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

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Projects

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
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

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

Project 1: E. coli, To Do

Order Isoamyl Acetate to smell [VV] -done
Order Isoamyl Acetate primers for 2 genes [VV] '-1 gene done
Check that coding mutagenesis reactions were successful (check gel, do transformation...tomorrow: miniprep, digest, gel) [AG/KB, everyone]; nanodrop [SP]
Hook up each biobrick coding region to a Rbs, Promotor, and Terminator [tomorrow]
PCR out the stationary phase promoter from E. coli genome once primers arrive [SP] -done
Make a plate of our new indole-negative E. coli strain YYC912 [KB] -done
Make a liquid culture of our new indole-negative E. coli strain YYC912 [tomorrow] -done
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
Continue email follow-ups, etc. [KB]

Project 2: Methylobacteria/Pseudomonas, To Do

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
order an appropriate methylobacterium strain for lab work
learn everything about working with/growing methylobacteria
research biofilters and construction
get pUCP22 (or something similar)
pUCP22 is an E. coli to Pseudomonas shuttle vector
- email Herbert Schweizer
- search for more sources of a similar vector
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.