to integrate promoter-gw-lux fragment onto chromosome of e.coli
TRANSCRIPT
To integrate promoter-GW-lux fragment onto chromosome of E.coli
Purpose: To alleviate the problem associated with variable copy no. of plasmid carrying the
promoter GW-lux fragment. This will in turn provide robust results in promoter assays
Urja BhattLovett Lab, Jan-Jun 2016
Brandeis University
PCR to amplify the GW-lux fragment from pDEW201GW plasmid with Forward primer (Pac1 site) and Reverse Primer (Xho1 site)
Taq Polymerase
Amplified GW-lux fragment & Disintegration of pDEW201GWlux
Digestion of destination vector (pGRG25) and GW-lux fragment with Pac1 and Xho1 and Ligation of GW-lux fragment to the cut vector
Resultant pGRG25-pDEW201GWlux plasmid
Electroporation of pGRG25-
pDEW201GWlux into E.coli
Plating the transformants onto selective
agar plates
1
Extraction of plasmid DNA from colonies on selective plate
2
Choosing Restriction sites to confirm identity of plasmid
MW: 2-Log DNA Ladder
1: pDEW201GW-NdeI
2: pDEW201GW-SwaI
3: pGRG25-pDEW201GWlux-NdeI
4: pGRG25-pDEW201GWlux-SwaI
5: pGRG25-NdeI 6: pGRG25-SwaI 7: 2-Log DNA Ladder
Restriction Digestion Confirmation of pGRG25-pDEW201GWlux from transformants
Sequence confirmation of plasmid from transformants
Using Gateway Technology to integrate desired promoter sequence onto pGRG25-pDEW201GWlux
Integration of iraD promoter sequence onto pGRG25-Gwlux to form pGRG25GWlux-piraD plasmid
E.coli
GenomePlasmid
Transposition of iraD promoter-GWlux fragment onto chromosome of E.coli
Integration of iraD promoter-GWlux fragment onto chromosome of E.coli
E.coli
Test the activity of piraD promoter in different strain
backgrounds and in presence or absence of DNA damaging agent using elaborate promoter assay
References
1. http://www.bio.miami.edu/dana/250/25008_7.html
2. http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/ch10s05.html
3. SnapGene Software for creating Graphics