From 2006.igem.org

Revision as of 03:11, 5 November 2005

=== 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

The Project: 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: A process through which cells can exchange genetic material such as plasmids. Conjugal plasmids exist within the cell and transcription of certain genes causes conjugal characteristics to be expressed within the host cell. Thus the conjugating plasmid can be passed between cells and keep itself alive while often providing symbiotic benefits to its host such as antibiotic immunities. The process of conjugation is induced by a cascade of genes that starts with TraJ. The TraJ signal is then propagated to downstream genes which cause the formation of various conjugal characteristics on the host cell's surface. The host cell then uses the structures formed on the cell surface to attach itself to a different cell and undergoes the process of rolling circle replication. During rolling circle replication, genetic material from the host is nicked at the Origin of Transfer (OriT), after which it undergoes unwinding and replication of a new strand inside of the recipient cell.

Riboregulator: Based on F. Isaacs and J.J. Collin's riboregulator model <5>.

Implementation

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.

Cell #1 (F type cell): 1-A Non-mobile synthetic F plasmid: Begins the conjugation signal, which it sends to plasmid B. Also contains the CFP tag which identifies the host cell as "F-type"
, and always produces mRNA 'key 2' which unlocks RNA lock 2 https://webfiles.berkeley.edu/carnive/public_html/fPicture1.png

1-B - Non-mobile almost-wild F plasmid: Contains all F-plasmid genes EXCEPT OriTf, TraJf
. Plasmid receives and propagates the conjugation signal from TraJf in plasmid 1-A and sends the signal to OriTf in 1-C

1-C - Mobile F plasmid: Contains the OriTf site which receives signal from plasmid 1-B. This plasmid then leaves the host cell and enters the conjugating recipient cell. Holds encrypted message (produce cI --> turn on GFP to signify "message 1 received") secured by RNA lock 1.
https://webfiles.berkeley.edu/carnive/public_html/fPicture2.png

Cell #2 (R type cell) 2-A Non-mobile synthetic R plasmid: Always produces mRNA 'key1'. Thus when it receives 'lock1' (sent by mobile plasmid 1-C) it can open the latter and produce cI, which will activate plasmid 1-C (turn on GFP, "message 1 received") and simultaneously activate TraJr (start R conjugation cascade)
https://webfiles.berkeley.edu/carnive/public_html/rPicture1.png

2-B Non-mobile almost-wild R plasmid: Just like 1-B, contains all of the wild type R-plasmid EXCEPT OriTr and TraJr. Propagates TraJr signal from 2-A and sends it to OriTr

2-C Mobile R plasmid: Contains the OriTr site, which receives signal from plasmid 2-B. This plasmid then leaves the host cell and submits its message back into cell #1
https://webfiles.berkeley.edu/carnive/public_html/rPicture2.png

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"