From 2006.igem.org

Revision as of 23:40, 4 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

Addressable Bacterial Communication

We are working on building addressable bacterial communication via conjugation. 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. This mobilized plasmid is sent to cells in the vicinity upon induction of the pBadAraC-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.

Protocols

Restrictions

   * 35 microliters of your favourite plasmid (make sure to record the concentration.  Should be above around 80 ng/microliter) 
   * 5 microliters of 10X BSA
   * 5 microliters of NEB buffer (check the chart).
   * 1.3-1.5 microliters of each delicious restriction enzyme 

Lamda-Red Protocol (from Datsenko and Wanner)

   * PCR TraJ? and OriT? from pKD3 purified plasmid with primers TJFlamF?/TJFlamR? and ORFlamF?/ORFlamR?
   * Run on 2microliters on gel and look for 1000bp fragments
   * PCR purify, elute in 40microliters of EB, digest overnight with Dpn I
   * Gel purify and elute in 30microliters of EB
   * Grow pox38 x pKD46 conjugate overnight in Amp Kan at 30C
   * Prepare appropriate number of 5mL LB tubes with Amp Kan + 2 for OD testing
   * Using 20% w/v arabinose at JAG's bench add 37.5microliters to each 5mL tube for 10mM
   * (If using larger culture flask add 7.5microliters of 20% w/v arabinose for every 1mL of culture)
   * Innoculate 50microliters of overnight culture per 5mL tube
   * Incubate in 30C shaker
   * Last time took about 2.5 hours to reach OD of approximately .45 so probably good to check at 1 and 2 hours
   * Ideal to stop at OD 0.4, be sure to have an ice bath ready before then and precool microcentrifuge to 4C
   * Rest of steps must be at 4C
   * At OD 0.4 place all cultures on ice
   * Spin down in 2mL eppendorfs at top speed 5min per spin removing supernatant
   * After spinning down all 5mL, wash with 10% ice cold glycerol. (50mL falcon tube in JAG's fridge)
   * Once with 1.5mL of glycerol, then twice with 750microliters. Spin 2 min each time, pouring out supernatant
   * The last time you pour out the supernatant there should be enough liquid and cells left to electroporate with (about 40-50 microliters)
   * Add 2 microliters of gel purified PCR product that has been restricted with Dpn? I to electrocompetent cells and transform
   * Place tubes in 37C <--I guess 37 is okay - for one hour and plate 150microliters on prewarmed Kan Chlor plates at 30C

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"