Team:UNAM Genomics Mexico/Results/OR

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https://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/OR
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{{:Template:Team:UNAM_Genomics_Mexico/webhtml| content=
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__NOTOC__
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<br />
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<center><h1>'''A3- LasB (GFP REPORTER) OR GATE'''</h1></center>
 +
<br />
 +
<br />
 +
This construct was designed to function as an OR logic gate. Our system consists in two different promoters (A3 and
 +
LasB) expressing the same reporter gene (GFP). LasB (BBa_R0079) promoter (used by NYMU-Taipei 2009, Northwestern 2011, Tokyo-tech iGEM 2011 and Tsinghua-A 2011) requires both an activator protein, LasR, and an N-acylhomoserine lactone compound termed ''Pseudomonas'' autoinducer (PAI). A3 promoter is activated by P4, a regulatory protein from phage φ29 from ''Bacillus subtilis''. In this way, if our target cell receive either LasR, P4 or both proteins, it will express GFP protein and we will have an OR gate.<br />
 +
<br />
 +
To obtain our final construct we required the following Biological Parts:<br />
 +
<br />
 +
<center>
 +
<table border="0"  height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10">
 +
<tr>
 +
<td id="contentcolumn" align= "center"><br />[[File:Unamgenomicsconstruccion3.png | 850px]]</td>
 +
</tr>
 +
</table>
 +
</center>
 +
 
 +
<br />
 +
<br />
 +
'''Obtained from the Registry''':<br />
 +
<br />
 +
[http://partsregistry.org/Part:BBa_K143001 BBa_K143001 (AmyE 5’)]<br />
 +
[http://partsregistry.org/Part:BBa_K143002 BBa_K143002 (AmyE 3’)]<br />
 +
[http://partsregistry.org/Part:BBa_R0079 BBa_R0079 (LasB)]<br />
 +
[http://partsregistry.org/Part:BBa_B0014 BBa_B0014 (Double Terminator)]<br />
 +
<br />
 +
'''Obtained from Margarita Salas Ph.D.’s Group''':<br />
 +
<br />
 +
A3 from phage phi29 from plasmid pFRC54.<br />
 +
<br />
 +
'''Omega cassette from plasmid pHP45Ω.'''<br />
 +
<br />
 +
'''GFP reporter gene'''<br />
 +
<br />
 +
'''We designed the following primers to add the RBS site to GusA''':<br />
 +
<br />
 +
'''GusA'''<br />
 +
<br />
 +
UPPER 5'-3'<br />
 +
PREFIX+RBS+SPACER+GUSA<br />
 +
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG atgttacgtcctgta<br />
 +
<br />
 +
LOWER 5'-3'<br />
 +
SUFIX+GUSA<br />
 +
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA tcattgtttgcctcc<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 />
 +
'''A3''' <br />
 +
<br />
 +
UPPER 5'-3'<br />
 +
PREFIX+A3<br />
 +
5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG taactttttgcaaga  3'<br />
 +
<br />
 +
LOWER 5'-3'<br />
 +
SUFIX+A3<br />
 +
5'GTTTCTTCCTGCAGCGGCCGCTACTAGTA ctacttaattatacc 3'<br />
 +
<br />
 +
<br />
 +
'''OMEGA CASSETTE'''<br />
 +
<br />
 +
PREFIX+OMEGA CASSETTE(45 bp)<br />
 +
5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG CCGGGGATCCGGTGA 3'<br />
 +
<br />
 +
SUFIX+OMEGA CASSETTE (44 bp)<br />
 +
5' GTTTCTTCCTGCAGCGGCCGCTACTAGTA CCGGGGATCCGGTGA 3'<br />
 +
 
 +
'''This A3-LasB OR team had to build the following constructs'''<br />
 +
<br />
 +
<center>
 +
<table border="0"  height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10">
 +
<tr>
 +
<td id="contentcolumn" align= "center"><br />[[File:UnamgenomicsresultadosOr1.png | x100px]]</td>
 +
</tr>
 +
<tr>
 +
<td id="contentcolumn" align= "center"><br />[[File:UnamgenomicsresultsOr2.png | x100px]]</td>
 +
</tr>
 +
<tr>
 +
<td id="contentcolumn" align= "center"><br />[[File:UnamgenomicsresultsOr3.png | x100px]]</td>
 +
</tr>
 +
</table>
 +
</center>
 +
 
 +
So we could obtain the final product:<br />
 +
<br />
 +
 
 +
<center>
 +
<table border="0"  height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10">
 +
<tr>
 +
<td id="contentcolumn" align= "center"><br />[[File:Unamgenomicsconstruccion3.png | 850px]]</td>
 +
</tr>
 +
</table>
 +
</center>
 +
 
 +
 
 +
 
 +
What we have up now is:
 +
 
 +
 
 +
<center>
 +
<table border="0"  height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10">
 +
<tr>
 +
<td id="contentcolumn" align= "center"><br />[[File:UnamgenomicsOrwhatwehave.png| 850px]]</td>
 +
</tr>
 +
</table>
 +
</center>
 +
::::::::::::::::::::::::::::::::::::::[[File:UnamgenomcisUp.png|right | 120px |link=Team:UNAM_Genomics_Mexico/Results/OR#A3-_LasB_.28GFP_REPORTER.29_OR_GATE]]
 +
'''Please see our wetlab notebook in the clicking the following image:'''<br /><br />
 +
 
 +
<center>
 +
<table border="0"  height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="10">
 +
<tr>
 +
<td id="contentcolumn" align= "center"><br />[[File:UnamgenomicsOr.png|200px| link=Team:UNAM_Genomics_Mexico/Notebook/OR]]<p>'''OR gate Notebook'''</p></td>
 +
</tr>
 +
</table>
 +
</center>
 +
<br />
 +
<br />
 +
<br />
 +
 
 +
 
 +
}}

Latest revision as of 00:29, 26 October 2012


UNAM-Genomics_Mexico


A3- LasB (GFP REPORTER) OR GATE



This construct was designed to function as an OR logic gate. Our system consists in two different promoters (A3 and LasB) expressing the same reporter gene (GFP). LasB (BBa_R0079) promoter (used by NYMU-Taipei 2009, Northwestern 2011, Tokyo-tech iGEM 2011 and Tsinghua-A 2011) requires both an activator protein, LasR, and an N-acylhomoserine lactone compound termed Pseudomonas autoinducer (PAI). A3 promoter is activated by P4, a regulatory protein from phage φ29 from Bacillus subtilis. In this way, if our target cell receive either LasR, P4 or both proteins, it will express GFP protein and we will have an OR gate.

To obtain our final construct we required the following Biological Parts:


Unamgenomicsconstruccion3.png



Obtained from the Registry:

[http://partsregistry.org/Part:BBa_K143001 BBa_K143001 (AmyE 5’)]
[http://partsregistry.org/Part:BBa_K143002 BBa_K143002 (AmyE 3’)]
[http://partsregistry.org/Part:BBa_R0079 BBa_R0079 (LasB)]
[http://partsregistry.org/Part:BBa_B0014 BBa_B0014 (Double Terminator)]

Obtained from Margarita Salas Ph.D.’s Group:

A3 from phage phi29 from plasmid pFRC54.

Omega cassette from plasmid pHP45Ω.

GFP reporter gene

We designed the following primers to add the RBS site to GusA:

GusA

UPPER 5'-3'
PREFIX+RBS+SPACER+GUSA
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG atgttacgtcctgta

LOWER 5'-3'
SUFIX+GUSA
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA tcattgtttgcctcc


We also designed the following primers to obtain A3 from phage phi29 from plasmid pFRC54 and Omega cassette from plasmid pHP45Ω:

A3

UPPER 5'-3'
PREFIX+A3
5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG taactttttgcaaga 3'

LOWER 5'-3'
SUFIX+A3
5'GTTTCTTCCTGCAGCGGCCGCTACTAGTA ctacttaattatacc 3'


OMEGA CASSETTE

PREFIX+OMEGA CASSETTE(45 bp)
5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG CCGGGGATCCGGTGA 3'

SUFIX+OMEGA CASSETTE (44 bp)
5' GTTTCTTCCTGCAGCGGCCGCTACTAGTA CCGGGGATCCGGTGA 3'

This A3-LasB OR team had to build the following constructs


UnamgenomicsresultadosOr1.png

UnamgenomicsresultsOr2.png

UnamgenomicsresultsOr3.png

So we could obtain the final product:


Unamgenomicsconstruccion3.png


What we have up now is:



UnamgenomicsOrwhatwehave.png
UnamgenomcisUp.png

Please see our wetlab notebook in the clicking the following image:


UnamgenomicsOr.png

OR gate Notebook




Retrieved from "http://2012.igem.org/Team:UNAM_Genomics_Mexico/Results/OR"