iGEM Bioware

iGEM Bioware

Week 9

Arsenic Bioremediation

Got ArsR into plasmid backbone and began site directed mutagenesis.   using NEB competent cells significantly improved efficiency.

PCR of ArsB and LamB is still not working--Any suggestions?  Both these proteins are vital to our project. 

Ordered new primers for RpoS promoter and ArsR Promoter with EcoRI cut site to increase yield and efficiency.

Also worked to get constitutive promoters in front of gas vesicles biobrick but getting lawns after transformations. 

Expected Sizes:

pRpoS438: 438 bppRpoS232: 232 bpArsB: 1290 bpLamBF1R: ~1.3 kbLamBF2R: ~1.3 kbArsR fusion: ~350 bpArsR: 354 bppArsRF1R1: 540 bppArsRF1R2: 547 bppArsRF2R1: 293 bppArsRF2R2: 300 bp

pRpoS438 pRpoS

232

ArsBLamBF1R

LamBF2R ArsR 

Fusion

ArsR pArsRF1R1

pArsRF1R2

pArsRF2R1

pArsRF2R2 (-)

~500 bp

Expected Size:ArsR insert: ~600 bp

ArsR1

ArsR21 kb

ladder(-)

500 bp

ArsR in Backbone

Transformation efficiency highly improved by using NEB cells instead of our own competent cells

Gold

Finally got golT out of Salmonella after using genomic prep kit

Still haven't been able to get gesABCo going to order primers for gesABC proteins individually

instead of trying to get the entire operon Did a digestion/ligation yesterday of golT, golB and golS

o going to transform and plate them Monday since we can't come in during the weekend

Gel of PCRExpected Sizes:

golT = 2300bp

golB = 200bp

golS = 500bp

gesABC = 6000bp

PCR - genomic and PCR purification template

We keep getting weird bands for gesABC and aren't getting the correct 6kb band, so we are going to order primers for ABC individually

Digestions - XbaI + SpeI

Decoder Update

Transformation efficiencies increased significantly using NEB 5a and Turbo electrocompetent cells.

Still searching for Gen5 software for HT Synergy Plate Reader

Matt is working on putting together a light cannon

Dippin' Dots Fundraiser

Selling again this afternoon from 12:30-3:30 in front of the Union

We had good sales Wednesday despite the rain, selling 45 cups!