From 2014.igem.org

Revision as of 21:13, 16 October 2014

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Synthesis of the gene coding for the SLPs(BBa_K1317002)

Initial strategy :

We tried to assemble the gene coding for the SLPs from 8 oligonucleotids with homolog regions with the Gibson Assembly.
First of all, consensus sequences for the spider silk were identified and our own protein was designed. Then, the nucleotidic sequence using the peptidic sequence was determined. We had to pay attention because our proteic sequence is made of repeted motifs.

2 different methods were used with the Gibson Assembly1: in one step at 50°C or with cycles of denaturation at 95°C and annealing at 50°C (figure 1). The enzyme used was the Phusion® High Fidelity Polymerase.

img2 Our 8 oligo weren’t assembled with these 2 methods, so another method was used : the PCR-Fusion2.
This method is made of different steps using the Phusion® High Fidelity Polymerase (picture 2). In a first step fragments were joining two by two, then fragments 1-2 were joined to fragments 3-4 and a PCR is achieved using fragments 1 and 4 as primers. The same method was used for fragments 5-6 and 7-8.
Finally, fragments 1-2-3-4 were assembled to fragments 5-6-7-8 and a PCR was also achieved using the fragments 1 and 8 as primers.

img3 This method wasn’t successful because fragments 6 and 7 were unable to join. Therefore, new fragments were designed with a different homolog region. The fragment 8 that added only 2 nucleotids was suppressed and these 2 nucleotids were added on fragments 7.

img4 Then, a new strategy was used (picture 3). The two first steps are common but then, fragments 5-6 are joined to fragments 1-2-3-4 and the PCR is made with the fragments 1 and 6. Finally the fragment 7 is added and a PCR is also achieved. img5 This method enable the assembly of the 7 fragments (picture 4). A fragment of 318 bp was expected on the electrophoresis gel.

img6

Reference :

[1] https://www.neb.com/tools-and-resources/feature-articles/gibson-assembly-building-a-synthetic-biology-toolset

[2] Shevchuk N.A., Bryksin A.V., Nusinovich Y.A., Cabello F.C., Sutherland M. et Ladisch S. Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously (2004) Nucleic Acids Res., 32(2), 19

Part:BBa_K1317001 : CDS for resilin-like polypeptide (RLP)

This part is the coding sequence for the resilin like polypeptide. This sequence was assembled from a consensus of the proresilin exon 1 from Drosophila melanogaster. This protein, in insects, allows resistance and elasticity used for jumping, flapping... The synthetic gene encodes a synthetic protein "resilin-like". The repeated aminoacids allow to retrieve these properties of resistance, resilience and elasticity, but with a minimal pattern of the original protein, which is more suited for downstream applications. It can be used to produce wire presenting these properties after wet-spinning.

Sequence and Features