Introduction

For our project, we decided to submit seven BioBricks to the iGEM Registry in the pSB1C3 plasmid backbone. Our submitted parts can be divided into two separate collections (Names as Parts collection one and Part Collection two). Parts collection one is associated with Engineered biofilm subunit CsgA, while Parts collection two is relevant to construction of hydrogenase gene clusters.

All of the constructs are protein-coding parts have been confirmed with gene sequences, demonstrated with expected functionalities, and thus it would be easy to handle for other users.Specifically, in order to make hydrogenase expression more productive, we optimized the gene sequences of 5 hydrogenase parts with the help of OptimumGeneTM (GenScript). We changed the codon usage bias in E. Coli by upgrading the CAI (Codon Adaptation Index) from a low level (around 0.30) to 0.97

Characterizations of the parts are included on the webpage of the parts section, but for more information about the submitted parts in real use, please go to project page

Part Collection

1. Part Collection One (Engineered Biofilm Subunit CsgA with SpyCatcher and HisTag)

a) In order to realize covalent link of two proteins, we make use of SpyTag and SpyCatcher system (see our Linkage System on Biofilm Page). The SpyTag is fused to HydA of the hydrogenase system, thus a SpyCatcher on CsgA would enable Spy-tagged Hydrogenase to be covalently attached to CsgA In the meantime, to endow biofilm CsgA protein with additional functionalities (such as specific binding to QDs or Nanorods in our case), we append one or two HisTag at the N- or/and C-terminus of the CsgA-spycatcher protein. This lead to two new constructs, HisTag-CsgA-SpyCatcher-HisTag and HisTag-CsgA-SpyCatcher (the former being sequence confirmed and submitted).

b) To determine if expression of the two proteins is successful, we constructed mCherry-SpyTag to test the activity of the SpyCatcher on the CsgA. mCherry-SpyTag is thus submitted as BBa_K2132003, sequence confirmed.