Team:CIDEB-UANL Mexico/project aroma

Since the beginning of iGEM project, the use of fluorescent reporters has been used in each one of the proposed projects in previous years, trying to test the theoretical presence of other proteins in E. coli. For our iGEM 2014 project, this module proposed to promote the usage of aroma reporters, instead of fluorescent ones.

Description

In the module, WinterGreen, the coding region, is the most important part. Normally, it catalyzes the conversion of salicylic acid into methyl salicylate. The protein is excreted by the bacteria and when salicylic acid is added to the medium, a chemical reaction takes place and produces methyl salicylate, which is responsible for the wintergreen odor.

But in this module, it will be regulated by temperature with the use of the RNA thermometer. When adding salicylic acid to the bacteria in a 32° Celsius environment, the production of the WinterGreen protein will begin.

Device

Originally, WinterGreen was thought to be the reporter of the Capture module, but in order to prove the function of this gene and also the one in charge of the capture of sodium ions (NhaS), we decided to separate the full device into the actual modules of Capture and Aroma, as seen in the figure below.

This device is composed by the following parts (see figure 3): (1) a constitutive promoter, (2) a ribo-switch, also called RNA thermometer; used to regulate the WinterGreen-odor protein production through temperature, (3) a Wintergreen-odor enzyme generator, used to allow the production of methyl salicylate, induced by salicylic acid, and (4) a terminator. All of these parts are ligated by an 8-bp scar (TACTAGAG).

Figure 3. Aroma Module

These 4 genetic parts form the Aroma device of the project. The full device's length is 1,251bp (including restriction sites).

Parts of the module

Justification

Different to fluorescent reporters, this module was made in order to (in the future) perform as an aroma reporter and also to test the correct function of the bacteria, for its future usage as a new reporter and functional part (CDS). It is desired to use this part in the project to replace the red fluorescent protein (RFP) in the Capture module. But it was preferable to test it apart to demonstrate its effectiveness. Similarly, this piece is also helpful when the biofilter is assembled, because when performing the filtration by silica, WinterGreen can demonstrate the presence of bacteria in the beads.

The team added the RNA thermometer in the device for regulating the production of the aroma. Another reason for selecting the RNA thermometer as a regulator was to continue the CIDEB UANL 2013 work with it.

Other teams that used RNA thermometer and BSMT1

RNA thermometer

TUDelft 2008: Temperature-sensing bacteria that changescolor at different temperatures; as a temperature reporter system inlarge-scale fermentations, or as a temperature-inducible protein productionsystem.

Figure 4. RNA Thermometer circuit, excerpted from TUDelft 2008 team

VictoriaBC 2009: NAND logic gate using the ribo-key/ribo-lock system designed by Berkeley 2006 team , producing RFP except when the cells are grown in the presence of both arabinose and IPTG, also coupling fluorescent outputs with the ribo-thermometers made by TUDelft 2008 team.

iGEM_CIDEB 2013: Production of Vip3ca3, which acts as a pesticide protein, regulated by specific temperatures in order to avoid overproduction and it will show activity against target organisms Coleoptera and Lepidoptera.

Wintergreen

MIT 2006: This device produces methyl salicylate in the presence of salicylic acid. Methyl salicylate smells strongly of mint (wintergreen). Production of methyl salicylate was verified both by scent and by gas chromatography: E. coli with no WGD did not produce methyl salicylate when SA was added to the medium, while E. coli with the WGD did produce methyl salicylate when SA was added to the medium.

Figure 6.Wintergreen odor enzyme (BSMT1) generator circuit by MIT 2006

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Bibliography/References