Difference between revisions of "Part:BBa K314300"

Revision as of 18:44, 25 October 2010

T6SS fosmid

Type VI secretion system from Pseudomonas aeruginosa in a pCC2Fos CopyControl Fosmid (Chloramphenicol Resistant).

The [http://en.wikipedia.org/wiki/Fosmid fosmid] (very large plasmid that contains a region of genomic DNA from a particular organism) encodes the HSI-I Type VI Secretion (T6SS) locus of Pseudomonas aeruginosa. The T6SS is encoded in two operons divergently transcribed bidirectionally from a promoter region. This region has been engineered using homologous recombination to replace native promoters with robust T7 promoters. The two promoters are contained in the same intergenic region, with one on the positive strand and one on the negative. When activated, they drive transcription in both directions, transcribing both T6SS operons.

The system allows for protein delivery directly from the donor cell into the periplasm or cytosol of the recepient organism. While we are using this system to deliver a toxin, other protein effectors might be used for a range of applications. This system is meant to be used with T6SS substrates that are expressed off of BioBrick plasmids. Control of the T6SS activity will be at the level of controlling T6SS substrates, since the number of genes in the T6SS is large.

We are using this plasmid to express a Type VI Secretion System in the BL21(DE3) strain of E. coli which has an IPTG inducible T7 RNA polymerase. The purpose of the system is to create a probiotic system, creating a secretion system for a toxin/antitoxin system (BBa_K314200-BBa_K3142003). This was used in the [http://2010.igem.org/Team:Washington 2010 Washington iGEM team project].

This is also on the design page notes, but worth mentioning here:

Note that this plasmid is for the purpose of making a strain that expresses the Type VI Secretion System - since the secretion system is complex and requires many genes which must be expressed together, BioBricking these genes individually does not make sense. Thus we have this part in a fosmid that can be expressed in the same strain as traditional BioBrick plasmids, such that proteins targeted for secretion can be expressed as BioBricks and then secreted by the secretion system. This is in line with iGEM, as the secretion system is present to essentially generate a background strain, then the user defines the proteins to be secreted by choosing the BioBricks and regulating them accordingly, allowing for a system that can be engineered to have a variety of different behaviors.

Western blot for Type VI Secretion Protein. As a control the native fosmid with P. aeruginosa promoters was expressed in P. aeruginosa. A band showing expressing of T6SS was seen. The engineered fosmid with T7 promoters, K314300, was expressed in E. coli. Expression was induced using 0.5 mM IPTG. A band was seen corresponding to T6SS protein, indicating that the T6SS is being expressed.

Sequence and Features