Part:BBa_K4212036:Design
ChiS3
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1912
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1912
Illegal NheI site found at 1011 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1912
Illegal BamHI site found at 1897 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1912
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1912
- 1000COMPATIBLE WITH RFC[1000]
Part Design
Our system consists in a fusion membrane protein, comprising of three parts: the anchor protein (a structural protein naturally embedded in the spore’s membrane), the linker and the chitinase, located outside the membrane. (Fig. 1a). A computational approach was chosen to identify the optimal linker length and aminoacid composition, and having defined such parameters a comparative analysis was done across different anchor proteins to identify the most suitable one (Fig. 1b).
Design Notes
This part consists the fusion protein consisted of CotG and ChiS, in which we combined these parts into level 1 Golden Gate Construct. Our dry lab team predicted the functional model of this chimera and our wet lab intended to test the effectiveness of this model. We added a flexible linker to this construct to test if it works better than the construct without the linker. We tried out different linkers to compare their effectiveness.Promoter hyper-spank is an inducible promoter, which was designed for high-level protein expression in B.subtilis and it was previously used by other iGEM teams. The addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG) can induce gene expression under the control of the promoter hyperspank. tL3S2P21 is a strong terminator, which helps to increase the production of desired protein.
Source
Synthetic construct.