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 bp pRpoS232: 232 bp ArsB: 1290 bp LamBF1R: ~1.3 kb LamBF2R: ~1.3 kb ArsR fusion: ~350 bp ArsR: 354 bp pArsRF1R1: 540 bp pArsRF1R2: 547 bp pArsRF2R1: 293 bp pArsRF2R2: 300 bp ArsB LamB F2R pRpoS 438 ArsR pArsR F1R1 pArsR F1R2 pRpoS 232 LamB F1R ArsR  Fusion ~500 bp pArsR F2R1 pArsR F2R2 (-)

(-) Expected Size: ArsR insert: ~600 bp ArsR 1 ArsR 2 1 kb ladder   ArsR 1 ArsR 2 (-) 1 kb ladder Expected Size: ArsR insert: ~600 bp 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 gesABC 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 going to transform and plate them Monday since we can't come in during the weekend

Gel of PCR Expected 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 We had good sales Wednesday despite the rain, selling 45 cups! Selling again this afternoon from 12:30-3:30 in front of the Union