Team:USP UNIFESP-Brazil/Experiments

AlgAranha Team USP_UNIFESP-Brazil

iGEM 2016




Experiments

Assembly of spider silk gene sequence: Getting ready for expression!

Assembly by cut&paste

As we had trouble with our USER-specific primers request and could not start with the USER method, we started an alternative way to polymerize the MaSp1 monomers. Based on the Nature protocol of Teulé et al(ref) we designed a cut and paste technique, to duplicate the MaSp1 sequence at each round.

Telué and collaborators use a cloning vector with a restriction site in its resistance gene and two complementary but non regenerable restriction sites, one at each side of the insert, which is the sequence that is going to be duplicated. The vector is cut in two parallel separated digestions, at one side of the insert and at the resistance gene, and on the other side of the insert and also at the resistance gene. In both digestions, two fragments will be generated: one that contains the insert and a part of the resistance gene, and one that does not contain the insert. Ligating the insert-containing fragments of the two different digestions together results in the duplication of the insert in a vector with a complete resistance gene.

In our case we used the same vector, that would be transformed into our production organism, Chlamydomonas reinhardtii. The pJP22 has a ScaI restriction site in the ampicillin resistance gene and two compatible but non regenerable restriction sites at the MCS: BglII and BamHI, so we went on and cloned the spider silk monomer MaSp1 Type2(link to part). Then, following the scheme of Teulé and collaborators(ref figure), we cut in parallel digestions the pJP22-MaSp1t2 with ScaI and either BamHI or BglII(ref figure). The insert-containing fragments were then ligated together and transformed into E. coli DH5α cells

Positive results

We were able to purify a PfuX7 polymerase and standardize a working PCR protocol

We were able to design and clone in pSB1C3 a protein domain part (BBa_K2136002) and a working gene expression cassete for microalgae (BBa_K2136010) !

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