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

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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. Dichloromethane metabolism and C1 utilization genes in Methylobacterium strains. Microbiology (Reading). 2002 Jun;148(Pt 6):1915-1922. DOI:10.1099/00221287-148-6-1915 | PubMed ID:12055310 | HubMed [yes]
  2. Brenner V, Holubová I, Benada O, and Hubácek J. Characterization of new plasmids from methylotrophic bacteria. Antonie Van Leeuwenhoek. 1991 Jul;60(1):43-8. DOI:10.1007/BF00580440 | PubMed ID:1796807 | HubMed [yes2]
  3. La Roche SD and Leisinger T. Identification of dcmR, the regulatory gene governing expression of dichloromethane dehalogenase in Methylobacterium sp. strain DM4. J Bacteriol. 1991 Nov;173(21):6714-21. DOI:10.1128/jb.173.21.6714-6721.1991 | PubMed ID:1938878 | HubMed [yes3]
  4. Vannelli T, Studer A, Kertesz M, and Leisinger T. Chloromethane Metabolism by Methylobacterium sp. Strain CM4. Appl Environ Microbiol. 1998 May 1;64(5):1933-6. DOI:10.1128/AEM.64.5.1933-1936.1998 | PubMed ID:9572975 | HubMed [yes4]
  5. Marx CJ and Lidstrom ME. Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria. Microbiology (Reading). 2001 Aug;147(Pt 8):2065-2075. DOI:10.1099/00221287-147-8-2065 | PubMed ID:11495985 | 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. Highly efficient biotransformation of eugenol to ferulic acid and further conversion to vanillin in recombinant strains of Escherichia coli. Appl Environ Microbiol. 2003 Nov;69(11):6569-76. DOI:10.1128/AEM.69.11.6569-6576.2003 | PubMed ID:14602615 | HubMed [Overhage03]
  1. Plaggenborg R, Overhage J, Steinbüchel A, and Priefert H. Functional analyses of genes involved in the metabolism of ferulic acid in Pseudomonas putida KT2440. Appl Microbiol Biotechnol. 2003 Jun;61(5-6):528-35. DOI:10.1007/s00253-003-1260-4 | PubMed ID:12764569 | HubMed [Plaggenborg03]

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

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