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Team:OUC-China/Project Background
From 2014.igem.org
Background
Double Plasmids
Overview
Construction of plasmid is an efficient way to make research deeply into microbiology. Generally, we induce the bacteria into competent cells, thereby the cell could accept the functional plasmid. However, not all bacteria can be induced into competent cells successfully. Under natural conditions, plasmids which depend on genes of themselves can be delivered by conjugating.
Broad-host-range (BHR) conjugative plasmids have been used as a simple and efficient way of transferring DNA between various species of bacteria. During conjugation, DNA is transferred indirectly from a donor cell to a recipient cell. Proteins involved in establishing cell-cell contacts and in the transferring process of DNA are encoded on conjugative plasmids. And on this basis we can construct conjugational plasmids that carry the sequence of functional gene.
Our project aims to construct a double plasmids system, one is a mini plasmid carrying the oriT sequence and the other carries the transfer regions of the broad-host-range IncP plasmid RP4, moreover, the two plasmids we construct are compatible. In the transfer system, the large one doesn’t carry OriT region while it carries the transfer regions tra and trb, due to the lack of Orit sequence, the large plasmid can’t transfer between cells, thus the recipient strains don’t have the ability of conjugation. In the meantime, the other plasmid carrying OriT can transfer under the expression of the tra1 and tra2.
RP4 plasmid
Plasmid RP4 is a resistance plasmid with remarkable features. It was isolated in 1969 from a Pseudomonas aeruginosa strain and it carries genes of ampicillin, kanamycin and tetracycline resistance and for its own replication, transfer and host fertility regulation. It belongs to the incompatibility group IncPa and is distinguished from other plasmids by its very broad host range. Broad-host-range IncPa plasmid RP4 mediates DNA transfer between virtually any Gram-negative bacterial species, as well as some Gram-positive bacteria.
Mpf
Previous studies have shown that DNA transfer by bacterial conjugation requires a mating pair formation (Mpf) system that specifies functions for establishing the physical contact between the donor and the recipient cell and for DNA transport across membranes [1]. During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes between the donor and the recipient cell. For example the IncPa plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 both of which contribute to Mpf revealed typical features of mainly inner membrane proteins.[2] Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction.[3] The Tra2 core region consists of 11 open reading frames, trbB, trbC, trbD, trbE, trbF, trbG, trbH, trbI, trbJ, trbK, and trbL , several of which (trbB, trbC, trbE, trbG, and trbL) were identified as components belonging to the Mpf system. Conjugative transfer of the promiscuous IncPa plasmid RP4 starts at a unique site designated the transfer origin (oriT).[5]
Picture from Grahn A M, Haase J, Bamford D H, et al. Components of the RP4 conjugative transfer apparatus form an envelope structure bridging inner and outer membranes of donor cells: implications for related macromolecule transport systems[J]. Journal of bacteriology, 2000, 182(6): 1564-1574.
