Team:Panama/24 July 2010

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 sequence. 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'