,X<br /><br />
11 AraC+ Ω+AmyE 3’ E,X<br /><br />
12 AraC+ Ω+AmyE 3’ E,X<br /><br />•Ran a gel with A3 PCR [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (26) <br /><br />
<br />
•It was the second time we didn’t obtain a band from A3’s PCR.<br />
<br />
•Probably because it had been missing the buffer 2’s ethanol (“blue lid”) from the PCR purification. <br /><br />
<br />
•Ran a gel woth E0040+B0014 X,P 4 to extract <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | Gen extraction protocol]]. <br /><br />
<br />
•Did an A3 PCR. <br /><br />
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::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
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=SEPTEMBER=<br />
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<li><br />
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<div class='captionrosa'><br />
<p class='captionInside'>1.A3 PCR 1. <br /><br />
2.A3 PCR 2. <br /><br />
3.E0040+B0014 X,P 1. <br /><br />
4.E0040+B0014 X,P 2. <br /><br />
5.Ω+AmyE 3’ dephosphorylated S,P. <br /><br />
6.Ω+AmyE 3’ dephosphorylated S,P II. <br /><br />
7.97 dephosphorylated S,P. <br /><br />
8.98 dephosphorylated S,P. <br /><br />
9.99 dephosphorylated S,P. <br /><br />
10-13. Arac+ Ω+AmyE 3’ dephosphorylated E,X 12. <br /><br />
14. 1 kb Ladder. <br /><br />
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</li><br />
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<h2>09/01/12</h2><br /><br />
•Ran a gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (28) <br /><br />
<br />
•Ligated E0040+B0014 X,P 2 +promoter S,P dephosphorylated; K143002 X,P+ Ω+AmyE 3’ dephosphorylated S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<h2>09/03/12</h2><br /><br />
<br />
•Digested B0079+GusA E,S 3,4,5; PVeg+XylR E,S 1,5,7,10,13,16 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>09/04/12</h2><br /><br />
•Ran gel with digestions: <br /><br />
B0079+GusA E,S 3,4,5<br /><br />
PVeg+XylR E,S 1,5,7,10,13,16 <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Purified A3 PCR 1,2. <br /><br />
<br />
•Ran a gel of A3 PCR <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] and digested with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•From ligations: PVeg+E0040+B0014, PBad/PXyl+E0040+B0014, B0079+E0040+B0014 only colonies grew in PBad/PXyl+E0040+B0014.<br />
<br />
•Plated colonies that grew in a new plate. <br /><br />
<br />
•Made liquid cultures of these <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•Repeated the ones that did not grow. <br /><br />
<br />
•Digested E0040+B0014 4 with X,P[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
-BBa_J23101 18E plate 1 plasmid BBa_J61002_2948 bp Amp+<br /><br />
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<li><br />
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<div class='captionaqua'><br />
<p class='captionInside'>24 PBad/PXyl+E0040+B0014 tubes we extracted <br /><br />
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<div class='captionmoradoclaro'><br />
<p class='captionInside'>1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
Extracted band from 1. and 2<br />
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<div class='captionmorado'><br />
<p class='captionInside'>Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S <br />
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<br />
<h2>09/05/12</h2><br /><br />
•From the 24 PBad/PXyl+E0040+B0014 tubes we extracted plasmids (29) and ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Extracted plasmid from 2 tubes of E0040+B0014. <br /><br />
<br />
•Ran a gel with yesterday’s digestions: <br /><br />
1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
<br />
•Extracted band from 1. and 2. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Purified A3 PCR from 2 0.6ml tubes 1 and 2. <br /><br />
<br />
•1 colony grew from ligation pVeg+E0040+B0014. <br /><br />
<br />
•We streaked this in another plate and did liquid cultures <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•To do: <br /><br />
•Ligate B0079 S,P dephosphorylated + E0040+B0014 X,P and Ω+AmyE 3’ S,P dephosphorylated+ K143001 X,P. <br /><br />
•Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S 10,13. (31) <br /><br />
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<div class='captionazul'><br />
<p class='captionInside'>•A3 PCR and gel A3 PCR X,P <br />
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<h2>09/06/12</h2><br /><br />
<br />
•A3 PCR and gel A3 PCR X,P 1,2. <br /><br />
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<br />
<h2>09/14/12</h2><br /><br />
<br />
•Digested Ω+AmyE 3’ 11,13,18, and 22 with E,X. <br /><br />
•Ligate: <br /><br />
PVeg S,P dephosphorylated+XylR X,P. <br /><br />
PBad p Xyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
pSB13C3 E,P dephosphorylated + A3 PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + GusA PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + XylR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pVeg E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pBad pXyl E,P. <br /><br />
pSB13C3 E,P dephosphorylated + Ω PCR E,P. <br /><br />
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<h2>09/16/12</h2><br /><br />
• Colonies in these plates grew: <br /><br />
- R0079+E0040+B0014 <br /><br />
- pBad/pXyl <br /><br />
- XylR <br /><br />
- pVeg <br /><br />
- A3+E0040+B0014 from colonies 1,3,4,5,6,7,8,9,10,11,12,13,14,15,17 <br /><br />
- pVeg+E0040+B0014 1,2,3,4,5,6,8,9,10,11,12,13,14,15,16 <br /><br />
- pBad/pXyl+E0040+B0014 2 tubes from the same correct colony. <br /><br />
- pVeg+XylR 1,2 <br /><br />
- pBad/pXyl+pSB1C3 1,2 <br /><br />
- XylR+pSB1C3 1,2 <br /><br />
- pVeg+pSB1C3 1,2 <br /><br />
- A3+pSB1C3 1,2 <br /><br />
- GusA+pSB1C3 1,2 <br /><br />
- omegacassette+pSB1C3 1,2 <br /><br />
<br /><br />
• Ran gel from the plasmid extractions that we did yesterday: <br /><br />
- A3+E0040+B0014 1-16 <br /><br />
- pVeg+E0040+B0014 1-16, 18 <br /><br />
- omega cassette+AmyE 3’ 11,13,18,22 <br /><br />
• Digested R0079+E0040+B0014 with E,S. to ligate with AmyE 3’ E,x dephosphorylated. <br /><br />
• Digested omega cassette with E,P. <br /><br />
• Digested pSB1C3 with E,P. <br /><br />
• Made liquid cultures from AmyE 5’ and AmyE 3’. <br /><br />
• Did plasmid extraction from pSB1C3 colonies 3 and 6. <br /><br />
<br /><br />
<br />
<h2>09/17/12</h2><br /><br />
• Digested A3+E0040+B0014 colonies 2,3,4,5,6,11 and 16 with E,S. <br /><br />
• Digested pVeg+E0040+B0014 colonies 1,2,3,4,5,9 and 10 with E,S. <br /><br />
• Dephosphorylated omega cassette+AmyE 3’ colonies 13,18 and 22 digested with E,X. <br /><br />
• Digested pBad/pXyl and pVeg with S,P. Then dephosphorylated. <br /><br />
• Digested AmyE 3’ with E,X. Then dephosphorylated. <br /><br />
<br /><br />
<br />
<h2>09/18/12</h2><br /><br />
<br /><br />
• Ran gel with: <br /><br />
1. A3 2 E,S <br /><br />
2. A3 3 E,S <br /><br />
3. A3 4 E,S <br /><br />
4. A3 5 E,S <br /><br />
5. A3 6 E,S <br /><br />
6. A3 11 E,S <br /><br />
7. A3 16 E,S <br /><br />
8. E0040+B0014 colony 1 E,X <br /><br />
9. AmyE 5’ E,P <br /><br />
10. AmyE 3’ E,X <br /><br />
11. Omega cassette E,S <br /><br />
12. pSB1C3 E,P <br /><br />
13. ladder <br /><br />
Bottom: <br /><br />
1. pVeg 1 E,S <br /><br />
2. pVeg 2 E,S <br /><br />
3. pVeg 3 E,S <br /><br />
4. pVeg 4 E,S <br /><br />
5. pVeg 5 E,S <br /><br />
6. pVeg 9 E,S <br /><br />
7. pVeg 10 E,S <br /><br />
8. pVeg S,P dephosphorylated <br /><br />
9. pVeg 2 S,P <br /><br />
10. pBad/pXyl S,P <br /><br />
11. XylR X,P <br /><br />
12. R0079+GFP+TT E,S <br /><br />
13. R0079 S,P <br /><br />
14. Ladder <br /><br />
• Dephosphorylated GFP+TT E,X; AmyE 5’ E,X; pSB1C3 E,P; pVeg 2 S,P; pBad/pXyl S,P and R0079 S,P. <br /><br />
<br /><br />
<h2>09/19/12</h2><br /><br />
<br /><br />
• Did PCR from A3 and a 1/50 dilution. <br /><br />
• Ran gel with: <br /><br />
1. pVeg 3 E,P. <br /><br />
2. pVeg 9 E,P. <br /><br />
3. pVeg 10 E,P. <br /><br />
4. pBAd/pXyl 3 E,P. <br /><br />
5. pBAd/pXyl 5 E,P. <br /><br />
6. pBAd/pXyl 1 E,P. <br /><br />
7. A3 3 E,P. <br /><br />
8. A3 4 E,P. <br /><br />
9. XylR 1 E,P. <br /><br />
10. XylR 3 E,P. <br /><br />
11. XylR 8 E,P. <br /><br />
12. pSB1C3 6 E,P. <br /><br />
13. pSB1C3 6 E,P. <br /><br />
14. AmyE 5’ E,S. <br /><br />
15. AmyE 5’ E,S. <br /><br />
16. A3 1/50 PCR. <br /><br />
17. A3 PCR. <br /><br />
18. Ladder <br /><br />
• We did transformations: <br /><br />
- 2 of R0079+GusA <br /><br />
- R0079+GusA control <br /><br />
- 2 of A3+pSB1AK3 <br /><br />
- A3+pSB1AK3 control <br /><br />
- 2 of omega cassette+pSB1C3 <br /><br />
- 2 of GusA+pSB1C3 <br /><br />
• Digested: <br /><br />
- pVeg+pSB1C3 <br /><br />
- pBad/pXyl+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
- A3+pSB1C3 <br /><br />
With E,P. <br /><br />
• Ran gel with these digestions. <br /><br />
<br /><br />
<h2>09/19/12</h2><br /><br />
• Made liquids cultures from: <br /><br />
- Glicerol of AmyE 5’ <br /><br />
- Glicerol of AmyE 3’ <br /><br />
- pBad/pXyl+pSB1C3 colony 5 <br /><br />
• Made liquid cultures from yesterday transformations: <br /><br />
- R0079+GusA 2 and control <br /><br />
- A3+pSB1AK3 2 and control <br /><br />
- Omega cassette+pSB1C3 2 <br /><br />
- GusA+pSB1C3 2 <br /><br />
• We transformed yesterday ligations: <br /><br />
- pBad/pXyl+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
- pVeg+pSB1C3 <br /><br />
- A3+pSB1C3 <br /><br />
• Purified A3 PCR with kit, and then, digested with E,P and E,S. <br /><br />
• Ran gel <br /><br />
<br /><br />
<h2>09/20/12</h2><br /><br />
<br /><br />
• Made liquid cultures from transformations: <br /><br />
- Omega cassette+pSB1C3 colony 7 <br /><br />
- 2 of GusA+pSB1C3 colony 32 <br /><br />
- R0079+GusA colony 12 <br /><br />
• Digested A3 1/50 PCR with E,P and E,S. <br /><br />
• Made liquid cultures from R0079+E0040+B0014; pBad/pXyl+E0040+B0014; pVeg+E0040+B0014; A3+E0040+B0014. <br /><br />
• Ligated: <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P <br /><br />
- A3 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
<br /><br />
<h2>09/21/12</h2><br /><br />
<br /><br />
• Ran gel with: <br /><br />
1. R0079+GFP+TT 1 <br /><br />
2. R0079+GFP+TT <br /><br />
3. pBad/pXyl+pSB1C3 1 <br /><br />
4. pBad/pXyl+pSB1C3 <br /><br />
5. omega cassette+psB1C3 1 <br /><br />
6. omega cassette+psB1C3 <br /><br />
7. pSB1C3 E,P 1 <br /><br />
8. pSB1C3 E,P <br /><br />
9. pSB1C3 E <br /><br />
10. pSB1C3 P <br /><br />
11. A3 1/50 PCR E,P <br /><br />
<br /><br />
<br />
<h2>09/22/12</h2><br /><br />
• Ran gel with lysis extractions: <br /><br />
1. GusA+pSB1C3 1 <br /><br />
2. GusA+pSB1C3 2 <br /><br />
3. GusA+pSB1C3 3 <br /><br />
4. GusA+pSB1C3 4 <br /><br />
5. GusA+pSB1C3 5 <br /><br />
6. GusA+pSB1C3 6 <br /><br />
7. GusA+pSB1C3 7 <br /><br />
8. GusA+pSB1C3 8 <br /><br />
9. GusA+pSB1C3 9 <br /><br />
10. GusA+pSB1C3 10 <br /><br />
11. GusA+pSB1C3 11 <br /><br />
12. GusA+pSB1C3 12 <br /><br />
13. pSB1C3 <br /><br />
14. A3+E0040+B0014 1 <br /><br />
15. A3+E0040+B0014 2 <br /><br />
16. A3+E0040+B0014 3 <br /><br />
17. A3+E0040+B0014 4 <br /><br />
18. A3+E0040+B0014 5 <br /><br />
19. A3+E0040+B0014 6 <br /><br />
20. A3+E0040+B0014 7 <br /><br />
21. A3+E0040+B0014 8 <br /><br />
22. A3+E0040+B0014 9 <br /><br />
23. pVeg+E0040+B0014 3 <br /><br />
24. pVeg+E0040+B0014 4 <br /><br />
25. pVeg+E0040+B0014 5 <br /><br />
26. pVeg+E0040+B0014 6 <br /><br />
27. pVeg+E0040+B0014 7 <br /><br />
28. pVeg+E0040+B0014 8 <br /><br />
29. pVeg+E0040+B0014 9 <br /><br />
30. pVeg <br /><br />
31. pBad/pXyl+E0040+B0014 1 <br /><br />
32. pBad/pXyl+E0040+B0014 2 <br /><br />
33. pBad/pXyl+E0040+B0014 3 <br /><br />
34. pBad/pXyl+E0040+B0014 4 <br /><br />
35. pBad/pXyl+E0040+B0014 5 <br /><br />
36. pBad/pXyl+E0040+B0014 6 <br /><br />
37. pBad/pXyl+E0040+B0014 7 <br /><br />
38. pBad/pXyl+E0040+B0014 8 <br /><br />
39. pBad/pXyl+E0040+B0014 9 <br /><br />
40. pBad/pXyl+E0040+B0014 10 <br /><br />
41. pBad/pXyl <br /><br />
42. pVeg+XylR 1 <br /><br />
43. pVeg+XylR 2 <br /><br />
44. pVeg+XylR 3 <br /><br />
45. pVeg+XylR 4 <br /><br />
46. pVeg+XylR 5 <br /><br />
47. pVeg+XylR 6 <br /><br />
48. pVeg+XylR 7 <br /><br />
49. pVeg+XylR 8 <br /><br />
50. omega cassette <br /><br />
<br /><br />
• Ran gel with yesterday digestions: <br /><br />
1. R0079+GFP+TT E,P <br /><br />
2. R0079+GFP+TT E,P <br /><br />
3. Omega cassette+pSB1C3 E,P <br /><br />
4. Omega cassette+pSB1C3 E,P <br /><br />
5. Ladder <br /><br />
6. pBad/pXyl+pSB1C3 <br /><br />
7. pBad/pXyl+pSB1C3 <br /><br />
8. pSB1C3 6 E,P. <br /><br />
9. A3 1/50 PCR E,S <br /><br />
10. pBbak C011 <br /><br />
11. pBbak C012 <br /><br />
12. ladder <br /><br />
<br /><br />
• Did pellet from R0079+GusA. <br /><br />
• Did plasmid extraction from omega cassette+pSB1C3 colonies 1,4,6 and 7. <br /><br />
• Digested them with E,P. <br /><br />
• Digested: <br /><br />
- GusA+pSB1C3 1,3,4,10 and 11 <br /><br />
- A3+E0040+B0014 2,4,7 and 9 <br /><br />
- pVeg+E0040+B0014 4,5 and 6 <br /><br />
- pBad/pXyl+E0040+B0014 3,7 and 10 <br /><br />
- pVeg+XylR 2,4 and 5 <br /><br />
with E,P. <br /><br />
• Made liquid cultures from pVeg+pSB1C3 <br /><br />
• Ligated: <br /><br />
- XylR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- pVeg E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- A3 1/50 PCR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- A3 1/50 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
<br /><br />
<h2>09/23/12</h2><br /><br />
<br /><br />
• Ran gel with yesterday digestions: <br /><br />
1. Omega cassette+pSB1C3 1 E,P <br /><br />
2. Omega cassette+pSB1C3 4 E,P <br /><br />
3. Omega cassette+pSB1C3 6 E,P <br /><br />
4. Omega cassette+pSB1C3 7 E,P <br /><br />
5. GusA+pSB1C3 1 E,P <br /><br />
6. GusA+pSB1C3 3 E,P <br /><br />
7. GusA+pSB1C3 4 E,P <br /><br />
8. GusA+pSB1C3 10 E,P <br /><br />
9. GusA+pSB1C3 11 E,P <br /><br />
10. pSB1C3 E,P dephosphorylated <br /><br />
11. pVeg+XylR 2 E,P <br /><br />
12. pVeg+XylR 4 E,P <br /><br />
13. pVeg+XylR 5 E,P <br /><br />
14. pVeg S,P dephosphorylated <br /><br />
15. pVeg+GFP+TT 4 E,P. <br /><br />
16. pVeg+GFP+TT 5 E,P. <br /><br />
17. pVeg+GFP+TT 6 E,P. <br /><br />
18. ladder <br /><br />
Bottom <br /><br />
1. pBad/pXyl+GFP+TT 3 E,P <br /><br />
2. pBad/pXyl+GFP+TT 7 E,P <br /><br />
3. pBad/pXyl+GFP+TT 10 E,P <br /><br />
4. pBad/pXyl S,P dephosphorylated <br /><br />
5. A3+GFP+TT 2 E,P <br /><br />
6. A3+GFP+TT 4 E,P <br /><br />
7. A3+GFP+TT 7 E,P <br /><br />
8. A3+GFP+TT 9 E,P <br /><br />
9. Ladder <br /><br />
<br /><br />
<h2>09/24/12</h2><br /><br />
<br /><br />
• Did PCR from omega cassette+pSB1C3 colonies 1,4,6 and 7. <br /><br />
• Made 1/50 dilutions from these PCR products. <br /><br />
• Ran gel with: <br /><br />
1. Omega cassette+pSB1C3 1/50 PCR 1 <br /><br />
2. Omega cassette+pSB1C3 PCR 1 <br /><br />
3. Omega cassette+pSB1C3 1/50 PCR 4 <br /><br />
4. Omega cassette+pSB1C3 PCR 4 <br /><br />
5. Omega cassette+pSB1C3 1/50 PCR 6 <br /><br />
6. Omega cassette+pSB1C3 PCR 6 <br /><br />
7. Omega cassette+pSB1C3 1/50 PCR 7 <br /><br />
8. Omega cassette+pSB1C3 PCR 7 <br /><br />
9. GusA+pSB1C3 1 P <br /><br />
10. GusA+pSB1C3 3 P <br /><br />
11. GusA+pSB1C3 4 P <br /><br />
12. GusA+pSB1C3 10 P <br /><br />
13. GusA+pSB1C3 11 P <br /><br />
14. pVeg+pSB1C3 6 E,P <br /><br />
15. pVeg+pSB1C3 5 E,P <br /><br />
16. pVeg+pSB1C3 4 E,P <br /><br />
17. pVeg+pSB1C3 3 E,P <br /><br />
18. pVeg+pSB1C3 2 E,P <br /><br />
19. pVeg+pSB1C3 1 E,P <br /><br />
20. ladder <br /><br />
<br />
• From yesterday transformations, these didn’t grow: <br /><br />
- pBad/pXyl+E0040+B0014 1 and 2. <br /><br />
- A3+E0040+B0014. <br /><br />
• Made liquid cultures from transformations that did grow: <br /><br />
- pVeg+E0040+B0014. <br /><br />
- pVeg+XylR. <br /><br />
- A3+pSB1C3 <br /><br />
- pVeg+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
<br /><br />
<h2>09/26/12</h2><br /><br />
<br /><br />
• Did plasmid extractions form liquids cultures: <br /><br />
- 8 of pVeg+pSB1C3 <br /><br />
- 8 of A3 <br /><br />
- 8 of XylR <br /><br />
• Digested them with E,P. <br /><br />
• Ran gel with: <br /><br />
1. Ladder <br /><br />
2. XylR+pSB1C3 1 E,P <br /><br />
3. XylR+pSB1C3 2 E,P <br /><br />
4. XylR+pSB1C3 3 E,P <br /><br />
5. pVeg+pSB1C3 1 E,P <br /><br />
6. pVeg+pSB1C3 3 E,P <br /><br />
7. pVeg+pSB1C3 4 E,P <br /><br />
8. pVeg+pSB1C3 5 E,P <br /><br />
9. pVeg+pSB1C3 6 E,P <br /><br />
10. pVeg+pSB1C3 8 E,P <br /><br />
11. pSB1C3 6 E,P dephosphorylated <br /><br />
12. A3+pSB1C3 1 E,P <br /><br />
13. A3+pSB1C3 3 E,P <br /><br />
14. A3+pSB1C3 4 E,P <br /><br />
15. A3+pSB1C3 5 E,P <br /><br />
16. A3+pSB1C3 6 E,P <br /><br />
17. A3+pSB1C3 8 E,P <br /><br />
18. Ladder <br /><br />
19. P4+E0040+B0014 1 E,X <br /><br />
20. P4+E0040+B0014 7 E,X <br /><br />
<br /><br />
<h2>09/27/12</h2><br /><br />
<br /><br />
• Digested pVeg+XylR colonies 1,4,5 and 6 in pJ209 plasmid with E,S for 3 hours. <br /><br />
• Digested pVeg+E0040+B0014 colonies 7,9,10 and 11 in pasmid pJ209 with E,P. <br /><br />
<br /><br />
<h2>10/09/12</h2><br /><br />
<br /><br />
• Ligated: <br /><br />
- A3 1/50 PCR E,P + pSB1C3 6 E,P dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
- P4+CI+E1010+B0014 E,X dephosphorylated + AmyE 5’+pBad/pXyl E,S. <br /><br />
<br /><br />
<h2>10/10/12</h2><br /><br />
<br /><br />
• Ligated: <br /><br />
- GusA PCR E,P + pSB1C3 6 E,P dephosphorylated. <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- A3 1/50 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
• Digested R0079+E0040+B0014 with E,S. <br /><br />
• Digested AmyE 3’ with E,X. Then dephosphorylated. <br /><br />
• Digested pVeg and R0079, A3 with X,P. <br /><br />
• Digested pBad/pSB1C3 with S,P. <br /><br />
<br /><br />
<h2>10/11/12</h2><br /><br />
<br /><br />
• Did plasmid extraction from liquid cultures: <br /><br />
- 2 of AmyE 5’ <br /><br />
- 2 of AmyE 3’ <br /><br />
- 2 of E0040+B0014 <br /><br />
• Ran gel with: <br /><br />
1. A3 1/50 PCR X,P <br /><br />
2. A3 1/50 PCR X,P <br /><br />
3. A3+pSB1C3 6 X,P <br /><br />
4. A3+pSB1C3 6 X,P <br /><br />
5. A3+pSB1C3 8 X,P <br /><br />
6. A3+pSB1C3 8 X,P <br /><br />
7. pVeg X,P <br /><br />
8. pVeg 96 X,P <br /><br />
9. pVeg 96 X,P <br /><br />
10. pVeg+pSB1C3 1 X,P <br /><br />
11. pVeg+pSB1C3 1 X,P <br /><br />
12. pVeg+pSB1C3 3 X,P <br /><br />
13. pVeg+pSB1C3 3 X,P <br /><br />
14. R0079 X,P <br /><br />
15. Ladder <br /><br />
<br /><br />
<h2>10/12/12</h2><br /><br />
<br /><br />
• Ligated:<br />
- AmyE 5’+pBad/pXyl 10 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- AmyE 5’+pBad/pXyl 11 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- AmyE 5’+pBad/pXyl 12 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
- A3 1/50 PCR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
<br /><br />
<h2>10/12/12</h2><br /><br />
<br /><br />
• Digested AmyE 5’ with S,P <br /><br />
• Dephosphorylated it. <br /><br />
• Ligated: <br /><br />
- AmyE 5’ S,P dephosphorylated with <br /><br />
- R0079 X,P <br /><br />
- pVeg 96 X,P <br /><br />
- A3 1/50 PCR X,P <br /><br />
- A3+pSB1C3 6 X,P <br /><br />
- R0079+E0040+B0014 <br /><br />
• Ligated pBad/pXyl+pSB1C3 S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
• Ran gel with: <br /><br />
1. A3+pSB1C3 1 <br /><br />
2. A3+pSB1C3 2 <br /><br />
3. A3+pSB1C3 3 <br /><br />
4. A3+pSB1C3 4 <br /><br />
5. A3+pSB1C3 5 <br /><br />
6. A3+pSB1C3 6 <br /><br />
7. A3+pSB1C3 7 <br /><br />
8. A3+pSB1C3 8 <br /><br />
9. A3+pSB1C3 9 <br /><br />
10. A3+pSB1C3 10 <br /><br />
11. A3+pSB1C3 11 <br /><br />
12. A3+pSB1C3 6 control <br /><br />
13. AmyE 5’+P4+CI+E1010+B0014 1 <br /><br />
14. AmyE 5’+P4+CI+E1010+B0014 2 <br /><br />
15. AmyE 5’+P4+CI+E1010+B0014 3 <br /><br />
16. AmyE 5’+P4+CI+E1010+B0014 4 <br /><br />
17. AmyE 5’+P4+CI+E1010+B0014 5 <br /><br />
18. AmyE 5’+P4+CI+E1010+B0014 6 <br /><br />
19. AmyE 5’+P4+CI+E1010+B0014 7 <br /><br />
20. P4+CI+E1010+B0014 1 control <br /><br />
<br /><br />
<h2>10/09/12</h2><br /><br />
<br /><br />
• Ligate: <br /><br />
K143001+pBad/pXyl + p4+CI+E1010+TT E,X dephosphorylated. <br /><br />
• Ligate: <br /><br />
pVeg S,P dephosphorylated + XylR X,P. <br /><br />
• Ligate: <br /><br />
A3 PCR 1/50 E,P + pSB1C3 E,P dephosphorylated <br /><br />
<br /><br />
<h2>10/13/12</h2><br /><br />
<br /><br />
• Digested AmyE 5’ with S,P. Then dephosphorylated. <br /><br />
• Ligate: <br /><br />
AmyE 5’ S,P dephosphorylated + <br /><br />
- R0079 X,P <br /><br />
- pVeg 96 X,P <br /><br />
- A3 PCR 1/50 X,P <br /><br />
- A3+pSB1C3 colony 6 X,P <br /><br />
- R0079+E0040+B0014 E,X <br /><br />
• Ligate: <br /><br />
pBad/pXyl+pSB1C3 S,P dephosphorylated + E0040+B0014 X,P <br /><br />
<br />
• Ran a gel with: <br /><br />
1. A3+pSB1C3 1 <br /><br />
2. A3+pSB1C3 2 <br /><br />
3. A3+pSB1C3 3 <br /><br />
4. A3+pSB1C3 4 <br /><br />
5. A3+pSB1C3 5 <br /><br />
6. A3+pSB1C3 6 <br /><br />
7. A3+pSB1C3 7 <br /><br />
8. A3+pSB1C3 8 <br /><br />
9. A3+pSB1C3 9 <br /><br />
10. A3+pSB1C3 10 <br /><br />
11. A3+pSB1C3 11 <br /><br />
12. A3+pSB1C3 6 control <br /><br />
13. AmyE 5’+P4+CI+E1010+B0014 8 <br /><br />
14. AmyE 5’+P4+CI+E1010+B0014 9 <br /><br />
15. AmyE 5’+P4+CI+E1010+B0014 10 <br /><br />
16. P4+CI+E1010+B0014 1 control <br /><br />
17. pVeg+XylR 1 <br /><br />
18. pVeg+XylR 2 <br /><br />
19. pVeg+XylR 3 <br /><br />
20. pVeg+XylR 4 <br /><br />
21. pVeg+XylR 5 <br /><br />
22. pVeg+XylR 6 <br /><br />
23. pVeg+XylR 7 <br /><br />
24. pVeg+XylR 8 <br /><br />
25. pVeg+XylR 10 <br /><br />
26. pVeg+XylR 11 <br /><br />
27. pVeg+XylR 12 <br /><br />
28. pVeg 96 control <br /><br />
<br />
• Ran gel with: <br /><br />
1. A3 1/50 PCR X,P <br /><br />
2. A3+pSB1C3 6 X,P <br /><br />
3. pVeg 96 X,P <br /><br />
4. pVeg+pSB1C3 1 X,P <br /><br />
5. pVeg+pSB1C3 3 X,P <br /><br />
6. R0079 X,P <br /><br />
7. pBad/pXyl+pSB1C3 S,P dephosphorylated <br /><br />
8. ladder <br /><br />
9. Digested: <br /><br />
- A3+pSB1C3 colonies 3,4,5 and 10 with E,P. <br /><br />
- AmyE 5’+pBad/pXyl+P4+CI+E1010*B0014 colonies 1,2,7 and 8 with E,P. <br /><br />
- pVeg+XylR 1,4,5 and 12 with E,P. <br /><br />
• Ran gel with: <br /><br />
1. GFP+TT 1 X,P <br /><br />
2. GFP+TT 1 X,P <br /><br />
3. GFP+TT 1 E,S <br /><br />
4. GFP+TT 1 E,S <br /><br />
5. GFP+TT 2 X,P <br /><br />
6. GFP+TT 2 X,P <br /><br />
7. GFP+TT 2 E,S <br /><br />
8. GFP+TT 2 E,S <br /><br />
9. Ladder <br /><br />
10. A3 1/50 PCR X,P <br /><br />
11. A3+pSB1C3 6 X,P <br /><br />
12. pVeg 96 X,P <br /><br />
13. pVeg+pSB1C3 1 X,P <br /><br />
14. R0079 X,P <br /><br />
15. AmyE 5’ S,P <br /><br />
<br />
<h2>10/16/12</h2><br /><br />
• Made liquid cultures from yesterday transformations: <br /><br />
- AmyE 5’+R0079 <br /><br />
- AmyE 5’+R0079_2 <br /><br />
- AmyE 5’+A3 <br /><br />
- AmyE 5’+pVeg <br /><br />
- pBad/pXyl+E0040+B0014 in pSB1C3 plasmid. <br /><br />
• Digested: <br /><br />
- AmyE 5’+pBad/pXyl+P4+CI+E1010+B0014 colonies 1,2,7 and 8 with E,P. <br /><br />
- pVeg+XylR 1,4,5 and 12 with E,P. <br /><br />
- R0079+GFP+TT and R0079+GFP+TT_2 with X,P. <br /><br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/ANDSugar
Team:UNAM Genomics Mexico/Notebook/ANDSugar
2012-10-27T03:48:11Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Arabinose/Xylose AND Gate Notebook'''</h1></center><br />
<br /><br />
<br /><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="20"><br />
<tr><br />
<td id="leftcolumn2"><center>2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JUNE | '''JUNE''']] </center> <br /><br />
<br /><br />
2.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F07.2F12 |06/07/12]] <br /><br />
2.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F12.2F12 |06/12/12]] <br /><br />
2.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F13.2F12 |06/13/12]] <br /><br />
2.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F14.2F12 |06/14/12]] <br /><br />
2.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F15.2F12 |06/15/12]] <br /><br />
2.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F18.2F12 |06/18/12]] <br /><br />
2.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F19.2F12 |06/19/12]] <br /><br />
2.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F22.2F12 |06/22/12]] <br /><br />
2.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F25.2F12 |06/25/12]] <br /><br />
2.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F26.2F12 |06/26/12]] <br /><br />
2.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F27.2F12 |06/27/12]] <br /><br />
2.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F29.2F12 |06/29/12]] <br /><br />
</td><br />
<br />
<td id="contentcolumn2"><center>3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JULY | '''JULY''']] </center> <br /><br />
<br /><br />
3.1[[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F03.2F12 | 07/03/12]] <br /><br />
3.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F04.2F12 | 07/04/12]] <br /><br />
3.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F06.2F12 | 07/06/12]] <br /><br />
3.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F07.2F12 | 07/07/12]] <br /><br />
3.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F08.2F12 | 07/08/12]] <br /><br />
3.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F09.2F12 | 07/09/12]] <br /><br />
3.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F10.2F12 | 07/10/12]] <br /><br />
3.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F11.2F12 | 07/11/12]] <br /><br />
3.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F12.2F12 | 07/12/12]] <br /><br />
3.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F13.2F12 | 07/13/12]] <br /><br />
3.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F14.2F12 | 07/14/12]] <br /><br />
3.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F16.2F12 | 07/16/12]] <br /><br />
3.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F17.2F12 | 07/17/12]] <br /><br />
3.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F18.2F12 | 07/18/12]] <br /><br />
3.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F19.2F12 | 07/19/12]] <br /><br />
3.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F20.2F12 | 07/20/12]] <br /><br />
3.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F23.2F12 | 07/23/12]] <br /><br />
3.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F24.2F12 | 07/24/12]] <br /><br />
3.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F25.2F12 | 07/25/12]] <br /><br />
3.20 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F26.2F12 | 07/26/12]] <br /><br />
3.21 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F27.2F12 | 07/27/12]] <br /><br />
3.22 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F30.2F12 | 07/30/12]] <br /><br />
3.23 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F31.2F12 | 07/31/12]] <br /><br />
</td><br />
<br />
<td id="rightcolumn2"><center>4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#AUGUST | '''AUGUST''']] </center> <br /><br />
<br /><br />
4.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F01.2F12 | 08/01/12]] <br /><br />
4.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F02.2F12 | 08/02/12]] <br /><br />
4.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F03.2F12 | 08/03/12]] <br /><br />
4.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F06.2F12 | 08/06/12]] <br /><br />
4.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F08.2F12 | 08/08/12]] <br /><br />
4.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F09.2F12 | 08/09/12]] <br /><br />
4.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F10.2F12 | 08/10/12]] <br /><br />
4.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F13.2F12 | 08/13/12]] <br /><br />
4.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F16.2F12 | 08/16/12]] <br /><br />
4.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F20.2F12 | 08/20/12]] <br /><br />
4.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F21.2F12 | 08/21/12]] <br /><br />
4.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F22.2F12 | 08/22/12]] <br /><br />
4.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F23.2F12 | 08/23/12]] <br /><br />
4.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F24.2F12 | 08/24/12]] <br /><br />
4.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F25.2F12 | 08/25/12]] <br /><br />
4.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F28.2F12 | 08/28/12]] <br /><br />
4.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F29.2F12 | 08/29/12]] <br /><br />
4.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F30.2F12 | 08/30/12]] <br /><br />
4.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F31.2F12 | 08/31/12]] <br /><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2"><center>5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#SEPTEMBER | '''SEPTEMBER''']] </center> <br /><br />
<br /><br />
5.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F01.2F12 | 09/01/12]] <br /><br />
5.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F02.2F12 | 09/03/12]] <br /><br />
5.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F04.2F12 | 09/04/12]] <br /><br />
5.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F05.2F12 | 09/05/12]] <br /><br />
5.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F06.2F12 | 09/06/12]] <br /><br />
5.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F14.2F12 | 09/14/12]] <br /><br />
5.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F16.2F12 | 09/16/12]]<br /><br />
5.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F17.2F12 | 09/17/12]]<br /><br />
5.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F18.2F12 | 09/18/12]]<br /><br />
5.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F19.2F12 | 09/19/12]]<br /><br />
5.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F19.2F12 | 09/19/12]]<br /><br />
5.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F20.2F12 | 09/20/12]]<br /><br />
5.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F21.2F12 | 09/21/12]]<br /><br />
5.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F22.2F12 | 09/22/12]]<br /><br />
5.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F23.2F12 | 09/23/12]]<br /><br />
5.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F24.2F12 | 09/24/12]]<br /><br />
5.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F26.2F12 | 09/26/12]]<br /><br />
5.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F27.2F12 | 09/27/12]]<br /><br />
<br />
<br />
<br />
</td><br />
<br />
<td id="contentcolumn2"><br />
'''OCTOBER'''<br /><br />
5.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F09.2F12 | 10/09/12]]<br /><br />
5.20 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F10.2F12 | 10/10/12]]<br /><br />
5.21 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F11.2F12 | 10/11/12]]<br /><br />
5.22 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F12.2F12 | 10/12/12]]<br /><br />
5.23 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F13.2F12 | 10/13/12]]<br /><br />
</td><br />
<br />
<td id="rightcolumn2"><center>[[File:UnmagenomicsBacteria sugar.png | 190px]]</center><p> '''On hover the images to see descriptions'''</p><td/><br />
</tr><br />
</table><br />
<br />
<br />
=JUNE=<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html> <br />
<h2>06/07/12</h2><br /><br />
PY BROTH 10g salt/L<br /><br />
PSB2K3 kanamicin<br /><br />
We transformed RFP E1010. <br /><br />
plate 1 18F<br /><br />
plate 2 17E<br /><br />
Stock 50 mg/mL<br /><br />
''E.coli'' 1/1000<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
We made liquid cultures with colonies [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
We extracted plasmids [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PLASMID_EXTRACTION_2 | PLASMID EXTRACTION PROTOCOL]]. <br /><br />
We ran a gel to check the extraction [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<h2>06/12/12</h2><br /><br />
We made glycerols of the bacteria <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed plasmid pHp45 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/38/UnamgenomicsandsugarBitacora_2.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>06/13/12</h2><br /><br />
We ran gel and extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] and lysed. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2d/UnamgenomicsandsugarBitacora_3.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
We extracted from gel<br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/14/12</h2><br /><br />
We extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made glycerols of pHp45 Ω [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450<br />
PSB4A5 Am (ampicillin) 1I BBa_J04450<br />
AraC BBa_C0080 <br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
2011 14L plate 1 pSB2K3 Km+<br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/5/56/UnamgenomicsandsugarBitacora_5.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
pHp45 Ω with E/P<br /><br />
We extracted from gel<br /><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/15/12</h2><br /><br />
> Digested pHp45 Ω with E/P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
> We extracted plasmid with kit. <br /><br />
> Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (5) <br /><br />
>Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br /><br />
ARAC<br /><br />
>From the overnight plate we prepared liquid culture [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
>We left them incubating overnight. <br /><br />
>We left a plate (LB Km DH5α C0080 and a control). <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<h2>06/18/12</h2><br /><br />
>Plasmid extraction AraC with kit. <br /><br />
>C0080-psB2K3 915 bp<br /><br />
>Streaked 4 LB Km 30 plates DH5α psB2K3. <br /><br />
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red. <br /><br />
>Ran gel with Spr/Strr (eppendorf -20ºC) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
>Made glycerols from one tube of DH5α C0080 Km 50 -> 04. <br /><br />
>Made glycerols from one tube of DH5α PSB4A5 Amp 100 -> 05 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]]. <br /><br />
>Digested C0080 X,S. <br /><br />
>Digested B0014 E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/5/52/UnamgenomicsandsugarBitacora_6.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
Digested B0014 with E, P and with E,X<br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<br /><br />
<br />
<h2>06/19/12</h2><br /><br />
>Digested B0014 with E, P and with E,X. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
>Ran gels with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation. <br /><br />
C0080 is in the first lane. <br /><br />
>Extracted from gel C0080 X,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Made glycerols from 4 plates LB Km DH5α pSB2K3 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
>Take out AmyE 5’ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
AmyE 5’ 18K plate 3 2010, 2011, 2012<br /><br />
AmyE 3’ 18M plate 3 2010, 2011, 2012<br /><br />
>Digested B0014 with E,X and E,P again [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<h2>06/22/12</h2><br /><br />
<br />
>Ran gels with digestions B0014 with E,X and E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
<br />
>Dephosphated B0014 E,X and B0014 E,P. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<h2>06/25/12</h2><br /><br />
>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
<br />
<h2>06/26/12</h2><br /><br />
>Ran gel with psB2K3 and psB4A5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
<h2>06/27/12</h2><br /><br />
>Transformed with plasmid B0079 1576 bp psB1A2 12A [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011<br /><br />
AmyE 5’ BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011<br /><br />
AmyE 3’ BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011<br /><br />
AmyE 5’ grew 2 colonies. <br /><br />
<br />
<h2>06/29/12</h2><br /><br />
>We did a DH5α K143001 Km30 Amp 100 glycerol [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] 07. <br /><br />
>We plated twice DH5α K143002 Km 30 Amp 100 + control and twice DH5α B0079 Amp 100 + control<br /><br />
These were both plated in 2 plates each. <br /><br />
They were left overnight since they had not grown by 7:00 pm so the next day glycerols were made. <br /><br />
>Liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
2 tubes DH5α K143001 Km30 Amp 100 <br /><br />
2 tubes DH5α K143002 Km30 Amp 100 <br /><br />
2 tubes DH5α B0079 Amp 100 <br /><br />
1 tube LB Km 30 Amp 100 control<br /><br />
1 tube LB Amp 100 control<br /><br />
>From the 6 tubes we extracted plasmid from kit. <br /><br />
<br /><br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=JULY=<br />
<br />
07/02/12<br /><br />
>Digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /><br />
>PCR’s<br /><br />
•AraC<br /><br />
•Cassete ΩSpr/Strr <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PCR_Protocol_.2850.C2.B5l.29 | PCR PROTOCOL]] <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/be/UnamgenomicsandsugarBitacora_8.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>After Cassete ΩSpr/Strr PCR we ran a gel <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsandsugarBitacora_9.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/03/12</h2><br /><br />
>After Cassete ΩSpr/Strr PCR we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (8) <br /><br />
>Ran gel with digestions from yesterday. (9) <br /><br />
>Did band extractions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Stored at -20ºC. <br /><br />
>Did PCR to Cassete ΩSpr/Strr to add RBS and prefix/suffix. <br /><br />
>Left digesting with E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/03/UnamgenomicsandsugarBitacora_9.1.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/04/12</h2><br /><br />
>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1). <br /><br />
>Ligated Cassete ΩSpr/Strr +K143002 dephosphated(digested with E,X and E,S). <br /><br />
>Transformed ligation and left overnight plated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs. <br /><br />
>Digested with PstI [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<h2>07/06/12</h2><br /><br />
4 LB Km30 Spec60 DH5α ΩSpr/Strr +K143002 still have not grown. <br /><br />
Ran a gel with yesterday’s digestions to chek if they were done properly (10) <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
Ran another gel with the rest of the samples. <br /><br />
Extracted GusA fragment <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
Repeated ligation ΩSpr/Strr +K143002 E,X dephosphated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Repeated ΩSpr/Strr PCR. <br /><br />
<br /><br />
<br /><br />
<h2>07/07/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002 . <br /><br />
<br /><br />
<br /><br />
<br />
<h2>07/08/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002. <br /><br />
<br /><br />
<br /><br />
<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/13/UnamgenomicsandsugarBitacora_11.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b2/UnamgenomicsandsugarBitacora_11.1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1 PCR omega P<br /><br />
2 PCR omega I<br /><br />
3 PCR AraC P<br /><br />
4 PCR AraC I<br /><br />
5 ladder 1 Kb<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>07/09/12</h2><br /><br />
From yesterday’s transformations only one colony grew. <br /><br />
From the previous transformation only 2 colonies grew. <br /><br />
These 3 were streaked in 3 plates: <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km 30 Sp 100 control. <br /><br />
Did liquid cultures in 3 tubes: <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km30 Sp 100 control. <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
Ran a gel with: (11) <br /><br />
GusA P<br /><br />
PBBR1 GusA<br /><br />
Ω E PCR P<br /><br />
Ω PCR P<br /><br />
Ω PCR I<br /><br />
Ω E<br /><br />
Ω PCR I<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] <br /><br />
Did GusA primers dissolution for PCR. <br /><br />
GusA PCR <br /><br />
Gel Extraction by kit of lanes 3 and 5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
'''PCR omega P, PCR omega I, PCR AraC P, PCR AraC I'''<br /><br />
Add 10 μl of each primer (LW and UP). <br /><br />
Add 3 μl of plasmid (P). <br /><br />
Add 30.4 μl buffer. <br /><br />
Add 5 μl Mg. <br /><br />
Add 8 μl DNTp’s. <br /><br />
Add 42.6μl H2O miliQ. <br /> <br />
Add 1 μl RTTG polymerase. <br /><br />
Centrifuge (spin) 8 secs. <br /><br />
Add vegetable oil till the eppendorf is full. <br /><br />
Place eppendorf 1 mL in thermocycler. <br /><br />
Run PCR with program “BERNA”. <br /><br /><br />
<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/4b/UnamgenomicsandsugarBitacora_11.2.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/10/12</h2><br /><br />
•GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S (2 times)[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTIONS]]. (11.2) <br /><br />
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<br /><br />
<br />
<h2>07/11/12</h2><br /><br />
•GusA PCR digestions with E,S; Ω+AmyE 3’ with E,X [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•T.V. AraC PCR+ Ω+AmyE 3’ E,X dephosphated ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/0a/UnamgenomicsandsugarBitacora_12.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1. ladder. <br /><br />
2. GusA PCR E,S. <br /><br />
3. Ω+AmyE 3’ with E,X. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/12/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: (12) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Transformed AraC PCR+ Ω+AmyE 3’ ligation in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•From pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid. <br /><br />
•Made glycerols from 2 LB Km 30 Sp100 DH5α Ω+AmyE 3’ plates and from 1 LB Amp100 pfrc54 (A3) plates with control[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
•Ligated GusA PCR with B0014 E,X desphophorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
•Digested Pfrc54 (A3) with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•Desphophorylated Ω+AmyE 3’ E,X. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/0f/UnamgenomicsandsugarBitacora_13.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>•Ran gel with pfrc54 S,P<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/13/12</h2><br /><br /><br />
•Ran gel with pfrc54 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (13) <br /><br />
•Transformation of GusA PCR + B0014 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Transformed with GFP E0040 psBIA2. <br /><br />
•Liquid Cultures with ligation: AraC+ Ω+AmyE 3’ for plasmid extraction at night [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•''B. Subtitils'' competent cells. <br /><br />
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<h2>07/14/12</h2><br /><br /><br />
•From 24 AraC+ Ω+AmyE 3’ LB Km 30 Sp 100 DH5α tubes: <br /><br />
•Extracted pellets<br /><br />
•Extracted plasmids. <br /><br />
•Ran gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br /><br />
•From transformed DH5α km 30: <br /><br />
GusA+B0014 DH5α Km50 24 pellets <br /><br />
E0040 DH5α LB Amp100 <br /><br />
From these two we: <br /><br />
•Did liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Extracted plasmid. <br /><br />
•From the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ran gel with this transformation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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<br /><br />
<br /><br />
<h2>07/16/12</h2><br /><br /><br />
•Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
•Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_14.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>07/17/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (14) <br /><br />
•The gel we ran didn’t work, probably because the agarose was not prepared correctly. <br /><br />
•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<br /><br />
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<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/db/Bitacora_15.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<br />
<h2>07/18/12</h2><br /><br /><br />
•From yesterday’s digestions we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (15) <br /><br /><br />
•We made liquid cultures of AraC+ Ω+AmyE 3’ 12, 15, 21 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br /><br />
•We ligated AraC PCR E,S dephosphated with + Ω+AmyE 3’ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
•We digested 1 + Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/19/12</h2><br /><br /><br />
•We ran We digested 1 + Ω+AmyE 3’ E,P with Miguel’s enzymes and with Abiel’s enzymes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
•We transformed with ligations AraC+ Ω+AmyE 3’ E and + Ω+AmyE 3’ E [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br /><br />
•Extracted plasmids from AraC+ Ω+AmyE 3’ 12,15,21. <br /><br /><br />
•Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f3/UnamgenomicsandsugarBitacora_16.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/20/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (16) <br /><br />
•Transformed GusA+B0014 in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Made liquid cultures from AraC+ Ω+AmyE 3’s transformation LB Km30 Sp 100 and control LB Km 30 Sp100 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<h2>07/23/12</h2><br /><br /><br />
•From 24 GusA+B0014 tubes (-) we didn’t do anything. <br /><br />
•From 4 AraC+ Ω+AmyE 3’ we extracted plasmid. <br /><br />
•Digested from the 4 AraC+ Ω+AmyE 3’ E,X; X; P and GusA PCR with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/24/12</h2><br /><br /><br />
•Digested B0014 E with X<br />
B0014 X with E<br /><br />
•Digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<h2>07/25/12</h2><br /><br /><br />
•Ran gel of digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Joined B0014 E with X B0014 X with E digestions. <br /><br />
•Made liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. Transformed [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•From LasR DH5α make liquid cultures and plate [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Digested Ω+AmyE 3’ with X,P; E,S; C0080 X,P; E,S; S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Ligated Gus PCR X,P with B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/84/UnamgenomicsandsugarBitacora_17.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1.Ω+AmyE 3’ X,P<br /><br />
2. Ω+AmyE 3’ E,S<br /><br />
3.C0080 X,P<br /><br />
4.C0080 E,S<br /><br />
5.C0080 S,P<br /><br />
6.ladder<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/26/12</h2><br /><br />
•From C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5α C0179 LB Cb 100 (two cultures) and a control without bacteria [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•We extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguel’s transformation), Puc, B.S. control and PSB4AE. <br /><br />
<br />
•Due to problems with the way we did the transformations of ligations we repeated them: <br /><br />
GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
<br />
GusAPCR X,P+ B0079 S,P dephosphorylated (-)<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•AraC+ Ω+AmyE 3’ S,P 1,2,3,4<br /><br />
•K143002 X,P<br /><br />
•AraC+ Ω S,P<br /><br />
•C0179 X,S<br /><br />
<br />
<br />
<h2>07/27/12</h2><br /><br /><br />
Ligated AraC+ Ω S,P dephosphorylated + K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<h2>07/30/12</h2><br /><br /><br />
•Transformed with ligations: <br /><br />
•AraC+ Ω dephosphprylated +K143002 X,P<br /><br />
•GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
•GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
•Transformed the following sythesis: <br /><br />
•91996 Pveg 140 bp<br /><br />
•91997 ArsR-CzrA_promoter 1 194 bp<br /><br />
•91998 ArsR-CzrA_promoter 2 221 bp<br /><br />
•91999 ArsR-CzrA_promoter 3 213 bp<br /><br />
•92000 pBad-pXyl 387 bp<br /><br />
•92001 XylR 1117pb<br /><br />
•92002 CI_pro_(NAND_INHIBITOR) 774 <br /><br />
•These synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked.<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Make liquid cultures of the following transformations for tomorrow [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]: <br /><br />
•AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+B0079<br /><br />
•Synthesis<br /><br />
<br />
<h2>07/31/12</h2><br /><br /><br />
Extracted plasmids from yesterday’s cultures and from K143002 cultures that were in the other lab. <br /><br />
<br /><br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=AUGUST=<br />
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<h2>08/01/12</h2><br /><br /><br />
Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b1/UnamgenomicsandsugarBitacora_17.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1 PCR GusA I<br /><br />
2 PCR GusA I<br /><br />
3 PCR GusA P<br /><br />
4 PCR GusA P<br /><br />
5 ladder 1 Kb<br /></p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/02/12</h2><br /><br /><br />
•Extracted plasmids from liquid cultures: <br /><br />
AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+BBR1<br /><br />
<br />
PCR GusA, GusA I, PCR GusA P, PCR GusA I<br /><br />
<br />
•Add 10 μl of each primer (LW and UP). <br /><br />
•Add 3 μl of plasmid (P). <br /><br />
•Add 30.4 μl buffer. <br /><br />
•Add 5 μl Mg. <br /><br />
•Add 8 μl DNTp’s. <br /><br />
•Add 42.6μl H2O miliQ. <br /><br />
•Add 1 μl RTTG polymerase. <br /><br />
•Centrifuge (spin) 8 secs. <br /><br />
•Add mineral oil till the eppendorf is full. <br /><br />
•Place eppendorf 1 mL in thermocycler. <br /><br />
•Run PCR with program “BERNA”. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/82/UnamgenomicsandsugarBitacora_18.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.A3+GusA 1<br /><br />
4.A3+GusA 2<br /><br />
5.A3+GusA 3<br /><br />
6.P GusA<br /><br />
7.P GusA 2<br /><br />
8.Ladder<br /><br />
9.01<br /><br />
10.06<br /><br />
11.96<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<br /><br />
<br />
<h2>08/03/12</h2><br /><br /><br />
• Ran a gel with: (18) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Ran another gel to extract with: <br /><br />
1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.00<br /><br />
4.01<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]: <br /> <br />
<br />
• Ω+K143002 E,X; A3+GusA E,S; GusAPCR X,P <br /><br />
<br />
•Extracted GusAPCR X,P digestion [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Left the following ligation: AraC+ Ω+K143002 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/17/UnamgenomicsandsugarBitacora_19.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3+GusA 2<br /><br />
2.A3+GusA 3<br /><br />
3. Ω+AmyE 3’ 2<br /><br />
4. Ω+AmyE 3’ 3<br /><br />
5.XylR X,P<br /><br />
6.pBad-pXyl X,P<br /><br />
7.GusA PCR X,P<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
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<br />
<h2>08/06/12</h2><br /><br /><br />
•Made liquid cultures of Ω+K143002 (2 cultures+1 control) and AraC+ Ω+K143002 (2 cultures+1 control) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ligated GusAPCR X,P+B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
•Ran a Gel with: (19) <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/8b/UnamgenomicsandsugarBitacora_19.1.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/08/12</h2><br /><br />
BBa_B0040 6I psB1A2 Amp+ plate 1<br /><br />
Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (19.1) <br /><br />
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<h2>08/09/12</h2><br /><br />
•From yesterday’s PCR’s : <br /><br />
• E0040 plasmid 1<br /><br />
E0040 plasmid 2<br /><br />
E0040 digested 1<br /><br />
E0040 digested 2<br /><br />
We purified with PCR kit<br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Made 14 PCR’s from AraC+ Ω+AmyE 3’. <br /><br />
•From B0079+GusA ligation and B0040 transformation:<br />
•Grew colonies. <br /><br />
•Made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Streaked these in a new plate. <br /><br />
•Diluted plasmid E0080 2 1/50. <br /><br />
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<h2>08/10/12</h2><br /><br />
•Yesterday’s gel did not come out as expected so we repeated the PCR. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/e/ef/UnamgenomicsandsugarBitacora_20.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.AraC+ Ω+AmyE 3’ <br /><br />
2-14.AraC+ Ω+AmyE 3’ <br /><br />
15.AraC+ Ω 1<br /><br />
16.AraC+ Ω 1<br /><br />
17.AraC+ Ω 1<br /><br />
18.PCR 1 GFP<br /><br />
19.PCR GFP<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<h2>08/13/12</h2><br /><br />
•After running a gel with 14 PCR’s AraC+ Ω+AmyE 3’ and it did not come out as expected, we ran a gel with the ligation AraC+ Ω+AmyE 3’ to select candidate (that weight the same as the ligation) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (20) <br /><br />
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•Extracted pasmids form liquid cultures: B0049, B0079+GusA. <br /><br />
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•AraC+ Ω+AmyE 3’ undigested didn’t run in the gel as expected, it ran similarly to AraC+ Ω<br /><br />
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• We repeated the ligation AraC+ Ω S,P dephosphorylated+K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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• GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.<br />
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• We ran a gel to extract. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f1/UnamgenomicsandsugarBitacora_22.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>1.6 AraC+ Ω+AmyE 3’ E with P. <br /><br /><br />
2.8 AraC+ Ω+AmyE 3’ E with P. <br /><br />
3.11 AraC+ Ω+AmyE 3’ E with P. <br /><br />
4.12 AraC+ Ω+AmyE 3’ E with P. <br /><br />
5.6 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
6.8 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
7.11 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
8.12 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
9.00 pBad/pXyl with S,P. <br /><br />
10.Ladder. <br /><br />
11.E0040 PCR E,S to extract the correct band. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/16/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: <br /><br />
<br />
•Digested: <br /><br />
ArSR-CzrA 97 with S,P<br /><br />
ArSR-CzrA 98 with S,P<br /><br />
ArSR-CzrA 99 with S,P<br /><br />
E0040 PCR with E,S<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
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<h2>08/20/12</h2><br /><br />
•Ligated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/21/12</h2><br /><br />
•Joined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated. <br /><br />
•Extracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR. <br /><br />
•Digested Ω+AmyE 3’ with E,X and + Ω+AmyE 3’ II with E,X (obtained these two from glycerols stored at -80ºC [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/29/UnamgenomicsandsugarBitacora_22.1.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested K143001+PBad, pXyl with E,S <br /> <br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/1c/UnamgenomicsandsugarBitacora_23.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>1.K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
4.A3 PCR<br /><br />
5.A3 PCR<br /><br />
6.E0040 PCR E,S<br /><br />
7.B0014 E,X dephosphorylated<br /><br />
8. Ω+AmyE 3’ E,X <br /><br />
9. Ω+AmyE 3’ E,X II<br /><br />
10.Ladder<br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/22/12</h2><br /><br />
•We did 2 PCR’s for A3. <br /><br />
<br />
•Digested K143001+PBad, pXyl with E,S <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (22.1) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did band extraction of: <br /><br />
1. K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
•Made liquid cultures from E1010+Boo14 that the other lab gave us and did 2 plates for glycerol Km+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Did 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ Ω+AmyE 3’ 6,8,11,12 Km+ and Spectinomycin+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Transformed ligations: <br /><br />
B0079+GusA Amp+<br /><br />
Pveg+XylR Chloramphenicol+<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]].. <br /><br />
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<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/78/UnamgenomicsandsugarBitacora_23.01.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1. Ω+AmyE 3’ E,X 6.2<br /><br />
2.Ω+AmyE 3’ II E,X<br /><br />
3.97 ArsR-CzrA<br /><br />
4.98 ArsR-CzrA<br /><br />
5.99 ArsR-CzrA<br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/23/12</h2><br /><br />
•The negative controls were contaminated. We didn’t manipulate the ones that had grown because of this. <br /><br />
•Ran gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
<br />
<br />
<h2>08/24/12</h2><br /><br />
From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ Ω+AmyE 3’ 6 and 8 we streaked 2 plates for glycerol of each ligation. <br />Repeated ligation B0079+GusA. <br /><br />
<br />
Ligated E0040 PCR E,S+B0014 E,X dephospohylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
Made 2 new plates from Pveg+XylR ligation and liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<h2>08/25/12</h2><br /><br />
Extracted pellet from 16 tubes of pVeg+XylR that were left overnight. <br /><br />
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Ligated B0079+GusA and B0040+B0014 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
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<h2>08/28/12</h2><br /><br />
<br />
Made liquid cultures from transformations tht were left overnight <br />
-R0079+GusA<br /><br />
-R0079+GusA (-)<br /><br />
-E0040+B0014<br /><br />
-E0040+B0014 (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/25/UnamgenomicsandsugarBitacora_23.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. A3 PCR 1. <br /><br />
2. A3 PCR 2. <br /><br />
3. Ω+AmyE 3’ S,P *.<br /><br />
4. Ω+AmyE 3’ S,P . <br /><br />
5. Pveg+XylR E,S 2. <br /><br />
6. Pveg+XylR E,S 3. <br /><br />
7. Pveg+XylR E,S 4. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/29/12</h2><br /><br />
Digested Pveg+XylR 2,3,4 with E,S. <br /><br />
<br />
Digested Ω+AmyE 3’ with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTION]]. <br /><br />
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Ran a PCR with A3 PCR (2 .6 ml tubes). <br /><br />
<br />
Ran a gel with <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2e/UnamgenomicsandsugarBitacoraBitacora_24.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1-23. E0040 + B0014 colonies<br /><br />
24-29. B0079+GusA 1<br /><br />
30. B0079<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/30/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (24) <br /><br />
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<br />
•Digested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='thumbnailWrapper'><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/3d/UnamgenomicsandsugarBitacora_25.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1-3B0079+GusA E,S 3. <br /><br />
4-9. PVeg+XylR E,S 1. <br /><br />
10-17. E0040+B0014 X,P 3. <br /><br />
18. 1 kb ladder. <br /><br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/d2/UnamgenomicsandsugarBitacora_26.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3 PCR. <br /><br />
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</li><br />
</ul><br />
</div><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7b/UnamgenomicsandsugarBitacora_27.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>E0040+B0014 X,P 4 to extract<br /><br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
<h2>08/31/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (25) <br /><br />
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<br />
•Dephosphorylated: <br /><br />
Ω+AmyE 3’ S,P <br /><br />
Ω+AmyE 3’ II S,P <br /><br />
6 AraC+ Ω+AmyE 3’ E,X<br /><br />
8 AraC+ Ω+AmyE 3’ E,X<br /><br />
11 AraC+ Ω+AmyE 3’ E,X<br /><br />
12 AraC+ Ω+AmyE 3’ E,X<br /><br />•Ran a gel with A3 PCR [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (26) <br /><br />
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•It was the second time we didn’t obtain a band from A3’s PCR.<br />
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•Probably because it had been missing the buffer 2’s ethanol (“blue lid”) from the PCR purification. <br /><br />
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•Ran a gel woth E0040+B0014 X,P 4 to extract <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | Gen extraction protocol]]. <br /><br />
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•Did an A3 PCR. <br /><br />
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<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
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=SEPTEMBER=<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b8/UnamgenomicsandsugarBitacora_28.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.A3 PCR 1. <br /><br />
2.A3 PCR 2. <br /><br />
3.E0040+B0014 X,P 1. <br /><br />
4.E0040+B0014 X,P 2. <br /><br />
5.Ω+AmyE 3’ dephosphorylated S,P. <br /><br />
6.Ω+AmyE 3’ dephosphorylated S,P II. <br /><br />
7.97 dephosphorylated S,P. <br /><br />
8.98 dephosphorylated S,P. <br /><br />
9.99 dephosphorylated S,P. <br /><br />
10-13. Arac+ Ω+AmyE 3’ dephosphorylated E,X 12. <br /><br />
14. 1 kb Ladder. <br /><br />
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</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
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<br />
<h2>09/01/12</h2><br /><br />
•Ran a gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (28) <br /><br />
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•Ligated E0040+B0014 X,P 2 +promoter S,P dephosphorylated; K143002 X,P+ Ω+AmyE 3’ dephosphorylated S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<br /><br />
<br />
<h2>09/03/12</h2><br /><br />
<br />
•Digested B0079+GusA E,S 3,4,5; PVeg+XylR E,S 1,5,7,10,13,16 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<br />
<h2>09/04/12</h2><br /><br />
•Ran gel with digestions: <br /><br />
B0079+GusA E,S 3,4,5<br /><br />
PVeg+XylR E,S 1,5,7,10,13,16 <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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•Purified A3 PCR 1,2. <br /><br />
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•Ran a gel of A3 PCR <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] and digested with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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•From ligations: PVeg+E0040+B0014, PBad/PXyl+E0040+B0014, B0079+E0040+B0014 only colonies grew in PBad/PXyl+E0040+B0014.<br />
<br />
•Plated colonies that grew in a new plate. <br /><br />
<br />
•Made liquid cultures of these <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•Repeated the ones that did not grow. <br /><br />
<br />
•Digested E0040+B0014 4 with X,P[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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•Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
-BBa_J23101 18E plate 1 plasmid BBa_J61002_2948 bp Amp+<br /><br />
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<p class='captionInside'>24 PBad/PXyl+E0040+B0014 tubes we extracted <br /><br />
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<p class='captionInside'>1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
Extracted band from 1. and 2<br />
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<p class='captionInside'>Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S <br />
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<h2>09/05/12</h2><br /><br />
•From the 24 PBad/PXyl+E0040+B0014 tubes we extracted plasmids (29) and ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Extracted plasmid from 2 tubes of E0040+B0014. <br /><br />
<br />
•Ran a gel with yesterday’s digestions: <br /><br />
1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
<br />
•Extracted band from 1. and 2. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Purified A3 PCR from 2 0.6ml tubes 1 and 2. <br /><br />
<br />
•1 colony grew from ligation pVeg+E0040+B0014. <br /><br />
<br />
•We streaked this in another plate and did liquid cultures <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•To do: <br /><br />
•Ligate B0079 S,P dephosphorylated + E0040+B0014 X,P and Ω+AmyE 3’ S,P dephosphorylated+ K143001 X,P. <br /><br />
•Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S 10,13. (31) <br /><br />
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<p class='captionInside'>•A3 PCR and gel A3 PCR X,P <br />
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<h2>09/06/12</h2><br /><br />
<br />
•A3 PCR and gel A3 PCR X,P 1,2. <br /><br />
<br />
<br />
<h2>09/14/12</h2><br /><br />
<br />
•Digested Ω+AmyE 3’ 11,13,18, and 22 with E,X. <br /><br />
•Ligate: <br /><br />
PVeg S,P dephosphorylated+XylR X,P. <br /><br />
PBad p Xyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
pSB13C3 E,P dephosphorylated + A3 PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + GusA PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + XylR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pVeg E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pBad pXyl E,P. <br /><br />
pSB13C3 E,P dephosphorylated + Ω PCR E,P. <br /><br />
<br /><br />
<br />
<br /><br />
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<br /><br />
<h2>09/16/12</h2><br /><br />
• Colonies in these plates grew: <br /><br />
- R0079+E0040+B0014 <br /><br />
- pBad/pXyl <br /><br />
- XylR <br /><br />
- pVeg <br /><br />
- A3+E0040+B0014 from colonies 1,3,4,5,6,7,8,9,10,11,12,13,14,15,17 <br /><br />
- pVeg+E0040+B0014 1,2,3,4,5,6,8,9,10,11,12,13,14,15,16 <br /><br />
- pBad/pXyl+E0040+B0014 2 tubes from the same correct colony. <br /><br />
- pVeg+XylR 1,2 <br /><br />
- pBad/pXyl+pSB1C3 1,2 <br /><br />
- XylR+pSB1C3 1,2 <br /><br />
- pVeg+pSB1C3 1,2 <br /><br />
- A3+pSB1C3 1,2 <br /><br />
- GusA+pSB1C3 1,2 <br /><br />
- omegacassette+pSB1C3 1,2 <br /><br />
<br /><br />
• Ran gel from the plasmid extractions that we did yesterday: <br /><br />
- A3+E0040+B0014 1-16 <br /><br />
- pVeg+E0040+B0014 1-16, 18 <br /><br />
- omega cassette+AmyE 3’ 11,13,18,22 <br /><br />
• Digested R0079+E0040+B0014 with E,S. to ligate with AmyE 3’ E,x dephosphorylated. <br /><br />
• Digested omega cassette with E,P. <br /><br />
• Digested pSB1C3 with E,P. <br /><br />
• Made liquid cultures from AmyE 5’ and AmyE 3’. <br /><br />
• Did plasmid extraction from pSB1C3 colonies 3 and 6. <br /><br />
<br /><br />
<br />
<h2>09/17/12</h2><br /><br />
• Digested A3+E0040+B0014 colonies 2,3,4,5,6,11 and 16 with E,S. <br /><br />
• Digested pVeg+E0040+B0014 colonies 1,2,3,4,5,9 and 10 with E,S. <br /><br />
• Dephosphorylated omega cassette+AmyE 3’ colonies 13,18 and 22 digested with E,X. <br /><br />
• Digested pBad/pXyl and pVeg with S,P. Then dephosphorylated. <br /><br />
• Digested AmyE 3’ with E,X. Then dephosphorylated. <br /><br />
<br /><br />
<br />
<h2>09/18/12</h2><br /><br />
<br /><br />
• Ran gel with: <br /><br />
1. A3 2 E,S <br /><br />
2. A3 3 E,S <br /><br />
3. A3 4 E,S <br /><br />
4. A3 5 E,S <br /><br />
5. A3 6 E,S <br /><br />
6. A3 11 E,S <br /><br />
7. A3 16 E,S <br /><br />
8. E0040+B0014 colony 1 E,X <br /><br />
9. AmyE 5’ E,P <br /><br />
10. AmyE 3’ E,X <br /><br />
11. Omega cassette E,S <br /><br />
12. pSB1C3 E,P <br /><br />
13. ladder <br /><br />
Bottom: <br /><br />
1. pVeg 1 E,S <br /><br />
2. pVeg 2 E,S <br /><br />
3. pVeg 3 E,S <br /><br />
4. pVeg 4 E,S <br /><br />
5. pVeg 5 E,S <br /><br />
6. pVeg 9 E,S <br /><br />
7. pVeg 10 E,S <br /><br />
8. pVeg S,P dephosphorylated <br /><br />
9. pVeg 2 S,P <br /><br />
10. pBad/pXyl S,P <br /><br />
11. XylR X,P <br /><br />
12. R0079+GFP+TT E,S <br /><br />
13. R0079 S,P <br /><br />
14. Ladder <br /><br />
• Dephosphorylated GFP+TT E,X; AmyE 5’ E,X; pSB1C3 E,P; pVeg 2 S,P; pBad/pXyl S,P and R0079 S,P. <br /><br />
<br /><br />
<h2>09/19/12</h2><br /><br />
<br /><br />
• Did PCR from A3 and a 1/50 dilution. <br /><br />
• Ran gel with: <br /><br />
1. pVeg 3 E,P. <br /><br />
2. pVeg 9 E,P. <br /><br />
3. pVeg 10 E,P. <br /><br />
4. pBAd/pXyl 3 E,P. <br /><br />
5. pBAd/pXyl 5 E,P. <br /><br />
6. pBAd/pXyl 1 E,P. <br /><br />
7. A3 3 E,P. <br /><br />
8. A3 4 E,P. <br /><br />
9. XylR 1 E,P. <br /><br />
10. XylR 3 E,P. <br /><br />
11. XylR 8 E,P. <br /><br />
12. pSB1C3 6 E,P. <br /><br />
13. pSB1C3 6 E,P. <br /><br />
14. AmyE 5’ E,S. <br /><br />
15. AmyE 5’ E,S. <br /><br />
16. A3 1/50 PCR. <br /><br />
17. A3 PCR. <br /><br />
18. Ladder <br /><br />
• We did transformations: <br /><br />
- 2 of R0079+GusA <br /><br />
- R0079+GusA control <br /><br />
- 2 of A3+pSB1AK3 <br /><br />
- A3+pSB1AK3 control <br /><br />
- 2 of omega cassette+pSB1C3 <br /><br />
- 2 of GusA+pSB1C3 <br /><br />
• Digested: <br /><br />
- pVeg+pSB1C3 <br /><br />
- pBad/pXyl+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
- A3+pSB1C3 <br /><br />
With E,P. <br /><br />
• Ran gel with these digestions. <br /><br />
<br /><br />
<h2>09/19/12</h2><br /><br />
• Made liquids cultures from: <br /><br />
- Glicerol of AmyE 5’ <br /><br />
- Glicerol of AmyE 3’ <br /><br />
- pBad/pXyl+pSB1C3 colony 5 <br /><br />
• Made liquid cultures from yesterday transformations: <br /><br />
- R0079+GusA 2 and control <br /><br />
- A3+pSB1AK3 2 and control <br /><br />
- Omega cassette+pSB1C3 2 <br /><br />
- GusA+pSB1C3 2 <br /><br />
• We transformed yesterday ligations: <br /><br />
- pBad/pXyl+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
- pVeg+pSB1C3 <br /><br />
- A3+pSB1C3 <br /><br />
• Purified A3 PCR with kit, and then, digested with E,P and E,S. <br /><br />
• Ran gel <br /><br />
<br /><br />
<h2>09/20/12</h2><br /><br />
<br /><br />
• Made liquid cultures from transformations: <br /><br />
- Omega cassette+pSB1C3 colony 7 <br /><br />
- 2 of GusA+pSB1C3 colony 32 <br /><br />
- R0079+GusA colony 12 <br /><br />
• Digested A3 1/50 PCR with E,P and E,S. <br /><br />
• Made liquid cultures from R0079+E0040+B0014; pBad/pXyl+E0040+B0014; pVeg+E0040+B0014; A3+E0040+B0014. <br /><br />
• Ligated: <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P <br /><br />
- A3 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
<br /><br />
<h2>09/21/12</h2><br /><br />
<br /><br />
• Ran gel with: <br /><br />
1. R0079+GFP+TT 1 <br /><br />
2. R0079+GFP+TT <br /><br />
3. pBad/pXyl+pSB1C3 1 <br /><br />
4. pBad/pXyl+pSB1C3 <br /><br />
5. omega cassette+psB1C3 1 <br /><br />
6. omega cassette+psB1C3 <br /><br />
7. pSB1C3 E,P 1 <br /><br />
8. pSB1C3 E,P <br /><br />
9. pSB1C3 E <br /><br />
10. pSB1C3 P <br /><br />
11. A3 1/50 PCR E,P <br /><br />
<br /><br />
<br />
<h2>09/22/12</h2><br /><br />
• Ran gel with lysis extractions: <br /><br />
1. GusA+pSB1C3 1 <br /><br />
2. GusA+pSB1C3 2 <br /><br />
3. GusA+pSB1C3 3 <br /><br />
4. GusA+pSB1C3 4 <br /><br />
5. GusA+pSB1C3 5 <br /><br />
6. GusA+pSB1C3 6 <br /><br />
7. GusA+pSB1C3 7 <br /><br />
8. GusA+pSB1C3 8 <br /><br />
9. GusA+pSB1C3 9 <br /><br />
10. GusA+pSB1C3 10 <br /><br />
11. GusA+pSB1C3 11 <br /><br />
12. GusA+pSB1C3 12 <br /><br />
13. pSB1C3 <br /><br />
14. A3+E0040+B0014 1 <br /><br />
15. A3+E0040+B0014 2 <br /><br />
16. A3+E0040+B0014 3 <br /><br />
17. A3+E0040+B0014 4 <br /><br />
18. A3+E0040+B0014 5 <br /><br />
19. A3+E0040+B0014 6 <br /><br />
20. A3+E0040+B0014 7 <br /><br />
21. A3+E0040+B0014 8 <br /><br />
22. A3+E0040+B0014 9 <br /><br />
23. pVeg+E0040+B0014 3 <br /><br />
24. pVeg+E0040+B0014 4 <br /><br />
25. pVeg+E0040+B0014 5 <br /><br />
26. pVeg+E0040+B0014 6 <br /><br />
27. pVeg+E0040+B0014 7 <br /><br />
28. pVeg+E0040+B0014 8 <br /><br />
29. pVeg+E0040+B0014 9 <br /><br />
30. pVeg <br /><br />
31. pBad/pXyl+E0040+B0014 1 <br /><br />
32. pBad/pXyl+E0040+B0014 2 <br /><br />
33. pBad/pXyl+E0040+B0014 3 <br /><br />
34. pBad/pXyl+E0040+B0014 4 <br /><br />
35. pBad/pXyl+E0040+B0014 5 <br /><br />
36. pBad/pXyl+E0040+B0014 6 <br /><br />
37. pBad/pXyl+E0040+B0014 7 <br /><br />
38. pBad/pXyl+E0040+B0014 8 <br /><br />
39. pBad/pXyl+E0040+B0014 9 <br /><br />
40. pBad/pXyl+E0040+B0014 10 <br /><br />
41. pBad/pXyl <br /><br />
42. pVeg+XylR 1 <br /><br />
43. pVeg+XylR 2 <br /><br />
44. pVeg+XylR 3 <br /><br />
45. pVeg+XylR 4 <br /><br />
46. pVeg+XylR 5 <br /><br />
47. pVeg+XylR 6 <br /><br />
48. pVeg+XylR 7 <br /><br />
49. pVeg+XylR 8 <br /><br />
50. omega cassette <br /><br />
<br /><br />
• Ran gel with yesterday digestions: <br /><br />
1. R0079+GFP+TT E,P <br /><br />
2. R0079+GFP+TT E,P <br /><br />
3. Omega cassette+pSB1C3 E,P <br /><br />
4. Omega cassette+pSB1C3 E,P <br /><br />
5. Ladder <br /><br />
6. pBad/pXyl+pSB1C3 <br /><br />
7. pBad/pXyl+pSB1C3 <br /><br />
8. pSB1C3 6 E,P. <br /><br />
9. A3 1/50 PCR E,S <br /><br />
10. pBbak C011 <br /><br />
11. pBbak C012 <br /><br />
12. ladder <br /><br />
<br /><br />
• Did pellet from R0079+GusA. <br /><br />
• Did plasmid extraction from omega cassette+pSB1C3 colonies 1,4,6 and 7. <br /><br />
• Digested them with E,P. <br /><br />
• Digested: <br /><br />
- GusA+pSB1C3 1,3,4,10 and 11 <br /><br />
- A3+E0040+B0014 2,4,7 and 9 <br /><br />
- pVeg+E0040+B0014 4,5 and 6 <br /><br />
- pBad/pXyl+E0040+B0014 3,7 and 10 <br /><br />
- pVeg+XylR 2,4 and 5 <br /><br />
with E,P. <br /><br />
• Made liquid cultures from pVeg+pSB1C3 <br /><br />
• Ligated: <br /><br />
- XylR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- pVeg E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- A3 1/50 PCR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- A3 1/50 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
<br /><br />
<h2>09/23/12</h2><br /><br />
<br /><br />
• Ran gel with yesterday digestions: <br /><br />
1. Omega cassette+pSB1C3 1 E,P <br /><br />
2. Omega cassette+pSB1C3 4 E,P <br /><br />
3. Omega cassette+pSB1C3 6 E,P <br /><br />
4. Omega cassette+pSB1C3 7 E,P <br /><br />
5. GusA+pSB1C3 1 E,P <br /><br />
6. GusA+pSB1C3 3 E,P <br /><br />
7. GusA+pSB1C3 4 E,P <br /><br />
8. GusA+pSB1C3 10 E,P <br /><br />
9. GusA+pSB1C3 11 E,P <br /><br />
10. pSB1C3 E,P dephosphorylated <br /><br />
11. pVeg+XylR 2 E,P <br /><br />
12. pVeg+XylR 4 E,P <br /><br />
13. pVeg+XylR 5 E,P <br /><br />
14. pVeg S,P dephosphorylated <br /><br />
15. pVeg+GFP+TT 4 E,P. <br /><br />
16. pVeg+GFP+TT 5 E,P. <br /><br />
17. pVeg+GFP+TT 6 E,P. <br /><br />
18. ladder <br /><br />
Bottom <br /><br />
1. pBad/pXyl+GFP+TT 3 E,P <br /><br />
2. pBad/pXyl+GFP+TT 7 E,P <br /><br />
3. pBad/pXyl+GFP+TT 10 E,P <br /><br />
4. pBad/pXyl S,P dephosphorylated <br /><br />
5. A3+GFP+TT 2 E,P <br /><br />
6. A3+GFP+TT 4 E,P <br /><br />
7. A3+GFP+TT 7 E,P <br /><br />
8. A3+GFP+TT 9 E,P <br /><br />
9. Ladder <br /><br />
<br /><br />
<h2>09/24/12</h2><br /><br />
<br /><br />
• Did PCR from omega cassette+pSB1C3 colonies 1,4,6 and 7. <br /><br />
• Made 1/50 dilutions from these PCR products. <br /><br />
• Ran gel with: <br /><br />
1. Omega cassette+pSB1C3 1/50 PCR 1 <br /><br />
2. Omega cassette+pSB1C3 PCR 1 <br /><br />
3. Omega cassette+pSB1C3 1/50 PCR 4 <br /><br />
4. Omega cassette+pSB1C3 PCR 4 <br /><br />
5. Omega cassette+pSB1C3 1/50 PCR 6 <br /><br />
6. Omega cassette+pSB1C3 PCR 6 <br /><br />
7. Omega cassette+pSB1C3 1/50 PCR 7 <br /><br />
8. Omega cassette+pSB1C3 PCR 7 <br /><br />
9. GusA+pSB1C3 1 P <br /><br />
10. GusA+pSB1C3 3 P <br /><br />
11. GusA+pSB1C3 4 P <br /><br />
12. GusA+pSB1C3 10 P <br /><br />
13. GusA+pSB1C3 11 P <br /><br />
14. pVeg+pSB1C3 6 E,P <br /><br />
15. pVeg+pSB1C3 5 E,P <br /><br />
16. pVeg+pSB1C3 4 E,P <br /><br />
17. pVeg+pSB1C3 3 E,P <br /><br />
18. pVeg+pSB1C3 2 E,P <br /><br />
19. pVeg+pSB1C3 1 E,P <br /><br />
20. ladder <br /><br />
<br />
• From yesterday transformations, these didn’t grow: <br /><br />
- pBad/pXyl+E0040+B0014 1 and 2. <br /><br />
- A3+E0040+B0014. <br /><br />
• Made liquid cultures from transformations that did grow: <br /><br />
- pVeg+E0040+B0014. <br /><br />
- pVeg+XylR. <br /><br />
- A3+pSB1C3 <br /><br />
- pVeg+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
<br /><br />
<h2>09/26/12</h2><br /><br />
<br /><br />
• Did plasmid extractions form liquids cultures: <br /><br />
- 8 of pVeg+pSB1C3 <br /><br />
- 8 of A3 <br /><br />
- 8 of XylR <br /><br />
• Digested them with E,P. <br /><br />
• Ran gel with: <br /><br />
1. Ladder <br /><br />
2. XylR+pSB1C3 1 E,P <br /><br />
3. XylR+pSB1C3 2 E,P <br /><br />
4. XylR+pSB1C3 3 E,P <br /><br />
5. pVeg+pSB1C3 1 E,P <br /><br />
6. pVeg+pSB1C3 3 E,P <br /><br />
7. pVeg+pSB1C3 4 E,P <br /><br />
8. pVeg+pSB1C3 5 E,P <br /><br />
9. pVeg+pSB1C3 6 E,P <br /><br />
10. pVeg+pSB1C3 8 E,P <br /><br />
11. pSB1C3 6 E,P dephosphorylated <br /><br />
12. A3+pSB1C3 1 E,P <br /><br />
13. A3+pSB1C3 3 E,P <br /><br />
14. A3+pSB1C3 4 E,P <br /><br />
15. A3+pSB1C3 5 E,P <br /><br />
16. A3+pSB1C3 6 E,P <br /><br />
17. A3+pSB1C3 8 E,P <br /><br />
18. Ladder <br /><br />
19. P4+E0040+B0014 1 E,X <br /><br />
20. P4+E0040+B0014 7 E,X <br /><br />
<br /><br />
<h2>09/27/12</h2><br /><br />
<br /><br />
• Digested pVeg+XylR colonies 1,4,5 and 6 in pJ209 plasmid with E,S for 3 hours. <br /><br />
• Digested pVeg+E0040+B0014 colonies 7,9,10 and 11 in pasmid pJ209 with E,P. <br /><br />
<br /><br />
<h2>10/09/12</h2><br /><br />
<br /><br />
• Ligated: <br /><br />
- A3 1/50 PCR E,P + pSB1C3 6 E,P dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
- P4+CI+E1010+B0014 E,X dephosphorylated + AmyE 5’+pBad/pXyl E,S. <br /><br />
<br /><br />
<h2>10/10/12</h2><br /><br />
<br /><br />
• Ligated: <br /><br />
- GusA PCR E,P + pSB1C3 6 E,P dephosphorylated. <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- A3 1/50 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
• Digested R0079+E0040+B0014 with E,S. <br /><br />
• Digested AmyE 3’ with E,X. Then dephosphorylated. <br /><br />
• Digested pVeg and R0079, A3 with X,P. <br /><br />
• Digested pBad/pSB1C3 with S,P. <br /><br />
<br /><br />
<h2>10/11/12</h2><br /><br />
<br /><br />
• Did plasmid extraction from liquid cultures: <br /><br />
- 2 of AmyE 5’ <br /><br />
- 2 of AmyE 3’ <br /><br />
- 2 of E0040+B0014 <br /><br />
• Ran gel with: <br /><br />
1. A3 1/50 PCR X,P <br /><br />
2. A3 1/50 PCR X,P <br /><br />
3. A3+pSB1C3 6 X,P <br /><br />
4. A3+pSB1C3 6 X,P <br /><br />
5. A3+pSB1C3 8 X,P <br /><br />
6. A3+pSB1C3 8 X,P <br /><br />
7. pVeg X,P <br /><br />
8. pVeg 96 X,P <br /><br />
9. pVeg 96 X,P <br /><br />
10. pVeg+pSB1C3 1 X,P <br /><br />
11. pVeg+pSB1C3 1 X,P <br /><br />
12. pVeg+pSB1C3 3 X,P <br /><br />
13. pVeg+pSB1C3 3 X,P <br /><br />
14. R0079 X,P <br /><br />
15. Ladder <br /><br />
<br /><br />
<h2>10/12/12</h2><br /><br />
<br /><br />
• Ligated:<br />
- AmyE 5’+pBad/pXyl 10 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- AmyE 5’+pBad/pXyl 11 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- AmyE 5’+pBad/pXyl 12 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
- A3 1/50 PCR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
<br /><br />
<h2>10/12/12</h2><br /><br />
<br /><br />
• Digested AmyE 5’ with S,P <br /><br />
• Dephosphorylated it. <br /><br />
• Ligated: <br /><br />
- AmyE 5’ S,P dephosphorylated with <br /><br />
- R0079 X,P <br /><br />
- pVeg 96 X,P <br /><br />
- A3 1/50 PCR X,P <br /><br />
- A3+pSB1C3 6 X,P <br /><br />
- R0079+E0040+B0014 <br /><br />
• Ligated pBad/pXyl+pSB1C3 S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
• Ran gel with: <br /><br />
1. A3+pSB1C3 1 <br /><br />
2. A3+pSB1C3 2 <br /><br />
3. A3+pSB1C3 3 <br /><br />
4. A3+pSB1C3 4 <br /><br />
5. A3+pSB1C3 5 <br /><br />
6. A3+pSB1C3 6 <br /><br />
7. A3+pSB1C3 7 <br /><br />
8. A3+pSB1C3 8 <br /><br />
9. A3+pSB1C3 9 <br /><br />
10. A3+pSB1C3 10 <br /><br />
11. A3+pSB1C3 11 <br /><br />
12. A3+pSB1C3 6 control <br /><br />
13. AmyE 5’+P4+CI+E1010+B0014 1 <br /><br />
14. AmyE 5’+P4+CI+E1010+B0014 2 <br /><br />
15. AmyE 5’+P4+CI+E1010+B0014 3 <br /><br />
16. AmyE 5’+P4+CI+E1010+B0014 4 <br /><br />
17. AmyE 5’+P4+CI+E1010+B0014 5 <br /><br />
18. AmyE 5’+P4+CI+E1010+B0014 6 <br /><br />
19. AmyE 5’+P4+CI+E1010+B0014 7 <br /><br />
20. P4+CI+E1010+B0014 1 control <br /><br />
<br /><br />
<h2>10/09/12</h2><br /><br />
<br /><br />
• Ligate: <br /><br />
K143001+pBad/pXyl + p4+CI+E1010+TT E,X dephosphorylated. <br /><br />
• Ligate: <br /><br />
pVeg S,P dephosphorylated + XylR X,P. <br /><br />
• Ligate: <br /><br />
A3 PCR 1/50 E,P + pSB1C3 E,P dephosphorylated <br /><br />
<br /><br />
<h2>10/13/12</h2><br /><br />
<br /><br />
• Digested AmyE 5’ with S,P. Then dephosphorylated. <br /><br />
• Ligate: <br /><br />
AmyE 5’ S,P dephosphorylated + <br /><br />
- R0079 X,P <br /><br />
- pVeg 96 X,P <br /><br />
- A3 PCR 1/50 X,P <br /><br />
- A3+pSB1C3 colony 6 X,P <br /><br />
- R0079+E0040+B0014 E,X <br /><br />
• Ligate: <br /><br />
pBad/pXyl+pSB1C3 S,P dephosphorylated + E0040+B0014 X,P <br /><br />
<br />
• Ran a gel with: <br /><br />
1. A3+pSB1C3 1 <br /><br />
2. A3+pSB1C3 2 <br /><br />
3. A3+pSB1C3 3 <br /><br />
4. A3+pSB1C3 4 <br /><br />
5. A3+pSB1C3 5 <br /><br />
6. A3+pSB1C3 6 <br /><br />
7. A3+pSB1C3 7 <br /><br />
8. A3+pSB1C3 8 <br /><br />
9. A3+pSB1C3 9 <br /><br />
10. A3+pSB1C3 10 <br /><br />
11. A3+pSB1C3 11 <br /><br />
12. A3+pSB1C3 6 control <br /><br />
13. AmyE 5’+P4+CI+E1010+B0014 8 <br /><br />
14. AmyE 5’+P4+CI+E1010+B0014 9 <br /><br />
15. AmyE 5’+P4+CI+E1010+B0014 10 <br /><br />
16. P4+CI+E1010+B0014 1 control <br /><br />
17. pVeg+XylR 1 <br /><br />
18. pVeg+XylR 2 <br /><br />
19. pVeg+XylR 3 <br /><br />
20. pVeg+XylR 4 <br /><br />
21. pVeg+XylR 5 <br /><br />
22. pVeg+XylR 6 <br /><br />
23. pVeg+XylR 7 <br /><br />
24. pVeg+XylR 8 <br /><br />
25. pVeg+XylR 10 <br /><br />
26. pVeg+XylR 11 <br /><br />
27. pVeg+XylR 12 <br /><br />
28. pVeg 96 control <br /><br />
<br />
• Ran gel with: <br /><br />
1. A3 1/50 PCR X,P <br /><br />
2. A3+pSB1C3 6 X,P <br /><br />
3. pVeg 96 X,P <br /><br />
4. pVeg+pSB1C3 1 X,P <br /><br />
5. pVeg+pSB1C3 3 X,P <br /><br />
6. R0079 X,P <br /><br />
7. pBad/pXyl+pSB1C3 S,P dephosphorylated <br /><br />
8. ladder <br /><br />
9. Digested: <br /><br />
- A3+pSB1C3 colonies 3,4,5 and 10 with E,P. <br /><br />
- AmyE 5’+pBad/pXyl+P4+CI+E1010*B0014 colonies 1,2,7 and 8 with E,P. <br /><br />
- pVeg+XylR 1,4,5 and 12 with E,P. <br /><br />
• Ran gel with: <br /><br />
1. GFP+TT 1 X,P <br /><br />
2. GFP+TT 1 X,P <br /><br />
3. GFP+TT 1 E,S <br /><br />
4. GFP+TT 1 E,S <br /><br />
5. GFP+TT 2 X,P <br /><br />
6. GFP+TT 2 X,P <br /><br />
7. GFP+TT 2 E,S <br /><br />
8. GFP+TT 2 E,S <br /><br />
9. Ladder <br /><br />
10. A3 1/50 PCR X,P <br /><br />
11. A3+pSB1C3 6 X,P <br /><br />
12. pVeg 96 X,P <br /><br />
13. pVeg+pSB1C3 1 X,P <br /><br />
14. R0079 X,P <br /><br />
15. AmyE 5’ S,P <br /><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/ANDSugar
Team:UNAM Genomics Mexico/Notebook/ANDSugar
2012-10-27T03:47:26Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__TOC__<br />
<br /><br />
<center><h1>'''Arabinose/Xylose AND Gate Notebook'''</h1></center><br />
<br /><br />
<br /><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="20"><br />
<tr><br />
<td id="leftcolumn2"><center>2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JUNE | '''JUNE''']] </center> <br /><br />
<br /><br />
2.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F07.2F12 |06/07/12]] <br /><br />
2.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F12.2F12 |06/12/12]] <br /><br />
2.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F13.2F12 |06/13/12]] <br /><br />
2.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F14.2F12 |06/14/12]] <br /><br />
2.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F15.2F12 |06/15/12]] <br /><br />
2.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F18.2F12 |06/18/12]] <br /><br />
2.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F19.2F12 |06/19/12]] <br /><br />
2.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F22.2F12 |06/22/12]] <br /><br />
2.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F25.2F12 |06/25/12]] <br /><br />
2.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F26.2F12 |06/26/12]] <br /><br />
2.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F27.2F12 |06/27/12]] <br /><br />
2.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F29.2F12 |06/29/12]] <br /><br />
</td><br />
<br />
<td id="contentcolumn2"><center>3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JULY | '''JULY''']] </center> <br /><br />
<br /><br />
3.1[[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F03.2F12 | 07/03/12]] <br /><br />
3.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F04.2F12 | 07/04/12]] <br /><br />
3.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F06.2F12 | 07/06/12]] <br /><br />
3.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F07.2F12 | 07/07/12]] <br /><br />
3.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F08.2F12 | 07/08/12]] <br /><br />
3.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F09.2F12 | 07/09/12]] <br /><br />
3.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F10.2F12 | 07/10/12]] <br /><br />
3.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F11.2F12 | 07/11/12]] <br /><br />
3.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F12.2F12 | 07/12/12]] <br /><br />
3.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F13.2F12 | 07/13/12]] <br /><br />
3.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F14.2F12 | 07/14/12]] <br /><br />
3.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F16.2F12 | 07/16/12]] <br /><br />
3.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F17.2F12 | 07/17/12]] <br /><br />
3.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F18.2F12 | 07/18/12]] <br /><br />
3.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F19.2F12 | 07/19/12]] <br /><br />
3.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F20.2F12 | 07/20/12]] <br /><br />
3.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F23.2F12 | 07/23/12]] <br /><br />
3.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F24.2F12 | 07/24/12]] <br /><br />
3.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F25.2F12 | 07/25/12]] <br /><br />
3.20 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F26.2F12 | 07/26/12]] <br /><br />
3.21 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F27.2F12 | 07/27/12]] <br /><br />
3.22 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F30.2F12 | 07/30/12]] <br /><br />
3.23 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F31.2F12 | 07/31/12]] <br /><br />
</td><br />
<br />
<td id="rightcolumn2"><center>4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#AUGUST | '''AUGUST''']] </center> <br /><br />
<br /><br />
4.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F01.2F12 | 08/01/12]] <br /><br />
4.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F02.2F12 | 08/02/12]] <br /><br />
4.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F03.2F12 | 08/03/12]] <br /><br />
4.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F06.2F12 | 08/06/12]] <br /><br />
4.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F08.2F12 | 08/08/12]] <br /><br />
4.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F09.2F12 | 08/09/12]] <br /><br />
4.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F10.2F12 | 08/10/12]] <br /><br />
4.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F13.2F12 | 08/13/12]] <br /><br />
4.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F16.2F12 | 08/16/12]] <br /><br />
4.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F20.2F12 | 08/20/12]] <br /><br />
4.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F21.2F12 | 08/21/12]] <br /><br />
4.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F22.2F12 | 08/22/12]] <br /><br />
4.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F23.2F12 | 08/23/12]] <br /><br />
4.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F24.2F12 | 08/24/12]] <br /><br />
4.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F25.2F12 | 08/25/12]] <br /><br />
4.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F28.2F12 | 08/28/12]] <br /><br />
4.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F29.2F12 | 08/29/12]] <br /><br />
4.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F30.2F12 | 08/30/12]] <br /><br />
4.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F31.2F12 | 08/31/12]] <br /><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2"><center>5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#SEPTEMBER | '''SEPTEMBER''']] </center> <br /><br />
<br /><br />
5.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F01.2F12 | 09/01/12]] <br /><br />
5.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F02.2F12 | 09/03/12]] <br /><br />
5.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F04.2F12 | 09/04/12]] <br /><br />
5.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F05.2F12 | 09/05/12]] <br /><br />
5.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F06.2F12 | 09/06/12]] <br /><br />
5.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F14.2F12 | 09/14/12]] <br /><br />
5.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F16.2F12 | 09/16/12]]<br /><br />
5.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F17.2F12 | 09/17/12]]<br /><br />
5.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F18.2F12 | 09/18/12]]<br /><br />
5.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F19.2F12 | 09/19/12]]<br /><br />
5.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F19.2F12 | 09/19/12]]<br /><br />
5.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F20.2F12 | 09/20/12]]<br /><br />
5.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F21.2F12 | 09/21/12]]<br /><br />
5.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F22.2F12 | 09/22/12]]<br /><br />
5.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F23.2F12 | 09/23/12]]<br /><br />
5.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F24.2F12 | 09/24/12]]<br /><br />
5.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F26.2F12 | 09/26/12]]<br /><br />
5.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F27.2F12 | 09/27/12]]<br /><br />
<br />
<br />
<br />
</td><br />
<br />
<td id="contentcolumn2"><br />
'''OCTOBER'''<br /><br />
5.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F09.2F12 | 10/09/12]]<br /><br />
5.20 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F10.2F12 | 10/10/12]]<br /><br />
5.21 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F11.2F12 | 10/11/12]]<br /><br />
5.22 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F12.2F12 | 10/12/12]]<br /><br />
5.23 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#10.2F13.2F12 | 10/13/12]]<br /><br />
</td><br />
<br />
<td id="rightcolumn2"><center>[[File:UnmagenomicsBacteria sugar.png | 190px]]</center><p> '''On hover the images to see descriptions'''</p><td/><br />
</tr><br />
</table><br />
<br />
<br />
=JUNE=<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html> <br />
<h2>06/07/12</h2><br /><br />
PY BROTH 10g salt/L<br /><br />
PSB2K3 kanamicin<br /><br />
We transformed RFP E1010. <br /><br />
plate 1 18F<br /><br />
plate 2 17E<br /><br />
Stock 50 mg/mL<br /><br />
''E.coli'' 1/1000<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
We made liquid cultures with colonies [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
We extracted plasmids [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PLASMID_EXTRACTION_2 | PLASMID EXTRACTION PROTOCOL]]. <br /><br />
We ran a gel to check the extraction [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<h2>06/12/12</h2><br /><br />
We made glycerols of the bacteria <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed plasmid pHp45 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/38/UnamgenomicsandsugarBitacora_2.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>06/13/12</h2><br /><br />
We ran gel and extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] and lysed. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2d/UnamgenomicsandsugarBitacora_3.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
We extracted from gel<br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/14/12</h2><br /><br />
We extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made glycerols of pHp45 Ω [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450<br />
PSB4A5 Am (ampicillin) 1I BBa_J04450<br />
AraC BBa_C0080 <br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
2011 14L plate 1 pSB2K3 Km+<br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/5/56/UnamgenomicsandsugarBitacora_5.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
pHp45 Ω with E/P<br /><br />
We extracted from gel<br /><br />
</li><br />
</ul><br />
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<h2>06/15/12</h2><br /><br />
> Digested pHp45 Ω with E/P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
> We extracted plasmid with kit. <br /><br />
> Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (5) <br /><br />
>Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
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ARAC<br /><br />
>From the overnight plate we prepared liquid culture [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
>We left them incubating overnight. <br /><br />
>We left a plate (LB Km DH5α C0080 and a control). <br /><br />
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<h2>06/18/12</h2><br /><br />
>Plasmid extraction AraC with kit. <br /><br />
>C0080-psB2K3 915 bp<br /><br />
>Streaked 4 LB Km 30 plates DH5α psB2K3. <br /><br />
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red. <br /><br />
>Ran gel with Spr/Strr (eppendorf -20ºC) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
>Made glycerols from one tube of DH5α C0080 Km 50 -> 04. <br /><br />
>Made glycerols from one tube of DH5α PSB4A5 Amp 100 -> 05 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]]. <br /><br />
>Digested C0080 X,S. <br /><br />
>Digested B0014 E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/5/52/UnamgenomicsandsugarBitacora_6.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
Digested B0014 with E, P and with E,X<br />
</li><br />
</ul><br />
</div><br />
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<br />
<h2>06/19/12</h2><br /><br />
>Digested B0014 with E, P and with E,X. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
>Ran gels with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation. <br /><br />
C0080 is in the first lane. <br /><br />
>Extracted from gel C0080 X,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Made glycerols from 4 plates LB Km DH5α pSB2K3 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
>Take out AmyE 5’ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
AmyE 5’ 18K plate 3 2010, 2011, 2012<br /><br />
AmyE 3’ 18M plate 3 2010, 2011, 2012<br /><br />
>Digested B0014 with E,X and E,P again [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>06/22/12</h2><br /><br />
<br />
>Ran gels with digestions B0014 with E,X and E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
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>Dephosphated B0014 E,X and B0014 E,P. <br /><br />
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<h2>06/25/12</h2><br /><br />
>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
<br />
<h2>06/26/12</h2><br /><br />
>Ran gel with psB2K3 and psB4A5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
<h2>06/27/12</h2><br /><br />
>Transformed with plasmid B0079 1576 bp psB1A2 12A [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011<br /><br />
AmyE 5’ BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011<br /><br />
AmyE 3’ BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011<br /><br />
AmyE 5’ grew 2 colonies. <br /><br />
<br />
<h2>06/29/12</h2><br /><br />
>We did a DH5α K143001 Km30 Amp 100 glycerol [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] 07. <br /><br />
>We plated twice DH5α K143002 Km 30 Amp 100 + control and twice DH5α B0079 Amp 100 + control<br /><br />
These were both plated in 2 plates each. <br /><br />
They were left overnight since they had not grown by 7:00 pm so the next day glycerols were made. <br /><br />
>Liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
2 tubes DH5α K143001 Km30 Amp 100 <br /><br />
2 tubes DH5α K143002 Km30 Amp 100 <br /><br />
2 tubes DH5α B0079 Amp 100 <br /><br />
1 tube LB Km 30 Amp 100 control<br /><br />
1 tube LB Amp 100 control<br /><br />
>From the 6 tubes we extracted plasmid from kit. <br /><br />
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<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=JULY=<br />
<br />
07/02/12<br /><br />
>Digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /><br />
>PCR’s<br /><br />
•AraC<br /><br />
•Cassete ΩSpr/Strr <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PCR_Protocol_.2850.C2.B5l.29 | PCR PROTOCOL]] <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/b/be/UnamgenomicsandsugarBitacora_8.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>After Cassete ΩSpr/Strr PCR we ran a gel <br />
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</li><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsandsugarBitacora_9.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /> <br />
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</li><br />
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</div><br />
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<br />
<h2>07/03/12</h2><br /><br />
>After Cassete ΩSpr/Strr PCR we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (8) <br /><br />
>Ran gel with digestions from yesterday. (9) <br /><br />
>Did band extractions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Stored at -20ºC. <br /><br />
>Did PCR to Cassete ΩSpr/Strr to add RBS and prefix/suffix. <br /><br />
>Left digesting with E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/0/03/UnamgenomicsandsugarBitacora_9.1.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
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</li><br />
</ul><br />
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<br />
<h2>07/04/12</h2><br /><br />
>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1). <br /><br />
>Ligated Cassete ΩSpr/Strr +K143002 dephosphated(digested with E,X and E,S). <br /><br />
>Transformed ligation and left overnight plated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs. <br /><br />
>Digested with PstI [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>07/06/12</h2><br /><br />
4 LB Km30 Spec60 DH5α ΩSpr/Strr +K143002 still have not grown. <br /><br />
Ran a gel with yesterday’s digestions to chek if they were done properly (10) <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
Ran another gel with the rest of the samples. <br /><br />
Extracted GusA fragment <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
Repeated ligation ΩSpr/Strr +K143002 E,X dephosphated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Repeated ΩSpr/Strr PCR. <br /><br />
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<h2>07/07/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002 . <br /><br />
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<h2>07/08/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/13/UnamgenomicsandsugarBitacora_11.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
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</li><br />
</ul><br />
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<img src='https://static.igem.org/mediawiki/2012/b/b2/UnamgenomicsandsugarBitacora_11.1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1 PCR omega P<br /><br />
2 PCR omega I<br /><br />
3 PCR AraC P<br /><br />
4 PCR AraC I<br /><br />
5 ladder 1 Kb<br /><br />
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</li><br />
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<h2>07/09/12</h2><br /><br />
From yesterday’s transformations only one colony grew. <br /><br />
From the previous transformation only 2 colonies grew. <br /><br />
These 3 were streaked in 3 plates: <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km 30 Sp 100 control. <br /><br />
Did liquid cultures in 3 tubes: <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km30 Sp 100 control. <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
Ran a gel with: (11) <br /><br />
GusA P<br /><br />
PBBR1 GusA<br /><br />
Ω E PCR P<br /><br />
Ω PCR P<br /><br />
Ω PCR I<br /><br />
Ω E<br /><br />
Ω PCR I<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] <br /><br />
Did GusA primers dissolution for PCR. <br /><br />
GusA PCR <br /><br />
Gel Extraction by kit of lanes 3 and 5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
'''PCR omega P, PCR omega I, PCR AraC P, PCR AraC I'''<br /><br />
Add 10 μl of each primer (LW and UP). <br /><br />
Add 3 μl of plasmid (P). <br /><br />
Add 30.4 μl buffer. <br /><br />
Add 5 μl Mg. <br /><br />
Add 8 μl DNTp’s. <br /><br />
Add 42.6μl H2O miliQ. <br /> <br />
Add 1 μl RTTG polymerase. <br /><br />
Centrifuge (spin) 8 secs. <br /><br />
Add vegetable oil till the eppendorf is full. <br /><br />
Place eppendorf 1 mL in thermocycler. <br /><br />
Run PCR with program “BERNA”. <br /><br /><br />
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<img src='https://static.igem.org/mediawiki/2012/4/4b/UnamgenomicsandsugarBitacora_11.2.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S <br /><br />
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</li><br />
</ul><br />
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<br />
<h2>07/10/12</h2><br /><br />
•GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S (2 times)[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTIONS]]. (11.2) <br /><br />
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<h2>07/11/12</h2><br /><br />
•GusA PCR digestions with E,S; Ω+AmyE 3’ with E,X [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•T.V. AraC PCR+ Ω+AmyE 3’ E,X dephosphated ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<div class='captionrojo'><br />
<p class='captionInside'>1. ladder. <br /><br />
2. GusA PCR E,S. <br /><br />
3. Ω+AmyE 3’ with E,X. <br /><br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>07/12/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: (12) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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•Transformed AraC PCR+ Ω+AmyE 3’ ligation in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
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•From pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid. <br /><br />
•Made glycerols from 2 LB Km 30 Sp100 DH5α Ω+AmyE 3’ plates and from 1 LB Amp100 pfrc54 (A3) plates with control[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
•Ligated GusA PCR with B0014 E,X desphophorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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•Digested Pfrc54 (A3) with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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•Desphophorylated Ω+AmyE 3’ E,X. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/0f/UnamgenomicsandsugarBitacora_13.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>•Ran gel with pfrc54 S,P<br /><br />
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</li><br />
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</div><br />
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</html><br />
<br />
<h2>07/13/12</h2><br /><br /><br />
•Ran gel with pfrc54 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (13) <br /><br />
•Transformation of GusA PCR + B0014 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Transformed with GFP E0040 psBIA2. <br /><br />
•Liquid Cultures with ligation: AraC+ Ω+AmyE 3’ for plasmid extraction at night [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•''B. Subtitils'' competent cells. <br /><br />
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<h2>07/14/12</h2><br /><br /><br />
•From 24 AraC+ Ω+AmyE 3’ LB Km 30 Sp 100 DH5α tubes: <br /><br />
•Extracted pellets<br /><br />
•Extracted plasmids. <br /><br />
•Ran gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br /><br />
•From transformed DH5α km 30: <br /><br />
GusA+B0014 DH5α Km50 24 pellets <br /><br />
E0040 DH5α LB Amp100 <br /><br />
From these two we: <br /><br />
•Did liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Extracted plasmid. <br /><br />
•From the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ran gel with this transformation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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<h2>07/16/12</h2><br /><br /><br />
•Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
•Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_14.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>07/17/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (14) <br /><br />
•The gel we ran didn’t work, probably because the agarose was not prepared correctly. <br /><br />
•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
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<br /><br />
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<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/db/Bitacora_15.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<br />
<h2>07/18/12</h2><br /><br /><br />
•From yesterday’s digestions we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (15) <br /><br /><br />
•We made liquid cultures of AraC+ Ω+AmyE 3’ 12, 15, 21 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br /><br />
•We ligated AraC PCR E,S dephosphated with + Ω+AmyE 3’ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
•We digested 1 + Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/19/12</h2><br /><br /><br />
•We ran We digested 1 + Ω+AmyE 3’ E,P with Miguel’s enzymes and with Abiel’s enzymes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
•We transformed with ligations AraC+ Ω+AmyE 3’ E and + Ω+AmyE 3’ E [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br /><br />
•Extracted plasmids from AraC+ Ω+AmyE 3’ 12,15,21. <br /><br /><br />
•Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f3/UnamgenomicsandsugarBitacora_16.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/20/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (16) <br /><br />
•Transformed GusA+B0014 in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Made liquid cultures from AraC+ Ω+AmyE 3’s transformation LB Km30 Sp 100 and control LB Km 30 Sp100 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<h2>07/23/12</h2><br /><br /><br />
•From 24 GusA+B0014 tubes (-) we didn’t do anything. <br /><br />
•From 4 AraC+ Ω+AmyE 3’ we extracted plasmid. <br /><br />
•Digested from the 4 AraC+ Ω+AmyE 3’ E,X; X; P and GusA PCR with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/24/12</h2><br /><br /><br />
•Digested B0014 E with X<br />
B0014 X with E<br /><br />
•Digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<h2>07/25/12</h2><br /><br /><br />
•Ran gel of digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Joined B0014 E with X B0014 X with E digestions. <br /><br />
•Made liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. Transformed [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•From LasR DH5α make liquid cultures and plate [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Digested Ω+AmyE 3’ with X,P; E,S; C0080 X,P; E,S; S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Ligated Gus PCR X,P with B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/84/UnamgenomicsandsugarBitacora_17.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1.Ω+AmyE 3’ X,P<br /><br />
2. Ω+AmyE 3’ E,S<br /><br />
3.C0080 X,P<br /><br />
4.C0080 E,S<br /><br />
5.C0080 S,P<br /><br />
6.ladder<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/26/12</h2><br /><br />
•From C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5α C0179 LB Cb 100 (two cultures) and a control without bacteria [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•We extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguel’s transformation), Puc, B.S. control and PSB4AE. <br /><br />
<br />
•Due to problems with the way we did the transformations of ligations we repeated them: <br /><br />
GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
<br />
GusAPCR X,P+ B0079 S,P dephosphorylated (-)<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•AraC+ Ω+AmyE 3’ S,P 1,2,3,4<br /><br />
•K143002 X,P<br /><br />
•AraC+ Ω S,P<br /><br />
•C0179 X,S<br /><br />
<br />
<br />
<h2>07/27/12</h2><br /><br /><br />
Ligated AraC+ Ω S,P dephosphorylated + K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<h2>07/30/12</h2><br /><br /><br />
•Transformed with ligations: <br /><br />
•AraC+ Ω dephosphprylated +K143002 X,P<br /><br />
•GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
•GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
•Transformed the following sythesis: <br /><br />
•91996 Pveg 140 bp<br /><br />
•91997 ArsR-CzrA_promoter 1 194 bp<br /><br />
•91998 ArsR-CzrA_promoter 2 221 bp<br /><br />
•91999 ArsR-CzrA_promoter 3 213 bp<br /><br />
•92000 pBad-pXyl 387 bp<br /><br />
•92001 XylR 1117pb<br /><br />
•92002 CI_pro_(NAND_INHIBITOR) 774 <br /><br />
•These synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked.<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Make liquid cultures of the following transformations for tomorrow [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]: <br /><br />
•AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+B0079<br /><br />
•Synthesis<br /><br />
<br />
<h2>07/31/12</h2><br /><br /><br />
Extracted plasmids from yesterday’s cultures and from K143002 cultures that were in the other lab. <br /><br />
<br /><br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=AUGUST=<br />
<br />
<h2>08/01/12</h2><br /><br /><br />
Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b1/UnamgenomicsandsugarBitacora_17.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1 PCR GusA I<br /><br />
2 PCR GusA I<br /><br />
3 PCR GusA P<br /><br />
4 PCR GusA P<br /><br />
5 ladder 1 Kb<br /></p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/02/12</h2><br /><br /><br />
•Extracted plasmids from liquid cultures: <br /><br />
AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+BBR1<br /><br />
<br />
PCR GusA, GusA I, PCR GusA P, PCR GusA I<br /><br />
<br />
•Add 10 μl of each primer (LW and UP). <br /><br />
•Add 3 μl of plasmid (P). <br /><br />
•Add 30.4 μl buffer. <br /><br />
•Add 5 μl Mg. <br /><br />
•Add 8 μl DNTp’s. <br /><br />
•Add 42.6μl H2O miliQ. <br /><br />
•Add 1 μl RTTG polymerase. <br /><br />
•Centrifuge (spin) 8 secs. <br /><br />
•Add mineral oil till the eppendorf is full. <br /><br />
•Place eppendorf 1 mL in thermocycler. <br /><br />
•Run PCR with program “BERNA”. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/82/UnamgenomicsandsugarBitacora_18.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.A3+GusA 1<br /><br />
4.A3+GusA 2<br /><br />
5.A3+GusA 3<br /><br />
6.P GusA<br /><br />
7.P GusA 2<br /><br />
8.Ladder<br /><br />
9.01<br /><br />
10.06<br /><br />
11.96<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
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<br /><br />
<br />
<h2>08/03/12</h2><br /><br /><br />
• Ran a gel with: (18) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Ran another gel to extract with: <br /><br />
1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.00<br /><br />
4.01<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]: <br /> <br />
<br />
• Ω+K143002 E,X; A3+GusA E,S; GusAPCR X,P <br /><br />
<br />
•Extracted GusAPCR X,P digestion [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Left the following ligation: AraC+ Ω+K143002 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/17/UnamgenomicsandsugarBitacora_19.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3+GusA 2<br /><br />
2.A3+GusA 3<br /><br />
3. Ω+AmyE 3’ 2<br /><br />
4. Ω+AmyE 3’ 3<br /><br />
5.XylR X,P<br /><br />
6.pBad-pXyl X,P<br /><br />
7.GusA PCR X,P<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/06/12</h2><br /><br /><br />
•Made liquid cultures of Ω+K143002 (2 cultures+1 control) and AraC+ Ω+K143002 (2 cultures+1 control) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ligated GusAPCR X,P+B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
•Ran a Gel with: (19) <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/8b/UnamgenomicsandsugarBitacora_19.1.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/08/12</h2><br /><br />
BBa_B0040 6I psB1A2 Amp+ plate 1<br /><br />
Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (19.1) <br /><br />
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<h2>08/09/12</h2><br /><br />
•From yesterday’s PCR’s : <br /><br />
• E0040 plasmid 1<br /><br />
E0040 plasmid 2<br /><br />
E0040 digested 1<br /><br />
E0040 digested 2<br /><br />
We purified with PCR kit<br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Made 14 PCR’s from AraC+ Ω+AmyE 3’. <br /><br />
•From B0079+GusA ligation and B0040 transformation:<br />
•Grew colonies. <br /><br />
•Made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Streaked these in a new plate. <br /><br />
•Diluted plasmid E0080 2 1/50. <br /><br />
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<br />
<br /><br />
<br />
<h2>08/10/12</h2><br /><br />
•Yesterday’s gel did not come out as expected so we repeated the PCR. <br /><br />
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<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/e/ef/UnamgenomicsandsugarBitacora_20.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.AraC+ Ω+AmyE 3’ <br /><br />
2-14.AraC+ Ω+AmyE 3’ <br /><br />
15.AraC+ Ω 1<br /><br />
16.AraC+ Ω 1<br /><br />
17.AraC+ Ω 1<br /><br />
18.PCR 1 GFP<br /><br />
19.PCR GFP<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<h2>08/13/12</h2><br /><br />
•After running a gel with 14 PCR’s AraC+ Ω+AmyE 3’ and it did not come out as expected, we ran a gel with the ligation AraC+ Ω+AmyE 3’ to select candidate (that weight the same as the ligation) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (20) <br /><br />
<br />
•Extracted pasmids form liquid cultures: B0049, B0079+GusA. <br /><br />
<br />
•AraC+ Ω+AmyE 3’ undigested didn’t run in the gel as expected, it ran similarly to AraC+ Ω<br /><br />
<br />
• We repeated the ligation AraC+ Ω S,P dephosphorylated+K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
• GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.<br />
<br />
• We ran a gel to extract. <br /><br />
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<br />
<br /><br />
<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f1/UnamgenomicsandsugarBitacora_22.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>1.6 AraC+ Ω+AmyE 3’ E with P. <br /><br /><br />
2.8 AraC+ Ω+AmyE 3’ E with P. <br /><br />
3.11 AraC+ Ω+AmyE 3’ E with P. <br /><br />
4.12 AraC+ Ω+AmyE 3’ E with P. <br /><br />
5.6 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
6.8 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
7.11 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
8.12 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
9.00 pBad/pXyl with S,P. <br /><br />
10.Ladder. <br /><br />
11.E0040 PCR E,S to extract the correct band. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/16/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: <br /><br />
<br />
•Digested: <br /><br />
ArSR-CzrA 97 with S,P<br /><br />
ArSR-CzrA 98 with S,P<br /><br />
ArSR-CzrA 99 with S,P<br /><br />
E0040 PCR with E,S<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
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<br /><br />
<br />
<br />
<h2>08/20/12</h2><br /><br />
•Ligated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/21/12</h2><br /><br />
•Joined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated. <br /><br />
•Extracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR. <br /><br />
•Digested Ω+AmyE 3’ with E,X and + Ω+AmyE 3’ II with E,X (obtained these two from glycerols stored at -80ºC [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/29/UnamgenomicsandsugarBitacora_22.1.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested K143001+PBad, pXyl with E,S <br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/1c/UnamgenomicsandsugarBitacora_23.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>1.K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
4.A3 PCR<br /><br />
5.A3 PCR<br /><br />
6.E0040 PCR E,S<br /><br />
7.B0014 E,X dephosphorylated<br /><br />
8. Ω+AmyE 3’ E,X <br /><br />
9. Ω+AmyE 3’ E,X II<br /><br />
10.Ladder<br />
</p><br />
</li><br />
</ul><br />
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<br />
<h2>08/22/12</h2><br /><br />
•We did 2 PCR’s for A3. <br /><br />
<br />
•Digested K143001+PBad, pXyl with E,S <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (22.1) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did band extraction of: <br /><br />
1. K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
•Made liquid cultures from E1010+Boo14 that the other lab gave us and did 2 plates for glycerol Km+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Did 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ Ω+AmyE 3’ 6,8,11,12 Km+ and Spectinomycin+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Transformed ligations: <br /><br />
B0079+GusA Amp+<br /><br />
Pveg+XylR Chloramphenicol+<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]].. <br /><br />
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<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/78/UnamgenomicsandsugarBitacora_23.01.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1. Ω+AmyE 3’ E,X 6.2<br /><br />
2.Ω+AmyE 3’ II E,X<br /><br />
3.97 ArsR-CzrA<br /><br />
4.98 ArsR-CzrA<br /><br />
5.99 ArsR-CzrA<br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/23/12</h2><br /><br />
•The negative controls were contaminated. We didn’t manipulate the ones that had grown because of this. <br /><br />
•Ran gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
<br />
<br />
<h2>08/24/12</h2><br /><br />
From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ Ω+AmyE 3’ 6 and 8 we streaked 2 plates for glycerol of each ligation. <br />Repeated ligation B0079+GusA. <br /><br />
<br />
Ligated E0040 PCR E,S+B0014 E,X dephospohylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
Made 2 new plates from Pveg+XylR ligation and liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>08/25/12</h2><br /><br />
Extracted pellet from 16 tubes of pVeg+XylR that were left overnight. <br /><br />
<br />
Ligated B0079+GusA and B0040+B0014 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/28/12</h2><br /><br />
<br />
Made liquid cultures from transformations tht were left overnight <br />
-R0079+GusA<br /><br />
-R0079+GusA (-)<br /><br />
-E0040+B0014<br /><br />
-E0040+B0014 (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/25/UnamgenomicsandsugarBitacora_23.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. A3 PCR 1. <br /><br />
2. A3 PCR 2. <br /><br />
3. Ω+AmyE 3’ S,P *.<br /><br />
4. Ω+AmyE 3’ S,P . <br /><br />
5. Pveg+XylR E,S 2. <br /><br />
6. Pveg+XylR E,S 3. <br /><br />
7. Pveg+XylR E,S 4. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/29/12</h2><br /><br />
Digested Pveg+XylR 2,3,4 with E,S. <br /><br />
<br />
Digested Ω+AmyE 3’ with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTION]]. <br /><br />
<br />
Ran a PCR with A3 PCR (2 .6 ml tubes). <br /><br />
<br />
Ran a gel with <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
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<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2e/UnamgenomicsandsugarBitacoraBitacora_24.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1-23. E0040 + B0014 colonies<br /><br />
24-29. B0079+GusA 1<br /><br />
30. B0079<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/30/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (24) <br /><br />
<br />
<br />
•Digested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/3d/UnamgenomicsandsugarBitacora_25.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1-3B0079+GusA E,S 3. <br /><br />
4-9. PVeg+XylR E,S 1. <br /><br />
10-17. E0040+B0014 X,P 3. <br /><br />
18. 1 kb ladder. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/d2/UnamgenomicsandsugarBitacora_26.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3 PCR. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7b/UnamgenomicsandsugarBitacora_27.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>E0040+B0014 X,P 4 to extract<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>08/31/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (25) <br /><br />
<br />
<br />
•Dephosphorylated: <br /><br />
Ω+AmyE 3’ S,P <br /><br />
Ω+AmyE 3’ II S,P <br /><br />
6 AraC+ Ω+AmyE 3’ E,X<br /><br />
8 AraC+ Ω+AmyE 3’ E,X<br /><br />
11 AraC+ Ω+AmyE 3’ E,X<br /><br />
12 AraC+ Ω+AmyE 3’ E,X<br /><br />•Ran a gel with A3 PCR [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (26) <br /><br />
<br />
•It was the second time we didn’t obtain a band from A3’s PCR.<br />
<br />
•Probably because it had been missing the buffer 2’s ethanol (“blue lid”) from the PCR purification. <br /><br />
<br />
•Ran a gel woth E0040+B0014 X,P 4 to extract <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | Gen extraction protocol]]. <br /><br />
<br />
•Did an A3 PCR. <br /><br />
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<br /><br />
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<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=SEPTEMBER=<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b8/UnamgenomicsandsugarBitacora_28.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.A3 PCR 1. <br /><br />
2.A3 PCR 2. <br /><br />
3.E0040+B0014 X,P 1. <br /><br />
4.E0040+B0014 X,P 2. <br /><br />
5.Ω+AmyE 3’ dephosphorylated S,P. <br /><br />
6.Ω+AmyE 3’ dephosphorylated S,P II. <br /><br />
7.97 dephosphorylated S,P. <br /><br />
8.98 dephosphorylated S,P. <br /><br />
9.99 dephosphorylated S,P. <br /><br />
10-13. Arac+ Ω+AmyE 3’ dephosphorylated E,X 12. <br /><br />
14. 1 kb Ladder. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>09/01/12</h2><br /><br />
•Ran a gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (28) <br /><br />
<br />
•Ligated E0040+B0014 X,P 2 +promoter S,P dephosphorylated; K143002 X,P+ Ω+AmyE 3’ dephosphorylated S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<br /><br />
<br />
<h2>09/03/12</h2><br /><br />
<br />
•Digested B0079+GusA E,S 3,4,5; PVeg+XylR E,S 1,5,7,10,13,16 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>09/04/12</h2><br /><br />
•Ran gel with digestions: <br /><br />
B0079+GusA E,S 3,4,5<br /><br />
PVeg+XylR E,S 1,5,7,10,13,16 <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Purified A3 PCR 1,2. <br /><br />
<br />
•Ran a gel of A3 PCR <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] and digested with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•From ligations: PVeg+E0040+B0014, PBad/PXyl+E0040+B0014, B0079+E0040+B0014 only colonies grew in PBad/PXyl+E0040+B0014.<br />
<br />
•Plated colonies that grew in a new plate. <br /><br />
<br />
•Made liquid cultures of these <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•Repeated the ones that did not grow. <br /><br />
<br />
•Digested E0040+B0014 4 with X,P[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
-BBa_J23101 18E plate 1 plasmid BBa_J61002_2948 bp Amp+<br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/c1/UnamgenomicsandsugarBitacora_29.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>24 PBad/PXyl+E0040+B0014 tubes we extracted <br /><br />
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</li><br />
</ul><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/a/a4/UnamgenomicsandsugarBitacora_30.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
Extracted band from 1. and 2<br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7d/UnamgenomicsandsugarBitacora_31.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S <br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>09/05/12</h2><br /><br />
•From the 24 PBad/PXyl+E0040+B0014 tubes we extracted plasmids (29) and ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Extracted plasmid from 2 tubes of E0040+B0014. <br /><br />
<br />
•Ran a gel with yesterday’s digestions: <br /><br />
1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
<br />
•Extracted band from 1. and 2. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Purified A3 PCR from 2 0.6ml tubes 1 and 2. <br /><br />
<br />
•1 colony grew from ligation pVeg+E0040+B0014. <br /><br />
<br />
•We streaked this in another plate and did liquid cultures <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•To do: <br /><br />
•Ligate B0079 S,P dephosphorylated + E0040+B0014 X,P and Ω+AmyE 3’ S,P dephosphorylated+ K143001 X,P. <br /><br />
•Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S 10,13. (31) <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/26/UnamgenomicsandsugarBitacora_32.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>•A3 PCR and gel A3 PCR X,P <br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
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<br /><br />
<br />
<h2>09/06/12</h2><br /><br />
<br />
•A3 PCR and gel A3 PCR X,P 1,2. <br /><br />
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<br />
<h2>09/14/12</h2><br /><br />
<br />
•Digested Ω+AmyE 3’ 11,13,18, and 22 with E,X. <br /><br />
•Ligate: <br /><br />
PVeg S,P dephosphorylated+XylR X,P. <br /><br />
PBad p Xyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
pSB13C3 E,P dephosphorylated + A3 PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + GusA PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + XylR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pVeg E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pBad pXyl E,P. <br /><br />
pSB13C3 E,P dephosphorylated + Ω PCR E,P. <br /><br />
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<h2>09/16/12</h2><br /><br />
• Colonies in these plates grew: <br /><br />
- R0079+E0040+B0014 <br /><br />
- pBad/pXyl <br /><br />
- XylR <br /><br />
- pVeg <br /><br />
- A3+E0040+B0014 from colonies 1,3,4,5,6,7,8,9,10,11,12,13,14,15,17 <br /><br />
- pVeg+E0040+B0014 1,2,3,4,5,6,8,9,10,11,12,13,14,15,16 <br /><br />
- pBad/pXyl+E0040+B0014 2 tubes from the same correct colony. <br /><br />
- pVeg+XylR 1,2 <br /><br />
- pBad/pXyl+pSB1C3 1,2 <br /><br />
- XylR+pSB1C3 1,2 <br /><br />
- pVeg+pSB1C3 1,2 <br /><br />
- A3+pSB1C3 1,2 <br /><br />
- GusA+pSB1C3 1,2 <br /><br />
- omegacassette+pSB1C3 1,2 <br /><br />
<br /><br />
• Ran gel from the plasmid extractions that we did yesterday: <br /><br />
- A3+E0040+B0014 1-16 <br /><br />
- pVeg+E0040+B0014 1-16, 18 <br /><br />
- omega cassette+AmyE 3’ 11,13,18,22 <br /><br />
• Digested R0079+E0040+B0014 with E,S. to ligate with AmyE 3’ E,x dephosphorylated. <br /><br />
• Digested omega cassette with E,P. <br /><br />
• Digested pSB1C3 with E,P. <br /><br />
• Made liquid cultures from AmyE 5’ and AmyE 3’. <br /><br />
• Did plasmid extraction from pSB1C3 colonies 3 and 6. <br /><br />
<br /><br />
<br />
<h2>09/17/12</h2><br /><br />
• Digested A3+E0040+B0014 colonies 2,3,4,5,6,11 and 16 with E,S. <br /><br />
• Digested pVeg+E0040+B0014 colonies 1,2,3,4,5,9 and 10 with E,S. <br /><br />
• Dephosphorylated omega cassette+AmyE 3’ colonies 13,18 and 22 digested with E,X. <br /><br />
• Digested pBad/pXyl and pVeg with S,P. Then dephosphorylated. <br /><br />
• Digested AmyE 3’ with E,X. Then dephosphorylated. <br /><br />
<br /><br />
<br />
<h2>09/18/12</h2><br /><br />
<br /><br />
• Ran gel with: <br /><br />
1. A3 2 E,S <br /><br />
2. A3 3 E,S <br /><br />
3. A3 4 E,S <br /><br />
4. A3 5 E,S <br /><br />
5. A3 6 E,S <br /><br />
6. A3 11 E,S <br /><br />
7. A3 16 E,S <br /><br />
8. E0040+B0014 colony 1 E,X <br /><br />
9. AmyE 5’ E,P <br /><br />
10. AmyE 3’ E,X <br /><br />
11. Omega cassette E,S <br /><br />
12. pSB1C3 E,P <br /><br />
13. ladder <br /><br />
Bottom: <br /><br />
1. pVeg 1 E,S <br /><br />
2. pVeg 2 E,S <br /><br />
3. pVeg 3 E,S <br /><br />
4. pVeg 4 E,S <br /><br />
5. pVeg 5 E,S <br /><br />
6. pVeg 9 E,S <br /><br />
7. pVeg 10 E,S <br /><br />
8. pVeg S,P dephosphorylated <br /><br />
9. pVeg 2 S,P <br /><br />
10. pBad/pXyl S,P <br /><br />
11. XylR X,P <br /><br />
12. R0079+GFP+TT E,S <br /><br />
13. R0079 S,P <br /><br />
14. Ladder <br /><br />
• Dephosphorylated GFP+TT E,X; AmyE 5’ E,X; pSB1C3 E,P; pVeg 2 S,P; pBad/pXyl S,P and R0079 S,P. <br /><br />
<br /><br />
<h2>09/19/12</h2><br /><br />
<br /><br />
• Did PCR from A3 and a 1/50 dilution. <br /><br />
• Ran gel with: <br /><br />
1. pVeg 3 E,P. <br /><br />
2. pVeg 9 E,P. <br /><br />
3. pVeg 10 E,P. <br /><br />
4. pBAd/pXyl 3 E,P. <br /><br />
5. pBAd/pXyl 5 E,P. <br /><br />
6. pBAd/pXyl 1 E,P. <br /><br />
7. A3 3 E,P. <br /><br />
8. A3 4 E,P. <br /><br />
9. XylR 1 E,P. <br /><br />
10. XylR 3 E,P. <br /><br />
11. XylR 8 E,P. <br /><br />
12. pSB1C3 6 E,P. <br /><br />
13. pSB1C3 6 E,P. <br /><br />
14. AmyE 5’ E,S. <br /><br />
15. AmyE 5’ E,S. <br /><br />
16. A3 1/50 PCR. <br /><br />
17. A3 PCR. <br /><br />
18. Ladder <br /><br />
• We did transformations: <br /><br />
- 2 of R0079+GusA <br /><br />
- R0079+GusA control <br /><br />
- 2 of A3+pSB1AK3 <br /><br />
- A3+pSB1AK3 control <br /><br />
- 2 of omega cassette+pSB1C3 <br /><br />
- 2 of GusA+pSB1C3 <br /><br />
• Digested: <br /><br />
- pVeg+pSB1C3 <br /><br />
- pBad/pXyl+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
- A3+pSB1C3 <br /><br />
With E,P. <br /><br />
• Ran gel with these digestions. <br /><br />
<br /><br />
<h2>09/19/12</h2><br /><br />
• Made liquids cultures from: <br /><br />
- Glicerol of AmyE 5’ <br /><br />
- Glicerol of AmyE 3’ <br /><br />
- pBad/pXyl+pSB1C3 colony 5 <br /><br />
• Made liquid cultures from yesterday transformations: <br /><br />
- R0079+GusA 2 and control <br /><br />
- A3+pSB1AK3 2 and control <br /><br />
- Omega cassette+pSB1C3 2 <br /><br />
- GusA+pSB1C3 2 <br /><br />
• We transformed yesterday ligations: <br /><br />
- pBad/pXyl+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
- pVeg+pSB1C3 <br /><br />
- A3+pSB1C3 <br /><br />
• Purified A3 PCR with kit, and then, digested with E,P and E,S. <br /><br />
• Ran gel <br /><br />
<br /><br />
<h2>09/20/12</h2><br /><br />
<br /><br />
• Made liquid cultures from transformations: <br /><br />
- Omega cassette+pSB1C3 colony 7 <br /><br />
- 2 of GusA+pSB1C3 colony 32 <br /><br />
- R0079+GusA colony 12 <br /><br />
• Digested A3 1/50 PCR with E,P and E,S. <br /><br />
• Made liquid cultures from R0079+E0040+B0014; pBad/pXyl+E0040+B0014; pVeg+E0040+B0014; A3+E0040+B0014. <br /><br />
• Ligated: <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P <br /><br />
- A3 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
<br /><br />
<h2>09/21/12</h2><br /><br />
<br /><br />
• Ran gel with: <br /><br />
1. R0079+GFP+TT 1 <br /><br />
2. R0079+GFP+TT <br /><br />
3. pBad/pXyl+pSB1C3 1 <br /><br />
4. pBad/pXyl+pSB1C3 <br /><br />
5. omega cassette+psB1C3 1 <br /><br />
6. omega cassette+psB1C3 <br /><br />
7. pSB1C3 E,P 1 <br /><br />
8. pSB1C3 E,P <br /><br />
9. pSB1C3 E <br /><br />
10. pSB1C3 P <br /><br />
11. A3 1/50 PCR E,P <br /><br />
<br /><br />
<br />
<h2>09/22/12</h2><br /><br />
• Ran gel with lysis extractions: <br /><br />
1. GusA+pSB1C3 1 <br /><br />
2. GusA+pSB1C3 2 <br /><br />
3. GusA+pSB1C3 3 <br /><br />
4. GusA+pSB1C3 4 <br /><br />
5. GusA+pSB1C3 5 <br /><br />
6. GusA+pSB1C3 6 <br /><br />
7. GusA+pSB1C3 7 <br /><br />
8. GusA+pSB1C3 8 <br /><br />
9. GusA+pSB1C3 9 <br /><br />
10. GusA+pSB1C3 10 <br /><br />
11. GusA+pSB1C3 11 <br /><br />
12. GusA+pSB1C3 12 <br /><br />
13. pSB1C3 <br /><br />
14. A3+E0040+B0014 1 <br /><br />
15. A3+E0040+B0014 2 <br /><br />
16. A3+E0040+B0014 3 <br /><br />
17. A3+E0040+B0014 4 <br /><br />
18. A3+E0040+B0014 5 <br /><br />
19. A3+E0040+B0014 6 <br /><br />
20. A3+E0040+B0014 7 <br /><br />
21. A3+E0040+B0014 8 <br /><br />
22. A3+E0040+B0014 9 <br /><br />
23. pVeg+E0040+B0014 3 <br /><br />
24. pVeg+E0040+B0014 4 <br /><br />
25. pVeg+E0040+B0014 5 <br /><br />
26. pVeg+E0040+B0014 6 <br /><br />
27. pVeg+E0040+B0014 7 <br /><br />
28. pVeg+E0040+B0014 8 <br /><br />
29. pVeg+E0040+B0014 9 <br /><br />
30. pVeg <br /><br />
31. pBad/pXyl+E0040+B0014 1 <br /><br />
32. pBad/pXyl+E0040+B0014 2 <br /><br />
33. pBad/pXyl+E0040+B0014 3 <br /><br />
34. pBad/pXyl+E0040+B0014 4 <br /><br />
35. pBad/pXyl+E0040+B0014 5 <br /><br />
36. pBad/pXyl+E0040+B0014 6 <br /><br />
37. pBad/pXyl+E0040+B0014 7 <br /><br />
38. pBad/pXyl+E0040+B0014 8 <br /><br />
39. pBad/pXyl+E0040+B0014 9 <br /><br />
40. pBad/pXyl+E0040+B0014 10 <br /><br />
41. pBad/pXyl <br /><br />
42. pVeg+XylR 1 <br /><br />
43. pVeg+XylR 2 <br /><br />
44. pVeg+XylR 3 <br /><br />
45. pVeg+XylR 4 <br /><br />
46. pVeg+XylR 5 <br /><br />
47. pVeg+XylR 6 <br /><br />
48. pVeg+XylR 7 <br /><br />
49. pVeg+XylR 8 <br /><br />
50. omega cassette <br /><br />
<br /><br />
• Ran gel with yesterday digestions: <br /><br />
1. R0079+GFP+TT E,P <br /><br />
2. R0079+GFP+TT E,P <br /><br />
3. Omega cassette+pSB1C3 E,P <br /><br />
4. Omega cassette+pSB1C3 E,P <br /><br />
5. Ladder <br /><br />
6. pBad/pXyl+pSB1C3 <br /><br />
7. pBad/pXyl+pSB1C3 <br /><br />
8. pSB1C3 6 E,P. <br /><br />
9. A3 1/50 PCR E,S <br /><br />
10. pBbak C011 <br /><br />
11. pBbak C012 <br /><br />
12. ladder <br /><br />
<br /><br />
• Did pellet from R0079+GusA. <br /><br />
• Did plasmid extraction from omega cassette+pSB1C3 colonies 1,4,6 and 7. <br /><br />
• Digested them with E,P. <br /><br />
• Digested: <br /><br />
- GusA+pSB1C3 1,3,4,10 and 11 <br /><br />
- A3+E0040+B0014 2,4,7 and 9 <br /><br />
- pVeg+E0040+B0014 4,5 and 6 <br /><br />
- pBad/pXyl+E0040+B0014 3,7 and 10 <br /><br />
- pVeg+XylR 2,4 and 5 <br /><br />
with E,P. <br /><br />
• Made liquid cultures from pVeg+pSB1C3 <br /><br />
• Ligated: <br /><br />
- XylR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- pVeg E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- A3 1/50 PCR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- A3 1/50 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
<br /><br />
<h2>09/23/12</h2><br /><br />
<br /><br />
• Ran gel with yesterday digestions: <br /><br />
1. Omega cassette+pSB1C3 1 E,P <br /><br />
2. Omega cassette+pSB1C3 4 E,P <br /><br />
3. Omega cassette+pSB1C3 6 E,P <br /><br />
4. Omega cassette+pSB1C3 7 E,P <br /><br />
5. GusA+pSB1C3 1 E,P <br /><br />
6. GusA+pSB1C3 3 E,P <br /><br />
7. GusA+pSB1C3 4 E,P <br /><br />
8. GusA+pSB1C3 10 E,P <br /><br />
9. GusA+pSB1C3 11 E,P <br /><br />
10. pSB1C3 E,P dephosphorylated <br /><br />
11. pVeg+XylR 2 E,P <br /><br />
12. pVeg+XylR 4 E,P <br /><br />
13. pVeg+XylR 5 E,P <br /><br />
14. pVeg S,P dephosphorylated <br /><br />
15. pVeg+GFP+TT 4 E,P. <br /><br />
16. pVeg+GFP+TT 5 E,P. <br /><br />
17. pVeg+GFP+TT 6 E,P. <br /><br />
18. ladder <br /><br />
Bottom <br /><br />
1. pBad/pXyl+GFP+TT 3 E,P <br /><br />
2. pBad/pXyl+GFP+TT 7 E,P <br /><br />
3. pBad/pXyl+GFP+TT 10 E,P <br /><br />
4. pBad/pXyl S,P dephosphorylated <br /><br />
5. A3+GFP+TT 2 E,P <br /><br />
6. A3+GFP+TT 4 E,P <br /><br />
7. A3+GFP+TT 7 E,P <br /><br />
8. A3+GFP+TT 9 E,P <br /><br />
9. Ladder <br /><br />
<br /><br />
<h2>09/24/12</h2><br /><br />
<br /><br />
• Did PCR from omega cassette+pSB1C3 colonies 1,4,6 and 7. <br /><br />
• Made 1/50 dilutions from these PCR products. <br /><br />
• Ran gel with: <br /><br />
1. Omega cassette+pSB1C3 1/50 PCR 1 <br /><br />
2. Omega cassette+pSB1C3 PCR 1 <br /><br />
3. Omega cassette+pSB1C3 1/50 PCR 4 <br /><br />
4. Omega cassette+pSB1C3 PCR 4 <br /><br />
5. Omega cassette+pSB1C3 1/50 PCR 6 <br /><br />
6. Omega cassette+pSB1C3 PCR 6 <br /><br />
7. Omega cassette+pSB1C3 1/50 PCR 7 <br /><br />
8. Omega cassette+pSB1C3 PCR 7 <br /><br />
9. GusA+pSB1C3 1 P <br /><br />
10. GusA+pSB1C3 3 P <br /><br />
11. GusA+pSB1C3 4 P <br /><br />
12. GusA+pSB1C3 10 P <br /><br />
13. GusA+pSB1C3 11 P <br /><br />
14. pVeg+pSB1C3 6 E,P <br /><br />
15. pVeg+pSB1C3 5 E,P <br /><br />
16. pVeg+pSB1C3 4 E,P <br /><br />
17. pVeg+pSB1C3 3 E,P <br /><br />
18. pVeg+pSB1C3 2 E,P <br /><br />
19. pVeg+pSB1C3 1 E,P <br /><br />
20. ladder <br /><br />
<br />
• From yesterday transformations, these didn’t grow: <br /><br />
- pBad/pXyl+E0040+B0014 1 and 2. <br /><br />
- A3+E0040+B0014. <br /><br />
• Made liquid cultures from transformations that did grow: <br /><br />
- pVeg+E0040+B0014. <br /><br />
- pVeg+XylR. <br /><br />
- A3+pSB1C3 <br /><br />
- pVeg+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
<br /><br />
<h2>09/26/12</h2><br /><br />
<br /><br />
• Did plasmid extractions form liquids cultures: <br /><br />
- 8 of pVeg+pSB1C3 <br /><br />
- 8 of A3 <br /><br />
- 8 of XylR <br /><br />
• Digested them with E,P. <br /><br />
• Ran gel with: <br /><br />
1. Ladder <br /><br />
2. XylR+pSB1C3 1 E,P <br /><br />
3. XylR+pSB1C3 2 E,P <br /><br />
4. XylR+pSB1C3 3 E,P <br /><br />
5. pVeg+pSB1C3 1 E,P <br /><br />
6. pVeg+pSB1C3 3 E,P <br /><br />
7. pVeg+pSB1C3 4 E,P <br /><br />
8. pVeg+pSB1C3 5 E,P <br /><br />
9. pVeg+pSB1C3 6 E,P <br /><br />
10. pVeg+pSB1C3 8 E,P <br /><br />
11. pSB1C3 6 E,P dephosphorylated <br /><br />
12. A3+pSB1C3 1 E,P <br /><br />
13. A3+pSB1C3 3 E,P <br /><br />
14. A3+pSB1C3 4 E,P <br /><br />
15. A3+pSB1C3 5 E,P <br /><br />
16. A3+pSB1C3 6 E,P <br /><br />
17. A3+pSB1C3 8 E,P <br /><br />
18. Ladder <br /><br />
19. P4+E0040+B0014 1 E,X <br /><br />
20. P4+E0040+B0014 7 E,X <br /><br />
<br /><br />
<h2>09/27/12</h2><br /><br />
<br /><br />
• Digested pVeg+XylR colonies 1,4,5 and 6 in pJ209 plasmid with E,S for 3 hours. <br /><br />
• Digested pVeg+E0040+B0014 colonies 7,9,10 and 11 in pasmid pJ209 with E,P. <br /><br />
<br /><br />
<h2>10/09/12</h2><br /><br />
<br /><br />
• Ligated: <br /><br />
- A3 1/50 PCR E,P + pSB1C3 6 E,P dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
- P4+CI+E1010+B0014 E,X dephosphorylated + AmyE 5’+pBad/pXyl E,S. <br /><br />
<br /><br />
<h2>10/10/12</h2><br /><br />
<br /><br />
• Ligated: <br /><br />
- GusA PCR E,P + pSB1C3 6 E,P dephosphorylated. <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- A3 1/50 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
• Digested R0079+E0040+B0014 with E,S. <br /><br />
• Digested AmyE 3’ with E,X. Then dephosphorylated. <br /><br />
• Digested pVeg and R0079, A3 with X,P. <br /><br />
• Digested pBad/pSB1C3 with S,P. <br /><br />
<br /><br />
<h2>10/11/12</h2><br /><br />
<br /><br />
• Did plasmid extraction from liquid cultures: <br /><br />
- 2 of AmyE 5’ <br /><br />
- 2 of AmyE 3’ <br /><br />
- 2 of E0040+B0014 <br /><br />
• Ran gel with: <br /><br />
1. A3 1/50 PCR X,P <br /><br />
2. A3 1/50 PCR X,P <br /><br />
3. A3+pSB1C3 6 X,P <br /><br />
4. A3+pSB1C3 6 X,P <br /><br />
5. A3+pSB1C3 8 X,P <br /><br />
6. A3+pSB1C3 8 X,P <br /><br />
7. pVeg X,P <br /><br />
8. pVeg 96 X,P <br /><br />
9. pVeg 96 X,P <br /><br />
10. pVeg+pSB1C3 1 X,P <br /><br />
11. pVeg+pSB1C3 1 X,P <br /><br />
12. pVeg+pSB1C3 3 X,P <br /><br />
13. pVeg+pSB1C3 3 X,P <br /><br />
14. R0079 X,P <br /><br />
15. Ladder <br /><br />
<br /><br />
<h2>10/12/12</h2><br /><br />
<br /><br />
• Ligated:<br />
- AmyE 5’+pBad/pXyl 10 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- AmyE 5’+pBad/pXyl 11 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- AmyE 5’+pBad/pXyl 12 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
- A3 1/50 PCR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
<br /><br />
<h2>10/12/12</h2><br /><br />
<br /><br />
• Digested AmyE 5’ with S,P <br /><br />
• Dephosphorylated it. <br /><br />
• Ligated: <br /><br />
- AmyE 5’ S,P dephosphorylated with <br /><br />
- R0079 X,P <br /><br />
- pVeg 96 X,P <br /><br />
- A3 1/50 PCR X,P <br /><br />
- A3+pSB1C3 6 X,P <br /><br />
- R0079+E0040+B0014 <br /><br />
• Ligated pBad/pXyl+pSB1C3 S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
• Ran gel with: <br /><br />
1. A3+pSB1C3 1 <br /><br />
2. A3+pSB1C3 2 <br /><br />
3. A3+pSB1C3 3 <br /><br />
4. A3+pSB1C3 4 <br /><br />
5. A3+pSB1C3 5 <br /><br />
6. A3+pSB1C3 6 <br /><br />
7. A3+pSB1C3 7 <br /><br />
8. A3+pSB1C3 8 <br /><br />
9. A3+pSB1C3 9 <br /><br />
10. A3+pSB1C3 10 <br /><br />
11. A3+pSB1C3 11 <br /><br />
12. A3+pSB1C3 6 control <br /><br />
13. AmyE 5’+P4+CI+E1010+B0014 1 <br /><br />
14. AmyE 5’+P4+CI+E1010+B0014 2 <br /><br />
15. AmyE 5’+P4+CI+E1010+B0014 3 <br /><br />
16. AmyE 5’+P4+CI+E1010+B0014 4 <br /><br />
17. AmyE 5’+P4+CI+E1010+B0014 5 <br /><br />
18. AmyE 5’+P4+CI+E1010+B0014 6 <br /><br />
19. AmyE 5’+P4+CI+E1010+B0014 7 <br /><br />
20. P4+CI+E1010+B0014 1 control <br /><br />
<br /><br />
<h2>10/09/12</h2><br /><br />
<br /><br />
• Ligate: <br /><br />
K143001+pBad/pXyl + p4+CI+E1010+TT E,X dephosphorylated. <br /><br />
• Ligate: <br /><br />
pVeg S,P dephosphorylated + XylR X,P. <br /><br />
• Ligate: <br /><br />
A3 PCR 1/50 E,P + pSB1C3 E,P dephosphorylated <br /><br />
<br /><br />
<h2>10/13/12</h2><br /><br />
<br /><br />
• Digested AmyE 5’ with S,P. Then dephosphorylated. <br /><br />
• Ligate: <br /><br />
AmyE 5’ S,P dephosphorylated + <br /><br />
- R0079 X,P <br /><br />
- pVeg 96 X,P <br /><br />
- A3 PCR 1/50 X,P <br /><br />
- A3+pSB1C3 colony 6 X,P <br /><br />
- R0079+E0040+B0014 E,X <br /><br />
• Ligate: <br /><br />
pBad/pXyl+pSB1C3 S,P dephosphorylated + E0040+B0014 X,P <br /><br />
<br />
• Ran a gel with: <br /><br />
1. A3+pSB1C3 1 <br /><br />
2. A3+pSB1C3 2 <br /><br />
3. A3+pSB1C3 3 <br /><br />
4. A3+pSB1C3 4 <br /><br />
5. A3+pSB1C3 5 <br /><br />
6. A3+pSB1C3 6 <br /><br />
7. A3+pSB1C3 7 <br /><br />
8. A3+pSB1C3 8 <br /><br />
9. A3+pSB1C3 9 <br /><br />
10. A3+pSB1C3 10 <br /><br />
11. A3+pSB1C3 11 <br /><br />
12. A3+pSB1C3 6 control <br /><br />
13. AmyE 5’+P4+CI+E1010+B0014 8 <br /><br />
14. AmyE 5’+P4+CI+E1010+B0014 9 <br /><br />
15. AmyE 5’+P4+CI+E1010+B0014 10 <br /><br />
16. P4+CI+E1010+B0014 1 control <br /><br />
17. pVeg+XylR 1 <br /><br />
18. pVeg+XylR 2 <br /><br />
19. pVeg+XylR 3 <br /><br />
20. pVeg+XylR 4 <br /><br />
21. pVeg+XylR 5 <br /><br />
22. pVeg+XylR 6 <br /><br />
23. pVeg+XylR 7 <br /><br />
24. pVeg+XylR 8 <br /><br />
25. pVeg+XylR 10 <br /><br />
26. pVeg+XylR 11 <br /><br />
27. pVeg+XylR 12 <br /><br />
28. pVeg 96 control <br /><br />
<br />
• Ran gel with: <br /><br />
1. A3 1/50 PCR X,P <br /><br />
2. A3+pSB1C3 6 X,P <br /><br />
3. pVeg 96 X,P <br /><br />
4. pVeg+pSB1C3 1 X,P <br /><br />
5. pVeg+pSB1C3 3 X,P <br /><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/ANDSugar
Team:UNAM Genomics Mexico/Notebook/ANDSugar
2012-10-27T03:38:14Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__TOC__<br />
<br /><br />
<center><h1>'''Arabinose/Xylose AND Gate Notebook'''</h1></center><br />
<br /><br />
<br /><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="20"><br />
<tr><br />
<td id="leftcolumn2"><center>2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JUNE | '''JUNE''']] </center> <br /><br />
<br /><br />
2.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F07.2F12 |06/07/12]] <br /><br />
2.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F12.2F12 |06/12/12]] <br /><br />
2.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F13.2F12 |06/13/12]] <br /><br />
2.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F14.2F12 |06/14/12]] <br /><br />
2.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F15.2F12 |06/15/12]] <br /><br />
2.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F18.2F12 |06/18/12]] <br /><br />
2.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F19.2F12 |06/19/12]] <br /><br />
2.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F22.2F12 |06/22/12]] <br /><br />
2.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F25.2F12 |06/25/12]] <br /><br />
2.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F26.2F12 |06/26/12]] <br /><br />
2.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F27.2F12 |06/27/12]] <br /><br />
2.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F29.2F12 |06/29/12]] <br /><br />
</td><br />
<br />
<td id="contentcolumn2"><center>3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JULY | '''JULY''']] </center> <br /><br />
<br /><br />
3.1[[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F03.2F12 | 07/03/12]] <br /><br />
3.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F04.2F12 | 07/04/12]] <br /><br />
3.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F06.2F12 | 07/06/12]] <br /><br />
3.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F07.2F12 | 07/07/12]] <br /><br />
3.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F08.2F12 | 07/08/12]] <br /><br />
3.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F09.2F12 | 07/09/12]] <br /><br />
3.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F10.2F12 | 07/10/12]] <br /><br />
3.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F11.2F12 | 07/11/12]] <br /><br />
3.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F12.2F12 | 07/12/12]] <br /><br />
3.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F13.2F12 | 07/13/12]] <br /><br />
3.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F14.2F12 | 07/14/12]] <br /><br />
3.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F16.2F12 | 07/16/12]] <br /><br />
3.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F17.2F12 | 07/17/12]] <br /><br />
3.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F18.2F12 | 07/18/12]] <br /><br />
3.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F19.2F12 | 07/19/12]] <br /><br />
3.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F20.2F12 | 07/20/12]] <br /><br />
3.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F23.2F12 | 07/23/12]] <br /><br />
3.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F24.2F12 | 07/24/12]] <br /><br />
3.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F25.2F12 | 07/25/12]] <br /><br />
3.20 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F26.2F12 | 07/26/12]] <br /><br />
3.21 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F27.2F12 | 07/27/12]] <br /><br />
3.22 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F30.2F12 | 07/30/12]] <br /><br />
3.23 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F31.2F12 | 07/31/12]] <br /><br />
</td><br />
<br />
<td id="rightcolumn2"><center>4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#AUGUST | '''AUGUST''']] </center> <br /><br />
<br /><br />
4.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F01.2F12 | 08/01/12]] <br /><br />
4.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F02.2F12 | 08/02/12]] <br /><br />
4.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F03.2F12 | 08/03/12]] <br /><br />
4.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F06.2F12 | 08/06/12]] <br /><br />
4.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F08.2F12 | 08/08/12]] <br /><br />
4.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F09.2F12 | 08/09/12]] <br /><br />
4.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F10.2F12 | 08/10/12]] <br /><br />
4.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F13.2F12 | 08/13/12]] <br /><br />
4.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F16.2F12 | 08/16/12]] <br /><br />
4.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F20.2F12 | 08/20/12]] <br /><br />
4.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F21.2F12 | 08/21/12]] <br /><br />
4.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F22.2F12 | 08/22/12]] <br /><br />
4.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F23.2F12 | 08/23/12]] <br /><br />
4.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F24.2F12 | 08/24/12]] <br /><br />
4.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F25.2F12 | 08/25/12]] <br /><br />
4.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F28.2F12 | 08/28/12]] <br /><br />
4.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F29.2F12 | 08/29/12]] <br /><br />
4.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F30.2F12 | 08/30/12]] <br /><br />
4.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F31.2F12 | 08/31/12]] <br /><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2"><center>5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#SEPTEMBER | '''SEPTEMBER''']] </center> <br /><br />
<br /><br />
5.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F01.2F12 | 09/01/12]] <br /><br />
5.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F02.2F12 | 09/03/12]] <br /><br />
5.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F04.2F12 | 09/04/12]] <br /><br />
5.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F05.2F12 | 09/05/12]] <br /><br />
5.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F06.2F12 | 09/06/12]] <br /><br />
5.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F14.2F12 | 09/14/12]] <br /><br />
<br />
</td><br />
<br />
<td id="contentcolumn2"><center>[[File:UnmagenomicsBacteria sugar.png | 190px]]</center><br />
</td><br />
<br />
<td id="rightcolumn2"><p> '''On hover the images to see descriptions'''</p><td/><br />
</tr><br />
</table><br />
<br />
<br />
=JUNE=<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html> <br />
<h2>06/07/12</h2><br /><br />
PY BROTH 10g salt/L<br /><br />
PSB2K3 kanamicin<br /><br />
We transformed RFP E1010. <br /><br />
plate 1 18F<br /><br />
plate 2 17E<br /><br />
Stock 50 mg/mL<br /><br />
''E.coli'' 1/1000<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
We made liquid cultures with colonies [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
We extracted plasmids [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PLASMID_EXTRACTION_2 | PLASMID EXTRACTION PROTOCOL]]. <br /><br />
We ran a gel to check the extraction [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<h2>06/12/12</h2><br /><br />
We made glycerols of the bacteria <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed plasmid pHp45 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/38/UnamgenomicsandsugarBitacora_2.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>06/13/12</h2><br /><br />
We ran gel and extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] and lysed. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2d/UnamgenomicsandsugarBitacora_3.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
We extracted from gel<br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/14/12</h2><br /><br />
We extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made glycerols of pHp45 Ω [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450<br />
PSB4A5 Am (ampicillin) 1I BBa_J04450<br />
AraC BBa_C0080 <br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
2011 14L plate 1 pSB2K3 Km+<br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/5/56/UnamgenomicsandsugarBitacora_5.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
pHp45 Ω with E/P<br /><br />
We extracted from gel<br /><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/15/12</h2><br /><br />
> Digested pHp45 Ω with E/P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
> We extracted plasmid with kit. <br /><br />
> Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (5) <br /><br />
>Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br /><br />
ARAC<br /><br />
>From the overnight plate we prepared liquid culture [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
>We left them incubating overnight. <br /><br />
>We left a plate (LB Km DH5α C0080 and a control). <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<h2>06/18/12</h2><br /><br />
>Plasmid extraction AraC with kit. <br /><br />
>C0080-psB2K3 915 bp<br /><br />
>Streaked 4 LB Km 30 plates DH5α psB2K3. <br /><br />
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red. <br /><br />
>Ran gel with Spr/Strr (eppendorf -20ºC) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
>Made glycerols from one tube of DH5α C0080 Km 50 -> 04. <br /><br />
>Made glycerols from one tube of DH5α PSB4A5 Amp 100 -> 05 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]]. <br /><br />
>Digested C0080 X,S. <br /><br />
>Digested B0014 E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/5/52/UnamgenomicsandsugarBitacora_6.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
Digested B0014 with E, P and with E,X<br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<br /><br />
<br />
<h2>06/19/12</h2><br /><br />
>Digested B0014 with E, P and with E,X. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
>Ran gels with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation. <br /><br />
C0080 is in the first lane. <br /><br />
>Extracted from gel C0080 X,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Made glycerols from 4 plates LB Km DH5α pSB2K3 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
>Take out AmyE 5’ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
AmyE 5’ 18K plate 3 2010, 2011, 2012<br /><br />
AmyE 3’ 18M plate 3 2010, 2011, 2012<br /><br />
>Digested B0014 with E,X and E,P again [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<h2>06/22/12</h2><br /><br />
<br />
>Ran gels with digestions B0014 with E,X and E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
<br />
>Dephosphated B0014 E,X and B0014 E,P. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<h2>06/25/12</h2><br /><br />
>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
<br />
<h2>06/26/12</h2><br /><br />
>Ran gel with psB2K3 and psB4A5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
<h2>06/27/12</h2><br /><br />
>Transformed with plasmid B0079 1576 bp psB1A2 12A [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011<br /><br />
AmyE 5’ BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011<br /><br />
AmyE 3’ BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011<br /><br />
AmyE 5’ grew 2 colonies. <br /><br />
<br />
<h2>06/29/12</h2><br /><br />
>We did a DH5α K143001 Km30 Amp 100 glycerol [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] 07. <br /><br />
>We plated twice DH5α K143002 Km 30 Amp 100 + control and twice DH5α B0079 Amp 100 + control<br /><br />
These were both plated in 2 plates each. <br /><br />
They were left overnight since they had not grown by 7:00 pm so the next day glycerols were made. <br /><br />
>Liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
2 tubes DH5α K143001 Km30 Amp 100 <br /><br />
2 tubes DH5α K143002 Km30 Amp 100 <br /><br />
2 tubes DH5α B0079 Amp 100 <br /><br />
1 tube LB Km 30 Amp 100 control<br /><br />
1 tube LB Amp 100 control<br /><br />
>From the 6 tubes we extracted plasmid from kit. <br /><br />
<br /><br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=JULY=<br />
<br />
07/02/12<br /><br />
>Digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /><br />
>PCR’s<br /><br />
•AraC<br /><br />
•Cassete ΩSpr/Strr <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PCR_Protocol_.2850.C2.B5l.29 | PCR PROTOCOL]] <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/be/UnamgenomicsandsugarBitacora_8.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>After Cassete ΩSpr/Strr PCR we ran a gel <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsandsugarBitacora_9.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/03/12</h2><br /><br />
>After Cassete ΩSpr/Strr PCR we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (8) <br /><br />
>Ran gel with digestions from yesterday. (9) <br /><br />
>Did band extractions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Stored at -20ºC. <br /><br />
>Did PCR to Cassete ΩSpr/Strr to add RBS and prefix/suffix. <br /><br />
>Left digesting with E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/03/UnamgenomicsandsugarBitacora_9.1.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/04/12</h2><br /><br />
>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1). <br /><br />
>Ligated Cassete ΩSpr/Strr +K143002 dephosphated(digested with E,X and E,S). <br /><br />
>Transformed ligation and left overnight plated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs. <br /><br />
>Digested with PstI [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>07/06/12</h2><br /><br />
4 LB Km30 Spec60 DH5α ΩSpr/Strr +K143002 still have not grown. <br /><br />
Ran a gel with yesterday’s digestions to chek if they were done properly (10) <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
Ran another gel with the rest of the samples. <br /><br />
Extracted GusA fragment <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
Repeated ligation ΩSpr/Strr +K143002 E,X dephosphated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Repeated ΩSpr/Strr PCR. <br /><br />
<br /><br />
<br /><br />
<h2>07/07/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002 . <br /><br />
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<br /><br />
<br />
<h2>07/08/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/13/UnamgenomicsandsugarBitacora_11.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
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</li><br />
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<div class='captionnaranja'><br />
<p class='captionInside'>1 PCR omega P<br /><br />
2 PCR omega I<br /><br />
3 PCR AraC P<br /><br />
4 PCR AraC I<br /><br />
5 ladder 1 Kb<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>07/09/12</h2><br /><br />
From yesterday’s transformations only one colony grew. <br /><br />
From the previous transformation only 2 colonies grew. <br /><br />
These 3 were streaked in 3 plates: <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km 30 Sp 100 control. <br /><br />
Did liquid cultures in 3 tubes: <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km30 Sp 100 control. <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
Ran a gel with: (11) <br /><br />
GusA P<br /><br />
PBBR1 GusA<br /><br />
Ω E PCR P<br /><br />
Ω PCR P<br /><br />
Ω PCR I<br /><br />
Ω E<br /><br />
Ω PCR I<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] <br /><br />
Did GusA primers dissolution for PCR. <br /><br />
GusA PCR <br /><br />
Gel Extraction by kit of lanes 3 and 5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
'''PCR omega P, PCR omega I, PCR AraC P, PCR AraC I'''<br /><br />
Add 10 μl of each primer (LW and UP). <br /><br />
Add 3 μl of plasmid (P). <br /><br />
Add 30.4 μl buffer. <br /><br />
Add 5 μl Mg. <br /><br />
Add 8 μl DNTp’s. <br /><br />
Add 42.6μl H2O miliQ. <br /> <br />
Add 1 μl RTTG polymerase. <br /><br />
Centrifuge (spin) 8 secs. <br /><br />
Add vegetable oil till the eppendorf is full. <br /><br />
Place eppendorf 1 mL in thermocycler. <br /><br />
Run PCR with program “BERNA”. <br /><br /><br />
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<img src='https://static.igem.org/mediawiki/2012/4/4b/UnamgenomicsandsugarBitacora_11.2.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/10/12</h2><br /><br />
•GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S (2 times)[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTIONS]]. (11.2) <br /><br />
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<br /><br />
<br />
<h2>07/11/12</h2><br /><br />
•GusA PCR digestions with E,S; Ω+AmyE 3’ with E,X [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•T.V. AraC PCR+ Ω+AmyE 3’ E,X dephosphated ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/0a/UnamgenomicsandsugarBitacora_12.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1. ladder. <br /><br />
2. GusA PCR E,S. <br /><br />
3. Ω+AmyE 3’ with E,X. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/12/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: (12) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Transformed AraC PCR+ Ω+AmyE 3’ ligation in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•From pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid. <br /><br />
•Made glycerols from 2 LB Km 30 Sp100 DH5α Ω+AmyE 3’ plates and from 1 LB Amp100 pfrc54 (A3) plates with control[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
•Ligated GusA PCR with B0014 E,X desphophorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
•Digested Pfrc54 (A3) with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•Desphophorylated Ω+AmyE 3’ E,X. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/0f/UnamgenomicsandsugarBitacora_13.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>•Ran gel with pfrc54 S,P<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
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<br />
<h2>07/13/12</h2><br /><br /><br />
•Ran gel with pfrc54 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (13) <br /><br />
•Transformation of GusA PCR + B0014 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Transformed with GFP E0040 psBIA2. <br /><br />
•Liquid Cultures with ligation: AraC+ Ω+AmyE 3’ for plasmid extraction at night [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•''B. Subtitils'' competent cells. <br /><br />
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<h2>07/14/12</h2><br /><br /><br />
•From 24 AraC+ Ω+AmyE 3’ LB Km 30 Sp 100 DH5α tubes: <br /><br />
•Extracted pellets<br /><br />
•Extracted plasmids. <br /><br />
•Ran gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br /><br />
•From transformed DH5α km 30: <br /><br />
GusA+B0014 DH5α Km50 24 pellets <br /><br />
E0040 DH5α LB Amp100 <br /><br />
From these two we: <br /><br />
•Did liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Extracted plasmid. <br /><br />
•From the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ran gel with this transformation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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<br /><br />
<br /><br />
<h2>07/16/12</h2><br /><br /><br />
•Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
•Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_14.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>07/17/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (14) <br /><br />
•The gel we ran didn’t work, probably because the agarose was not prepared correctly. <br /><br />
•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/db/Bitacora_15.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br /><br />
<br />
<h2>07/18/12</h2><br /><br /><br />
•From yesterday’s digestions we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (15) <br /><br /><br />
•We made liquid cultures of AraC+ Ω+AmyE 3’ 12, 15, 21 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br /><br />
•We ligated AraC PCR E,S dephosphated with + Ω+AmyE 3’ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
•We digested 1 + Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/19/12</h2><br /><br /><br />
•We ran We digested 1 + Ω+AmyE 3’ E,P with Miguel’s enzymes and with Abiel’s enzymes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
•We transformed with ligations AraC+ Ω+AmyE 3’ E and + Ω+AmyE 3’ E [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br /><br />
•Extracted plasmids from AraC+ Ω+AmyE 3’ 12,15,21. <br /><br /><br />
•Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f3/UnamgenomicsandsugarBitacora_16.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/20/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (16) <br /><br />
•Transformed GusA+B0014 in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Made liquid cultures from AraC+ Ω+AmyE 3’s transformation LB Km30 Sp 100 and control LB Km 30 Sp100 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<h2>07/23/12</h2><br /><br /><br />
•From 24 GusA+B0014 tubes (-) we didn’t do anything. <br /><br />
•From 4 AraC+ Ω+AmyE 3’ we extracted plasmid. <br /><br />
•Digested from the 4 AraC+ Ω+AmyE 3’ E,X; X; P and GusA PCR with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/24/12</h2><br /><br /><br />
•Digested B0014 E with X<br />
B0014 X with E<br /><br />
•Digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>07/25/12</h2><br /><br /><br />
•Ran gel of digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Joined B0014 E with X B0014 X with E digestions. <br /><br />
•Made liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. Transformed [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•From LasR DH5α make liquid cultures and plate [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Digested Ω+AmyE 3’ with X,P; E,S; C0080 X,P; E,S; S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Ligated Gus PCR X,P with B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<li><br />
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<div class='captionazul'><br />
<p class='captionInside'>1.Ω+AmyE 3’ X,P<br /><br />
2. Ω+AmyE 3’ E,S<br /><br />
3.C0080 X,P<br /><br />
4.C0080 E,S<br /><br />
5.C0080 S,P<br /><br />
6.ladder<br /><br />
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</li><br />
</ul><br />
</div><br />
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<br />
<h2>07/26/12</h2><br /><br />
•From C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5α C0179 LB Cb 100 (two cultures) and a control without bacteria [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•We extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguel’s transformation), Puc, B.S. control and PSB4AE. <br /><br />
<br />
•Due to problems with the way we did the transformations of ligations we repeated them: <br /><br />
GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
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GusAPCR X,P+ B0079 S,P dephosphorylated (-)<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•AraC+ Ω+AmyE 3’ S,P 1,2,3,4<br /><br />
•K143002 X,P<br /><br />
•AraC+ Ω S,P<br /><br />
•C0179 X,S<br /><br />
<br />
<br />
<h2>07/27/12</h2><br /><br /><br />
Ligated AraC+ Ω S,P dephosphorylated + K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<h2>07/30/12</h2><br /><br /><br />
•Transformed with ligations: <br /><br />
•AraC+ Ω dephosphprylated +K143002 X,P<br /><br />
•GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
•GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
•Transformed the following sythesis: <br /><br />
•91996 Pveg 140 bp<br /><br />
•91997 ArsR-CzrA_promoter 1 194 bp<br /><br />
•91998 ArsR-CzrA_promoter 2 221 bp<br /><br />
•91999 ArsR-CzrA_promoter 3 213 bp<br /><br />
•92000 pBad-pXyl 387 bp<br /><br />
•92001 XylR 1117pb<br /><br />
•92002 CI_pro_(NAND_INHIBITOR) 774 <br /><br />
•These synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked.<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Make liquid cultures of the following transformations for tomorrow [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]: <br /><br />
•AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+B0079<br /><br />
•Synthesis<br /><br />
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<h2>07/31/12</h2><br /><br /><br />
Extracted plasmids from yesterday’s cultures and from K143002 cultures that were in the other lab. <br /><br />
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::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=AUGUST=<br />
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<h2>08/01/12</h2><br /><br /><br />
Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
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<div class='captiongray'><br />
<p class='captionInside'>1 PCR GusA I<br /><br />
2 PCR GusA I<br /><br />
3 PCR GusA P<br /><br />
4 PCR GusA P<br /><br />
5 ladder 1 Kb<br /></p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/02/12</h2><br /><br /><br />
•Extracted plasmids from liquid cultures: <br /><br />
AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+BBR1<br /><br />
<br />
PCR GusA, GusA I, PCR GusA P, PCR GusA I<br /><br />
<br />
•Add 10 μl of each primer (LW and UP). <br /><br />
•Add 3 μl of plasmid (P). <br /><br />
•Add 30.4 μl buffer. <br /><br />
•Add 5 μl Mg. <br /><br />
•Add 8 μl DNTp’s. <br /><br />
•Add 42.6μl H2O miliQ. <br /><br />
•Add 1 μl RTTG polymerase. <br /><br />
•Centrifuge (spin) 8 secs. <br /><br />
•Add mineral oil till the eppendorf is full. <br /><br />
•Place eppendorf 1 mL in thermocycler. <br /><br />
•Run PCR with program “BERNA”. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/82/UnamgenomicsandsugarBitacora_18.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.A3+GusA 1<br /><br />
4.A3+GusA 2<br /><br />
5.A3+GusA 3<br /><br />
6.P GusA<br /><br />
7.P GusA 2<br /><br />
8.Ladder<br /><br />
9.01<br /><br />
10.06<br /><br />
11.96<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br /><br />
<br /><br />
<br />
<h2>08/03/12</h2><br /><br /><br />
• Ran a gel with: (18) <br /><br />
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[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Ran another gel to extract with: <br /><br />
1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.00<br /><br />
4.01<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]: <br /> <br />
<br />
• Ω+K143002 E,X; A3+GusA E,S; GusAPCR X,P <br /><br />
<br />
•Extracted GusAPCR X,P digestion [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Left the following ligation: AraC+ Ω+K143002 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/17/UnamgenomicsandsugarBitacora_19.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3+GusA 2<br /><br />
2.A3+GusA 3<br /><br />
3. Ω+AmyE 3’ 2<br /><br />
4. Ω+AmyE 3’ 3<br /><br />
5.XylR X,P<br /><br />
6.pBad-pXyl X,P<br /><br />
7.GusA PCR X,P<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/06/12</h2><br /><br /><br />
•Made liquid cultures of Ω+K143002 (2 cultures+1 control) and AraC+ Ω+K143002 (2 cultures+1 control) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ligated GusAPCR X,P+B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
•Ran a Gel with: (19) <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/8b/UnamgenomicsandsugarBitacora_19.1.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/08/12</h2><br /><br />
BBa_B0040 6I psB1A2 Amp+ plate 1<br /><br />
Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (19.1) <br /><br />
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<br />
<h2>08/09/12</h2><br /><br />
•From yesterday’s PCR’s : <br /><br />
• E0040 plasmid 1<br /><br />
E0040 plasmid 2<br /><br />
E0040 digested 1<br /><br />
E0040 digested 2<br /><br />
We purified with PCR kit<br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Made 14 PCR’s from AraC+ Ω+AmyE 3’. <br /><br />
•From B0079+GusA ligation and B0040 transformation:<br />
•Grew colonies. <br /><br />
•Made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Streaked these in a new plate. <br /><br />
•Diluted plasmid E0080 2 1/50. <br /><br />
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<br />
<h2>08/10/12</h2><br /><br />
•Yesterday’s gel did not come out as expected so we repeated the PCR. <br /><br />
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<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/e/ef/UnamgenomicsandsugarBitacora_20.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.AraC+ Ω+AmyE 3’ <br /><br />
2-14.AraC+ Ω+AmyE 3’ <br /><br />
15.AraC+ Ω 1<br /><br />
16.AraC+ Ω 1<br /><br />
17.AraC+ Ω 1<br /><br />
18.PCR 1 GFP<br /><br />
19.PCR GFP<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<h2>08/13/12</h2><br /><br />
•After running a gel with 14 PCR’s AraC+ Ω+AmyE 3’ and it did not come out as expected, we ran a gel with the ligation AraC+ Ω+AmyE 3’ to select candidate (that weight the same as the ligation) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (20) <br /><br />
<br />
•Extracted pasmids form liquid cultures: B0049, B0079+GusA. <br /><br />
<br />
•AraC+ Ω+AmyE 3’ undigested didn’t run in the gel as expected, it ran similarly to AraC+ Ω<br /><br />
<br />
• We repeated the ligation AraC+ Ω S,P dephosphorylated+K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
• GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.<br />
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• We ran a gel to extract. <br /><br />
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<div class='thumbnailWrapper'><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f1/UnamgenomicsandsugarBitacora_22.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>1.6 AraC+ Ω+AmyE 3’ E with P. <br /><br /><br />
2.8 AraC+ Ω+AmyE 3’ E with P. <br /><br />
3.11 AraC+ Ω+AmyE 3’ E with P. <br /><br />
4.12 AraC+ Ω+AmyE 3’ E with P. <br /><br />
5.6 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
6.8 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
7.11 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
8.12 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
9.00 pBad/pXyl with S,P. <br /><br />
10.Ladder. <br /><br />
11.E0040 PCR E,S to extract the correct band. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/16/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: <br /><br />
<br />
•Digested: <br /><br />
ArSR-CzrA 97 with S,P<br /><br />
ArSR-CzrA 98 with S,P<br /><br />
ArSR-CzrA 99 with S,P<br /><br />
E0040 PCR with E,S<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
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<h2>08/20/12</h2><br /><br />
•Ligated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/21/12</h2><br /><br />
•Joined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated. <br /><br />
•Extracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR. <br /><br />
•Digested Ω+AmyE 3’ with E,X and + Ω+AmyE 3’ II with E,X (obtained these two from glycerols stored at -80ºC [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/29/UnamgenomicsandsugarBitacora_22.1.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested K143001+PBad, pXyl with E,S <br /> <br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/1c/UnamgenomicsandsugarBitacora_23.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>1.K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
4.A3 PCR<br /><br />
5.A3 PCR<br /><br />
6.E0040 PCR E,S<br /><br />
7.B0014 E,X dephosphorylated<br /><br />
8. Ω+AmyE 3’ E,X <br /><br />
9. Ω+AmyE 3’ E,X II<br /><br />
10.Ladder<br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/22/12</h2><br /><br />
•We did 2 PCR’s for A3. <br /><br />
<br />
•Digested K143001+PBad, pXyl with E,S <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (22.1) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did band extraction of: <br /><br />
1. K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
•Made liquid cultures from E1010+Boo14 that the other lab gave us and did 2 plates for glycerol Km+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Did 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ Ω+AmyE 3’ 6,8,11,12 Km+ and Spectinomycin+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Transformed ligations: <br /><br />
B0079+GusA Amp+<br /><br />
Pveg+XylR Chloramphenicol+<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]].. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/78/UnamgenomicsandsugarBitacora_23.01.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1. Ω+AmyE 3’ E,X 6.2<br /><br />
2.Ω+AmyE 3’ II E,X<br /><br />
3.97 ArsR-CzrA<br /><br />
4.98 ArsR-CzrA<br /><br />
5.99 ArsR-CzrA<br />
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</li><br />
</ul><br />
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<br />
<h2>08/23/12</h2><br /><br />
•The negative controls were contaminated. We didn’t manipulate the ones that had grown because of this. <br /><br />
•Ran gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
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<h2>08/24/12</h2><br /><br />
From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ Ω+AmyE 3’ 6 and 8 we streaked 2 plates for glycerol of each ligation. <br />Repeated ligation B0079+GusA. <br /><br />
<br />
Ligated E0040 PCR E,S+B0014 E,X dephospohylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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Made 2 new plates from Pveg+XylR ligation and liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<h2>08/25/12</h2><br /><br />
Extracted pellet from 16 tubes of pVeg+XylR that were left overnight. <br /><br />
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Ligated B0079+GusA and B0040+B0014 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<h2>08/28/12</h2><br /><br />
<br />
Made liquid cultures from transformations tht were left overnight <br />
-R0079+GusA<br /><br />
-R0079+GusA (-)<br /><br />
-E0040+B0014<br /><br />
-E0040+B0014 (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/25/UnamgenomicsandsugarBitacora_23.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. A3 PCR 1. <br /><br />
2. A3 PCR 2. <br /><br />
3. Ω+AmyE 3’ S,P *.<br /><br />
4. Ω+AmyE 3’ S,P . <br /><br />
5. Pveg+XylR E,S 2. <br /><br />
6. Pveg+XylR E,S 3. <br /><br />
7. Pveg+XylR E,S 4. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/29/12</h2><br /><br />
Digested Pveg+XylR 2,3,4 with E,S. <br /><br />
<br />
Digested Ω+AmyE 3’ with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTION]]. <br /><br />
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Ran a PCR with A3 PCR (2 .6 ml tubes). <br /><br />
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Ran a gel with <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/2/2e/UnamgenomicsandsugarBitacoraBitacora_24.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1-23. E0040 + B0014 colonies<br /><br />
24-29. B0079+GusA 1<br /><br />
30. B0079<br /><br />
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</li><br />
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</html><br />
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<h2>08/30/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (24) <br /><br />
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•Digested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='captionnaranja'><br />
<p class='captionInside'>1-3B0079+GusA E,S 3. <br /><br />
4-9. PVeg+XylR E,S 1. <br /><br />
10-17. E0040+B0014 X,P 3. <br /><br />
18. 1 kb ladder. <br /><br />
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</li><br />
</ul><br />
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<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/d2/UnamgenomicsandsugarBitacora_26.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3 PCR. <br /><br />
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</li><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7b/UnamgenomicsandsugarBitacora_27.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>E0040+B0014 X,P 4 to extract<br /><br />
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</li><br />
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<h2>08/31/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (25) <br /><br />
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•Dephosphorylated: <br /><br />
Ω+AmyE 3’ S,P <br /><br />
Ω+AmyE 3’ II S,P <br /><br />
6 AraC+ Ω+AmyE 3’ E,X<br /><br />
8 AraC+ Ω+AmyE 3’ E,X<br /><br />
11 AraC+ Ω+AmyE 3’ E,X<br /><br />
12 AraC+ Ω+AmyE 3’ E,X<br /><br />•Ran a gel with A3 PCR [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (26) <br /><br />
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•It was the second time we didn’t obtain a band from A3’s PCR.<br />
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•Probably because it had been missing the buffer 2’s ethanol (“blue lid”) from the PCR purification. <br /><br />
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•Ran a gel woth E0040+B0014 X,P 4 to extract <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | Gen extraction protocol]]. <br /><br />
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•Did an A3 PCR. <br /><br />
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=SEPTEMBER=<br />
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<p class='captionInside'>1.A3 PCR 1. <br /><br />
2.A3 PCR 2. <br /><br />
3.E0040+B0014 X,P 1. <br /><br />
4.E0040+B0014 X,P 2. <br /><br />
5.Ω+AmyE 3’ dephosphorylated S,P. <br /><br />
6.Ω+AmyE 3’ dephosphorylated S,P II. <br /><br />
7.97 dephosphorylated S,P. <br /><br />
8.98 dephosphorylated S,P. <br /><br />
9.99 dephosphorylated S,P. <br /><br />
10-13. Arac+ Ω+AmyE 3’ dephosphorylated E,X 12. <br /><br />
14. 1 kb Ladder. <br /><br />
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<h2>09/01/12</h2><br /><br />
•Ran a gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (28) <br /><br />
<br />
•Ligated E0040+B0014 X,P 2 +promoter S,P dephosphorylated; K143002 X,P+ Ω+AmyE 3’ dephosphorylated S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<h2>09/03/12</h2><br /><br />
<br />
•Digested B0079+GusA E,S 3,4,5; PVeg+XylR E,S 1,5,7,10,13,16 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>09/04/12</h2><br /><br />
•Ran gel with digestions: <br /><br />
B0079+GusA E,S 3,4,5<br /><br />
PVeg+XylR E,S 1,5,7,10,13,16 <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Purified A3 PCR 1,2. <br /><br />
<br />
•Ran a gel of A3 PCR <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] and digested with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•From ligations: PVeg+E0040+B0014, PBad/PXyl+E0040+B0014, B0079+E0040+B0014 only colonies grew in PBad/PXyl+E0040+B0014.<br />
<br />
•Plated colonies that grew in a new plate. <br /><br />
<br />
•Made liquid cultures of these <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•Repeated the ones that did not grow. <br /><br />
<br />
•Digested E0040+B0014 4 with X,P[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
-BBa_J23101 18E plate 1 plasmid BBa_J61002_2948 bp Amp+<br /><br />
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<div class='captionaqua'><br />
<p class='captionInside'>24 PBad/PXyl+E0040+B0014 tubes we extracted <br /><br />
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<p class='captionInside'>1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
Extracted band from 1. and 2<br />
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<div class='captionmorado'><br />
<p class='captionInside'>Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S <br />
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<h2>09/05/12</h2><br /><br />
•From the 24 PBad/PXyl+E0040+B0014 tubes we extracted plasmids (29) and ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Extracted plasmid from 2 tubes of E0040+B0014. <br /><br />
<br />
•Ran a gel with yesterday’s digestions: <br /><br />
1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
<br />
•Extracted band from 1. and 2. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Purified A3 PCR from 2 0.6ml tubes 1 and 2. <br /><br />
<br />
•1 colony grew from ligation pVeg+E0040+B0014. <br /><br />
<br />
•We streaked this in another plate and did liquid cultures <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•To do: <br /><br />
•Ligate B0079 S,P dephosphorylated + E0040+B0014 X,P and Ω+AmyE 3’ S,P dephosphorylated+ K143001 X,P. <br /><br />
•Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S 10,13. (31) <br /><br />
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<div class='captionazul'><br />
<p class='captionInside'>•A3 PCR and gel A3 PCR X,P <br />
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<br />
<h2>09/06/12</h2><br /><br />
<br />
•A3 PCR and gel A3 PCR X,P 1,2. <br /><br />
<br />
<br />
<h2>09/14/12</h2><br /><br />
<br />
•Digested Ω+AmyE 3’ 11,13,18, and 22 with E,X. <br /><br />
•Ligate: <br /><br />
PVeg S,P dephosphorylated+XylR X,P. <br /><br />
PBad p Xyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
pSB13C3 E,P dephosphorylated + A3 PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + GusA PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + XylR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pVeg E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pBad pXyl E,P. <br /><br />
pSB13C3 E,P dephosphorylated + Ω PCR E,P. <br /><br />
<br /><br />
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<br /><br />
<h2>09/16/12</h2><br /><br />
• Colonies in these plates grew: <br /><br />
- R0079+E0040+B0014 <br /><br />
- pBad/pXyl <br /><br />
- XylR <br /><br />
- pVeg <br /><br />
- A3+E0040+B0014 from colonies 1,3,4,5,6,7,8,9,10,11,12,13,14,15,17 <br /><br />
- pVeg+E0040+B0014 1,2,3,4,5,6,8,9,10,11,12,13,14,15,16 <br /><br />
- pBad/pXyl+E0040+B0014 2 tubes from the same correct colony. <br /><br />
- pVeg+XylR 1,2 <br /><br />
- pBad/pXyl+pSB1C3 1,2 <br /><br />
- XylR+pSB1C3 1,2 <br /><br />
- pVeg+pSB1C3 1,2 <br /><br />
- A3+pSB1C3 1,2 <br /><br />
- GusA+pSB1C3 1,2 <br /><br />
- omegacassette+pSB1C3 1,2 <br /><br />
<br /><br />
• Ran gel from the plasmid extractions that we did yesterday: <br /><br />
- A3+E0040+B0014 1-16 <br /><br />
- pVeg+E0040+B0014 1-16, 18 <br /><br />
- omega cassette+AmyE 3’ 11,13,18,22 <br /><br />
• Digested R0079+E0040+B0014 with E,S. to ligate with AmyE 3’ E,x dephosphorylated. <br /><br />
• Digested omega cassette with E,P. <br /><br />
• Digested pSB1C3 with E,P. <br /><br />
• Made liquid cultures from AmyE 5’ and AmyE 3’. <br /><br />
• Did plasmid extraction from pSB1C3 colonies 3 and 6. <br /><br />
<br /><br />
<br />
<h2>09/17/12</h2><br /><br />
• Digested A3+E0040+B0014 colonies 2,3,4,5,6,11 and 16 with E,S. <br /><br />
• Digested pVeg+E0040+B0014 colonies 1,2,3,4,5,9 and 10 with E,S. <br /><br />
• Dephosphorylated omega cassette+AmyE 3’ colonies 13,18 and 22 digested with E,X. <br /><br />
• Digested pBad/pXyl and pVeg with S,P. Then dephosphorylated. <br /><br />
• Digested AmyE 3’ with E,X. Then dephosphorylated. <br /><br />
<br /><br />
<br />
<h2>09/18/12</h2><br /><br />
<br /><br />
• Ran gel with: <br /><br />
1. A3 2 E,S <br /><br />
2. A3 3 E,S <br /><br />
3. A3 4 E,S <br /><br />
4. A3 5 E,S <br /><br />
5. A3 6 E,S <br /><br />
6. A3 11 E,S <br /><br />
7. A3 16 E,S <br /><br />
8. E0040+B0014 colony 1 E,X <br /><br />
9. AmyE 5’ E,P <br /><br />
10. AmyE 3’ E,X <br /><br />
11. Omega cassette E,S <br /><br />
12. pSB1C3 E,P <br /><br />
13. ladder <br /><br />
Bottom: <br /><br />
1. pVeg 1 E,S <br /><br />
2. pVeg 2 E,S <br /><br />
3. pVeg 3 E,S <br /><br />
4. pVeg 4 E,S <br /><br />
5. pVeg 5 E,S <br /><br />
6. pVeg 9 E,S <br /><br />
7. pVeg 10 E,S <br /><br />
8. pVeg S,P dephosphorylated <br /><br />
9. pVeg 2 S,P <br /><br />
10. pBad/pXyl S,P <br /><br />
11. XylR X,P <br /><br />
12. R0079+GFP+TT E,S <br /><br />
13. R0079 S,P <br /><br />
14. Ladder <br /><br />
• Dephosphorylated GFP+TT E,X; AmyE 5’ E,X; pSB1C3 E,P; pVeg 2 S,P; pBad/pXyl S,P and R0079 S,P. <br /><br />
<br /><br />
<h2>09/19/12</h2><br /><br />
<br /><br />
• Did PCR from A3 and a 1/50 dilution. <br /><br />
• Ran gel with: <br /><br />
1. pVeg 3 E,P. <br /><br />
2. pVeg 9 E,P. <br /><br />
3. pVeg 10 E,P. <br /><br />
4. pBAd/pXyl 3 E,P. <br /><br />
5. pBAd/pXyl 5 E,P. <br /><br />
6. pBAd/pXyl 1 E,P. <br /><br />
7. A3 3 E,P. <br /><br />
8. A3 4 E,P. <br /><br />
9. XylR 1 E,P. <br /><br />
10. XylR 3 E,P. <br /><br />
11. XylR 8 E,P. <br /><br />
12. pSB1C3 6 E,P. <br /><br />
13. pSB1C3 6 E,P. <br /><br />
14. AmyE 5’ E,S. <br /><br />
15. AmyE 5’ E,S. <br /><br />
16. A3 1/50 PCR. <br /><br />
17. A3 PCR. <br /><br />
18. Ladder <br /><br />
• We did transformations: <br /><br />
- 2 of R0079+GusA <br /><br />
- R0079+GusA control <br /><br />
- 2 of A3+pSB1AK3 <br /><br />
- A3+pSB1AK3 control <br /><br />
- 2 of omega cassette+pSB1C3 <br /><br />
- 2 of GusA+pSB1C3 <br /><br />
• Digested: <br /><br />
- pVeg+pSB1C3 <br /><br />
- pBad/pXyl+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
- A3+pSB1C3 <br /><br />
With E,P. <br /><br />
• Ran gel with these digestions. <br /><br />
<br /><br />
<h2>09/19/12</h2><br /><br />
• Made liquids cultures from: <br /><br />
- Glicerol of AmyE 5’ <br /><br />
- Glicerol of AmyE 3’ <br /><br />
- pBad/pXyl+pSB1C3 colony 5 <br /><br />
• Made liquid cultures from yesterday transformations: <br /><br />
- R0079+GusA 2 and control <br /><br />
- A3+pSB1AK3 2 and control <br /><br />
- Omega cassette+pSB1C3 2 <br /><br />
- GusA+pSB1C3 2 <br /><br />
• We transformed yesterday ligations: <br /><br />
- pBad/pXyl+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
- pVeg+pSB1C3 <br /><br />
- A3+pSB1C3 <br /><br />
• Purified A3 PCR with kit, and then, digested with E,P and E,S. <br /><br />
• Ran gel <br /><br />
<br /><br />
<h2>09/20/12</h2><br /><br />
<br /><br />
• Made liquid cultures from transformations: <br /><br />
- Omega cassette+pSB1C3 colony 7 <br /><br />
- 2 of GusA+pSB1C3 colony 32 <br /><br />
- R0079+GusA colony 12 <br /><br />
• Digested A3 1/50 PCR with E,P and E,S. <br /><br />
• Made liquid cultures from R0079+E0040+B0014; pBad/pXyl+E0040+B0014; pVeg+E0040+B0014; A3+E0040+B0014. <br /><br />
• Ligated: <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P <br /><br />
- A3 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
<br /><br />
<h2>09/21/12</h2><br /><br />
<br /><br />
• Ran gel with: <br /><br />
1. R0079+GFP+TT 1 <br /><br />
2. R0079+GFP+TT <br /><br />
3. pBad/pXyl+pSB1C3 1 <br /><br />
4. pBad/pXyl+pSB1C3 <br /><br />
5. omega cassette+psB1C3 1 <br /><br />
6. omega cassette+psB1C3 <br /><br />
7. pSB1C3 E,P 1 <br /><br />
8. pSB1C3 E,P <br /><br />
9. pSB1C3 E <br /><br />
10. pSB1C3 P <br /><br />
11. A3 1/50 PCR E,P <br /><br />
<br /><br />
<br />
<h2>09/22/12</h2><br /><br />
• Ran gel with lysis extractions: <br /><br />
1. GusA+pSB1C3 1 <br /><br />
2. GusA+pSB1C3 2 <br /><br />
3. GusA+pSB1C3 3 <br /><br />
4. GusA+pSB1C3 4 <br /><br />
5. GusA+pSB1C3 5 <br /><br />
6. GusA+pSB1C3 6 <br /><br />
7. GusA+pSB1C3 7 <br /><br />
8. GusA+pSB1C3 8 <br /><br />
9. GusA+pSB1C3 9 <br /><br />
10. GusA+pSB1C3 10 <br /><br />
11. GusA+pSB1C3 11 <br /><br />
12. GusA+pSB1C3 12 <br /><br />
13. pSB1C3 <br /><br />
14. A3+E0040+B0014 1 <br /><br />
15. A3+E0040+B0014 2 <br /><br />
16. A3+E0040+B0014 3 <br /><br />
17. A3+E0040+B0014 4 <br /><br />
18. A3+E0040+B0014 5 <br /><br />
19. A3+E0040+B0014 6 <br /><br />
20. A3+E0040+B0014 7 <br /><br />
21. A3+E0040+B0014 8 <br /><br />
22. A3+E0040+B0014 9 <br /><br />
23. pVeg+E0040+B0014 3 <br /><br />
24. pVeg+E0040+B0014 4 <br /><br />
25. pVeg+E0040+B0014 5 <br /><br />
26. pVeg+E0040+B0014 6 <br /><br />
27. pVeg+E0040+B0014 7 <br /><br />
28. pVeg+E0040+B0014 8 <br /><br />
29. pVeg+E0040+B0014 9 <br /><br />
30. pVeg <br /><br />
31. pBad/pXyl+E0040+B0014 1 <br /><br />
32. pBad/pXyl+E0040+B0014 2 <br /><br />
33. pBad/pXyl+E0040+B0014 3 <br /><br />
34. pBad/pXyl+E0040+B0014 4 <br /><br />
35. pBad/pXyl+E0040+B0014 5 <br /><br />
36. pBad/pXyl+E0040+B0014 6 <br /><br />
37. pBad/pXyl+E0040+B0014 7 <br /><br />
38. pBad/pXyl+E0040+B0014 8 <br /><br />
39. pBad/pXyl+E0040+B0014 9 <br /><br />
40. pBad/pXyl+E0040+B0014 10 <br /><br />
41. pBad/pXyl <br /><br />
42. pVeg+XylR 1 <br /><br />
43. pVeg+XylR 2 <br /><br />
44. pVeg+XylR 3 <br /><br />
45. pVeg+XylR 4 <br /><br />
46. pVeg+XylR 5 <br /><br />
47. pVeg+XylR 6 <br /><br />
48. pVeg+XylR 7 <br /><br />
49. pVeg+XylR 8 <br /><br />
50. omega cassette <br /><br />
<br /><br />
• Ran gel with yesterday digestions: <br /><br />
1. R0079+GFP+TT E,P <br /><br />
2. R0079+GFP+TT E,P <br /><br />
3. Omega cassette+pSB1C3 E,P <br /><br />
4. Omega cassette+pSB1C3 E,P <br /><br />
5. Ladder <br /><br />
6. pBad/pXyl+pSB1C3 <br /><br />
7. pBad/pXyl+pSB1C3 <br /><br />
8. pSB1C3 6 E,P. <br /><br />
9. A3 1/50 PCR E,S <br /><br />
10. pBbak C011 <br /><br />
11. pBbak C012 <br /><br />
12. ladder <br /><br />
<br /><br />
• Did pellet from R0079+GusA. <br /><br />
• Did plasmid extraction from omega cassette+pSB1C3 colonies 1,4,6 and 7. <br /><br />
• Digested them with E,P. <br /><br />
• Digested: <br /><br />
- GusA+pSB1C3 1,3,4,10 and 11 <br /><br />
- A3+E0040+B0014 2,4,7 and 9 <br /><br />
- pVeg+E0040+B0014 4,5 and 6 <br /><br />
- pBad/pXyl+E0040+B0014 3,7 and 10 <br /><br />
- pVeg+XylR 2,4 and 5 <br /><br />
with E,P. <br /><br />
• Made liquid cultures from pVeg+pSB1C3 <br /><br />
• Ligated: <br /><br />
- XylR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- pVeg E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- A3 1/50 PCR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- A3 1/50 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
<br /><br />
<h2>09/23/12</h2><br /><br />
<br /><br />
• Ran gel with yesterday digestions: <br /><br />
1. Omega cassette+pSB1C3 1 E,P <br /><br />
2. Omega cassette+pSB1C3 4 E,P <br /><br />
3. Omega cassette+pSB1C3 6 E,P <br /><br />
4. Omega cassette+pSB1C3 7 E,P <br /><br />
5. GusA+pSB1C3 1 E,P <br /><br />
6. GusA+pSB1C3 3 E,P <br /><br />
7. GusA+pSB1C3 4 E,P <br /><br />
8. GusA+pSB1C3 10 E,P <br /><br />
9. GusA+pSB1C3 11 E,P <br /><br />
10. pSB1C3 E,P dephosphorylated <br /><br />
11. pVeg+XylR 2 E,P <br /><br />
12. pVeg+XylR 4 E,P <br /><br />
13. pVeg+XylR 5 E,P <br /><br />
14. pVeg S,P dephosphorylated <br /><br />
15. pVeg+GFP+TT 4 E,P. <br /><br />
16. pVeg+GFP+TT 5 E,P. <br /><br />
17. pVeg+GFP+TT 6 E,P. <br /><br />
18. ladder <br /><br />
Bottom <br /><br />
1. pBad/pXyl+GFP+TT 3 E,P <br /><br />
2. pBad/pXyl+GFP+TT 7 E,P <br /><br />
3. pBad/pXyl+GFP+TT 10 E,P <br /><br />
4. pBad/pXyl S,P dephosphorylated <br /><br />
5. A3+GFP+TT 2 E,P <br /><br />
6. A3+GFP+TT 4 E,P <br /><br />
7. A3+GFP+TT 7 E,P <br /><br />
8. A3+GFP+TT 9 E,P <br /><br />
9. Ladder <br /><br />
<br /><br />
<h2>09/24/12</h2><br /><br />
<br /><br />
• Did PCR from omega cassette+pSB1C3 colonies 1,4,6 and 7. <br /><br />
• Made 1/50 dilutions from these PCR products. <br /><br />
• Ran gel with: <br /><br />
1. Omega cassette+pSB1C3 1/50 PCR 1 <br /><br />
2. Omega cassette+pSB1C3 PCR 1 <br /><br />
3. Omega cassette+pSB1C3 1/50 PCR 4 <br /><br />
4. Omega cassette+pSB1C3 PCR 4 <br /><br />
5. Omega cassette+pSB1C3 1/50 PCR 6 <br /><br />
6. Omega cassette+pSB1C3 PCR 6 <br /><br />
7. Omega cassette+pSB1C3 1/50 PCR 7 <br /><br />
8. Omega cassette+pSB1C3 PCR 7 <br /><br />
9. GusA+pSB1C3 1 P <br /><br />
10. GusA+pSB1C3 3 P <br /><br />
11. GusA+pSB1C3 4 P <br /><br />
12. GusA+pSB1C3 10 P <br /><br />
13. GusA+pSB1C3 11 P <br /><br />
14. pVeg+pSB1C3 6 E,P <br /><br />
15. pVeg+pSB1C3 5 E,P <br /><br />
16. pVeg+pSB1C3 4 E,P <br /><br />
17. pVeg+pSB1C3 3 E,P <br /><br />
18. pVeg+pSB1C3 2 E,P <br /><br />
19. pVeg+pSB1C3 1 E,P <br /><br />
20. ladder <br /><br />
<br />
• From yesterday transformations, these didn’t grow: <br /><br />
- pBad/pXyl+E0040+B0014 1 and 2. <br /><br />
- A3+E0040+B0014. <br /><br />
• Made liquid cultures from transformations that did grow: <br /><br />
- pVeg+E0040+B0014. <br /><br />
- pVeg+XylR. <br /><br />
- A3+pSB1C3 <br /><br />
- pVeg+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
<br /><br />
<h2>09/26/12</h2><br /><br />
<br /><br />
• Did plasmid extractions form liquids cultures: <br /><br />
- 8 of pVeg+pSB1C3 <br /><br />
- 8 of A3 <br /><br />
- 8 of XylR <br /><br />
• Digested them with E,P. <br /><br />
• Ran gel with: <br /><br />
1. Ladder <br /><br />
2. XylR+pSB1C3 1 E,P <br /><br />
3. XylR+pSB1C3 2 E,P <br /><br />
4. XylR+pSB1C3 3 E,P <br /><br />
5. pVeg+pSB1C3 1 E,P <br /><br />
6. pVeg+pSB1C3 3 E,P <br /><br />
7. pVeg+pSB1C3 4 E,P <br /><br />
8. pVeg+pSB1C3 5 E,P <br /><br />
9. pVeg+pSB1C3 6 E,P <br /><br />
10. pVeg+pSB1C3 8 E,P <br /><br />
11. pSB1C3 6 E,P dephosphorylated <br /><br />
12. A3+pSB1C3 1 E,P <br /><br />
13. A3+pSB1C3 3 E,P <br /><br />
14. A3+pSB1C3 4 E,P <br /><br />
15. A3+pSB1C3 5 E,P <br /><br />
16. A3+pSB1C3 6 E,P <br /><br />
17. A3+pSB1C3 8 E,P <br /><br />
18. Ladder <br /><br />
19. P4+E0040+B0014 1 E,X <br /><br />
20. P4+E0040+B0014 7 E,X <br /><br />
<br /><br />
<h2>09/27/12</h2><br /><br />
<br /><br />
• Digested pVeg+XylR colonies 1,4,5 and 6 in pJ209 plasmid with E,S for 3 hours. <br /><br />
• Digested pVeg+E0040+B0014 colonies 7,9,10 and 11 in pasmid pJ209 with E,P. <br /><br />
<br /><br />
<h2>10/09/12</h2><br /><br />
<br /><br />
• Ligated: <br /><br />
- A3 1/50 PCR E,P + pSB1C3 6 E,P dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
- P4+CI+E1010+B0014 E,X dephosphorylated + AmyE 5’+pBad/pXyl E,S. <br /><br />
<br /><br />
<h2>10/10/12</h2><br /><br />
<br /><br />
• Ligated: <br /><br />
- GusA PCR E,P + pSB1C3 6 E,P dephosphorylated. <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- A3 1/50 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
• Digested R0079+E0040+B0014 with E,S. <br /><br />
• Digested AmyE 3’ with E,X. Then dephosphorylated. <br /><br />
• Digested pVeg and R0079, A3 with X,P. <br /><br />
• Digested pBad/pSB1C3 with S,P. <br /><br />
<br /><br />
<h2>10/11/12</h2><br /><br />
<br /><br />
• Did plasmid extraction from liquid cultures: <br /><br />
- 2 of AmyE 5’ <br /><br />
- 2 of AmyE 3’ <br /><br />
- 2 of E0040+B0014 <br /><br />
• Ran gel with: <br /><br />
1. A3 1/50 PCR X,P <br /><br />
2. A3 1/50 PCR X,P <br /><br />
3. A3+pSB1C3 6 X,P <br /><br />
4. A3+pSB1C3 6 X,P <br /><br />
5. A3+pSB1C3 8 X,P <br /><br />
6. A3+pSB1C3 8 X,P <br /><br />
7. pVeg X,P <br /><br />
8. pVeg 96 X,P <br /><br />
9. pVeg 96 X,P <br /><br />
10. pVeg+pSB1C3 1 X,P <br /><br />
11. pVeg+pSB1C3 1 X,P <br /><br />
12. pVeg+pSB1C3 3 X,P <br /><br />
13. pVeg+pSB1C3 3 X,P <br /><br />
14. R0079 X,P <br /><br />
15. Ladder <br /><br />
<br /><br />
<h2>10/12/12</h2><br /><br />
<br /><br />
• Ligated:<br />
- AmyE 5’+pBad/pXyl 10 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- AmyE 5’+pBad/pXyl 11 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- AmyE 5’+pBad/pXyl 12 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
- A3 1/50 PCR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
<br /><br />
<h2>10/12/12</h2><br /><br />
<br /><br />
• Digested AmyE 5’ with S,P <br /><br />
• Dephosphorylated it. <br /><br />
• Ligated: <br /><br />
- AmyE 5’ S,P dephosphorylated with <br /><br />
- R0079 X,P <br /><br />
- pVeg 96 X,P <br /><br />
- A3 1/50 PCR X,P <br /><br />
- A3+pSB1C3 6 X,P <br /><br />
- R0079+E0040+B0014 <br /><br />
• Ligated pBad/pXyl+pSB1C3 S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
• Ran gel with: <br /><br />
1. A3+pSB1C3 1 <br /><br />
2. A3+pSB1C3 2 <br /><br />
3. A3+pSB1C3 3 <br /><br />
4. A3+pSB1C3 4 <br /><br />
5. A3+pSB1C3 5 <br /><br />
6. A3+pSB1C3 6 <br /><br />
7. A3+pSB1C3 7 <br /><br />
8. A3+pSB1C3 8 <br /><br />
9. A3+pSB1C3 9 <br /><br />
10. A3+pSB1C3 10 <br /><br />
11. A3+pSB1C3 11 <br /><br />
12. A3+pSB1C3 6 control <br /><br />
13. AmyE 5’+P4+CI+E1010+B0014 1 <br /><br />
14. AmyE 5’+P4+CI+E1010+B0014 2 <br /><br />
15. AmyE 5’+P4+CI+E1010+B0014 3 <br /><br />
16. AmyE 5’+P4+CI+E1010+B0014 4 <br /><br />
17. AmyE 5’+P4+CI+E1010+B0014 5 <br /><br />
18. AmyE 5’+P4+CI+E1010+B0014 6 <br /><br />
19. AmyE 5’+P4+CI+E1010+B0014 7 <br /><br />
20. P4+CI+E1010+B0014 1 control <br /><br />
<br /><br />
<h2>10/09/12</h2><br /><br />
<br /><br />
• Ligate: <br /><br />
K143001+pBad/pXyl + p4+CI+E1010+TT E,X dephosphorylated. <br /><br />
• Ligate: <br /><br />
pVeg S,P dephosphorylated + XylR X,P. <br /><br />
• Ligate: <br /><br />
A3 PCR 1/50 E,P + pSB1C3 E,P dephosphorylated <br /><br />
<br /><br />
<h2>10/13/12</h2><br /><br />
<br /><br />
• Digested AmyE 5’ with S,P. Then dephosphorylated. <br /><br />
• Ligate: <br /><br />
AmyE 5’ S,P dephosphorylated + <br /><br />
- R0079 X,P <br /><br />
- pVeg 96 X,P <br /><br />
- A3 PCR 1/50 X,P <br /><br />
- A3+pSB1C3 colony 6 X,P <br /><br />
- R0079+E0040+B0014 E,X <br /><br />
• Ligate: <br /><br />
pBad/pXyl+pSB1C3 S,P dephosphorylated + E0040+B0014 X,P <br /><br />
<br />
• Ran a gel with: <br /><br />
1. A3+pSB1C3 1 <br /><br />
2. A3+pSB1C3 2 <br /><br />
3. A3+pSB1C3 3 <br /><br />
4. A3+pSB1C3 4 <br /><br />
5. A3+pSB1C3 5 <br /><br />
6. A3+pSB1C3 6 <br /><br />
7. A3+pSB1C3 7 <br /><br />
8. A3+pSB1C3 8 <br /><br />
9. A3+pSB1C3 9 <br /><br />
10. A3+pSB1C3 10 <br /><br />
11. A3+pSB1C3 11 <br /><br />
12. A3+pSB1C3 6 control <br /><br />
13. AmyE 5’+P4+CI+E1010+B0014 8 <br /><br />
14. AmyE 5’+P4+CI+E1010+B0014 9 <br /><br />
15. AmyE 5’+P4+CI+E1010+B0014 10 <br /><br />
16. P4+CI+E1010+B0014 1 control <br /><br />
17. pVeg+XylR 1 <br /><br />
18. pVeg+XylR 2 <br /><br />
19. pVeg+XylR 3 <br /><br />
20. pVeg+XylR 4 <br /><br />
21. pVeg+XylR 5 <br /><br />
22. pVeg+XylR 6 <br /><br />
23. pVeg+XylR 7 <br /><br />
24. pVeg+XylR 8 <br /><br />
25. pVeg+XylR 10 <br /><br />
26. pVeg+XylR 11 <br /><br />
27. pVeg+XylR 12 <br /><br />
28. pVeg 96 control <br /><br />
<br />
• Ran gel with: <br /><br />
1. A3 1/50 PCR X,P <br /><br />
2. A3+pSB1C3 6 X,P <br /><br />
3. pVeg 96 X,P <br /><br />
4. pVeg+pSB1C3 1 X,P <br /><br />
5. pVeg+pSB1C3 3 X,P <br /><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/ANDSugar
Team:UNAM Genomics Mexico/Notebook/ANDSugar
2012-10-27T03:37:23Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Arabinose/Xylose AND Gate Notebook'''</h1></center><br />
<br /><br />
<br /><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="20"><br />
<tr><br />
<td id="leftcolumn2"><center>2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JUNE | '''JUNE''']] </center> <br /><br />
<br /><br />
2.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F07.2F12 |06/07/12]] <br /><br />
2.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F12.2F12 |06/12/12]] <br /><br />
2.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F13.2F12 |06/13/12]] <br /><br />
2.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F14.2F12 |06/14/12]] <br /><br />
2.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F15.2F12 |06/15/12]] <br /><br />
2.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F18.2F12 |06/18/12]] <br /><br />
2.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F19.2F12 |06/19/12]] <br /><br />
2.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F22.2F12 |06/22/12]] <br /><br />
2.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F25.2F12 |06/25/12]] <br /><br />
2.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F26.2F12 |06/26/12]] <br /><br />
2.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F27.2F12 |06/27/12]] <br /><br />
2.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F29.2F12 |06/29/12]] <br /><br />
</td><br />
<br />
<td id="contentcolumn2"><center>3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JULY | '''JULY''']] </center> <br /><br />
<br /><br />
3.1[[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F03.2F12 | 07/03/12]] <br /><br />
3.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F04.2F12 | 07/04/12]] <br /><br />
3.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F06.2F12 | 07/06/12]] <br /><br />
3.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F07.2F12 | 07/07/12]] <br /><br />
3.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F08.2F12 | 07/08/12]] <br /><br />
3.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F09.2F12 | 07/09/12]] <br /><br />
3.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F10.2F12 | 07/10/12]] <br /><br />
3.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F11.2F12 | 07/11/12]] <br /><br />
3.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F12.2F12 | 07/12/12]] <br /><br />
3.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F13.2F12 | 07/13/12]] <br /><br />
3.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F14.2F12 | 07/14/12]] <br /><br />
3.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F16.2F12 | 07/16/12]] <br /><br />
3.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F17.2F12 | 07/17/12]] <br /><br />
3.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F18.2F12 | 07/18/12]] <br /><br />
3.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F19.2F12 | 07/19/12]] <br /><br />
3.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F20.2F12 | 07/20/12]] <br /><br />
3.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F23.2F12 | 07/23/12]] <br /><br />
3.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F24.2F12 | 07/24/12]] <br /><br />
3.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F25.2F12 | 07/25/12]] <br /><br />
3.20 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F26.2F12 | 07/26/12]] <br /><br />
3.21 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F27.2F12 | 07/27/12]] <br /><br />
3.22 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F30.2F12 | 07/30/12]] <br /><br />
3.23 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F31.2F12 | 07/31/12]] <br /><br />
</td><br />
<br />
<td id="rightcolumn2"><center>4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#AUGUST | '''AUGUST''']] </center> <br /><br />
<br /><br />
4.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F01.2F12 | 08/01/12]] <br /><br />
4.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F02.2F12 | 08/02/12]] <br /><br />
4.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F03.2F12 | 08/03/12]] <br /><br />
4.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F06.2F12 | 08/06/12]] <br /><br />
4.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F08.2F12 | 08/08/12]] <br /><br />
4.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F09.2F12 | 08/09/12]] <br /><br />
4.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F10.2F12 | 08/10/12]] <br /><br />
4.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F13.2F12 | 08/13/12]] <br /><br />
4.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F16.2F12 | 08/16/12]] <br /><br />
4.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F20.2F12 | 08/20/12]] <br /><br />
4.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F21.2F12 | 08/21/12]] <br /><br />
4.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F22.2F12 | 08/22/12]] <br /><br />
4.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F23.2F12 | 08/23/12]] <br /><br />
4.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F24.2F12 | 08/24/12]] <br /><br />
4.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F25.2F12 | 08/25/12]] <br /><br />
4.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F28.2F12 | 08/28/12]] <br /><br />
4.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F29.2F12 | 08/29/12]] <br /><br />
4.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F30.2F12 | 08/30/12]] <br /><br />
4.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F31.2F12 | 08/31/12]] <br /><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2"><center>5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#SEPTEMBER | '''SEPTEMBER''']] </center> <br /><br />
<br /><br />
5.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F01.2F12 | 09/01/12]] <br /><br />
5.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F02.2F12 | 09/03/12]] <br /><br />
5.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F04.2F12 | 09/04/12]] <br /><br />
5.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F05.2F12 | 09/05/12]] <br /><br />
5.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F06.2F12 | 09/06/12]] <br /><br />
5.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F14.2F12 | 09/14/12]] <br /><br />
<br />
</td><br />
<br />
<td id="contentcolumn2"><center>[[File:UnmagenomicsBacteria sugar.png | 190px]]</center><br />
</td><br />
<br />
<td id="rightcolumn2"><p> '''On hover the images to see descriptions'''</p><td/><br />
</tr><br />
</table><br />
<br />
<br />
=JUNE=<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html> <br />
<h2>06/07/12</h2><br /><br />
PY BROTH 10g salt/L<br /><br />
PSB2K3 kanamicin<br /><br />
We transformed RFP E1010. <br /><br />
plate 1 18F<br /><br />
plate 2 17E<br /><br />
Stock 50 mg/mL<br /><br />
''E.coli'' 1/1000<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
We made liquid cultures with colonies [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
We extracted plasmids [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PLASMID_EXTRACTION_2 | PLASMID EXTRACTION PROTOCOL]]. <br /><br />
We ran a gel to check the extraction [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<h2>06/12/12</h2><br /><br />
We made glycerols of the bacteria <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed plasmid pHp45 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/38/UnamgenomicsandsugarBitacora_2.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>06/13/12</h2><br /><br />
We ran gel and extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] and lysed. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
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<div class='captionrojo'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
We extracted from gel<br />
</li><br />
</ul><br />
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<h2>06/14/12</h2><br /><br />
We extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made glycerols of pHp45 Ω [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450<br />
PSB4A5 Am (ampicillin) 1I BBa_J04450<br />
AraC BBa_C0080 <br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
2011 14L plate 1 pSB2K3 Km+<br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/5/56/UnamgenomicsandsugarBitacora_5.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
pHp45 Ω with E/P<br /><br />
We extracted from gel<br /><br />
</li><br />
</ul><br />
</div><br />
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<h2>06/15/12</h2><br /><br />
> Digested pHp45 Ω with E/P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
> We extracted plasmid with kit. <br /><br />
> Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (5) <br /><br />
>Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
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ARAC<br /><br />
>From the overnight plate we prepared liquid culture [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
>We left them incubating overnight. <br /><br />
>We left a plate (LB Km DH5α C0080 and a control). <br /><br />
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<h2>06/18/12</h2><br /><br />
>Plasmid extraction AraC with kit. <br /><br />
>C0080-psB2K3 915 bp<br /><br />
>Streaked 4 LB Km 30 plates DH5α psB2K3. <br /><br />
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red. <br /><br />
>Ran gel with Spr/Strr (eppendorf -20ºC) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
>Made glycerols from one tube of DH5α C0080 Km 50 -> 04. <br /><br />
>Made glycerols from one tube of DH5α PSB4A5 Amp 100 -> 05 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]]. <br /><br />
>Digested C0080 X,S. <br /><br />
>Digested B0014 E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/5/52/UnamgenomicsandsugarBitacora_6.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
Digested B0014 with E, P and with E,X<br />
</li><br />
</ul><br />
</div><br />
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</html><br />
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<br /><br />
<br />
<h2>06/19/12</h2><br /><br />
>Digested B0014 with E, P and with E,X. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
>Ran gels with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation. <br /><br />
C0080 is in the first lane. <br /><br />
>Extracted from gel C0080 X,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Made glycerols from 4 plates LB Km DH5α pSB2K3 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
>Take out AmyE 5’ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
AmyE 5’ 18K plate 3 2010, 2011, 2012<br /><br />
AmyE 3’ 18M plate 3 2010, 2011, 2012<br /><br />
>Digested B0014 with E,X and E,P again [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>06/22/12</h2><br /><br />
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>Ran gels with digestions B0014 with E,X and E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
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>Dephosphated B0014 E,X and B0014 E,P. <br /><br />
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<h2>06/25/12</h2><br /><br />
>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
<br />
<h2>06/26/12</h2><br /><br />
>Ran gel with psB2K3 and psB4A5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
<h2>06/27/12</h2><br /><br />
>Transformed with plasmid B0079 1576 bp psB1A2 12A [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011<br /><br />
AmyE 5’ BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011<br /><br />
AmyE 3’ BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011<br /><br />
AmyE 5’ grew 2 colonies. <br /><br />
<br />
<h2>06/29/12</h2><br /><br />
>We did a DH5α K143001 Km30 Amp 100 glycerol [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] 07. <br /><br />
>We plated twice DH5α K143002 Km 30 Amp 100 + control and twice DH5α B0079 Amp 100 + control<br /><br />
These were both plated in 2 plates each. <br /><br />
They were left overnight since they had not grown by 7:00 pm so the next day glycerols were made. <br /><br />
>Liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
2 tubes DH5α K143001 Km30 Amp 100 <br /><br />
2 tubes DH5α K143002 Km30 Amp 100 <br /><br />
2 tubes DH5α B0079 Amp 100 <br /><br />
1 tube LB Km 30 Amp 100 control<br /><br />
1 tube LB Amp 100 control<br /><br />
>From the 6 tubes we extracted plasmid from kit. <br /><br />
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<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=JULY=<br />
<br />
07/02/12<br /><br />
>Digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /><br />
>PCR’s<br /><br />
•AraC<br /><br />
•Cassete ΩSpr/Strr <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PCR_Protocol_.2850.C2.B5l.29 | PCR PROTOCOL]] <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/b/be/UnamgenomicsandsugarBitacora_8.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>After Cassete ΩSpr/Strr PCR we ran a gel <br />
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</li><br />
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<div class='thumbnailWrapper'><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsandsugarBitacora_9.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /> <br />
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</li><br />
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</div><br />
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</html><br />
<br />
<h2>07/03/12</h2><br /><br />
>After Cassete ΩSpr/Strr PCR we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (8) <br /><br />
>Ran gel with digestions from yesterday. (9) <br /><br />
>Did band extractions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Stored at -20ºC. <br /><br />
>Did PCR to Cassete ΩSpr/Strr to add RBS and prefix/suffix. <br /><br />
>Left digesting with E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/0/03/UnamgenomicsandsugarBitacora_9.1.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
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</li><br />
</ul><br />
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</html><br />
<br />
<h2>07/04/12</h2><br /><br />
>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1). <br /><br />
>Ligated Cassete ΩSpr/Strr +K143002 dephosphated(digested with E,X and E,S). <br /><br />
>Transformed ligation and left overnight plated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs. <br /><br />
>Digested with PstI [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>07/06/12</h2><br /><br />
4 LB Km30 Spec60 DH5α ΩSpr/Strr +K143002 still have not grown. <br /><br />
Ran a gel with yesterday’s digestions to chek if they were done properly (10) <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
Ran another gel with the rest of the samples. <br /><br />
Extracted GusA fragment <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
Repeated ligation ΩSpr/Strr +K143002 E,X dephosphated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Repeated ΩSpr/Strr PCR. <br /><br />
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<h2>07/07/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002 . <br /><br />
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<h2>07/08/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/13/UnamgenomicsandsugarBitacora_11.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
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</li><br />
</ul><br />
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<img src='https://static.igem.org/mediawiki/2012/b/b2/UnamgenomicsandsugarBitacora_11.1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1 PCR omega P<br /><br />
2 PCR omega I<br /><br />
3 PCR AraC P<br /><br />
4 PCR AraC I<br /><br />
5 ladder 1 Kb<br /><br />
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</li><br />
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<h2>07/09/12</h2><br /><br />
From yesterday’s transformations only one colony grew. <br /><br />
From the previous transformation only 2 colonies grew. <br /><br />
These 3 were streaked in 3 plates: <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km 30 Sp 100 control. <br /><br />
Did liquid cultures in 3 tubes: <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km30 Sp 100 control. <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
Ran a gel with: (11) <br /><br />
GusA P<br /><br />
PBBR1 GusA<br /><br />
Ω E PCR P<br /><br />
Ω PCR P<br /><br />
Ω PCR I<br /><br />
Ω E<br /><br />
Ω PCR I<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] <br /><br />
Did GusA primers dissolution for PCR. <br /><br />
GusA PCR <br /><br />
Gel Extraction by kit of lanes 3 and 5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
'''PCR omega P, PCR omega I, PCR AraC P, PCR AraC I'''<br /><br />
Add 10 μl of each primer (LW and UP). <br /><br />
Add 3 μl of plasmid (P). <br /><br />
Add 30.4 μl buffer. <br /><br />
Add 5 μl Mg. <br /><br />
Add 8 μl DNTp’s. <br /><br />
Add 42.6μl H2O miliQ. <br /> <br />
Add 1 μl RTTG polymerase. <br /><br />
Centrifuge (spin) 8 secs. <br /><br />
Add vegetable oil till the eppendorf is full. <br /><br />
Place eppendorf 1 mL in thermocycler. <br /><br />
Run PCR with program “BERNA”. <br /><br /><br />
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<img src='https://static.igem.org/mediawiki/2012/4/4b/UnamgenomicsandsugarBitacora_11.2.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S <br /><br />
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</li><br />
</ul><br />
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</html><br />
<br />
<h2>07/10/12</h2><br /><br />
•GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S (2 times)[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTIONS]]. (11.2) <br /><br />
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<h2>07/11/12</h2><br /><br />
•GusA PCR digestions with E,S; Ω+AmyE 3’ with E,X [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•T.V. AraC PCR+ Ω+AmyE 3’ E,X dephosphated ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/0/0a/UnamgenomicsandsugarBitacora_12.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1. ladder. <br /><br />
2. GusA PCR E,S. <br /><br />
3. Ω+AmyE 3’ with E,X. <br /><br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>07/12/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: (12) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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•Transformed AraC PCR+ Ω+AmyE 3’ ligation in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
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•From pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid. <br /><br />
•Made glycerols from 2 LB Km 30 Sp100 DH5α Ω+AmyE 3’ plates and from 1 LB Amp100 pfrc54 (A3) plates with control[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
•Ligated GusA PCR with B0014 E,X desphophorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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•Digested Pfrc54 (A3) with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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•Desphophorylated Ω+AmyE 3’ E,X. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/0f/UnamgenomicsandsugarBitacora_13.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>•Ran gel with pfrc54 S,P<br /><br />
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</li><br />
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</div><br />
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</html><br />
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<h2>07/13/12</h2><br /><br /><br />
•Ran gel with pfrc54 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (13) <br /><br />
•Transformation of GusA PCR + B0014 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Transformed with GFP E0040 psBIA2. <br /><br />
•Liquid Cultures with ligation: AraC+ Ω+AmyE 3’ for plasmid extraction at night [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•''B. Subtitils'' competent cells. <br /><br />
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<h2>07/14/12</h2><br /><br /><br />
•From 24 AraC+ Ω+AmyE 3’ LB Km 30 Sp 100 DH5α tubes: <br /><br />
•Extracted pellets<br /><br />
•Extracted plasmids. <br /><br />
•Ran gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br /><br />
•From transformed DH5α km 30: <br /><br />
GusA+B0014 DH5α Km50 24 pellets <br /><br />
E0040 DH5α LB Amp100 <br /><br />
From these two we: <br /><br />
•Did liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Extracted plasmid. <br /><br />
•From the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ran gel with this transformation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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<h2>07/16/12</h2><br /><br /><br />
•Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
•Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_14.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>07/17/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (14) <br /><br />
•The gel we ran didn’t work, probably because the agarose was not prepared correctly. <br /><br />
•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/db/Bitacora_15.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<br />
<h2>07/18/12</h2><br /><br /><br />
•From yesterday’s digestions we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (15) <br /><br /><br />
•We made liquid cultures of AraC+ Ω+AmyE 3’ 12, 15, 21 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br /><br />
•We ligated AraC PCR E,S dephosphated with + Ω+AmyE 3’ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
•We digested 1 + Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/19/12</h2><br /><br /><br />
•We ran We digested 1 + Ω+AmyE 3’ E,P with Miguel’s enzymes and with Abiel’s enzymes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
•We transformed with ligations AraC+ Ω+AmyE 3’ E and + Ω+AmyE 3’ E [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br /><br />
•Extracted plasmids from AraC+ Ω+AmyE 3’ 12,15,21. <br /><br /><br />
•Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f3/UnamgenomicsandsugarBitacora_16.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/20/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (16) <br /><br />
•Transformed GusA+B0014 in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Made liquid cultures from AraC+ Ω+AmyE 3’s transformation LB Km30 Sp 100 and control LB Km 30 Sp100 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<h2>07/23/12</h2><br /><br /><br />
•From 24 GusA+B0014 tubes (-) we didn’t do anything. <br /><br />
•From 4 AraC+ Ω+AmyE 3’ we extracted plasmid. <br /><br />
•Digested from the 4 AraC+ Ω+AmyE 3’ E,X; X; P and GusA PCR with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/24/12</h2><br /><br /><br />
•Digested B0014 E with X<br />
B0014 X with E<br /><br />
•Digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<h2>07/25/12</h2><br /><br /><br />
•Ran gel of digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Joined B0014 E with X B0014 X with E digestions. <br /><br />
•Made liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. Transformed [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•From LasR DH5α make liquid cultures and plate [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Digested Ω+AmyE 3’ with X,P; E,S; C0080 X,P; E,S; S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Ligated Gus PCR X,P with B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/84/UnamgenomicsandsugarBitacora_17.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1.Ω+AmyE 3’ X,P<br /><br />
2. Ω+AmyE 3’ E,S<br /><br />
3.C0080 X,P<br /><br />
4.C0080 E,S<br /><br />
5.C0080 S,P<br /><br />
6.ladder<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/26/12</h2><br /><br />
•From C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5α C0179 LB Cb 100 (two cultures) and a control without bacteria [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•We extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguel’s transformation), Puc, B.S. control and PSB4AE. <br /><br />
<br />
•Due to problems with the way we did the transformations of ligations we repeated them: <br /><br />
GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
<br />
GusAPCR X,P+ B0079 S,P dephosphorylated (-)<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•AraC+ Ω+AmyE 3’ S,P 1,2,3,4<br /><br />
•K143002 X,P<br /><br />
•AraC+ Ω S,P<br /><br />
•C0179 X,S<br /><br />
<br />
<br />
<h2>07/27/12</h2><br /><br /><br />
Ligated AraC+ Ω S,P dephosphorylated + K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<h2>07/30/12</h2><br /><br /><br />
•Transformed with ligations: <br /><br />
•AraC+ Ω dephosphprylated +K143002 X,P<br /><br />
•GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
•GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
•Transformed the following sythesis: <br /><br />
•91996 Pveg 140 bp<br /><br />
•91997 ArsR-CzrA_promoter 1 194 bp<br /><br />
•91998 ArsR-CzrA_promoter 2 221 bp<br /><br />
•91999 ArsR-CzrA_promoter 3 213 bp<br /><br />
•92000 pBad-pXyl 387 bp<br /><br />
•92001 XylR 1117pb<br /><br />
•92002 CI_pro_(NAND_INHIBITOR) 774 <br /><br />
•These synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked.<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Make liquid cultures of the following transformations for tomorrow [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]: <br /><br />
•AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+B0079<br /><br />
•Synthesis<br /><br />
<br />
<h2>07/31/12</h2><br /><br /><br />
Extracted plasmids from yesterday’s cultures and from K143002 cultures that were in the other lab. <br /><br />
<br /><br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=AUGUST=<br />
<br />
<h2>08/01/12</h2><br /><br /><br />
Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b1/UnamgenomicsandsugarBitacora_17.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1 PCR GusA I<br /><br />
2 PCR GusA I<br /><br />
3 PCR GusA P<br /><br />
4 PCR GusA P<br /><br />
5 ladder 1 Kb<br /></p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/02/12</h2><br /><br /><br />
•Extracted plasmids from liquid cultures: <br /><br />
AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+BBR1<br /><br />
<br />
PCR GusA, GusA I, PCR GusA P, PCR GusA I<br /><br />
<br />
•Add 10 μl of each primer (LW and UP). <br /><br />
•Add 3 μl of plasmid (P). <br /><br />
•Add 30.4 μl buffer. <br /><br />
•Add 5 μl Mg. <br /><br />
•Add 8 μl DNTp’s. <br /><br />
•Add 42.6μl H2O miliQ. <br /><br />
•Add 1 μl RTTG polymerase. <br /><br />
•Centrifuge (spin) 8 secs. <br /><br />
•Add mineral oil till the eppendorf is full. <br /><br />
•Place eppendorf 1 mL in thermocycler. <br /><br />
•Run PCR with program “BERNA”. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/82/UnamgenomicsandsugarBitacora_18.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.A3+GusA 1<br /><br />
4.A3+GusA 2<br /><br />
5.A3+GusA 3<br /><br />
6.P GusA<br /><br />
7.P GusA 2<br /><br />
8.Ladder<br /><br />
9.01<br /><br />
10.06<br /><br />
11.96<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
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<br /><br />
<br />
<h2>08/03/12</h2><br /><br /><br />
• Ran a gel with: (18) <br /><br />
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[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Ran another gel to extract with: <br /><br />
1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.00<br /><br />
4.01<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]: <br /> <br />
<br />
• Ω+K143002 E,X; A3+GusA E,S; GusAPCR X,P <br /><br />
<br />
•Extracted GusAPCR X,P digestion [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
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•Left the following ligation: AraC+ Ω+K143002 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/17/UnamgenomicsandsugarBitacora_19.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3+GusA 2<br /><br />
2.A3+GusA 3<br /><br />
3. Ω+AmyE 3’ 2<br /><br />
4. Ω+AmyE 3’ 3<br /><br />
5.XylR X,P<br /><br />
6.pBad-pXyl X,P<br /><br />
7.GusA PCR X,P<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/06/12</h2><br /><br /><br />
•Made liquid cultures of Ω+K143002 (2 cultures+1 control) and AraC+ Ω+K143002 (2 cultures+1 control) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ligated GusAPCR X,P+B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
•Ran a Gel with: (19) <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/8b/UnamgenomicsandsugarBitacora_19.1.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/08/12</h2><br /><br />
BBa_B0040 6I psB1A2 Amp+ plate 1<br /><br />
Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (19.1) <br /><br />
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<h2>08/09/12</h2><br /><br />
•From yesterday’s PCR’s : <br /><br />
• E0040 plasmid 1<br /><br />
E0040 plasmid 2<br /><br />
E0040 digested 1<br /><br />
E0040 digested 2<br /><br />
We purified with PCR kit<br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Made 14 PCR’s from AraC+ Ω+AmyE 3’. <br /><br />
•From B0079+GusA ligation and B0040 transformation:<br />
•Grew colonies. <br /><br />
•Made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Streaked these in a new plate. <br /><br />
•Diluted plasmid E0080 2 1/50. <br /><br />
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<h2>08/10/12</h2><br /><br />
•Yesterday’s gel did not come out as expected so we repeated the PCR. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/e/ef/UnamgenomicsandsugarBitacora_20.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.AraC+ Ω+AmyE 3’ <br /><br />
2-14.AraC+ Ω+AmyE 3’ <br /><br />
15.AraC+ Ω 1<br /><br />
16.AraC+ Ω 1<br /><br />
17.AraC+ Ω 1<br /><br />
18.PCR 1 GFP<br /><br />
19.PCR GFP<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<h2>08/13/12</h2><br /><br />
•After running a gel with 14 PCR’s AraC+ Ω+AmyE 3’ and it did not come out as expected, we ran a gel with the ligation AraC+ Ω+AmyE 3’ to select candidate (that weight the same as the ligation) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (20) <br /><br />
<br />
•Extracted pasmids form liquid cultures: B0049, B0079+GusA. <br /><br />
<br />
•AraC+ Ω+AmyE 3’ undigested didn’t run in the gel as expected, it ran similarly to AraC+ Ω<br /><br />
<br />
• We repeated the ligation AraC+ Ω S,P dephosphorylated+K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
• GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.<br />
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• We ran a gel to extract. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f1/UnamgenomicsandsugarBitacora_22.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>1.6 AraC+ Ω+AmyE 3’ E with P. <br /><br /><br />
2.8 AraC+ Ω+AmyE 3’ E with P. <br /><br />
3.11 AraC+ Ω+AmyE 3’ E with P. <br /><br />
4.12 AraC+ Ω+AmyE 3’ E with P. <br /><br />
5.6 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
6.8 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
7.11 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
8.12 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
9.00 pBad/pXyl with S,P. <br /><br />
10.Ladder. <br /><br />
11.E0040 PCR E,S to extract the correct band. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/16/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: <br /><br />
<br />
•Digested: <br /><br />
ArSR-CzrA 97 with S,P<br /><br />
ArSR-CzrA 98 with S,P<br /><br />
ArSR-CzrA 99 with S,P<br /><br />
E0040 PCR with E,S<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
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<br />
<h2>08/20/12</h2><br /><br />
•Ligated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/21/12</h2><br /><br />
•Joined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated. <br /><br />
•Extracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR. <br /><br />
•Digested Ω+AmyE 3’ with E,X and + Ω+AmyE 3’ II with E,X (obtained these two from glycerols stored at -80ºC [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/29/UnamgenomicsandsugarBitacora_22.1.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested K143001+PBad, pXyl with E,S <br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/1c/UnamgenomicsandsugarBitacora_23.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>1.K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
4.A3 PCR<br /><br />
5.A3 PCR<br /><br />
6.E0040 PCR E,S<br /><br />
7.B0014 E,X dephosphorylated<br /><br />
8. Ω+AmyE 3’ E,X <br /><br />
9. Ω+AmyE 3’ E,X II<br /><br />
10.Ladder<br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/22/12</h2><br /><br />
•We did 2 PCR’s for A3. <br /><br />
<br />
•Digested K143001+PBad, pXyl with E,S <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (22.1) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did band extraction of: <br /><br />
1. K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
•Made liquid cultures from E1010+Boo14 that the other lab gave us and did 2 plates for glycerol Km+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Did 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ Ω+AmyE 3’ 6,8,11,12 Km+ and Spectinomycin+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Transformed ligations: <br /><br />
B0079+GusA Amp+<br /><br />
Pveg+XylR Chloramphenicol+<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]].. <br /><br />
<br /><br />
<br />
<br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/78/UnamgenomicsandsugarBitacora_23.01.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1. Ω+AmyE 3’ E,X 6.2<br /><br />
2.Ω+AmyE 3’ II E,X<br /><br />
3.97 ArsR-CzrA<br /><br />
4.98 ArsR-CzrA<br /><br />
5.99 ArsR-CzrA<br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/23/12</h2><br /><br />
•The negative controls were contaminated. We didn’t manipulate the ones that had grown because of this. <br /><br />
•Ran gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
<br />
<br />
<h2>08/24/12</h2><br /><br />
From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ Ω+AmyE 3’ 6 and 8 we streaked 2 plates for glycerol of each ligation. <br />Repeated ligation B0079+GusA. <br /><br />
<br />
Ligated E0040 PCR E,S+B0014 E,X dephospohylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
Made 2 new plates from Pveg+XylR ligation and liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>08/25/12</h2><br /><br />
Extracted pellet from 16 tubes of pVeg+XylR that were left overnight. <br /><br />
<br />
Ligated B0079+GusA and B0040+B0014 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/28/12</h2><br /><br />
<br />
Made liquid cultures from transformations tht were left overnight <br />
-R0079+GusA<br /><br />
-R0079+GusA (-)<br /><br />
-E0040+B0014<br /><br />
-E0040+B0014 (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/25/UnamgenomicsandsugarBitacora_23.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. A3 PCR 1. <br /><br />
2. A3 PCR 2. <br /><br />
3. Ω+AmyE 3’ S,P *.<br /><br />
4. Ω+AmyE 3’ S,P . <br /><br />
5. Pveg+XylR E,S 2. <br /><br />
6. Pveg+XylR E,S 3. <br /><br />
7. Pveg+XylR E,S 4. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/29/12</h2><br /><br />
Digested Pveg+XylR 2,3,4 with E,S. <br /><br />
<br />
Digested Ω+AmyE 3’ with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTION]]. <br /><br />
<br />
Ran a PCR with A3 PCR (2 .6 ml tubes). <br /><br />
<br />
Ran a gel with <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2e/UnamgenomicsandsugarBitacoraBitacora_24.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1-23. E0040 + B0014 colonies<br /><br />
24-29. B0079+GusA 1<br /><br />
30. B0079<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/30/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (24) <br /><br />
<br />
<br />
•Digested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/3d/UnamgenomicsandsugarBitacora_25.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1-3B0079+GusA E,S 3. <br /><br />
4-9. PVeg+XylR E,S 1. <br /><br />
10-17. E0040+B0014 X,P 3. <br /><br />
18. 1 kb ladder. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/d2/UnamgenomicsandsugarBitacora_26.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3 PCR. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7b/UnamgenomicsandsugarBitacora_27.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>E0040+B0014 X,P 4 to extract<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>08/31/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (25) <br /><br />
<br />
<br />
•Dephosphorylated: <br /><br />
Ω+AmyE 3’ S,P <br /><br />
Ω+AmyE 3’ II S,P <br /><br />
6 AraC+ Ω+AmyE 3’ E,X<br /><br />
8 AraC+ Ω+AmyE 3’ E,X<br /><br />
11 AraC+ Ω+AmyE 3’ E,X<br /><br />
12 AraC+ Ω+AmyE 3’ E,X<br /><br />•Ran a gel with A3 PCR [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (26) <br /><br />
<br />
•It was the second time we didn’t obtain a band from A3’s PCR.<br />
<br />
•Probably because it had been missing the buffer 2’s ethanol (“blue lid”) from the PCR purification. <br /><br />
<br />
•Ran a gel woth E0040+B0014 X,P 4 to extract <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | Gen extraction protocol]]. <br /><br />
<br />
•Did an A3 PCR. <br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=SEPTEMBER=<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b8/UnamgenomicsandsugarBitacora_28.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.A3 PCR 1. <br /><br />
2.A3 PCR 2. <br /><br />
3.E0040+B0014 X,P 1. <br /><br />
4.E0040+B0014 X,P 2. <br /><br />
5.Ω+AmyE 3’ dephosphorylated S,P. <br /><br />
6.Ω+AmyE 3’ dephosphorylated S,P II. <br /><br />
7.97 dephosphorylated S,P. <br /><br />
8.98 dephosphorylated S,P. <br /><br />
9.99 dephosphorylated S,P. <br /><br />
10-13. Arac+ Ω+AmyE 3’ dephosphorylated E,X 12. <br /><br />
14. 1 kb Ladder. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>09/01/12</h2><br /><br />
•Ran a gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (28) <br /><br />
<br />
•Ligated E0040+B0014 X,P 2 +promoter S,P dephosphorylated; K143002 X,P+ Ω+AmyE 3’ dephosphorylated S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<br /><br />
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<br /><br />
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<br /><br />
<br />
<h2>09/03/12</h2><br /><br />
<br />
•Digested B0079+GusA E,S 3,4,5; PVeg+XylR E,S 1,5,7,10,13,16 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>09/04/12</h2><br /><br />
•Ran gel with digestions: <br /><br />
B0079+GusA E,S 3,4,5<br /><br />
PVeg+XylR E,S 1,5,7,10,13,16 <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Purified A3 PCR 1,2. <br /><br />
<br />
•Ran a gel of A3 PCR <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] and digested with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•From ligations: PVeg+E0040+B0014, PBad/PXyl+E0040+B0014, B0079+E0040+B0014 only colonies grew in PBad/PXyl+E0040+B0014.<br />
<br />
•Plated colonies that grew in a new plate. <br /><br />
<br />
•Made liquid cultures of these <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•Repeated the ones that did not grow. <br /><br />
<br />
•Digested E0040+B0014 4 with X,P[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
-BBa_J23101 18E plate 1 plasmid BBa_J61002_2948 bp Amp+<br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/c1/UnamgenomicsandsugarBitacora_29.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>24 PBad/PXyl+E0040+B0014 tubes we extracted <br /><br />
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</li><br />
</ul><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/a/a4/UnamgenomicsandsugarBitacora_30.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
Extracted band from 1. and 2<br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7d/UnamgenomicsandsugarBitacora_31.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S <br />
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</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>09/05/12</h2><br /><br />
•From the 24 PBad/PXyl+E0040+B0014 tubes we extracted plasmids (29) and ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Extracted plasmid from 2 tubes of E0040+B0014. <br /><br />
<br />
•Ran a gel with yesterday’s digestions: <br /><br />
1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
<br />
•Extracted band from 1. and 2. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Purified A3 PCR from 2 0.6ml tubes 1 and 2. <br /><br />
<br />
•1 colony grew from ligation pVeg+E0040+B0014. <br /><br />
<br />
•We streaked this in another plate and did liquid cultures <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•To do: <br /><br />
•Ligate B0079 S,P dephosphorylated + E0040+B0014 X,P and Ω+AmyE 3’ S,P dephosphorylated+ K143001 X,P. <br /><br />
•Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S 10,13. (31) <br /><br />
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<div class='thumbnailWrapper'><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/26/UnamgenomicsandsugarBitacora_32.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>•A3 PCR and gel A3 PCR X,P <br />
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</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br /><br />
<br />
<h2>09/06/12</h2><br /><br />
<br />
•A3 PCR and gel A3 PCR X,P 1,2. <br /><br />
<br />
<br />
<h2>09/14/12</h2><br /><br />
<br />
•Digested Ω+AmyE 3’ 11,13,18, and 22 with E,X. <br /><br />
•Ligate: <br /><br />
PVeg S,P dephosphorylated+XylR X,P. <br /><br />
PBad p Xyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
pSB13C3 E,P dephosphorylated + A3 PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + GusA PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + XylR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pVeg E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pBad pXyl E,P. <br /><br />
pSB13C3 E,P dephosphorylated + Ω PCR E,P. <br /><br />
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<h2>09/16/12</h2><br /><br />
• Colonies in these plates grew: <br /><br />
- R0079+E0040+B0014 <br /><br />
- pBad/pXyl <br /><br />
- XylR <br /><br />
- pVeg <br /><br />
- A3+E0040+B0014 from colonies 1,3,4,5,6,7,8,9,10,11,12,13,14,15,17 <br /><br />
- pVeg+E0040+B0014 1,2,3,4,5,6,8,9,10,11,12,13,14,15,16 <br /><br />
- pBad/pXyl+E0040+B0014 2 tubes from the same correct colony. <br /><br />
- pVeg+XylR 1,2 <br /><br />
- pBad/pXyl+pSB1C3 1,2 <br /><br />
- XylR+pSB1C3 1,2 <br /><br />
- pVeg+pSB1C3 1,2 <br /><br />
- A3+pSB1C3 1,2 <br /><br />
- GusA+pSB1C3 1,2 <br /><br />
- omegacassette+pSB1C3 1,2 <br /><br />
<br /><br />
• Ran gel from the plasmid extractions that we did yesterday: <br /><br />
- A3+E0040+B0014 1-16 <br /><br />
- pVeg+E0040+B0014 1-16, 18 <br /><br />
- omega cassette+AmyE 3’ 11,13,18,22 <br /><br />
• Digested R0079+E0040+B0014 with E,S. to ligate with AmyE 3’ E,x dephosphorylated. <br /><br />
• Digested omega cassette with E,P. <br /><br />
• Digested pSB1C3 with E,P. <br /><br />
• Made liquid cultures from AmyE 5’ and AmyE 3’. <br /><br />
• Did plasmid extraction from pSB1C3 colonies 3 and 6. <br /><br />
<br /><br />
<br />
<h2>09/17/12</h2><br /><br />
• Digested A3+E0040+B0014 colonies 2,3,4,5,6,11 and 16 with E,S. <br /><br />
• Digested pVeg+E0040+B0014 colonies 1,2,3,4,5,9 and 10 with E,S. <br /><br />
• Dephosphorylated omega cassette+AmyE 3’ colonies 13,18 and 22 digested with E,X. <br /><br />
• Digested pBad/pXyl and pVeg with S,P. Then dephosphorylated. <br /><br />
• Digested AmyE 3’ with E,X. Then dephosphorylated. <br /><br />
<br /><br />
<br />
<h2>09/18/12</h2><br /><br />
<br /><br />
• Ran gel with: <br /><br />
1. A3 2 E,S <br /><br />
2. A3 3 E,S <br /><br />
3. A3 4 E,S <br /><br />
4. A3 5 E,S <br /><br />
5. A3 6 E,S <br /><br />
6. A3 11 E,S <br /><br />
7. A3 16 E,S <br /><br />
8. E0040+B0014 colony 1 E,X <br /><br />
9. AmyE 5’ E,P <br /><br />
10. AmyE 3’ E,X <br /><br />
11. Omega cassette E,S <br /><br />
12. pSB1C3 E,P <br /><br />
13. ladder <br /><br />
Bottom: <br /><br />
1. pVeg 1 E,S <br /><br />
2. pVeg 2 E,S <br /><br />
3. pVeg 3 E,S <br /><br />
4. pVeg 4 E,S <br /><br />
5. pVeg 5 E,S <br /><br />
6. pVeg 9 E,S <br /><br />
7. pVeg 10 E,S <br /><br />
8. pVeg S,P dephosphorylated <br /><br />
9. pVeg 2 S,P <br /><br />
10. pBad/pXyl S,P <br /><br />
11. XylR X,P <br /><br />
12. R0079+GFP+TT E,S <br /><br />
13. R0079 S,P <br /><br />
14. Ladder <br /><br />
• Dephosphorylated GFP+TT E,X; AmyE 5’ E,X; pSB1C3 E,P; pVeg 2 S,P; pBad/pXyl S,P and R0079 S,P. <br /><br />
<br /><br />
<h2>09/19/12</h2><br /><br />
<br /><br />
• Did PCR from A3 and a 1/50 dilution. <br /><br />
• Ran gel with: <br /><br />
1. pVeg 3 E,P. <br /><br />
2. pVeg 9 E,P. <br /><br />
3. pVeg 10 E,P. <br /><br />
4. pBAd/pXyl 3 E,P. <br /><br />
5. pBAd/pXyl 5 E,P. <br /><br />
6. pBAd/pXyl 1 E,P. <br /><br />
7. A3 3 E,P. <br /><br />
8. A3 4 E,P. <br /><br />
9. XylR 1 E,P. <br /><br />
10. XylR 3 E,P. <br /><br />
11. XylR 8 E,P. <br /><br />
12. pSB1C3 6 E,P. <br /><br />
13. pSB1C3 6 E,P. <br /><br />
14. AmyE 5’ E,S. <br /><br />
15. AmyE 5’ E,S. <br /><br />
16. A3 1/50 PCR. <br /><br />
17. A3 PCR. <br /><br />
18. Ladder <br /><br />
• We did transformations: <br /><br />
- 2 of R0079+GusA <br /><br />
- R0079+GusA control <br /><br />
- 2 of A3+pSB1AK3 <br /><br />
- A3+pSB1AK3 control <br /><br />
- 2 of omega cassette+pSB1C3 <br /><br />
- 2 of GusA+pSB1C3 <br /><br />
• Digested: <br /><br />
- pVeg+pSB1C3 <br /><br />
- pBad/pXyl+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
- A3+pSB1C3 <br /><br />
With E,P. <br /><br />
• Ran gel with these digestions. <br /><br />
<br /><br />
<h2>09/19/12</h2><br /><br />
• Made liquids cultures from: <br /><br />
- Glicerol of AmyE 5’ <br /><br />
- Glicerol of AmyE 3’ <br /><br />
- pBad/pXyl+pSB1C3 colony 5 <br /><br />
• Made liquid cultures from yesterday transformations: <br /><br />
- R0079+GusA 2 and control <br /><br />
- A3+pSB1AK3 2 and control <br /><br />
- Omega cassette+pSB1C3 2 <br /><br />
- GusA+pSB1C3 2 <br /><br />
• We transformed yesterday ligations: <br /><br />
- pBad/pXyl+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
- pVeg+pSB1C3 <br /><br />
- A3+pSB1C3 <br /><br />
• Purified A3 PCR with kit, and then, digested with E,P and E,S. <br /><br />
• Ran gel <br /><br />
<br /><br />
<h2>09/20/12</h2><br /><br />
<br /><br />
• Made liquid cultures from transformations: <br /><br />
- Omega cassette+pSB1C3 colony 7 <br /><br />
- 2 of GusA+pSB1C3 colony 32 <br /><br />
- R0079+GusA colony 12 <br /><br />
• Digested A3 1/50 PCR with E,P and E,S. <br /><br />
• Made liquid cultures from R0079+E0040+B0014; pBad/pXyl+E0040+B0014; pVeg+E0040+B0014; A3+E0040+B0014. <br /><br />
• Ligated: <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P <br /><br />
- A3 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
<br /><br />
<h2>09/21/12</h2><br /><br />
<br /><br />
• Ran gel with: <br /><br />
1. R0079+GFP+TT 1 <br /><br />
2. R0079+GFP+TT <br /><br />
3. pBad/pXyl+pSB1C3 1 <br /><br />
4. pBad/pXyl+pSB1C3 <br /><br />
5. omega cassette+psB1C3 1 <br /><br />
6. omega cassette+psB1C3 <br /><br />
7. pSB1C3 E,P 1 <br /><br />
8. pSB1C3 E,P <br /><br />
9. pSB1C3 E <br /><br />
10. pSB1C3 P <br /><br />
11. A3 1/50 PCR E,P <br /><br />
<br /><br />
<br />
<h2>09/22/12</h2><br /><br />
• Ran gel with lysis extractions: <br /><br />
1. GusA+pSB1C3 1 <br /><br />
2. GusA+pSB1C3 2 <br /><br />
3. GusA+pSB1C3 3 <br /><br />
4. GusA+pSB1C3 4 <br /><br />
5. GusA+pSB1C3 5 <br /><br />
6. GusA+pSB1C3 6 <br /><br />
7. GusA+pSB1C3 7 <br /><br />
8. GusA+pSB1C3 8 <br /><br />
9. GusA+pSB1C3 9 <br /><br />
10. GusA+pSB1C3 10 <br /><br />
11. GusA+pSB1C3 11 <br /><br />
12. GusA+pSB1C3 12 <br /><br />
13. pSB1C3 <br /><br />
14. A3+E0040+B0014 1 <br /><br />
15. A3+E0040+B0014 2 <br /><br />
16. A3+E0040+B0014 3 <br /><br />
17. A3+E0040+B0014 4 <br /><br />
18. A3+E0040+B0014 5 <br /><br />
19. A3+E0040+B0014 6 <br /><br />
20. A3+E0040+B0014 7 <br /><br />
21. A3+E0040+B0014 8 <br /><br />
22. A3+E0040+B0014 9 <br /><br />
23. pVeg+E0040+B0014 3 <br /><br />
24. pVeg+E0040+B0014 4 <br /><br />
25. pVeg+E0040+B0014 5 <br /><br />
26. pVeg+E0040+B0014 6 <br /><br />
27. pVeg+E0040+B0014 7 <br /><br />
28. pVeg+E0040+B0014 8 <br /><br />
29. pVeg+E0040+B0014 9 <br /><br />
30. pVeg <br /><br />
31. pBad/pXyl+E0040+B0014 1 <br /><br />
32. pBad/pXyl+E0040+B0014 2 <br /><br />
33. pBad/pXyl+E0040+B0014 3 <br /><br />
34. pBad/pXyl+E0040+B0014 4 <br /><br />
35. pBad/pXyl+E0040+B0014 5 <br /><br />
36. pBad/pXyl+E0040+B0014 6 <br /><br />
37. pBad/pXyl+E0040+B0014 7 <br /><br />
38. pBad/pXyl+E0040+B0014 8 <br /><br />
39. pBad/pXyl+E0040+B0014 9 <br /><br />
40. pBad/pXyl+E0040+B0014 10 <br /><br />
41. pBad/pXyl <br /><br />
42. pVeg+XylR 1 <br /><br />
43. pVeg+XylR 2 <br /><br />
44. pVeg+XylR 3 <br /><br />
45. pVeg+XylR 4 <br /><br />
46. pVeg+XylR 5 <br /><br />
47. pVeg+XylR 6 <br /><br />
48. pVeg+XylR 7 <br /><br />
49. pVeg+XylR 8 <br /><br />
50. omega cassette <br /><br />
<br /><br />
• Ran gel with yesterday digestions: <br /><br />
1. R0079+GFP+TT E,P <br /><br />
2. R0079+GFP+TT E,P <br /><br />
3. Omega cassette+pSB1C3 E,P <br /><br />
4. Omega cassette+pSB1C3 E,P <br /><br />
5. Ladder <br /><br />
6. pBad/pXyl+pSB1C3 <br /><br />
7. pBad/pXyl+pSB1C3 <br /><br />
8. pSB1C3 6 E,P. <br /><br />
9. A3 1/50 PCR E,S <br /><br />
10. pBbak C011 <br /><br />
11. pBbak C012 <br /><br />
12. ladder <br /><br />
<br /><br />
• Did pellet from R0079+GusA. <br /><br />
• Did plasmid extraction from omega cassette+pSB1C3 colonies 1,4,6 and 7. <br /><br />
• Digested them with E,P. <br /><br />
• Digested: <br /><br />
- GusA+pSB1C3 1,3,4,10 and 11 <br /><br />
- A3+E0040+B0014 2,4,7 and 9 <br /><br />
- pVeg+E0040+B0014 4,5 and 6 <br /><br />
- pBad/pXyl+E0040+B0014 3,7 and 10 <br /><br />
- pVeg+XylR 2,4 and 5 <br /><br />
with E,P. <br /><br />
• Made liquid cultures from pVeg+pSB1C3 <br /><br />
• Ligated: <br /><br />
- XylR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- pVeg E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- A3 1/50 PCR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- A3 1/50 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
<br /><br />
<h2>09/23/12</h2><br /><br />
<br /><br />
• Ran gel with yesterday digestions: <br /><br />
1. Omega cassette+pSB1C3 1 E,P <br /><br />
2. Omega cassette+pSB1C3 4 E,P <br /><br />
3. Omega cassette+pSB1C3 6 E,P <br /><br />
4. Omega cassette+pSB1C3 7 E,P <br /><br />
5. GusA+pSB1C3 1 E,P <br /><br />
6. GusA+pSB1C3 3 E,P <br /><br />
7. GusA+pSB1C3 4 E,P <br /><br />
8. GusA+pSB1C3 10 E,P <br /><br />
9. GusA+pSB1C3 11 E,P <br /><br />
10. pSB1C3 E,P dephosphorylated <br /><br />
11. pVeg+XylR 2 E,P <br /><br />
12. pVeg+XylR 4 E,P <br /><br />
13. pVeg+XylR 5 E,P <br /><br />
14. pVeg S,P dephosphorylated <br /><br />
15. pVeg+GFP+TT 4 E,P. <br /><br />
16. pVeg+GFP+TT 5 E,P. <br /><br />
17. pVeg+GFP+TT 6 E,P. <br /><br />
18. ladder <br /><br />
Bottom <br /><br />
1. pBad/pXyl+GFP+TT 3 E,P <br /><br />
2. pBad/pXyl+GFP+TT 7 E,P <br /><br />
3. pBad/pXyl+GFP+TT 10 E,P <br /><br />
4. pBad/pXyl S,P dephosphorylated <br /><br />
5. A3+GFP+TT 2 E,P <br /><br />
6. A3+GFP+TT 4 E,P <br /><br />
7. A3+GFP+TT 7 E,P <br /><br />
8. A3+GFP+TT 9 E,P <br /><br />
9. Ladder <br /><br />
<br /><br />
<h2>09/24/12</h2><br /><br />
<br /><br />
• Did PCR from omega cassette+pSB1C3 colonies 1,4,6 and 7. <br /><br />
• Made 1/50 dilutions from these PCR products. <br /><br />
• Ran gel with: <br /><br />
1. Omega cassette+pSB1C3 1/50 PCR 1 <br /><br />
2. Omega cassette+pSB1C3 PCR 1 <br /><br />
3. Omega cassette+pSB1C3 1/50 PCR 4 <br /><br />
4. Omega cassette+pSB1C3 PCR 4 <br /><br />
5. Omega cassette+pSB1C3 1/50 PCR 6 <br /><br />
6. Omega cassette+pSB1C3 PCR 6 <br /><br />
7. Omega cassette+pSB1C3 1/50 PCR 7 <br /><br />
8. Omega cassette+pSB1C3 PCR 7 <br /><br />
9. GusA+pSB1C3 1 P <br /><br />
10. GusA+pSB1C3 3 P <br /><br />
11. GusA+pSB1C3 4 P <br /><br />
12. GusA+pSB1C3 10 P <br /><br />
13. GusA+pSB1C3 11 P <br /><br />
14. pVeg+pSB1C3 6 E,P <br /><br />
15. pVeg+pSB1C3 5 E,P <br /><br />
16. pVeg+pSB1C3 4 E,P <br /><br />
17. pVeg+pSB1C3 3 E,P <br /><br />
18. pVeg+pSB1C3 2 E,P <br /><br />
19. pVeg+pSB1C3 1 E,P <br /><br />
20. ladder <br /><br />
<br />
• From yesterday transformations, these didn’t grow: <br /><br />
- pBad/pXyl+E0040+B0014 1 and 2. <br /><br />
- A3+E0040+B0014. <br /><br />
• Made liquid cultures from transformations that did grow: <br /><br />
- pVeg+E0040+B0014. <br /><br />
- pVeg+XylR. <br /><br />
- A3+pSB1C3 <br /><br />
- pVeg+pSB1C3 <br /><br />
- XylR+pSB1C3 <br /><br />
<br /><br />
<h2>09/26/12</h2><br /><br />
<br /><br />
• Did plasmid extractions form liquids cultures: <br /><br />
- 8 of pVeg+pSB1C3 <br /><br />
- 8 of A3 <br /><br />
- 8 of XylR <br /><br />
• Digested them with E,P. <br /><br />
• Ran gel with: <br /><br />
1. Ladder <br /><br />
2. XylR+pSB1C3 1 E,P <br /><br />
3. XylR+pSB1C3 2 E,P <br /><br />
4. XylR+pSB1C3 3 E,P <br /><br />
5. pVeg+pSB1C3 1 E,P <br /><br />
6. pVeg+pSB1C3 3 E,P <br /><br />
7. pVeg+pSB1C3 4 E,P <br /><br />
8. pVeg+pSB1C3 5 E,P <br /><br />
9. pVeg+pSB1C3 6 E,P <br /><br />
10. pVeg+pSB1C3 8 E,P <br /><br />
11. pSB1C3 6 E,P dephosphorylated <br /><br />
12. A3+pSB1C3 1 E,P <br /><br />
13. A3+pSB1C3 3 E,P <br /><br />
14. A3+pSB1C3 4 E,P <br /><br />
15. A3+pSB1C3 5 E,P <br /><br />
16. A3+pSB1C3 6 E,P <br /><br />
17. A3+pSB1C3 8 E,P <br /><br />
18. Ladder <br /><br />
19. P4+E0040+B0014 1 E,X <br /><br />
20. P4+E0040+B0014 7 E,X <br /><br />
<br /><br />
<h2>09/27/12</h2><br /><br />
<br /><br />
• Digested pVeg+XylR colonies 1,4,5 and 6 in pJ209 plasmid with E,S for 3 hours. <br /><br />
• Digested pVeg+E0040+B0014 colonies 7,9,10 and 11 in pasmid pJ209 with E,P. <br /><br />
<br /><br />
<h2>10/09/12</h2><br /><br />
<br /><br />
• Ligated: <br /><br />
- A3 1/50 PCR E,P + pSB1C3 6 E,P dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
- P4+CI+E1010+B0014 E,X dephosphorylated + AmyE 5’+pBad/pXyl E,S. <br /><br />
<br /><br />
<h2>10/10/12</h2><br /><br />
<br /><br />
• Ligated: <br /><br />
- GusA PCR E,P + pSB1C3 6 E,P dephosphorylated. <br /><br />
- pBad/pXyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- pVeg S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
- A3 1/50 PCR E,S + E0040+B0014 E,X dephosphorylated. <br /><br />
• Digested R0079+E0040+B0014 with E,S. <br /><br />
• Digested AmyE 3’ with E,X. Then dephosphorylated. <br /><br />
• Digested pVeg and R0079, A3 with X,P. <br /><br />
• Digested pBad/pSB1C3 with S,P. <br /><br />
<br /><br />
<h2>10/11/12</h2><br /><br />
<br /><br />
• Did plasmid extraction from liquid cultures: <br /><br />
- 2 of AmyE 5’ <br /><br />
- 2 of AmyE 3’ <br /><br />
- 2 of E0040+B0014 <br /><br />
• Ran gel with: <br /><br />
1. A3 1/50 PCR X,P <br /><br />
2. A3 1/50 PCR X,P <br /><br />
3. A3+pSB1C3 6 X,P <br /><br />
4. A3+pSB1C3 6 X,P <br /><br />
5. A3+pSB1C3 8 X,P <br /><br />
6. A3+pSB1C3 8 X,P <br /><br />
7. pVeg X,P <br /><br />
8. pVeg 96 X,P <br /><br />
9. pVeg 96 X,P <br /><br />
10. pVeg+pSB1C3 1 X,P <br /><br />
11. pVeg+pSB1C3 1 X,P <br /><br />
12. pVeg+pSB1C3 3 X,P <br /><br />
13. pVeg+pSB1C3 3 X,P <br /><br />
14. R0079 X,P <br /><br />
15. Ladder <br /><br />
<br /><br />
<h2>10/12/12</h2><br /><br />
<br /><br />
• Ligated:<br />
- AmyE 5’+pBad/pXyl 10 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- AmyE 5’+pBad/pXyl 11 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- AmyE 5’+pBad/pXyl 12 E,S + P4+CI+E1010+TT E,X dephosphorylated. <br /><br />
- pVeg S,P dephosphorylated + XylR X,P. <br /><br />
- A3 1/50 PCR E,P + pSB1C3 E,P dephosphorylated. <br /><br />
<br /><br />
<h2>10/12/12</h2><br /><br />
<br /><br />
• Digested AmyE 5’ with S,P <br /><br />
• Dephosphorylated it. <br /><br />
• Ligated: <br /><br />
- AmyE 5’ S,P dephosphorylated with <br /><br />
- R0079 X,P <br /><br />
- pVeg 96 X,P <br /><br />
- A3 1/50 PCR X,P <br /><br />
- A3+pSB1C3 6 X,P <br /><br />
- R0079+E0040+B0014 <br /><br />
• Ligated pBad/pXyl+pSB1C3 S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
• Ran gel with: <br /><br />
1. A3+pSB1C3 1 <br /><br />
2. A3+pSB1C3 2 <br /><br />
3. A3+pSB1C3 3 <br /><br />
4. A3+pSB1C3 4 <br /><br />
5. A3+pSB1C3 5 <br /><br />
6. A3+pSB1C3 6 <br /><br />
7. A3+pSB1C3 7 <br /><br />
8. A3+pSB1C3 8 <br /><br />
9. A3+pSB1C3 9 <br /><br />
10. A3+pSB1C3 10 <br /><br />
11. A3+pSB1C3 11 <br /><br />
12. A3+pSB1C3 6 control <br /><br />
13. AmyE 5’+P4+CI+E1010+B0014 1 <br /><br />
14. AmyE 5’+P4+CI+E1010+B0014 2 <br /><br />
15. AmyE 5’+P4+CI+E1010+B0014 3 <br /><br />
16. AmyE 5’+P4+CI+E1010+B0014 4 <br /><br />
17. AmyE 5’+P4+CI+E1010+B0014 5 <br /><br />
18. AmyE 5’+P4+CI+E1010+B0014 6 <br /><br />
19. AmyE 5’+P4+CI+E1010+B0014 7 <br /><br />
20. P4+CI+E1010+B0014 1 control <br /><br />
<br /><br />
<h2>10/09/12</h2><br /><br />
<br /><br />
• Ligate: <br /><br />
K143001+pBad/pXyl + p4+CI+E1010+TT E,X dephosphorylated. <br /><br />
• Ligate: <br /><br />
pVeg S,P dephosphorylated + XylR X,P. <br /><br />
• Ligate: <br /><br />
A3 PCR 1/50 E,P + pSB1C3 E,P dephosphorylated <br /><br />
<br /><br />
<h2>10/13/12</h2><br /><br />
<br /><br />
• Digested AmyE 5’ with S,P. Then dephosphorylated. <br /><br />
• Ligate: <br /><br />
AmyE 5’ S,P dephosphorylated + <br /><br />
- R0079 X,P <br /><br />
- pVeg 96 X,P <br /><br />
- A3 PCR 1/50 X,P <br /><br />
- A3+pSB1C3 colony 6 X,P <br /><br />
- R0079+E0040+B0014 E,X <br /><br />
• Ligate: <br /><br />
pBad/pXyl+pSB1C3 S,P dephosphorylated + E0040+B0014 X,P <br /><br />
<br />
• Ran a gel with: <br /><br />
1. A3+pSB1C3 1 <br /><br />
2. A3+pSB1C3 2 <br /><br />
3. A3+pSB1C3 3 <br /><br />
4. A3+pSB1C3 4 <br /><br />
5. A3+pSB1C3 5 <br /><br />
6. A3+pSB1C3 6 <br /><br />
7. A3+pSB1C3 7 <br /><br />
8. A3+pSB1C3 8 <br /><br />
9. A3+pSB1C3 9 <br /><br />
10. A3+pSB1C3 10 <br /><br />
11. A3+pSB1C3 11 <br /><br />
12. A3+pSB1C3 6 control <br /><br />
13. AmyE 5’+P4+CI+E1010+B0014 8 <br /><br />
14. AmyE 5’+P4+CI+E1010+B0014 9 <br /><br />
15. AmyE 5’+P4+CI+E1010+B0014 10 <br /><br />
16. P4+CI+E1010+B0014 1 control <br /><br />
17. pVeg+XylR 1 <br /><br />
18. pVeg+XylR 2 <br /><br />
19. pVeg+XylR 3 <br /><br />
20. pVeg+XylR 4 <br /><br />
21. pVeg+XylR 5 <br /><br />
22. pVeg+XylR 6 <br /><br />
23. pVeg+XylR 7 <br /><br />
24. pVeg+XylR 8 <br /><br />
25. pVeg+XylR 10 <br /><br />
26. pVeg+XylR 11 <br /><br />
27. pVeg+XylR 12 <br /><br />
28. pVeg 96 control <br /><br />
<br />
• Ran gel with: <br /><br />
1. A3 1/50 PCR X,P <br /><br />
2. A3+pSB1C3 6 X,P <br /><br />
3. pVeg 96 X,P <br /><br />
4. pVeg+pSB1C3 1 X,P <br /><br />
5. pVeg+pSB1C3 3 X,P <br /><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/ANDSugar
Team:UNAM Genomics Mexico/Notebook/ANDSugar
2012-10-27T03:30:45Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Arabinose/Xylose AND Gate Notebook'''</h1></center><br />
<br /><br />
<br /><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="20"><br />
<tr><br />
<td id="leftcolumn2"><center>2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JUNE | '''JUNE''']] </center> <br /><br />
<br /><br />
2.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F07.2F12 |06/07/12]] <br /><br />
2.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F12.2F12 |06/12/12]] <br /><br />
2.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F13.2F12 |06/13/12]] <br /><br />
2.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F14.2F12 |06/14/12]] <br /><br />
2.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F15.2F12 |06/15/12]] <br /><br />
2.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F18.2F12 |06/18/12]] <br /><br />
2.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F19.2F12 |06/19/12]] <br /><br />
2.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F22.2F12 |06/22/12]] <br /><br />
2.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F25.2F12 |06/25/12]] <br /><br />
2.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F26.2F12 |06/26/12]] <br /><br />
2.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F27.2F12 |06/27/12]] <br /><br />
2.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F29.2F12 |06/29/12]] <br /><br />
</td><br />
<br />
<td id="contentcolumn2"><center>3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JULY | '''JULY''']] </center> <br /><br />
<br /><br />
3.1[[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F03.2F12 | 07/03/12]] <br /><br />
3.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F04.2F12 | 07/04/12]] <br /><br />
3.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F06.2F12 | 07/06/12]] <br /><br />
3.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F07.2F12 | 07/07/12]] <br /><br />
3.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F08.2F12 | 07/08/12]] <br /><br />
3.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F09.2F12 | 07/09/12]] <br /><br />
3.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F10.2F12 | 07/10/12]] <br /><br />
3.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F11.2F12 | 07/11/12]] <br /><br />
3.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F12.2F12 | 07/12/12]] <br /><br />
3.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F13.2F12 | 07/13/12]] <br /><br />
3.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F14.2F12 | 07/14/12]] <br /><br />
3.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F16.2F12 | 07/16/12]] <br /><br />
3.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F17.2F12 | 07/17/12]] <br /><br />
3.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F18.2F12 | 07/18/12]] <br /><br />
3.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F19.2F12 | 07/19/12]] <br /><br />
3.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F20.2F12 | 07/20/12]] <br /><br />
3.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F23.2F12 | 07/23/12]] <br /><br />
3.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F24.2F12 | 07/24/12]] <br /><br />
3.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F25.2F12 | 07/25/12]] <br /><br />
3.20 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F26.2F12 | 07/26/12]] <br /><br />
3.21 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F27.2F12 | 07/27/12]] <br /><br />
3.22 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F30.2F12 | 07/30/12]] <br /><br />
3.23 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F31.2F12 | 07/31/12]] <br /><br />
</td><br />
<br />
<td id="rightcolumn2"><center>4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#AUGUST | '''AUGUST''']] </center> <br /><br />
<br /><br />
4.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F01.2F12 | 08/01/12]] <br /><br />
4.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F02.2F12 | 08/02/12]] <br /><br />
4.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F03.2F12 | 08/03/12]] <br /><br />
4.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F06.2F12 | 08/06/12]] <br /><br />
4.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F08.2F12 | 08/08/12]] <br /><br />
4.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F09.2F12 | 08/09/12]] <br /><br />
4.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F10.2F12 | 08/10/12]] <br /><br />
4.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F13.2F12 | 08/13/12]] <br /><br />
4.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F16.2F12 | 08/16/12]] <br /><br />
4.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F20.2F12 | 08/20/12]] <br /><br />
4.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F21.2F12 | 08/21/12]] <br /><br />
4.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F22.2F12 | 08/22/12]] <br /><br />
4.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F23.2F12 | 08/23/12]] <br /><br />
4.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F24.2F12 | 08/24/12]] <br /><br />
4.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F25.2F12 | 08/25/12]] <br /><br />
4.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F28.2F12 | 08/28/12]] <br /><br />
4.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F29.2F12 | 08/29/12]] <br /><br />
4.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F30.2F12 | 08/30/12]] <br /><br />
4.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F31.2F12 | 08/31/12]] <br /><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2"><center>5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#SEPTEMBER | '''SEPTEMBER''']] </center> <br /><br />
<br /><br />
5.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F01.2F12 | 09/01/12]] <br /><br />
5.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F02.2F12 | 09/03/12]] <br /><br />
5.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F04.2F12 | 09/04/12]] <br /><br />
5.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F05.2F12 | 09/05/12]] <br /><br />
5.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F06.2F12 | 09/06/12]] <br /><br />
5.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F14.2F12 | 09/14/12]] <br /><br />
<br />
</td><br />
<br />
<td id="contentcolumn2"><center>[[File:UnmagenomicsBacteria sugar.png | 190px]]</center><br />
</td><br />
<br />
<td id="rightcolumn2"><p> '''On hover the images to see descriptions'''</p><td/><br />
</tr><br />
</table><br />
<br />
<br />
=JUNE=<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html> <br />
<h2>06/07/12</h2><br /><br />
PY BROTH 10g salt/L<br /><br />
PSB2K3 kanamicin<br /><br />
We transformed RFP E1010. <br /><br />
plate 1 18F<br /><br />
plate 2 17E<br /><br />
Stock 50 mg/mL<br /><br />
''E.coli'' 1/1000<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
We made liquid cultures with colonies [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
We extracted plasmids [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PLASMID_EXTRACTION_2 | PLASMID EXTRACTION PROTOCOL]]. <br /><br />
We ran a gel to check the extraction [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<h2>06/12/12</h2><br /><br />
We made glycerols of the bacteria <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed plasmid pHp45 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/38/UnamgenomicsandsugarBitacora_2.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>06/13/12</h2><br /><br />
We ran gel and extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] and lysed. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2d/UnamgenomicsandsugarBitacora_3.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
We extracted from gel<br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/14/12</h2><br /><br />
We extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made glycerols of pHp45 Ω [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450<br />
PSB4A5 Am (ampicillin) 1I BBa_J04450<br />
AraC BBa_C0080 <br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
2011 14L plate 1 pSB2K3 Km+<br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/5/56/UnamgenomicsandsugarBitacora_5.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
pHp45 Ω with E/P<br /><br />
We extracted from gel<br /><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/15/12</h2><br /><br />
> Digested pHp45 Ω with E/P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
> We extracted plasmid with kit. <br /><br />
> Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (5) <br /><br />
>Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br /><br />
ARAC<br /><br />
>From the overnight plate we prepared liquid culture [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
>We left them incubating overnight. <br /><br />
>We left a plate (LB Km DH5α C0080 and a control). <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<h2>06/18/12</h2><br /><br />
>Plasmid extraction AraC with kit. <br /><br />
>C0080-psB2K3 915 bp<br /><br />
>Streaked 4 LB Km 30 plates DH5α psB2K3. <br /><br />
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red. <br /><br />
>Ran gel with Spr/Strr (eppendorf -20ºC) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
>Made glycerols from one tube of DH5α C0080 Km 50 -> 04. <br /><br />
>Made glycerols from one tube of DH5α PSB4A5 Amp 100 -> 05 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]]. <br /><br />
>Digested C0080 X,S. <br /><br />
>Digested B0014 E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/5/52/UnamgenomicsandsugarBitacora_6.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
Digested B0014 with E, P and with E,X<br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<br /><br />
<br />
<h2>06/19/12</h2><br /><br />
>Digested B0014 with E, P and with E,X. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
>Ran gels with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation. <br /><br />
C0080 is in the first lane. <br /><br />
>Extracted from gel C0080 X,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Made glycerols from 4 plates LB Km DH5α pSB2K3 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
>Take out AmyE 5’ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
AmyE 5’ 18K plate 3 2010, 2011, 2012<br /><br />
AmyE 3’ 18M plate 3 2010, 2011, 2012<br /><br />
>Digested B0014 with E,X and E,P again [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<h2>06/22/12</h2><br /><br />
<br />
>Ran gels with digestions B0014 with E,X and E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
<br />
>Dephosphated B0014 E,X and B0014 E,P. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<h2>06/25/12</h2><br /><br />
>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
<br />
<h2>06/26/12</h2><br /><br />
>Ran gel with psB2K3 and psB4A5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
<h2>06/27/12</h2><br /><br />
>Transformed with plasmid B0079 1576 bp psB1A2 12A [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011<br /><br />
AmyE 5’ BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011<br /><br />
AmyE 3’ BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011<br /><br />
AmyE 5’ grew 2 colonies. <br /><br />
<br />
<h2>06/29/12</h2><br /><br />
>We did a DH5α K143001 Km30 Amp 100 glycerol [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] 07. <br /><br />
>We plated twice DH5α K143002 Km 30 Amp 100 + control and twice DH5α B0079 Amp 100 + control<br /><br />
These were both plated in 2 plates each. <br /><br />
They were left overnight since they had not grown by 7:00 pm so the next day glycerols were made. <br /><br />
>Liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
2 tubes DH5α K143001 Km30 Amp 100 <br /><br />
2 tubes DH5α K143002 Km30 Amp 100 <br /><br />
2 tubes DH5α B0079 Amp 100 <br /><br />
1 tube LB Km 30 Amp 100 control<br /><br />
1 tube LB Amp 100 control<br /><br />
>From the 6 tubes we extracted plasmid from kit. <br /><br />
<br /><br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=JULY=<br />
<br />
07/02/12<br /><br />
>Digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /><br />
>PCR’s<br /><br />
•AraC<br /><br />
•Cassete ΩSpr/Strr <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PCR_Protocol_.2850.C2.B5l.29 | PCR PROTOCOL]] <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/be/UnamgenomicsandsugarBitacora_8.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>After Cassete ΩSpr/Strr PCR we ran a gel <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsandsugarBitacora_9.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/03/12</h2><br /><br />
>After Cassete ΩSpr/Strr PCR we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (8) <br /><br />
>Ran gel with digestions from yesterday. (9) <br /><br />
>Did band extractions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Stored at -20ºC. <br /><br />
>Did PCR to Cassete ΩSpr/Strr to add RBS and prefix/suffix. <br /><br />
>Left digesting with E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/03/UnamgenomicsandsugarBitacora_9.1.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/04/12</h2><br /><br />
>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1). <br /><br />
>Ligated Cassete ΩSpr/Strr +K143002 dephosphated(digested with E,X and E,S). <br /><br />
>Transformed ligation and left overnight plated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs. <br /><br />
>Digested with PstI [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>07/06/12</h2><br /><br />
4 LB Km30 Spec60 DH5α ΩSpr/Strr +K143002 still have not grown. <br /><br />
Ran a gel with yesterday’s digestions to chek if they were done properly (10) <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
Ran another gel with the rest of the samples. <br /><br />
Extracted GusA fragment <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
Repeated ligation ΩSpr/Strr +K143002 E,X dephosphated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Repeated ΩSpr/Strr PCR. <br /><br />
<br /><br />
<br /><br />
<h2>07/07/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002 . <br /><br />
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<br /><br />
<br />
<h2>07/08/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/13/UnamgenomicsandsugarBitacora_11.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b2/UnamgenomicsandsugarBitacora_11.1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1 PCR omega P<br /><br />
2 PCR omega I<br /><br />
3 PCR AraC P<br /><br />
4 PCR AraC I<br /><br />
5 ladder 1 Kb<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>07/09/12</h2><br /><br />
From yesterday’s transformations only one colony grew. <br /><br />
From the previous transformation only 2 colonies grew. <br /><br />
These 3 were streaked in 3 plates: <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km 30 Sp 100 control. <br /><br />
Did liquid cultures in 3 tubes: <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km30 Sp 100 control. <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
Ran a gel with: (11) <br /><br />
GusA P<br /><br />
PBBR1 GusA<br /><br />
Ω E PCR P<br /><br />
Ω PCR P<br /><br />
Ω PCR I<br /><br />
Ω E<br /><br />
Ω PCR I<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] <br /><br />
Did GusA primers dissolution for PCR. <br /><br />
GusA PCR <br /><br />
Gel Extraction by kit of lanes 3 and 5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
'''PCR omega P, PCR omega I, PCR AraC P, PCR AraC I'''<br /><br />
Add 10 μl of each primer (LW and UP). <br /><br />
Add 3 μl of plasmid (P). <br /><br />
Add 30.4 μl buffer. <br /><br />
Add 5 μl Mg. <br /><br />
Add 8 μl DNTp’s. <br /><br />
Add 42.6μl H2O miliQ. <br /> <br />
Add 1 μl RTTG polymerase. <br /><br />
Centrifuge (spin) 8 secs. <br /><br />
Add vegetable oil till the eppendorf is full. <br /><br />
Place eppendorf 1 mL in thermocycler. <br /><br />
Run PCR with program “BERNA”. <br /><br /><br />
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<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/4b/UnamgenomicsandsugarBitacora_11.2.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/10/12</h2><br /><br />
•GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S (2 times)[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTIONS]]. (11.2) <br /><br />
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<br /><br />
<br />
<h2>07/11/12</h2><br /><br />
•GusA PCR digestions with E,S; Ω+AmyE 3’ with E,X [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•T.V. AraC PCR+ Ω+AmyE 3’ E,X dephosphated ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/0a/UnamgenomicsandsugarBitacora_12.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1. ladder. <br /><br />
2. GusA PCR E,S. <br /><br />
3. Ω+AmyE 3’ with E,X. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/12/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: (12) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Transformed AraC PCR+ Ω+AmyE 3’ ligation in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•From pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid. <br /><br />
•Made glycerols from 2 LB Km 30 Sp100 DH5α Ω+AmyE 3’ plates and from 1 LB Amp100 pfrc54 (A3) plates with control[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
•Ligated GusA PCR with B0014 E,X desphophorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
•Digested Pfrc54 (A3) with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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•Desphophorylated Ω+AmyE 3’ E,X. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/0f/UnamgenomicsandsugarBitacora_13.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>•Ran gel with pfrc54 S,P<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>07/13/12</h2><br /><br /><br />
•Ran gel with pfrc54 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (13) <br /><br />
•Transformation of GusA PCR + B0014 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Transformed with GFP E0040 psBIA2. <br /><br />
•Liquid Cultures with ligation: AraC+ Ω+AmyE 3’ for plasmid extraction at night [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•''B. Subtitils'' competent cells. <br /><br />
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<h2>07/14/12</h2><br /><br /><br />
•From 24 AraC+ Ω+AmyE 3’ LB Km 30 Sp 100 DH5α tubes: <br /><br />
•Extracted pellets<br /><br />
•Extracted plasmids. <br /><br />
•Ran gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br /><br />
•From transformed DH5α km 30: <br /><br />
GusA+B0014 DH5α Km50 24 pellets <br /><br />
E0040 DH5α LB Amp100 <br /><br />
From these two we: <br /><br />
•Did liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Extracted plasmid. <br /><br />
•From the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ran gel with this transformation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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<br /><br />
<h2>07/16/12</h2><br /><br /><br />
•Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
•Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_14.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>07/17/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (14) <br /><br />
•The gel we ran didn’t work, probably because the agarose was not prepared correctly. <br /><br />
•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/db/Bitacora_15.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br /><br />
<br />
<h2>07/18/12</h2><br /><br /><br />
•From yesterday’s digestions we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (15) <br /><br /><br />
•We made liquid cultures of AraC+ Ω+AmyE 3’ 12, 15, 21 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br /><br />
•We ligated AraC PCR E,S dephosphated with + Ω+AmyE 3’ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
•We digested 1 + Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/19/12</h2><br /><br /><br />
•We ran We digested 1 + Ω+AmyE 3’ E,P with Miguel’s enzymes and with Abiel’s enzymes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
•We transformed with ligations AraC+ Ω+AmyE 3’ E and + Ω+AmyE 3’ E [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br /><br />
•Extracted plasmids from AraC+ Ω+AmyE 3’ 12,15,21. <br /><br /><br />
•Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f3/UnamgenomicsandsugarBitacora_16.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/20/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (16) <br /><br />
•Transformed GusA+B0014 in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Made liquid cultures from AraC+ Ω+AmyE 3’s transformation LB Km30 Sp 100 and control LB Km 30 Sp100 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<h2>07/23/12</h2><br /><br /><br />
•From 24 GusA+B0014 tubes (-) we didn’t do anything. <br /><br />
•From 4 AraC+ Ω+AmyE 3’ we extracted plasmid. <br /><br />
•Digested from the 4 AraC+ Ω+AmyE 3’ E,X; X; P and GusA PCR with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<h2>07/24/12</h2><br /><br /><br />
•Digested B0014 E with X<br />
B0014 X with E<br /><br />
•Digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>07/25/12</h2><br /><br /><br />
•Ran gel of digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Joined B0014 E with X B0014 X with E digestions. <br /><br />
•Made liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. Transformed [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•From LasR DH5α make liquid cultures and plate [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Digested Ω+AmyE 3’ with X,P; E,S; C0080 X,P; E,S; S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Ligated Gus PCR X,P with B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/84/UnamgenomicsandsugarBitacora_17.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1.Ω+AmyE 3’ X,P<br /><br />
2. Ω+AmyE 3’ E,S<br /><br />
3.C0080 X,P<br /><br />
4.C0080 E,S<br /><br />
5.C0080 S,P<br /><br />
6.ladder<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>07/26/12</h2><br /><br />
•From C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5α C0179 LB Cb 100 (two cultures) and a control without bacteria [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•We extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguel’s transformation), Puc, B.S. control and PSB4AE. <br /><br />
<br />
•Due to problems with the way we did the transformations of ligations we repeated them: <br /><br />
GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
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GusAPCR X,P+ B0079 S,P dephosphorylated (-)<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•AraC+ Ω+AmyE 3’ S,P 1,2,3,4<br /><br />
•K143002 X,P<br /><br />
•AraC+ Ω S,P<br /><br />
•C0179 X,S<br /><br />
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<h2>07/27/12</h2><br /><br /><br />
Ligated AraC+ Ω S,P dephosphorylated + K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<h2>07/30/12</h2><br /><br /><br />
•Transformed with ligations: <br /><br />
•AraC+ Ω dephosphprylated +K143002 X,P<br /><br />
•GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
•GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
•Transformed the following sythesis: <br /><br />
•91996 Pveg 140 bp<br /><br />
•91997 ArsR-CzrA_promoter 1 194 bp<br /><br />
•91998 ArsR-CzrA_promoter 2 221 bp<br /><br />
•91999 ArsR-CzrA_promoter 3 213 bp<br /><br />
•92000 pBad-pXyl 387 bp<br /><br />
•92001 XylR 1117pb<br /><br />
•92002 CI_pro_(NAND_INHIBITOR) 774 <br /><br />
•These synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked.<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Make liquid cultures of the following transformations for tomorrow [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]: <br /><br />
•AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+B0079<br /><br />
•Synthesis<br /><br />
<br />
<h2>07/31/12</h2><br /><br /><br />
Extracted plasmids from yesterday’s cultures and from K143002 cultures that were in the other lab. <br /><br />
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::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
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=AUGUST=<br />
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<h2>08/01/12</h2><br /><br /><br />
Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b1/UnamgenomicsandsugarBitacora_17.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1 PCR GusA I<br /><br />
2 PCR GusA I<br /><br />
3 PCR GusA P<br /><br />
4 PCR GusA P<br /><br />
5 ladder 1 Kb<br /></p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/02/12</h2><br /><br /><br />
•Extracted plasmids from liquid cultures: <br /><br />
AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+BBR1<br /><br />
<br />
PCR GusA, GusA I, PCR GusA P, PCR GusA I<br /><br />
<br />
•Add 10 μl of each primer (LW and UP). <br /><br />
•Add 3 μl of plasmid (P). <br /><br />
•Add 30.4 μl buffer. <br /><br />
•Add 5 μl Mg. <br /><br />
•Add 8 μl DNTp’s. <br /><br />
•Add 42.6μl H2O miliQ. <br /><br />
•Add 1 μl RTTG polymerase. <br /><br />
•Centrifuge (spin) 8 secs. <br /><br />
•Add mineral oil till the eppendorf is full. <br /><br />
•Place eppendorf 1 mL in thermocycler. <br /><br />
•Run PCR with program “BERNA”. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/82/UnamgenomicsandsugarBitacora_18.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.A3+GusA 1<br /><br />
4.A3+GusA 2<br /><br />
5.A3+GusA 3<br /><br />
6.P GusA<br /><br />
7.P GusA 2<br /><br />
8.Ladder<br /><br />
9.01<br /><br />
10.06<br /><br />
11.96<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
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<br /><br />
<br />
<h2>08/03/12</h2><br /><br /><br />
• Ran a gel with: (18) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Ran another gel to extract with: <br /><br />
1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.00<br /><br />
4.01<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]: <br /> <br />
<br />
• Ω+K143002 E,X; A3+GusA E,S; GusAPCR X,P <br /><br />
<br />
•Extracted GusAPCR X,P digestion [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Left the following ligation: AraC+ Ω+K143002 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/17/UnamgenomicsandsugarBitacora_19.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3+GusA 2<br /><br />
2.A3+GusA 3<br /><br />
3. Ω+AmyE 3’ 2<br /><br />
4. Ω+AmyE 3’ 3<br /><br />
5.XylR X,P<br /><br />
6.pBad-pXyl X,P<br /><br />
7.GusA PCR X,P<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/06/12</h2><br /><br /><br />
•Made liquid cultures of Ω+K143002 (2 cultures+1 control) and AraC+ Ω+K143002 (2 cultures+1 control) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ligated GusAPCR X,P+B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
•Ran a Gel with: (19) <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/8b/UnamgenomicsandsugarBitacora_19.1.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/08/12</h2><br /><br />
BBa_B0040 6I psB1A2 Amp+ plate 1<br /><br />
Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (19.1) <br /><br />
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<br />
<h2>08/09/12</h2><br /><br />
•From yesterday’s PCR’s : <br /><br />
• E0040 plasmid 1<br /><br />
E0040 plasmid 2<br /><br />
E0040 digested 1<br /><br />
E0040 digested 2<br /><br />
We purified with PCR kit<br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Made 14 PCR’s from AraC+ Ω+AmyE 3’. <br /><br />
•From B0079+GusA ligation and B0040 transformation:<br />
•Grew colonies. <br /><br />
•Made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Streaked these in a new plate. <br /><br />
•Diluted plasmid E0080 2 1/50. <br /><br />
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<br /><br />
<br />
<h2>08/10/12</h2><br /><br />
•Yesterday’s gel did not come out as expected so we repeated the PCR. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/e/ef/UnamgenomicsandsugarBitacora_20.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.AraC+ Ω+AmyE 3’ <br /><br />
2-14.AraC+ Ω+AmyE 3’ <br /><br />
15.AraC+ Ω 1<br /><br />
16.AraC+ Ω 1<br /><br />
17.AraC+ Ω 1<br /><br />
18.PCR 1 GFP<br /><br />
19.PCR GFP<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<h2>08/13/12</h2><br /><br />
•After running a gel with 14 PCR’s AraC+ Ω+AmyE 3’ and it did not come out as expected, we ran a gel with the ligation AraC+ Ω+AmyE 3’ to select candidate (that weight the same as the ligation) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (20) <br /><br />
<br />
•Extracted pasmids form liquid cultures: B0049, B0079+GusA. <br /><br />
<br />
•AraC+ Ω+AmyE 3’ undigested didn’t run in the gel as expected, it ran similarly to AraC+ Ω<br /><br />
<br />
• We repeated the ligation AraC+ Ω S,P dephosphorylated+K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
• GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.<br />
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• We ran a gel to extract. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f1/UnamgenomicsandsugarBitacora_22.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>1.6 AraC+ Ω+AmyE 3’ E with P. <br /><br /><br />
2.8 AraC+ Ω+AmyE 3’ E with P. <br /><br />
3.11 AraC+ Ω+AmyE 3’ E with P. <br /><br />
4.12 AraC+ Ω+AmyE 3’ E with P. <br /><br />
5.6 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
6.8 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
7.11 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
8.12 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
9.00 pBad/pXyl with S,P. <br /><br />
10.Ladder. <br /><br />
11.E0040 PCR E,S to extract the correct band. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/16/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: <br /><br />
<br />
•Digested: <br /><br />
ArSR-CzrA 97 with S,P<br /><br />
ArSR-CzrA 98 with S,P<br /><br />
ArSR-CzrA 99 with S,P<br /><br />
E0040 PCR with E,S<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
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<h2>08/20/12</h2><br /><br />
•Ligated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/21/12</h2><br /><br />
•Joined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated. <br /><br />
•Extracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR. <br /><br />
•Digested Ω+AmyE 3’ with E,X and + Ω+AmyE 3’ II with E,X (obtained these two from glycerols stored at -80ºC [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/29/UnamgenomicsandsugarBitacora_22.1.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested K143001+PBad, pXyl with E,S <br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/1c/UnamgenomicsandsugarBitacora_23.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>1.K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
4.A3 PCR<br /><br />
5.A3 PCR<br /><br />
6.E0040 PCR E,S<br /><br />
7.B0014 E,X dephosphorylated<br /><br />
8. Ω+AmyE 3’ E,X <br /><br />
9. Ω+AmyE 3’ E,X II<br /><br />
10.Ladder<br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/22/12</h2><br /><br />
•We did 2 PCR’s for A3. <br /><br />
<br />
•Digested K143001+PBad, pXyl with E,S <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (22.1) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did band extraction of: <br /><br />
1. K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
•Made liquid cultures from E1010+Boo14 that the other lab gave us and did 2 plates for glycerol Km+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Did 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ Ω+AmyE 3’ 6,8,11,12 Km+ and Spectinomycin+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Transformed ligations: <br /><br />
B0079+GusA Amp+<br /><br />
Pveg+XylR Chloramphenicol+<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]].. <br /><br />
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<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/78/UnamgenomicsandsugarBitacora_23.01.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1. Ω+AmyE 3’ E,X 6.2<br /><br />
2.Ω+AmyE 3’ II E,X<br /><br />
3.97 ArsR-CzrA<br /><br />
4.98 ArsR-CzrA<br /><br />
5.99 ArsR-CzrA<br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
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<h2>08/23/12</h2><br /><br />
•The negative controls were contaminated. We didn’t manipulate the ones that had grown because of this. <br /><br />
•Ran gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
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<h2>08/24/12</h2><br /><br />
From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ Ω+AmyE 3’ 6 and 8 we streaked 2 plates for glycerol of each ligation. <br />Repeated ligation B0079+GusA. <br /><br />
<br />
Ligated E0040 PCR E,S+B0014 E,X dephospohylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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Made 2 new plates from Pveg+XylR ligation and liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<h2>08/25/12</h2><br /><br />
Extracted pellet from 16 tubes of pVeg+XylR that were left overnight. <br /><br />
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Ligated B0079+GusA and B0040+B0014 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<h2>08/28/12</h2><br /><br />
<br />
Made liquid cultures from transformations tht were left overnight <br />
-R0079+GusA<br /><br />
-R0079+GusA (-)<br /><br />
-E0040+B0014<br /><br />
-E0040+B0014 (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/25/UnamgenomicsandsugarBitacora_23.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. A3 PCR 1. <br /><br />
2. A3 PCR 2. <br /><br />
3. Ω+AmyE 3’ S,P *.<br /><br />
4. Ω+AmyE 3’ S,P . <br /><br />
5. Pveg+XylR E,S 2. <br /><br />
6. Pveg+XylR E,S 3. <br /><br />
7. Pveg+XylR E,S 4. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/29/12</h2><br /><br />
Digested Pveg+XylR 2,3,4 with E,S. <br /><br />
<br />
Digested Ω+AmyE 3’ with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTION]]. <br /><br />
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Ran a PCR with A3 PCR (2 .6 ml tubes). <br /><br />
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Ran a gel with <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2e/UnamgenomicsandsugarBitacoraBitacora_24.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1-23. E0040 + B0014 colonies<br /><br />
24-29. B0079+GusA 1<br /><br />
30. B0079<br /><br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
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<h2>08/30/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (24) <br /><br />
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•Digested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/3/3d/UnamgenomicsandsugarBitacora_25.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1-3B0079+GusA E,S 3. <br /><br />
4-9. PVeg+XylR E,S 1. <br /><br />
10-17. E0040+B0014 X,P 3. <br /><br />
18. 1 kb ladder. <br /><br />
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</li><br />
</ul><br />
</div><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/d2/UnamgenomicsandsugarBitacora_26.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3 PCR. <br /><br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7b/UnamgenomicsandsugarBitacora_27.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>E0040+B0014 X,P 4 to extract<br /><br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
<h2>08/31/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (25) <br /><br />
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•Dephosphorylated: <br /><br />
Ω+AmyE 3’ S,P <br /><br />
Ω+AmyE 3’ II S,P <br /><br />
6 AraC+ Ω+AmyE 3’ E,X<br /><br />
8 AraC+ Ω+AmyE 3’ E,X<br /><br />
11 AraC+ Ω+AmyE 3’ E,X<br /><br />
12 AraC+ Ω+AmyE 3’ E,X<br /><br />•Ran a gel with A3 PCR [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (26) <br /><br />
<br />
•It was the second time we didn’t obtain a band from A3’s PCR.<br />
<br />
•Probably because it had been missing the buffer 2’s ethanol (“blue lid”) from the PCR purification. <br /><br />
<br />
•Ran a gel woth E0040+B0014 X,P 4 to extract <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | Gen extraction protocol]]. <br /><br />
<br />
•Did an A3 PCR. <br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=SEPTEMBER=<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b8/UnamgenomicsandsugarBitacora_28.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.A3 PCR 1. <br /><br />
2.A3 PCR 2. <br /><br />
3.E0040+B0014 X,P 1. <br /><br />
4.E0040+B0014 X,P 2. <br /><br />
5.Ω+AmyE 3’ dephosphorylated S,P. <br /><br />
6.Ω+AmyE 3’ dephosphorylated S,P II. <br /><br />
7.97 dephosphorylated S,P. <br /><br />
8.98 dephosphorylated S,P. <br /><br />
9.99 dephosphorylated S,P. <br /><br />
10-13. Arac+ Ω+AmyE 3’ dephosphorylated E,X 12. <br /><br />
14. 1 kb Ladder. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>09/01/12</h2><br /><br />
•Ran a gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (28) <br /><br />
<br />
•Ligated E0040+B0014 X,P 2 +promoter S,P dephosphorylated; K143002 X,P+ Ω+AmyE 3’ dephosphorylated S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<h2>09/03/12</h2><br /><br />
<br />
•Digested B0079+GusA E,S 3,4,5; PVeg+XylR E,S 1,5,7,10,13,16 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>09/04/12</h2><br /><br />
•Ran gel with digestions: <br /><br />
B0079+GusA E,S 3,4,5<br /><br />
PVeg+XylR E,S 1,5,7,10,13,16 <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Purified A3 PCR 1,2. <br /><br />
<br />
•Ran a gel of A3 PCR <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] and digested with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•From ligations: PVeg+E0040+B0014, PBad/PXyl+E0040+B0014, B0079+E0040+B0014 only colonies grew in PBad/PXyl+E0040+B0014.<br />
<br />
•Plated colonies that grew in a new plate. <br /><br />
<br />
•Made liquid cultures of these <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•Repeated the ones that did not grow. <br /><br />
<br />
•Digested E0040+B0014 4 with X,P[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
-BBa_J23101 18E plate 1 plasmid BBa_J61002_2948 bp Amp+<br /><br />
<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/c1/UnamgenomicsandsugarBitacora_29.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>24 PBad/PXyl+E0040+B0014 tubes we extracted <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/a/a4/UnamgenomicsandsugarBitacora_30.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
Extracted band from 1. and 2<br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7d/UnamgenomicsandsugarBitacora_31.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S <br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>09/05/12</h2><br /><br />
•From the 24 PBad/PXyl+E0040+B0014 tubes we extracted plasmids (29) and ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Extracted plasmid from 2 tubes of E0040+B0014. <br /><br />
<br />
•Ran a gel with yesterday’s digestions: <br /><br />
1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
<br />
•Extracted band from 1. and 2. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Purified A3 PCR from 2 0.6ml tubes 1 and 2. <br /><br />
<br />
•1 colony grew from ligation pVeg+E0040+B0014. <br /><br />
<br />
•We streaked this in another plate and did liquid cultures <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•To do: <br /><br />
•Ligate B0079 S,P dephosphorylated + E0040+B0014 X,P and Ω+AmyE 3’ S,P dephosphorylated+ K143001 X,P. <br /><br />
•Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S 10,13. (31) <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/26/UnamgenomicsandsugarBitacora_32.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>•A3 PCR and gel A3 PCR X,P <br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br /><br />
<br />
<h2>09/06/12</h2><br /><br />
<br />
•A3 PCR and gel A3 PCR X,P 1,2. <br /><br />
<br />
<br />
<h2>09/14/12</h2><br /><br />
<br />
•Digested Ω+AmyE 3’ 11,13,18, and 22 with E,X. <br /><br />
•Ligate: <br /><br />
PVeg S,P dephosphorylated+XylR X,P. <br /><br />
PBad p Xyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
pSB13C3 E,P dephosphorylated + A3 PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + GusA PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + XylR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pVeg E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pBad pXyl E,P. <br /><br />
pSB13C3 E,P dephosphorylated + Ω PCR E,P. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/ANDSugar
Team:UNAM Genomics Mexico/Notebook/ANDSugar
2012-10-27T03:27:56Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Arabinose/Xylose AND Gate Notebook'''</h1></center><br />
<br /><br />
<br /><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="20"><br />
<tr><br />
<td id="leftcolumn2"><center>2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JUNE | '''JUNE''']] </center> <br /><br />
<br /><br />
2.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F07.2F12 |06/07/12]] <br /><br />
2.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F12.2F12 |06/12/12]] <br /><br />
2.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F13.2F12 |06/13/12]] <br /><br />
2.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F14.2F12 |06/14/12]] <br /><br />
2.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F15.2F12 |06/15/12]] <br /><br />
2.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F18.2F12 |06/18/12]] <br /><br />
2.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F19.2F12 |06/19/12]] <br /><br />
2.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F22.2F12 |06/22/12]] <br /><br />
2.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F25.2F12 |06/25/12]] <br /><br />
2.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F26.2F12 |06/26/12]] <br /><br />
2.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F27.2F12 |06/27/12]] <br /><br />
2.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F29.2F12 |06/29/12]] <br /><br />
</td><br />
<br />
<td id="contentcolumn2"><center>3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JULY | '''JULY''']] </center> <br /><br />
<br /><br />
3.1[[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F03.2F12 | 07/03/12]] <br /><br />
3.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F04.2F12 | 07/04/12]] <br /><br />
3.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F06.2F12 | 07/06/12]] <br /><br />
3.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F07.2F12 | 07/07/12]] <br /><br />
3.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F08.2F12 | 07/08/12]] <br /><br />
3.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F09.2F12 | 07/09/12]] <br /><br />
3.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F10.2F12 | 07/10/12]] <br /><br />
3.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F11.2F12 | 07/11/12]] <br /><br />
3.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F12.2F12 | 07/12/12]] <br /><br />
3.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F13.2F12 | 07/13/12]] <br /><br />
3.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F14.2F12 | 07/14/12]] <br /><br />
3.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F16.2F12 | 07/16/12]] <br /><br />
3.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F17.2F12 | 07/17/12]] <br /><br />
3.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F18.2F12 | 07/18/12]] <br /><br />
3.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F19.2F12 | 07/19/12]] <br /><br />
3.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F20.2F12 | 07/20/12]] <br /><br />
3.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F23.2F12 | 07/23/12]] <br /><br />
3.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F24.2F12 | 07/24/12]] <br /><br />
3.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F25.2F12 | 07/25/12]] <br /><br />
3.20 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F26.2F12 | 07/26/12]] <br /><br />
3.21 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F27.2F12 | 07/27/12]] <br /><br />
3.22 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F30.2F12 | 07/30/12]] <br /><br />
3.23 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F31.2F12 | 07/31/12]] <br /><br />
</td><br />
<br />
<td id="rightcolumn2"><center>4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#AUGUST | '''AUGUST''']] </center> <br /><br />
<br /><br />
4.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F01.2F12 | 08/01/12]] <br /><br />
4.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F02.2F12 | 08/02/12]] <br /><br />
4.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F03.2F12 | 08/03/12]] <br /><br />
4.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F06.2F12 | 08/06/12]] <br /><br />
4.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F08.2F12 | 08/08/12]] <br /><br />
4.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F09.2F12 | 08/09/12]] <br /><br />
4.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F10.2F12 | 08/10/12]] <br /><br />
4.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F13.2F12 | 08/13/12]] <br /><br />
4.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F16.2F12 | 08/16/12]] <br /><br />
4.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F20.2F12 | 08/20/12]] <br /><br />
4.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F21.2F12 | 08/21/12]] <br /><br />
4.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F22.2F12 | 08/22/12]] <br /><br />
4.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F23.2F12 | 08/23/12]] <br /><br />
4.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F24.2F12 | 08/24/12]] <br /><br />
4.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F25.2F12 | 08/25/12]] <br /><br />
4.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F28.2F12 | 08/28/12]] <br /><br />
4.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F29.2F12 | 08/29/12]] <br /><br />
4.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F30.2F12 | 08/30/12]] <br /><br />
4.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F31.2F12 | 08/31/12]] <br /><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2"><center>5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#SEPTEMBER | '''SEPTEMBER''']] </center> <br /><br />
<br /><br />
5.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F01.2F12 | 09/01/12]] <br /><br />
5.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F02.2F12 | 09/03/12]] <br /><br />
5.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F04.2F12 | 09/04/12]] <br /><br />
5.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F05.2F12 | 09/05/12]] <br /><br />
5.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F06.2F12 | 09/06/12]] <br /><br />
5.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F14.2F12 | 09/14/12]] <br /><br />
<br />
</td><br />
<br />
<td id="contentcolumn2"><center>[[File:UnmagenomicsBacteria sugar.png | 190px]]</center><br />
</td><br />
<br />
<td id="rightcolumn2"><p> '''On hover the images to see descriptions'''</p><td/><br />
</tr><br />
</table><br />
<br />
<br />
=JUNE=<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html> <br />
<h2>06/07/12</h2><br /><br />
PY BROTH 10g salt/L<br /><br />
PSB2K3 kanamicin<br /><br />
We transformed RFP E1010. <br /><br />
plate 1 18F<br /><br />
plate 2 17E<br /><br />
Stock 50 mg/mL<br /><br />
''E.coli'' 1/1000<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
We made liquid cultures with colonies [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
We extracted plasmids [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PLASMID_EXTRACTION_2 | PLASMID EXTRACTION PROTOCOL]]. <br /><br />
We ran a gel to check the extraction [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<h2>06/12/12</h2><br /><br />
We made glycerols of the bacteria <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed plasmid pHp45 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/38/UnamgenomicsandsugarBitacora_2.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>06/13/12</h2><br /><br />
We ran gel and extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] and lysed. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2d/UnamgenomicsandsugarBitacora_3.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
We extracted from gel<br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/14/12</h2><br /><br />
We extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made glycerols of pHp45 Ω [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450<br />
PSB4A5 Am (ampicillin) 1I BBa_J04450<br />
AraC BBa_C0080 <br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
2011 14L plate 1 pSB2K3 Km+<br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
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<div class='captionverde'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
pHp45 Ω with E/P<br /><br />
We extracted from gel<br /><br />
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<h2>06/15/12</h2><br /><br />
> Digested pHp45 Ω with E/P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
> We extracted plasmid with kit. <br /><br />
> Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (5) <br /><br />
>Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
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ARAC<br /><br />
>From the overnight plate we prepared liquid culture [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
>We left them incubating overnight. <br /><br />
>We left a plate (LB Km DH5α C0080 and a control). <br /><br />
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<h2>06/18/12</h2><br /><br />
>Plasmid extraction AraC with kit. <br /><br />
>C0080-psB2K3 915 bp<br /><br />
>Streaked 4 LB Km 30 plates DH5α psB2K3. <br /><br />
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red. <br /><br />
>Ran gel with Spr/Strr (eppendorf -20ºC) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
>Made glycerols from one tube of DH5α C0080 Km 50 -> 04. <br /><br />
>Made glycerols from one tube of DH5α PSB4A5 Amp 100 -> 05 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]]. <br /><br />
>Digested C0080 X,S. <br /><br />
>Digested B0014 E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='captionrosa'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
Digested B0014 with E, P and with E,X<br />
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<h2>06/19/12</h2><br /><br />
>Digested B0014 with E, P and with E,X. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
>Ran gels with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation. <br /><br />
C0080 is in the first lane. <br /><br />
>Extracted from gel C0080 X,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Made glycerols from 4 plates LB Km DH5α pSB2K3 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
>Take out AmyE 5’ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
AmyE 5’ 18K plate 3 2010, 2011, 2012<br /><br />
AmyE 3’ 18M plate 3 2010, 2011, 2012<br /><br />
>Digested B0014 with E,X and E,P again [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>06/22/12</h2><br /><br />
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>Ran gels with digestions B0014 with E,X and E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
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>Dephosphated B0014 E,X and B0014 E,P. <br /><br />
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<h2>06/25/12</h2><br /><br />
>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
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<h2>06/26/12</h2><br /><br />
>Ran gel with psB2K3 and psB4A5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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<h2>06/27/12</h2><br /><br />
>Transformed with plasmid B0079 1576 bp psB1A2 12A [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011<br /><br />
AmyE 5’ BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011<br /><br />
AmyE 3’ BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011<br /><br />
AmyE 5’ grew 2 colonies. <br /><br />
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<h2>06/29/12</h2><br /><br />
>We did a DH5α K143001 Km30 Amp 100 glycerol [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] 07. <br /><br />
>We plated twice DH5α K143002 Km 30 Amp 100 + control and twice DH5α B0079 Amp 100 + control<br /><br />
These were both plated in 2 plates each. <br /><br />
They were left overnight since they had not grown by 7:00 pm so the next day glycerols were made. <br /><br />
>Liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
2 tubes DH5α K143001 Km30 Amp 100 <br /><br />
2 tubes DH5α K143002 Km30 Amp 100 <br /><br />
2 tubes DH5α B0079 Amp 100 <br /><br />
1 tube LB Km 30 Amp 100 control<br /><br />
1 tube LB Amp 100 control<br /><br />
>From the 6 tubes we extracted plasmid from kit. <br /><br />
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::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
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=JULY=<br />
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07/02/12<br /><br />
>Digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /><br />
>PCR’s<br /><br />
•AraC<br /><br />
•Cassete ΩSpr/Strr <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PCR_Protocol_.2850.C2.B5l.29 | PCR PROTOCOL]] <br /><br />
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<div class='captionmoradoclaro'><br />
<p class='captionInside'>After Cassete ΩSpr/Strr PCR we ran a gel <br />
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<div class='captionmorado'><br />
<p class='captionInside'>B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /> <br />
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</li><br />
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<br />
<h2>07/03/12</h2><br /><br />
>After Cassete ΩSpr/Strr PCR we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (8) <br /><br />
>Ran gel with digestions from yesterday. (9) <br /><br />
>Did band extractions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Stored at -20ºC. <br /><br />
>Did PCR to Cassete ΩSpr/Strr to add RBS and prefix/suffix. <br /><br />
>Left digesting with E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='captionazul'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
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</li><br />
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<h2>07/04/12</h2><br /><br />
>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1). <br /><br />
>Ligated Cassete ΩSpr/Strr +K143002 dephosphated(digested with E,X and E,S). <br /><br />
>Transformed ligation and left overnight plated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs. <br /><br />
>Digested with PstI [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>07/06/12</h2><br /><br />
4 LB Km30 Spec60 DH5α ΩSpr/Strr +K143002 still have not grown. <br /><br />
Ran a gel with yesterday’s digestions to chek if they were done properly (10) <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
Ran another gel with the rest of the samples. <br /><br />
Extracted GusA fragment <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
Repeated ligation ΩSpr/Strr +K143002 E,X dephosphated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Repeated ΩSpr/Strr PCR. <br /><br />
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<h2>07/07/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002 . <br /><br />
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<h2>07/08/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002. <br /><br />
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<div class='captiongray'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
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</li><br />
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<div class='captionnaranja'><br />
<p class='captionInside'>1 PCR omega P<br /><br />
2 PCR omega I<br /><br />
3 PCR AraC P<br /><br />
4 PCR AraC I<br /><br />
5 ladder 1 Kb<br /><br />
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<h2>07/09/12</h2><br /><br />
From yesterday’s transformations only one colony grew. <br /><br />
From the previous transformation only 2 colonies grew. <br /><br />
These 3 were streaked in 3 plates: <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km 30 Sp 100 control. <br /><br />
Did liquid cultures in 3 tubes: <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km30 Sp 100 control. <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
Ran a gel with: (11) <br /><br />
GusA P<br /><br />
PBBR1 GusA<br /><br />
Ω E PCR P<br /><br />
Ω PCR P<br /><br />
Ω PCR I<br /><br />
Ω E<br /><br />
Ω PCR I<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] <br /><br />
Did GusA primers dissolution for PCR. <br /><br />
GusA PCR <br /><br />
Gel Extraction by kit of lanes 3 and 5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
'''PCR omega P, PCR omega I, PCR AraC P, PCR AraC I'''<br /><br />
Add 10 μl of each primer (LW and UP). <br /><br />
Add 3 μl of plasmid (P). <br /><br />
Add 30.4 μl buffer. <br /><br />
Add 5 μl Mg. <br /><br />
Add 8 μl DNTp’s. <br /><br />
Add 42.6μl H2O miliQ. <br /> <br />
Add 1 μl RTTG polymerase. <br /><br />
Centrifuge (spin) 8 secs. <br /><br />
Add vegetable oil till the eppendorf is full. <br /><br />
Place eppendorf 1 mL in thermocycler. <br /><br />
Run PCR with program “BERNA”. <br /><br /><br />
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<div class='captionverde'><br />
<p class='captionInside'>GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S <br /><br />
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</li><br />
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<br />
<h2>07/10/12</h2><br /><br />
•GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S (2 times)[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTIONS]]. (11.2) <br /><br />
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<h2>07/11/12</h2><br /><br />
•GusA PCR digestions with E,S; Ω+AmyE 3’ with E,X [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•T.V. AraC PCR+ Ω+AmyE 3’ E,X dephosphated ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<div class='captionrojo'><br />
<p class='captionInside'>1. ladder. <br /><br />
2. GusA PCR E,S. <br /><br />
3. Ω+AmyE 3’ with E,X. <br /><br />
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</li><br />
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<h2>07/12/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: (12) <br /><br />
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[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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•Transformed AraC PCR+ Ω+AmyE 3’ ligation in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
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•From pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid. <br /><br />
•Made glycerols from 2 LB Km 30 Sp100 DH5α Ω+AmyE 3’ plates and from 1 LB Amp100 pfrc54 (A3) plates with control[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
•Ligated GusA PCR with B0014 E,X desphophorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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•Digested Pfrc54 (A3) with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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•Desphophorylated Ω+AmyE 3’ E,X. <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/0/0f/UnamgenomicsandsugarBitacora_13.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>•Ran gel with pfrc54 S,P<br /><br />
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</li><br />
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<h2>07/13/12</h2><br /><br /><br />
•Ran gel with pfrc54 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (13) <br /><br />
•Transformation of GusA PCR + B0014 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Transformed with GFP E0040 psBIA2. <br /><br />
•Liquid Cultures with ligation: AraC+ Ω+AmyE 3’ for plasmid extraction at night [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•''B. Subtitils'' competent cells. <br /><br />
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<h2>07/14/12</h2><br /><br /><br />
•From 24 AraC+ Ω+AmyE 3’ LB Km 30 Sp 100 DH5α tubes: <br /><br />
•Extracted pellets<br /><br />
•Extracted plasmids. <br /><br />
•Ran gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br /><br />
•From transformed DH5α km 30: <br /><br />
GusA+B0014 DH5α Km50 24 pellets <br /><br />
E0040 DH5α LB Amp100 <br /><br />
From these two we: <br /><br />
•Did liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Extracted plasmid. <br /><br />
•From the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ran gel with this transformation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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<h2>07/16/12</h2><br /><br /><br />
•Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
•Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='captionrosa'><br />
<p class='captionInside'>Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
Ω+AmyE 3’ E,P <br /><br />
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</li><br />
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<h2>07/17/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (14) <br /><br />
•The gel we ran didn’t work, probably because the agarose was not prepared correctly. <br /><br />
•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/d/db/Bitacora_15.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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<br />
<h2>07/18/12</h2><br /><br /><br />
•From yesterday’s digestions we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (15) <br /><br /><br />
•We made liquid cultures of AraC+ Ω+AmyE 3’ 12, 15, 21 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br /><br />
•We ligated AraC PCR E,S dephosphated with + Ω+AmyE 3’ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
•We digested 1 + Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/19/12</h2><br /><br /><br />
•We ran We digested 1 + Ω+AmyE 3’ E,P with Miguel’s enzymes and with Abiel’s enzymes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
•We transformed with ligations AraC+ Ω+AmyE 3’ E and + Ω+AmyE 3’ E [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br /><br />
•Extracted plasmids from AraC+ Ω+AmyE 3’ 12,15,21. <br /><br /><br />
•Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f3/UnamgenomicsandsugarBitacora_16.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P <br /><br />
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</li><br />
</ul><br />
</div><br />
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<br />
<h2>07/20/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (16) <br /><br />
•Transformed GusA+B0014 in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Made liquid cultures from AraC+ Ω+AmyE 3’s transformation LB Km30 Sp 100 and control LB Km 30 Sp100 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<h2>07/23/12</h2><br /><br /><br />
•From 24 GusA+B0014 tubes (-) we didn’t do anything. <br /><br />
•From 4 AraC+ Ω+AmyE 3’ we extracted plasmid. <br /><br />
•Digested from the 4 AraC+ Ω+AmyE 3’ E,X; X; P and GusA PCR with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/24/12</h2><br /><br /><br />
•Digested B0014 E with X<br />
B0014 X with E<br /><br />
•Digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<h2>07/25/12</h2><br /><br /><br />
•Ran gel of digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Joined B0014 E with X B0014 X with E digestions. <br /><br />
•Made liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. Transformed [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•From LasR DH5α make liquid cultures and plate [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Digested Ω+AmyE 3’ with X,P; E,S; C0080 X,P; E,S; S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Ligated Gus PCR X,P with B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/84/UnamgenomicsandsugarBitacora_17.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1.Ω+AmyE 3’ X,P<br /><br />
2. Ω+AmyE 3’ E,S<br /><br />
3.C0080 X,P<br /><br />
4.C0080 E,S<br /><br />
5.C0080 S,P<br /><br />
6.ladder<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>07/26/12</h2><br /><br />
•From C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5α C0179 LB Cb 100 (two cultures) and a control without bacteria [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•We extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguel’s transformation), Puc, B.S. control and PSB4AE. <br /><br />
<br />
•Due to problems with the way we did the transformations of ligations we repeated them: <br /><br />
GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
<br />
GusAPCR X,P+ B0079 S,P dephosphorylated (-)<br /><br />
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GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•AraC+ Ω+AmyE 3’ S,P 1,2,3,4<br /><br />
•K143002 X,P<br /><br />
•AraC+ Ω S,P<br /><br />
•C0179 X,S<br /><br />
<br />
<br />
<h2>07/27/12</h2><br /><br /><br />
Ligated AraC+ Ω S,P dephosphorylated + K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<h2>07/30/12</h2><br /><br /><br />
•Transformed with ligations: <br /><br />
•AraC+ Ω dephosphprylated +K143002 X,P<br /><br />
•GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
•GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
•Transformed the following sythesis: <br /><br />
•91996 Pveg 140 bp<br /><br />
•91997 ArsR-CzrA_promoter 1 194 bp<br /><br />
•91998 ArsR-CzrA_promoter 2 221 bp<br /><br />
•91999 ArsR-CzrA_promoter 3 213 bp<br /><br />
•92000 pBad-pXyl 387 bp<br /><br />
•92001 XylR 1117pb<br /><br />
•92002 CI_pro_(NAND_INHIBITOR) 774 <br /><br />
•These synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked.<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Make liquid cultures of the following transformations for tomorrow [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]: <br /><br />
•AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+B0079<br /><br />
•Synthesis<br /><br />
<br />
<h2>07/31/12</h2><br /><br /><br />
Extracted plasmids from yesterday’s cultures and from K143002 cultures that were in the other lab. <br /><br />
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::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
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=AUGUST=<br />
<br />
<h2>08/01/12</h2><br /><br /><br />
Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b1/UnamgenomicsandsugarBitacora_17.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1 PCR GusA I<br /><br />
2 PCR GusA I<br /><br />
3 PCR GusA P<br /><br />
4 PCR GusA P<br /><br />
5 ladder 1 Kb<br /></p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/02/12</h2><br /><br /><br />
•Extracted plasmids from liquid cultures: <br /><br />
AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+BBR1<br /><br />
<br />
PCR GusA, GusA I, PCR GusA P, PCR GusA I<br /><br />
<br />
•Add 10 μl of each primer (LW and UP). <br /><br />
•Add 3 μl of plasmid (P). <br /><br />
•Add 30.4 μl buffer. <br /><br />
•Add 5 μl Mg. <br /><br />
•Add 8 μl DNTp’s. <br /><br />
•Add 42.6μl H2O miliQ. <br /><br />
•Add 1 μl RTTG polymerase. <br /><br />
•Centrifuge (spin) 8 secs. <br /><br />
•Add mineral oil till the eppendorf is full. <br /><br />
•Place eppendorf 1 mL in thermocycler. <br /><br />
•Run PCR with program “BERNA”. <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/8/82/UnamgenomicsandsugarBitacora_18.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.A3+GusA 1<br /><br />
4.A3+GusA 2<br /><br />
5.A3+GusA 3<br /><br />
6.P GusA<br /><br />
7.P GusA 2<br /><br />
8.Ladder<br /><br />
9.01<br /><br />
10.06<br /><br />
11.96<br /><br />
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</li><br />
</ul><br />
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<br />
<h2>08/03/12</h2><br /><br /><br />
• Ran a gel with: (18) <br /><br />
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[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Ran another gel to extract with: <br /><br />
1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.00<br /><br />
4.01<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]: <br /> <br />
<br />
• Ω+K143002 E,X; A3+GusA E,S; GusAPCR X,P <br /><br />
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•Extracted GusAPCR X,P digestion [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
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•Left the following ligation: AraC+ Ω+K143002 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/17/UnamgenomicsandsugarBitacora_19.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3+GusA 2<br /><br />
2.A3+GusA 3<br /><br />
3. Ω+AmyE 3’ 2<br /><br />
4. Ω+AmyE 3’ 3<br /><br />
5.XylR X,P<br /><br />
6.pBad-pXyl X,P<br /><br />
7.GusA PCR X,P<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
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<br />
<h2>08/06/12</h2><br /><br /><br />
•Made liquid cultures of Ω+K143002 (2 cultures+1 control) and AraC+ Ω+K143002 (2 cultures+1 control) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ligated GusAPCR X,P+B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
•Ran a Gel with: (19) <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/8b/UnamgenomicsandsugarBitacora_19.1.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P <br /><br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/08/12</h2><br /><br />
BBa_B0040 6I psB1A2 Amp+ plate 1<br /><br />
Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (19.1) <br /><br />
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<h2>08/09/12</h2><br /><br />
•From yesterday’s PCR’s : <br /><br />
• E0040 plasmid 1<br /><br />
E0040 plasmid 2<br /><br />
E0040 digested 1<br /><br />
E0040 digested 2<br /><br />
We purified with PCR kit<br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Made 14 PCR’s from AraC+ Ω+AmyE 3’. <br /><br />
•From B0079+GusA ligation and B0040 transformation:<br />
•Grew colonies. <br /><br />
•Made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Streaked these in a new plate. <br /><br />
•Diluted plasmid E0080 2 1/50. <br /><br />
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<h2>08/10/12</h2><br /><br />
•Yesterday’s gel did not come out as expected so we repeated the PCR. <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/e/ef/UnamgenomicsandsugarBitacora_20.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.AraC+ Ω+AmyE 3’ <br /><br />
2-14.AraC+ Ω+AmyE 3’ <br /><br />
15.AraC+ Ω 1<br /><br />
16.AraC+ Ω 1<br /><br />
17.AraC+ Ω 1<br /><br />
18.PCR 1 GFP<br /><br />
19.PCR GFP<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<h2>08/13/12</h2><br /><br />
•After running a gel with 14 PCR’s AraC+ Ω+AmyE 3’ and it did not come out as expected, we ran a gel with the ligation AraC+ Ω+AmyE 3’ to select candidate (that weight the same as the ligation) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (20) <br /><br />
<br />
•Extracted pasmids form liquid cultures: B0049, B0079+GusA. <br /><br />
<br />
•AraC+ Ω+AmyE 3’ undigested didn’t run in the gel as expected, it ran similarly to AraC+ Ω<br /><br />
<br />
• We repeated the ligation AraC+ Ω S,P dephosphorylated+K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
• GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.<br />
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• We ran a gel to extract. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f1/UnamgenomicsandsugarBitacora_22.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>1.6 AraC+ Ω+AmyE 3’ E with P. <br /><br /><br />
2.8 AraC+ Ω+AmyE 3’ E with P. <br /><br />
3.11 AraC+ Ω+AmyE 3’ E with P. <br /><br />
4.12 AraC+ Ω+AmyE 3’ E with P. <br /><br />
5.6 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
6.8 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
7.11 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
8.12 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
9.00 pBad/pXyl with S,P. <br /><br />
10.Ladder. <br /><br />
11.E0040 PCR E,S to extract the correct band. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<br />
<h2>08/16/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: <br /><br />
<br />
•Digested: <br /><br />
ArSR-CzrA 97 with S,P<br /><br />
ArSR-CzrA 98 with S,P<br /><br />
ArSR-CzrA 99 with S,P<br /><br />
E0040 PCR with E,S<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
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<br />
<h2>08/20/12</h2><br /><br />
•Ligated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/21/12</h2><br /><br />
•Joined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated. <br /><br />
•Extracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR. <br /><br />
•Digested Ω+AmyE 3’ with E,X and + Ω+AmyE 3’ II with E,X (obtained these two from glycerols stored at -80ºC [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/29/UnamgenomicsandsugarBitacora_22.1.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested K143001+PBad, pXyl with E,S <br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/1c/UnamgenomicsandsugarBitacora_23.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>1.K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
4.A3 PCR<br /><br />
5.A3 PCR<br /><br />
6.E0040 PCR E,S<br /><br />
7.B0014 E,X dephosphorylated<br /><br />
8. Ω+AmyE 3’ E,X <br /><br />
9. Ω+AmyE 3’ E,X II<br /><br />
10.Ladder<br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/22/12</h2><br /><br />
•We did 2 PCR’s for A3. <br /><br />
<br />
•Digested K143001+PBad, pXyl with E,S <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (22.1) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did band extraction of: <br /><br />
1. K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
•Made liquid cultures from E1010+Boo14 that the other lab gave us and did 2 plates for glycerol Km+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Did 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ Ω+AmyE 3’ 6,8,11,12 Km+ and Spectinomycin+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Transformed ligations: <br /><br />
B0079+GusA Amp+<br /><br />
Pveg+XylR Chloramphenicol+<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]].. <br /><br />
<br /><br />
<br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/78/UnamgenomicsandsugarBitacora_23.01.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1. Ω+AmyE 3’ E,X 6.2<br /><br />
2.Ω+AmyE 3’ II E,X<br /><br />
3.97 ArsR-CzrA<br /><br />
4.98 ArsR-CzrA<br /><br />
5.99 ArsR-CzrA<br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/23/12</h2><br /><br />
•The negative controls were contaminated. We didn’t manipulate the ones that had grown because of this. <br /><br />
•Ran gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
<br />
<br />
<h2>08/24/12</h2><br /><br />
From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ Ω+AmyE 3’ 6 and 8 we streaked 2 plates for glycerol of each ligation. <br />Repeated ligation B0079+GusA. <br /><br />
<br />
Ligated E0040 PCR E,S+B0014 E,X dephospohylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
Made 2 new plates from Pveg+XylR ligation and liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>08/25/12</h2><br /><br />
Extracted pellet from 16 tubes of pVeg+XylR that were left overnight. <br /><br />
<br />
Ligated B0079+GusA and B0040+B0014 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/28/12</h2><br /><br />
<br />
Made liquid cultures from transformations tht were left overnight <br />
-R0079+GusA<br /><br />
-R0079+GusA (-)<br /><br />
-E0040+B0014<br /><br />
-E0040+B0014 (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/25/UnamgenomicsandsugarBitacora_23.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. A3 PCR 1. <br /><br />
2. A3 PCR 2. <br /><br />
3. Ω+AmyE 3’ S,P *.<br /><br />
4. Ω+AmyE 3’ S,P . <br /><br />
5. Pveg+XylR E,S 2. <br /><br />
6. Pveg+XylR E,S 3. <br /><br />
7. Pveg+XylR E,S 4. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/29/12</h2><br /><br />
Digested Pveg+XylR 2,3,4 with E,S. <br /><br />
<br />
Digested Ω+AmyE 3’ with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTION]]. <br /><br />
<br />
Ran a PCR with A3 PCR (2 .6 ml tubes). <br /><br />
<br />
Ran a gel with <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2e/UnamgenomicsandsugarBitacoraBitacora_24.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1-23. E0040 + B0014 colonies<br /><br />
24-29. B0079+GusA 1<br /><br />
30. B0079<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/30/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (24) <br /><br />
<br />
<br />
•Digested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/3d/UnamgenomicsandsugarBitacora_25.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1-3B0079+GusA E,S 3. <br /><br />
4-9. PVeg+XylR E,S 1. <br /><br />
10-17. E0040+B0014 X,P 3. <br /><br />
18. 1 kb ladder. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/d2/UnamgenomicsandsugarBitacora_26.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3 PCR. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7b/UnamgenomicsandsugarBitacora_27.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>E0040+B0014 X,P 4 to extract<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>08/31/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (25) <br /><br />
<br />
<br />
•Dephosphorylated: <br /><br />
Ω+AmyE 3’ S,P <br /><br />
Ω+AmyE 3’ II S,P <br /><br />
6 AraC+ Ω+AmyE 3’ E,X<br /><br />
8 AraC+ Ω+AmyE 3’ E,X<br /><br />
11 AraC+ Ω+AmyE 3’ E,X<br /><br />
12 AraC+ Ω+AmyE 3’ E,X<br /><br />•Ran a gel with A3 PCR [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (26) <br /><br />
<br />
•It was the second time we didn’t obtain a band from A3’s PCR.<br />
<br />
•Probably because it had been missing the buffer 2’s ethanol (“blue lid”) from the PCR purification. <br /><br />
<br />
•Ran a gel woth E0040+B0014 X,P 4 to extract <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | Gen extraction protocol]]. <br /><br />
<br />
•Did an A3 PCR. <br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=SEPTEMBER=<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b8/UnamgenomicsandsugarBitacora_28.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.A3 PCR 1. <br /><br />
2.A3 PCR 2. <br /><br />
3.E0040+B0014 X,P 1. <br /><br />
4.E0040+B0014 X,P 2. <br /><br />
5.Ω+AmyE 3’ dephosphorylated S,P. <br /><br />
6.Ω+AmyE 3’ dephosphorylated S,P II. <br /><br />
7.97 dephosphorylated S,P. <br /><br />
8.98 dephosphorylated S,P. <br /><br />
9.99 dephosphorylated S,P. <br /><br />
10-13. Arac+ Ω+AmyE 3’ dephosphorylated E,X 12. <br /><br />
14. 1 kb Ladder. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>09/01/12</h2><br /><br />
•Ran a gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (28) <br /><br />
<br />
•Ligated E0040+B0014 X,P 2 +promoter S,P dephosphorylated; K143002 X,P+ Ω+AmyE 3’ dephosphorylated S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<h2>09/03/12</h2><br /><br />
<br />
•Digested B0079+GusA E,S 3,4,5; PVeg+XylR E,S 1,5,7,10,13,16 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>09/04/12</h2><br /><br />
•Ran gel with digestions: <br /><br />
B0079+GusA E,S 3,4,5<br /><br />
PVeg+XylR E,S 1,5,7,10,13,16 <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Purified A3 PCR 1,2. <br /><br />
<br />
•Ran a gel of A3 PCR <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] and digested with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•From ligations: PVeg+E0040+B0014, PBad/PXyl+E0040+B0014, B0079+E0040+B0014 only colonies grew in PBad/PXyl+E0040+B0014.<br />
<br />
•Plated colonies that grew in a new plate. <br /><br />
<br />
•Made liquid cultures of these <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•Repeated the ones that did not grow. <br /><br />
<br />
•Digested E0040+B0014 4 with X,P[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
-BBa_J23101 18E plate 1 plasmid BBa_J61002_2948 bp Amp+<br /><br />
<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/c1/UnamgenomicsandsugarBitacora_29.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>24 PBad/PXyl+E0040+B0014 tubes we extracted <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/a/a4/UnamgenomicsandsugarBitacora_30.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
Extracted band from 1. and 2<br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7d/UnamgenomicsandsugarBitacora_31.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S <br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>09/05/12</h2><br /><br />
•From the 24 PBad/PXyl+E0040+B0014 tubes we extracted plasmids (29) and ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Extracted plasmid from 2 tubes of E0040+B0014. <br /><br />
<br />
•Ran a gel with yesterday’s digestions: <br /><br />
1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
<br />
•Extracted band from 1. and 2. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Purified A3 PCR from 2 0.6ml tubes 1 and 2. <br /><br />
<br />
•1 colony grew from ligation pVeg+E0040+B0014. <br /><br />
<br />
•We streaked this in another plate and did liquid cultures <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•To do: <br /><br />
•Ligate B0079 S,P dephosphorylated + E0040+B0014 X,P and Ω+AmyE 3’ S,P dephosphorylated+ K143001 X,P. <br /><br />
•Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S 10,13. (31) <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/26/UnamgenomicsandsugarBitacora_32.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>•A3 PCR and gel A3 PCR X,P <br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br /><br />
<br />
<h2>09/06/12</h2><br /><br />
<br />
•A3 PCR and gel A3 PCR X,P 1,2. <br /><br />
<br />
<br />
<h2>09/14/12</h2><br /><br />
<br />
•Digested Ω+AmyE 3’ 11,13,18, and 22 with E,X. <br /><br />
•Ligate: <br /><br />
PVeg S,P dephosphorylated+XylR X,P. <br /><br />
PBad p Xyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
pSB13C3 E,P dephosphorylated + A3 PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + GusA PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + XylR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pVeg E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pBad pXyl E,P. <br /><br />
pSB13C3 E,P dephosphorylated + Ω PCR E,P. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/ANDSugar
Team:UNAM Genomics Mexico/Notebook/ANDSugar
2012-10-27T03:16:05Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Arabinose/Xylose AND Gate Notebook'''</h1></center><br />
<br /><br />
<br /><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="20"><br />
<tr><br />
<td id="leftcolumn2"><center>2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JUNE | '''JUNE''']] </center> <br /><br />
<br /><br />
2.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F07.2F12 |06/07/12]] <br /><br />
2.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F12.2F12 |06/12/12]] <br /><br />
2.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F13.2F12 |06/13/12]] <br /><br />
2.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F14.2F12 |06/14/12]] <br /><br />
2.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F15.2F12 |06/15/12]] <br /><br />
2.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F18.2F12 |06/18/12]] <br /><br />
2.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F19.2F12 |06/19/12]] <br /><br />
2.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F22.2F12 |06/22/12]] <br /><br />
2.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F25.2F12 |06/25/12]] <br /><br />
2.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F26.2F12 |06/26/12]] <br /><br />
2.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F27.2F12 |06/27/12]] <br /><br />
2.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F29.2F12 |06/29/12]] <br /><br />
</td><br />
<br />
<td id="contentcolumn2"><center>3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JULY | '''JULY''']] </center> <br /><br />
<br /><br />
3.1[[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F03.2F12 | 07/03/12]] <br /><br />
3.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F04.2F12 | 07/04/12]] <br /><br />
3.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F06.2F12 | 07/06/12]] <br /><br />
3.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F07.2F12 | 07/07/12]] <br /><br />
3.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F08.2F12 | 07/08/12]] <br /><br />
3.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F09.2F12 | 07/09/12]] <br /><br />
3.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F10.2F12 | 07/10/12]] <br /><br />
3.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F11.2F12 | 07/11/12]] <br /><br />
3.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F12.2F12 | 07/12/12]] <br /><br />
3.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F13.2F12 | 07/13/12]] <br /><br />
3.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F14.2F12 | 07/14/12]] <br /><br />
3.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F16.2F12 | 07/16/12]] <br /><br />
3.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F17.2F12 | 07/17/12]] <br /><br />
3.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F18.2F12 | 07/18/12]] <br /><br />
3.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F19.2F12 | 07/19/12]] <br /><br />
3.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F20.2F12 | 07/20/12]] <br /><br />
3.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F23.2F12 | 07/23/12]] <br /><br />
3.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F24.2F12 | 07/24/12]] <br /><br />
3.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F25.2F12 | 07/25/12]] <br /><br />
3.20 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F26.2F12 | 07/26/12]] <br /><br />
3.21 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F27.2F12 | 07/27/12]] <br /><br />
3.22 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F30.2F12 | 07/30/12]] <br /><br />
3.23 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F31.2F12 | 07/31/12]] <br /><br />
</td><br />
<br />
<td id="rightcolumn2"><center>4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#AUGUST | '''AUGUST''']] </center> <br /><br />
<br /><br />
4.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F01.2F12 | 08/01/12]] <br /><br />
4.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F02.2F12 | 08/02/12]] <br /><br />
4.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F03.2F12 | 08/03/12]] <br /><br />
4.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F06.2F12 | 08/06/12]] <br /><br />
4.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F08.2F12 | 08/08/12]] <br /><br />
4.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F09.2F12 | 08/09/12]] <br /><br />
4.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F10.2F12 | 08/10/12]] <br /><br />
4.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F13.2F12 | 08/13/12]] <br /><br />
4.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F16.2F12 | 08/16/12]] <br /><br />
4.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F20.2F12 | 08/20/12]] <br /><br />
4.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F21.2F12 | 08/21/12]] <br /><br />
4.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F22.2F12 | 08/22/12]] <br /><br />
4.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F23.2F12 | 08/23/12]] <br /><br />
4.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F24.2F12 | 08/24/12]] <br /><br />
4.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F25.2F12 | 08/25/12]] <br /><br />
4.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F28.2F12 | 08/28/12]] <br /><br />
4.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F29.2F12 | 08/29/12]] <br /><br />
4.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F30.2F12 | 08/30/12]] <br /><br />
4.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F31.2F12 | 08/31/12]] <br /><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2"><center>5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#SEPTEMBER | '''SEPTEMBER''']] </center> <br /><br />
<br /><br />
5.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F01.2F12 | 09/01/12]] <br /><br />
5.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F02.2F12 | 09/03/12]] <br /><br />
5.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F04.2F12 | 09/04/12]] <br /><br />
5.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F05.2F12 | 09/05/12]] <br /><br />
5.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F06.2F12 | 09/06/12]] <br /><br />
5.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F14.2F12 | 09/14/12]] <br /><br />
<br />
</td><br />
<br />
<td id="contentcolumn2"><center>[[File:UnmagenomicsBacteria sugar.png | 190px]]</center><br />
</td><br />
<br />
<td id="rightcolumn2"><p> '''On hover the images to see descriptions'''</p><td/><br />
</tr><br />
</table><br />
<br />
<br />
=JUNE=<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html> <br />
<h2>06/07/12</h2><br /><br />
PY BROTH 10g salt/L<br /><br />
PSB2K3 kanamicin<br /><br />
We transformed RFP E1010. <br /><br />
plate 1 18F<br /><br />
plate 2 17E<br /><br />
Stock 50 mg/mL<br /><br />
''E.coli'' 1/1000<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
We made liquid cultures with colonies [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
We extracted plasmids [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PLASMID_EXTRACTION_2 | PLASMID EXTRACTION PROTOCOL]]. <br /><br />
We ran a gel to check the extraction [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<h2>06/12/12</h2><br /><br />
We made glycerols of the bacteria <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed plasmid pHp45 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/38/UnamgenomicsandsugarBitacora_2.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>06/13/12</h2><br /><br />
We ran gel and extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] and lysed [LYSIS PROTOCOL]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2d/UnamgenomicsandsugarBitacora_3.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
We extracted from gel<br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/14/12</h2><br /><br />
We extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made glycerols of pHp45 Ω [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450<br />
PSB4A5 Am (ampicillin) 1I BBa_J04450<br />
AraC BBa_C0080 <br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
2011 14L plate 1 pSB2K3 Km+<br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/5/56/UnamgenomicsandsugarBitacora_5.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
pHp45 Ω with E/P<br /><br />
We extracted from gel<br /><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/15/12</h2><br /><br />
> Digested pHp45 Ω with E/P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
> We extracted plasmid with kit. <br /><br />
> Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (5) <br /><br />
>Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br /><br />
ARAC<br /><br />
>From the overnight plate we prepared liquid culture [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
>We left them incubating overnight. <br /><br />
>We left a plate (LB Km DH5α C0080 and a control). <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<h2>06/18/12</h2><br /><br />
>Plasmid extraction AraC with kit. <br /><br />
>C0080-psB2K3 915 bp<br /><br />
>Streaked 4 LB Km 30 plates DH5α psB2K3. <br /><br />
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red. <br /><br />
>Ran gel with Spr/Strr (eppendorf -20ºC) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
>Made glycerols from one tube of DH5α C0080 Km 50 -> 04. <br /><br />
>Made glycerols from one tube of DH5α PSB4A5 Amp 100 -> 05 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]]. <br /><br />
>Digested C0080 X,S. <br /><br />
>Digested B0014 E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/5/52/UnamgenomicsandsugarBitacora_6.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
Digested B0014 with E, P and with E,X<br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<br /><br />
<br />
<h2>06/19/12</h2><br /><br />
>Digested B0014 with E, P and with E,X. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
>Ran gels with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation. <br /><br />
C0080 is in the first lane. <br /><br />
>Extracted from gel C0080 X,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Made glycerols from 4 plates LB Km DH5α pSB2K3 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
>Take out AmyE 5’ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
AmyE 5’ 18K plate 3 2010, 2011, 2012<br /><br />
AmyE 3’ 18M plate 3 2010, 2011, 2012<br /><br />
>Digested B0014 with E,X and E,P again [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<h2>06/22/12</h2><br /><br />
<br />
>Ran gels with digestions B0014 with E,X and E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
<br />
>Dephosphated B0014 E,X and B0014 E,P [DESPHOPHORYLATION PROTOCOL]. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<h2>06/25/12</h2><br /><br />
>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
<br />
<h2>06/26/12</h2><br /><br />
>Ran gel with psB2K3 and psB4A5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
<h2>06/27/12</h2><br /><br />
>Transformed with plasmid B0079 1576 bp psB1A2 12A [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011<br /><br />
AmyE 5’ BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011<br /><br />
AmyE 3’ BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011<br /><br />
AmyE 5’ grew 2 colonies. <br /><br />
<br />
<h2>06/29/12</h2><br /><br />
>We did a DH5α K143001 Km30 Amp 100 glycerol [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] 07. <br /><br />
>We plated twice DH5α K143002 Km 30 Amp 100 + control and twice DH5α B0079 Amp 100 + control<br /><br />
These were both plated in 2 plates each. <br /><br />
They were left overnight since they had not grown by 7:00 pm so the next day glycerols were made. <br /><br />
>Liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
2 tubes DH5α K143001 Km30 Amp 100 <br /><br />
2 tubes DH5α K143002 Km30 Amp 100 <br /><br />
2 tubes DH5α B0079 Amp 100 <br /><br />
1 tube LB Km 30 Amp 100 control<br /><br />
1 tube LB Amp 100 control<br /><br />
>From the 6 tubes we extracted plasmid from kit. <br /><br />
<br /><br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=JULY=<br />
<br />
07/02/12<br /><br />
>Digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /><br />
>PCR’s<br /><br />
•AraC<br /><br />
•Cassete ΩSpr/Strr <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PCR_Protocol_.2850.C2.B5l.29 | PCR PROTOCOL]] <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/be/UnamgenomicsandsugarBitacora_8.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>After Cassete ΩSpr/Strr PCR we ran a gel <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsandsugarBitacora_9.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/03/12</h2><br /><br />
>After Cassete ΩSpr/Strr PCR we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (8) <br /><br />
>Ran gel with digestions from yesterday. (9) <br /><br />
>Did band extractions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Stored at -20ºC. <br /><br />
>Did PCR to Cassete ΩSpr/Strr to add RBS and prefix/suffix [PCR PROTOCOL]. <br /><br />
>Left digesting with E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/03/UnamgenomicsandsugarBitacora_9.1.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/04/12</h2><br /><br />
>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1) [DESPHOPHORYLATION PROTOCOL]. <br /><br />
>Ligated Cassete ΩSpr/Strr +K143002 dephosphated(digested with E,X and E,S) LIGATION PROTOCOL]. <br /><br />
>Transformed ligation and left overnight plated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
>Digested with PstI [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<h2>07/06/12</h2><br /><br />
4 LB Km30 Spec60 DH5α ΩSpr/Strr +K143002 still have not grown. <br /><br />
Ran a gel with yesterday’s digestions to chek if they were done properly (10) <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
Ran another gel with the rest of the samples. <br /><br />
Extracted GusA fragment <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
Repeated ligation ΩSpr/Strr +K143002 E,X dephosphated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Repeated ΩSpr/Strr PCR. <br /><br />
<br /><br />
<br /><br />
<h2>07/07/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002 . <br /><br />
<br /><br />
<br /><br />
<br />
<h2>07/08/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002. <br /><br />
<br /><br />
<br /><br />
<br />
<br />
<html><br />
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<img src='https://static.igem.org/mediawiki/2012/1/13/UnamgenomicsandsugarBitacora_11.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
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<div class='captionnaranja'><br />
<p class='captionInside'>1 PCR omega P<br /><br />
2 PCR omega I<br /><br />
3 PCR AraC P<br /><br />
4 PCR AraC I<br /><br />
5 ladder 1 Kb<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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<h2>07/09/12</h2><br /><br />
From yesterday’s transformations only one colony grew. <br /><br />
From the previous transformation only 2 colonies grew. <br /><br />
These 3 were streaked in 3 plates: <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km 30 Sp 100 control. <br /><br />
Did liquid cultures in 3 tubes: <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km30 Sp 100 control. <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
Ran a gel with: (11) <br /><br />
GusA P<br /><br />
PBBR1 GusA<br /><br />
Ω E PCR P<br /><br />
Ω PCR P<br /><br />
Ω PCR I<br /><br />
Ω E<br /><br />
Ω PCR I<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] <br /><br />
Did GusA primers dissolution for PCR. <br /><br />
GusA PCR <br /><br />
[PCR PROTOCOL] <br /><br />
Gel Extraction by kit of lanes 3 and 5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
'''PCR omega P, PCR omega I, PCR AraC P, PCR AraC I'''<br /><br />
Add 10 μl of each primer (LW and UP). <br /><br />
Add 3 μl of plasmid (P). <br /><br />
Add 30.4 μl buffer. <br /><br />
Add 5 μl Mg. <br /><br />
Add 8 μl DNTp’s. <br /><br />
Add 42.6μl H2O miliQ. <br /> <br />
Add 1 μl RTTG polymerase. <br /><br />
Centrifuge (spin) 8 secs. <br /><br />
Add vegetable oil till the eppendorf is full. <br /><br />
Place eppendorf 1 mL in thermocycler. <br /><br />
Run PCR with program “BERNA”. <br /><br /><br />
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<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/4b/UnamgenomicsandsugarBitacora_11.2.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/10/12</h2><br /><br />
•GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S (2 times)[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTIONS]]. (11.2) <br /><br />
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<h2>07/11/12</h2><br /><br />
•GusA PCR digestions with E,S; Ω+AmyE 3’ with E,X [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•T.V. AraC PCR+ Ω+AmyE 3’ E,X dephosphated ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/0a/UnamgenomicsandsugarBitacora_12.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1. ladder. <br /><br />
2. GusA PCR E,S. <br /><br />
3. Ω+AmyE 3’ with E,X. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/12/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: (12) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Transformed AraC PCR+ Ω+AmyE 3’ ligation in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•From pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
•Made glycerols from 2 LB Km 30 Sp100 DH5α Ω+AmyE 3’ plates and from 1 LB Amp100 pfrc54 (A3) plates with control[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
•Ligated GusA PCR with B0014 E,X desphophorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
•Digested Pfrc54 (A3) with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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•Desphophorylated Ω+AmyE 3’ E,X [DESPHOPHORYLATION PROTOCOL]. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/0/0f/UnamgenomicsandsugarBitacora_13.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>•Ran gel with pfrc54 S,P<br /><br />
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</li><br />
</ul><br />
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<br />
<h2>07/13/12</h2><br /><br /><br />
•Ran gel with pfrc54 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (13) <br /><br />
•Transformation of GusA PCR + B0014 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Transformed with GFP E0040 psBIA2. <br /><br />
•Liquid Cultures with ligation: AraC+ Ω+AmyE 3’ for plasmid extraction at night [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•''B. Subtitils'' competent cells [''B.Subtilis'' group protocol]. <br /><br />
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<h2>07/14/12</h2><br /><br /><br />
•From 24 AraC+ Ω+AmyE 3’ LB Km 30 Sp 100 DH5α tubes: <br /><br />
•Extracted pellets<br /><br />
•Extracted plasmids [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
•Ran gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br /><br />
•From transformed DH5α km 30: <br /><br />
GusA+B0014 DH5α Km50 24 pellets <br /><br />
E0040 DH5α LB Amp100 <br /><br />
From these two we: <br /><br />
•Did liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Extracted plasmid [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
•From the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ran gel with this transformation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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<br /><br />
<br /><br />
<h2>07/16/12</h2><br /><br /><br />
•Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
•Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_14.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>07/17/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (14) <br /><br />
•The gel we ran didn’t work, probably because the agarose was not prepared correctly. <br /><br />
•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/db/Bitacora_15.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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<br /><br />
<br />
<h2>07/18/12</h2><br /><br /><br />
•From yesterday’s digestions we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (15) <br /><br /><br />
•We made liquid cultures of AraC+ Ω+AmyE 3’ 12, 15, 21 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br /><br />
•We ligated AraC PCR E,S dephosphated with + Ω+AmyE 3’ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
•We digested 1 + Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/19/12</h2><br /><br /><br />
•We ran We digested 1 + Ω+AmyE 3’ E,P with Miguel’s enzymes and with Abiel’s enzymes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
•We transformed with ligations AraC+ Ω+AmyE 3’ E and + Ω+AmyE 3’ E [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br /><br />
•Extracted plasmids from AraC+ Ω+AmyE 3’ 12,15,21 [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br /><br />
•Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<img src='https://static.igem.org/mediawiki/2012/f/f3/UnamgenomicsandsugarBitacora_16.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P <br /><br />
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</li><br />
</ul><br />
</div><br />
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<br />
<h2>07/20/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (16) <br /><br />
•Transformed GusA+B0014 in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Made liquid cultures from AraC+ Ω+AmyE 3’s transformation LB Km30 Sp 100 and control LB Km 30 Sp100 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<h2>07/23/12</h2><br /><br /><br />
•From 24 GusA+B0014 tubes (-) we didn’t do anything. <br /><br />
•From 4 AraC+ Ω+AmyE 3’ we extracted plasmid [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
•Digested from the 4 AraC+ Ω+AmyE 3’ E,X; X; P and GusA PCR with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<h2>07/24/12</h2><br /><br /><br />
•Digested B0014 E with X<br />
B0014 X with E<br /><br />
•Digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>07/25/12</h2><br /><br /><br />
•Ran gel of digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Joined B0014 E with X B0014 X with E digestions. <br /><br />
•Made liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. Transformed [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•From LasR DH5α make liquid cultures and plate [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Digested Ω+AmyE 3’ with X,P; E,S; C0080 X,P; E,S; S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Ligated Gus PCR X,P with B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/84/UnamgenomicsandsugarBitacora_17.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1.Ω+AmyE 3’ X,P<br /><br />
2. Ω+AmyE 3’ E,S<br /><br />
3.C0080 X,P<br /><br />
4.C0080 E,S<br /><br />
5.C0080 S,P<br /><br />
6.ladder<br /><br />
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</li><br />
</ul><br />
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<br />
<h2>07/26/12</h2><br /><br />
•From C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5α C0179 LB Cb 100 (two cultures) and a control without bacteria [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•We extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguel’s transformation), Puc, B.S. control and PSB4AE [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
<br />
•Due to problems with the way we did the transformations of ligations we repeated them: <br /><br />
GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
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GusAPCR X,P+ B0079 S,P dephosphorylated (-)<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
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GusAPCR X,P+ A3 S,P dephosphorylated (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•AraC+ Ω+AmyE 3’ S,P 1,2,3,4<br /><br />
•K143002 X,P<br /><br />
•AraC+ Ω S,P<br /><br />
•C0179 X,S<br /><br />
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<h2>07/27/12</h2><br /><br /><br />
Ligated AraC+ Ω S,P dephosphorylated + K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<h2>07/30/12</h2><br /><br /><br />
•Transformed with ligations: <br /><br />
•AraC+ Ω dephosphprylated +K143002 X,P<br /><br />
•GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
•GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
•Transformed the following sythesis: <br /><br />
•91996 Pveg 140 bp<br /><br />
•91997 ArsR-CzrA_promoter 1 194 bp<br /><br />
•91998 ArsR-CzrA_promoter 2 221 bp<br /><br />
•91999 ArsR-CzrA_promoter 3 213 bp<br /><br />
•92000 pBad-pXyl 387 bp<br /><br />
•92001 XylR 1117pb<br /><br />
•92002 CI_pro_(NAND_INHIBITOR) 774 <br /><br />
•These synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked.<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Make liquid cultures of the following transformations for tomorrow [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]: <br /><br />
•AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+B0079<br /><br />
•Synthesis<br /><br />
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<h2>07/31/12</h2><br /><br /><br />
Extracted plasmids from yesterday’s cultures and from K143002 cultures that were in the other lab [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
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::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
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=AUGUST=<br />
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<h2>08/01/12</h2><br /><br /><br />
Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
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<div class='captiongray'><br />
<p class='captionInside'>1 PCR GusA I<br /><br />
2 PCR GusA I<br /><br />
3 PCR GusA P<br /><br />
4 PCR GusA P<br /><br />
5 ladder 1 Kb<br /></p><br />
</li><br />
</ul><br />
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<br />
<h2>08/02/12</h2><br /><br /><br />
•Extracted plasmids from liquid cultures[PLASMID EXTRACTION (“soft” lysis) PROTOCOL]: <br /><br />
AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+BBR1<br /><br />
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PCR GusA, GusA I, PCR GusA P, PCR GusA I<br /><br />
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•Add 10 μl of each primer (LW and UP). <br /><br />
•Add 3 μl of plasmid (P). <br /><br />
•Add 30.4 μl buffer. <br /><br />
•Add 5 μl Mg. <br /><br />
•Add 8 μl DNTp’s. <br /><br />
•Add 42.6μl H2O miliQ. <br /><br />
•Add 1 μl RTTG polymerase. <br /><br />
•Centrifuge (spin) 8 secs. <br /><br />
•Add mineral oil till the eppendorf is full. <br /><br />
•Place eppendorf 1 mL in thermocycler. <br /><br />
•Run PCR with program “BERNA”. <br /><br />
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<div class='captionnaranja'><br />
<p class='captionInside'>1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.A3+GusA 1<br /><br />
4.A3+GusA 2<br /><br />
5.A3+GusA 3<br /><br />
6.P GusA<br /><br />
7.P GusA 2<br /><br />
8.Ladder<br /><br />
9.01<br /><br />
10.06<br /><br />
11.96<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<br /><br />
<br />
<h2>08/03/12</h2><br /><br /><br />
• Ran a gel with: (18) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Ran another gel to extract with: <br /><br />
1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.00<br /><br />
4.01<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]: <br /> <br />
<br />
• Ω+K143002 E,X; A3+GusA E,S; GusAPCR X,P <br /><br />
<br />
•Extracted GusAPCR X,P digestion [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Left the following ligation: AraC+ Ω+K143002 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<br /><br />
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<br /><br />
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<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/17/UnamgenomicsandsugarBitacora_19.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3+GusA 2<br /><br />
2.A3+GusA 3<br /><br />
3. Ω+AmyE 3’ 2<br /><br />
4. Ω+AmyE 3’ 3<br /><br />
5.XylR X,P<br /><br />
6.pBad-pXyl X,P<br /><br />
7.GusA PCR X,P<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/06/12</h2><br /><br /><br />
•Made liquid cultures of Ω+K143002 (2 cultures+1 control) and AraC+ Ω+K143002 (2 cultures+1 control) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ligated GusAPCR X,P+B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
•Ran a Gel with: (19) <br /><br />
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<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/8b/UnamgenomicsandsugarBitacora_19.1.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/08/12</h2><br /><br />
BBa_B0040 6I psB1A2 Amp+ plate 1<br /><br />
Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (19.1) <br /><br />
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<br /><br />
<br />
<h2>08/09/12</h2><br /><br />
•From yesterday’s PCR’s : <br /><br />
• E0040 plasmid 1<br /><br />
E0040 plasmid 2<br /><br />
E0040 digested 1<br /><br />
E0040 digested 2<br /><br />
We purified with PCR kit<br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Made 14 PCR’s from AraC+ Ω+AmyE 3’. <br /><br />
•From B0079+GusA ligation and B0040 transformation:<br />
•Grew colonies. <br /><br />
•Made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Streaked these in a new plate. <br /><br />
•Diluted plasmid E0080 2 1/50. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<h2>08/10/12</h2><br /><br />
•Yesterday’s gel did not come out as expected so we repeated the PCR. <br /><br />
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<br /><br />
<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/e/ef/UnamgenomicsandsugarBitacora_20.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.AraC+ Ω+AmyE 3’ <br /><br />
2-14.AraC+ Ω+AmyE 3’ <br /><br />
15.AraC+ Ω 1<br /><br />
16.AraC+ Ω 1<br /><br />
17.AraC+ Ω 1<br /><br />
18.PCR 1 GFP<br /><br />
19.PCR GFP<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<h2>08/13/12</h2><br /><br />
•After running a gel with 14 PCR’s AraC+ Ω+AmyE 3’ and it did not come out as expected, we ran a gel with the ligation AraC+ Ω+AmyE 3’ to select candidate (that weight the same as the ligation) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (20) <br /><br />
<br />
•Extracted pasmids form liquid cultures: B0049, B0079+GusA [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
<br />
•AraC+ Ω+AmyE 3’ undigested didn’t run in the gel as expected, it ran similarly to AraC+ Ω<br /><br />
<br />
• We repeated the ligation AraC+ Ω S,P dephosphorylated+K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
• GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.<br />
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• We ran a gel to extract. <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f1/UnamgenomicsandsugarBitacora_22.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>1.6 AraC+ Ω+AmyE 3’ E with P. <br /><br /><br />
2.8 AraC+ Ω+AmyE 3’ E with P. <br /><br />
3.11 AraC+ Ω+AmyE 3’ E with P. <br /><br />
4.12 AraC+ Ω+AmyE 3’ E with P. <br /><br />
5.6 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
6.8 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
7.11 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
8.12 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
9.00 pBad/pXyl with S,P. <br /><br />
10.Ladder. <br /><br />
11.E0040 PCR E,S to extract the correct band. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/16/12</h2><br /><br />
•Ran a gel with yesterday’s digestions [GEL ELECTROPHORESIS (22)PROTOCOL]: <br /><br />
<br />
•Digested: <br /><br />
ArSR-CzrA 97 with S,P<br /><br />
ArSR-CzrA 98 with S,P<br /><br />
ArSR-CzrA 99 with S,P<br /><br />
E0040 PCR with E,S<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
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<br /><br />
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<br />
<h2>08/20/12</h2><br /><br />
•Ligated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/21/12</h2><br /><br />
•Joined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated [DEPHOSPHORYLATION PROTOCOL]. <br /><br />
•Extracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
•Digested Ω+AmyE 3’ with E,X and + Ω+AmyE 3’ II with E,X (obtained these two from glycerols stored at -80ºC [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/29/UnamgenomicsandsugarBitacora_22.1.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested K143001+PBad, pXyl with E,S <br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/1c/UnamgenomicsandsugarBitacora_23.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>1.K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
4.A3 PCR<br /><br />
5.A3 PCR<br /><br />
6.E0040 PCR E,S<br /><br />
7.B0014 E,X dephosphorylated<br /><br />
8. Ω+AmyE 3’ E,X <br /><br />
9. Ω+AmyE 3’ E,X II<br /><br />
10.Ladder<br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/22/12</h2><br /><br />
•We did 2 PCR’s for A3 [PCR PROTOCOL]. <br /><br />
<br />
•Digested K143001+PBad, pXyl with E,S <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (22.1) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did band extraction of: <br /><br />
1. K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
•Made liquid cultures from E1010+Boo14 that the other lab gave us and did 2 plates for glycerol Km+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Did 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ Ω+AmyE 3’ 6,8,11,12 Km+ and Spectinomycin+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Transformed ligations: <br /><br />
B0079+GusA Amp+<br /><br />
Pveg+XylR Chloramphenicol+<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]].. <br /><br />
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<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/78/UnamgenomicsandsugarBitacora_23.01.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1. Ω+AmyE 3’ E,X 6.2<br /><br />
2.Ω+AmyE 3’ II E,X<br /><br />
3.97 ArsR-CzrA<br /><br />
4.98 ArsR-CzrA<br /><br />
5.99 ArsR-CzrA<br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/23/12</h2><br /><br />
•The negative controls were contaminated. We didn’t manipulate the ones that had grown because of this. <br /><br />
•Ran gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
<br />
<br />
<h2>08/24/12</h2><br /><br />
From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ Ω+AmyE 3’ 6 and 8 we streaked 2 plates for glycerol of each ligation. <br />Repeated ligation B0079+GusA. <br /><br />
<br />
Ligated E0040 PCR E,S+B0014 E,X dephospohylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
Made 2 new plates from Pveg+XylR ligation and liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>08/25/12</h2><br /><br />
Extracted pellet from 16 tubes of pVeg+XylR that were left overnight [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
<br />
Ligated B0079+GusA and B0040+B0014 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/28/12</h2><br /><br />
<br />
Made liquid cultures from transformations tht were left overnight <br />
-R0079+GusA<br /><br />
-R0079+GusA (-)<br /><br />
-E0040+B0014<br /><br />
-E0040+B0014 (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
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<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/25/UnamgenomicsandsugarBitacora_23.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. A3 PCR 1. <br /><br />
2. A3 PCR 2. <br /><br />
3. Ω+AmyE 3’ S,P *.<br /><br />
4. Ω+AmyE 3’ S,P . <br /><br />
5. Pveg+XylR E,S 2. <br /><br />
6. Pveg+XylR E,S 3. <br /><br />
7. Pveg+XylR E,S 4. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/29/12</h2><br /><br />
Digested Pveg+XylR 2,3,4 with E,S. <br /><br />
<br />
Digested Ω+AmyE 3’ with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTION]]. <br /><br />
<br />
Ran a PCR with A3 PCR (2 .6 ml tubes) [PCR PROTOCOL]. <br /><br />
<br />
Ran a gel with <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2e/UnamgenomicsandsugarBitacoraBitacora_24.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1-23. E0040 + B0014 colonies<br /><br />
24-29. B0079+GusA 1<br /><br />
30. B0079<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
<br />
<h2>08/30/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (24) <br /><br />
<br />
<br />
•Digested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='thumbnailWrapper'><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/3d/UnamgenomicsandsugarBitacora_25.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1-3B0079+GusA E,S 3. <br /><br />
4-9. PVeg+XylR E,S 1. <br /><br />
10-17. E0040+B0014 X,P 3. <br /><br />
18. 1 kb ladder. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/d2/UnamgenomicsandsugarBitacora_26.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3 PCR. <br /><br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7b/UnamgenomicsandsugarBitacora_27.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>E0040+B0014 X,P 4 to extract<br /><br />
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</li><br />
</ul><br />
</div><br />
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</html><br />
<h2>08/31/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (25) <br /><br />
<br />
<br />
•Dephosphorylated [DEPHOSPHORYLATION PROTOCOL]: <br /><br />
Ω+AmyE 3’ S,P <br /><br />
Ω+AmyE 3’ II S,P <br /><br />
6 AraC+ Ω+AmyE 3’ E,X<br /><br />
8 AraC+ Ω+AmyE 3’ E,X<br /><br />
11 AraC+ Ω+AmyE 3’ E,X<br /><br />
12 AraC+ Ω+AmyE 3’ E,X<br /><br />•Ran a gel with A3 PCR [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (26) <br /><br />
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•It was the second time we didn’t obtain a band from A3’s PCR.<br />
<br />
•Probably because it had been missing the buffer 2’s ethanol (“blue lid”) from the PCR purification. <br /><br />
<br />
•Ran a gel woth E0040+B0014 X,P 4 to extract [GEL (27) ELECTROPHORESIS PROTOCOL] <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | Gen extraction protocol]]. <br /><br />
<br />
•Did an A3 PCR [PCR PROTOCOL]. <br /><br />
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<br /><br />
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<br /><br />
<br />
<br /><br />
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<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=SEPTEMBER=<br />
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<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b8/UnamgenomicsandsugarBitacora_28.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.A3 PCR 1. <br /><br />
2.A3 PCR 2. <br /><br />
3.E0040+B0014 X,P 1. <br /><br />
4.E0040+B0014 X,P 2. <br /><br />
5.Ω+AmyE 3’ dephosphorylated S,P. <br /><br />
6.Ω+AmyE 3’ dephosphorylated S,P II. <br /><br />
7.97 dephosphorylated S,P. <br /><br />
8.98 dephosphorylated S,P. <br /><br />
9.99 dephosphorylated S,P. <br /><br />
10-13. Arac+ Ω+AmyE 3’ dephosphorylated E,X 12. <br /><br />
14. 1 kb Ladder. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
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</html><br />
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<br />
<h2>09/01/12</h2><br /><br />
•Ran a gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (28) <br /><br />
<br />
•Ligated E0040+B0014 X,P 2 +promoter S,P dephosphorylated; K143002 X,P+ Ω+AmyE 3’ dephosphorylated S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<h2>09/03/12</h2><br /><br />
<br />
•Digested B0079+GusA E,S 3,4,5; PVeg+XylR E,S 1,5,7,10,13,16 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>09/04/12</h2><br /><br />
•Ran gel with digestions: <br /><br />
B0079+GusA E,S 3,4,5<br /><br />
PVeg+XylR E,S 1,5,7,10,13,16 <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Purified A3 PCR 1,2. <br /><br />
<br />
•Ran a gel of A3 PCR <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] and digested with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•From ligations: PVeg+E0040+B0014, PBad/PXyl+E0040+B0014, B0079+E0040+B0014 only colonies grew in PBad/PXyl+E0040+B0014.<br />
<br />
•Plated colonies that grew in a new plate. <br /><br />
<br />
•Made liquid cultures of these <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•Repeated the ones that did not grow. <br /><br />
<br />
•Digested E0040+B0014 4 with X,P[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
-BBa_J23101 18E plate 1 plasmid BBa_J61002_2948 bp Amp+<br /><br />
<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/c1/UnamgenomicsandsugarBitacora_29.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>24 PBad/PXyl+E0040+B0014 tubes we extracted <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/a/a4/UnamgenomicsandsugarBitacora_30.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
Extracted band from 1. and 2<br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7d/UnamgenomicsandsugarBitacora_31.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S <br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>09/05/12</h2><br /><br />
•From the 24 PBad/PXyl+E0040+B0014 tubes we extracted plasmids (29) [PLASMID EXTRACTION (“soft” lysis) PROTOCOL] and ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Extracted plasmid from 2 tubes of E0040+B0014 [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
<br />
•Ran a gel with yesterday’s digestions [GEL ELECTROPHORESIS (30)PROTOCOL]: <br /><br />
1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
<br />
•Extracted band from 1. and 2. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Purified A3 PCR from 2 0.6ml tubes 1 and 2. <br /><br />
<br />
•1 colony grew from ligation pVeg+E0040+B0014. <br /><br />
<br />
•We streaked this in another plate and did liquid cultures <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•To do: <br /><br />
•Ligate B0079 S,P dephosphorylated + E0040+B0014 X,P and Ω+AmyE 3’ S,P dephosphorylated+ K143001 X,P. <br /><br />
•Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S 10,13. (31) <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/26/UnamgenomicsandsugarBitacora_32.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>•A3 PCR and gel A3 PCR X,P <br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br /><br />
<br />
<h2>09/06/12</h2><br /><br />
<br />
•A3 PCR and gel A3 PCR X,P 1,2 [PCR PROTOCOL] [GEL (32)ELECTROPHORESIS PROTOCOL]. <br /><br />
<br />
<br />
<h2>09/14/12</h2><br /><br />
<br />
•Digested Ω+AmyE 3’ 11,13,18, and 22 with E,X. <br /><br />
•Ligate: <br /><br />
PVeg S,P dephosphorylated+XylR X,P. <br /><br />
PBad p Xyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
pSB13C3 E,P dephosphorylated + A3 PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + GusA PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + XylR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pVeg E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pBad pXyl E,P. <br /><br />
pSB13C3 E,P dephosphorylated + Ω PCR E,P. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/ANDSugar
Team:UNAM Genomics Mexico/Notebook/ANDSugar
2012-10-27T03:14:55Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Arabinose/Xylose AND Gate Notebook'''</h1></center><br />
<br /><br />
<br /><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="20"><br />
<tr><br />
<td id="leftcolumn2"><center>2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JUNE | '''JUNE''']] </center> <br /><br />
<br /><br />
2.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F07.2F12 |06/07/12]] <br /><br />
2.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F12.2F12 |06/12/12]] <br /><br />
2.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F13.2F12 |06/13/12]] <br /><br />
2.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F14.2F12 |06/14/12]] <br /><br />
2.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F15.2F12 |06/15/12]] <br /><br />
2.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F18.2F12 |06/18/12]] <br /><br />
2.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F19.2F12 |06/19/12]] <br /><br />
2.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F22.2F12 |06/22/12]] <br /><br />
2.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F25.2F12 |06/25/12]] <br /><br />
2.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F26.2F12 |06/26/12]] <br /><br />
2.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F27.2F12 |06/27/12]] <br /><br />
2.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F29.2F12 |06/29/12]] <br /><br />
</td><br />
<br />
<td id="contentcolumn2"><center>3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JULY | '''JULY''']] </center> <br /><br />
<br /><br />
3.1[[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F03.2F12 | 07/03/12]] <br /><br />
3.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F04.2F12 | 07/04/12]] <br /><br />
3.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F06.2F12 | 07/06/12]] <br /><br />
3.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F07.2F12 | 07/07/12]] <br /><br />
3.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F08.2F12 | 07/08/12]] <br /><br />
3.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F09.2F12 | 07/09/12]] <br /><br />
3.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F10.2F12 | 07/10/12]] <br /><br />
3.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F11.2F12 | 07/11/12]] <br /><br />
3.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F12.2F12 | 07/12/12]] <br /><br />
3.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F13.2F12 | 07/13/12]] <br /><br />
3.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F14.2F12 | 07/14/12]] <br /><br />
3.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F16.2F12 | 07/16/12]] <br /><br />
3.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F17.2F12 | 07/17/12]] <br /><br />
3.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F18.2F12 | 07/18/12]] <br /><br />
3.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F19.2F12 | 07/19/12]] <br /><br />
3.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F20.2F12 | 07/20/12]] <br /><br />
3.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F23.2F12 | 07/23/12]] <br /><br />
3.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F24.2F12 | 07/24/12]] <br /><br />
3.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F25.2F12 | 07/25/12]] <br /><br />
3.20 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F26.2F12 | 07/26/12]] <br /><br />
3.21 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F27.2F12 | 07/27/12]] <br /><br />
3.22 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F30.2F12 | 07/30/12]] <br /><br />
3.23 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F31.2F12 | 07/31/12]] <br /><br />
</td><br />
<br />
<td id="rightcolumn2"><center>4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#AUGUST | '''AUGUST''']] </center> <br /><br />
<br /><br />
4.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F01.2F12 | 08/01/12]] <br /><br />
4.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F02.2F12 | 08/02/12]] <br /><br />
4.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F03.2F12 | 08/03/12]] <br /><br />
4.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F06.2F12 | 08/06/12]] <br /><br />
4.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F08.2F12 | 08/08/12]] <br /><br />
4.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F09.2F12 | 08/09/12]] <br /><br />
4.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F10.2F12 | 08/10/12]] <br /><br />
4.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F13.2F12 | 08/13/12]] <br /><br />
4.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F16.2F12 | 08/16/12]] <br /><br />
4.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F20.2F12 | 08/20/12]] <br /><br />
4.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F21.2F12 | 08/21/12]] <br /><br />
4.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F22.2F12 | 08/22/12]] <br /><br />
4.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F23.2F12 | 08/23/12]] <br /><br />
4.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F24.2F12 | 08/24/12]] <br /><br />
4.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F25.2F12 | 08/25/12]] <br /><br />
4.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F28.2F12 | 08/28/12]] <br /><br />
4.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F29.2F12 | 08/29/12]] <br /><br />
4.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F30.2F12 | 08/30/12]] <br /><br />
4.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F31.2F12 | 08/31/12]] <br /><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2"><center>5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#SEPTEMBER | '''SEPTEMBER''']] </center> <br /><br />
<br /><br />
5.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F01.2F12 | 09/01/12]] <br /><br />
5.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F02.2F12 | 09/03/12]] <br /><br />
5.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F04.2F12 | 09/04/12]] <br /><br />
5.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F05.2F12 | 09/05/12]] <br /><br />
5.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F06.2F12 | 09/06/12]] <br /><br />
5.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F14.2F12 | 09/14/12]] <br /><br />
<br />
</td><br />
<br />
<td id="contentcolumn2"><center>[[File:UnmagenomicsBacteria sugar.png | 190px]]</center><br />
</td><br />
<br />
<td id="rightcolumn2"><p> '''On hover the images to see descriptions'''</p><td/><br />
</tr><br />
</table><br />
<br />
<br />
=JUNE=<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html> <br />
<h2>06/07/12</h2><br /><br />
PY BROTH 10g salt/L<br /><br />
PSB2K3 kanamicin<br /><br />
We transformed RFP E1010. <br /><br />
plate 1 18F<br /><br />
plate 2 17E<br /><br />
Stock 50 mg/mL<br /><br />
''E.coli'' 1/1000<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
We made liquid cultures with colonies [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
We extracted plasmids [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PLASMID_EXTRACTION_2 | PLASMID EXTRACTION PROTOCOL]]. <br /><br />
We ran a gel to check the extraction [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<h2>06/12/12</h2><br /><br />
We made glycerols of the bacteria <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed plasmid pHp45 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/38/UnamgenomicsandsugarBitacora_2.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
2.E1010<br />
</div><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>06/13/12</h2><br /><br />
We ran gel and extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] and lysed [LYSIS PROTOCOL]. <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2d/UnamgenomicsandsugarBitacora_3.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
We extracted from gel<br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/14/12</h2><br /><br />
We extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br /><br />
We made glycerols of pHp45 Ω [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450<br />
PSB4A5 Am (ampicillin) 1I BBa_J04450<br />
AraC BBa_C0080 <br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
2011 14L plate 1 pSB2K3 Km+<br /><br />
2012 14L plate 1 pSB2K3 Km+<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/5/56/UnamgenomicsandsugarBitacora_5.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
pHp45 Ω with E/P<br /><br />
We extracted from gel<br /><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>06/15/12</h2><br /><br />
> Digested pHp45 Ω with E/P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
> We extracted plasmid with kit. <br /><br />
> Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (5) <br /><br />
>Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br /><br />
ARAC<br /><br />
>From the overnight plate we prepared liquid culture [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
>We left them incubating overnight. <br /><br />
>We left a plate (LB Km DH5α C0080 and a control). <br /><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<br /><br />
<br />
<br /><br />
<h2>06/18/12</h2><br /><br />
>Plasmid extraction AraC with kit. <br /><br />
>C0080-psB2K3 915 bp<br /><br />
>Streaked 4 LB Km 30 plates DH5α psB2K3. <br /><br />
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red. <br /><br />
>Ran gel with Spr/Strr (eppendorf -20ºC) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
>Made glycerols from one tube of DH5α C0080 Km 50 -> 04. <br /><br />
>Made glycerols from one tube of DH5α PSB4A5 Amp 100 -> 05 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]]. <br /><br />
>Digested C0080 X,S. <br /><br />
>Digested B0014 E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='captionrosa'><br />
<p class='captionInside'>1. 1 kb ladder <br /><br />
Digested B0014 with E, P and with E,X<br />
</li><br />
</ul><br />
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<br /><br />
<br /><br />
<br />
<h2>06/19/12</h2><br /><br />
>Digested B0014 with E, P and with E,X. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
>Ran gels with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation. <br /><br />
C0080 is in the first lane. <br /><br />
>Extracted from gel C0080 X,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Made glycerols from 4 plates LB Km DH5α pSB2K3 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
>Take out AmyE 5’ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
AmyE 5’ 18K plate 3 2010, 2011, 2012<br /><br />
AmyE 3’ 18M plate 3 2010, 2011, 2012<br /><br />
>Digested B0014 with E,X and E,P again [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>06/22/12</h2><br /><br />
<br />
>Ran gels with digestions B0014 with E,X and E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br /><br />
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>Dephosphated B0014 E,X and B0014 E,P [DESPHOPHORYLATION PROTOCOL]. <br /><br />
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<h2>06/25/12</h2><br /><br />
>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br /><br />
<br />
<h2>06/26/12</h2><br /><br />
>Ran gel with psB2K3 and psB4A5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
<h2>06/27/12</h2><br /><br />
>Transformed with plasmid B0079 1576 bp psB1A2 12A [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011<br /><br />
AmyE 5’ BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011<br /><br />
AmyE 3’ BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011<br /><br />
AmyE 5’ grew 2 colonies. <br /><br />
<br />
<h2>06/29/12</h2><br /><br />
>We did a DH5α K143001 Km30 Amp 100 glycerol [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] 07. <br /><br />
>We plated twice DH5α K143002 Km 30 Amp 100 + control and twice DH5α B0079 Amp 100 + control<br /><br />
These were both plated in 2 plates each. <br /><br />
They were left overnight since they had not grown by 7:00 pm so the next day glycerols were made. <br /><br />
>Liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
2 tubes DH5α K143001 Km30 Amp 100 <br /><br />
2 tubes DH5α K143002 Km30 Amp 100 <br /><br />
2 tubes DH5α B0079 Amp 100 <br /><br />
1 tube LB Km 30 Amp 100 control<br /><br />
1 tube LB Amp 100 control<br /><br />
>From the 6 tubes we extracted plasmid from kit. <br /><br />
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<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=JULY=<br />
<br />
07/02/12<br /><br />
>Digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /><br />
>PCR’s<br /><br />
•AraC<br /><br />
•Cassete ΩSpr/Strr <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PCR_Protocol_.2850.C2.B5l.29 | PCR PROTOCOL]] <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/be/UnamgenomicsandsugarBitacora_8.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>After Cassete ΩSpr/Strr PCR we ran a gel <br />
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</li><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsandsugarBitacora_9.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>B0079 digestion with S,P<br /><br />
K143001 with S,P<br /><br />
K143002 with S,P <br /> <br />
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</li><br />
</ul><br />
</div><br />
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<br />
<h2>07/03/12</h2><br /><br />
>After Cassete ΩSpr/Strr PCR we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (8) <br /><br />
>Ran gel with digestions from yesterday. (9) <br /><br />
>Did band extractions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
>Stored at -20ºC. <br /><br />
>Did PCR to Cassete ΩSpr/Strr to add RBS and prefix/suffix [PCR PROTOCOL]. <br /><br />
>Left digesting with E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<div class='captionazul'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
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</li><br />
</ul><br />
</div><br />
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<br />
<h2>07/04/12</h2><br /><br />
>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1) [DESPHOPHORYLATION PROTOCOL]. <br /><br />
>Ligated Cassete ΩSpr/Strr +K143002 dephosphated(digested with E,X and E,S) LIGATION PROTOCOL]. <br /><br />
>Transformed ligation and left overnight plated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
>Digested with PstI [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<h2>07/06/12</h2><br /><br />
4 LB Km30 Spec60 DH5α ΩSpr/Strr +K143002 still have not grown. <br /><br />
Ran a gel with yesterday’s digestions to chek if they were done properly (10) <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
Ran another gel with the rest of the samples. <br /><br />
Extracted GusA fragment <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
Repeated ligation ΩSpr/Strr +K143002 E,X dephosphated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Repeated ΩSpr/Strr PCR. <br /><br />
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<br /><br />
<h2>07/07/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002 . <br /><br />
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<br />
<h2>07/08/12</h2><br /><br />
Transformed DH5α ΩSpr/Strr +K143002 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/13/UnamgenomicsandsugarBitacora_11.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br /> <br />
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</li><br />
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</div><br />
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<img src='https://static.igem.org/mediawiki/2012/b/b2/UnamgenomicsandsugarBitacora_11.1.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1 PCR omega P<br /><br />
2 PCR omega I<br /><br />
3 PCR AraC P<br /><br />
4 PCR AraC I<br /><br />
5 ladder 1 Kb<br /><br />
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</li><br />
</ul><br />
</div><br />
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<h2>07/09/12</h2><br /><br />
From yesterday’s transformations only one colony grew. <br /><br />
From the previous transformation only 2 colonies grew. <br /><br />
These 3 were streaked in 3 plates: <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km 30 Sp 100 control. <br /><br />
Did liquid cultures in 3 tubes: <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 1. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 2. <br /><br />
ΩSpr/Strr +K143002 LB Km 30 Sp 100 3. <br /><br />
LB Km30 Sp 100 control. <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
Ran a gel with: (11) <br /><br />
GusA P<br /><br />
PBBR1 GusA<br /><br />
Ω E PCR P<br /><br />
Ω PCR P<br /><br />
Ω PCR I<br /><br />
Ω E<br /><br />
Ω PCR I<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] <br /><br />
Did GusA primers dissolution for PCR. <br /><br />
GusA PCR <br /><br />
[PCR PROTOCOL] <br /><br />
Gel Extraction by kit of lanes 3 and 5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
'''PCR omega P, PCR omega I, PCR AraC P, PCR AraC I'''<br /><br />
Add 10 μl of each primer (LW and UP). <br /><br />
Add 3 μl of plasmid (P). <br /><br />
Add 30.4 μl buffer. <br /><br />
Add 5 μl Mg. <br /><br />
Add 8 μl DNTp’s. <br /><br />
Add 42.6μl H2O miliQ. <br /> <br />
Add 1 μl RTTG polymerase. <br /><br />
Centrifuge (spin) 8 secs. <br /><br />
Add vegetable oil till the eppendorf is full. <br /><br />
Place eppendorf 1 mL in thermocycler. <br /><br />
Run PCR with program “BERNA”. <br /><br /><br />
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<img src='https://static.igem.org/mediawiki/2012/4/4b/UnamgenomicsandsugarBitacora_11.2.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S <br /><br />
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</li><br />
</ul><br />
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<br />
<h2>07/10/12</h2><br /><br />
•GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S (2 times)[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTIONS]]. (11.2) <br /><br />
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<h2>07/11/12</h2><br /><br />
•GusA PCR digestions with E,S; Ω+AmyE 3’ with E,X [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•T.V. AraC PCR+ Ω+AmyE 3’ E,X dephosphated ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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<div class='captionrojo'><br />
<p class='captionInside'>1. ladder. <br /><br />
2. GusA PCR E,S. <br /><br />
3. Ω+AmyE 3’ with E,X. <br /><br />
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</li><br />
</ul><br />
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<br />
<h2>07/12/12</h2><br /><br />
•Ran a gel with yesterday’s digestions: (12) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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•Transformed AraC PCR+ Ω+AmyE 3’ ligation in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
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•From pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
•Made glycerols from 2 LB Km 30 Sp100 DH5α Ω+AmyE 3’ plates and from 1 LB Amp100 pfrc54 (A3) plates with control[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
•Ligated GusA PCR with B0014 E,X desphophorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
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•Digested Pfrc54 (A3) with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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•Desphophorylated Ω+AmyE 3’ E,X [DESPHOPHORYLATION PROTOCOL]. <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/0/0f/UnamgenomicsandsugarBitacora_13.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>•Ran gel with pfrc54 S,P<br /><br />
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</li><br />
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<br />
<h2>07/13/12</h2><br /><br /><br />
•Ran gel with pfrc54 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (13) <br /><br />
•Transformation of GusA PCR + B0014 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Transformed with GFP E0040 psBIA2. <br /><br />
•Liquid Cultures with ligation: AraC+ Ω+AmyE 3’ for plasmid extraction at night [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•''B. Subtitils'' competent cells [''B.Subtilis'' group protocol]. <br /><br />
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<h2>07/14/12</h2><br /><br /><br />
•From 24 AraC+ Ω+AmyE 3’ LB Km 30 Sp 100 DH5α tubes: <br /><br />
•Extracted pellets<br /><br />
•Extracted plasmids [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
•Ran gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br /><br />
•From transformed DH5α km 30: <br /><br />
GusA+B0014 DH5α Km50 24 pellets <br /><br />
E0040 DH5α LB Amp100 <br /><br />
From these two we: <br /><br />
•Did liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Extracted plasmid [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
•From the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ran gel with this transformation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
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<h2>07/16/12</h2><br /><br /><br />
•Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
•Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_14.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>Digested E,P AraC+ Ω+AmyE 3’ <br /><br />
Ω+AmyE 3’ E,P <br /><br />
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</li><br />
</ul><br />
</div><br />
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<h2>07/17/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (14) <br /><br />
•The gel we ran didn’t work, probably because the agarose was not prepared correctly. <br /><br />
•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/db/Bitacora_15.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S<br /><br />
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</li><br />
</ul><br />
</div><br />
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<br />
<h2>07/18/12</h2><br /><br /><br />
•From yesterday’s digestions we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (15) <br /><br /><br />
•We made liquid cultures of AraC+ Ω+AmyE 3’ 12, 15, 21 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br /><br />
•We ligated AraC PCR E,S dephosphated with + Ω+AmyE 3’ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
•We digested 1 + Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/19/12</h2><br /><br /><br />
•We ran We digested 1 + Ω+AmyE 3’ E,P with Miguel’s enzymes and with Abiel’s enzymes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
•We transformed with ligations AraC+ Ω+AmyE 3’ E and + Ω+AmyE 3’ E [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br /><br />
•Extracted plasmids from AraC+ Ω+AmyE 3’ 12,15,21 [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br /><br />
•Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br /><br />
<br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f3/UnamgenomicsandsugarBitacora_16.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/20/12</h2><br /><br /><br />
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (16) <br /><br />
•Transformed GusA+B0014 in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•Made liquid cultures from AraC+ Ω+AmyE 3’s transformation LB Km30 Sp 100 and control LB Km 30 Sp100 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br /><br />
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<br /><br />
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<br /><br />
<br />
<br /><br />
<h2>07/23/12</h2><br /><br /><br />
•From 24 GusA+B0014 tubes (-) we didn’t do anything. <br /><br />
•From 4 AraC+ Ω+AmyE 3’ we extracted plasmid [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
•Digested from the 4 AraC+ Ω+AmyE 3’ E,X; X; P and GusA PCR with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<h2>07/24/12</h2><br /><br /><br />
•Digested B0014 E with X<br />
B0014 X with E<br /><br />
•Digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<h2>07/25/12</h2><br /><br /><br />
•Ran gel of digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Joined B0014 E with X B0014 X with E digestions. <br /><br />
•Made liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. Transformed [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
•From LasR DH5α make liquid cultures and plate [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Digested Ω+AmyE 3’ with X,P; E,S; C0080 X,P; E,S; S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•Ligated Gus PCR X,P with B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/84/UnamgenomicsandsugarBitacora_17.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1.Ω+AmyE 3’ X,P<br /><br />
2. Ω+AmyE 3’ E,S<br /><br />
3.C0080 X,P<br /><br />
4.C0080 E,S<br /><br />
5.C0080 S,P<br /><br />
6.ladder<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>07/26/12</h2><br /><br />
•From C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5α C0179 LB Cb 100 (two cultures) and a control without bacteria [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•We extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguel’s transformation), Puc, B.S. control and PSB4AE [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
<br />
•Due to problems with the way we did the transformations of ligations we repeated them: <br /><br />
GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
<br />
GusAPCR X,P+ B0079 S,P dephosphorylated (-)<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
<br />
GusAPCR X,P+ A3 S,P dephosphorylated (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
•AraC+ Ω+AmyE 3’ S,P 1,2,3,4<br /><br />
•K143002 X,P<br /><br />
•AraC+ Ω S,P<br /><br />
•C0179 X,S<br /><br />
<br />
<br />
<h2>07/27/12</h2><br /><br /><br />
Ligated AraC+ Ω S,P dephosphorylated + K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<h2>07/30/12</h2><br /><br /><br />
•Transformed with ligations: <br /><br />
•AraC+ Ω dephosphprylated +K143002 X,P<br /><br />
•GusAPCR X,P+ A3 S,P dephosphorylated<br /><br />
•GusAPCR X,P+ B0079 S,P dephosphorylated<br /><br />
•Transformed the following sythesis: <br /><br />
•91996 Pveg 140 bp<br /><br />
•91997 ArsR-CzrA_promoter 1 194 bp<br /><br />
•91998 ArsR-CzrA_promoter 2 221 bp<br /><br />
•91999 ArsR-CzrA_promoter 3 213 bp<br /><br />
•92000 pBad-pXyl 387 bp<br /><br />
•92001 XylR 1117pb<br /><br />
•92002 CI_pro_(NAND_INHIBITOR) 774 <br /><br />
•These synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked.<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
<br />
•Make liquid cultures of the following transformations for tomorrow [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]: <br /><br />
•AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+B0079<br /><br />
•Synthesis<br /><br />
<br />
<h2>07/31/12</h2><br /><br /><br />
Extracted plasmids from yesterday’s cultures and from K143002 cultures that were in the other lab [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
<br /><br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=AUGUST=<br />
<br />
<h2>08/01/12</h2><br /><br /><br />
Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br /><br />
<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b1/UnamgenomicsandsugarBitacora_17.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1 PCR GusA I<br /><br />
2 PCR GusA I<br /><br />
3 PCR GusA P<br /><br />
4 PCR GusA P<br /><br />
5 ladder 1 Kb<br /></p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/02/12</h2><br /><br /><br />
•Extracted plasmids from liquid cultures[PLASMID EXTRACTION (“soft” lysis) PROTOCOL]: <br /><br />
AraC+ Ω+K143002 <br /><br />
•GusA+A3<br /><br />
•GusA+BBR1<br /><br />
<br />
PCR GusA, GusA I, PCR GusA P, PCR GusA I<br /><br />
<br />
•Add 10 μl of each primer (LW and UP). <br /><br />
•Add 3 μl of plasmid (P). <br /><br />
•Add 30.4 μl buffer. <br /><br />
•Add 5 μl Mg. <br /><br />
•Add 8 μl DNTp’s. <br /><br />
•Add 42.6μl H2O miliQ. <br /><br />
•Add 1 μl RTTG polymerase. <br /><br />
•Centrifuge (spin) 8 secs. <br /><br />
•Add mineral oil till the eppendorf is full. <br /><br />
•Place eppendorf 1 mL in thermocycler. <br /><br />
•Run PCR with program “BERNA”. <br /><br />
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<br /><br />
<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/82/UnamgenomicsandsugarBitacora_18.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.A3+GusA 1<br /><br />
4.A3+GusA 2<br /><br />
5.A3+GusA 3<br /><br />
6.P GusA<br /><br />
7.P GusA 2<br /><br />
8.Ladder<br /><br />
9.01<br /><br />
10.06<br /><br />
11.96<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<br /><br />
<br />
<h2>08/03/12</h2><br /><br /><br />
• Ran a gel with: (18) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Ran another gel to extract with: <br /><br />
1.GusA PCR 1<br /><br />
2.GusA PCR 2<br /><br />
3.00<br /><br />
4.01<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]: <br /> <br />
<br />
• Ω+K143002 E,X; A3+GusA E,S; GusAPCR X,P <br /><br />
<br />
•Extracted GusAPCR X,P digestion [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Left the following ligation: AraC+ Ω+K143002 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br /><br />
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<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/17/UnamgenomicsandsugarBitacora_19.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3+GusA 2<br /><br />
2.A3+GusA 3<br /><br />
3. Ω+AmyE 3’ 2<br /><br />
4. Ω+AmyE 3’ 3<br /><br />
5.XylR X,P<br /><br />
6.pBad-pXyl X,P<br /><br />
7.GusA PCR X,P<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/06/12</h2><br /><br /><br />
•Made liquid cultures of Ω+K143002 (2 cultures+1 control) and AraC+ Ω+K143002 (2 cultures+1 control) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Ligated GusAPCR X,P+B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
•Ran a Gel with: (19) <br /><br />
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<br /><br />
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<br /><br />
<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/8/8b/UnamgenomicsandsugarBitacora_19.1.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/08/12</h2><br /><br />
BBa_B0040 6I psB1A2 Amp+ plate 1<br /><br />
Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (19.1) <br /><br />
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<br />
<h2>08/09/12</h2><br /><br />
•From yesterday’s PCR’s : <br /><br />
• E0040 plasmid 1<br /><br />
E0040 plasmid 2<br /><br />
E0040 digested 1<br /><br />
E0040 digested 2<br /><br />
We purified with PCR kit<br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Made 14 PCR’s from AraC+ Ω+AmyE 3’. <br /><br />
•From B0079+GusA ligation and B0040 transformation:<br />
•Grew colonies. <br /><br />
•Made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
•Streaked these in a new plate. <br /><br />
•Diluted plasmid E0080 2 1/50. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<h2>08/10/12</h2><br /><br />
•Yesterday’s gel did not come out as expected so we repeated the PCR. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/e/ef/UnamgenomicsandsugarBitacora_20.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.AraC+ Ω+AmyE 3’ <br /><br />
2-14.AraC+ Ω+AmyE 3’ <br /><br />
15.AraC+ Ω 1<br /><br />
16.AraC+ Ω 1<br /><br />
17.AraC+ Ω 1<br /><br />
18.PCR 1 GFP<br /><br />
19.PCR GFP<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<h2>08/13/12</h2><br /><br />
•After running a gel with 14 PCR’s AraC+ Ω+AmyE 3’ and it did not come out as expected, we ran a gel with the ligation AraC+ Ω+AmyE 3’ to select candidate (that weight the same as the ligation) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (20) <br /><br />
<br />
•Extracted pasmids form liquid cultures: B0049, B0079+GusA [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
<br />
•AraC+ Ω+AmyE 3’ undigested didn’t run in the gel as expected, it ran similarly to AraC+ Ω<br /><br />
<br />
• We repeated the ligation AraC+ Ω S,P dephosphorylated+K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
• GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.<br />
<br />
• We ran a gel to extract. <br /><br />
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<br /><br />
<br />
<br /><br />
<br />
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<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/f/f1/UnamgenomicsandsugarBitacora_22.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>1.6 AraC+ Ω+AmyE 3’ E with P. <br /><br /><br />
2.8 AraC+ Ω+AmyE 3’ E with P. <br /><br />
3.11 AraC+ Ω+AmyE 3’ E with P. <br /><br />
4.12 AraC+ Ω+AmyE 3’ E with P. <br /><br />
5.6 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
6.8 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
7.11 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
8.12 AraC+ Ω+AmyE 3’ with E,X. <br /><br />
9.00 pBad/pXyl with S,P. <br /><br />
10.Ladder. <br /><br />
11.E0040 PCR E,S to extract the correct band. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>08/16/12</h2><br /><br />
•Ran a gel with yesterday’s digestions [GEL ELECTROPHORESIS (22)PROTOCOL]: <br /><br />
<br />
•Digested: <br /><br />
ArSR-CzrA 97 with S,P<br /><br />
ArSR-CzrA 98 with S,P<br /><br />
ArSR-CzrA 99 with S,P<br /><br />
E0040 PCR with E,S<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
<br /><br />
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<br /><br />
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<br /><br />
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<br /><br />
<br />
<br /><br />
<br />
<br />
<h2>08/20/12</h2><br /><br />
•Ligated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/21/12</h2><br /><br />
•Joined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated [DEPHOSPHORYLATION PROTOCOL]. <br /><br />
•Extracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
•Digested Ω+AmyE 3’ with E,X and + Ω+AmyE 3’ II with E,X (obtained these two from glycerols stored at -80ºC [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/29/UnamgenomicsandsugarBitacora_22.1.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>Digested K143001+PBad, pXyl with E,S <br /> <br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/1/1c/UnamgenomicsandsugarBitacora_23.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>1.K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
4.A3 PCR<br /><br />
5.A3 PCR<br /><br />
6.E0040 PCR E,S<br /><br />
7.B0014 E,X dephosphorylated<br /><br />
8. Ω+AmyE 3’ E,X <br /><br />
9. Ω+AmyE 3’ E,X II<br /><br />
10.Ladder<br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/22/12</h2><br /><br />
•We did 2 PCR’s for A3 [PCR PROTOCOL]. <br /><br />
<br />
•Digested K143001+PBad, pXyl with E,S <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (22.1) <br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
•Did band extraction of: <br /><br />
1. K143001+PBad, pXyl E,S 10<br /><br />
2.K143001+PBad, pXyl E,S 11<br /><br />
3.K143001+PBad, pXyl E,S 12<br /><br />
<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
•Made liquid cultures from E1010+Boo14 that the other lab gave us and did 2 plates for glycerol Km+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Did 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ Ω+AmyE 3’ 6,8,11,12 Km+ and Spectinomycin+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br /><br />
<br />
•Transformed ligations: <br /><br />
B0079+GusA Amp+<br /><br />
Pveg+XylR Chloramphenicol+<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]].. <br /><br />
<br /><br />
<br />
<br /><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/78/UnamgenomicsandsugarBitacora_23.01.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>1. Ω+AmyE 3’ E,X 6.2<br /><br />
2.Ω+AmyE 3’ II E,X<br /><br />
3.97 ArsR-CzrA<br /><br />
4.98 ArsR-CzrA<br /><br />
5.99 ArsR-CzrA<br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/23/12</h2><br /><br />
•The negative controls were contaminated. We didn’t manipulate the ones that had grown because of this. <br /><br />
•Ran gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br /><br />
<br />
<br />
<h2>08/24/12</h2><br /><br />
From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ Ω+AmyE 3’ 6 and 8 we streaked 2 plates for glycerol of each ligation. <br />Repeated ligation B0079+GusA. <br /><br />
<br />
Ligated E0040 PCR E,S+B0014 E,X dephospohylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
Made 2 new plates from Pveg+XylR ligation and liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>08/25/12</h2><br /><br />
Extracted pellet from 16 tubes of pVeg+XylR that were left overnight [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
<br />
Ligated B0079+GusA and B0040+B0014 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br />
<h2>08/28/12</h2><br /><br />
<br />
Made liquid cultures from transformations tht were left overnight <br />
-R0079+GusA<br /><br />
-R0079+GusA (-)<br /><br />
-E0040+B0014<br /><br />
-E0040+B0014 (-)<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/25/UnamgenomicsandsugarBitacora_23.1.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1. A3 PCR 1. <br /><br />
2. A3 PCR 2. <br /><br />
3. Ω+AmyE 3’ S,P *.<br /><br />
4. Ω+AmyE 3’ S,P . <br /><br />
5. Pveg+XylR E,S 2. <br /><br />
6. Pveg+XylR E,S 3. <br /><br />
7. Pveg+XylR E,S 4. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/29/12</h2><br /><br />
Digested Pveg+XylR 2,3,4 with E,S. <br /><br />
<br />
Digested Ω+AmyE 3’ with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTION]]. <br /><br />
<br />
Ran a PCR with A3 PCR (2 .6 ml tubes) [PCR PROTOCOL]. <br /><br />
<br />
Ran a gel with <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/2e/UnamgenomicsandsugarBitacoraBitacora_24.jpg' height="300"/><br />
<div class='captiongray'><br />
<p class='captionInside'>1-23. E0040 + B0014 colonies<br /><br />
24-29. B0079+GusA 1<br /><br />
30. B0079<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>08/30/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (24) <br /><br />
<br />
<br />
•Digested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/3/3d/UnamgenomicsandsugarBitacora_25.jpg' height="300"/><br />
<div class='captionnaranja'><br />
<p class='captionInside'>1-3B0079+GusA E,S 3. <br /><br />
4-9. PVeg+XylR E,S 1. <br /><br />
10-17. E0040+B0014 X,P 3. <br /><br />
18. 1 kb ladder. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/d/d2/UnamgenomicsandsugarBitacora_26.jpg' height="300"/><br />
<div class='captionrojo'><br />
<p class='captionInside'>1.A3 PCR. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7b/UnamgenomicsandsugarBitacora_27.jpg' height="300"/><br />
<div class='captionverde'><br />
<p class='captionInside'>E0040+B0014 X,P 4 to extract<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<h2>08/31/12</h2><br /><br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (25) <br /><br />
<br />
<br />
•Dephosphorylated [DEPHOSPHORYLATION PROTOCOL]: <br /><br />
Ω+AmyE 3’ S,P <br /><br />
Ω+AmyE 3’ II S,P <br /><br />
6 AraC+ Ω+AmyE 3’ E,X<br /><br />
8 AraC+ Ω+AmyE 3’ E,X<br /><br />
11 AraC+ Ω+AmyE 3’ E,X<br /><br />
12 AraC+ Ω+AmyE 3’ E,X<br /><br />•Ran a gel with A3 PCR [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (26) <br /><br />
<br />
•It was the second time we didn’t obtain a band from A3’s PCR.<br />
<br />
•Probably because it had been missing the buffer 2’s ethanol (“blue lid”) from the PCR purification. <br /><br />
<br />
•Ran a gel woth E0040+B0014 X,P 4 to extract [GEL (27) ELECTROPHORESIS PROTOCOL] <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | Gen extraction protocol]]. <br /><br />
<br />
•Did an A3 PCR [PCR PROTOCOL]. <br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#Arabinose.2FXylose_AND_Gate_Notebook]]<br />
<br />
=SEPTEMBER=<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/b/b8/UnamgenomicsandsugarBitacora_28.jpg' height="300"/><br />
<div class='captionrosa'><br />
<p class='captionInside'>1.A3 PCR 1. <br /><br />
2.A3 PCR 2. <br /><br />
3.E0040+B0014 X,P 1. <br /><br />
4.E0040+B0014 X,P 2. <br /><br />
5.Ω+AmyE 3’ dephosphorylated S,P. <br /><br />
6.Ω+AmyE 3’ dephosphorylated S,P II. <br /><br />
7.97 dephosphorylated S,P. <br /><br />
8.98 dephosphorylated S,P. <br /><br />
9.99 dephosphorylated S,P. <br /><br />
10-13. Arac+ Ω+AmyE 3’ dephosphorylated E,X 12. <br /><br />
14. 1 kb Ladder. <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<br />
<h2>09/01/12</h2><br /><br />
•Ran a gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (28) <br /><br />
<br />
•Ligated E0040+B0014 X,P 2 +promoter S,P dephosphorylated; K143002 X,P+ Ω+AmyE 3’ dephosphorylated S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<h2>09/03/12</h2><br /><br />
<br />
•Digested B0079+GusA E,S 3,4,5; PVeg+XylR E,S 1,5,7,10,13,16 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
<br />
<h2>09/04/12</h2><br /><br />
•Ran gel with digestions: <br /><br />
B0079+GusA E,S 3,4,5<br /><br />
PVeg+XylR E,S 1,5,7,10,13,16 <br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Purified A3 PCR 1,2. <br /><br />
<br />
•Ran a gel of A3 PCR <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] and digested with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•From ligations: PVeg+E0040+B0014, PBad/PXyl+E0040+B0014, B0079+E0040+B0014 only colonies grew in PBad/PXyl+E0040+B0014.<br />
<br />
•Plated colonies that grew in a new plate. <br /><br />
<br />
•Made liquid cultures of these <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•Repeated the ones that did not grow. <br /><br />
<br />
•Digested E0040+B0014 4 with X,P[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
<br />
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
<br />
•Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
-BBa_J23101 18E plate 1 plasmid BBa_J61002_2948 bp Amp+<br /><br />
<br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/c1/UnamgenomicsandsugarBitacora_29.jpg' height="300"/><br />
<div class='captionaqua'><br />
<p class='captionInside'>24 PBad/PXyl+E0040+B0014 tubes we extracted <br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/a/a4/UnamgenomicsandsugarBitacora_30.jpg' height="300"/><br />
<div class='captionmoradoclaro'><br />
<p class='captionInside'>1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
Extracted band from 1. and 2<br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/7/7d/UnamgenomicsandsugarBitacora_31.jpg' height="300"/><br />
<div class='captionmorado'><br />
<p class='captionInside'>Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S <br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br />
<h2>09/05/12</h2><br /><br />
•From the 24 PBad/PXyl+E0040+B0014 tubes we extracted plasmids (29) [PLASMID EXTRACTION (“soft” lysis) PROTOCOL] and ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Extracted plasmid from 2 tubes of E0040+B0014 [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
<br />
•Ran a gel with yesterday’s digestions [GEL ELECTROPHORESIS (30)PROTOCOL]: <br /><br />
1.E0040+B0014 X,P<br /><br />
2.E0040+B0014 X,P<br /><br />
3.PVeg+XylR E<br /><br />
4.B0079+GusA S<br /><br />
5.1 kb plus ladder<br /><br />
<br />
•Extracted band from 1. and 2. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br /><br />
<br />
•Purified A3 PCR from 2 0.6ml tubes 1 and 2. <br /><br />
<br />
•1 colony grew from ligation pVeg+E0040+B0014. <br /><br />
<br />
•We streaked this in another plate and did liquid cultures <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
<br />
•To do: <br /><br />
•Ligate B0079 S,P dephosphorylated + E0040+B0014 X,P and Ω+AmyE 3’ S,P dephosphorylated+ K143001 X,P. <br /><br />
•Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S 10,13. (31) <br /><br />
<br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/2/26/UnamgenomicsandsugarBitacora_32.jpg' height="300"/><br />
<div class='captionazul'><br />
<p class='captionInside'>•A3 PCR and gel A3 PCR X,P <br />
<br /><br />
</p><br />
</li><br />
</ul><br />
</div><br />
<br /><br /><br />
</html><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<h2>09/06/12</h2><br /><br />
<br />
•A3 PCR and gel A3 PCR X,P 1,2 [PCR PROTOCOL] [GEL (32)ELECTROPHORESIS PROTOCOL]. <br /><br />
<br />
<br />
<h2>09/14/12</h2><br /><br />
<br />
•Digested Ω+AmyE 3’ 11,13,18, and 22 with E,X. <br /><br />
•Ligate: <br /><br />
PVeg S,P dephosphorylated+XylR X,P. <br /><br />
PBad p Xyl S,P dephosphorylated + E0040+B0014 X,P. <br /><br />
pSB13C3 E,P dephosphorylated + A3 PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + GusA PCR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + XylR E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pVeg E,P. <br /><br />
pSB13C3 E,P dephosphorylated + pBad pXyl E,P. <br /><br />
pSB13C3 E,P dephosphorylated + Ω PCR E,P. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
<br />
<br /><br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling/Sweet_AND
Team:UNAM Genomics Mexico/Modeling/Sweet AND
2012-10-27T03:08:14Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<center><h1>'''AND's Results'''</h1></center> <br />
<br /><br />
<br /><br />
<h1>'''Sweet AND'''</h1> <br />
<br /><br />
<br /><br />
<br />
Xylose and arabinose are sugars used by bacteria as carbon sources. XylR and AraC are<br />
transcriptional regulators modulated by xylose and arabinose. For xylose sensing, our hybrid promoter<br />
has as backbone made of the pBAD promoter plus a binding site for XylR. XylR represses <br />
transcription until xylose is present in the cell, a process similar to the metal sensor of ArsR and CzrA. As for arabinose, AraC represses transcription through DNA looping. When arabinose enters the cell, it dissolves the loop and transcription can take place. We conclude that arabinose and xylose are essential for the expression of genes under the pBAD/xylR promoter, thus acting as an AND Boolean operation. <br/><br/><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UNAM_GENOMICS_MEXICO_AND_bf_sweet.png]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
'''Repressors'''<br/><br/><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align= "center"><br />[[File:UGM_tv_stf.png]]<br /><br /><p><br />
</p></td><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UNAM GENOMICS MEXICO Heavy metal and2.jpg| 300px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
'''Inputs'''<br/><br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align= "center"><br />[[File:UGM_tv_si.png]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
The behavior of both ANDs predicted by our model is consistent with what we expected. From the perspective of the repressors, a higher level of PoPs (Polymerase Per Second, which is a measurement of the level of transcription) is reached when both repressor concentrations are 0. Reciprocally, PoPs start to fall when the concentration of repressors increases, reaching the highest level of repression (lower PoPs) at high concentration of both repressors. This behavior reminds us of the way a NOR behaves, because at low levels of repressor PoPs is high, but for repression, high concentration of one repressor is enough. However, we can't control (directly) the concentration of repressors, so the right way to look at the system is from the perspective of the Input: the metals or the sugars. <br/><br/><br />
If we looked PoPs as a function of the Input, it behaves as an AND logic gate, as can be seen in the continuous TRUTH TABLE. At lower concentrations of Inputs, the PoPs level is in a very repressed stage. If you keep increasing only one of the Inputs on only one of the axes (yes, axes is the plural of axis), there won’t be high activation until the addition of high concentrations on the other axis. PoPs will increase as the Input concentration increases in a direct proportion fashion. <br/><br/><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Modeling/Sweet_AND#Sweet_AND]]<br />
<h2>'''KAPPA'''</h2><br/><br/><br />
<br />
Sometimes people wonder why we used Kappa, a stochastic approach, to model our system, which was also described by deterministic differential equations. First of all, Kappa helped us come up with the concentration ranges entered in the differential equations (here, an exclamation of awe is usually heard, and with good reason). How did this come to happen? Well, the simulation in kappa is driven by the binding events and is dependent on the species concentrations, so using general kinetic rates we were able to see the dynamic behavior given by the architecture of the network. This allowed us to approximate the final species concentration and look for parameters missing in our ODE system, which was something useful, since we were as lost as a three-legged dog in a rodeo dance. <br/><br/><br />
So, first we ran the [https://static.igem.org/mediawiki/2012/f/fd/And_sugar.txt And Sugar kappa script] shifting the sugar initial concentrations in the single input burst and getting the average of 100 iterations to eliminate the noise in the simulation. After running some simulation time, we were happy to see that our "Sweet AND" behaved as expected, that is, as an AND. Besides, we were able to see the maximal concentration of the protein that would be downstream of our AND, and the time it took to reach it. As you can see, our simulation grid is not described in its entirety. This is because kappa can be computationally very expensive for non-atomic rules, so we chose to feed our ODE system with some parameters and scan the system in a broader manner instead of dying of old age waiting for Kappa (if only we had a supercluster to run things in...). Nevertheless, some behaviors can only be noticed by looking at the simulation grid. For example, the activation of P4 depending on the xylose and arabinose concentrations. As it becomes apparent through a meticulous and insightful analysis only doable by a really smart, handsome, and admirable people, the activation of P4 depends on the concentrations of both, xylose and arabinose. When only one of the two sugars has a high concentration, the activation is not as elevated as when the two have high concentrations. <br/><br/><br />
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<table border="1" cellpadding="2px" width="100%" height="600px"><br />
<tr><br />
<td rowspan="7">Xylose</td><br />
<td>500,000</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/0/08/UNAM-Genomics_Mexico_Ara500_xyl500.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/0/08/UNAM-Genomics_Mexico_Ara500_xyl500.png" alt="large" /><br />
</span></a></div></td><br />
</tr><br />
<br />
<tr><br />
<td>350,000</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/a/aa/UNAM-Genomics_Mexico_Ara100_xyl350.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/a/aa/UNAM-Genomics_Mexico_Ara100_xyl350.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>100,000</td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/7/7a/UNAM-Genomics_Mexico_Ara0_xyl100.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/7/7a/UNAM-Genomics_Mexico_Ara0_xyl100.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/9/97/UNAM-Genomics_Mexico_Ara100_xyl100.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/9/97/UNAM-Genomics_Mexico_Ara100_xyl100.png" alt="large" /></span></a></div></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/7/7c/UNAM-Genomics_Mexico_Ara350_xyl100.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/7/7c/UNAM-Genomics_Mexico_Ara350_xyl100.png" alt="large" /></span></a></div></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>75,000</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/c/c3/UNAM-Genomics_Mexico_Ara75_xyl75.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UNAM-Genomics_Mexico_Ara75_xyl75.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>50,000</td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/5/58/UNAM-Genomics_Mexico_Ara0_xyl50.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/5/58/UNAM-Genomics_Mexico_Ara0_xyl50.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/8/83/UNAM-Genomics_Mexico_Ara50_xyl50.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/8/83/UNAM-Genomics_Mexico_Ara50_xyl50.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>25,000</td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/6/6f/UNAM-Genomics_Mexico_Ara25_xyl25.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/6/6f/UNAM-Genomics_Mexico_Ara25_xyl25.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>0.0</td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/4/4e/UNAM-Genomics_Mexico_Ara0_xyl0.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/4/4e/UNAM-Genomics_Mexico_Ara0_xyl0.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/6/6d/UNAM-Genomics_Mexico_Ara50_xyl0.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/UNAM-Genomics_Mexico_Ara50_xyl0.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/4/4f/UNAM-Genomics_Mexico_Ara100_xyl0.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/4/4f/UNAM-Genomics_Mexico_Ara100_xyl0.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td></td><br />
<td></td><br />
<td>0.0</td><br />
<td>25,000</td><br />
<td>50,000</td><br />
<td>75,000</td><br />
<td>100,000</td><br />
<td>350,000</td><br />
<td height="14px">500,000</td><br />
</tr><br />
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<td></td><br />
<td colspan="7" height="14px">Arabinose</td><br />
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<br />
We can focus only in the dynamics of the downstream protein, instead of the dynamics of the whole system. This plot describes the time to reach steady state and maximal concentration of the AND downstream protein. It also describes what the maximal concentration of the AND protein is. All of this is based on a single input burst concentration.<br />
<br/><br/><br />
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<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:UNAM_GENOMICS_MEXICO_AND_downProt.png]]<br /><br /><p><br />
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</center><br />
<br/><br/><br />
<br />
<h2>'''Kind of input'''</h2><br/><br/><br />
<br />
<br />
The Endogenous system and Downstream protein dynamics is dependent on the kind of input. The inputs used by each AND have a unique effect in the response of the cell, because carbohydrates are degraded by the cell. When we add sugars to the sweet AND system, if they are added just like a discrete and singular dose, the cell will react to the alteration through the activation of the degradation machinery, and when the sugar pool gets empty, the cell achieves a similar state to the one before the sugar was introduced, that is, a return to pre-induction steady state. When the sugar addition is constant, the cell has a complete phenotypic change, reaching a new steady state.<br/><br/><br />
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<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:500ara_500xyl.png | 400px]]<br /><br /><p><br />
</p></td><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Continuando.png | 400px]]<br /><br /><p><br />
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</center><br />
<br/><br/><br />
<br />
<h2>'''Constitutive VS NO-Constitutive XylR expression'''</h2><br/><br/><br />
<br />
It has been reported that xylR has to be in high concentrations in order to repress in an effective manner. So we decided to look the dynamics of the AND with constitutive expression (The endogenous expression plus the pveg constitutive expression) of xylR vs No-Constitutive expression of xylR (just the endogenous expression). Our simulation says that for a finest logic behaviour it is needed the constitutive expression. Because when we compare the dynamics of the downstream protein (pink lines label with P4) when we add a single input burst of 100,000 arabinose molecules, the active araC concentration is depleted, so all the repression is given by xylR. But in the NO-constitutive expression AND the concentration of P4 goes significant higher than the AND with the constitutive expression. So we decided to express xylR constitutively in our constructions<br/><br/><br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:UNAM-Genomics_Mexico_Ara100_xyl0.png | 400px]]<br /><br /><p><br />
</p></td><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:NOCONSTIT.png | 400px]]<br /><br /><p><br />
</p></td><br />
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</center><br />
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::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Modeling/Sweet_AND#Sweet_AND]]<br />
<br />
== '''Transfer Function''' ==<br />
<br />
<br />
Our logic AND gates are fully dependent on the intracellular concentrations of the inducers (in this case, xylose and arabinose). To accomplish our duty in model, we therefore make use of the "Transfer Function", which requires taking into account the regulation and dynamics of the endogenous Bacillus subtilis influx-efflux intracellular sugar regulation system. The "Transfer Function" is going to be the "Black Box" that will give you the dynamic<br />
concentration through time of whatever is downstream of the "AND" when you<br />
feed it with input data. It doesn't matter if it’s a single input burst or a<br />
continuous input.<br />
<br/><br />
<br/><br />
So, our first task was to reconstruct the regulatory network of B. subtilis for the influx-efflux system. The regulation data was retrieved from several papers and databases like [http://bsubcyc.org/ http://bsubcyc.org/]<br />
<br/><br/><br />
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<br />
<center><br />
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<td id="rightcolumn2" align= "center"><br />[[File:UNAM_GENOMICS_MEXICO_Sweet_AND.png | 600px]]<br /><br /><p>Sweet AND gate: Arabinose and xylose are imported into the cell by the araE permease. The<br />
carbon metabolism system is highly regulated in order to optimize the consumption of sugars<br />
using the least amount of energy. XylR and AraR are repressors responsible for the regulation of genes involved in the metabolism and intake of xylose and arabinose, respectively. Although XylR is not well characterized, it’s hypothesized that XylR represses itself; some studies suggest a correlation between XylR and the concentration of xylose inside the cell. AraR not only represses the production of genes like araE or araA, but it also represses itself. AraA and xylA are isomerases that convert L-arabinose into L-ribulose and D-xylose into D-xylulose. XylR from B.subtilis is also used by our construction as a repressor. Under that constitutive promoter, AraC is also produced for the regulation of the hybrid promoter. The output of the AND is the production of a transcription factor, LasR or P4, and RFP.<br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:UNAM_GENOMICS_MEXICO_AND_3D.png | 850px]]<br /><br /><p><br />
</p></td><br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:UNAM_GENOMICS_MEXICO_AND_3D_3D.png | 850px]]<br /><br /><p><br />
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<br />
<br />
One observation worthy of mention came when we realized that the<br />
system was most repressed when there was scarce xylose in the system, not<br />
when there was none. On the contrary, when there is no xylose in the system,<br />
there is a very small expression gradient dependent on the concentration of<br />
arabinose, yet this gradient does not surpass a threshold where we could<br />
consider the AND as "ON".<br/><br/><br />
<br />
We are coupling an endogenous and an exogenous repressor to our AND. This<br />
feature can easily be explained by the fact that we are expressing the<br />
endogenous repressor XylR in a constitutive manner. This is due to the fact that<br />
it has been reported that it has to be in high concentrations in order to repress<br />
in an effective manner. When we add xylose to the medium, endogenous XylR<br />
gene increases its expression. This makes<br />
repression against the AND increase when xylose is found in the medium in<br />
small amounts. After the repression threshold is surpassed, xylose stops being<br />
a repressor and shifts to being an activator, so that an increase in its<br />
concentration augments the AND's downstream protein expression.<br/><br/><br />
<br />
<br />
The suggested amount of each input that activates the gate according to our model is:<br/><br/><br />
<br />
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<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_sug_c.png]]<br /><br /><p><br />
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</table><br />
</center><br />
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::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Modeling/Sweet_AND#Sweet_AND]]<br />
<br />
== '''Deterministic model''' ==<br />
<br />
'''[[Team:UNAM_Genomics_Mexico/Modeling/Parameters | General assumptions]]''':<br /><br />
<br />
Xylose and arabinose are part of the pool of molecules that can be used by B. subtilis as carbon sources. Among membrane transporters, affinity for particular kinds of sugars is often high; nevertheless AraE is the main permease responsible for the intake of arabinose, xylose, and galactose monosaccharides. AraE is down-regulated by AraR, which also represses some other genes involved in arabinose metabolism. AraR is inactivated by arabinose and by itself [1]. These little details evidentiate the fact that arabinose is required for xylose cellular influx.<br/><br/><br />
The concentrations of XylA and AraA depend on the concentration of their respective repressors, XylR and AraR, as well as their respective dissociation constants.<br/><br/><br />
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<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_O5.png]]<br /><br /><p><br />
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<br/><br/><br />
<br />
Intracellular xylose induces the production of genes involved in its metabolism, also through<br />
the inactivation of their correspondent repressor, XylR. XylA is the enzyme that catalyzes the<br />
conversion of D-xylose to D-xylulose. For arabinose, the isomerase that catalyzes the conversion of L-arabinose into L-ribulose is AraA [2].<br/><br/><br />
In that way, the amount of intracellular arabinose is defined as the amount introduced by AraE minus the amount converted to L-ribulose by AraA; for xylose the process is similar but the degradation depends on xylA. We assume that the rates of intake of both sugars by AraE are similar. For the degradation rates by AraA and XylA, they are defined by Michaelis-Menten kinetics, since this kind of equation takes into account the energy consumption, and also because it is a simple reaction with one step and one substrate.<br/><br/><br />
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<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_O6.png]]<br /><br /><p><br />
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<br />
As for the amount of XylR presented in the system, we had two sources of production. One is the endogenous production in Bacillus subtilis, while the other is the production by our exogenous construct inserted into the genome under the Pveg constitutive promoter. This construction was introduced to counteract the leakiness of the promoter under XylR repression [http://partsregistry.org/Part:BBa_K143036 BBa_K143036].<br/><br/><br />
<br />
For AraR, which is produced only through the endogenous B. subtilis production, we considered the self-repression and described its production through a Hill function dependent on the concentration of the protein[3].<br/><br/><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:UNAM_genomics_ODE7.png]]<br /><br /><p><br />
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<br />
<br />
The final TF involved in our system is AraC, from E. coli, which is produced in our construct by the Pveg promoter. Its concentration depends exclusively on the maximal rate of transcription, and the general degradation rate. Since the repression method used by AraC is DNA looping, we used the same approach as in [4], where the repression intensity of AraC was defined in terms of arabinose instead of AraC concentration. In this case, the Hill coefficient used was n=3, as reported in the same source as the best fitting value. Under the same assumption of independence between the two TF involved in the hybrid promoter, the total repression intensity is the repression given by AraC DNA looping plus the intensity of XylR, both modeled with a Hill function.<br/><br/><br />
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<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_O8.png|850px]]<br /><br /><p><br />
</p></td><br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_O9.png]]<br /><br /><p><br />
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</center><br />
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::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Modeling/Sweet_AND#Sweet_AND]]<br />
<br />
==''' Conclusions:''' ==<br />
<br />
<br />
Complex mixture of inputs of varying natures, plus their collective properties and regulation of the designed system, yield very interesting behaviors. First, we want to highlight the existence of two unique steady states that are derived from the kind of addition of the inputs: one is reached when after an initial addition of sugars, these are degraded (in this case, there is an interval of time that shows the increment of the downstream protein and then its production rate decays as the sugars are degraded until they reach the state preceding the addition of sugars) and the other is when the amount of sugars is constant in the environment (like when using a chemostat the cell reaches a new state where there’s an equilibrium between the sugar catabolism and the continuous production of the downstream TF in the AND).<br />
The last remark is about the contribution to the AND by each input. We could see that when we had null or low levels of xylose, the repression remained strong. In the case of arabinose, its concentration contributes to the max level of expression of the downstream TF and has an impact on the way curve of expression behaves. Our hypothesis is that the relevance of arabinose is due to the fact that it plays a role in the regulation of the transportes, which has as its job the introduction of both kind of sugars. Our hypothesis is based on the fact that we see in the gradient graphs the decay of the transporter, followed by a sudden increase of it.<br />
<br/><br />
<br/><br />
References<br/><br />
<br />
[1]Krispin O, Allmansberger R. The Bacillus subtilis AraE protein displays a broad substrate specificity for several different sugars. J Bacteriol. 1998 June; 180(12): 3250–3252. PMCID: PMC107832.<br/><br />
<br/><br />
[2]Gu Y, Ren C, Sun Z, Rodionov D A, Zhang W, Yang S, Yang C, Jiang W. Reconstruction of xylose utilization pathway and regulons in Firmicutes. BMC Genomics 2010, 11:255 doi:10.1186/1471-2164-11-25.<br/><br />
<br/><br />
[3]Sá-Nogueira I, Nogueira T V, Soares S, Lencastre H. The Bacillus subtilis L-arabinose (ara) operon: nucleotide sequence, genetic organization and expression. Microbiology. 1997. 143:957-969.<br/><br />
<br/><br />
[4]Megerle, Judith. Cell to cell variability of gene expression dynamics in inducible regulatory networks. Dissertation of Physics Faculty of Ludwig Maximilians University of Munich. January 2011.<br/><br />
<br/><br />
<br/><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling/Sweet_AND
Team:UNAM Genomics Mexico/Modeling/Sweet AND
2012-10-27T03:07:36Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<center><h1>'''AND's Results'''</h1></center> <br />
<br /><br />
<br /><br />
<h1>'''Sweet AND'''</h1> <br />
<br /><br />
<br /><br />
<br />
Xylose and arabinose are sugars used by bacteria as carbon sources. XylR and AraC are<br />
transcriptional regulators modulated by xylose and arabinose. For xylose sensing, our hybrid promoter<br />
has as backbone made of the pBAD promoter plus a binding site for XylR. XylR represses <br />
transcription until xylose is present in the cell, a process similar to the metal sensor of ArsR and CzrA. As for arabinose, AraC represses transcription through DNA looping. When arabinose enters the cell, it dissolves the loop and transcription can take place. We conclude that arabinose and xylose are essential for the expression of genes under the pBAD/xylR promoter, thus acting as an AND Boolean operation. <br/><br/><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UNAM_GENOMICS_MEXICO_AND_bf_sweet.png]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
'''Repressors'''<br/><br/><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align= "center"><br />[[File:UGM_tv_stf.png]]<br /><br /><p><br />
</p></td><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UNAM GENOMICS MEXICO Heavy metal and2.jpg| 400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
'''Inputs'''<br/><br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align= "center"><br />[[File:UGM_tv_si.png]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
The behavior of both ANDs predicted by our model is consistent with what we expected. From the perspective of the repressors, a higher level of PoPs (Polymerase Per Second, which is a measurement of the level of transcription) is reached when both repressor concentrations are 0. Reciprocally, PoPs start to fall when the concentration of repressors increases, reaching the highest level of repression (lower PoPs) at high concentration of both repressors. This behavior reminds us of the way a NOR behaves, because at low levels of repressor PoPs is high, but for repression, high concentration of one repressor is enough. However, we can't control (directly) the concentration of repressors, so the right way to look at the system is from the perspective of the Input: the metals or the sugars. <br/><br/><br />
If we looked PoPs as a function of the Input, it behaves as an AND logic gate, as can be seen in the continuous TRUTH TABLE. At lower concentrations of Inputs, the PoPs level is in a very repressed stage. If you keep increasing only one of the Inputs on only one of the axes (yes, axes is the plural of axis), there won’t be high activation until the addition of high concentrations on the other axis. PoPs will increase as the Input concentration increases in a direct proportion fashion. <br/><br/><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Modeling/Sweet_AND#Sweet_AND]]<br />
<h2>'''KAPPA'''</h2><br/><br/><br />
<br />
Sometimes people wonder why we used Kappa, a stochastic approach, to model our system, which was also described by deterministic differential equations. First of all, Kappa helped us come up with the concentration ranges entered in the differential equations (here, an exclamation of awe is usually heard, and with good reason). How did this come to happen? Well, the simulation in kappa is driven by the binding events and is dependent on the species concentrations, so using general kinetic rates we were able to see the dynamic behavior given by the architecture of the network. This allowed us to approximate the final species concentration and look for parameters missing in our ODE system, which was something useful, since we were as lost as a three-legged dog in a rodeo dance. <br/><br/><br />
So, first we ran the [https://static.igem.org/mediawiki/2012/f/fd/And_sugar.txt And Sugar kappa script] shifting the sugar initial concentrations in the single input burst and getting the average of 100 iterations to eliminate the noise in the simulation. After running some simulation time, we were happy to see that our "Sweet AND" behaved as expected, that is, as an AND. Besides, we were able to see the maximal concentration of the protein that would be downstream of our AND, and the time it took to reach it. As you can see, our simulation grid is not described in its entirety. This is because kappa can be computationally very expensive for non-atomic rules, so we chose to feed our ODE system with some parameters and scan the system in a broader manner instead of dying of old age waiting for Kappa (if only we had a supercluster to run things in...). Nevertheless, some behaviors can only be noticed by looking at the simulation grid. For example, the activation of P4 depending on the xylose and arabinose concentrations. As it becomes apparent through a meticulous and insightful analysis only doable by a really smart, handsome, and admirable people, the activation of P4 depends on the concentrations of both, xylose and arabinose. When only one of the two sugars has a high concentration, the activation is not as elevated as when the two have high concentrations. <br/><br/><br />
<br />
<br />
<br />
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<table border="1" cellpadding="2px" width="100%" height="600px"><br />
<tr><br />
<td rowspan="7">Xylose</td><br />
<td>500,000</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/0/08/UNAM-Genomics_Mexico_Ara500_xyl500.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/0/08/UNAM-Genomics_Mexico_Ara500_xyl500.png" alt="large" /><br />
</span></a></div></td><br />
</tr><br />
<br />
<tr><br />
<td>350,000</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/a/aa/UNAM-Genomics_Mexico_Ara100_xyl350.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/a/aa/UNAM-Genomics_Mexico_Ara100_xyl350.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>100,000</td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/7/7a/UNAM-Genomics_Mexico_Ara0_xyl100.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/7/7a/UNAM-Genomics_Mexico_Ara0_xyl100.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/9/97/UNAM-Genomics_Mexico_Ara100_xyl100.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/9/97/UNAM-Genomics_Mexico_Ara100_xyl100.png" alt="large" /></span></a></div></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/7/7c/UNAM-Genomics_Mexico_Ara350_xyl100.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/7/7c/UNAM-Genomics_Mexico_Ara350_xyl100.png" alt="large" /></span></a></div></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>75,000</td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/c/c3/UNAM-Genomics_Mexico_Ara75_xyl75.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UNAM-Genomics_Mexico_Ara75_xyl75.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>50,000</td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/5/58/UNAM-Genomics_Mexico_Ara0_xyl50.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/5/58/UNAM-Genomics_Mexico_Ara0_xyl50.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/8/83/UNAM-Genomics_Mexico_Ara50_xyl50.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/8/83/UNAM-Genomics_Mexico_Ara50_xyl50.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>25,000</td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/6/6f/UNAM-Genomics_Mexico_Ara25_xyl25.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/6/6f/UNAM-Genomics_Mexico_Ara25_xyl25.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>0.0</td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/4/4e/UNAM-Genomics_Mexico_Ara0_xyl0.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/4/4e/UNAM-Genomics_Mexico_Ara0_xyl0.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/6/6d/UNAM-Genomics_Mexico_Ara50_xyl0.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/6/6d/UNAM-Genomics_Mexico_Ara50_xyl0.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td><div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2012/4/4f/UNAM-Genomics_Mexico_Ara100_xyl0.png" alt="thumb" class="resize_thumb" /><span><br />
<img src="https://static.igem.org/mediawiki/2012/4/4f/UNAM-Genomics_Mexico_Ara100_xyl0.png" alt="large" /></span></a></div></td><br />
<td></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td></td><br />
<td></td><br />
<td>0.0</td><br />
<td>25,000</td><br />
<td>50,000</td><br />
<td>75,000</td><br />
<td>100,000</td><br />
<td>350,000</td><br />
<td height="14px">500,000</td><br />
</tr><br />
<br />
<tr><br />
<td></td><br />
<td></td><br />
<td colspan="7" height="14px">Arabinose</td><br />
</tr><br />
<br />
<br />
<br />
<br />
</table><br />
<br />
</html><br />
<br />
<br />
We can focus only in the dynamics of the downstream protein, instead of the dynamics of the whole system. This plot describes the time to reach steady state and maximal concentration of the AND downstream protein. It also describes what the maximal concentration of the AND protein is. All of this is based on a single input burst concentration.<br />
<br/><br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UNAM_GENOMICS_MEXICO_AND_downProt.png]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<h2>'''Kind of input'''</h2><br/><br/><br />
<br />
<br />
The Endogenous system and Downstream protein dynamics is dependent on the kind of input. The inputs used by each AND have a unique effect in the response of the cell, because carbohydrates are degraded by the cell. When we add sugars to the sweet AND system, if they are added just like a discrete and singular dose, the cell will react to the alteration through the activation of the degradation machinery, and when the sugar pool gets empty, the cell achieves a similar state to the one before the sugar was introduced, that is, a return to pre-induction steady state. When the sugar addition is constant, the cell has a complete phenotypic change, reaching a new steady state.<br/><br/><br />
<br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:500ara_500xyl.png | 400px]]<br /><br /><p><br />
</p></td><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Continuando.png | 400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<h2>'''Constitutive VS NO-Constitutive XylR expression'''</h2><br/><br/><br />
<br />
It has been reported that xylR has to be in high concentrations in order to repress in an effective manner. So we decided to look the dynamics of the AND with constitutive expression (The endogenous expression plus the pveg constitutive expression) of xylR vs No-Constitutive expression of xylR (just the endogenous expression). Our simulation says that for a finest logic behaviour it is needed the constitutive expression. Because when we compare the dynamics of the downstream protein (pink lines label with P4) when we add a single input burst of 100,000 arabinose molecules, the active araC concentration is depleted, so all the repression is given by xylR. But in the NO-constitutive expression AND the concentration of P4 goes significant higher than the AND with the constitutive expression. So we decided to express xylR constitutively in our constructions<br/><br/><br />
<br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UNAM-Genomics_Mexico_Ara100_xyl0.png | 400px]]<br /><br /><p><br />
</p></td><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:NOCONSTIT.png | 400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
<br />
<br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Modeling/Sweet_AND#Sweet_AND]]<br />
<br />
== '''Transfer Function''' ==<br />
<br />
<br />
Our logic AND gates are fully dependent on the intracellular concentrations of the inducers (in this case, xylose and arabinose). To accomplish our duty in model, we therefore make use of the "Transfer Function", which requires taking into account the regulation and dynamics of the endogenous Bacillus subtilis influx-efflux intracellular sugar regulation system. The "Transfer Function" is going to be the "Black Box" that will give you the dynamic<br />
concentration through time of whatever is downstream of the "AND" when you<br />
feed it with input data. It doesn't matter if it’s a single input burst or a<br />
continuous input.<br />
<br/><br />
<br/><br />
So, our first task was to reconstruct the regulatory network of B. subtilis for the influx-efflux system. The regulation data was retrieved from several papers and databases like [http://bsubcyc.org/ http://bsubcyc.org/]<br />
<br/><br/><br />
<br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UNAM_GENOMICS_MEXICO_Sweet_AND.png | 600px]]<br /><br /><p>Sweet AND gate: Arabinose and xylose are imported into the cell by the araE permease. The<br />
carbon metabolism system is highly regulated in order to optimize the consumption of sugars<br />
using the least amount of energy. XylR and AraR are repressors responsible for the regulation of genes involved in the metabolism and intake of xylose and arabinose, respectively. Although XylR is not well characterized, it’s hypothesized that XylR represses itself; some studies suggest a correlation between XylR and the concentration of xylose inside the cell. AraR not only represses the production of genes like araE or araA, but it also represses itself. AraA and xylA are isomerases that convert L-arabinose into L-ribulose and D-xylose into D-xylulose. XylR from B.subtilis is also used by our construction as a repressor. Under that constitutive promoter, AraC is also produced for the regulation of the hybrid promoter. The output of the AND is the production of a transcription factor, LasR or P4, and RFP.<br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br /><br />
<br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UNAM_GENOMICS_MEXICO_AND_3D.png | 850px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UNAM_GENOMICS_MEXICO_AND_3D_3D.png | 850px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br /><br />
<br />
<br />
One observation worthy of mention came when we realized that the<br />
system was most repressed when there was scarce xylose in the system, not<br />
when there was none. On the contrary, when there is no xylose in the system,<br />
there is a very small expression gradient dependent on the concentration of<br />
arabinose, yet this gradient does not surpass a threshold where we could<br />
consider the AND as "ON".<br/><br/><br />
<br />
We are coupling an endogenous and an exogenous repressor to our AND. This<br />
feature can easily be explained by the fact that we are expressing the<br />
endogenous repressor XylR in a constitutive manner. This is due to the fact that<br />
it has been reported that it has to be in high concentrations in order to repress<br />
in an effective manner. When we add xylose to the medium, endogenous XylR<br />
gene increases its expression. This makes<br />
repression against the AND increase when xylose is found in the medium in<br />
small amounts. After the repression threshold is surpassed, xylose stops being<br />
a repressor and shifts to being an activator, so that an increase in its<br />
concentration augments the AND's downstream protein expression.<br/><br/><br />
<br />
<br />
The suggested amount of each input that activates the gate according to our model is:<br/><br/><br />
<br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_sug_c.png]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Modeling/Sweet_AND#Sweet_AND]]<br />
<br />
== '''Deterministic model''' ==<br />
<br />
'''[[Team:UNAM_Genomics_Mexico/Modeling/Parameters | General assumptions]]''':<br /><br />
<br />
Xylose and arabinose are part of the pool of molecules that can be used by B. subtilis as carbon sources. Among membrane transporters, affinity for particular kinds of sugars is often high; nevertheless AraE is the main permease responsible for the intake of arabinose, xylose, and galactose monosaccharides. AraE is down-regulated by AraR, which also represses some other genes involved in arabinose metabolism. AraR is inactivated by arabinose and by itself [1]. These little details evidentiate the fact that arabinose is required for xylose cellular influx.<br/><br/><br />
The concentrations of XylA and AraA depend on the concentration of their respective repressors, XylR and AraR, as well as their respective dissociation constants.<br/><br/><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_O5.png]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
Intracellular xylose induces the production of genes involved in its metabolism, also through<br />
the inactivation of their correspondent repressor, XylR. XylA is the enzyme that catalyzes the<br />
conversion of D-xylose to D-xylulose. For arabinose, the isomerase that catalyzes the conversion of L-arabinose into L-ribulose is AraA [2].<br/><br/><br />
In that way, the amount of intracellular arabinose is defined as the amount introduced by AraE minus the amount converted to L-ribulose by AraA; for xylose the process is similar but the degradation depends on xylA. We assume that the rates of intake of both sugars by AraE are similar. For the degradation rates by AraA and XylA, they are defined by Michaelis-Menten kinetics, since this kind of equation takes into account the energy consumption, and also because it is a simple reaction with one step and one substrate.<br/><br/><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
As for the amount of XylR presented in the system, we had two sources of production. One is the endogenous production in Bacillus subtilis, while the other is the production by our exogenous construct inserted into the genome under the Pveg constitutive promoter. This construction was introduced to counteract the leakiness of the promoter under XylR repression [http://partsregistry.org/Part:BBa_K143036 BBa_K143036].<br/><br/><br />
<br />
For AraR, which is produced only through the endogenous B. subtilis production, we considered the self-repression and described its production through a Hill function dependent on the concentration of the protein[3].<br/><br/><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UNAM_genomics_ODE7.png]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
The final TF involved in our system is AraC, from E. coli, which is produced in our construct by the Pveg promoter. Its concentration depends exclusively on the maximal rate of transcription, and the general degradation rate. Since the repression method used by AraC is DNA looping, we used the same approach as in [4], where the repression intensity of AraC was defined in terms of arabinose instead of AraC concentration. In this case, the Hill coefficient used was n=3, as reported in the same source as the best fitting value. Under the same assumption of independence between the two TF involved in the hybrid promoter, the total repression intensity is the repression given by AraC DNA looping plus the intensity of XylR, both modeled with a Hill function.<br/><br/><br />
<br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_O8.png|850px]]<br /><br /><p><br />
</p></td><br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_O9.png]]<br /><br /><p><br />
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</table><br />
</center><br />
<br/><br/><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Modeling/Sweet_AND#Sweet_AND]]<br />
<br />
==''' Conclusions:''' ==<br />
<br />
<br />
Complex mixture of inputs of varying natures, plus their collective properties and regulation of the designed system, yield very interesting behaviors. First, we want to highlight the existence of two unique steady states that are derived from the kind of addition of the inputs: one is reached when after an initial addition of sugars, these are degraded (in this case, there is an interval of time that shows the increment of the downstream protein and then its production rate decays as the sugars are degraded until they reach the state preceding the addition of sugars) and the other is when the amount of sugars is constant in the environment (like when using a chemostat the cell reaches a new state where there’s an equilibrium between the sugar catabolism and the continuous production of the downstream TF in the AND).<br />
The last remark is about the contribution to the AND by each input. We could see that when we had null or low levels of xylose, the repression remained strong. In the case of arabinose, its concentration contributes to the max level of expression of the downstream TF and has an impact on the way curve of expression behaves. Our hypothesis is that the relevance of arabinose is due to the fact that it plays a role in the regulation of the transportes, which has as its job the introduction of both kind of sugars. Our hypothesis is based on the fact that we see in the gradient graphs the decay of the transporter, followed by a sudden increase of it.<br />
<br/><br />
<br/><br />
References<br/><br />
<br />
[1]Krispin O, Allmansberger R. The Bacillus subtilis AraE protein displays a broad substrate specificity for several different sugars. J Bacteriol. 1998 June; 180(12): 3250–3252. PMCID: PMC107832.<br/><br />
<br/><br />
[2]Gu Y, Ren C, Sun Z, Rodionov D A, Zhang W, Yang S, Yang C, Jiang W. Reconstruction of xylose utilization pathway and regulons in Firmicutes. BMC Genomics 2010, 11:255 doi:10.1186/1471-2164-11-25.<br/><br />
<br/><br />
[3]Sá-Nogueira I, Nogueira T V, Soares S, Lencastre H. The Bacillus subtilis L-arabinose (ara) operon: nucleotide sequence, genetic organization and expression. Microbiology. 1997. 143:957-969.<br/><br />
<br/><br />
[4]Megerle, Judith. Cell to cell variability of gene expression dynamics in inducible regulatory networks. Dissertation of Physics Faculty of Ludwig Maximilians University of Munich. January 2011.<br/><br />
<br/><br />
<br/><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Bacillus
Team:UNAM Genomics Mexico/Notebook/Bacillus
2012-10-27T02:55:24Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''''Bacillus subtilis'' Notebook'''</h1></center><br />
<br /><br />
<br /><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg"><br />
<tr><br />
<td id="contentcolumn" align= "center"><p>[[File:UnamgenomicsBacillus.png|200px]]</p></td><br />
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</table><br />
</center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg"><br />
<tr><br />
<td id="leftcolumn2" align= "center"><p>'''[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#July | July]]'''<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#10.2F07.2F12 | 10/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#11.2F07.2F12 | 11/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#12.2F07.2F12 | 12/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#13.2F07.2F12 | 13/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#16.2F07.2F12 | 16/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#17.2F07.2F12 | 17/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#18.2F07.2F12 | 18/07/12]]<br /></p></td><br />
<td id="contentcolumn2" align= "center"><p>'''[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#September | September]]'''<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#12.2F09.2F12 | 12/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#13.2F09.2F12 | 13/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#14.2F09.2F12 | 14/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#18.2F09.2F12 | 18/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#19.2F09.2F12 | 19/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#20.2F09.2F12 | 20/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#21.2F09.2F12 | 21/09/12]]<br /></p></td><br />
<td id="rightcolumn2" align= "center"><p>[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#October | '''October''']]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#22.2F10.2F12 | 22/10/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#23.2F10.2F12 | 23/10/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#24.2F10.2F12 | 24/10/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#25.2F10.2F12 | 25/10/12]]<br /></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<h1>July</h1><br />
<h2>10/07/12</h2><br /><br />
Plate WT Bacillus on LB media. <br /><br />
Prepare TFB1 & TFBII solutions, sterilize and store at 4°C. <br /><br />
Store sterile 1.5 ml eppendorf tubes at -20 °C. <br /><br />
Store sterile falcon tubes at -4°C. <br /><br /><br />
<h2>11/07/12</h2><br /><br />
Filter TFB solutions<br /><br />
Incubate the preculture at 37°C overnight. <br /><br />
<h2>12/07/12</h2><br /><br />
Incubate the preculture again because ir fell down<br /><br /><br />
<h2>13/07/12</h2><br /><br /><br />
Follow the RbCl2 competent cell protocol. <br /><br />
Transform Bacillus with pSB4A5<br /><br /><br />
<h2>16/07/12</h2><br /><br />
Our Bacillus subtilis transformation with psB4A5 failed; the only colony that grew was not pink, then we thought it was contamination and that our cells were not competent. <br /><br />
To elucidate what was the reason for what the bacteria did not grow, we plated our competent cells on LB media and transformed again but this time using puc19. <br /><br />
We also replated the only colony onto selective media. <br /><br />
We prepared all the solutions needed to make Bacillus subtilis competent according to Cambridge 2008 protocol (https://2008.igem.org/Team:Cambridge/Bacillus_subtilis_transformation) <br /><br />
<h2>17/07/12</h2><br /><br />
Competent cells are ok; they grew on LB media. <br /><br />
The replated colony grew but is not pink as it should be. Our hypothesis was that this part is for coli, so the expression in bacillus would be lower. To know if the bacillus were transformed or not, we extracted plasmid. <br /><br /><br />
<h2>18/07/12</h2><br /><br />
Today we ran a gel to see if Bacillus had the plasmid but it has not. <br /><br />
<br />
<br />
<h1>September</h1><br />
<h2>12/09/12</h2><br /><br />
We’ve made competent B.subtilis using the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | Two step transformation procedure]]<br />
We transformed our plasmid in E.coli RecA + by heatshock [Escherichia coli MC1061 heat shock transformation protocol]<br />
<h2>13/09/12</h2><br /><br />
Today we extracted plasmid by the Birbom –Doly method from our transformation in recA+, then we transformed bacillus with this plasmid.<br />
<h2>14/09/12</h2><br /><br />
Results:<br />
Bacillus subtilis were successfully transformed with pSB1AK3.<br />
<h2>18/09/12</h2><br /><br />
We’ve made competent B.subtilis using the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | Two step transformation procedure]]<br />
We also made competent E. coli RecA+ with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | CaCl2 procedure]].<br />
<h2>19/09/12</h2><br /><br />
We transformed RecA+ E.coli with 10 microliters [65.5 ng/microliter] of bBAK143079 plasmid.<br />
<h2>20/09/12</h2><br /><br />
As the transformation didn´t grow, we transformed again with a higher concentration of plasmid: <br />
20 microliters. <br /><br /><br />
<h2>21/09/12</h2><br /><br />
One of the petri dishes has 5 colonies; we made liquid culture to extract plasmid. <br /><br /><br />
<br />
<br />
<h1>October</h1><br /><br />
<h2>22/10/12</h2><br /><br />
We prepare the culture in a solid LB medium for the SEM Analysis ([[Team:UNAM_Genomics_Mexico/Notebook/Protocols#SEM_Analysis_.28Scanning_Electron_Microscope.29.5B1.5D | See the SEM Analysis Protocol]]). <br /><br />
<br /><br />
<h2>23/10/12</h2><br /><br />
We went to the Microscopy Department in UNAM to finish fixing the cells with osmium and with liquid CO2 and watch the nanotubes with the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#SEM_Analysis_.28Scanning_Electron_Microscope.29.5B1.5D | SEM]]. <br /><br />
'''Succesful Results!''' <br /><br />
<br /><br />
<h2>24/10/12</h2><br /><br />
We transform in the MC1061 RecA+ the 97 promoter plasmid with our '''Heavy Metal construction''' with our [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | Transformation Procedure]]. <br /><br />
'''Succesful Results!''' <br /><br />
<br /><br />
<h2>25/10/12</h2><br /><br />
We made a plasmid extraction from our cells, we ran a gel with it and transform it into B. subtilis with the <br />
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Two-step_Bacillus_subtilis_Transformation_Procedure | '''B. subtilis Tranformation Procedure''']]. <br /><br />
'''Succesful Results!''' <br /><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T02:51:40Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<center><h1>'''Welcome to our homepage'''</h1></center> <br />
<br /><br />
<table border="0" width="900px" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2"><p><br />
<h2>'''Bacillus booleanus'''</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
</p><br />
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</table><br />
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<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
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<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|510px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
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<td id="contentcolumn" align="center"><p><br />
<h1>Regional Jamboree!!!</h1><br />
[[File:Unamgenomicsbestwikihome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<br /><br />
[[File:Unamgenomicsbogotahome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<b>Regional Jamboree LOTS of FUN!</b><br />
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<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
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<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a></td><br />
</tr><br />
<tr><br />
<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
<br />
<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a></td><br />
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Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Bacillus
Team:UNAM Genomics Mexico/Notebook/Bacillus
2012-10-27T02:50:09Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''''Bacillus subtilis'' Notebook'''</h1></center><br />
<br /><br />
<br /><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg"><br />
<tr><br />
<td id="contentcolumn" align= "center"><p>[[File:UnamgenomicsBacillus.png|200px]]</p></td><br />
</tr><br />
</table><br />
</center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg"><br />
<tr><br />
<td id="leftcolumn2" align= "center"><p>'''[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#July | July]]'''<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#10.2F07.2F12 | 10/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#11.2F07.2F12 | 11/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#12.2F07.2F12 | 12/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#13.2F07.2F12 | 13/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#16.2F07.2F12 | 16/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#17.2F07.2F12 | 17/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#18.2F07.2F12 | 18/07/12]]<br /></p></td><br />
<td id="contentcolumn2" align= "center"><p>'''[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#September | September]]'''<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#12.2F09.2F12 | 12/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#13.2F09.2F12 | 13/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#14.2F09.2F12 | 14/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#18.2F09.2F12 | 18/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#19.2F09.2F12 | 19/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#20.2F09.2F12 | 20/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#21.2F09.2F12 | 21/09/12]]<br /></p></td><br />
<td id="rightcolumn2" align= "center"><p>[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#October | '''October''']]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#22.2F10.2F12 | 22/10/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#23.2F10.2F12 | 23/10/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#24.2F10.2F12 | 24/10/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#25.2F10.2F12 | 25/10/12]]<br /></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br /><br />
<br />
<br /><br />
<h1>July</h1><br />
<h2>10/07/12</h2><br /><br />
Plate WT Bacillus on LB media. <br /><br />
Prepare TFB1 & TFBII solutions, sterilize and store at 4°C. <br /><br />
Store sterile 1.5 ml eppendorf tubes at -20 °C. <br /><br />
Store sterile falcon tubes at -4°C. <br /><br /><br />
<h2>11/07/12</h2><br /><br />
Filter TFB solutions<br /><br />
Incubate the preculture at 37°C overnight. <br /><br />
<h2>12/07/12</h2><br /><br />
Incubate the preculture again because ir fell down<br /><br /><br />
<h2>13/07/12</h2><br /><br /><br />
Follow the RbCl2 competent cell protocol. <br /><br />
Transform Bacillus with pSB4A5<br /><br /><br />
<h2>16/07/12</h2><br /><br />
Our Bacillus subtilis transformation with psB4A5 failed; the only colony that grew was not pink, then we thought it was contamination and that our cells were not competent. <br /><br />
To elucidate what was the reason for what the bacteria did not grow, we plated our competent cells on LB media and transformed again but this time using puc19. <br /><br />
We also replated the only colony onto selective media. <br /><br />
We prepared all the solutions needed to make Bacillus subtilis competent according to Cambridge 2008 protocol (https://2008.igem.org/Team:Cambridge/Bacillus_subtilis_transformation) <br /><br />
<h2>17/07/12</h2><br /><br />
Competent cells are ok; they grew on LB media. <br /><br />
The replated colony grew but is not pink as it should be. Our hypothesis was that this part is for coli, so the expression in bacillus would be lower. To know if the bacillus were transformed or not, we extracted plasmid. <br /><br /><br />
<h2>18/07/12</h2><br /><br />
Today we ran a gel to see if Bacillus had the plasmid but it has not. <br /><br />
<br />
<br />
<h1>September</h1><br />
<h2>12/09/12</h2><br /><br />
We’ve made competent B.subtilis using the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | Two step transformation procedure]]<br />
We transformed our plasmid in E.coli RecA + by heatshock [Escherichia coli MC1061 heat shock transformation protocol]<br />
<h2>13/09/12</h2><br /><br />
Today we extracted plasmid by the Birbom –Doly method from our transformation in recA+, then we transformed bacillus with this plasmid.<br />
<h2>14/09/12</h2><br /><br />
Results:<br />
Bacillus subtilis were successfully transformed with pSB1AK3.<br />
<h2>18/09/12</h2><br /><br />
We’ve made competent B.subtilis using the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | Two step transformation procedure]]<br />
We also made competent E. coli RecA+ with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | CaCl2 procedure]].<br />
<h2>19/09/12</h2><br /><br />
We transformed RecA+ E.coli with 10 microliters [65.5 ng/microliter] of bBAK143079 plasmid.<br />
<h2>20/09/12</h2><br /><br />
As the transformation didn´t grow, we transformed again with a higher concentration of plasmid: <br />
20 microliters. <br /><br /><br />
<h2>21/09/12</h2><br /><br />
One of the petri dishes has 5 colonies; we made liquid culture to extract plasmid. <br /><br /><br />
<br />
<br />
<h1>October</h1><br /><br />
<h2>22/10/12</h2><br /><br />
We prepare the culture in a solid LB medium for the SEM Analysis (See the SEM Analysis Protocol). <br /><br />
<br /><br />
<h2>23/10/12</h2><br /><br />
We went to the Microscopy Department in UNAM to finish fixing the cells with osmium and with liquid CO2 and watch the nanotubes with the SEM. <br /><br />
'''Succesful Results!''' <br /><br />
<br /><br />
<h2>24/10/12</h2><br /><br />
We transform in the MC1061 RecA+ the 97 promoter plasmid with our '''Heavy Metal construction''' with our Transformation Procedure. <br /><br />
'''Succesful Results!''' <br /><br />
<br /><br />
<h2>25/10/12</h2><br /><br />
We made a plasmid extraction from our cells, we ran a gel with it and transform it into B. subtilis with the <br />
'''B. subtilis Tranformation Procedure'''. <br /><br />
'''Succesful Results!''' <br /><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Bacillus
Team:UNAM Genomics Mexico/Notebook/Bacillus
2012-10-27T02:49:02Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''''Bacillus subtilis'' Notebook'''</h1></center><br />
<br /><br />
<br />
<br /><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg"><br />
<tr><br />
<td id="leftcolumn2" align= "center"><p>'''[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#July | July]]'''<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#10.2F07.2F12 | 10/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#11.2F07.2F12 | 11/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#12.2F07.2F12 | 12/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#13.2F07.2F12 | 13/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#16.2F07.2F12 | 16/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#17.2F07.2F12 | 17/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#18.2F07.2F12 | 18/07/12]]<br /></p></td><br />
<td id="contentcolumn2" align= "center"><p>'''[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#September | September]]'''<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#12.2F09.2F12 | 12/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#13.2F09.2F12 | 13/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#14.2F09.2F12 | 14/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#18.2F09.2F12 | 18/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#19.2F09.2F12 | 19/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#20.2F09.2F12 | 20/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#21.2F09.2F12 | 21/09/12]]<br /></p></td><br />
<td id="rightcolumn2" align= "center"><p>[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#October | October]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#22.2F10.2F12 | 22/10/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#23.2F10.2F12 | 23/10/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#24.2F10.2F12 | 24/10/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#25.2F10.2F12 | 25/10/12]]<br /></p></td><br />
</tr><br />
</table><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg"><br />
<tr><br />
<td id="contentcolumn" align= "center"><p>[[File:UnamgenomicsBacillus.png|200px]]</p></td><br />
</tr><br />
</table><br />
</center><br />
<br />
<h1>July</h1><br />
<h2>10/07/12</h2><br /><br />
Plate WT Bacillus on LB media. <br /><br />
Prepare TFB1 & TFBII solutions, sterilize and store at 4°C. <br /><br />
Store sterile 1.5 ml eppendorf tubes at -20 °C. <br /><br />
Store sterile falcon tubes at -4°C. <br /><br /><br />
<h2>11/07/12</h2><br /><br />
Filter TFB solutions<br /><br />
Incubate the preculture at 37°C overnight. <br /><br />
<h2>12/07/12</h2><br /><br />
Incubate the preculture again because ir fell down<br /><br /><br />
<h2>13/07/12</h2><br /><br /><br />
Follow the RbCl2 competent cell protocol. <br /><br />
Transform Bacillus with pSB4A5<br /><br /><br />
<h2>16/07/12</h2><br /><br />
Our Bacillus subtilis transformation with psB4A5 failed; the only colony that grew was not pink, then we thought it was contamination and that our cells were not competent. <br /><br />
To elucidate what was the reason for what the bacteria did not grow, we plated our competent cells on LB media and transformed again but this time using puc19. <br /><br />
We also replated the only colony onto selective media. <br /><br />
We prepared all the solutions needed to make Bacillus subtilis competent according to Cambridge 2008 protocol (https://2008.igem.org/Team:Cambridge/Bacillus_subtilis_transformation) <br /><br />
<h2>17/07/12</h2><br /><br />
Competent cells are ok; they grew on LB media. <br /><br />
The replated colony grew but is not pink as it should be. Our hypothesis was that this part is for coli, so the expression in bacillus would be lower. To know if the bacillus were transformed or not, we extracted plasmid. <br /><br /><br />
<h2>18/07/12</h2><br /><br />
Today we ran a gel to see if Bacillus had the plasmid but it has not. <br /><br />
<br />
<br />
<h1>September</h1><br />
<h2>12/09/12</h2><br /><br />
We’ve made competent B.subtilis using the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | Two step transformation procedure]]<br />
We transformed our plasmid in E.coli RecA + by heatshock [Escherichia coli MC1061 heat shock transformation protocol]<br />
<h2>13/09/12</h2><br /><br />
Today we extracted plasmid by the Birbom –Doly method from our transformation in recA+, then we transformed bacillus with this plasmid.<br />
<h2>14/09/12</h2><br /><br />
Results:<br />
Bacillus subtilis were successfully transformed with pSB1AK3.<br />
<h2>18/09/12</h2><br /><br />
We’ve made competent B.subtilis using the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | Two step transformation procedure]]<br />
We also made competent E. coli RecA+ with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | CaCl2 procedure]].<br />
<h2>19/09/12</h2><br /><br />
We transformed RecA+ E.coli with 10 microliters [65.5 ng/microliter] of bBAK143079 plasmid.<br />
<h2>20/09/12</h2><br /><br />
As the transformation didn´t grow, we transformed again with a higher concentration of plasmid: <br />
20 microliters. <br /><br /><br />
<h2>21/09/12</h2><br /><br />
One of the petri dishes has 5 colonies; we made liquid culture to extract plasmid. <br /><br /><br />
<br />
<br />
<h1>October</h1><br /><br />
<h2>22/10/12</h2><br /><br />
We prepare the culture in a solid LB medium for the SEM Analysis (See the SEM Analysis Protocol). <br /><br />
<br /><br />
<h2>23/10/12</h2><br /><br />
We went to the Microscopy Department in UNAM to finish fixing the cells with osmium and with liquid CO2 and watch the nanotubes with the SEM. <br /><br />
'''Succesful Results!''' <br /><br />
<br /><br />
<h2>24/10/12</h2><br /><br />
We transform in the MC1061 RecA+ the 97 promoter plasmid with our '''Heavy Metal construction''' with our Transformation Procedure. <br /><br />
'''Succesful Results!''' <br /><br />
<br /><br />
<h2>25/10/12</h2><br /><br />
We made a plasmid extraction from our cells, we ran a gel with it and transform it into B. subtilis with the <br />
'''B. subtilis Tranformation Procedure'''. <br /><br />
'''Succesful Results!''' <br /><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Bacillus
Team:UNAM Genomics Mexico/Notebook/Bacillus
2012-10-27T02:42:15Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__TOC__<br />
<br /><br />
<center><h1>'''''Bacillus subtilis'' Notebook'''</h1></center><br />
<br /><br />
<br />
<br /><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg"><br />
<tr><br />
<td id="leftcolumn2" align= "center"><p>'''[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#July | July]]'''<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#10.2F07.2F12 | 10/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#11.2F07.2F12 | 11/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#12.2F07.2F12 | 12/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#13.2F07.2F12 | 13/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#16.2F07.2F12 | 16/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#17.2F07.2F12 | 17/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#18.2F07.2F12 | 18/07/12]]<br /></p></td><br />
<td id="contentcolumn2" align= "center"><p>'''[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#September | September]]'''<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#12.2F09.2F12 | 12/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#13.2F09.2F12 | 13/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#14.2F09.2F12 | 14/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#18.2F09.2F12 | 18/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#19.2F09.2F12 | 19/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#20.2F09.2F12 | 20/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#21.2F09.2F12 | 21/09/12]]<br /></p></td><br />
<td id="rightcolumn2" align= "center"><p>[[File:UnamgenomicsBacillus.png|200px]]</p></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<h1>July</h1><br />
<h2>10/07/12</h2><br /><br />
Plate WT Bacillus on LB media. <br /><br />
Prepare TFB1 & TFBII solutions, sterilize and store at 4°C. <br /><br />
Store sterile 1.5 ml eppendorf tubes at -20 °C. <br /><br />
Store sterile falcon tubes at -4°C. <br /><br /><br />
<h2>11/07/12</h2><br /><br />
Filter TFB solutions<br /><br />
Incubate the preculture at 37°C overnight. <br /><br />
<h2>12/07/12</h2><br /><br />
Incubate the preculture again because ir fell down<br /><br /><br />
<h2>13/07/12</h2><br /><br /><br />
Follow the RbCl2 competent cell protocol. <br /><br />
Transform Bacillus with pSB4A5<br /><br /><br />
<h2>16/07/12</h2><br /><br />
Our Bacillus subtilis transformation with psB4A5 failed; the only colony that grew was not pink, then we thought it was contamination and that our cells were not competent. <br /><br />
To elucidate what was the reason for what the bacteria did not grow, we plated our competent cells on LB media and transformed again but this time using puc19. <br /><br />
We also replated the only colony onto selective media. <br /><br />
We prepared all the solutions needed to make Bacillus subtilis competent according to Cambridge 2008 protocol (https://2008.igem.org/Team:Cambridge/Bacillus_subtilis_transformation) <br /><br />
<h2>17/07/12</h2><br /><br />
Competent cells are ok; they grew on LB media. <br /><br />
The replated colony grew but is not pink as it should be. Our hypothesis was that this part is for coli, so the expression in bacillus would be lower. To know if the bacillus were transformed or not, we extracted plasmid. <br /><br /><br />
<h2>18/07/12</h2><br /><br />
Today we ran a gel to see if Bacillus had the plasmid but it has not. <br /><br />
<br />
<br />
<h1>September</h1><br />
<h2>12/09/12</h2><br /><br />
We’ve made competent B.subtilis using the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | Two step transformation procedure]]<br />
We transformed our plasmid in E.coli RecA + by heatshock [Escherichia coli MC1061 heat shock transformation protocol]<br />
<h2>13/09/12</h2><br /><br />
Today we extracted plasmid by the Birbom –Doly method from our transformation in recA+, then we transformed bacillus with this plasmid.<br />
<h2>14/09/12</h2><br /><br />
Results:<br />
Bacillus subtilis were successfully transformed with pSB1AK3.<br />
<h2>18/09/12</h2><br /><br />
We’ve made competent B.subtilis using the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | Two step transformation procedure]]<br />
We also made competent E. coli RecA+ with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | CaCl2 procedure]].<br />
<h2>19/09/12</h2><br /><br />
We transformed RecA+ E.coli with 10 microliters [65.5 ng/microliter] of bBAK143079 plasmid.<br />
<h2>20/09/12</h2><br /><br />
As the transformation didn´t grow, we transformed again with a higher concentration of plasmid: <br />
20 microliters. <br /><br /><br />
<h2>21/09/12</h2><br /><br />
One of the petri dishes has 5 colonies; we made liquid culture to extract plasmid. <br /><br /><br />
<br />
<br />
<h1>October</h1><br /><br />
<h2>22/10/12</h2><br /><br />
We prepare the culture in a solid LB medium for the SEM Analysis (See the SEM Analysis Protocol). <br /><br />
<br /><br />
<h2>23/10/12</h2><br /><br />
We went to the Microscopy Department in UNAM to finish fixing the cells with osmium and with liquid CO2 and watch the nanotubes with the SEM. <br /><br />
'''Succesful Results!''' <br /><br />
<br /><br />
<h2>24/10/12</h2><br /><br />
We transform in the MC1061 RecA+ the 97 promoter plasmid with our '''Heavy Metal construction''' with our Transformation Procedure. <br /><br />
'''Succesful Results!''' <br /><br />
<br /><br />
<h2>25/10/12</h2><br /><br />
We made a plasmid extraction from our cells, we ran a gel with it and transform it into B. subtilis with the <br />
'''B. subtilis Tranformation Procedure'''. <br /><br />
'''Succesful Results!''' <br /><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Bacillus
Team:UNAM Genomics Mexico/Notebook/Bacillus
2012-10-27T02:40:53Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''''Bacillus subtilis'' Notebook'''</h1></center><br />
<br /><br />
<br />
<br /><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg"><br />
<tr><br />
<td id="leftcolumn2" align= "center"><p>'''[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#July | July]]'''<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#10.2F07.2F12 | 10/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#11.2F07.2F12 | 11/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#12.2F07.2F12 | 12/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#13.2F07.2F12 | 13/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#16.2F07.2F12 | 16/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#17.2F07.2F12 | 17/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#18.2F07.2F12 | 18/07/12]]<br /></p></td><br />
<td id="contentcolumn2" align= "center"><p>'''[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#September | September]]'''<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#12.2F09.2F12 | 12/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#13.2F09.2F12 | 13/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#14.2F09.2F12 | 14/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#18.2F09.2F12 | 18/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#19.2F09.2F12 | 19/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#20.2F09.2F12 | 20/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#21.2F09.2F12 | 21/09/12]]<br /></p></td><br />
<td id="rightcolumn2" align= "center"><p>[[File:UnamgenomicsBacillus.png|200px]]</p></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<h1>July</h1><br />
<h2>10/07/12</h2><br /><br />
Plate WT Bacillus on LB media. <br /><br />
Prepare TFB1 & TFBII solutions, sterilize and store at 4°C. <br /><br />
Store sterile 1.5 ml eppendorf tubes at -20 °C. <br /><br />
Store sterile falcon tubes at -4°C. <br /><br /><br />
<h2>11/07/12</h2><br /><br />
Filter TFB solutions<br /><br />
Incubate the preculture at 37°C overnight. <br /><br />
<h2>12/07/12</h2><br /><br />
Incubate the preculture again because ir fell down<br /><br /><br />
<h2>13/07/12</h2><br /><br /><br />
Follow the RbCl2 competent cell protocol. <br /><br />
Transform Bacillus with pSB4A5<br /><br /><br />
<h2>16/07/12</h2><br /><br />
Our Bacillus subtilis transformation with psB4A5 failed; the only colony that grew was not pink, then we thought it was contamination and that our cells were not competent. <br /><br />
To elucidate what was the reason for what the bacteria did not grow, we plated our competent cells on LB media and transformed again but this time using puc19. <br /><br />
We also replated the only colony onto selective media. <br /><br />
We prepared all the solutions needed to make Bacillus subtilis competent according to Cambridge 2008 protocol (https://2008.igem.org/Team:Cambridge/Bacillus_subtilis_transformation) <br /><br />
<h2>17/07/12</h2><br /><br />
Competent cells are ok; they grew on LB media. <br /><br />
The replated colony grew but is not pink as it should be. Our hypothesis was that this part is for coli, so the expression in bacillus would be lower. To know if the bacillus were transformed or not, we extracted plasmid. <br /><br /><br />
<h2>18/07/12</h2><br /><br />
Today we ran a gel to see if Bacillus had the plasmid but it has not. <br /><br />
<br />
<br />
<h1>September</h1><br />
<h2>12/09/12</h2><br /><br />
We’ve made competent B.subtilis using the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | Two step transformation procedure]]<br />
We transformed our plasmid in E.coli RecA + by heatshock [Escherichia coli MC1061 heat shock transformation protocol]<br />
<h2>13/09/12</h2><br /><br />
Today we extracted plasmid by the Birbom –Doly method from our transformation in recA+, then we transformed bacillus with this plasmid.<br />
<h2>14/09/12</h2><br /><br />
Results:<br />
Bacillus subtilis were successfully transformed with pSB1AK3.<br />
<h2>18/09/12</h2><br /><br />
We’ve made competent B.subtilis using the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | Two step transformation procedure]]<br />
We also made competent E. coli RecA+ with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | CaCl2 procedure]].<br />
<h2>19/09/12</h2><br /><br />
We transformed RecA+ E.coli with 10 microliters [65.5 ng/microliter] of bBAK143079 plasmid.<br />
<h2>20/09/12</h2><br /><br />
As the transformation didn´t grow, we transformed again with a higher concentration of plasmid: <br />
20 microliters. <br /><br /><br />
<h2>21/09/12</h2><br /><br />
One of the petri dishes has 5 colonies; we made liquid culture to extract plasmid. <br /><br /><br />
<br />
<br />
<h1>October</h1><br /><br />
<h2>22/10/12</h2><br /><br />
We prepare the culture in a solid LB medium for the SEM Analysis (See the SEM Analysis Protocol). <br /><br />
<br /><br />
<h2>23/10/12</h2><br /><br />
We went to the Microscopy Department in UNAM to finish fixing the cells with osmium and with liquid CO2 and watch the nanotubes with the SEM. <br /><br />
'''Succesful Results!''' <br /><br />
<br /><br />
<h2>24/10/12</h2><br /><br />
We transform in the MC1061 RecA+ the 97 promoter plasmid with our '''Heavy Metal construction''' with our Transformation Procedure. <br /><br />
'''Succesful Results!''' <br /><br />
<br /><br />
<h2>25/10/12</h2><br /><br />
We made a plasmid extraction from our cells, we ran a gel with it and transform it into B. subtilis with the <br />
'''B. subtilis Tranformation Procedure'''. <br /><br />
'''Succesful Results!''' <br /><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Bacillus
Team:UNAM Genomics Mexico/Notebook/Bacillus
2012-10-27T02:40:09Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''''Bacillus subtilis'' Notebook'''</h1></center><br />
<br /><br />
<br />
<br /><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg"><br />
<tr><br />
<td id="leftcolumn2" align= "center"><p>'''[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#July | July]]'''<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#10.2F07.2F12 | 10/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#11.2F07.2F12 | 11/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#12.2F07.2F12 | 12/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#13.2F07.2F12 | 13/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#16.2F07.2F12 | 16/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#17.2F07.2F12 | 17/07/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#18.2F07.2F12 | 18/07/12]]<br /></p></td><br />
<td id="contentcolumn2" align= "center"><p>'''[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#September | September]]'''<br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#12.2F09.2F12 | 12/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#13.2F09.2F12 | 13/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#14.2F09.2F12 | 14/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#18.2F09.2F12 | 18/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#19.2F09.2F12 | 19/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#20.2F09.2F12 | 20/09/12]]<br /><br />
[[Team:UNAM_Genomics_Mexico/Notebook/Bacillus#21.2F09.2F12 | 21/09/12]]<br /></p></td><br />
<td id="rightcolumn2" align= "center"><p>[[File:UnamgenomicsBacillus.png|200px]]</p></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<h1>July</h1><br />
<h2>10/07/12</h2><br /><br />
Plate WT Bacillus on LB media. <br /><br />
Prepare TFB1 & TFBII solutions, sterilize and store at 4°C. <br /><br />
Store sterile 1.5 ml eppendorf tubes at -20 °C. <br /><br />
Store sterile falcon tubes at -4°C. <br /><br /><br />
<h2>11/07/12</h2><br /><br />
Filter TFB solutions<br /><br />
Incubate the preculture at 37°C overnight. <br /><br />
<h2>12/07/12</h2><br /><br />
Incubate the preculture again because ir fell down<br /><br /><br />
<h2>13/07/12</h2><br /><br /><br />
Follow the RbCl2 competent cell protocol. <br /><br />
Transform Bacillus with pSB4A5<br /><br /><br />
<h2>16/07/12</h2><br /><br />
Our Bacillus subtilis transformation with psB4A5 failed; the only colony that grew was not pink, then we thought it was contamination and that our cells were not competent. <br /><br />
To elucidate what was the reason for what the bacteria did not grow, we plated our competent cells on LB media and transformed again but this time using puc19. <br /><br />
We also replated the only colony onto selective media. <br /><br />
We prepared all the solutions needed to make Bacillus subtilis competent according to Cambridge 2008 protocol (https://2008.igem.org/Team:Cambridge/Bacillus_subtilis_transformation) <br /><br />
<h2>17/07/12</h2><br /><br />
Competent cells are ok; they grew on LB media. <br /><br />
The replated colony grew but is not pink as it should be. Our hypothesis was that this part is for coli, so the expression in bacillus would be lower. To know if the bacillus were transformed or not, we extracted plasmid. <br /><br /><br />
<h2>18/07/12</h2><br /><br />
Today we ran a gel to see if Bacillus had the plasmid but it has not. <br /><br />
<br />
<br />
<h1>September</h1><br />
<h2>12/09/12</h2><br /><br />
We’ve made competent B.subtilis using the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | Two step transformation procedure]]<br />
We transformed our plasmid in E.coli RecA + by heatshock [Escherichia coli MC1061 heat shock transformation protocol]<br />
<h2>13/09/12</h2><br /><br />
Today we extracted plasmid by the Birbom –Doly method from our transformation in recA+, then we transformed bacillus with this plasmid.<br />
<h2>14/09/12</h2><br /><br />
Results:<br />
Bacillus subtilis were successfully transformed with pSB1AK3.<br />
<h2>18/09/12</h2><br /><br />
We’ve made competent B.subtilis using the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | Two step transformation procedure]]<br />
We also made competent E. coli RecA+ with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Escherichia_coli_MC1061_heat_shock_transformation_protocol | CaCl2 procedure]].<br />
<h2>19/09/12</h2><br /><br />
We transformed RecA+ E.coli with 10 microliters [65.5 ng/microliter] of bBAK143079 plasmid.<br />
<h2>20/09/12</h2><br /><br />
As the transformation didn´t grow, we transformed again with a higher concentration of plasmid: <br />
20 microliters. <br /><br /><br />
<h2>21/09/12</h2><br /><br />
One of the petri dishes has 5 colonies; we made liquid culture to extract plasmid. <br /><br /><br />
<br />
<br />
<h2>October</h2><br /><br />
<h2>22/10/12</h2><br /><br />
We prepare the culture in a solid LB medium for the SEM Analysis (See the SEM Analysis Protocol). <br /><br />
<br /><br />
<h2>23/10/12</h2><br /><br />
We went to the Microscopy Department in UNAM to finish fixing the cells with osmium and with liquid CO2 and watch the nanotubes with the SEM. <br /><br />
'''Succesful Results!''' <br /><br />
<br /><br />
<h2>24/10/12</h2><br /><br />
We transform in the MC1061 RecA+ the 97 promoter plasmid with our '''Heavy Metal construction''' with our Transformation Procedure. <br /><br />
'''Succesful Results!''' <br /><br />
<br /><br />
<h2>25/10/12</h2><br /><br />
We made a plasmid extraction from our cells, we ran a gel with it and transform it into B. subtilis with the <br />
'''B. subtilis Tranformation Procedure'''. <br /><br />
'''Succesful Results!''' <br /><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Parts
Team:UNAM Genomics Mexico/Parts
2012-10-27T02:29:36Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Our Parts'''</h1></center> <br />
<br /><br />
<br /><br />
<table border="0" width="850" cellspacing="15" bgcolor="transparent" cellpadding="10" id="tablecontentbg"><br />
<tr><br />
<br />
<td id="contentcolumn" align="center"><p><br /><br />
<br /><br />
<groupparts>iGEM012 UNAM_Genomics_Mexico</groupparts><br />
<br /><br />
<br /><br />
</p></td><br />
<br />
</tr><br />
</table><br />
<br /><br />
<br /><br />
<br />
<center><h1>'''Characterization'''</h1></center><br />
<br /><br />
<br /><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851004 <h1>'''Aminoglycoside antibiotic resistance Sm+ Spc+'''</h1>] <br />
<br /><br />
<br /><br />
The aminoglycoside antibiotic resistance gene (aadA +)of pHP45Ω was originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid. Plasmid pHP45Ω was constructed in Pierre Prentki and Henry M. Krisch, as stated in NCBI with this fragment. The addA sequence can be found in, it is confirmed to be the one contained in pHP45Ω and the complete sequence of pHP45Ω plasmid can be found in the registry references.<br />
<br />
Hawaii 2008 iGEM team attempted before to construct a similar cassette but didn’t succeed. For iGEM UNAM Genomics México 2012 project, the Ω Cassette was used in the design of an OR logic gate using a recently described new type of communication system between ''Bacillus Subtilis'' cells called Nanotubes.<br />
<br><br />
<br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete1.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
<br />
Second Repetition <br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete2.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851002 <h1>'''pBAD/pXyl promoter'''</h1>]<br/><br />
<br/><br />
The characterization of this construction was made on an E.coli strain in a semi quantitative attempt. In that way, we expected that only araC was repressing the promoter because the xylR binding sites corresponds to xylR of B. subtilis. Since it requires sugars for its expression, and the system is repressed by a metabolite from the pentose metabolism pathway, we used a minimal medium, M9 (Sambrook, 1989), but we changed the glucose for arginine as carbon source in order to get the lowest interference in the expression of GFP from another monosaccharides species in the medium. We use a gradient for both, xylose and arabinose on cultures at 0.1 O.D (540 nm). We could see that the expression of GFP increases as the amount of sugars added also increases. An amount of 0.01% (g/ml) of arabinose is enough for an increase of 4 times the basal expression, and the maximum production is approximately 5 times greater for the downstream genes with 0.1%(g/ml) of arabinose. Xylose gradient had a small contribution on the expression of GFP, which could be attributed to the partial similarity of the binding sites between xylR from B. subtilis and E. coli. The measurements were made with a filter fluorometer based in three different measures for each condition.<br/><br />
<br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM Pbadxyl.png|600px]]<br /><br /><p>Expression of GFP in the sugars gradient measured in fluorescence units<br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling/Parameters
Team:UNAM Genomics Mexico/Modeling/Parameters
2012-10-27T02:28:19Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<center><h1>'''Parameters and considerations'''</h1></center> <br />
<br /><br />
Our team made both a deterministic and a stochastic model. There is a strong emphasis on exploring the dynamics given by the system's structure and the individual component's parameters in either type of model. The parameters were obtained from articles and databases such as Harvard Medical School's [http://bionumbers.hms.harvard.edu BioNumbers]. Another important source of information was the general binding rates included in the [https://github.com/jkrivine/KaSim Kappa Simulator] manual.<br /><br />
<br /><br />
<br /><br />
'''General assumptions:'''<br /><br />
<br /><br />
●This model's scope is single cell. While the regulation steps were modeled through Hill equations, other reactions were treated under Michaelis-Menten assumptions, notably isomerizations and transport processes (i.e. those that involved energy consumption).<br /><br />
<br />
●The maximal transcription rate for the system species was obtained by dividing the maximum number of nucleotides processed by the RNA polymerase (i.e. maximum polymerase activity), by the length of the nucleotide sequence.<br /><br />
<br />
●Translational rates were obtained by getting the maximum number of amino acids added by the ribosome divided by the length of the protein in amino acids.<br /><br />
<br />
●The protein degradation rate is considered equal for all the protein species in the model.<br /><br />
<br />
●The degradation rate for mRNAs is equal for all the different species of mRNAs.<br /><br />
<br />
●The different binding sites of the same transcription factor have the same affinity to the TF.<br /><br />
<br />
●For each mRNA species, its concentration will be the sum of the production as affected by its respective promoter and the transcription factors that regulate it, minus the degradation of the mRNA at that time.<br /><br />
<br />
●For each protein, the change in its concentration depends in the amount of protein produced by translation minus the degradation rate of the protein.<br /><br />
<br />
●The Hill coefficients tend to be 2.<br /><br />
<br />
●The parameters of concentration were expressed in terms of molecules per cell (volume of a B.subtilis cell ~10^-14) and the time units were converted sec.<br /><br />
<br />
<h2>'''Parameters'''</h2><br /><br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_Par1.png|870px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_Par2.png|870px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_Par3.png|870px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_Par4.png|870px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM_Par5.png|870px]]<br /><br /><p>'''NOTE: mol refers to molecules, not the unit mol'''<br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br />
<br /><br />
<br /><br />
<br />
<br />
<br />
== '''References''' ==<br />
<br /><br />
<br />
[1]Rust, L.; E.C. Pcsi, B.H. Iglewski. Analysis of the Pseudomonas aeruginosa elastase (lasB) regulatory region.J. Bacteriol. February 1996 vol. 178 no. 4 1134-1140.<br /><br />
<br />
[2]Lyons T, David J E. Transport and storage of metal ions in Biology. Chapter V. "http://www.ffame.org/pubs/Transport%20and%20Storage%20of%20Metal%20Ions%20in%20Biology.pdf ".<br /><br />
<br />
[3]Fujita M, Tanaka K, Takahashi H, Amemura A. Transciption of hte principal sigma-factor genes, rpoD and rpoS, in Pseudomonas aeruginosa is controlled according to the growth phase. Mol Microbiology. 1994 Sep;13 (6):1071-7<br /><br />
<br />
[4]Laval Université. Chapitre 2 Régulation transcriptionnelle chez Pseudomonas aeruginosa. Collection Mémoires et théses électroniques. "http://archimede.bibl.ulaval.ca/archimede/fichiers/24237/ch02.html ".<br /><br />
<br />
[5]Kreuzer P, Gartner D, Allmansberger R, Hillen W. Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator.J Bacteriol. 1989 Jul ; 171(7): 3840-5<br /><br />
<br />
[6]Kreuzer P, Gärtner D, Allmansberger R, Hillen W. Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator. J Bacteriol. 1989 Jul;171(7):3840-5.<br /><br />
<br />
[7]Buchler N, Gerland U, Hwa T. On schemes of combinatorial transcription logic. PNAS April 29, 2003 vol. 100no. 9 5136-5141 DOI:10.1073<br /><br />
<br />
[8]Bhavsar A, Zhao X, Brown E. Development and characterization of a xylose-dependent systme for expression of cloned genes in Bacillus subtilis: Conditional complementation of a Teichoic acid mutant. Appl Environ Microbiol. 2001 January; 67(1): 403–410. doi: 10.1128/AEM.67.1.403-410.2001<br /><br />
<br />
[9]Moore C, Gablla A, Hui M, Ye R W, Helmann J D. Genetic and physiological responses of Bacillus subtillis to metal ion stress. Molecular Microbiology. July 2005 Vol 57(1): 27–40.<br /><br />
<br />
[10]Fujita M, Gonzáles-Pastor J E, Losick R. High- and low-threshold genes in the Spo0A regulon of Bacillus subtilis. J. Bacteriol. February 2005 vol. 187 no. 4 1357-1368. DOI: 10.1128/JB.187.4.1357-1368.2005.<br /><br />
<br />
[11]Klaus A, Hueck C, Hillen W. Catabolite repression of the Bacillus subtilis xyl operon involves a cis element functional in the context of a unrelated sequence, and glucose exerts additional xylR-dependent repression. J Bacteriol. 1994 March; 176(6): 1738–1745. PMCID: PMC205262<br /><br />
<br />
[12]Jarmer H, Larsen T S, et al. Sigma A recognition sites in the Bacillus subtilis genome. Microbiology. September 2001. Vol 147(9):2417-2424.<br /><br />
<br />
[13]Helmann J D. Sigma factors in gene expression. Nature. 2001. Encyclopedia of life sciences.<br /><br />
<br />
[14]Gärtner D, Geissendörfer, Hillen W. Expression of the Bacillus subtilis xyl operon is repressed at the level of transcription and is induced by xylose. J Bacteriol. 1988. 170(7):3102-3109.<br /><br />
<br />
[15]Bintu L, Buchler N, Garcia H G, Gerland U, Hwa T. Transcriptional regulation by the numbers: Models. Genetics & Development. Opinion. 2005. 15(2):116-124.DOI:10.1016<br /><br />
<br />
[16]Goryachev A B, Toh D J, Lee J. System analysis of a quorum sensing network: Design constraints imposed by the functional requirements, network topology and kinetic constraints. BioSystems. 2006. 83:178-187.<br /><br />
<br />
[17]Procházková K, Cermáková K, Pachl P, et al. Structure of the effector-binding domain of the arabinose repressor AraR from Bacillus subtilis.<br /><br />
<br />
[18]Megerle, Judith. Cell to cell variability of gene expression dynamics in inducible regulatory networks. Dissertation of Physics Faculty of Ludwig Maximilians University of Munich. January 2011.<br /><br />
<br />
[19]Krispin O, Allmansberger R. The Bacillus subtilis AraE protein displays a broad substrate specificity for several different sugars. J Bacteriol. 1998 June; 180(12): 3250–3252. PMCID: PMC107832.<br /><br />
<br />
[20]Gu Y, Ren C, Sun Z, Rodionov D A, Zhang W, Yang S, Yang C, Jiang W. Reconstruction of xylose utilization pathway and regulons in Firmicutes. BMC Genomics 2010, 11:255 doi:10.1186/1471-2164-11-25.<br /><br />
<br />
[21]Moore C, Helmann J D. Metal ion homeostasis in Bacillus subtilis. Microbiology. April 2005. 8(2):188-195 DOI: 10.1016/j.mib.2005.02.007<br /><br />
<br />
[22]Sá-Nogueira I, Nogueira T V, Soares S, Lencastre H. The Bacillus subtilis L-arabinose (ara) operon: nucleotide sequence, genetic organization and expression. Microbiology. 1997. 143:957-969.<br /><br />
<br />
[23]Chakravorty D, Wang B, Won Lee C, Giedroc D P, Merz K M. Simulations of allosteric motions in the zinc sensor CzrA. J. Am. Chem. Soc. 2012, 134(7):3367-3376.<br /><br />
<br /><br />
<br /><br />
<br /><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Attributions
Team:UNAM Genomics Mexico/Attributions
2012-10-27T02:27:25Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br />
<center> <h1>'''Attributions'''</h1> </center><br />
<br /><br /><br />
<br />
[[File:Unamgenomicsmexicoteamundergrads.JPG|870px|center|thumb|Here we are the Super UNAM Genomics TEAM :) everybody is a hard worker]]<br />
<br /><br /><br />
<br />
<br />
All work on this project was done by members of the UNAM_Genomics_Mexico's 2012 iGEM Team. Original constructs from which to obtain P4 gene and A3 promoter to Biobrick were supplied by Dr. Margarita Salas from Autonomous University of Madrid. ''Bacillus subtilis'' strains were provided by Dr. Ben-Yehuda from School of Medicine-IMRIC, Dr. Gabriela Olmedo from Center for Research and Advanced Studies of the National Polytechnic Institute (CINVESTAV,IPN) Campus Irapuato and Dr. Guillermo Gosset from Biotechnology Institute (IBT, UNAM). Supervision and direction was provided by members of the Center for Genomic Sciences, (CCG) however all work was done by the team, during the months of May 2012 to present. Most work was developed in the CCG from National Autonomous University of México (UNAM). Part of the work with ''Bacillus subtillis'' was done in CINVESTAV Campus Irapuato. Special thanks to Amhed Missael Vargas Velazquez for his support with MATLAB®, Dr. Otto Geiger for allowing us to use its lab at CCG to work with the OR and the Sweet AND and to Dr. Gabriela Olmedo for her help with ''Bacillus subtilis''. Also Dr. Fernando García from The Cellular Physiology Department of UNAM that helped us with the SEM Analysis and Alfonso Leija,PhD from the Functional Genomics of Eukaryotes Research Program at Center of Genomic Sciences, who helped us with parts characterization.<br />
<br />
<br /><br /><br />
<center><h1>'''Our Instructors'''</h1><br /></center><br /><br /><br />
<br /><br /><br />
<br />
[[File:Unamgenomicsmexicoteamadvisors.JPG|870px|center|thumb|Our instructors and advisors ready to be shoot by a Biobrick squad]]<br /><br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamDavid.jpg| 400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''David Romero-Camarena'''<br />This guy is our instructor<br />•Main Instructor<br /><br />
</p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamResendis.jpg| 400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Osbaldo Resendis-Antonio'''<br />This guy is our instructor<br />• Modeling instructor<br /></p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Attributions#Attributions]]<br />
<center><h1>'''Our Advisors'''</h1><br /></center><br /><br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamHector.JPG|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Hector F. Medina'''<br />This guy is our modeling advisor<br />Hector is well known in the iGEM dark side as "Mamma Raven". He is also awesome modeling... just look at his photo!</p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamRogelio.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Rogelio Hernandez Tamayo'''<br />This guy is our wet-lab advisor<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsmexicoteamMiguel.JPG|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Miguel Angel Wences Guzman'''<br />This guy is our wet-lab advisor<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamOsam.jpg| 400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Fares Osam Yáñez Cuña'''<br />This guy is our wet-lab advisor<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamPepe_con_ursus.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Jose Antonio Alonso-Pavon'''<br />This guy is our Ethics and Human Practices advisor<br />"Pepe" has a master degree in Bioethics</p></td><br />
</tr><br />
<br />
</table><br />
</center><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Attributions#Attributions]]<br />
<center> <h1>'''Meet our team'''</h1> </center><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:Unamgenomicsteamabiel.JPG|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Abiel Trevino Garza'''<br />• Project design<br />
•Part design<br /><br />
•Wiki design<br /><br />
•Wiki implementation<br /><br />
•Human practices/Video edition<br /><br />
•Human practices/Recording<br /><br />
•Human practices/ Biosintetizarte 2.0<br /><br />
•Human practices/ Symposium Participation<br /><br />
•Funds raising <br /><br />
•Public relations<br /><br />
•Wet lab Sweet AND<br /><br />
</p> <br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamMariange.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Maria Angelica Bravo'''<br /> <br />
•Project design<br /><br />
•Part design<br /><br />
•Public relations<br /><br />
•Human practices/ video script and edition<br /><br />
•Human practices/ Symposium Participation<br /><br />
•Wet lab/ ''Bacillus subtilis''<br /><br />
•Wet lab/ SEM analysis<br /><br />
<br /></p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamDiego.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Diego Rodríguez'''<br />•Project design<br /><br />
•Part design<br /><br />
•Modeling<br /><br />
•Wet lab/ sweet AND<br /><br />
•Wet lab/ OR<br /><br />
<br /></p></td><br />
</tr><br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamWera.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Dulce Beatriz Vargas Landin'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ Heavy-Metal AND<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsmexicoteamJonathan.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Jonathan Padilla Gómez'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ Sweet AND<br /><br />
•Wet lab / OR<br /><br />
•Human practices/ Symposium Participation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamKaren.jpeg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Ana Karen Mojica Avila'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ Heavy-Metal AND<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamBenja.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Benjamín Hernández Rodríguez'''<br />•Project design<br /><br />
•Part design<br /><br />
•Human practices/ video script<br /><br />
•Human practices/ video recording<br /><br />
•Part documentation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamDiana.JPG|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Diana Vera Cruz'''<br />• Project design<br />
•Part design<br /><br />
•Modeling<br /><br />
•Human practices/ video script<br /><br />
•Human practices/ video recording<br /><br />
•Human practices/ Symposium Participation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamLessonN7.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Roberto Tirado Magallanes'''<br />•Project design<br /><br />
•Part design<br /><br />
•Modeling<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsmexicotemLaura.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Laura Teresa Jimenez Barrón'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ ''Bacillus subtillis''<br /><br />
•Wet lab/ SEM analysis<br /><br />
•Part characterization<br /><br />
•The sweet voice of Miss. Cohnnie<br /><br />
•Human practices/ Symposium Participation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamRebe.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Rebeca Borges Monroy'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ Heavy-Metal AND<br /><br />
•Human Practices/ Video Edition<br /><br />
•Human practices/ Symposium Participation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Attributions#Attributions]]<br />
<br />
<br />
Each team must clearly attribute work done by the team on this page. They must distinguish work done by the team from work done by others, including the host labs, advisors, instructors, graduate students, and postgraduate masters students.<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Attributions
Team:UNAM Genomics Mexico/Attributions
2012-10-27T02:25:20Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br />
<center> <h1>'''Attributions'''</h1> </center><br />
<br /><br /><br />
<br />
[[File:Unamgenomicsmexicoteamundergrads.JPG|870px|center|thumb|Here we are the Super UNAM Genomics TEAM :) everybody is a hard worker]]<br />
<br /><br /><br />
<br />
<br />
All work on this project was done by members of the UNAM_Genomics_Mexico's 2012 iGEM Team. Original constructs from which to obtain P4 gene and A3 promoter to Biobrick were supplied by Dr. Margarita Salas from Autonomous University of Madrid. ''Bacillus subtilis'' strains were provided by Dr. Ben-Yehuda from School of Medicine-IMRIC, Dr. Gabriela Olmedo from Center for Research and Advanced Studies of the National Polytechnic Institute (CINVESTAV,IPN) Campus Irapuato and Dr. Guillermo Gosset from Biotechnology Institute (IBT, UNAM). Supervision and direction was provided by members of the Center for Genomic Sciences, (CCG) however all work was done by the team, during the months of May 2012 to present. Most work was developed in the CCG from National Autonomous University of México (UNAM). Part of the work with ''Bacillus subtillis'' was done in CINVESTAV Campus Irapuato. Special thanks to Amhed Missael Vargas Velazquez for his support with MATLAB®, Dr. Otto Geiger for allowing us to use its lab at CCG to work with the OR and the Sweet AND and to Dr. Gabriela Olmedo for her help with ''Bacillus subtilis''. Also Dr. Fernando García from The Cellular Physiology Department of UNAM that helped us with the SEM Analysis. Also PhD Alfonso Leija from Functional Genomics of Eukaryotes Research Program at Center of Genomic Sciences, who help us with parts characterization.<br />
<br />
<br /><br /><br />
<center><h1>'''Our Instructors'''</h1><br /></center><br /><br /><br />
<br /><br /><br />
<br />
[[File:Unamgenomicsmexicoteamadvisors.JPG|870px|center|thumb|Our instructors and advisors ready to be shoot by a Biobrick squad]]<br /><br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamDavid.jpg| 400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''David Romero-Camarena'''<br />This guy is our instructor<br />•Main Instructor<br /><br />
</p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamResendis.jpg| 400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Osbaldo Resendis-Antonio'''<br />This guy is our instructor<br />• Modeling instructor<br /></p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Attributions#Attributions]]<br />
<center><h1>'''Our Advisors'''</h1><br /></center><br /><br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamHector.JPG|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Hector F. Medina'''<br />This guy is our modeling advisor<br />Hector is well known in the iGEM dark side as "Mamma Raven". He is also awesome modeling... just look at his photo!</p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamRogelio.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Rogelio Hernandez Tamayo'''<br />This guy is our wet-lab advisor<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsmexicoteamMiguel.JPG|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Miguel Angel Wences Guzman'''<br />This guy is our wet-lab advisor<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamOsam.jpg| 400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Fares Osam Yáñez Cuña'''<br />This guy is our wet-lab advisor<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamPepe_con_ursus.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Jose Antonio Alonso-Pavon'''<br />This guy is our Ethics and Human Practices advisor<br />"Pepe" has a master degree in Bioethics</p></td><br />
</tr><br />
<br />
</table><br />
</center><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Attributions#Attributions]]<br />
<center> <h1>'''Meet our team'''</h1> </center><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:Unamgenomicsteamabiel.JPG|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Abiel Trevino Garza'''<br />• Project design<br />
•Part design<br /><br />
•Wiki design<br /><br />
•Wiki implementation<br /><br />
•Human practices/Video edition<br /><br />
•Human practices/Recording<br /><br />
•Human practices/ Biosintetizarte 2.0<br /><br />
•Human practices/ Symposium Participation<br /><br />
•Funds raising <br /><br />
•Public relations<br /><br />
•Wet lab Sweet AND<br /><br />
</p> <br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamMariange.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Maria Angelica Bravo'''<br /> <br />
•Project design<br /><br />
•Part design<br /><br />
•Public relations<br /><br />
•Human practices/ video script and edition<br /><br />
•Human practices/ Symposium Participation<br /><br />
•Wet lab/ ''Bacillus subtilis''<br /><br />
•Wet lab/ SEM analysis<br /><br />
<br /></p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamDiego.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Diego Rodríguez'''<br />•Project design<br /><br />
•Part design<br /><br />
•Modeling<br /><br />
•Wet lab/ sweet AND<br /><br />
•Wet lab/ OR<br /><br />
<br /></p></td><br />
</tr><br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamWera.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Dulce Beatriz Vargas Landin'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ Heavy-Metal AND<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsmexicoteamJonathan.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Jonathan Padilla Gómez'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ Sweet AND<br /><br />
•Wet lab / OR<br /><br />
•Human practices/ Symposium Participation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamKaren.jpeg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Ana Karen Mojica Avila'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ Heavy-Metal AND<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamBenja.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Benjamín Hernández Rodríguez'''<br />•Project design<br /><br />
•Part design<br /><br />
•Human practices/ video script<br /><br />
•Human practices/ video recording<br /><br />
•Part documentation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamDiana.JPG|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Diana Vera Cruz'''<br />• Project design<br />
•Part design<br /><br />
•Modeling<br /><br />
•Human practices/ video script<br /><br />
•Human practices/ video recording<br /><br />
•Human practices/ Symposium Participation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamLessonN7.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Roberto Tirado Magallanes'''<br />•Project design<br /><br />
•Part design<br /><br />
•Modeling<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsmexicotemLaura.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Laura Teresa Jimenez Barrón'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ ''Bacillus subtillis''<br /><br />
•Wet lab/ SEM analysis<br /><br />
•Part characterization<br /><br />
•The sweet voice of Miss. Cohnnie<br /><br />
•Human practices/ Symposium Participation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamRebe.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Rebeca Borges Monroy'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ Heavy-Metal AND<br /><br />
•Human Practices/ Video Edition<br /><br />
•Human practices/ Symposium Participation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Attributions#Attributions]]<br />
<br />
<br />
Each team must clearly attribute work done by the team on this page. They must distinguish work done by the team from work done by others, including the host labs, advisors, instructors, graduate students, and postgraduate masters students.<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Parts
Team:UNAM Genomics Mexico/Parts
2012-10-27T02:22:58Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Our Parts'''</h1></center> <br />
<br /><br />
<br /><br />
<table border="0" width="850" cellspacing="15" bgcolor="transparent" cellpadding="10" id="tablecontentbg"><br />
<tr><br />
<br />
<td id="contentcolumn" align="center"><p><br /><br />
<br /><br />
<groupparts>iGEM012 UNAM_Genomics_Mexico</groupparts><br />
<br /><br />
<br /><br />
</p></td><br />
<br />
</tr><br />
</table><br />
<br /><br />
<br /><br />
<br />
<center><h1>'''Characterization'''</h1></center><br />
<br /><br />
<br /><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851004 <h1>'''Aminoglycoside antibiotic resistance Sm+ Spc+'''</h1>] <br />
<br /><br />
<br /><br />
The aminoglycoside antibiotic resistance gene (aadA +)of pHP45Ω was originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid. Plasmid pHP45Ω was constructed in Pierre Prentki and Henry M. Krisch, as stated in NCBI with this fragment. The addA sequence can be found in, it is confirmed to be the one contained in pHP45Ω and the complete sequence of pHP45Ω plasmid can be found in the registry references.<br />
<br />
Hawaii 2008 iGEM team attempted before to construct a similar cassette but didn’t succeed. For iGEM UNAM Genomics México 2012 project, the Ω Cassette was used in the design of an OR logic gate using a recently described new type of communication system between ''Bacillus Subtilis'' cells called Nanotubes.<br />
<br><br />
<br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete1.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
<br />
Second Repetition <br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete2.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851002 <h1>'''XylR/pBAD promoter'''</h1>]<br/><br />
<br/><br />
The characterization of this construction was made on an E.coli strain in a semi quantitative attempt. In that way, we expected that only araC was repressing the promoter because the xylR binding sites corresponds to xylR of B. subtilis. Since it requires sugars for its expression, and the system is repressed by a metabolite from the pentose metabolism pathway, we used a minimal medium, M9 (Sambrook, 1989), but we changed the glucose for arginine as carbon source in order to get the lowest interference in the expression of GFP from another monosaccharides species in the medium. We use a gradient for both, xylose and arabinose on cultures at 0.1 O.D (540 nm). We could see that the expression of GFP increases as the amount of sugars added also increases. An amount of 0.01% (g/ml) of arabinose is enough for an increase of 4 times the basal expression, and the maximum production is approximately 5 times greater for the downstream genes with 0.1%(g/ml) of arabinose. Xylose gradient had a small contribution on the expression of GFP, which could be attributed to the partial similarity of the binding sites between xylR from B. subtilis and E. coli. The measurements were made with a filter fluorometer based in three different measures for each condition.<br/><br />
<br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM Pbadxyl.png|600px]]<br /><br /><p>Expression of GFP in the sugars gradient measured in fluorescence units<br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Parts
Team:UNAM Genomics Mexico/Parts
2012-10-27T02:22:26Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Our Parts'''</h1></center> <br />
<br /><br />
<br /><br />
<br />
<br />
<table border="0" width="850" cellspacing="15" bgcolor="transparent" cellpadding="10" id="tablecontentbg"><br />
<tr><br />
<br />
<td id="contentcolumn" align="center"><p><br /><br />
<br /><br />
<groupparts>iGEM012 UNAM_Genomics_Mexico</groupparts><br />
<br /><br />
<br /><br />
</p></td><br />
<br />
</tr><br />
</table><br />
<br />
<br />
<center><h1>'''Characterization'''</h1></center><br />
<br /><br />
<br /><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851004 <h1>'''Aminoglycoside antibiotic resistance Sm+ Spc+'''</h1>] <br />
<br />
The aminoglycoside antibiotic resistance gene (aadA +)of pHP45Ω was originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid. Plasmid pHP45Ω was constructed in Pierre Prentki and Henry M. Krisch, as stated in NCBI with this fragment. The addA sequence can be found in, it is confirmed to be the one contained in pHP45Ω and the complete sequence of pHP45Ω plasmid can be found in the registry references.<br />
<br />
Hawaii 2008 iGEM team attempted before to construct a similar cassette but didn’t succeed. For iGEM UNAM Genomics México 2012 project, the Ω Cassette was used in the design of an OR logic gate using a recently described new type of communication system between ''Bacillus Subtilis'' cells called Nanotubes.<br />
<br><br />
<br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete1.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
<br />
Second Repetition <br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete2.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851002 <h1>'''XylR/pBAD promoter'''</h1>]<br/><br />
<br/><br />
The characterization of this construction was made on an E.coli strain in a semi quantitative attempt. In that way, we expected that only araC was repressing the promoter because the xylR binding sites corresponds to xylR of B. subtilis. Since it requires sugars for its expression, and the system is repressed by a metabolite from the pentose metabolism pathway, we used a minimal medium, M9 (Sambrook, 1989), but we changed the glucose for arginine as carbon source in order to get the lowest interference in the expression of GFP from another monosaccharides species in the medium. We use a gradient for both, xylose and arabinose on cultures at 0.1 O.D (540 nm). We could see that the expression of GFP increases as the amount of sugars added also increases. An amount of 0.01% (g/ml) of arabinose is enough for an increase of 4 times the basal expression, and the maximum production is approximately 5 times greater for the downstream genes with 0.1%(g/ml) of arabinose. Xylose gradient had a small contribution on the expression of GFP, which could be attributed to the partial similarity of the binding sites between xylR from B. subtilis and E. coli. The measurements were made with a filter fluorometer based in three different measures for each condition.<br/><br />
<br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM Pbadxyl.png|600px]]<br /><br /><p>Expression of GFP in the sugars gradient measured in fluorescence units<br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Parts
Team:UNAM Genomics Mexico/Parts
2012-10-27T02:17:52Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Our Parts'''</h1></center> <br />
<br /><br />
<br /><br />
<br />
<br />
<table border="0" width="850" cellspacing="15" bgcolor="transparent" cellpadding="10" id="tablecontentbg"><br />
<tr><br />
<br />
<td id="contentcolumn" align="center"><p><br /><br />
<br /><br />
<groupparts>iGEM012 UNAM_Genomics_Mexico</groupparts><br />
<br /><br />
<br /><br />
</p></td><br />
<br />
</tr><br />
</table><br />
<br />
<br />
<center><h1>'''Characterization'''</h1></center><br />
<br /><br />
<br /><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851004 <h1>'''Aminoglycoside antibiotic resistance Sm+ Spc+'''</h1>] <br />
<br />
The aminoglycoside antibiotic resistance gene (aadA +)of pHP45Ω was originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid. Plasmid pHP45Ω was constructed in Pierre Prentki and Henry M. Krisch, as stated in NCBI with this fragment. The addA sequence can be found in, it is confirmed to be the one contained in pHP45Ω and the complete sequence of pHP45Ω plasmid can be found in the registry references.<br />
<br />
Hawaii 2008 iGEM team attempted before to construct a similar cassette but didn’t succeed. For iGEM UNAM Genomics México 2012 project, the Ω Cassette was used in the design of an OR logic gate using a recently described new type of communication system between ''Bacillus Subtilis'' cells called Nanotubes.<br />
<br><br />
<br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete1.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
<br />
Second Repetition <br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete2.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851002 <h1>XylR/pBAD promoter</h1>]<br/><br />
<br/><br />
The characterization of this construction was made on an E.coli strain in a semi quantitative attempt. In that way, we expected that only araC was repressing the promoter because the xylR binding sites corresponds to xylR of B. subtilis. Since it requires sugars for its expression, and the system is repressed by a metabolite from the pentose metabolism pathway, we used a minimal medium, M9 (Sambrook, 1989), but we changed the glucose for arginine as carbon source in order to get the lowest interference in the expression of GFP from another monosaccharides species in the medium. We use a gradient for both, xylose and arabinose on cultures at 0.1 O.D (540 nm). We could see that the expression of GFP increases as the amount of sugars added also increases. An amount of 0.01% (g/ml) of arabinose is enough for an increase of 4 times the basal expression, and the maximum production is approximately 5 times greater for the downstream genes with 0.1%(g/ml) of arabinose. Xylose gradient had a small contribution on the expression of GFP, which could be attributed to the partial similarity of the binding sites between xylR from B. subtilis and E. coli. The measurements were made with a filter fluorometer based in three different measures for each condition.<br/><br />
<br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM Pbadxyl.png|600px]]<br /><br /><p>Expression of GFP in the sugars gradient measured in fluorescence units<br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Parts
Team:UNAM Genomics Mexico/Parts
2012-10-27T02:17:30Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Our Parts'''</h1></center> <br />
<br /><br />
<br /><br />
<br />
<br />
<table border="0" width="850" cellspacing="15" bgcolor="transparent" cellpadding="10" id="tablecontentbg"><br />
<tr><br />
<br />
<td id="contentcolumn" align="center"><p><br /><br />
<br /><br />
<groupparts>iGEM012 UNAM_Genomics_Mexico</groupparts><br />
<br /><br />
<br /><br />
</p></td><br />
<br />
</tr><br />
</table><br />
<br />
<br />
<center><h1>'''Characterization'''</h1></center><br />
<br /><br />
<br /><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851004 <h1>'''Aminoglycoside antibiotic resistance Sm+ Spc+'''</h1>] <br />
<br />
The aminoglycoside antibiotic resistance gene (aadA +)of pHP45Ω was originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid. Plasmid pHP45Ω was constructed in Pierre Prentki and Henry M. Krisch, as stated in NCBI with this fragment. The addA sequence can be found in, it is confirmed to be the one contained in pHP45Ω and the complete sequence of pHP45Ω plasmid can be found in the registry references.<br />
<br />
Hawaii 2008 iGEM team attempted before to construct a similar cassette but didn’t succeed. For iGEM UNAM Genomics México 2012 project, the Ω Cassette was used in the design of an OR logic gate using a recently described new type of communication system between ''Bacillus Subtilis'' cells called Nanotubes.<br />
<br><br />
<br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete1.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
<br />
Second Repetition <br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete2.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851002 <h1>XylR/pBAD promoter</h1>]<br/><br />
<br/><br />
The characterization of this construction was made on an E.coli strain in a semi quantitative attempt. In that way, we expected that only araC was repressing the promoter because the xylR binding sites corresponds to xylR of B. subtilis. Since it requires sugars for its expression, and the system is repressed by a metabolite from the pentose metabolism pathway, we used a minimal medium, M9 (Sambrook, 1989), but we changed the glucose for arginine as carbon source in order to get the lowest interference in the expression of GFP from another monosaccharides species in the medium. We use a gradient for both, xylose and arabinose on cultures at 0.1 O.D (540 nm). We could see that the expression of GFP increases as the amount of sugars added also increases. An amount of 0.01% (g/ml) of arabinose is enough for an increase of 4 times the basal expression, and the maximum production is approximately 5 times greater for the downstream genes with 0.1%(g/ml) of arabinose. Xylose gradient had a small contribution on the expression of GFP, which could be attributed to the partial similarity of the binding sites between xylR from B. subtilis and E. coli. The measurements were made with a filter fluorometer based in three different measures for each condition.<br/><br />
<br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM Pbadxyl.png|400px]]<br /><br /><p>Expression of GFP in the sugars gradient measured in fluorescence units<br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Parts
Team:UNAM Genomics Mexico/Parts
2012-10-27T02:15:48Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Our Parts'''</h1></center> <br />
<br /><br />
<br /><br />
<br />
<br />
<table border="0" width="850" cellspacing="15" bgcolor="transparent" cellpadding="10" id="tablecontentbg"><br />
<tr><br />
<br />
<td id="contentcolumn" align="center"><p><br /><br />
<br /><br />
<groupparts>iGEM012 UNAM_Genomics_Mexico</groupparts><br />
<br /><br />
<br /><br />
</p></td><br />
<br />
</tr><br />
</table><br />
<br />
<br />
<center><h1>'''Characterization'''</h1></center><br />
<br /><br />
<br /><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851004 <h1>'''Aminoglycoside antibiotic resistance Sm+ Spc+'''</h1>] <br />
<br />
The aminoglycoside antibiotic resistance gene (aadA +)of pHP45Ω was originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid. Plasmid pHP45Ω was constructed in Pierre Prentki and Henry M. Krisch, as stated in NCBI with this fragment. The addA sequence can be found in, it is confirmed to be the one contained in pHP45Ω and the complete sequence of pHP45Ω plasmid can be found in the registry references.<br />
<br />
Hawaii 2008 iGEM team attempted before to construct a similar cassette but didn’t succeed. For iGEM UNAM Genomics México 2012 project, the Ω Cassette was used in the design of an OR logic gate using a recently described new type of communication system between ''Bacillus Subtilis'' cells called Nanotubes.<br />
<br><br />
<br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete1.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
<br />
Second Repetition <br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete2.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851002 <h1>XylR/pBAD promoter</h1>]<br/><br />
<br/><br />
The characterization of this construction was made on an E.coli strain in a semi quantitative attempt. In that way, we expected that only araC was repressing the promoter because the xylR binding sites corresponds to xylR of B. subtilis. Since it requires sugars for its expression, and the system is repressed by a metabolite from the pentose metabolism pathway, we used a minimal medium, M9 (Sambrook, 1989), but we changed the glucose for arginine as carbon source in order to get the lowest interference in the expression of GFP from another monosaccharides species in the medium. We use a gradient for both, xylose and arabinose on cultures at 0.1 O.D (540 nm). We could see that the expression of GFP increases as the amount of sugars added also increases. An amount of 0.01% (g/ml) of arabinose is enough for an increase of 4 times the basal expression, and the maximum production is approximately 5 times greater for the downstream genes with 0.1%(g/ml) of arabinose. Xylose gradient had a small contribution on the expression of GFP, which could be attributed to the partial similarity of the binding sites between xylR from B. subtilis and E. coli. The measurements were made with a filter fluorometer based in three different measures for each condition.<br/><br />
<br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM Pbadxyl.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Parts
Team:UNAM Genomics Mexico/Parts
2012-10-27T02:15:31Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
<br />
<br /><br />
<center><h1>'''Our Parts'''</h1></center> <br />
<br /><br />
<br /><br />
<br />
<br />
<table border="0" width="850" cellspacing="15" bgcolor="transparent" cellpadding="10" id="tablecontentbg"><br />
<tr><br />
<br />
<td id="contentcolumn" align="center"><p><br /><br />
<br /><br />
<groupparts>iGEM012 UNAM_Genomics_Mexico</groupparts><br />
<br /><br />
<br /><br />
</p></td><br />
<br />
</tr><br />
</table><br />
<br />
<br />
<center><h1>'''Characterization'''</h1></center><br />
<br /><br />
<br /><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851004 <h1>'''Aminoglycoside antibiotic resistance Sm+ Spc+'''</h1>] <br />
<br />
The aminoglycoside antibiotic resistance gene (aadA +)of pHP45Ω was originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid. Plasmid pHP45Ω was constructed in Pierre Prentki and Henry M. Krisch, as stated in NCBI with this fragment. The addA sequence can be found in, it is confirmed to be the one contained in pHP45Ω and the complete sequence of pHP45Ω plasmid can be found in the registry references.<br />
<br />
Hawaii 2008 iGEM team attempted before to construct a similar cassette but didn’t succeed. For iGEM UNAM Genomics México 2012 project, the Ω Cassette was used in the design of an OR logic gate using a recently described new type of communication system between ''Bacillus Subtilis'' cells called Nanotubes.<br />
<br><br />
<br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete1.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
<br />
Second Repetition <br><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:Unamgenomicsomegacassete2.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K851002 <h1>XylR/pBAD promoter</h1>]<br/><br />
<br/><br />
The characterization of this construction was made on an E.coli strain in a semi quantitative attempt. In that way, we expected that only araC was repressing the promoter because the xylR binding sites corresponds to xylR of B. subtilis. Since it requires sugars for its expression, and the system is repressed by a metabolite from the pentose metabolism pathway, we used a minimal medium, M9 (Sambrook, 1989), but we changed the glucose for arginine as carbon source in order to get the lowest interference in the expression of GFP from another monosaccharides species in the medium. We use a gradient for both, xylose and arabinose on cultures at 0.1 O.D (540 nm). We could see that the expression of GFP increases as the amount of sugars added also increases. An amount of 0.01% (g/ml) of arabinose is enough for an increase of 4 times the basal expression, and the maximum production is approximately 5 times greater for the downstream genes with 0.1%(g/ml) of arabinose. Xylose gradient had a small contribution on the expression of GFP, which could be attributed to the partial similarity of the binding sites between xylR from B. subtilis and E. coli. The measurements were made with a filter fluorometer based in three different measures for each condition.<br/><br />
<br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UGM Pbadxyl.png|400px]]<br /><br /><p><br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Attributions
Team:UNAM Genomics Mexico/Attributions
2012-10-27T02:05:24Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br />
<center> <h1>'''Attributions'''</h1> </center><br />
<br /><br /><br />
<br />
[[File:Unamgenomicsmexicoteamundergrads.JPG|870px|center|thumb|Here we are the Super UNAM Genomics TEAM :) everybody is a hard worker]]<br />
<br /><br /><br />
<br />
<br />
All work on this project was done by members of the UNAM_Genomics_Mexico's 2012 iGEM Team. Original constructs from which to obtain P4 gene and A3 promoter to Biobrick were supplied by Dr. Margarita Salas from Autonomous University of Madrid. ''Bacillus subtilis'' strains were provided by Dr. Ben-Yehuda from School of Medicine-IMRIC, Dr. Gabriela Olmedo from Center for Research and Advanced Studies of the National Polytechnic Institute (CINVESTAV,IPN) Campus Irapuato and Dr. Guillermo Gosset from Biotechnology Institute (IBT, UNAM). Supervision and direction was provided by members of the Center for Genomic Sciences, (CCG) however all work was done by the team, during the months of May 2012 to present. Most work was developed in the CCG from National Autonomous University of México (UNAM). Part of the work with ''Bacillus subtillis'' was done in CINVESTAV Campus Irapuato. Special thanks to Amhed Missael Vargas Velazquez for his support with MATLAB®, Dr. Otto Geiger for allowing us to use its lab at CCG to work with the OR and the Sweet AND and to Dr. Gabriela Olmedo for her help with ''Bacillus subtilis''. Also Dr. Fernando García from The Cellular Physiology Department of UNAM that helped us with the SEM Analysis.<br />
<br />
<br /><br /><br />
<center><h1>'''Our Instructors'''</h1><br /></center><br /><br /><br />
<br /><br /><br />
<br />
[[File:Unamgenomicsmexicoteamadvisors.JPG|870px|center|thumb|Our instructors and advisors ready to be shoot by a Biobrick squad]]<br /><br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamDavid.jpg| 400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''David Romero-Camarena'''<br />This guy is our instructor<br />•Main Instructor<br /><br />
</p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamResendis.jpg| 400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Osbaldo Resendis-Antonio'''<br />This guy is our instructor<br />• Modeling instructor<br /></p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Attributions#Attributions]]<br />
<center><h1>'''Our Advisors'''</h1><br /></center><br /><br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamHector.JPG|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Hector F. Medina'''<br />This guy is our modeling advisor<br />Hector is well known in the iGEM dark side as "Mamma Raven". He is also awesome modeling... just look at his photo!</p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamRogelio.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Rogelio Hernandez Tamayo'''<br />This guy is our wet-lab advisor<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsmexicoteamMiguel.JPG|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Miguel Angel Wences Guzman'''<br />This guy is our wet-lab advisor<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamOsam.jpg| 400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Fares Osam Yáñez Cuña'''<br />This guy is our wet-lab advisor<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamPepe_con_ursus.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Jose Antonio Alonso-Pavon'''<br />This guy is our Ethics and Human Practices advisor<br />"Pepe" has a master degree in Bioethics</p></td><br />
</tr><br />
<br />
</table><br />
</center><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Attributions#Attributions]]<br />
<center> <h1>'''Meet our team'''</h1> </center><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:Unamgenomicsteamabiel.JPG|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Abiel Trevino Garza'''<br />• Project design<br />
•Part design<br /><br />
•Wiki design<br /><br />
•Wiki implementation<br /><br />
•Human practices/Video edition<br /><br />
•Human practices/Recording<br /><br />
•Human practices/ Biosintetizarte 2.0<br /><br />
•Human practices/ Symposium Participation<br /><br />
•Funds raising <br /><br />
•Public relations<br /><br />
•Wet lab Sweet AND<br /><br />
</p> <br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamMariange.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Maria Angelica Bravo'''<br /> <br />
•Project design<br /><br />
•Part design<br /><br />
•Public relations<br /><br />
•Human practices/ video script and edition<br /><br />
•Human practices/ Symposium Participation<br /><br />
•Wet lab/ ''Bacillus subtilis''<br /><br />
•Wet lab/ SEM analysis<br /><br />
<br /></p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamDiego.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Diego Rodríguez'''<br />•Project design<br /><br />
•Part design<br /><br />
•Modeling<br /><br />
•Wet lab/ sweet AND<br /><br />
•Wet lab/ OR<br /><br />
<br /></p></td><br />
</tr><br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamWera.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Dulce Beatriz Vargas Landin'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ Heavy-Metal AND<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsmexicoteamJonathan.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Jonathan Padilla Gómez'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ Sweet AND<br /><br />
•Wet lab / OR<br /><br />
•Human practices/ Symposium Participation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamKaren.jpeg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Ana Karen Mojica Avila'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ Heavy-Metal AND<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamBenja.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Benjamín Hernández Rodríguez'''<br />•Project design<br /><br />
•Part design<br /><br />
•Human practices/ video script<br /><br />
•Human practices/ video recording<br /><br />
•Part documentation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamDiana.JPG|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Diana Vera Cruz'''<br />• Project design<br />
•Part design<br /><br />
•Modeling<br /><br />
•Human practices/ video script<br /><br />
•Human practices/ video recording<br /><br />
•Human practices/ Symposium Participation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamLessonN7.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Roberto Tirado Magallanes'''<br />•Project design<br /><br />
•Part design<br /><br />
•Modeling<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsmexicotemLaura.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Laura Teresa Jimenez Barrón'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ ''Bacillus subtillis''<br /><br />
•Wet lab/ SEM analysis<br /><br />
•Part characterization<br /><br />
•The sweet voice of Miss. Cohnnie<br /><br />
•Human practices/ Symposium Participation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
<tr><br />
<td id="leftcolumn2" align="center"><br />[[File:UnamgenomicsteamRebe.jpg|400px]]<br /><br /></td><br />
<td id="rightcolumn2" align="center"><br /><p>'''Rebeca Borges Monroy'''<br />•Project design<br /><br />
•Part design<br /><br />
•Wet lab/ Heavy-Metal AND<br /><br />
•Human Practices/ Video Edition<br /><br />
•Human practices/ Symposium Participation<br /><br />
<br /></p></td><br />
</tr><br />
<br />
</table><br />
</center><br />
<br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Attributions#Attributions]]<br />
<br />
<br />
Each team must clearly attribute work done by the team on this page. They must distinguish work done by the team from work done by others, including the host labs, advisors, instructors, graduate students, and postgraduate masters students.<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Partners
Team:UNAM Genomics Mexico/HumanPractices/Partners
2012-10-27T02:00:50Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Our Partners, Sponsors and Friends.'''</h1></center> <br />
<br />
<br />
<br />
<br /><br />
<br /><br />
We would like to send a shout out to the people whose help and support helped us in the realization of this project.<br />
<br /><br />
<br /><br />
[http://tamaulipas.gob.mx/ '''Government of the State of Tamaulipas'''], [http://itca.gob.mx/ '''Instituto Tamaulipeco para la Cultura y las Artes'''] and the [http://www.cotacyt.gob.mx/ '''COTACYT'''] '''Consejo Tamaulipeco de Ciencia y Tecnología''' for their support and sponsorship with BioSintetizArte and their hosting of the exhibitions and the talks in the future.<br /><br />
<br /><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UnamgenomicsTamaulipas.png|400px|link=http://tamaulipas.gob.mx/]]<br /><br /><p>[http://tamaulipas.gob.mx/ '''Government of the State of Tamaulipas''']<br />
</p></td><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UnamgenomicsLOGO ITC.jpg|200px|link=http://itca.gob.mx/]]<br /><br /><p>[http://itca.gob.mx/ '''Instituto Tamaulipeco para la Cultura y las Artes''']<br />
</p></td><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UnamgenomicsCropperCapture.png|200px|link=http://www.cotacyt.gob.mx/]]<br /><br /><p>[http://www.cotacyt.gob.mx/ '''COTACYT''']<br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br /><br />
The non-profit organizations [http://www.masciencia.org '''Más Ciencia por México'''] and [http://acmor.org '''Academia de Ciencias de Morelos'''] for their support in the outreach strategy and finding places to present our talks.<br /><br />
<br /><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UnamgenomicsMCPM.png|200px|link=http://www.masciencia.org]]<br /><br /><p>[http://www.masciencia.org '''Más Ciencia por México''']<br />
</p></td><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UnamgenomicsmexicoLogoacmor.jpg|200px|link=http://acmor.org]]<br /><br /><p>[http://acmor.org '''Academia de Ciencias de Morelos''']<br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br />
To our university and our program, the [http://www.ccg.unam.mx/en '''Center for Genomic Sciences'''] and [http://www.lcg.unam.mx/ '''Licenciatura en Ciencias Genómicas'''] for their everlasting support in every possible way!<br /><br />
<br /><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UnamgenomicsCCG Logo HR.jpg |200px|link=http://www.ccg.unam.mx/en]] <br /><br /><p>[http://www.ccg.unam.mx/en '''Center for Genomic Sciences''']<br />
</p></td><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UnamgneomcisGenomicas.jpg|200px|link=http://www.lcg.unam.mx/]]<br /><br /><p>[http://www.lcg.unam.mx/ '''Licenciatura en Ciencias Genómicas''']<br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br />
<br /><br />
We would like to thank profoundly the following people that, at some point of the project, helped us with their perspectives, ideas, connections, and their helping hands.<br />
<br /><br />
:* A big thank you to our panelists, Julia, Martín, David and Alejandra, for accepting the invitation to be part of this proyect<br />
:* Karla Cedano and everyone at Inno-ba, for their precise and insightful comments, as well as helping with the diffusion of our events with the press.<br />
:* Meztli Matadamas, for helping us with our videos and bearing with us through our moments of sheer stress.<br />
<br /><br />
<br /><br />
=='''Podcast Collaboration'''==<br />
<br /><br />
We collaborated with the teams of the University of [[Team:Cornell/outreach/awareness | '''Cornell''']], [[Team:SDU-Denmark/collaboration/podcast | '''SDU-Denmark''']] and [[Team:UIUC-Illinois/Outreach | '''University of Illinois''']], in the production of a podcast about some of the Ethical, Social, and Environmental implications of<br />
large-scale genetic engineering projects in modern society by creating a series of videos accessible <br />
to the general public. Our topic was the Genetic Modified Organisms.<br /><br />
<br /><br />
Our task was to present a video of "Genetic Modified Organism", in which we explain what is happening<br />
nowadays with it, and some implications that this topic has in the society. We tried to explain it in a way<br />
that everyone understands the idea.<br /><br />
<br /><br />
Our video focused on transgenic organisms. We wanted to give simple examples so that the general audience could<br />
learn about how genetic engineering techniques are being used to satisfy increasing demands of food, how they<br />
might help cure diseases, and produce medicine in large amounts to reduce costs. We also included alternatives of<br />
how animals are being used without genetic modification, for example, by taking advantage of their dung as a <br />
non-poisonous repellent.We want to thank them for this opportunity. You can watch the video here:<br />
<br /><br />
<br /><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2"><iframe width="850" height="638" src="http://www.youtube.com/embed/DkEegUWK_vs" frameborder="0" allowfullscreen></iframe> <p>Transgenic Organisms </p></td><br />
</tr><br />
</table><br />
</html><br />
<br /><br />
=='''Collaboration with the University of Munchen'''==<br />
<br /><br />
We were awarded the “most famous [[Team:TU_Munich/Results/RFC | “'''Original Bavarian Collaboration-Medal'''”]] for answering the survey about Bioparts they sent us.<br />
<br /><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />{{Team:TU_Munich/Badge}}<br /><br /><p>Fiends TEAM<br />
</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br/><br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T01:53:27Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<center><h1>'''Welcome to our homepage'''</h1></center> <br />
<br /><br />
<table border="0" width="900px" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2"><p><br />
<br /><br />
<h2>'''Bacillus booleanus'''</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
</p><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|510px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
<td id="contentcolumn" align="center"><p><br />
<h1>Regional Jamboree!!!</h1><br />
[[File:Unamgenomicsbestwikihome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<br /><br />
[[File:Unamgenomicsbogotahome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<b>Regional Jamboree LOTS of FUN!</b><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
<br />
<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a></td><br />
</tr><br />
<tr><br />
<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
<br />
<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a></td><br />
</tr><br />
</table><br />
</html><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T01:49:48Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<center><h1>'''Welcome to our homepage'''</h1></center> <br />
<br /><br />
<table border="0" width="900px" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2"><p><br />
<br /><br />
<h2>'''Bacillus booleanus'''</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
</p><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|510px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
<td id="contentcolumn" align="center"><p><br />
<h1>Regional Jamboree!!!</h1><br />
[[File:Unamgenomicsbestwikihome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<br /><br />
[[File:Unamgenomicsbogotahome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<b>Regional Jamboree LOTS of FUN!</b><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
<br />
<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
<br />
<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T01:47:36Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<center><h1>'''Welcome to our homepage'''</h1></center> <br />
<br /><br />
<table border="0" width="900px" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2"><p><br />
<br /><br />
<h2>'''Bacillus booleanus'''</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
</p><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|510px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
<td id="contentcolumn" align="center"><p><br />
<h1>Regional Jamboree!!!</h1><br />
[[File:Unamgenomicsbestwikihome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<br /><br />
[[File:Unamgenomicsbogotahome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<b>Regional Jamboree LOTS of FUN!</b><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
<br />
<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
<br />
<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
<br />
<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T01:47:01Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<center><h1>'''Welcome to our homepage'''</h1></center> <br />
<br /><br />
<table border="0" width="900px" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2"><p><br />
<br /><br />
<h2>Bacillus booleanus</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
</p><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|510px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
<td id="contentcolumn" align="center"><p><br />
<h1>Regional Jamboree!!!</h1><br />
[[File:Unamgenomicsbestwikihome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<br /><br />
[[File:Unamgenomicsbogotahome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<b>Regional Jamboree LOTS of FUN!</b><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
<br />
<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
<br />
<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
<br />
<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T01:45:30Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<center><h1>'''Welcome to our homepage'''</h1></center> <br />
<br /><br />
<br /><br />
<br />
<table border="0" width="900px" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn2"><p><br />
<br /><br />
<h2>Bacillus booleanus</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
</p><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|510px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
<td id="contentcolumn" align="center"><p><br />
<h1>Regional Jamboree!!!</h1><br />
[[File:Unamgenomicsbestwikihome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<br /><br />
[[File:Unamgenomicsbogotahome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<b>Regional Jamboree LOTS of FUN!</b><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
<br />
<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
<br />
<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
<br />
<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T01:44:57Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Welcome to our homepage'''</h1></center> <br />
<br /><br />
<br /><br />
<br />
<br />
<table border="0" width="900px" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn2"><p><br />
<br /><br />
<h2>Bacillus booleanus</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
</p><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|510px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
<td id="contentcolumn" align="center"><p><br />
<h1>Regional Jamboree!!!</h1><br />
[[File:Unamgenomicsbestwikihome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<br /><br />
[[File:Unamgenomicsbogotahome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<b>Regional Jamboree LOTS of FUN!</b><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
<br />
<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
<br />
<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
<br />
<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T01:43:34Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Welcome to our homepage'''</h1></center> <br />
<br /><br />
<br /><br />
<br />
<br />
<table border="0" width="900px" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn2"><p><br />
<br /><br />
<h2>Bacillus booleanus</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
</p><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
<br />
<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
<br />
<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|510px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
<td id="contentcolumn" align="center"><p><br />
<h1>Regional Jamboree!!!</h1><br />
[[File:Unamgenomicsbestwikihome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<br /><br />
[[File:Unamgenomicsbogotahome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<b>Regional Jamboree LOT of FUN!</b><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
<br />
<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T01:39:23Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Welcome to our homepage'''</h1></center> <br />
<br /><br />
<br /><br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
<br />
<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
<br />
<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<table border="0" width="900px" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn"><p><br />
<h1>Project description</h1><br />
<br /><br />
<h2>BACILLUS BOOLEANUS</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
</p><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|510px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
<td id="contentcolumn" align="center"><p><br />
<h1>Regional Jamboree!!!</h1><br />
[[File:Unamgenomicsbestwikihome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<br /><br />
[[File:Unamgenomicsbogotahome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<b>Regional Jamboree LOT of FUN!</b><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
<br />
<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T01:37:41Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Welcome to our homepage'''</h1></center> <br />
<br /><br />
<br /><br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
<br />
<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
<br />
<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<table border="0" width="900px" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn"><p><br />
<h1>Project description</h1><br />
<br /><br />
<h2>BACILLUS BOOLEANUS</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
</p><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|500px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
<td id="contentcolumn" align="center"><p><br />
<h1>Regional Jamboree!!!</h1><br />
[[File:Unamgenomicsbestwikihome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<br /><br />
[[File:Unamgenomicsbogotahome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<b>More info here</b><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
<br />
<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T01:36:24Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Welcome to our homepage'''</h1></center> <br />
<br /><br />
<br /><br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
<br />
<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
<br />
<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<table border="0" width="900px" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn"><p><br />
<h1>Project description</h1><br />
<br /><br />
<h2>BACILLUS BOOLEANUS</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
</p><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|400px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:Unamgenomicsbestwikihome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
[[File:Unamgenomicsbogotahome.JPG|300px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<b>More info here</b><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
<br />
<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T01:35:22Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Welcome to our homepage'''</h1></center> <br />
<br /><br />
<br /><br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
<br />
<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
<br />
<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<table border="0" width="900px" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn"><p><br />
<h1>Project description</h1><br />
<br /><br />
<h2>BACILLUS BOOLEANUS</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
</p><br />
</td><br />
</tr><br />
<br />
</table><br />
<br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|400px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:Unamgenomicsbogotahome.JPG|400px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
[[File:Unamgenomicsbogotahome.JPG|400px|link=Team:UNAM_Genomics_Mexico/Notebook/Photos]]<br /><br />
<b>More info here</b><br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
<br />
<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T01:33:16Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''Welcome to our homepage'''</h1></center> <br />
<br /><br />
<br /><br />
<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
<br />
<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
<br />
<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a</td><br />
</tr><br />
</table><br />
</html><br />
<br />
<table border="0" width="900px" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn"><p><br />
<h1>Project description</h1><br />
<br /><br />
<h2>BACILLUS BOOLEANUS</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
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<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|400px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|400px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
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<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
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<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a</td><br />
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Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico
Team:UNAM Genomics Mexico
2012-10-27T01:32:38Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
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<center><h1>'''Welcome to our homepage'''</h1></center> <br />
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<td id="leftcolumn2"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="190"/><br /><br /><p><b>Nanotubes</b></p></a></td><br />
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<td id="contentcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription"><img src="https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsLogiv.jpg" alt="some_text" height="200"/><br /><br /><p><b>The System Logic</b></p></a></td><br />
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<td id="rightcolumn2" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/BiosintetizarteEN"><img src="https://static.igem.org/mediawiki/2012/f/f1/CRATELBIOSINTEtizartechico.jpg" alt="some_text" height="170"/><br /><br /><p><b>Call</b></p></a</td><br />
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<h1>Project description</h1><br />
<br /><br />
<h2>BACILLUS BOOLEANUS</h2> <br />
<br /><br />
A logic gate is an idealized (or physical) device implementing a Boolean function that performs a logic operation on one or more logic inputs and produces a single logic output. A single logic gate is not a computer, many of them are necessary and they need to communicate with each other to compute stuff. In this way, a complex logic system can be created, like a computer. The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Taking these two main ideas, it could be possible to create a “biological computer”. Bacillus booleanus is a project that wants to link several Boolean operations to make the beginnings of a biological computer. How does everything work? We are working on the creation of different strains of Bacillus subtilis. Each one will be able to perform a single Boolean operation, just like transistors do in an electronic computer. As we mentioned, our bacteria need to communicate to achieve trascendence, but how could this be possible? In 2011 Ben-Yehuda et. al. identified a type of bacterial communication mediated by nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. By using these nanotubes, our bacteria will be capable to communicate with others, creating complex networks of logic gates. Using this, it could be possible to develop a complex network of operators to regulate, for example, a synthetic metabolic pathway.<br />
</p><br />
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<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|450px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
<td id="contentcolumn" align="center"><p><br />
<h1>We found Nanotubes!!!</h1><br />
[[File:UnamgenomicsNanocirculorojo.jpg|450px|link=Team:UNAM_Genomics_Mexico/Results/Nanotubes]]<br />
<b>More info here</b><br />
</p><br />
</td><br />
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<html><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="leftcolumn3"align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/HumanPractices/Who_is_Mrs._Cohnnie_PhD?"><img src="https://static.igem.org/mediawiki/2012/6/60/UnamgenomicsmissLogomujer.png" alt="some_text" height="190"/><br /><br /><p><b>Meet Mrs. Lupita Cohnnie, PhD</b></p></a></td><br />
<br />
<td id="contentcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Notebook/Videos"><img src="https://static.igem.org/mediawiki/2012/b/b7/UnamgenomicsVideos.png" alt="some_text" height="200"/><br /><br /><p><b>Our Videos</b></p></a></td><br />
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<td id="rightcolumn3" align="center"><a href="https://2012.igem.org/Team:UNAM_Genomics_Mexico/Modeling"><img src="https://static.igem.org/mediawiki/2012/e/e2/UnamgenomicsModelwelcome.png" alt="some_text" height="170"/><br /><br /><p><b>Our Model</b></p></a</td><br />
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Abieltega
http://2012.igem.org/File:Unamgenomicsbestwikihome.JPG
File:Unamgenomicsbestwikihome.JPG
2012-10-27T01:30:44Z
<p>Abieltega: </p>
<hr />
<div></div>
Abieltega
http://2012.igem.org/File:Unamgenomicsbogotahome.JPG
File:Unamgenomicsbogotahome.JPG
2012-10-27T01:29:17Z
<p>Abieltega: </p>
<hr />
<div></div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription
Team:UNAM Genomics Mexico/Project/DeeperDescription
2012-10-27T00:58:09Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
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<center><h1>'''Deep Description'''</h1></center><br />
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Our boolean constructions are two, the AND & OR, for it to form a system, but how we will connect this two operations? With the nanotubes that ''Bacillus subtilis'' form. <br /><br />
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== '''Why B. subtilis?''' ==<br />
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We found a paper in which Ben-Yehuda et. al. demostrated that ''Bacillus subtilis'' form nanotubes between them, ''Escherichia coli'' and ''Staphylococcus aureus''. And they were able to get through a GFP and some other smaller molecules. So, we decided to use this connection to form the principles of a molecular computer. How? The output of the first operation will the input for the next one, and that was what we did. Also, there is not many work in the'' B. subtilis'' at the iGEM competition, so we decided to standardize the protocols of competent cells and transformation, and to send biobricks that ''B. subtilis'' could transform and integrate for them to work correctly [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Bacillus_subtilis_Protocols | (new standard protocol)]]. <br /><br /><br />
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<p class='captionInside'>2011 Gyanendra P. Dubey, Sigal Ben-Yehuda. Intercellular Nanotubes Mediate Bacterial Communication. Cell, 2011; 144 (4): 590 DOI:10.1016/j.cell.2011.01.015<br /></p><br />
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<p class='captionInside'>2011 Gyanendra P. Dubey, Sigal Ben-Yehuda. Intercellular Nanotubes Mediate Bacterial Communication. Cell, 2011; 144 (4): 590 DOI:10.1016/j.cell.2011.01.015<br /></p><br />
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== '''How does the AND work?''' ==<br />
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The AND operator in general only gives an output when the two inputs recognized in the operator are present. In the design, we considered the possibility that the constitutive promoter could continue with transcription even after getting to the double terminator and to avoid this, we designed the AND construction with the inducible promoter before the constitutive one. <br /><br />
We have two types of AND: <br /><br />
•'''The Heavy Metals AND''': The promoters CzrA/ArsR have something in common, they both sense cadmium. When the two promoters detect cadmium, they would not repress anymore and start transcription of P4 or LasR, and we will know it is working because our bacterium will be red, because of our reporter gene (RFP). The promoters are based in the CadA promoter plus a binding site for ArsR for get a response that depend in both, CzrA and ArsR. <br /><br />
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<td id="contentcolumnwhite" align= "center"><br />[[File:UnamgenomicsCadmiopromotores.png | 430px ]]<br /><br /><p>Our CzrA/ArsR </p></td><br />
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•'''CzrA/ArsR''': This transcription factors are involved in metal homeostasis, each one had affinity for a set of metals[1], the intersection of both sensors is Cadmium[2][3], in this case the proposed input metals will be Zinc for CzrA and Arsenic for ArsR. This TFs acts as repressors in the absence of metals, when there is metal in the cell, the metal binds to the transcription factor and make a conformation change that dissolves the affinity of the protein for their binding sites and let the promoter free to recruit RNA polymerase to start transcription[1]. <br /><br />
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We have three''' CzrA/ArsR promoters''', one of them was designed by Newcastle team in 2009 ([http://partsregistry.org/Part:BBa_K174015 Part BBa_K174015]), the two remaining were design by our team based on the previous by Newcastle. Why we need more than one promoter? The part BBa_K174015 has no reported experience, so it could work as it should or not and the way this cadmium sensor was developed allow us to play with the combinatory of binding sites and with the -10 and -35 boxes. The cadmium sensor of Newcastle is -35, -10 and binding sites, our promoters are:<br /><br />
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'''Promoter 1''': attta, -35,-10, ArsR binding site overlapping -35, CsrA binding site overlapping TSS, rbs+atg.<br /><br />
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'''Promoter 2''': -35, -10, ArsR binding site overlapping after TSS, CzrA overlapping TSS, rbs+atg.<br /><br />
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In this way, by designing different versions of the “same” part we can reach the most optimal biobrick.<br /><br />
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•'''P4''': It´s a transcription factor from ''phi29 phage'', which is a phage of ''B. subtilis'', it is the activator of the A3 promoter[4,5]. <br /><br />
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•'''LasR''': It is another activator transcription factor, its origin is ''Pseudomonas aeruginosa'' PAO1, its correspondent promoter is LasB[20]. <br /><br />
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•'''CI''': This protein is a repressor from Lambda phage. <br /><br />
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•'''RFP''': highly engineered mutant of red fluorescent protein from ''Discosoma striata'' (coral), this is our reporter gene[6]. <br /><br />
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•'''Omega cassette''': The Omega Cassette provides resistance to Spectinomycin and Streptomycin; it was obtained from plasmid pHP45omega[7,8]. Specifically, the resistance is provided by the aminoglycoside antibiotic resistance gene (aadA +) originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid[8]. <br /><br />
<br />
•'''AmyE''': Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus. This is achieved by using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence([http://partsregistry.org/Part:BBa_K143001 BBa_K143001]) can be added to the front of a BioBrick construct and the 3' integration sequence specific for this locus ([http://partsregistry.org/Part:BBa_K143002 BBa_K143002]) to the rear of the Biobrick construct to allow integration of the BioBrick construct into the chromosome of the Gram-positive bacterium B. subtilis at the amyE locus[18,19]. <br /><br />
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<center><br />
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<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsConstruccion1.png| x115px |link=Team:UNAM_Genomics_Mexico/Results/AND#CzrA-ArsR_AND_GATE]]<p>'''Heavy metal AND Results'''</p></td><br />
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•'''The pBAD/pXyl AND''': The promoter pBAD is activated with arabinose, and pXyl with xylose. When both the promoters are activated it starts the transcription of P4 or LasR, and we will know it is working because our bacteria will be red, because of the reporter gene (RFP). <br /><br />
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•'''pBAD''': It´s the promoter under the sigma factor 70 from ''E. coli'', which is analogous to the sigma A factor of B. subtilis. It’s repressed by AraC and induced by arabinose, which triggers the rearrangement of the AraC monomers binding for the recruitment of RNA polymerase[9,10]. <br /><br />
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•'''pXyl''': It´s a promoter in B. subtilis which is regulated by Xylose and XylR[11,12]. In the endogenous systems, pXyl is the promoter for a set of genes involved in xylose metabolism[12]. <br /><br />
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•P4: It´s a transcription factor from phi29 phage, which is a phage of B. subtilis, it is the activator of the A3 promoter[4,5]. <br /><br />
<br />
•'''LasR''': It is another activator transcription factor, its origin is ''Pseudomonas aeruginosa'' PAO1, its correspondent promoter is LasB[20]. <br /><br />
<br />
•'''CI''': This protein is a repressor from Lambda phage. <br /><br />
<br />
•'''RFP''': highly engineered mutant of red fluorescent protein from ''Discosoma striata'' (coral), this is our reporter gene[6]. <br /><br />
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•'''Pveg''': This is a constitutive promoter from ''B. subtilis'' regulates by the sigma factor sigma A[13]. <br /><br />
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•'''XylR''': This is a repressor transcription factor which in the presence of xylose is inactivated by a conformational change triggered by the binding with xylose that induces the dissociation of the XylR from its binding site[14]. <br /><br />
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•'''AraC''': It´s one ''E.coli'' repressor transcription factor. In the absence of arabinose, araC binds to two binding sites located in the pBAD promoter and through the binding of both monomers od AraC a DNA looping is made which blocks the transcription of the genes under that promoter[15]. When arabinose is present in the medium. <br /> <br />
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•'''Omega cassette''': The Omega Cassette provides resistance to Spectinomycin and Streptomycin; it was obtained from plasmid pHP45omega[7,8]. Specifically, the resistance is provided by the aminoglycoside antibiotic resistance gene (aadA +) originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid[8]. <br /><br />
<br />
•'''AmyE''': Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus. This is achieved by using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence([http://partsregistry.org/Part:BBa_K143001BBa_K143001]) can be added to the front of a BioBrick construct and the 3' integration sequence specific for this locus ([http://partsregistry.org/Part:BBa_K143002 BBa_K143002]) to the rear of the Biobrick construct to allow integration of the BioBrick construct into the chromosome of the Gram-positive bacterium B. subtilis at the amyE locus[18,19]. <br /><br />
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<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="leftcolumn3" align= "center"><br />[[File:Unamgenomicsdeepconstruccion 2.png| x115px |link=Team:UNAM_Genomics_Mexico/Results/AND#ARABINOSE-XYLOSE_AND_GATE]]<p>'''Sugar AND Results'''</p></td><br />
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<h2>'''How does the OR work?'''</h2><br /><br />
•The OR operator gives an output when one, or both, of the inputs recognized by the operator are present. Our OR works with the A3 and LasB promoter, that need the P4 and LasR TF, respectively. When they are active, they transcribe GFP, and we can notice it is working because of the fluorescence that GFP produces.. <br /><br />
<br />
•'''LasB:''' Promoter regulated positively by LasR in ''P. aeruginosa''[21]. <br /><br />
<br />
•'''A3''': Promoter regulated positively by P4 from phi29 phage[4,16,17]. <br /><br />
<br />
•'''GFP:''' The GFP reporter gene encodes green fluorescent protein derived from jellyfish ''Aequeora victoria'' wild-type GFP[22]. <br /><br />
<br />
•'''Omega cassette:''' The Omega Cassette provides resistance to Spectinomycin and Streptomycin; it was obtained from plasmid pHP45omega[7,8]. Specifically, the resistance is provided by the aminoglycoside antibiotic resistance gene (aadA +) originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid[8]. <br /><br />
<br />
•'''AmyE:''' Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus. This is achieved by using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence([http://partsregistry.org/Part:BBa_K143001 BBa_K143001]) can be added to the front of a BioBrick construct and the 3' integration sequence specific for this locus ([http://partsregistry.org/Part:BBa_K143001 BBa_K143002]) to the rear of the Biobrick construct to allow integration of the BioBrick construct into the chromosome of the Gram-positive bacterium B. subtilis at the amyE locus [18,19]. <br /><br />
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<center><br />
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<td id="contentcolumn" align= "center"><br />[[File:Unamgenomicsconstruccion3.png | 850px|link=Team:UNAM_Genomics_Mexico/Results/OR]]</td><br />
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::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Project/DeeperDescription#Deep_Description]]<br />
<center>[[File:Unamgenomicsdeepamye.jpg]]</center><br />
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[1] Busenlehner LS, Pennella MA, Giedroc DP (2003). The SmtB/ArsR family of metalloregulatory transcriptional repressors: Structural insights into prokaryotic metal resistance. FEMS Microbiol Rev , 27:131-143. <br /><br />
[2] Charles M Moore and John D Helmann(2005). Metal ion homeostasis in ''Bacillus subtilis''. Current Opinion in Microbiology, 8:188–195. <br /><br />
[3] Moore CM, Gaballa A, Hui M, Ye RW, Helmann JD (2005). Genetic and physiological responses of ''Bacillus subtilis'' to metal ion stress. Mol Microbiol(1) , 27–40. <br /><br />
[4] Camacho A & Salas M (2010) DNA bending and looping in the transcriptional control of bacteriophage ϕ29. FEMS Microbiol Rev. 34(5):828-841. <br /><br />
[5] Rojo F, Mencía M, Monsalve M & Salas M (1998) Transcription activation and repression by interaction of a regulator with the a subunit of RNA polymerase: the model of phage ϕ29 protein p4. Nucleic Acid Re 60: 29–46<br /><br />
[6] http://partsregistry.org/Part:BBa_E1010<br /><br />
[7] Pierre Prentki, Anna Bind and Andrée Epstein (1991). Plasmid vectors for selecting ISI-promoted deletions in cloned DNA: sequence analysis of the omega interposon. Gene, 103:17-23.<br />
[8] Pierre Prentki and Henry M. Krisch (1984). In vitro insertional mutagenesis with a selectable DNA fragment. Gene, 29:303-313<br /><br />
[9] Lee, N. (1980) Molecular Aspects of ara Regulation. In The Operon, J. H. Miller and W. S. Reznikoff, eds. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory), pp. 389-410. <br /><br />
[10] Lee, N., Francklyn, C., and Hamilton, E. P. (1987). Arabinose-Induced Binding of AraC Protein to araI2 Activates the araBAD Operon Promoter. Proc. Natl. Acad. Sci. USA 84, 8814-8818. <br /><br />
[11] Shamanna, D. K., and K. E. Sanderson. 1979. Genetics and regulation of D-xylose utilization in Salmonella typhimurium LT2. J. Bacteriol. 139:71-79. <br /><br />
[12] D Gartner, M Geissendorfer, & W Hillen(1988). Expression of the ''Bacillus subtilis'' xyl Operon Is Repressed at the Level of Transcription and Is Induced by Xylose J Bacteriol 170:7,3102-3109.<br />
[13] Moran CP, Lang N, LeGrice SF, Lee G, Stephens M, Sonenshein AL, Pero J, Losick R (1982). Nucleotide sequences that signal the initiation of transcription and translation in ''Bacillus subtilis''. Mol Gen Genet; 186(3): 339-46 <br /><br />
[14] Kreuzer P, Gärtner D, Allmansberger R, Hillen W (1989). Identification and sequence analysis of the ''Bacillus subtilis'' W23 xylR gene and xyl operator. J Bacteriol. Jul;171(7):3840-5. <br /><br />
[15] Schlief, R. (2000). Regulation of the L-arabinose operon of ''Escherichia coli''. Trends in Genetics. 16(12):559-565. <br /><br />
[16] Sogo JM, Inciarte MR, Corral J, Viñuela E & Salas M(1979) RNA polymerase binding sites and transcription map of the DNA of ''Bacillus subtilis'' phage ϕ29. J Mol Biol 127: 411–436.<br />
[17] Nuez B, Rojo F & SalasM(1992) Phage ϕ29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein–protein contact. P Natl Acad Sci USA 89: 11401–11405. <br /><br />
[18]Cutting, S M.; Vander-Horn, P B. Genetic analysis. In: Harwood C R, Cutting S M. , editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons, Ltd.; 1990. pp. 27–74. <br /><br />
[19] http://partsregistry.org/Part:BBa_K143001 <br /><br />
[20]http://regtransbase.lbl.gov/cgi-bin/regtransbasepage=regulatorinfo&type=site&g<br />
uid=76697<br /><br />
[21]Analysis of the Pseudomonas aeruginosa elastase (lasB) regulatory region. L Rust, E C Pesci and B H Iglewski, J. Bacteriol. February 1996 vol. 178 no. 4 1134-1140 <br /><br />
[22] http://partsregistry.org/wiki/index.php/Part:BBa_E0040<br /><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription
Team:UNAM Genomics Mexico/Project/DeeperDescription
2012-10-27T00:56:35Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br />
<center><h1>'''Deep Description'''</h1></center><br />
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Our boolean constructions are two, the AND & OR, for it to form a system, but how we will connect this two operations? With the nanotubes that ''Bacillus subtilis'' form. <br /><br />
<br /><br />
<br />
== '''Why B. subtilis?''' ==<br />
<br /><br />
We found a paper in which Ben-Yehuda et. al. demostrated that ''Bacillus subtilis'' form nanotubes between them, ''Escherichia coli'' and ''Staphylococcus aureus''. And they were able to get through a GFP and some other smaller molecules. So, we decided to use this connection to form the principles of a molecular computer. How? The output of the first operation will the input for the next one, and that was what we did. Also, there is not many work in the'' B. subtilis'' at the iGEM competition, so we decided to standardize the protocols of competent cells and transformation, and to send biobricks that ''B. subtilis'' could transform and integrate for them to work correctly [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Bacillus_subtilis_Protocols | (new standard protocol)]]. <br /><br /><br />
<center><br />
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<img src='https://static.igem.org/mediawiki/2012/6/6a/Unamgenomicsdeepdescriptionbacillus1.png' height="200" /><br />
<div class='captiongray'><br />
<p class='captionInside'>2011 Gyanendra P. Dubey, Sigal Ben-Yehuda. Intercellular Nanotubes Mediate Bacterial Communication. Cell, 2011; 144 (4): 590 DOI:10.1016/j.cell.2011.01.015<br /></p><br />
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<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/c9/Unamgenomicsdeepdescriptionbacillus2.jpg' height="200" /><br />
<div class='captionnaranja'><br />
<p class='captionInside'>2011 Gyanendra P. Dubey, Sigal Ben-Yehuda. Intercellular Nanotubes Mediate Bacterial Communication. Cell, 2011; 144 (4): 590 DOI:10.1016/j.cell.2011.01.015<br /></p><br />
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== '''How does the AND work?''' ==<br />
<br /><br />
<br /><br />
The AND operator in general only gives an output when the two inputs recognized in the operator are present. In the design, we considered the possibility that the constitutive promoter could continue with transcription even after getting to the double terminator and to avoid this, we designed the AND construction with the inducible promoter before the constitutive one. <br /><br />
We have two types of AND: <br /><br />
•'''The Heavy Metals AND''': The promoters CzrA/ArsR have something in common, they both sense cadmium. When the two promoters detect cadmium, they would not repress anymore and start transcription of P4 or LasR, and we will know it is working because our bacterium will be red, because of our reporter gene (RFP). The promoters are based in the CadA promoter plus a binding site for ArsR for get a response that depend in both, CzrA and ArsR. <br /><br />
<br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10" align="right"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UnamgenomicsCadmiopromotores.png | 430px ]]<br /><br /><p>Our CzrA/ArsR </p></td><br />
</tr><br />
</table><br />
<br />
•'''CzrA/ArsR''': This transcription factors are involved in metal homeostasis, each one had affinity for a set of metals[1], the intersection of both sensors is Cadmium[2][3], in this case the proposed input metals will be Zinc for CzrA and Arsenic for ArsR. This TFs acts as repressors in the absence of metals, when there is metal in the cell, the metal binds to the transcription factor and make a conformation change that dissolves the affinity of the protein for their binding sites and let the promoter free to recruit RNA polymerase to start transcription[1]. <br /><br />
<br />
We have three''' CzrA/ArsR promoters''', one of them was designed by Newcastle team in 2009 ([http://partsregistry.org/Part:BBa_K174015 Part BBa_K174015]), the two remaining were design by our team based on the previous by Newcastle. Why we need more than one promoter? The part BBa_K174015 has no reported experience, so it could work as it should or not and the way this cadmium sensor was developed allow us to play with the combinatory of binding sites and with the -10 and -35 boxes. The cadmium sensor of Newcastle is -35, -10 and binding sites, our promoters are:<br /><br />
<br />
'''Promoter 1''': attta, -35,-10, ArsR binding site overlapping -35, CsrA binding site overlapping TSS, rbs+atg.<br /><br />
<br />
'''Promoter 2''': -35, -10, ArsR binding site overlapping after TSS, CzrA overlapping TSS, rbs+atg.<br /><br />
<br />
In this way, by designing different versions of the “same” part we can reach the most optimal biobrick.<br /><br />
<br />
•'''P4''': It´s a transcription factor from ''phi29 phage'', which is a phage of ''B. subtilis'', it is the activator of the A3 promoter[4,5]. <br /><br />
<br />
•'''LasR''': It is another activator transcription factor, its origin is ''Pseudomonas aeruginosa'' PAO1, its correspondent promoter is LasB[20]. <br /><br />
<br />
•'''CI''': This protein is a repressor from Lambda phage. <br /><br />
<br />
•'''RFP''': highly engineered mutant of red fluorescent protein from ''Discosoma striata'' (coral), this is our reporter gene[6]. <br /><br />
<br />
•'''Omega cassette''': The Omega Cassette provides resistance to Spectinomycin and Streptomycin; it was obtained from plasmid pHP45omega[7,8]. Specifically, the resistance is provided by the aminoglycoside antibiotic resistance gene (aadA +) originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid[8]. <br /><br />
<br />
•'''AmyE''': Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus. This is achieved by using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence([http://partsregistry.org/Part:BBa_K143001 BBa_K143001]) can be added to the front of a BioBrick construct and the 3' integration sequence specific for this locus ([http://partsregistry.org/Part:BBa_K143002 BBa_K143002]) to the rear of the Biobrick construct to allow integration of the BioBrick construct into the chromosome of the Gram-positive bacterium B. subtilis at the amyE locus[18,19]. <br /><br />
<br /><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsConstruccion1.png| x115px |link=Team:UNAM_Genomics_Mexico/Results/AND#CzrA-ArsR_AND_GATE]]<p>'''Heavy metal AND Results'''</p></td><br />
</tr><br />
</table><br />
</center><br />
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<br /><br />
<br />
•'''The pBAD/pXyl AND''': The promoter pBAD is activated with arabinose, and pXyl with xylose. When both the promoters are activated it starts the transcription of P4 or LasR, and we will know it is working because our bacteria will be red, because of the reporter gene (RFP). <br /><br />
<br />
•'''pBAD''': It´s the promoter under the sigma factor 70 from ''E. coli'', which is analogous to the sigma A factor of B. subtilis. It’s repressed by AraC and induced by arabinose, which triggers the rearrangement of the AraC monomers binding for the recruitment of RNA polymerase[9,10]. <br /><br />
<br />
•'''pXyl''': It´s a promoter in B. subtilis which is regulated by Xylose and XylR[11,12]. In the endogenous systems, pXyl is the promoter for a set of genes involved in xylose metabolism[12]. <br /><br />
<br />
•P4: It´s a transcription factor from phi29 phage, which is a phage of B. subtilis, it is the activator of the A3 promoter[4,5]. <br /><br />
<br />
•'''LasR''': It is another activator transcription factor, its origin is ''Pseudomonas aeruginosa'' PAO1, its correspondent promoter is LasB[20]. <br /><br />
<br />
•'''CI''': This protein is a repressor from Lambda phage. <br /><br />
<br />
•'''RFP''': highly engineered mutant of red fluorescent protein from ''Discosoma striata'' (coral), this is our reporter gene[6]. <br /><br />
<br />
•'''Pveg''': This is a constitutive promoter from ''B. subtilis'' regulates by the sigma factor sigma A[13]. <br /><br />
<br />
•'''XylR''': This is a repressor transcription factor which in the presence of xylose is inactivated by a conformational change triggered by the binding with xylose that induces the dissociation of the XylR from its binding site[14]. <br /><br />
<br />
•'''AraC''': It´s one ''E.coli'' repressor transcription factor. In the absence of arabinose, araC binds to two binding sites located in the pBAD promoter and through the binding of both monomers od AraC a DNA looping is made which blocks the transcription of the genes under that promoter[15]. When arabinose is present in the medium. <br /> <br />
<br />
•'''Omega cassette''': The Omega Cassette provides resistance to Spectinomycin and Streptomycin; it was obtained from plasmid pHP45omega[7,8]. Specifically, the resistance is provided by the aminoglycoside antibiotic resistance gene (aadA +) originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid[8]. <br /><br />
<br />
•'''AmyE''': Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus. This is achieved by using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence([http://partsregistry.org/Part:BBa_K143001BBa_K143001]) can be added to the front of a BioBrick construct and the 3' integration sequence specific for this locus ([http://partsregistry.org/Part:BBa_K143002 BBa_K143002]) to the rear of the Biobrick construct to allow integration of the BioBrick construct into the chromosome of the Gram-positive bacterium B. subtilis at the amyE locus[18,19]. <br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3" align= "center"><br />[[File:Unamgenomicsdeepconstruccion 2.png| x115px |link=Team:UNAM_Genomics_Mexico/Results/AND#ARABINOSE-XYLOSE_AND_GATE]]<p>'''Sugar AND Results'''</p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br />
<br /><br />
<br />
<h2>'''How does the Or work?'''</h2><br /><br />
•The OR operator gives an output when one, or both, of the inputs recognized by the operator are present. Our OR works with the A3 and LasB promoter, that need the P4 and LasR TF, respectively. When they are active, they transcribe GFP, and we can notice it is working because of the fluorescence that GFP produces.. <br /><br />
<br />
•'''LasB:''' Promoter regulated positively by LasR in ''P. aeruginosa''[21]. <br /><br />
<br />
•'''A3''': Promoter regulated positively by P4 from phi29 phage[4,16,17]. <br /><br />
<br />
•'''GFP:''' The GFP reporter gene encodes green fluorescent protein derived from jellyfish ''Aequeora victoria'' wild-type GFP[22]. <br /><br />
<br />
•'''Omega cassette:''' The Omega Cassette provides resistance to Spectinomycin and Streptomycin; it was obtained from plasmid pHP45omega[7,8]. Specifically, the resistance is provided by the aminoglycoside antibiotic resistance gene (aadA +) originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid[8]. <br /><br />
<br />
•'''AmyE:''' Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus. This is achieved by using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence([http://partsregistry.org/Part:BBa_K143001 BBa_K143001]) can be added to the front of a BioBrick construct and the 3' integration sequence specific for this locus ([http://partsregistry.org/Part:BBa_K143001 BBa_K143002]) to the rear of the Biobrick construct to allow integration of the BioBrick construct into the chromosome of the Gram-positive bacterium B. subtilis at the amyE locus [18,19]. <br /><br />
<br /><br />
<br />
<br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align= "center"><br />[[File:Unamgenomicsconstruccion3.png | 850px|link=Team:UNAM_Genomics_Mexico/Results/OR]]</td><br />
</tr><br />
</table><br />
</center><br />
<br />
<br /><br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Project/DeeperDescription#Deep_Description]]<br />
<center>[[File:Unamgenomicsdeepamye.jpg]]</center><br />
<br /><br />
<br /><br />
<br />
[1] Busenlehner LS, Pennella MA, Giedroc DP (2003). The SmtB/ArsR family of metalloregulatory transcriptional repressors: Structural insights into prokaryotic metal resistance. FEMS Microbiol Rev , 27:131-143. <br /><br />
[2] Charles M Moore and John D Helmann(2005). Metal ion homeostasis in ''Bacillus subtilis''. Current Opinion in Microbiology, 8:188–195. <br /><br />
[3] Moore CM, Gaballa A, Hui M, Ye RW, Helmann JD (2005). Genetic and physiological responses of ''Bacillus subtilis'' to metal ion stress. Mol Microbiol(1) , 27–40. <br /><br />
[4] Camacho A & Salas M (2010) DNA bending and looping in the transcriptional control of bacteriophage ϕ29. FEMS Microbiol Rev. 34(5):828-841. <br /><br />
[5] Rojo F, Mencía M, Monsalve M & Salas M (1998) Transcription activation and repression by interaction of a regulator with the a subunit of RNA polymerase: the model of phage ϕ29 protein p4. Nucleic Acid Re 60: 29–46<br /><br />
[6] http://partsregistry.org/Part:BBa_E1010<br /><br />
[7] Pierre Prentki, Anna Bind and Andrée Epstein (1991). Plasmid vectors for selecting ISI-promoted deletions in cloned DNA: sequence analysis of the omega interposon. Gene, 103:17-23.<br />
[8] Pierre Prentki and Henry M. Krisch (1984). In vitro insertional mutagenesis with a selectable DNA fragment. Gene, 29:303-313<br /><br />
[9] Lee, N. (1980) Molecular Aspects of ara Regulation. In The Operon, J. H. Miller and W. S. Reznikoff, eds. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory), pp. 389-410. <br /><br />
[10] Lee, N., Francklyn, C., and Hamilton, E. P. (1987). Arabinose-Induced Binding of AraC Protein to araI2 Activates the araBAD Operon Promoter. Proc. Natl. Acad. Sci. USA 84, 8814-8818. <br /><br />
[11] Shamanna, D. K., and K. E. Sanderson. 1979. Genetics and regulation of D-xylose utilization in Salmonella typhimurium LT2. J. Bacteriol. 139:71-79. <br /><br />
[12] D Gartner, M Geissendorfer, & W Hillen(1988). Expression of the ''Bacillus subtilis'' xyl Operon Is Repressed at the Level of Transcription and Is Induced by Xylose J Bacteriol 170:7,3102-3109.<br />
[13] Moran CP, Lang N, LeGrice SF, Lee G, Stephens M, Sonenshein AL, Pero J, Losick R (1982). Nucleotide sequences that signal the initiation of transcription and translation in ''Bacillus subtilis''. Mol Gen Genet; 186(3): 339-46 <br /><br />
[14] Kreuzer P, Gärtner D, Allmansberger R, Hillen W (1989). Identification and sequence analysis of the ''Bacillus subtilis'' W23 xylR gene and xyl operator. J Bacteriol. Jul;171(7):3840-5. <br /><br />
[15] Schlief, R. (2000). Regulation of the L-arabinose operon of ''Escherichia coli''. Trends in Genetics. 16(12):559-565. <br /><br />
[16] Sogo JM, Inciarte MR, Corral J, Viñuela E & Salas M(1979) RNA polymerase binding sites and transcription map of the DNA of ''Bacillus subtilis'' phage ϕ29. J Mol Biol 127: 411–436.<br />
[17] Nuez B, Rojo F & SalasM(1992) Phage ϕ29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein–protein contact. P Natl Acad Sci USA 89: 11401–11405. <br /><br />
[18]Cutting, S M.; Vander-Horn, P B. Genetic analysis. In: Harwood C R, Cutting S M. , editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons, Ltd.; 1990. pp. 27–74. <br /><br />
[19] http://partsregistry.org/Part:BBa_K143001 <br /><br />
[20]http://regtransbase.lbl.gov/cgi-bin/regtransbasepage=regulatorinfo&type=site&g<br />
uid=76697<br /><br />
[21]Analysis of the Pseudomonas aeruginosa elastase (lasB) regulatory region. L Rust, E C Pesci and B H Iglewski, J. Bacteriol. February 1996 vol. 178 no. 4 1134-1140 <br /><br />
[22] http://partsregistry.org/wiki/index.php/Part:BBa_E0040<br /><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/DeeperDescription
Team:UNAM Genomics Mexico/Project/DeeperDescription
2012-10-27T00:38:07Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br />
<center><h1>'''Deep Description'''</h1></center><br />
<br /><br />
<br /><br />
<br /><br />
<center><br />
<html><br />
<div class="prezi-player"><style type="text/css" media="screen">.prezi-player { width: 800px; } .prezi-player-links { text-align: center; }</style><object id="prezi_or0xn1xbzour" name="prezi_or0xn1xbzour" classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" width="800" height="600"><param name="movie" value="http://prezi.com/bin/preziloader.swf"/><param name="allowfullscreen" value="true"/><param name="allowFullScreenInteractive" value="true"/><param name="allowscriptaccess" value="always"/><param name="bgcolor" value="#ffffff"/><param name="flashvars" value="prezi_id=or0xn1xbzour&amp;lock_to_path=0&amp;color=ffffff&amp;autoplay=no&amp;autohide_ctrls=0"/><embed id="preziEmbed_or0xn1xbzour" name="preziEmbed_or0xn1xbzour" src="http://prezi.com/bin/preziloader.swf" type="application/x-shockwave-flash" allowfullscreen="true" allowFullScreenInteractive="true" allowscriptaccess="always" width="800" height="600" bgcolor="#ffffff" flashvars="prezi_id=or0xn1xbzour&amp;lock_to_path=0&amp;color=ffffff&amp;autoplay=no&amp;autohide_ctrls=0"></embed></object><div class="prezi-player-links"><p><a title="Copy of Project Description" href="http://prezi.com/or0xn1xbzour/copy-of-project-description/">Copy of Project Description</a> on <a href="http://prezi.com">Prezi</a></p></div></div><br />
</html><br />
</center><br />
<br />
<br /><br />
<br /><br />
<br /><br />
<br />
Our boolean constructions are two, the AND & OR, for it to form a system, but how we will connect this two operations? With the nanotubes that ''Bacillus subtilis'' form. <br /><br />
<br /><br />
<br />
== '''Why B. subtilis?''' ==<br />
<br /><br />
We found a paper in which Ben-Yehuda et. al. demostrated that ''Bacillus subtilis'' form nanotubes between them, ''Escherichia coli'' and ''Staphylococcus aureus''. And they were able to get through a GFP and some other smaller molecules. So, we decided to use this connection to form the principles of a molecular computer. How? The output of the first operation will the input for the next one, and that was what we did. Also, there is not many work in the'' B. subtilis'' at the iGEM competition, so we decided to standardize the protocols of competent cells and transformation, and to send biobricks that ''B. subtilis'' could transform and integrate for them to work correctly [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Bacillus_subtilis_Protocols | (new standard protocol)]]. <br /><br /><br />
<center><br />
<html><br />
<div class='thumbnailWrapper'><br />
<ul><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/6/6a/Unamgenomicsdeepdescriptionbacillus1.png' height="200" /><br />
<div class='captiongray'><br />
<p class='captionInside'>2011 Gyanendra P. Dubey, Sigal Ben-Yehuda. Intercellular Nanotubes Mediate Bacterial Communication. Cell, 2011; 144 (4): 590 DOI:10.1016/j.cell.2011.01.015<br /></p><br />
</div><br />
</li><br />
<li><br />
<img src='https://static.igem.org/mediawiki/2012/c/c9/Unamgenomicsdeepdescriptionbacillus2.jpg' height="200" /><br />
<div class='captionnaranja'><br />
<p class='captionInside'>2011 Gyanendra P. Dubey, Sigal Ben-Yehuda. Intercellular Nanotubes Mediate Bacterial Communication. Cell, 2011; 144 (4): 590 DOI:10.1016/j.cell.2011.01.015<br /></p><br />
</div><br />
<div class='clear'></div><br />
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== '''How does the AND work?''' ==<br />
<br /><br />
<br /><br />
The AND operator in general only gives an output when the two inputs recognized be the operator are present. In the design, we considered the possibility that the constitutive promoter could continue with transcription even after getting to the double terminator and to avoid this, we designed the AND construction with the inducible promoter before the constitutive one. <br /><br />
We have two types of AND: <br /><br />
•'''The Heavy Metals AND''': The promoters CzrA/ArsR have something in common, they both sense cadmium. When the two promoters detect cadmium, they would not repress anymore and start transcription of P4 or LasR, and we will know it is working because our bacterium will be red, because of our reporter gene (RFP). The promoters are based in the CadA promoter plus a binding site for ArsR for get a response that depend in both, CzrA and ArsR. <br /><br />
<br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10" align="right"><br />
<tr><br />
<td id="contentcolumnwhite" align= "center"><br />[[File:UnamgenomicsCadmiopromotores.png | 430px ]]<br /><br /><p>Our CzrA/ArsR </p></td><br />
</tr><br />
</table><br />
<br />
•'''CzrA/ArsR''': This transcription factors are involved in metal homeostasis, each one had affinity for a set of metals[1], the intersection of both sensors is Cadmium[2][3], in this case the proposed input metals will be Zinc for CzrA and Arsenic for ArsR. This TFs acts as repressors in the absence of metals, when there is metal in the cell, the metal binds to the transcription factor and make a conformation change that dissolves the affinity of the protein for their binding sites and let the promoter free to recruit RNA polymerase to start transcription[1]. <br /><br />
<br />
We have three''' CzrA/ArsR promoters''', one of them was designed by Newcastle team in 2009 ([http://partsregistry.org/Part:BBa_K174015 Part BBa_K174015]), the two remaining were design by our team based on the previous by Newcastle. Why we need more than one promoter? The part BBa_K174015 has no reported experience, so it could work as it should or not and the way this cadmium sensor was developed allow us to play with the combinatory of binding sites and with the -10 and -35 boxes. The cadmium sensor of Newcastle is -35, -10 and binding sites, our promoters are:<br /><br />
<br />
'''Promoter 1''': attta, -35,-10, ArsR binding site overlapping -35, CsrA binding site overlapping TSS, rbs+atg.<br /><br />
<br />
'''Promoter 2''': -35, -10, ArsR binding site overlapping after TSS, CzrA overlapping TSS, rbs+atg.<br /><br />
<br />
In this way, by designing different versions of the “same” part we can reach the most optimal biobrick.<br /><br />
<br />
•'''P4''': It´s a transcription factor from ''phi29 phage'', which is a phage of ''B. subtilis'', it is the activator of the A3 promoter[4,5]. <br /><br />
<br />
•'''LasR''': It is another activator transcription factor, its origin is ''Pseudomonas aeruginosa'' PAO1, its correspondent promoter is LasB[20]. <br /><br />
<br />
•'''CI''': This protein is a repressor from Lambda phage. <br /><br />
<br />
•'''RFP''': highly engineered mutant of red fluorescent protein from ''Discosoma striata'' (coral), this is our reporter gene[6]. <br /><br />
<br />
•'''Omega cassette''': The Omega Cassette provides resistance to Spectinomycin and Streptomycin; it was obtained from plasmid pHP45omega[7,8]. Specifically, the resistance is provided by the aminoglycoside antibiotic resistance gene (aadA +) originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid[8]. <br /><br />
<br />
•'''AmyE''': Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus. This is achieved by using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence([http://partsregistry.org/Part:BBa_K143001 BBa_K143001]) can be added to the front of a BioBrick construct and the 3' integration sequence specific for this locus ([http://partsregistry.org/Part:BBa_K143002 BBa_K143002]) to the rear of the Biobrick construct to allow integration of the BioBrick construct into the chromosome of the Gram-positive bacterium B. subtilis at the amyE locus[18,19]. <br /><br />
<br /><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsConstruccion1.png| x115px |link=Team:UNAM_Genomics_Mexico/Results/AND#CzrA-ArsR_AND_GATE]]<p>'''Heavy metal AND Results'''</p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br />
<br /><br />
<br />
•'''The pBAD/pXyl AND''': The promoter pBAD is activated with arabinose, and pXyl with xylose. When both the promoters are activated it starts the transcription of P4 or LasR, and we will know it is working because our bacteria will be red, because of the reporter gene (RFP). <br /><br />
<br />
•'''pBAD''': It´s the promoter under the sigma factor 70 from ''E. coli'', which is analogous to the sigma A factor of B. subtilis. It’s repressed by AraC and induced by arabinose, which triggers the rearrangement of the AraC monomers binding for the recruitment of RNA polymerase[9,10]. <br /><br />
<br />
•'''pXyl''': It´s a promoter in B. subtilis which is regulated by Xylose and XylR[11,12]. In the endogenous systems, pXyl is the promoter for a set of genes involved in xylose metabolism[12]. <br /><br />
<br />
•P4: It´s a transcription factor from phi29 phage, which is a phage of B. subtilis, it is the activator of the A3 promoter[4,5]. <br /><br />
<br />
•'''LasR''': It is another activator transcription factor, its origin is ''Pseudomonas aeruginosa'' PAO1, its correspondent promoter is LasB[20]. <br /><br />
<br />
•'''CI''': This protein is a repressor from Lambda phage. <br /><br />
<br />
•'''RFP''': highly engineered mutant of red fluorescent protein from ''Discosoma striata'' (coral), this is our reporter gene[6]. <br /><br />
<br />
•'''Pveg''': This is a constitutive promoter from ''B. subtilis'' regulates by the sigma factor sigma A[13]. <br /><br />
<br />
•'''XylR''': This is a repressor transcription factor which in the presence of xylose is inactivated by a conformational change triggered by the binding with xylose that induces the dissociation of the XylR from its binding site[14]. <br /><br />
<br />
•'''AraC''': It´s one ''E.coli'' repressor transcription factor. In the absence of arabinose, araC binds to two binding sites located in the pBAD promoter and through the binding of both monomers od AraC a DNA looping is made which blocks the transcription of the genes under that promoter[15]. When arabinose is present in the medium. <br /> <br />
<br />
•'''Omega cassette''': The Omega Cassette provides resistance to Spectinomycin and Streptomycin; it was obtained from plasmid pHP45omega[7,8]. Specifically, the resistance is provided by the aminoglycoside antibiotic resistance gene (aadA +) originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid[8]. <br /><br />
<br />
•'''AmyE''': Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus. This is achieved by using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence([http://partsregistry.org/Part:BBa_K143001BBa_K143001]) can be added to the front of a BioBrick construct and the 3' integration sequence specific for this locus ([http://partsregistry.org/Part:BBa_K143002 BBa_K143002]) to the rear of the Biobrick construct to allow integration of the BioBrick construct into the chromosome of the Gram-positive bacterium B. subtilis at the amyE locus[18,19]. <br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3" align= "center"><br />[[File:Unamgenomicsdeepconstruccion 2.png| x115px |link=Team:UNAM_Genomics_Mexico/Results/AND#ARABINOSE-XYLOSE_AND_GATE]]<p>'''Sugar AND Results'''</p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br />
<br /><br />
<br />
<h2>'''How does the Or work?'''</h2><br /><br />
•The OR operator gives an output when one, or both, of the inputs recognized by the operator are present. Our OR works with the A3 and LasB promoter, that need the P4 and LasR TF, respectively. When they are active, they transcribe GFP, and we can notice it is working because of the fluorescence that GFP produces.. <br /><br />
<br />
•'''LasB:''' Promoter regulated positively by LasR in ''P. aeruginosa''[21]. <br /><br />
<br />
•'''A3''': Promoter regulated positively by P4 from phi29 phage[4,16,17]. <br /><br />
<br />
•'''GFP:''' The GFP reporter gene encodes green fluorescent protein derived from jellyfish ''Aequeora victoria'' wild-type GFP[22]. <br /><br />
<br />
•'''Omega cassette:''' The Omega Cassette provides resistance to Spectinomycin and Streptomycin; it was obtained from plasmid pHP45omega[7,8]. Specifically, the resistance is provided by the aminoglycoside antibiotic resistance gene (aadA +) originally carried on a 1.7-kb PvuII-HindIII fragment from the R100.1 plasmid[8]. <br /><br />
<br />
•'''AmyE:''' Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus. This is achieved by using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence([http://partsregistry.org/Part:BBa_K143001 BBa_K143001]) can be added to the front of a BioBrick construct and the 3' integration sequence specific for this locus ([http://partsregistry.org/Part:BBa_K143001 BBa_K143002]) to the rear of the Biobrick construct to allow integration of the BioBrick construct into the chromosome of the Gram-positive bacterium B. subtilis at the amyE locus [18,19]. <br /><br />
<br /><br />
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<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="contentcolumn" align= "center"><br />[[File:Unamgenomicsconstruccion3.png | 850px|link=Team:UNAM_Genomics_Mexico/Results/OR]]</td><br />
</tr><br />
</table><br />
</center><br />
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<br /><br />
<br /><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Project/DeeperDescription#Deep_Description]]<br />
<center>[[File:Unamgenomicsdeepamye.jpg]]</center><br />
<br /><br />
<br /><br />
<br />
[1] Busenlehner LS, Pennella MA, Giedroc DP (2003). The SmtB/ArsR family of metalloregulatory transcriptional repressors: Structural insights into prokaryotic metal resistance. FEMS Microbiol Rev , 27:131-143. <br /><br />
[2] Charles M Moore and John D Helmann(2005). Metal ion homeostasis in ''Bacillus subtilis''. Current Opinion in Microbiology, 8:188–195. <br /><br />
[3] Moore CM, Gaballa A, Hui M, Ye RW, Helmann JD (2005). Genetic and physiological responses of ''Bacillus subtilis'' to metal ion stress. Mol Microbiol(1) , 27–40. <br /><br />
[4] Camacho A & Salas M (2010) DNA bending and looping in the transcriptional control of bacteriophage ϕ29. FEMS Microbiol Rev. 34(5):828-841. <br /><br />
[5] Rojo F, Mencía M, Monsalve M & Salas M (1998) Transcription activation and repression by interaction of a regulator with the a subunit of RNA polymerase: the model of phage ϕ29 protein p4. Nucleic Acid Re 60: 29–46<br /><br />
[6] http://partsregistry.org/Part:BBa_E1010<br /><br />
[7] Pierre Prentki, Anna Bind and Andrée Epstein (1991). Plasmid vectors for selecting ISI-promoted deletions in cloned DNA: sequence analysis of the omega interposon. Gene, 103:17-23.<br />
[8] Pierre Prentki and Henry M. Krisch (1984). In vitro insertional mutagenesis with a selectable DNA fragment. Gene, 29:303-313<br /><br />
[9] Lee, N. (1980) Molecular Aspects of ara Regulation. In The Operon, J. H. Miller and W. S. Reznikoff, eds. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory), pp. 389-410. <br /><br />
[10] Lee, N., Francklyn, C., and Hamilton, E. P. (1987). Arabinose-Induced Binding of AraC Protein to araI2 Activates the araBAD Operon Promoter. Proc. Natl. Acad. Sci. USA 84, 8814-8818. <br /><br />
[11] Shamanna, D. K., and K. E. Sanderson. 1979. Genetics and regulation of D-xylose utilization in Salmonella typhimurium LT2. J. Bacteriol. 139:71-79. <br /><br />
[12] D Gartner, M Geissendorfer, & W Hillen(1988). Expression of the ''Bacillus subtilis'' xyl Operon Is Repressed at the Level of Transcription and Is Induced by Xylose J Bacteriol 170:7,3102-3109.<br />
[13] Moran CP, Lang N, LeGrice SF, Lee G, Stephens M, Sonenshein AL, Pero J, Losick R (1982). Nucleotide sequences that signal the initiation of transcription and translation in ''Bacillus subtilis''. Mol Gen Genet; 186(3): 339-46 <br /><br />
[14] Kreuzer P, Gärtner D, Allmansberger R, Hillen W (1989). Identification and sequence analysis of the ''Bacillus subtilis'' W23 xylR gene and xyl operator. J Bacteriol. Jul;171(7):3840-5. <br /><br />
[15] Schlief, R. (2000). Regulation of the L-arabinose operon of ''Escherichia coli''. Trends in Genetics. 16(12):559-565. <br /><br />
[16] Sogo JM, Inciarte MR, Corral J, Viñuela E & Salas M(1979) RNA polymerase binding sites and transcription map of the DNA of ''Bacillus subtilis'' phage ϕ29. J Mol Biol 127: 411–436.<br />
[17] Nuez B, Rojo F & SalasM(1992) Phage ϕ29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein–protein contact. P Natl Acad Sci USA 89: 11401–11405. <br /><br />
[18]Cutting, S M.; Vander-Horn, P B. Genetic analysis. In: Harwood C R, Cutting S M. , editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons, Ltd.; 1990. pp. 27–74. <br /><br />
[19] http://partsregistry.org/Part:BBa_K143001 <br /><br />
[20]http://regtransbase.lbl.gov/cgi-bin/regtransbasepage=regulatorinfo&type=site&g<br />
uid=76697<br /><br />
[21]Analysis of the Pseudomonas aeruginosa elastase (lasB) regulatory region. L Rust, E C Pesci and B H Iglewski, J. Bacteriol. February 1996 vol. 178 no. 4 1134-1140 <br /><br />
[22] http://partsregistry.org/wiki/index.php/Part:BBa_E0040<br /><br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/AND
Team:UNAM Genomics Mexico/Results/AND
2012-10-27T00:36:39Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
__NOTOC__<br />
<br /><br />
<center><h1>'''AND's Results'''</h1></center> <br />
<br /><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsANDcadmio.png | 200px |link=Team:UNAM_Genomics_Mexico/Results/AND#CzrA-ArsR_AND_GATE]]<br /><br /><p>'''AND Heavy Metals'''</p></td><br />
<td id="leftcolumn3" align= "center"><br />[[File:UnamgenomicsANDarabinosa.png | 200px |link=Team:UNAM_Genomics_Mexico/Results/AND#ARABINOSE-XYLOSE_AND_GATE]]<p>'''AND Sugar''''</p></td><br />
</tr><br />
</table><br />
</center><br />
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<br/><br />
<br />
== '''CzrA-ArsR AND GATE''' ==<br />
<br/><br />
<br/><br />
'''HEAVY-METAL AND'''<br/><br />
<br/><br />
This construct was designed to function as a logic gate, an AND to be specific. This is due to the way the CzrA-ArsR promoter was designed. We used a system that sensed heavy metals in Bacillus subtilis, which was originally designed by the iGEM Newcastle team 2009 ([https://2009.igem.org/Team:Newcastle/Project#Cadmium_Sensing Newcastle University iGEM team 2009]). This system consists of a fused promoter, which includes both ArsR and CzrA binding sites. Arar and CzrA are metal sensing repressors. They both respond to cadmium, however silver, arsenic, or copper induces ArsR and zinc, cobalt, or nickel induces CzrA as well (Moore CM, Helmann JD. Metal ion homeostasis in Bacillus subtilis. Curr Opin Microbiol. 2005 Apr;8(2):188-95.). If we use two different metals, specific for each repressor we will have an AND gate. Besides Newcastle’s 2009 design, we designed two different fused promoters with the same binding sites but in different order to try different combinations that could make the system more efficient. <br/><br />
<br/><br />
<br/><br />
'''To obtain our final construct we required the following Biological Parts''':<br/><br />
<br/><br />
<br/><br />
'''Obtained from the registry:'''<br/><br />
[http://partsregistry.org/Part:BBa_K143001 BBa_K143001 (AmyE 5’)]<br/><br />
[http://partsregistry.org/Part:BBa_K143002 BBa_K143002] (AmyE 3’)<br/><br />
[http://partsregistry.org/Part:BBa_E1010 BBa_E1010 (RFP)]<br/><br />
[http://partsregistry.org/Part:BBa_B0014 BBa_B0014 (Terminator)]<br/><br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0179 BBa_C0179 (LasR)]<br/><br />
<br/><br />
<br/><br />
'''Synthesis Products:'''<br/><br />
CzrA-AsR 99<br/><br />
CzrA-AsR 98<br/><br />
CzrA-AsR 97<br/><br />
RBS-CI<br/><br />
<br/><br />
<br/><br />
'''Obtained from Margarita Salas Ph.D.’s Group:'''<br/><br />
P4 from phage phi29 from plasmid pRMn25.<br/><br />
<br/><br />
<br/><br />
'''Omega cassette from plasmid pHP45Ω.'''<br/><br />
<br/><br />
We designed the following primers to add the RBS site to BBa_C0179, BBa_E100 and P4.<br/><br />
<br/><br />
'''LASR_2.0_seq_registry'''<br/><br />
UPPER 5'-3'<br/><br />
PREFIJO+RBS+ESPACIADOR+LASR<br/><br />
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG atggccttggttgac<br/><br/><br />
LOWER 5'-3'<br/><br />
SUFIJO+LASR_<br/><br />
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA ttattagagagtaat<br/><br />
<br/><br />
'''RFP'''<br/><br />
UPPER 5'-3'<br/><br />
PREFIX+RBS+SPACER+RFP <br/><br />
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTTCCTCCGAA<br/><br />
<br/><br />
LOWER 5'-3'<br/><br />
SUFIX+RFP<br/><br />
GTTTCTTCCTGCAGCGGCCGCTACTAGTA TTATTAAGCACCGGT<br/><br />
<br/><br />
'''P4''' <br/><br />
PREFIX+RBS+SPACER+P4 <br/><br />
UPPER<br/><br />
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGCCTAAAACACAA<br/><br />
<br/><br />
SUFIX+P4<br/><br />
LOWER 5'-3'<br/><br />
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA CTACACCATACTTTT<br/><br />
<br/><br />
<br/><br />
This metal AND team had to build the following construct:<br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsAndmetals1.png| x90px]]</td><br />
</tr><br />
</table><br />
</center><br />
<br />
The other AND (pBad/pXyl) team was in charge of building this part. <br/><br />
<br/><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3" align= "center"><br />[[File:UnamgenomicsresultsOr3.png| x80px]]</td><br />
</tr><br />
</table><br />
</center><br />
<br />
<br />
'''So we obtain the final product:'''<br/><br />
<br/><br />
After the Regional Jamboree, we continued working very hard, and our work has paid off, these our new results:<br />
Amy,98,P4,CI,RFP,TT<br/><br />
Amy99,P4,CI,RFP,TT<br/><br />
Also we feel very proud and excited because we finally have one complete AND gate =D !!!!<br/><br />
AMY,97,P4,CI,RFP,TT,OMEGA,AMY3<br/><br />
<br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsConstruccion1.png| x120px]]</td><br />
</tr><br />
</table><br />
</center><br />
<br/><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsmexicoandResultspartsmetal.png| 800px]]<p>'''AmyE 5’_ArsR/CzrA 97-99, P4_CI, RFP_Terminator, P4_CI_RFP_Terminator and AmyE 5’_CzrA/ArsR 99-97_P4CI_RFP_Terminator'''</p></td><br />
</tr><br />
</table><br />
</center><br />
<br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Results/AND]]<br />
<br/><br />
<br/><br />
<br/><br />
<br />
== '''ARABINOSE-XYLOSE AND GATE''' ==<br />
<br/><br />
'''SWEET AND'''<br />
<br/><br />
<br/><br />
This construct was designed to function as a AND logic gate. This is due to the way the pBad/pXyl promoter was designed. We used a system that sensed l-arabinose in E.coli which was originally designed by Amelia Hardjasa and used by iGEM09_British_Columbia1,2. In the absence of arabinose, the repressor protein AraC (BBa_C0080) binds to the AraI1 operator site of pBAD and the upstream operator site AraO2, blocking transcription1. In the presence of arabinose, AraC binds to it and changes its conformation such that it interacts with the AraI1 and AraI2 operator sites, permitting transcription1. We also used a promoter inducible by xylose that has been designed for high expression in B.subtilis which was originally designed by James Chappell and used by iGEM08_Imperial_College. Xylose does not induce the promoter xylose directly, but requires the transcriptional regulator XylR (BBa_K143036). Our system consists in a fused promoter which includes both AraC and XylR binding sites. AraC and XylR are l-arabinose and xylose sensing, respectively, repressors. In this way, if we use these two inputs, each specific for each repressor we will have an AND gate.<br/><br />
To obtain our final construct we required the following Biological Parts:<br/><br />
<br/><br />
<br/><br />
'''Obtained from the Registry:'''<br/><br />
[http://partsregistry.org/Part:BBa_K143001 BBa_K143001 (AmyE 5’)]<br/><br />
[http://partsregistry.org/Part:BBa_K143002 BBa_K143002 (AmyE 3’)]<br/><br />
[http://partsregistry.org/Part:BBa_E1010 BBa_E1010 (RFP)]<br/><br />
[http://partsregistry.org/Part:BBa_B0014 BBa_B0014 (Terminator)]<br/><br />
[http://partsregistry.org/wiki/index.php/Part:BBa_C0080 BBa_C0080 (AraC)]<br/><br />
<br/><br />
'''Synthesis Products:'''<br/><br />
pBad/pXyl promoter<br/><br />
RBS XylR<br/><br />
<br />
'''Obtained from Margarita Salas Ph.D.’s Group:'''<br/><br />
A3 from phage phi29 from plasmid pFRC54.<br/><br />
<br/><br />
'''Omega cassette from plasmid pHP45Ω.'''<br/><br />
<br/><br />
We designed the following primers to add the RBS site to BBa_E1010 (RFP) and BBa_C0080 (AraC):<br/><br />
'''RFP'''<br/><br />
UPPER 5'-3'<br/><br />
PREFIX+RBS+SPACER+RFP <br/><br />
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTTCCTCCGAA<br/><br />
LOWER 5'-3'<br/><br />
SUFIX+RFP<br/><br />
GTTTCTTCCTGCAGCGGCCGCTACTAGTA TTATTAAGCACCGGT<br/><br />
<br/><br />
'''ARAC without LVA'''<br/><br />
UPPER 5'-3'<br/><br />
PREFIX+RBS+SPACER+ARAC<br/><br />
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTGAAGCGCAA<br/><br />
LOWER 5'-3'<br/><br />
SUFIX+ARAC<br/><br />
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA CAACTTGACGGCTAC<br/><br />
<br/><br />
We also designed the following primers to obtain A3 from phage phi29 from plasmid pFRC54 and Omega cassette from plasmid pHP45Ω:<br/><br />
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'''A3''' <br/><br />
UPPER 5'-3'<br/><br />
PREFIX+A3<br/><br />
5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG taactttttgcaaga 3'<br/><br />
LOWER 5'-3'<br/><br />
SUFIX+A3<br/><br />
5'GTTTCTTCCTGCAGCGGCCGCTACTAGTA ctacttaattatacc 3'<br/><br />
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<br/><br />
'''OMEGA CASSETTE''' <br/><br />
PREFIX+OMEGA CASSETTE(45 bp)<br/><br />
5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG CCGGGGATCCGGTGA 3'<br/><br />
<br/><br />
SUFIX+OMEGA CASSETTE (44 bp)<br/><br />
5' GTTTCTTCCTGCAGCGGCCGCTACTAGTA CCGGGGATCCGGTGA 3'<br/><br />
<br/><br />
<br/><br />
This arabinose-xylose AND team had to build the following constructs:<br/><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3" align= "center"><br />[[File:UnamgenomicsresutsAmypbadpxy.png | x90px]]<br /><br /><p></p></td><br />
</tr><br />
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<td id="leftcolumn3" align= "center"><br />[[File:UnamgenomicsresultsP4lasr.png | x90px]]<p></p></td><br />
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<td id="leftcolumn3" align= "center"><br />[[File:UnamgenomicsResultsabisugar1.png | x90px]]<p></p></td><br />
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</table><br />
</center><br />
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The other AND (heavy metals) team was in charge of building this part.<br/><br />
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<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsRfgstop.png| x90px]]<p>'''RFP_Terminator'''</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br />
<br/><br />
So we could obtain the final product:<br/><br />
<br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3" align= "center"><br />[[File:Unamgenomicsdeepconstruccion 2.png| x115px]]<p></p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br />
What we have up now is:<br/><br />
<br/><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3" align= "center"><br />[[File:UnamgenomicsResultssugar.png| 850px]]<p>'''AmyE 5’_pBad/pXyl, pVeg_RBS_XylR, Omega cassette_AmyE 3’, RBS_AraC_Omega cassette_AmyE 3’'''</p></td><br />
</tr><br />
</table><br />
</center><br />
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<br/><br />
We are working hard to obtain these last two constructs in order to finish it:<br/><br />
<br/><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="leftcolumn3" align= "center"><br />[[File:UnamgenomicsresultsSugarfalta.png| 850px]]<p>'''pVeg_RBS_XylR_RBS_AraC_Omega cassette_AmyE 3’, AmyE 5’_pBad/pXyl_RBS_P4/LasR_RBS_CI_RBS_RFP_Double Terminador'''</p></td><br />
</tr><br />
</table><br />
</center><br />
<br/><br />
<br/><br />
<br/><br />
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Results/AND]]<br />
<br />
'''Please see our wetlab notebook in the clicking the following image:'''<br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsANDcadmio.png | 200px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDMetal]]<br /><br /><p>'''AND Heavy Metals Notebook'''</p></td><br />
<br />
<td id="leftcolumn3" align= "center"><br />[[File:UnamgenomicsANDarabinosa.png | 200px |link=Team:UNAM_Genomics_Mexico/Notebook/ANDSugar]]<p>'''AND Sugar Notebook''''</p></td><br />
</tr><br />
</table><br />
</center><br />
== '''References''' ==<br />
<br/><br />
[1] Schlief, R. (2000). Regulation of the L-arabinose operon of Escherichia coli. Trends in Genetics. 16(12):559-565. <br/><br />
[2] Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, and Keasling JD. (2001). Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology. 147(12):3241-7. <br/><br />
<br/><br />
<br />
<br />
<br />
}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/Nanotubes
Team:UNAM Genomics Mexico/Results/Nanotubes
2012-10-27T00:32:21Z
<p>Abieltega: </p>
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<center><h1>'''Nanotubes'''</h1></center> <br />
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<td id="leftcolumn2" align="center"><img src="https://static.igem.org/mediawiki/2012/d/d2/Unamgenomicsnanotubes.jpg" alt="some_text" height="200"/><br /><br /><p>Nanotubes</p></td><br />
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<td id="contentcolumn2" align="center"><img src="https://static.igem.org/mediawiki/2012/9/96/Unamgenomicsnanotubes1.jpg" alt="some_text" height="200"/><br /><br /><p>Nanotubes</p></td><br />
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Just as people, bacteria need to communicate with others. For this goal there are many paths, and one of the most recent discoveries are Nanotubes that bridge neighboring cells, providing a network for exchange of cellular molecules within, and between species. Ben-Yehuda et al discovered these nanotubes in 2011. They show an extraordinary form of communication between ''Bacillus subtilis''. Our team was astonished because of the implications. In the article they described that GFP and calcein, two molecules which cannot leave the cytoplasm, can be transferred to neighboring cells in ''B. subtilis''. This means bacterium can share cytoplasm. The complex network of cells sharing cytoplasm that can be created was our main motivation to create Bacillus booleanus.<br /><br />
<br /><br />
Little is still known about nanotubes, and that's what makes them a very interesting subject of study. We are not the first iGEM team interested in working with them. In 2011 the [https://2011.igem.org/Team:Paris_Bettencourt Paris_Bettencourt] team worked with nanotubes, their goal being to characterize them<br /><br />
<br /><br />
To accomplish our goal of communicate our logic gates, we contacted [https://medicine.ekmd.huji.ac.il/En/Publications/ResearchersPages/pages/sigalb.aspx Ben Yehuda] group, and [https://2011.igem.org/Team:Paris_Bettencourt Paris Bettencourt iGEM 2011 team], to obtain protocols and work experience.<br /><br />
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<p class='captionInside'>2011 Gyanendra P. Dubey, Sigal Ben-Yehuda. Intercellular Nanotubes Mediate Bacterial Communication. Cell, 2011; 144 (4): 590 DOI:10.1016/j.cell.2011.01.015<br /></p><br />
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<p class='captionInside'>2011 Gyanendra P. Dubey, Sigal Ben-Yehuda. Intercellular Nanotubes Mediate Bacterial Communication. Cell, 2011; 144 (4): 590 DOI:10.1016/j.cell.2011.01.015<br /></p><br />
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== '''We worked to recreate the formation of nanotubes, and we did it!''' ==<br />
<br /><br /><br />
We are the second group that is able to see this nanotubes, Ben Yehuda's work team and us, and answering the question <br />
that [https://2011.igem.org/Team:Paris_Bettencourt Paris Bettencourt had in iGEM 2011], TUBE OR NOT TUBE? '''We said TUBE'''.<br />
<br /><br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsNanocirculorojo.jpg|800px| link=Team:UNAM_Genomics_Mexico/Notebook/Bacillus]]<p>'''PY79 cells were grown to midexponential phase, plated on LB agar, incubated for 6 hr at 37°C, and visualized by SEM Rej34 circles: (A) Bacillus subtilis cells(x 7,500). The red circles indicate intercellular nanotubes connecting neighboring cells. The scale bar represents 1 micrometer. '''</p></td><br />
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</center><br />
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We prepare our cells with the [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#SEM_Analysis_.28Scanning_Electron_Microscope.29.5B1.5D | SEM Analysis Protocol]], We fixed our cells in the right moment (6 hrs of growth at 37°C), that is when they are in the exponential growth phase, and then we watch them in the SEM (Scanning Electron Microscope) and we get different and amazing pictures. <br /><br /><br />
We got similar results to the article, tube length ranged from 0.25 micrometers to 0.9 micrometers, whereas width ranged approximately from 50 to 80 nm.<br />
<br /><br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsREJ35 valores.jpg|800px| link=Team:UNAM_Genomics_Mexico/Notebook/Bacillus]]<p>'''PY79 cells were grown to midexponential phase, plated on LB agar, incubated for 6 hr at 37°C, and visualized by SEM Rej35_valores:(A)Measurment of the length and width of the nanotubes connecting the (PY79) cells grown on solid LB medium. The scale bar represent 1 micrometer'''</p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br /><br />
<br />
We also got an amazing picture about the nanotubes in which we can see, that there are many nanotubes among the cells, <br />
and that they communicate between different nanotubes to the same cell, observing this amount of nanotubes, we could<br />
think that the probability of transfer will raise. <br /><br /><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsREJ48.jpg|800px| link=Team:UNAM_Genomics_Mexico/Notebook/Bacillus]]<p>'''PY79 cells were grown to midexponential phase, plated on LB agar, incubated for 6 hr at 37°C, and visualized by SEM Rej48: (A) Field of cells demonstrating the occurrence of a network of intercellular nanotubes (X 10,000). The scale bar represents 1 micrometer'''</p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br /><br />
<br />
<br /><br />
Slider captions<br /><br />
PY79 cells were grown to midexponential phase, plated on LB agar, incubated for 6 hr at 37°C, and visualized by SEM Rej43, 44, 46, 47. <br /> <br />
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::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Results/Nanotubes#Nanotubes]]<br />
<br />
'''Please see our wetlab notebook in the clicking the following image:'''<br /><br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsBacillus.png|200px| link=Team:UNAM_Genomics_Mexico/Notebook/Bacillus]]<p>'''Bacillus Notebook'''</p></td><br />
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</center><br />
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}}</div>
Abieltega
http://2012.igem.org/Team:UNAM_Genomics_Mexico/NewsPapers
Team:UNAM Genomics Mexico/NewsPapers
2012-10-27T00:13:22Z
<p>Abieltega: </p>
<hr />
<div>{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=<br />
<br />
<h1>'''We worked to recreate the formation of nanotubes, and we did it!'''</h1><br />
<br /><br /><br />
<br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
<tr><br />
<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsNanocirculorojo.jpg|800px| link=Team:UNAM_Genomics_Mexico/Notebook/Bacillus]]<p>'''PY79 cells were grown to midexponential phase, plated on LB agar, incubated for 6 hr at 37°C, and visualized by SEM Rej34 circles: (A) Bacillus subtilis cells(x 7,500). The red circles indicate intercellular nanotubes connecting neighboring cells. The scale bar represents 1 micrometer. '''</p></td><br />
</tr><br />
</table><br />
</center><br />
<br /><br /><br />
<br />
<h1>'''We obtain one AND final product:'''</h1><br/><br />
<br/><br />
<center><br />
<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="rightcolumn2" align= "center"><br />[[File:UnamgenomicsConstruccion1.png| x120px]]</td><br />
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</center><br />
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<h1><center>Compilation of newspapers where our BioSintetizARTE 2.0 call was published.</center></h1> <br />
<br /><br />
<br /><br />
The UNAM Genomics Mexico Team is pleased to introduce to you the BioSintetizARTE call, this call its quite important because our team is really concern about the important role of citizen involvement where all views can find expression and the diversity of languages specially through the universal language... the ART.<br />
<br /><br />
<br /><br />
We launch our call on August 1st all over Mexico trying to find all views we can get in order to have a more colorful landscape of Synthetic Biology. This are some of the newspapers where our call was published all over Mexico. <br />
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<table border="0" height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10"><br />
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<td id="leftcolumn2" align="center">[[File:Unamgenomicsmexiconewselmanana.JPG|850px| Newspaper "El Manana" published 1 august 2012 visible in all Mexico]]<br /><br /><p>Newspaper "El Manana" published 1 august 2012 visible in all Mexico</p></td><br />
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<td id="contentcolumn2" align="center">[[File:Unamgenomicsmexicoelsoldetampico.JPG|800px|Newspaper "El Sol de Tampico" published 1 august 2012 visible in North East of Mexico]]<br /><br /><p>Newspaper "El Sol de Tampico" published 1 august 2012 visible in North East of Mexico</p></td><br />
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<td id="rightcolumn2" align="center">[[File:Unamgenomicsmexiconewslajornada.JPG|850px|Newspaper "La Jornada" published 2 august 2012 visible in all Mexico is also one of the most widespread newspaper in Mexico]]<br /><br /><p>Newspaper "<br />
La Jornada" published 2 august 2012 visible in all Mexico is also one of the most widespread newspaper in Mexico</p></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
}}</div>
Abieltega