Part:BBa_K1763444:Design
Bombyx mori silk co-spinning module
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1448
Illegal BamHI site found at 221 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1439
Illegal BsaI.rc site found at 719
Design Notes
We decided to use a LacI promoter to regulate expression of the large protein construct in E. coli K12 laboratory strains, in lieu of utilizing a T7 promoter or constitutive Anderson family promoters. This is to maximize the protein yield of the co-spinning module without causing a tradeoff between regular cell growth and housekeeping protein synthesis. Additionally, we decided to use sfGFP as the reporter gene, instead of a standard mut3b GFP, due to the ability of sfGFP to function even when fused to large bulky protein domains (in this case, FibNT and FibCT). Lastly, we designed BsaI TypeIIS restriction sites flanking the reporter gene that when cut, generates sticky ends compatible with EcoRI and SpeI sites. This was created to allow iGEM teams to take protein domains of interest from the Standard Registry of Parts, and fuse them into the co-spinning module by a simple Digest and Ligate assembly to swap the sfGFP construct. However, the protein domain to be added must have the Silver Fusion prefix and suffix, to allow for in-frame cloning of the protein domain. Without this in-frame cloning, the scar sites of the tradition prefix and suffix will cause a frameshift mutation that mutate the translation product, and renders it non-functional.
Source
The LacI promoter (BBa_R0010), RBS (BBa_B0034), and super folder GFP (BBa_I746916) were derived from sequences found in the Standard Registry of Parts. The Bombyx mori silk fibroin heavy chain N-terminal (FibNT) and C-terminal (FibCT) domains were derived from the sequenced consensus genome of Bombyx mori silkworm species, and were synthesized using IDT gBlocks gene fragments.
References
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