1 / 15

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. 

nowles
Télécharger la présentation

iGEM Bioware

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. iGEM Bioware Week 9

  2. 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. 

  3. ArsB LamB F2R 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 ArsR pArsR F1R1 pArsR F1R2 pRpoS 232 LamB F1R ArsR  Fusion pRpoS 438 ~500 bp pArsR F2R1 pArsR F2R2 (-)

  4. ArsR 1 ArsR 2 (-) 1 kb ladder Expected Size: ArsR insert: ~600 bp 500 bp

  5. ArsR in Backbone Transformation efficiency highly improved by using NEB cells instead of our own competent cells

  6. 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

  7. Gel of PCR Expected Sizes: golT = 2300bp golB = 200bp golS = 500bp gesABC = 6000bp

  8. 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

  9. Digestions - XbaI + SpeI

  10. 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

  11. 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

More Related