Team:Caltech/Week 14

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Caltech iGEM 2011

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September 12

Miniprep of WT-F87A

Results

Miniprep concentrations:

Miniprep	Nanodrop conc. (ng/uL)
WT-F87A	118

September 13

Overexpression of his-tagged DDT-dechlorinase in pET protein expression vector in preparation for purification, at 20&degC and 37&degC
Quikchange of Wt-F87A with newly miniprepped DNA
Plates of results, + the rest of SOC

September 14

Protein purification of DDT-dechlorinase using Sigma-aldrich his-select protein purification columns
SDS gradient gel of purified products, as well as pellet, first flow-through, and first wash

Results

No visible colonies on plates

September 15

Finish staining SDS gel
Retransform quikchange pcr products
Make and autoclave M9 solution
Set up biofilm experiments using different substrates (glass beads vs. wood chips), media (dilute LB vs. M9), and conditions (20&degC vs. 37&deg C vs. shaking)

Results

eluted DDT dechlorinase band clearly is visible, size according to broad range ladder

September 16

Made desalting buffer (20mM Tris-HCl + 150mM NaCl)
Desalt DDT-dechlorinase buffer of imidazole to stabilize protein through dialysis
Made X-gal-IPTG plates for quikchange lacZ repair control