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.

Figure 2: PCR Fusion strategy to assemble the gene coding for the SLPs

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.

Table 2: Sequence of the new fragments

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.

Fig 3 : Strategy to assemble the CDS for the SLPs using the new fragments 6 and 7

This method enable the assembly of the 7 fragments (picture 4). A fragment of 318 bp was expected on the electrophoresis gel.

Figure 4 : Gel electrophoresis on 3% agarose

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

Design Notes

The gene used to assemble the biobrick has been provided by GenScript even if the assembling strategy succeeded It allows us to have the proper sequence without mutation and with sequencing results.
The strategy of our assembly is described below anyway. The gene was synthesized by ordering specific oligo with overlapping regions, and by adding a NheI site for further fusing proteins.
We used the pro-resilin exon 1 from Drosophila melanogaster. We identified a repetitive pattern, PSXXYGAP. So we blast it against the protein database of pro-resilin to check if the pattern was conserved within different species or specific to Drosophila.

We have taken XX randomly and we brought out conserved pattern so we used it to do our resilin like polypeptide with the following pattern: (RLP) MW-[(PSSSYGAP)(PSNSYGAP)(PSTSYGAP)(PVAYGAP)]3

The literature delivers some mechanical properties concerning native resilin as high resilience (<90%) that allows the protein to recover its initial folding after a high tensile stress. Thus it can be an interesting characteristic to show and use, so we wanted to test it on our RLP.

Source

References