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