Part:BBa_K1317002:Design
CDS of silk-like protein (SLP)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 312
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The gene was synthesized by ordering specific oligo with overlapping regions, and by adding a NheI site for the assembling.
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.
Our 8 oligos were not properly assembled with these 2 methods, so another method was used : the PCR-Fusion. This method consists of different steps using the Phusion® High Fidelity Polymerase (figure 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.
Source
major ampullate spidroin 1 (MaSp1) gene from Nephila clavipes
References
[2] Shevchuk N.A. et al. Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously (2004) Nucleic Acids Res., 32(2), 19