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 (
BBa_K2132001) and HisTag-CsgA-SpyCatcher (the former being sequence confirmed and submitted).
➤ CsgASpyCatcherHisTag (
E. coli) -
BBa_K2132001
➤ mCherry-SpyTag (
E. coli) -
BBa_K2132003
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.
2.Parts Collection Two (Hydrogenase gene clusters)
We utilize [FeFe] Hydrogenases originally from the bacterium Clostridium acetobutylicum (coding sequence: hydA,
BBa_K2132004 &
BBa_K2132005) to accept electrons and therefor enable catalytic production of hydrogen in our project. Synthesis of heterologous [FeFe] hydrogenase in
E. coli requires co-expression of HydE (coding sequence: hydE,
BBa_K2132006), HydF (coding sequence: hydF,
BBa_K2132007), and HydG (coding sequence: hydG,
BBa_K2132008).
In this collection, we sub-cloned the coding sequence into the pSB1C3 individually, with two appended tags at the N-terminus (His-tag to faciliate purification and TEV site as cleavable site for Histag cutting off). We have confirmed that the presence of the two tags won’t disrupt expression and normal functionalites of HydA.
➤ HydA with SpyCatcher, Histag and TEV site (
E. coli) -
BBa_K2132004
➤ HydA with SpyTag, Histag and TEV site (
E. coli) -
BBa_K2132005
➤ HydE with Histag and TEV site (
E. coli) -
BBa_K2132006
➤ HydF with Histag and TEV site (
E. coli) -
BBa_K2132007
➤ HydG with Histag and TEV site (
E. coli) -
BBa_K2132008
★Optimization of this collection
The original sequences of hydrogenase were found in www.genome.jp. With the help of OptimumGene™, We used the following parameters to optimize our gene sequences without changing their amino acids sequence: Codon usage bias, GC content, CpG dinucleotides content, mRNA secondary structure, Cryptic splicing sites, Premature PolyA sites, Internal chi sites and ribosomal binding sites, Negative CpG islands, RNA instability motif (ARE), Repeat sequences (direct repeat, reverse repeat, and Dyad repeat).
