Part:BBa_K3552026

GsPilA

prepilin peptidase-dependent protein B, a minor pilin. a bitopic inner membrane protein located in the cytosol of the inner membrane, with the length of 564 bp/ 187 aa.

The part collection includes: Parts that are different kinds of type 4 pilus: BBa_K3552000 BBa_K3552001 BBa_K3552002. Parts that are the generator of the type 4 pilus: BBa_K3552003 BBa_K3552004 BBa_K3552005 BBa_K3552006 BBa_K3552007 BBa_K3552008 BBa_K3552018 BBa_K3552019 BBa_K3552020 BBa_K3552021 BBa_K3552022 BBa_K3552023 BBa_K3552024 BBa_K3552025 BBa_K3552026 BBa_K3552027 BBa_K3552028 BBa_K3552029. Parts that are a complete circuit: BBa_K3552009 BBa_K3552010 BBa_K3552011 BBa_K3552012.

Our part collection can instruct other teams to designed new rechargeable pilus and substitution of different major pilin.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

Luna Rico, Areli et al. “Functional reconstitution of the type IVa pilus assembly system from enterohaemorrhagic Escherichia coli.” Molecular microbiology vol. 111,3 (2019): 732-749. doi:10.1111/mmi.14188

Usage and Biology

At min 61, the ppdAB-ygdB-ppdC operon encodes the four minor pilins. These minor pilins and their homologues in the family contain two conserved Cys pairs at similar positions. We used the ppd genes from the EHEC strain EDL933 as building blocks to reconstitute an artificial T4P assembly operon in non-pathogenic E.coli, DH5a. Neither deletion of ppdB nor overproduction of ppdABC results in defects in chemotais or flagellar motility. This gene is predicted to be an inner membrane protein.

Characterization

One of the possibilities that limit the pili yield could be the coupling of the genetic sequence of pili generator (BBa_K3552006 BBa_K3552007 and BBa_K3552008). When the genes in the clusters overlap each other and share a same sequence, it could be extremely inconvenient for us to modify the gene sequence such as RBSs (ribosomal binding sites) and conduct codon optimization for biosynthetic engineering purpose. In order to improve the yield of pili production, we designed and carried out experiments to decouple these gene clusters into singular genes with optimization of RBS in front of each gene. We suggest that the reassembled pili generator of decoupled gene segments would not decrease or might increase the pili productivity.

There are two steps in decoupling: 1) amplification of each genes; 2) codon optimization and point mutation. We first amplified all the 12 genes from the gene clusters. Then, we designed codon optimization to alter the RBS with a similar strength as the original one and carried out point mutations to eliminate the same sequence of two genes according to the design and therefore avoid the recombination of the genes back to the original form. In the design, we kept the beginning sequence of the later gene unchanged but altered the ending of the former gene. After every portions of the gene of generator was decoupled with targeting molecular masses, we reassembled all parts together directly to form a new plasmid with decoupled pili generator by using Gibson Assembly due to the time limitation. But the result of our Gibson Assembly is basically all negative.