Berkeley
From 2006.igem.org
Contents |
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 are working on building addressable bacterial communication via conjugation. Our construct consists of four different synthetic plasmids placed within communicating cells.
Background:
Conjugation:
1. Plasmid 1=Modified 'F' plasmid
This plasmid contains the wild-type phenotype of the F plasmid, including genes for normal replication, plasmid maintenance, and most importantly for us, conjugation. However, this cell has two conjugation proteins genes--the origin of transfer (OriT) and the
A message, in the form of a gene locked by the Isaacs et al. riboregulator, is transferred within a packet plasmid mobilized by F-plasmid conjugation.
A chemical signal (the binding of the ligand ara to the pBad promoter) begins the cascade for the mobilizable plasmid. This plasmid consists of a controlled TraJF conjugation regulator, expression of which triggers a cascade that constructs and uses F-plasmid conjugation machinery to transmit the packet plasmid. Addressing is achieved because the message can only be unlocked by cells containing a trans activating key which unlocks the hairpin formed over the RBS by the cis-repressed lock, 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.
Work Schedule
May-June: Synthetic Biology primer and project brainstorming
July: Theoretical project design and background research on bacterial conjugation and technique training
September: Project training continues, implementation begins
Relevant Papers
1. Balbás et al. "A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome"
2. Datsenko, Wanner, "One-step inactivation of chromosomal genes in escherichia coli k-12 using PCR products"
3. Haldimann, Wanner, "Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria"
4. Isaacs et al., "Engineered riboregulators enable post-transcriptional control of gene expression"
5. Jaenecke et al., "A stringently controlled expression system for analyzing lateral gene transfer between bacteria"
6. Knight, "Idempotent Vector Design for Standard Assembly of Biobricks"
7. Lawley et al., "F factor conjugation is a true type IV secretion system"
8. Lessl et al., "The Mating Pair Formation System of Plasmid RP4"
9. Miller et al., "F Factor Inhibition of Conjugal Transfer of broad host range plasmid RP4"
10. Martinez-Morales et al., "Chromosomal Integration of Heterologous DNA in Escherichia coli"
11. Wilkins, "Plasmid promiscuity - meeting the challenge of DNA immigration control"
