Quantitate the Constitutive Expression Level of GFP fused to pBR322 P1 Promoter

 

The goal of this experiment is to measure in a reproducible fashion the constitutive level of GFP when expressed in E coli. strain   using the P1 promoter of the medium copy number plasmid pBR322. The experiment involves creating a new vector pBR322+GFP(LVA) by inserting the GFP(LVA) coding sequence from pGFP(LVA) into pBR322 at the EcoRI restriction site (downstream of the P1 promoter), transforming the E coli with the new vector, growing the transformed strain in a selective medium, staining the cells, and observing the luminosity of individual cells using a flow cytometer. The experiments in Sections 3.1, 3.2, and 3.3 can be performed in parallel, followed by experiment in Section 3.4 and finally Section 4.2.

 
• Extract GFP(LVA) insert
  • Plate and inoculate BL21+pGFP(LVA)
  • Transform    with pGFP(LVA)
  • Miniprep    to extract plasmid
  • Restriction digest of pGFP(LVA) with HindIII
  • Separate digested GFP(LVA) insert with QIAEX agarose gel extraction kit
  • Second Attempt: Restriction Digest of pGFP(LVA)
  • Second Attempt: Separate digested GFP(LVA) insert with QIAEX agarose gel extraction kit
• Create additional pBR322 vector
  • Inoculate and Grow  
  • Transform    with pBR322, plate, and grow
  • Inoculate    into LB Amp liquid and grow
  • Miniprep    to extract plasmid
  • Restriction digest of pBR322 with EcoRI
  • Analytical Gel to Check pBR322 digest
  • Second Attempt: Restriction Digest of pBR322
  • Second Attempt: Separate digested pBR322 insert with QIAEX agarose gel extraction kit
• Create EcoRI to HindIII adapters
• Transform and grow colonies of  
  • Ligation of GFP(LVA), pBR322, and HindIII-EcoRI Adapters
  • Electroporate pBR322+GFP(LVA) into  , and Inoculate