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.Find the gene sequence in pubmed.

2.In which bacteria it is found.

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

4.We 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 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.

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

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 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.