Team:UNAM Genomics Mexico/Notebook/ANDMetal

From 2012.igem.org

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__TOC__
__TOC__
<br />
<br />
-
= May =
+
<h2>05/29/2012</h2><br />
-
<br />
+
 
-
==05/29/2012==
+
Our group is in charge of building part of the “and” construction. We started analyzing if the plasmid we have with P4 actually had what we needed. <br /><br />
-
<br />
+
The plasmid PRMn25 contains the protein P4. It has Amp100  resistance and comes in Escherichia coli  NFI. Cells were lysed to make sure the plasmid was present in these cells (5000bp). We ran a gel with 3 lysis, a sample from the plasmid PFRC54 (A3 promoter) and a sample of total DNA from the strain from which PFRC54 was obtained. <br /><br />
-
Our group is in charge of building part of the “and” construction. We started analyzing if the plasmid we have with P4 actually had what we needed.  
+
-
The plasmid PRMn25 contains the protein P4. It has Amp100  resistance and comes in Escherichia coli  NFI. Cells were lysed to make sure the plasmid was present in these cells (5000bp). We ran a gel with 3 lysis, a sample from the plasmid PFRC54 (A3 promoter) and a sample of total DNA from the strain from which PFRC54 was obtained.  
+
   
   
-
1. 10 kb ladder  
+
1. 10 kb ladder <br />
-
2. Lysis PRMn25 (Rebeca)
+
2. Lysis PRMn25 <br />
-
3. Lysis PRMn25 (Karen)
+
3. Lysis PRMn25 <br />
-
4. Lysis PRMn25 (Dulce)
+
4. Lysis PRMn25 <br />
-
5. Purified PFRC54(A3)
+
5. Purified PFRC54(A3) <br />
-
6. Total DNA (PFRC54)
+
6. Total DNA (PFRC54) <br />
-
==05/31/2012==
+
<h2>05/31/2012</h2><br />
-
<br />
+
Plasmid PRMn25 was digested with SpeI and EcoRI. We do not have the sequence of the plasmid, but there seem to be several restriction sites for SpeI and just one site for EcoRI. The digestions were left for 4 hours and they were plused in the microwave 3 times, 10 seconds each time. Only Dulce’s digestion worked, even though it was the “dirtiest” sample. <br /><br />
-
Plasmid PRMn25 was digested with SpeI and EcoRI. We do not have the sequence of the plasmid, but there seem to be several restriction sites for SpeI and just one site for EcoRI. The digestions were left for 4 hours and they were plused in the microwave 3 times, 10 seconds each time. Only Dulce’s digestion worked, even though it was the “dirtiest” sample.  
+
   
   
-
1. 10 kb ladder
+
1. 10 kb ladder<br />
-
2. SpeI PRMn25 digestion (Dulce)
+
2. SpeI PRMn25 digestion <br />
-
3. EcoRI PRMn25 digestion (Dulce)
+
3. EcoRI PRMn25 digestion <br />
-
4. PRMn25 lysis (Dulce)
+
4. PRMn25 lysis <br />
-
5. SpeI PRMn25 digestion (Karen)
+
5. SpeI PRMn25 digestion <br />
-
6. EcoRI PRMn25 digestion (Karen)
+
6. EcoRI PRMn25 digestion <br />
-
7. PRMn25 lysis (Karen)
+
7. PRMn25 lysis <br />
-
8. SpeI PRMn25 digestion (Rebeca)
+
8. SpeI PRMn25 digestion <br />
-
9. EcoRI PRMn25 digestion (Rebeca)
+
9. EcoRI PRMn25 digestion <br />
-
10. PRMn25 lysis (Rebeca)
+
10. PRMn25 lysis <br />
-
<br />
+
 
-
=June=
+
<h2>06/06/2012</h2><br />
-
<br />
+
We repeated the lysis of PRMn25. <br />
-
==06/06/2012==
+
Digestions (20 µl) <br />
-
We repeated the lysis of PRMn25.  
+
H2O 12 µl<br />
-
Digestions (20 l)
+
Enzime 1 µl<br />
-
H2O 12 l
+
Buffer 10x  2 µl<br />
-
Enzime 1 l
+
Plasmid 5 µl<br />
-
Buffer 10x  2 l
+
37ºC<br /><br />
-
Plasmid 5 l
+
PRMn25 was digested with EcoRi and BamHI. Since we don’t have the sequence we used these enzyme to confirm the restriction sites mentioned in Rojo et al. (1990). These sites were corroborated and the vector was linearized. We recycled the gel used for the PRMn25 lysis, which is why the gel seems out of phase. <br /><br />
-
37ºC
+
-
PRMn25 was digested with EcoRi and BamHI. Since we don’t have the sequence we used these enzyme to confirm the restriction sites mentioned in Rojo et al. (1990). These sites were corroborated and the vector was linearized. We recycled the gel used for the PRMn25 lysis, which is why the gel seems out of phase.  
+
   
   
-
1.10 kb ladder
+
1.10 kb ladder<br />
-
2. PRMn25 (P4) lysis 1
+
2. PRMn25 (P4) lysis 1<br />
-
3. PRMn25 (P4) lysis 2
+
3. PRMn25 (P4) lysis 2<br />
-
4. ---------------
+
4. ---------------<br />
-
5. 10 kb ladder
+
5. 10 kb ladder<br />
-
6. EcoRI PRMn25 digestion
+
6. EcoRI PRMn25 digestion<br />
-
7. BamHI PRMn25 digestion
+
7. BamHI PRMn25 digestion<br />
-
==06/08/12==
+
<h2>06/08/12</h2><br /><br />
-
<br />
+
The lysis worked so we transformed PRMn25 in DH5. We performed a lysis on these cells to see if they had been transformed correctly with PRMn25. <br /><br />
-
The lysis worked so we transformed PRMn25 in DH5. We performed a lysis on these cells to see if they had been transformed correctly with PRMn25.  
+
   
   
-
1. 10kb ladder
+
1. 10kb ladder<br />
-
2. PRMn25 (P4) lysis 1
+
2. PRMn25 (P4) lysis 1<br />
-
3. Cell lysis of cells transformed with lysis 1   
+
3. Cell lysis of cells transformed with lysis 1  <br />
-
4.Cell lysis of cells transformed with lysis 1   
+
4.Cell lysis of cells transformed with lysis 1  <br />
-
5. Cell lysis of cells transformed with lysis 1   
+
5. Cell lysis of cells transformed with lysis 1  <br />
-
6. Cell lysis of cells transformed with lysis 1   
+
6. Cell lysis of cells transformed with lysis 1  <br />
-
7. Cell lysis of cells transformed with lysis 1   
+
7. Cell lysis of cells transformed with lysis 1  <br />
-
8. Cell lysis of cells transformed with lysis 1   
+
8. Cell lysis of cells transformed with lysis 1  <br />
-
9. Cell lysis of cells transformed with lysis 1   
+
9. Cell lysis of cells transformed with lysis 1  <br />
-
10. Cell lysis of cells transformed with lysis 1   
+
10. Cell lysis of cells transformed with lysis 1  <br />
-
11. Cell lysis of cells transformed with lysis 1   
+
11. Cell lysis of cells transformed with lysis 1  <br />
-
12. Cell lysis of cells transformed with lysis 1   
+
12. Cell lysis of cells transformed with lysis 1  <br />
-
 
+
<br />
-
 
+
-
We also transformed PFRC54.
+
-
 
+
-
Transformation in DH5
+
-
Place DH5+5  μl DNA in ice at 4ºC.
+
-
42ºC 2 minutes.
+
-
Add 1 ml LB to the same tube.
+
-
1 hour at 37ºC x 9000 rpm.
+
-
 
+
-
 
+
-
 
+
-
 
+
-
==06/11/12==
+
<br />
<br />
-
We designed primers for our project. (Note from 07/10/12: we redesigned the primer for LasR, since the sequence was incorrect.)
+
We also transformed PFRC54.   [TRANSFORMATION PROTOCOL] <br />
-
OLIGOS 14/06/12
+
-
LASR
 
-
UPPER 5'-3'
+
<h2>06/11/12</h2><br />
 +
We designed primers for our project. (Note from 07/10/12: we redesigned the primer for LasR, since the sequence was incorrect.) <br />
 +
OLIGOS 14/06/12<br />
 +
LASR<br />
 +
UPPER 5'-3'<br />
 +
PREFIX+RBS+SPACER+LASR<br />
-
PREFIJO+RBS+ESPACIADOR+LASR
+
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG  ATGGCATTAGTAGAT<br /><br />
-
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG  ATGGCATTAGTAGAT
+
LOWER 5'-3'<br />
-
LOWER 5'-3'
+
SUFIX+LASR<br />
-
 
+
-
SUFIJO+LASR
+
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA TCATAATGTAATTAA
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA TCATAATGTAATTAA
 +
P4 5'-3'<br />
 +
PREFIX+RBS+SPACER+P4 <br />
 +
upper<br />
-
P4 5'-3'
+
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGCCTAAAACACAA<br />
-
PREFIJO+RBS+ESPACIADOR+P4  
+
SUFIX+P4<br />
-
upper
+
lower 5'-3'<br />
-
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGCCTAAAACACAA
+
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA CTACACCATACTTTT<br />
 +
A3 (PROMOTER) <br />
-
SUFIJO+P4
+
UPPER 5'-3'<br />
-
lower 5'-3'
+
PREFIX+A3<br />
-
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA CTACACCATACTTTT
+
5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG taactttttgcaaga  3'<br />
 +
LOWER 5'-3'<br />
 +
SUFIX+A3<br />
 +
5'GTTTCTTCCTGCAGCGGCCGCTACTAGTA ctacttaattatacc 3'<br />
 +
RFP<br />
-
A3 (PROMOTOR)
+
UPPER 5'-3'<br />
-
UPPER 5'-3'
+
PREFIX+RBS+SPACER+RFP<br /> 
-
PREFIJO+A3
+
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTTCCTCCGAA<br />
-
5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG taactttttgcaaga  3'
+
LOWER 5'-3'<br />
-
LOWER 5'-3'
+
SUFIX+RFP<br />
-
SUFIJO+A3
+
GTTTCTTCCTGCAGCGGCCGCTACTAGTA TTATTAAGCACCGGT<br />
-
5'GTTTCTTCCTGCAGCGGCCGCTACTAGTA ctacttaattatacc 3'
 
 +
GUSA<br />
 +
UPPER 5'-3'<br />
 +
PREFIX+RBS+SPACER+GUSA<br />
-
RFP
+
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG atgttacgtcctgta<br />
-
UPPER 5'-3'
+
LOWER 5'-3'<br />
-
PREFIJO+RBS+ESPACIADOR+RFP 
+
SUFIX+GUSA<br />
-
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTTCCTCCGAA
+
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA tcattgtttgcctcc<br />
-
LOWER 5'-3'
 
-
SUFIJO+RFP
 
-
GTTTCTTCCTGCAGCGGCCGCTACTAGTA TTATTAAGCACCGGT
+
ARAC without LVA (version 2 registry part: BBa_C0080) <br />
 +
UPPER 5'-3'<br />
 +
PREFIX+RBS+SPACER+ARAC<br />
-
GUSA
+
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTGAAGCGCAA<br />
-
UPPER 5'-3'
+
LOWER 5'-3'<br />
-
PREFIJO+RBS+ESPACIADOR+GUSA
+
SUFIX+ARAC<br />
-
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG atgttacgtcctgta
+
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA CAACTTGACGGCTAC<br />
-
LOWER 5'-3'
+
<h2>06/14/2012</h2><br />
-
 
+
We obtained the terminator from the 2012 distribution (plate 2 24C) and we transformed the DNA in DH5. The terminator (BBa_B0014) comes in the PSBIAK3 plasmid with resistance to Amp and Km. The RFP (BBa_E1010) comes in the plasmid PSBI2K3 with resistance to Amp and we obtained it from the 2012 distribution plate 2 (17E). We obtained the purified plasmid of the RFP from the AraC AND team. (Diego, Jonathan, and Abiel). <br />
-
SUFIJO+GUSA
+
-
 
+
-
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA tcattgtttgcctcc
+
-
 
+
-
 
+
-
 
+
-
ARAC sin LVA (version 2 registry parte: BBa_C0080)
+
-
 
+
-
UPPER 5'-3'
+
-
 
+
-
PREFIJO+RBS+ESPACIADOR+ARAC
+
-
 
+
-
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTGAAGCGCAA
+
-
 
+
-
LOWER 5'-3'
+
-
 
+
-
SUFIJO+ARAC
+
-
 
+
-
GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA CAACTTGACGGCTAC
+
-
 
+
-
==06/14/2012==
+
-
<br />
+
-
We obtained the terminator from the 2012 distribution (plate 2 24C) and we transformed the DNA in DH5. The terminator (BBa_B0014) comes in the PSBIAK3 plasmid with resistance to Amp and Km. The RFP (BBa_E1010) comes in the plasmid PSBI2K3 with resistance to Amp and we obtained it from the 2012 distribution plate 2 (17E). We obtained the purified plasmid of the RFP from the AraC AND team. (Diego, Jonathan, and Abiel).
+
   
   
-
1. 1 kb ladder.
+
1. 1 kb ladder. <br />
-
2. BBa_B0014 (1) – terminator
+
2. BBa_B0014 (1) – terminator<br />
-
3. BBa_B0014 (4) – terminator
+
3. BBa_B0014 (4) – terminator<br />
-
4. BBa_E1010 (RFP) Lysis
+
4. BBa_E1010 (RFP) Lysis<br />
-
5. purified BBa_E1010 PSB12K3  (AraC AND team)
+
5. purified BBa_E1010 PSB12K3  (AraC AND team) <br />
-
6. 700 bp ladder
+
6. 700 bp ladder<br />
-
==06/15/12==
+
<h2>06/15/12</h2><br />
-
<br />
+
Due to the failed digestions, we did the RFP and terminator lysis again. <br />
-
Due to the failed digestions, we did the RFP and terminator lysis again.
+
   
   
-
1. 1 kb ladder
+
1. 1 kb ladder<br />
-
2. Terminator lysis (BBa_B0014)
+
2. Terminator lysis (BBa_B0014) <br />
-
3. RFP lysis (BBa_E1010)
+
3. RFP lysis (BBa_E1010) <br />
-
The AraC team has the terminator plasmid purified, we are thinking of using that one.
+
The AraC team has the terminator plasmid purified, we are thinking of using that one. <br />
-
 
+
-
==06/18/12==
+
-
<br />
+
-
We used the RFP that we transformed and the RFP that the AraC team did as a positive control. The terminator still looks strange, since we can’t observe supercoling which is normally observed.
+
-
We are waiting for our primers, and the team will be going to a math modeling course for a week.
+
-
+
-
 
+
-
1. 700 bp ladder
+
-
2. BBa_B0014 lysis (terminator)
+
-
3. BBa_E1010 lysis (RFP)
+
-
4. BBa_E1010 lysis (RFP AraC team)
+
-
5. 1 kb ladder
+
-
 
+
-
<br />
+
-
=July=
+
-
==07/02/12==
+
-
<br />
+
-
We did 2 PCRs, since the primers have arrived. We purified by miniprep RFP and P4. With this PCR we will add the prefix (EcoRI and XbaI) and RBS to the beginning of the sequence, and suffix (restiction sites for SpeI asn PstI).
+
-
 
+
-
RFP
+
-
 
+
-
Water  29.5 μl
+
-
Buffer  5 μl
+
-
Up  2.5 μl
+
-
Lw 2.5 μl
+
-
MgCl2  1 μl 
+
-
DNA 0.5 μl
+
-
TaqPol 1 μl
+
-
DNTP’s 8 μl
+
-
 
+
-
P4
+
-
 
+
-
Water  29.5 μl
+
-
Buffer  5 μl
+
-
Up  2.5 μl
+
-
Lw 2.5 μl
+
-
MgCl2  1 μl l
+
-
DNA 0.5 μl
+
-
TaqPol 1 μl
+
-
DNTP’s 8 μl
+
-
 
+
-
TM’s
+
-
RFP UP 78ºC
+
-
RFP LW      75.5 ºC
+
-
P4 UP    73.6 ºC
+
-
P4 LW 73.9ºC
+
-
 
+
-
 
+
-
Taking into account both TM’s, we used the same amplification program for both, thermocycle B progam 16EM.
+
-
3 step
+
-
95ºC 4 min
+
-
95ºC 1 min
+
-
55ºC 1 min
+
-
70ºC 1 min
+
-
goto 2:4  30 times
+
-
72ºC 5 min
+
-
We ran a gel and we observed fragments that correspond to the RFP and P4, we can also observe other fragments which means we will have to purify the bands.
+
-
+
-
1. 1 kb ladder
+
-
2. PCR RFP
+
-
3. negative control PCR RFP
+
-
4. P4 PCR
+
-
5. negative control PCR P4
+
-
 
+
-
==07/03/12==
+
-
<br />
+
-
We used Roche kit for band purification.
+
-
 
+
-
+
-
1. 1 kb ladder
+
-
2. PCR product P4
+
-
3. PCR product RFP
+
-
 
+
-
1. Cut the band of the fragment you wish to purify.
+
-
2. 400 l of Binding Buffer, 10 min. at 65ºC with shaker so that the agarose melts.
+
-
3. 200 l deisopropanol after 5 min in the shaker.
+
-
4. Pass through column and centrifuge. Throw out supernatant.
+
-
5. 700 l wash buffer, centrifuge, throw it out, centrifuge.
+
-
6. Pass column to clean tube, add 40 l de elution buffer.
+
-
 
+
-
1. 1kb Ladder
+
-
2. P4 band
+
-
3. RFP band
+
-
 
+
-
1. 1kb ladder
+
-
2. P4 purification
+
-
3. RFP purification
+
-
 
+
-
The P4 purification turned out slightly dirty and the RFP turned out completely dirty. We repeated the purifications by loading the previously “purified” sample and repeating the procedure.
+
-
 
+
-
==07/03/12==
+
-
<br />
+
-
1. Ladder 1 kb
+
-
2. Purified P4
+
-
3. Purified RFP
+
-
We used Roche kit to purify the band again.
+
-
 
+
-
1. 1kb ladder
+
-
2. cut P4
+
-
3. cut RFP
+
-
 
+
-
The RFP came out fine, but P4 was degraded, we will have to repeat P4’s PCR. (Gel is recycled, which is why image is blurry).
+
-
We transformed LasR from 2010 distribution in DH5.
+
-
 
+
-
==07/04/2012==
+
-
<br />
+
-
 
+
-
1. Degraded P4.
+
-
2. Purified RFP.
+
-
3. 1kb Ladder.
+
-
We repeated the gel where we checked the re-purification, and indeed we need to repeat P4’s PCR.
+
-
 
+
-
1. 1 kb ladder
+
-
2. P4 PCR (1)
+
-
3. P4 PCR (2)
+
-
4. Negative control P4 PCR
+
-
+
-
The PCR worked, but a band purification will be needed. We fused all of the PCR product and loaded it.
+
-
 
+
-
1. 1 kb ladder
+
-
2. P4 PCR product
+
-
 
+
-
1. 1 kb ladder.
+
-
2. Cut P4.
+
-
 
+
-
1. P4 purified from band
+
-
2. 1 kb ladder
+
-
 
+
-
We grew bacterial culture in antibiotics for LasR lysis.
+
-
 
+
-
Rfp+terminator ligation
+
-
Buffer 3.5 μl
+
-
RFP 15 μl
+
-
Terminator 8 μl
+
-
T4 Ligase 1 μl
+
-
H2O 7.5 μl
+
-
total 35 μl
+
-
At 22ºC
+
-
 
+
-
==07/05/12==
+
-
<br />
+
-
+
-
1. 1 kb ladder
+
-
2. LasR lysis
+
-
3. LasR lysis
+
-
4. LasR lysis
+
-
5. LasR lysis
+
-
6. LasR lysis
+
-
7. LasR lysis
+
-
8. LasR lysis
+
-
9. LasR lysis
+
-
Cells were transformed with DH5 with RFP+terminator ligation.
+
-
 
+
-
1. 1 kb ladder
+
-
2. Terminator digested with EcoRI and XbaI/ dephosphatated
+
-
3. RFP digested with EcoRI and SpeI.
+
-
LasR lysis were digested. We ran a gel with LasR PCRs to add RBS prefix and suffix.
+
-
1. XbaI SpeI LasR digestion (3 hours)
+
-
2. XbaI SpeI LasR digestion (3 hours)
+
-
3. XbaI SpeI LasR digestion (3 hours)
+
-
4. XbaI SpeI LasR digestion (3 hours)
+
-
5. 1 kb ladder
+
-
6. LasR PCR
+
-
7. LasR PCR
+
-
8. LasR PCR
+
-
9. LasR PCR
+
-
10. Negative control LasR PCR
+
-
<br />
+
-
==06/07/12==
+
-
<br />
+
-
We ran LasR’s PCRs again, the last PCR didn’t work, which is why we lowered the temperature to 65ºC. We left the digestions all night.
+
-
 
+
-
PCR
+
-
95ºC 4 min
+
-
95ºC 1 min
+
-
55ºC 1 min
+
-
65ºC 1 min
+
-
go to 2:4 3 times
+
-
72ºC 5 min
+
-
 
+
-
1. LasR PCR
+
-
2.  LasR PCR
+
-
3. LasR PCR
+
-
4. LasR PCR
+
-
5. LasR PCR
+
-
6. LasR PCR
+
-
7. LasR PCR
+
-
8. LasR PCR
+
-
9. Negative control LasR PCR
+
-
10. 1 kb ladder
+
-
11. X/S LasR digestion
+
-
12. X/S LasR digestion
+
-
13. X/S LasR digestion
+
-
14. X/S LasR digestion
+
-
15. X/S LasR digestion
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Since they didn’t work, we decided to do it directly from the distribution from 2012 and 2010.
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We noticed our mistake…. The sequence we designed our primers for was an incorrect sequence!
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Revision as of 19:54, 17 September 2012


UNAM-Genomics_Mexico


Cadmium/Heavy metals AND Gate




Nanotubes!! The logic Random info


Contents


05/29/2012


Our group is in charge of building part of the “and” construction. We started analyzing if the plasmid we have with P4 actually had what we needed.

The plasmid PRMn25 contains the protein P4. It has Amp100 resistance and comes in Escherichia coli NFI. Cells were lysed to make sure the plasmid was present in these cells (5000bp). We ran a gel with 3 lysis, a sample from the plasmid PFRC54 (A3 promoter) and a sample of total DNA from the strain from which PFRC54 was obtained.


1. 10 kb ladder
2. Lysis PRMn25
3. Lysis PRMn25
4. Lysis PRMn25
5. Purified PFRC54(A3)
6. Total DNA (PFRC54)

05/31/2012


Plasmid PRMn25 was digested with SpeI and EcoRI. We do not have the sequence of the plasmid, but there seem to be several restriction sites for SpeI and just one site for EcoRI. The digestions were left for 4 hours and they were plused in the microwave 3 times, 10 seconds each time. Only Dulce’s digestion worked, even though it was the “dirtiest” sample.


1. 10 kb ladder
2. SpeI PRMn25 digestion
3. EcoRI PRMn25 digestion
4. PRMn25 lysis
5. SpeI PRMn25 digestion
6. EcoRI PRMn25 digestion
7. PRMn25 lysis
8. SpeI PRMn25 digestion
9. EcoRI PRMn25 digestion
10. PRMn25 lysis


06/06/2012


We repeated the lysis of PRMn25.
Digestions (20 µl)
H2O 12 µl
Enzime 1 µl
Buffer 10x 2 µl
Plasmid 5 µl
37ºC

PRMn25 was digested with EcoRi and BamHI. Since we don’t have the sequence we used these enzyme to confirm the restriction sites mentioned in Rojo et al. (1990). These sites were corroborated and the vector was linearized. We recycled the gel used for the PRMn25 lysis, which is why the gel seems out of phase.

1.10 kb ladder
2. PRMn25 (P4) lysis 1
3. PRMn25 (P4) lysis 2
4. ---------------
5. 10 kb ladder
6. EcoRI PRMn25 digestion
7. BamHI PRMn25 digestion

06/08/12



The lysis worked so we transformed PRMn25 in DH5. We performed a lysis on these cells to see if they had been transformed correctly with PRMn25.


1. 10kb ladder
2. PRMn25 (P4) lysis 1
3. Cell lysis of cells transformed with lysis 1
4.Cell lysis of cells transformed with lysis 1
5. Cell lysis of cells transformed with lysis 1
6. Cell lysis of cells transformed with lysis 1
7. Cell lysis of cells transformed with lysis 1
8. Cell lysis of cells transformed with lysis 1
9. Cell lysis of cells transformed with lysis 1
10. Cell lysis of cells transformed with lysis 1
11. Cell lysis of cells transformed with lysis 1
12. Cell lysis of cells transformed with lysis 1


We also transformed PFRC54. [TRANSFORMATION PROTOCOL]



06/11/12


We designed primers for our project. (Note from 07/10/12: we redesigned the primer for LasR, since the sequence was incorrect.)
OLIGOS 14/06/12
LASR
UPPER 5'-3'
PREFIX+RBS+SPACER+LASR

GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCATTAGTAGAT

LOWER 5'-3'

SUFIX+LASR

GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA TCATAATGTAATTAA


P4 5'-3'

PREFIX+RBS+SPACER+P4

upper

GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGCCTAAAACACAA

SUFIX+P4

lower 5'-3'

GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA CTACACCATACTTTT
A3 (PROMOTER)

UPPER 5'-3'

PREFIX+A3

5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG taactttttgcaaga 3'

LOWER 5'-3'

SUFIX+A3

5'GTTTCTTCCTGCAGCGGCCGCTACTAGTA ctacttaattatacc 3'

RFP

UPPER 5'-3'

PREFIX+RBS+SPACER+RFP

GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTTCCTCCGAA

LOWER 5'-3'

SUFIX+RFP

GTTTCTTCCTGCAGCGGCCGCTACTAGTA TTATTAAGCACCGGT


GUSA

UPPER 5'-3'

PREFIX+RBS+SPACER+GUSA

GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG atgttacgtcctgta

LOWER 5'-3'

SUFIX+GUSA

GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA tcattgtttgcctcc


ARAC without LVA (version 2 registry part: BBa_C0080)

UPPER 5'-3'

PREFIX+RBS+SPACER+ARAC

GTTTCTTCGAATTCGCGGCCGCTTCTAGAG AAAGGTGGTGAA TACTAG ATGGCTGAAGCGCAA

LOWER 5'-3'

SUFIX+ARAC

GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTA CAACTTGACGGCTAC

06/14/2012


We obtained the terminator from the 2012 distribution (plate 2 24C) and we transformed the DNA in DH5. The terminator (BBa_B0014) comes in the PSBIAK3 plasmid with resistance to Amp and Km. The RFP (BBa_E1010) comes in the plasmid PSBI2K3 with resistance to Amp and we obtained it from the 2012 distribution plate 2 (17E). We obtained the purified plasmid of the RFP from the AraC AND team. (Diego, Jonathan, and Abiel).


1. 1 kb ladder.
2. BBa_B0014 (1) – terminator
3. BBa_B0014 (4) – terminator
4. BBa_E1010 (RFP) Lysis
5. purified BBa_E1010 PSB12K3 (AraC AND team)
6. 700 bp ladder

06/15/12


Due to the failed digestions, we did the RFP and terminator lysis again.

1. 1 kb ladder
2. Terminator lysis (BBa_B0014)
3. RFP lysis (BBa_E1010)
The AraC team has the terminator plasmid purified, we are thinking of using that one.

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