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Every research project has its bugs. The exponential phase dependent wintergreen odor generator produced methyl salicylate roughly linearly with respect to the density of cells in culture. This matched the trend of the constitutive promoter, meaning that the exponential phase dependent control device failed to limit isoamyl acetate production to exponential phase.
Bacterial Odor Generators
Now that their odor generators were working as expected, the team needed to replace the constitutive promoters with promoters only active during the appropriate phase of cell growth. Recall that the team wanted to design a system that produces wintergreen odor during exponential phase and banana odor during stationary phase. To accomplish this they fitted their odor generators with growth phase dependent transcriptional devices. For the banana odor generator they used the promoter pOsmY which is switched on during the high osmotic pressure present during stationary phase. Thus, ATF1 and isoamyl acetate are only produced during stationary phase. Their solution for the wintergreen odor generator was a bit different. They again used the pOsmY promoter, but they also added a transcriptional inverter. The inverter reverses the signal of the pOsmY promoter, so that BSMT1 is produced when the pOsmY promoter is turned off during the low osmotic pressure of exponential phase and not produced when the pOsmY promoter is turned on during stationary phase.
Image Credit: Benjah bmm27 and Mschel (via Wikimedia)
My last post described composite parts and gave a couple brief examples. Now I would like to talk in depth about a cool example of composite parts: bacterial odor generators. When I say bacterial odor generators, I mean a system that produces an odorant compound not naturally produced by E. coli. After all, E. coli doesn't need to be programmed to smell like feces it already does! This post is first and foremost a description of the Eau d'E. coli project done by Reshma Shetty and the 2006 MIT iGEM team. I hope you will enjoy this opportunity to learn about a real and interesting research project in this field.
References and Further Reading:
When their systems were fully assembled, the team grew two separate cultures. One culture contained the cells with the wintergreen odor generator as well as a supplement of salicylic acid. The second contained a supplement of isoamyl alcohol and the cells with the banana odor generator. Human subjects were generally able to distinguish which culture was generating which odorant, and the team confirmed that the appropriate odorant was produced using gas chromatography.
The Eau d'E. coli project aimed to program E. coli to smell like wintergreen during the exponential phase of bacterial growth, when nutrients are plentiful in culture and cells divide exponentially, and like bananas during the stationary phase of growth when nutrients begin to run out and growth slows. Accomplishing this task required several intermediate steps. First the team needed to choose a strain of E. coli lacking its native fecal odor so that their odor generators would be detectable. The team chose the strain YYC912, which has a mutation in the tnaA gene responsible for producing the foul smelling molecule indole.
Registry of Standard Biological Parts.
Using their odor free strain, the team began designing and building their odor generators. The odorants responsible for wintergreen and banana odor are methyl salicylate and isoamyl acetate, respectively. The team chose the enzyme BSMT1 to generate methyl salicylate for their wintergreen odor generator. BSMT1 converts salicylic acid, which was added to the bacterial cultures, to methyl salicylate. They chose the enzyme ATF1 for their banana odor Cheap Women Canada Goose Thompson Jacket Black Ireland generator. ATF1 converts isoamyl alcohol added to cultures to isoamyl acetate, or banana odor. The team assembled their odor generators using BioBrick parts. They chose an existing promoter ("switch") from the registry to control the production of the odorants. In this early stage they chose a constitutive promoter, meaning that the "switch" is always on and the odorants are continuously produced. Since no BSMT1 or ATF1 BioBricks existed, the team had to modify existing genes coding for the two enzymes into BioBrick parts adhering to the BioBrick standard.
Shetty, R. P. Applying Engineering Principles to the Design and Construction of Transcriptional Devices. Department of Biological Engineering, MIT (2008).
MIT 2006 iGEM Team. MIT 2006 iGEM Wiki.
This is a great example of synthetic biology at work. Engineering bacterial odor seems like a frivolous pursuit, but there are a number of important direct and indirect applications. In her dissertation, Dr. Shetty pointed out that odor engineering could be combined with off gas sampling of bioreactors (which is already common practice) to gain information about bacterial metabolism in an industrial setting very quickly. In a more indirect sense, engineering whole metabolic pathways will certainly be an important component of the anticipated synthetic biology revolution in this century. Programming bacteria to generate useful compounds such as bioplastics often requires extensive metabolic engineering and at the moment remains a tricky business.