Team:Panama/24 July 2010

July 24

Development of a genetically engineered E. coli that can produces the rhamnosyltranferase 1, for the production of a biosurfactant.

Steps to follow:

1.Find the gene sequences in pubmed.

2.In which bacteria is found.

3.After we found the gene, we designed two different set of primers.

4.Look for the sequences of the restriction enzymes E, X, P and S, inside the gene sequence.

5.If we found in the gene sequence a recognition sequence for the restriction enzymes.

6. So we should look for a mutagenesis protocol or kit.

7.We choose the Stratagene lightning mutagenesis kit. We also design three different set of primers, because the base pairs that surround the PstI unwanted sequence were different.

We found a paper which describes the Rhab1, the one we need for our project. This gene is found in Pseudomonas aureginosa We looked for the gene sequence en GeneBank, and this it is:

Rhla (Gene ID: 878955) LOCUS NC_002516 888 bp DNA

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

Well we found the gene; we analysed and look for the sequence of the restriction sites of E, X, P and S. And sadly we found the sequence of the PstI. That definitely is a setback, but thanks to mutagenesis, we can solve this problem. We design a set of primers, for the amplification of the Rhabl gene. For mutagenesis of our gene we choose the Stratagene lightning mutagenesis kit, and we also design another set of primers. Before this we analysed the gene and check the reading frame and verify which base pair we can be change without changing de amino acids sequence. Here it is the gene sequences, in red the codons we need to mutate.