IGEM:MIT/2006/Notebook/2006-6-14

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06/14/06 Lab

  1. Made 2 glycerols of each of 9 liquid cultures (18 total) from 6-13-06 (BAMT,SAMT,BSMT [A,B,C])
    • Added one ml of culture to each 1ml tube of glycerol
  2. Made 9 minipreps of the 9 (BAMT,SAMT,BSMT [A,B,C]) (method outlined 6-13-06)
    • note: while centrifuging (after adding N3), the lid of the tube containing the lysed BAMT-A cells fell off and the tub got stuck in the centrifuge. We pulled it out with tweezers and kept going, but if something goes wrong with this sample later, this might be why...
  3. Prepared DNA samples for sequencing
    • 12 μL sample consisted of 3.2 pmol of primer [either T7 (.2 μL of 100ng/μL) or TφR (1 μL of 4 μM)] and ~300ng of plasmid DNA.
  4. Ran experiment to determine if the cells that grew w/ BAMT last night will express the enzyme and produce methyl benzoate. (we also added SA to see if there were some SAMT cells in the culture)
Control (45mL LB + 5 mL BL21 cells from Reshma) BAMT (45mL LB w/ KAN + 5 mL BAMT cells from culture that grew last night)
** not induced
induced induced
** 1.5 μL SA, not induced
1.5 μL SA, induced 1.5 μL SA, induced
** 2 μL BA, not induced
2 μL BA, induced 2 μL BA, induced
** 400 μL BA, induced [OD600 = .76 when we added IPTG]
** 400 μL SA, induced [OD600 = .62 when we added IPTG]

To do

  • I've contacted User:Curt about use of GC. -Austin
  • Project summary?

Notes

Resources on gas chromatography-olfactometry

  1. EtiƩvant PX, Callement G, Langlois D, Issanchou S, and Coquibus N. . pmid:10564037. PubMed HubMed [Etievant99]
  2. van Ruth SM. . pmid:11377272. PubMed HubMed [Ruth01]
All Medline abstracts: PubMed HubMed

Applications Research

Light Sensor

The light sensing device is a combination of four parts, I15008, I15009, I15010, and R0082. The device is on the featured parts page below:

http://parts.mit.edu/registry/index.php/Featured_Parts:Light_Sensor

Also in the registry is M30109, which would make up strictly the light sensing device of the system. However, no physical DNA is listed as being present in this part's entry in the registry. The part description is below:

<bbpart>BBa_M30109</bbpart>

  • RS 09:09, 14 June 2006 (EDT): Natalie got this part synthesized by DNA2.0. It probably hasn't been submitted to the registry yet. She'd likely be happy to give it to us. Note that I think this part has an additional inverter in it. It is not clear if this inverter works.

Heat Sensor

There are two kinds of heat-sensitive promoters that are well described in openwetware: cold shock and heat shock promoters. Here is a description of some of them characterized by the UCSF team last year:

"Only four promoters (barely) survived the screen. By far, the best promoter is hybB, which controlls the hydrogenase II operon. It is clearly active at temperatures lower than 30oC and is off at temperatures higher than 30oC. Two other cold-shock promoters also showed a T-dependent response: ansB and cspA_x. AnsB controls an asparginase operon. CspA is part of the classical cold shock response. It's mRNA has a toxic leader, which is also supposed to participate in adaptation. We removed this sequence, which we denote with an 'x.' Finally, we have had mixed success with the heat-shock htpG promoter, which is part of the classical heat-shock response. It is known to produce a pulse first, but it is unique in that it (in the literature) comes to a T-dependent steady-state."

Although I did not find which parts they are in the registry, I am fairly certain that the parts are in the registry. This device would probably be the easiest to get working since the each device (cold sensor and hot sensor) merely consist of one part, a promoter.

  • Austin 10:20, 14 June 2006 (EDT): As of the end of Fall 2005, these parts were not in the registry (the Harvard MCB100 class wanted to use them). I asked Chris Voigt and he seemed to indicate they wouldn't be put in and we should synthesize them (they are really short anyway).

Quorum Sensor

F1610 coupled with F2621 or F2622 can act as a quorum-sensing device. I do not believe that this is the easiest way to develop the quorum sensing system, but it is well-characterized. I thought that there were quorum promoters which would make the construction of such a system easier, but I am not sure.

  • BC 16:33, 14 June 2006 (EDT): To the best of my knowledge, no one has found a quorum sensing system in E. coli so these quorum sensing systems seem to be the best currently available and Ron Weiss from Princeton has used them to good effect.
  1. Basu S, Gerchman Y, Collins CH, Arnold FH, and Weiss R. . pmid:15858574. PubMed HubMed [Weiss]

Write-Up

Summary

Looking back on the project outline on 6/5/06, we are on track in testing out the different scent systems. There are currently a number of items to troubleshoot. Some issues include the strength of the "natural" bacterial smell, lack of supposed scent, and the poor growth of BSMT colonies. While we are still exploring possiblities, we want to pin down a [number of] route(s) for applications within the coming week.

Current Progress

  1. Tested out smell system with BAMT and BA against control
    • Seems like the indole is a bit overpowering
  2. Prep work
    • Designed and ordered primers
    • Sequencing the current cultures

Projected Summer Timeline

June

  • Get BAMT/SAMT/BSMT systems (BSB from now on?) working
  • Suppress indole
  • List/determine applications to pursue

July

  • Design/construct the appropriate controls
  • Test the controls
  • Incorporate controls with the scent systems

August

  • Implement the entire systems

Sept-Nov

  • Continue to build/refine final application(s)
  • Look into tweaking certain details (biochem pathways etc.)
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