To integrate promoter-GW-lux fragment onto chromosome of E.coli
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Transcript of To integrate promoter-GW-lux fragment onto chromosome of E.coli
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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
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PCR to amplify the GW-lux fragment from pDEW201GW plasmid with Forward primer (Pac1 site) and Reverse Primer (Xho1 site)
Taq Polymerase
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Amplified GW-lux fragment & Disintegration of pDEW201GWlux
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Digestion of destination vector (pGRG25) and GW-lux fragment with Pac1 and Xho1 and Ligation of GW-lux fragment to the cut vector
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Resultant pGRG25-pDEW201GWlux plasmid
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Electroporation of pGRG25-
pDEW201GWlux into E.coli
Plating the transformants onto selective
agar plates
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Extraction of plasmid DNA from colonies on selective plate
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Choosing Restriction sites to confirm identity of plasmid
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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
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Sequence confirmation of plasmid from transformants
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Using Gateway Technology to integrate desired promoter sequence onto pGRG25-pDEW201GWlux
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Integration of iraD promoter sequence onto pGRG25-Gwlux to form pGRG25GWlux-piraD plasmid
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E.coli
GenomePlasmid
Transposition of iraD promoter-GWlux fragment onto chromosome of E.coli
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Integration of iraD promoter-GWlux fragment onto chromosome of E.coli
E.coli
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Test the activity of piraD promoter in different strain
backgrounds and in presence or absence of DNA damaging agent using elaborate promoter assay
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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