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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.
Transfection
overview
At present, lots of methods can realize eukaryotic cell transfection. Such as using lentivirus, lipidosome and particle bombardment. Although these methods had been used in many labs, they also had their own disadvantages. For example, producing lentivirus is time-waste and expensive. Using lipidosome is easier, but it may cause toxic reaction. Particle bombardment is too expensive and hard to operate for many labs. Because of the complex environment in fishes' alimentary canal, a kind of engineering bacteria was used to transfer our new design plasmid by conjugation with intestinal flora. After the conjugation between engineering and entrenched bacteria, the TAT-H4 protein will be produced. TAT-PTD is a kind of penetrating peptide which has been thorough study. Its main job is moving into the cell by endocytosis. Histone H4 is a kind of the histone protein. It can interaction with the negatively charged phosphate groups in DNA interaction, so that it can combined with them. We devised a new fusion protein called TAT-H4 to achieve combination and transfection. The protein will combine our plasmid and prepare for releasing to the external environment. To achieve release, we used iGEM08_UC_Berkeley BBa_ k112808,iGEM2006_BerkeleyBBa_J23106,iGEM09_British_Columbia BBa_ k206000,iGEM04_Antiquity BBa_P0440 to structure our own self-lysis device. Once the fusion protein has accumulated to the proper concentration, the bacteria will lyse by induction. Then the complex will transfect into fish cells.
TAT-PTD
Protein transduction domains (PTDs) is a kind of small molecule polypeptide substance which can pass through mammals’ plasma membrane. This kind of material has been widely used in every field of protein treatment. TAT-PTD is a kind of penetrating peptide which has been thoroughly studied . It is a polypeptide domain derived from a TAT protein within HIV - 1 virus . TAT-PTD is made up of 11 amino acids. It is encoded by YGRKKRRQRRR, composed of several strongly basic amino acid sequence, and is the key to complete roles in membrane peptides.
In the current study, some researchers found that histones can mediate gene transfer (histone transfection) effectively. H4 is one of the histones which, are rich in positively charged forms. It can interact with the negatively charged phosphate groups in DNA interaction, so that it can be combined with them.
图The TAT-H4 fusion protein we designed can help the TAT combine with plasmids on H4 histones to enter cells, and putting the plasmid into cells for, expressing the plasmid.
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