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iGEM Summer Week 6  

iGEM Summer Week 6  . 7.2.2010. Overview. Arsenic System. Parts we have: lamB, Gas Vesicles, Constitutive Promoters Parts we need: Metallothionein, ArsR (transcription factor), ArsB (efflux) Plasmid Backbone:      Minipreped 4 plasmids each with RFP and a different resistance.

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iGEM Summer Week 6  

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  1. iGEM Summer Week 6   7.2.2010

  2. Overview

  3. Arsenic System Parts we have: lamB, Gas Vesicles, Constitutive Promoters Parts we need: Metallothionein, ArsR (transcription factor), ArsB (efflux) Plasmid Backbone:      Minipreped 4 plasmids each with RFP and a different resistance.     Digested each with E,P and X,S

  4. Gel of Digests 1kb 3kb 2kb 1.5kb 1kb 0.5kb

  5. Gel of Gas Vesicles, Promoters, and PAL (membrane protein) Ligation 1: J23100 + Gas Vesicles Ligation 2: J23119 + Gas Vesicles Ligation 3: PAL into pSB1T3 3kb 2kb 1.5kb 1kb 0.5kb *We shouldn't have used ~2,000ng of pSB2K3 for the gel.

  6. Gold We have: golB, golS We need: golT, gesABC and to synthesize the golS-recognized promoter We are still trying to PCR out golT and gesABC from salmonella • Salmonella enterica serovar Typhimurium 14028s We are also cloning golB and golS into backbones

  7. Decoder Last week we were able to clone out MicA, MicF, OmpA, OmpF, and Hfq. Our digestions/ligations have not been working though.  This last week we have been trying to clone them out again and it hasn't been working.  We currently are running tests on our digestion materials and PCR materials to make sure there isn't contamination and that everything works. 

  8. Lock and Key 1 2 3 4 5678 9 10                         1 2  3 4 5 6789 10 1    23   4   56 Red - Lock1 Black - RFP Purple - Key1Yellow - Lock3Orange - GFPGreen - No Template Black(Empty) - pBad/araC Double Digest/(1)Single Digest/(2)Single Digest/No Enzyme

  9. Contamination?pSB1C3 - Invitrogen Digest 1-1kb ladder 2-full digest 3-EcoRI digest 4-PstI digest 5-Template 6-No Template 7-1kb ladder 8-empty 9-empty 10-empty

  10. Miscellaneous Volleyball tournament - hopefully on July 17th Dip'n Dots worked well.  Promega is sending supplies and hopefully Midsci will too!

  11. Questions • Any recommendations for a stationary phase promoter?  • protein gel materials? • How should we test that our protein fusions are in the membrane? • How can we assay for the presence of metals?

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