From 2006.igem.org

Berkeley iGEM Team

Professors:

Adam Arkin
Jay Keasling

GSIs:

Jonathan Goler
Justyn Jaworski

Members:

Michael Chen
Vlad Goldenberg
Stephen Handley
Melissa Li
Jonathan Sternberg
Jay Su
Eddie Wang
Gabriel Wu

Addressable Bacterial Communication

We've been working on addressable bacterial communication via conjugation. The message being transferred is a gene locked using the Isaacs et al. riboregulator, and is sent in a packet mobilized by F-plasmid conjugation. This mobilized plasmid is sent to cells in the vicinity upon induction of the pBadAraC-controlled TraJF conjugation regulatory protein, expression of which triggers a cascade that constructs and uses F-plasmid conjugation machinery to send the packet plasmid. The message can only be unlocked by cells containing a trans activating key which acts to unlock the hairpin formed over the RBS by the cis-repressed riboregulator, where addressability is achieved by varying a 5 nucleotide region shared by the locks and keys. Upon receipt of the packet plasmid, the recipient cell turns on its own RP2-based conjugation machinery to send a similar acknowledgement packet back to the original cell, containing a genetic message locked and opened by a second addressed lock/key pair.

We have used the lambda-red protocol to knock out the TraJ gene on the F plasmid so as to have total control over transfer via the pBadAraC promoter. Additionally, by knocking out the OriT nick region, we have marooned the F plasmid and its transfer machinery in the original cell so as to ensure only the packet is being sent.

Relevant Papers

Balbás et al. "A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome"
Datsenko, Wanner, "One-step inactivation of chromosomal genes in escherichia coli k-12 using PCR products"
Haldimann, Wanner, "Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria"
Isaacs et al., "Engineered riboregulators enable post-transcriptional control of gene expression" Jaenecke et al., "A stringently controlled expression system for analyzing lateral gene transfer between bacteria"
Knight, "Idempotent Vector Design for Standard Assembly of Biobricks"
Lawley et al., "F factor conjugation is a true type IV secretion system"
Lessl et al., "The Mating Pair Formation System of Plasmid RP4"
Miller et al., "F Factor Inhibition of Conjugal Transfer of broad host range plasmid RP4"
Martinez-Morales et al., "Chromosomal Integration of Heterologous DNA in Escherichia coli"
Wilkins, "Plasmid promiscuity - meeting the challenge of DNA immigration control"