Melb:Plan:GFP Fluorescent reporter


Revision as of 07:53, 11 August 2007 by Patricia (Talk | contribs)

LacZ reporter

  1. Ligate LacZ coding region to gentamycin RBS part. (Done?)(1 day)
  2. Transform and select for appropriate construct using Kan/Gen plate. (Done?)(1 day + overnight)
  3. Liquid culture, miniprep and screen via restriction digest (EcoRI and SpeI), digest double terminator (P1 1G) simultaneously (EcoRI and XbaI) (overnight + 1 day)
  4. Make glycerol stock of RBS-LacZ containing cells. (same day)
  5. Ligate the RBS-LacZ construct into double terminator plasmid. (same day)
  6. Transform ligation and select for construct using Gen/Amp plate. (1 day + overnight)
  7. Liquid culture, miniprep and confirm using EcoRI/Pst digest (overnight + 1 day)
  8. prepare glycerol stock (same day)

This construct can then be ligated to appropriate promoters for use as a test harness with the substrate 3,4-cyclohexenoesculetin--D-galactopyranoside (S-Gal). For information on conditions see supplementary info from the following paper:

Nature 438, 441-442 (24 November 2005) Synthetic biology: Engineering Escherichia coli to see light Anselm Levskaya1, Aaron A. Chevalier2, Jeffrey J. Tabor2, Zachary Booth Simpson2, Laura A. Lavery2, Matthew Levy2, Eric A. Davidson2, Alexander Scouras2, Andrew D. Ellington2,3, Edward M. Marcotte2,3 and Christopher A. Voigt1,4,5

Test harnesses needed:

  • OmpR promoter: Ligate to promoter (3 days) and express in Envz+ cells under osmotic stress (time?)
  • Red light system: Above construct in Envz- cells with red light system
  • Blue light system: Ligate to Promoter responding to ComA (3 days), in Envz- cells constitutively expressing ComA and Blue light receptors (time?)
  • AND gate: Use above construct in Envz- cells constitutively expressing Blue light receptor and Red light receptor with ComA expression under the control of OmpR promoter. (time?)