IGEM:MIT/2006/Notebook/2006-7-11

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Contents

Projects

  1. E. coli lab strain
    • We want to engineer a pleasant perfuming E. coli system that (with no exogenous action or input) can grow and smell when it reaches stationary phase
      • all substrates will be biosynthesized by cells
      • stationary phase controls gene expression
      • pleasant smell does not need to compete with indole's unpleasant smell
  2. Methylobacteria and/or Pseudomonas fluorescens biofilter
    • combine our perfuming power into bacteria that already mop-up smelly compounds
    • colonize a biofilter with our engineered bacterial system

Project 1: E. coli, Progress

  1. Biobricks of SAMT, BAMT, BSMT, ATF1 in sequencing process
  2. 1 smell is detectable/GC quantifiable in SAMT and BSMT cell cultures
  3. pUC18-derivative plasmid for SA biosynthesis via pchBA is definitely coming soon
  4. 1 indole knock-out strain has been recieved
  5. Stationary phase promoter primers have been ordered

Project 1: E. coli, To Do

  1. Order Isoamyl Acetate to smell [VV] -done
  2. Order Isoamyl Acetate primers for 2 genes [VV] '-1 gene done
  3. Check that coding mutagenesis reactions were successful (check gel, do transformation...tomorrow: miniprep, digest, gel) [AG/KB, everyone]; nanodrop [SP]
  4. Hook up each biobrick coding region to a Rbs, Promotor, and Terminator [tomorrow]
  5. PCR out the stationary phase promoter from E. coli genome once primers arrive [SP] -done
  6. Make a plate of our new indole-negative E. coli strain YYC912 [KB] -done
  7. Make a liquid culture of our new indole-negative E. coli strain YYC912 [tomorrow] -done
  8. Set up an overnight culture as a GC control for tomorrow [VV] -done
    • Does it smell noticably different from regular indole-positive E. coli?? YES, -done
    • If yes, (make competent?) and transform with Dudareva SAMT plasmid and make GC measurements of methyl salicylate
    • transform with our biobrick plasmids
  9. Continue email follow-ups, etc. [KB]

Project 2: Methylobacteria/Pseudomonas, To Do

  1. get pWUBR (or something similar)
    • PWUBR is an E. coli to Methylobacteria shuttle vector
    • email J. Hubacek and T. Leisinger
    • search for more sources of a similar vector
  2. order an appropriate methylobacterium strain for lab work
  3. learn everything about working with/growing methylobacteria
  4. research biofilters and construction
  5. get pUCP22 (or something similar)
  6. pUCP22 is an E. coli to Pseudomonas shuttle vector
    • email Herbert Schweizer
    • search for more sources of a similar vector
  7. learn everything about working with/growing pseudomonas

Hey Kate...these are the papers that I found and started looking at:[1, 2]. [2] inidcates that they used conjugation to transfer their plasmids from E. coli to M. dicloromethanicum. The plasmid--S17-1--seems to be a special mobilization system. I am reading more on this.

Update: OK. So it seems that several studies have been done in which this E. coli S17-1 mobilization plasmid was used [2, 3, 4], the majority of which were done by the Leisinger group in Switzerland. Leisinger et. al cite this reference each time they discuss conjugal transfer of the S17-1 plasmid from E. coli to Methylobacterium in their methods.

Update2: Paper [5] looks interesting.

  1. Kayser MF, Ucurum Z, and Vuilleumier S. . pmid:12055310. PubMed HubMed [yes]
  2. Brenner V, Holubová I, Benada O, and Hubácek J. . pmid:1796807. PubMed HubMed [yes2]
  3. La Roche SD and Leisinger T. . pmid:1938878. PubMed HubMed [yes3]
  4. Vannelli T, Studer A, Kertesz M, and Leisinger T. . pmid:9572975. PubMed HubMed [yes4]
  5. Marx CJ and Lidstrom ME. . pmid:11495985. PubMed HubMed [yes5]
All Medline abstracts: PubMed HubMed

Wet Lab Work Completed Today

1. DpnI digest of site-mutagenized BAMT and ATF1

2. Gel of site-mutagenized BAMT and ATF1

3. PCR cleanup of site-mutagenized BAMT and ATF1

4. PCRed osmY out of E. coli genome

Protocol: 45 uL PCR Master Mix

2.5 uL E. coli genome (~100 copies)

1.25 uL each primer (25 uM)

The steps were:

(a) 94deg for :30 (b) 55deg for :30 (c)68deg for 2:15, then 72deg for 10:00

5. Transformed site-mutagenized BAMT and ATF1 into competent cells

Vanillin?

  1. Overhage J, Steinbüchel A, and Priefert H. . pmid:14602615. PubMed HubMed [Overhage03]
  1. Plaggenborg R, Overhage J, Steinbüchel A, and Priefert H. . pmid:12764569. PubMed HubMed [Plaggenborg03]

http://www.scielo.br/scielo.php?pid=S1517-83822003000500037&script=sci_arttext

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