Team:Panama/24 July 2010

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==='''July 24'''===
==='''July 24'''===
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Development of a genetically engineered ''E. coli'' that can produce rhamnosyltranferase 1, for the production of a biosurfactant.
+
Finally, we define our project!
-
Steps to follow:
+
Our goal is the development of a genetically engineered ''E. coli'' that can produce rhamnosyltranferase 1 enzyme.
 +
These are the steps we follow for our project design:
 +
 +
1. First we look for information regarding the biosurfactant production and we found a paper that describes the Rhlab gene.
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1.Find the gene sequence in pubmed.
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2. This gene is found in the ''Pseudomonas aeruginosa''.
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2.In which bacteria it is found.
+
3. We search the Rhlab gene sequence in GeneBank, here are the accession numbers:
 +
Rhla (Gene ID: 878955)
 +
LOCUS NC_002516 888 bp DNA
 +
 
 +
Rhlb (Gene ID: 878954)
 +
LOCUS NC_002516 1281 bp DNA
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3.After we found the gene, we designed two different set of primers.
+
4. The next step was the RhlAB gene sequence analysis, we found THREE PstI recognition sequenceS. After this we designed two different set of primers for the RhlAB amplification from the ''P. aeruginosa'' genomic DNA.  
-
4.We look for the sequences of the restriction enzymes E, X, P and S, inside the gene sequence.
+
FORWARD
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5.If we found in the gene sequence a recognition sequence for the restriction enzymes.
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a. RhT-F1a
 +
  5'- GCG ATA GCT GTT TGC CTG TT -3'
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6.So we should look for a mutagenesis protocol or kit.
+
b. RhT-F1b
 +
  5'- GTT TGC CTG TTC GAA AAT T -3'
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7.We chose the Stratagene lightning mutagenesis kit. We also designed three different set of primers, because the base pairs that surround the PstI unwanted sequence were different.
+
REVERSE
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We found a paper which describes the Rhab1, the one we need for our project. This gene is found in ''Pseudomona aureginosa''.
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c. RhT-R2a
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We looked for the gene sequence en GeneBank:
+
  5'- TGG CAA CCC TAT CTG TTA TGC -3'
-
Rhla (Gene ID: 878955)
+
d. RhT-2b
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LOCUS NC_002516 888 bp DNA
+
  5'- CGA TAC GGC AAA ATC ATG G -3'
-
Rhlb (Gene ID: 878954)
+
5. We began an extensive research for an easy, fast and cheap mutagenesis kit. Thankfully we found the QuikChange Lightning MultiSite-Directed Mutagenesis Kit (Catalog #210513 and #210515)
-
LOCUS NC_002516  1281 bp DNA
+
 
 +
6. For the mutagenesis protocol we also designed three different set of primers (we use the QuickChange Primer design tool from Agilent Technologies), because the base pairs that surround the PstI unwanted sequence were different. Nevertheless, before we design the primers we analyzed the Rhlab codon sequence, because we want to performed silent mutations (change the codon but not the amino acid). We follow the manufacturer guidelines for the primer design, which were:  
 +
 
 +
a. The three primers used for simultaneous mutagenesis must anneal to the same strand of the template plasmid.
 +
 
 +
b. Primers should be between 25 and 45 bases in length, with a melting temperature (Tm) of ≥75°C
 +
 
 +
c. The desired point mutation or degenerate codon should be close to the middle of the primer with ~10–15 bases of template-complementary sequence on both sides.
 +
 
 +
The mutagenesis sequences primers are:
 +
 
 +
a. RhT-g45a
 +
  5'- CTC GAC CGA ACA CCT ACA GGG CGA CTT CTA C -3'
 +
 
 +
b. RhT-c62t
 +
  5'- ACC TGC CGC ACT GTA GTT TCT CGC GGG -3'
 +
 
 +
c. RhT-g159a
 +
  5'- CGA ATC ACG TGC TAC AGC GCG CCT ACG -3'
-
Well we found the gene; we analyzed and looked for the sequence of the restriction sites of E, X, P and S. Sadly we found the sequence of the PstI. That definitely is a setback, but thanks to mutagenesis, we can solve this problem. We designed a set of primers, for the amplification of the Rhabl gene. For mutagenesis of our gene we chose the Stratagene lightning mutagenesis kit, and we also designed another set of primers. Before this we analyzed the gene and checked the reading frame and verified which base pair we can change without changing de amino acids sequence.
 
{{:Team:Panama/calendar2}}
{{:Team:Panama/calendar2}}

Latest revision as of 02:13, 28 October 2010

iGEM Panama

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July 24

Finally, we define our project!

Our goal is the development of a genetically engineered E. coli that can produce rhamnosyltranferase 1 enzyme. These are the steps we follow for our project design:

1. First we look for information regarding the biosurfactant production and we found a paper that describes the Rhlab gene.

2. This gene is found in the Pseudomonas aeruginosa.

3. We search the Rhlab gene sequence in GeneBank, here are the accession numbers: Rhla (Gene ID: 878955) LOCUS NC_002516 888 bp DNA

Rhlb (Gene ID: 878954) LOCUS NC_002516 1281 bp DNA

4. The next step was the RhlAB gene sequence analysis, we found THREE PstI recognition sequenceS. After this we designed two different set of primers for the RhlAB amplification from the P. aeruginosa genomic DNA.

FORWARD

a. RhT-F1a 

  5'- GCG ATA GCT GTT TGC CTG TT -3'

b. RhT-F1b 

  5'- GTT TGC CTG TTC GAA AAT T -3'

REVERSE

c. RhT-R2a 

  5'- TGG CAA CCC TAT CTG TTA TGC -3'

d. RhT-2b 

  5'- CGA TAC GGC AAA ATC ATG G -3'

5. We began an extensive research for an easy, fast and cheap mutagenesis kit. Thankfully we found the QuikChange Lightning MultiSite-Directed Mutagenesis Kit (Catalog #210513 and #210515)

6. For the mutagenesis protocol we also designed three different set of primers (we use the QuickChange Primer design tool from Agilent Technologies), because the base pairs that surround the PstI unwanted sequence were different. Nevertheless, before we design the primers we analyzed the Rhlab codon sequence, because we want to performed silent mutations (change the codon but not the amino acid). We follow the manufacturer guidelines for the primer design, which were:

a. The three primers used for simultaneous mutagenesis must anneal to the same strand of the template plasmid.

b. Primers should be between 25 and 45 bases in length, with a melting temperature (Tm) of ≥75°C

c. The desired point mutation or degenerate codon should be close to the middle of the primer with ~10–15 bases of template-complementary sequence on both sides.

The mutagenesis sequences primers are:

a. RhT-g45a 

  5'- CTC GAC CGA ACA CCT ACA GGG CGA CTT CTA C -3'

b. RhT-c62t 

  5'- ACC TGC CGC ACT GTA GTT TCT CGC GGG -3'

c. RhT-g159a 

  5'- CGA ATC ACG TGC TAC AGC GCG CCT ACG -3'


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Retrieved from "http://2010.igem.org/Team:Panama/24_July_2010"