Team:Brown/Notebook Meetings/6-22-09


Advising Meeting Gary, Adrian, Diana

June 22, 2009

SFH 218

  • 9:12 AM: Meeting starts
  • 9:12: Group 1 progress. Received and purified the DNA in pBluescript plasmid. Now trying to move to expression vector. Indu got the endonucleases and cut the DNA out. Will run gel and isolate today.
  • 9:15: Gary suggests to continue digest and send away for sequence. Use same primers as promoter regions to sequence.
  • 9:17: For sequencing, 5:00PM is the last time here, 6:00 at Kinko’s, 7:00 downtown. Sp6, t7, t3 are the three main primers used. Gary will meet with us later to show how sequencing samples can be sent.
  • 9:21: There is a little extra fat on the oblique’s of the protein, we will have to get rid of this later.
  • 9:23: Discussed how to get rid of creature growing in the oven. Definitely autoclave biohazard materials. After autoclaving, spray with ethanol.
  • 9:27: Looking ahead for group 1, after confirming sequence, we plan to put the DNA segment into pNotat. Gary will give us the cells. Always good to keep a stock, Gary will help us on protocol for freezing cells.
  • 9:29: Eventually, will test the protein binding with a nickel column. 6-His tag binds to nickel column. Goal for next week: Digest for HBP, sequence back, made primers, amplified sequence of interest, putting it into pGem (allows for easy cloning of PCR products).
  • 9:32: Also when ordering primers, order the biobrick primers.
  • 9:33: Group 2 progress. From team secretion, switching for S. aureus to S. epidermidis. Call Chris Harwood for his approval before ordering S. epidermidis.
  • 9:35: Looking ahead, when putting on secretion sequence – Gunnar suggested putting it a few amino acids downstream. How should we do this?
  • 9:37: Use ß-lactamase to test secretion. Measure both how much is in the cells and how much is in the media with an immunoblot.
  • 9:40: Also make PCR primers for quorum sensing gene, hooking up to ccdb gene. This enables cell death to initiate once population reaching a certain point. Eli explains the Quorum sensing cascade. Diana has a concern that at a low level ccdb could be produced, killing the cell when we don’t want to.
  • 9:44: Major goals, ordering S. epidermidis, isolate DNA. Concern – if you’re never turning on the kill switch, there is no pressure to keep it accurate, high mutation rate. Make signal peptide via Geneart. Ligate the signal peptide to GFP. Place into S. epi.
  • 9:49: Adrian points out that Geneart might take a long time. Gary suggests PCRing another secretion signal of a gene that is already in the cell. Look for labs to get DNA.
  • 9:52: Group 3 progress. The Collins lab is sending us the pIKE107 plasmid and maybe the bacterial strain (stab agar).
  • 9:54: Once we transform pIKE107 into E. Coli (w LacI knockout), test it like in the Collins paper (in hopes of generating figures), and finally replace GFP with EV131. Ultimate goal is to place cassette in new organism.
  • 9:58: To Biobrick: most basic functional unit (i.e. promoter), and the final cassette.
  • 9:59: We have to study how to do FACS, and obtaining a strain of E.coli with lacI KO.
  • 10:04: Is there a concern with replicating exactly what Collins did in his paper and biobricking those parts? We should go and visit BU.
  • 10:06: Need to order IPTG and aTc. (Diana may have a stock of a tetracycline analog).
  • 10:09: Test how change in unit size (if we want multiple HBPs) will affect the bistable switch mechanics. Modeling this would be useful.
  • 10:10: Adrian asks: Codon table shifts in ticks or S. aureus? (ex: is AUU Methionine, or is certain tRNA in greater quantities in one type?) Could add tRNAs or alter the sequence of the protein to use the abundant tRNAs.
  • 10:12: Consult the clinical trial and figure out how they got that much EV131.
  • 10:13: John Cumbers is coming by next week, we should definitely plan a meeting with him. Gary will schedule something.
  • 10:15: End of meeting. Go to work.