Difference between revisions of "Part:BBa K314204"

Revision as of 18:11, 24 October 2010

p4T6S::T7

A [http://en.wikipedia.org/wiki/Fosmid fosmid] (F' BAC) (very large plasmid that contains a region of genomic DNA from a particular organism) which encodes the HSI-I Type VI Secretion (T6SS) locus of Pseudomonas aeruginosa.

This fosmid contains both major operons of the T6SS, divergently transcribed. The original plasmid regulated the expression of the two operons via complex Pseudomonas aeruginosa transcriptional regulation. We engineered the regulatory locus of K314204, using homologous recombination, to replace native promoters with two divergent, 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.

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].

The system allows for protein delivery directly from the donor cell into the periplasm or cytosol of the recipient organism. While we are using this system to deliver a toxin, other protein effectors might be used for a range of applications.

Sequence and Features