From 2006.igem.org

Contents 1 Berkeley iGEM Team 1.1 Members: 1.2 Advisors: 1.3 GSIs: 2 Protocols 3 Work Schedule and Progress 4 The Project: Addressable Bacterial Communication 4.1 Background: 4.2 Implementation 5 Relevant Papers 6 Thanks to

Berkeley iGEM Team

Members:

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

Advisors:

Professor Adam Arkin

Professor Jay Keasling

GSIs:

Jonathan Goler

Justyn Jaworski

Protocols

Berkeley Protocols

Work Schedule and Progress

Berkeley Work Schedule and Progress

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 is a process through which cells can exchange genetic material on plasmids. Conjugal plasmids (in our case incF and incP plasmids) carry the machinery necessary to transfer themselves in the form of mating pair formation (mpf) and DNA transfer (dtr) genes. Conjugation is under the control of the TraJ regulatory protein, which when expressed induces a cascade that results in the formation of a pore by mpf genes and then subsequent nicking, rolling circle replication and transfer of one strand of the plasmid by the relaxosome complex and other dtr proteins. The relaxosome nicks the plasmid at the OriT region and then covalently attaches one of its subunits to the 5' end of the plasmid DNA, and by doing so it is able to drag the plasmid across the pore formed by the mpf machinery by means of a coupling protein. Upon reaching its destination, the single strand of plasmid DNA is recircularized and a complement strand is synthesized by transferred primases.

The riboregulator we are using is a biobricked version of the Isaacs riboregulator <5>, where we have added biobricks sites and designated an addressing region in the cis-repressed locks and the trans-activating keys.

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.

Genetic Construct

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The following is an outline of our genetic construct. The construct exists as 3 separate plasmids (designated A,B,C for convenience) for 2 different types of cells ("F type" and "R type" respectively).

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

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. Frost, Ipp en-Ihler, Skurray. Microbial Rev 1994. Analysis and Sequence of the F Plasmid [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=7915817&query_hl=2&itool=pubmed_docsum Pubmed]
   
4. Haldimann, Wanner, "Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria"
   
5. Isaacs, Dwyer DJ, Ding C, Pervouchine DD, Cantor CR, Collins JJ. "Engineered riboregulators enable post-transcriptional control of gene expression" [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15208640&query_hl=7&itool=pubmed_docsum Pubmed] [http://www.nature.com/nbt/journal/v22/n7/abs/nbt986.html;jsessionid=ABB92F041E994E3B8E4A5213699C3CB0 Journal]
   
6. Jaenecke et al., "A stringently controlled expression system for analyzing lateral gene transfer between bacteria"
   
7. Knight, "Idempotent Vector Design for Standard Assembly of Biobricks"
   
8. Lawley et al., "F factor conjugation is a true type IV secretion system"
   
9. Lessl et al., "The Mating Pair Formation System of Plasmid RP4"
   
10. Miller et al., "F Factor Inhibition of Conjugal Transfer of broad host range plasmid RP4"
    
11. Martinez-Morales et al., "Chromosomal Integration of Heterologous DNA in Escherichia coli"
    
12. Wilkins, "Plasmid promiscuity - meeting the challenge of DNA immigration control"

Thanks to

(in no particular order) Team Connie, Team John Dueber, Doug Pitera, Mario Ouelet, sequencing elf, Roti truck