IGEM:MIT/2006/Notebook/2006-6-15

Progress from 6/14/06

1. Sequencing results came back- I checked BAMT-A-T7 and it matched strongly with snapdragon BAMT. Cool!

Lab 6/15/06

1. Created fresh cultures for smell test

Control (49mL LB + 1 mL BL21 cells from Reshma)	BAMT (49mL LB w/ KAN + 1 mL BAMT cells from culture that grew last night)
induced at start	induced at start
**	200 μL SA, induced at start
**	200 μL BA, induced at start

1. Started VENT PCR for ATF1
   • Materials
     • 5 μL ThermoPol buffer, 0.5 μL 100mM dNTP, 0.5 μL VENT polymerase, 39 μL water (6x for master mix)
     • 1 μL template, 2 μL 25 μM primer1 (same for primer2)
   • Controls
     • Negative: no template (-t); no primer (-p)
     • Positive:same template (+t); new primer, new template (+pt)
   • Used PCR settings detailed on machine as IGEMPCR

Indigo idea (KB)

okay, so I want to know what people think about oxidizing indole into indigo with hopes that our new good smell compounds will not be so overpowered by the bad fecal smell of indole

genbank:AF306552

here's some initial links:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1372930&dopt=Abstract

http://www.science.siu.edu/microbiology/micr421/chapter12.html

http://aem.asm.org/cgi/content/abstract/63/11/4287

here are some papers that might be of interest (The Bhushan one seems cool):

1. Drewlo S, Brämer CO, Madkour M, Mayer F, and Steinbüchel A. Cloning and expression of a Ralstonia eutropha HF39 gene mediating indigo formation in Escherichia coli. Appl Environ Microbiol. 2001 Apr;67(4):1964-9. DOI:10.1128/AEM.67.4.1964-1969.2001 |
2. Hart S, Koch KR, and Woods DR. Identification of indigo-related pigments produced by Escherichia coli containing a cloned Rhodococcus gene. J Gen Microbiol. 1992 Jan;138(1):211-6. DOI:10.1099/00221287-138-1-211 |
3. Bhushan B, Samanta SK, and Jain RK. Indigo production by naphthalene-degrading bacteria. Lett Appl Microbiol. 2000 Jul;31(1):5-9. DOI:10.1046/j.1472-765x.2000.00754.x |

• Austin 11:25, 15 June 2006 (EDT): According to this http://www.fullpassport.com/Trip2001/Diary/dec-13.html the indigo smell supposedly repels mosquitos and might repel people. Don't know what the specific smell is.
• RS 11:48, 15 June 2006 (EDT): There's some talk of it having anti-bacterial properties on this forum. No idea if this is true or how much indigo must be around to be toxic. But Kate already found the enzyme expressed in bacteria right? So it can't be too bad.

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.

4. Basu S, Gerchman Y, Collins CH, Arnold FH, and Weiss R. A synthetic multicellular system for programmed pattern formation. Nature. 2005 Apr 28;434(7037):1130-4. DOI:10.1038/nature03461 |

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
   • New Idea for indole: Lets try oxidizing it into indigo? gene for reacition is in GenBank, has been done and documented in numerous papers, might eliminate fecal smell of indole, we would get fancy blue bacteria as an added bonus!!
   • What about seeing if we can get smell from plated cultures rather than liquid cultures? or try minimal media?
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
• Create BioBricks from the genes that we have received.

July

• Design/construct the appropriate controls
  • Substrate concentration, media type, etc.
• 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.)

To do

• Start fresh cultures for smell test
  • dilute back further this time
  • Perhaps we can cut back on the number of controls? This step is just to check for smell.
• Try hexane extraction?
• If primers come in, we can do a PCR
• 4pm "big picture" meeting with all advisors

Meeting Notes [and what needs to be done!]

Meeting Summary

We summarized our work to date, and discussed the direction that our research should take in the coming weeks / months. Some good questions were asked...

• Do we want to try minimal media instead of LB to cut down on the background stench? (we could also limit tryptophan present in the media; perhaps this would cut down on the indole?)
• We could try adding methyl benzoate to the culture to see how much we'll need to detect a pleasant scent. Is the concentration we'll need toxic??
• Are we still looking into biosynthesis of precursors? If not...why not?
• Can we inhibit the enzymes that cause indole production?
• Why do E. coli smell bad?
• Would expressing the enzymes in yeast would be more effective?
• How can we split up the project to make work more efficient?
• Can we make a "projects page" on the wiki that provides a project overview (which contains links to more detailed descriptions)?

Work to be done in general!!

1. Determine toxicity of precursors (benzoic acid, salicylic acid, isoamyl alcohol) and products (methyl benzoate, methyl salicylate, isoamyl acetate) [Determining the "smellable" concentration of product goes along with this]
2. Try using minimal media instead of L.B., and try plating cultures.
3. Do further research into what smells bad in E. coli, and if it's indole, look into the elimination of indole.
4. Take a second look at biosynthesis of precursors!
5. Fix the wiki and figure out gas chromotography.
6. Get DNA synthesized for heat promoter, and think about more applications
7. Transform our plasmids into yeast

Work to be done tomorrow!!

1. PCR biobricks [SAMT, BAMT, BSMT...the sequences from the plasmids that were transformed into Top10 cells came out OK]
2. Mutagenesis PCR for ATF1
3. Transform plasmids isolated from Top10 cells into BL21 competent cells (and make our own BL21 competent cells)
4. If we get methyl benzoate or methyl salicylate, try to determine the concentration necessary to detect a scent.