Difference between revisions of "Part:BBa K3552005"

Revision as of 05:08, 26 October 2020

ppdA-ppdB-ygdB-ppdC-gspO(gene from DH5a)

Prepilin peptidase-dependent protein A, a minor pilin. A bitopic inner membrane protein located in the inner membrane, with the length of 471 bp/156 aa. 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. YgdB is a domain-containing protein with a length of 408 bp/135 aa. prepilin peptidase-dependent protein C, a bitopic inner membrane protein located in the inner membrane, with the length of 324 bp/ 107 aa. GspO plays a role in type II pseudopili formation by removing the leader sequence from substrate proteins and subsequently methylating the alpha-amino group of the newly exposed N-terminal phenylalanine. Substrates include proteins required for biogenesis of the type II general secretory apparatus. This part is in the part collection where we have 13 genes that code for the proteins.

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

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

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 ppdC nor overproduction of ppdABC results in defects in chemotais or flagellar motility. This gene is predicted to be an inner membrane protein. The gene gspO, one of the two prepilin peptidase genes in E.coli K-12, is the last gene of the gsp operon encoding the chitinase-specific type 2 secretion system. In EHEC, almost all gsp genes have been lost during evolution, leaving only the gspO fragment encoding the prepilin peptidase catalytic domain. Additionally, EHEC strains have the gspO homologue etpO within a plsmid-encoded type 2 secretion system cluster. This gene is also a part of the generator of the major pilin PilA.

Characterization

We first uses the bacteria DH5a as the template and obtain the DNA fragment of 12 genes. We verified the practicability of the generator through the western blot of pilA in vivo which confirmed the usage of the generator because the PilA is able to display outside the cell.