Difference between revisions of "Part:BBa K3552005"

Latest revision as of 13:44, 27 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

Construction

We amplified each gene cluster of pili generator (BBa_K3552003, BBa_K3552004 and BBa_K3552005) from E.coli DH5a. Then, due to the long length of each gene cluster, we initially assembled all segments we obtained separately on pSB1CJ vector by Gibson Assembly to form three sub-plasmid: 1)BBa_K3552000 and BBa_K3552003 , 2)BBa_K3552004 , and 3)BBa_K3552005. Additionally, we constructed another sub-plasmid with pET28a vector for the backbone of our major plasmid. Finally, four sub-plasmids were assembled through Golden Gate Assembly to form the final major plasmid of Gs pilA with pili generator, BBa_K3552009.

Since using the pili generator from the strain Enterohaemorrhagic Escherichia Coli (EHEC) will give a better production of T4P, we conducted point mutation on three the sub-plasmids we constructed before (BBa_K3552003, BBa_K3552004 and BBa_K3552005) for further construction, deriving the original gene sequence into that of EDL933, obtaining three new sub-plasmids: BBa_K3552006, BBa_K3552007 and BBa_K3552008. We then conducted the same assembling method to assemble three new sub-plasmids on pET28a vector into a new major plasmid possesses Gs pilA with EHEC pili generator, BBa_K3552010.

Pili production

In order to compare the function of two types of generator, generator similar to EHEC genes and generator from DH5a genes, we carried out a pili production analysis. In this analysis, we determined the yield of three types of pili production and measured the effects of two variables on pili production: cultivation time and mobile oxygen presence. We did two repeated experiments under the same environmental conditions for cultivation except for the variables. To further prove that the use of the EDL933 pili generator could result in a better yield of pili, we designed a new plasmid of Gs pili with a DH5a generator, and compare the pili yield of two plasmids.

For the first experiment, We prepared 32 plates, 8 plates for each group; half of the plates were sealed with laboratory films to limit the mobility of oxygen and cultured at 30-celsius degrees. We added 8 plates in the second group for an additional measurement in 72h growth. In the first experiment, we set two groups of time variables: 24h vs 48h cultivation time. We took two plates for each sample group for every 24h. The results showed that all four samples had a better yield of pili after 48h cultivation comparing to 24h. Furthermore, we wanted to discover whether the pili production will increase as the time of cultivation increases beyond 48h. In the second experiment, we did a repeat experiment with an additional group of 72h cultivation time. The results were similar to that of the first experiment, showing the same trend of better yield in 48h than 24h cultivation, and that 24h cultured plates basically have no pili produced. The 72h cultivation might not have the highest yield and parts of the sample have high pili production after 48h period. For Gs and Pa pilA, 48h cultivation will be better than 72h in pili production while the trend of Gm pilA and Gs pilA with DH5a generator goes the opposite. But these results would have a certain degree of uncertainty.