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.

How is the Aroma module composed?

This gene is composed by the following parts (see figure 1A): (1) a constitutive promoter, (2) a RNA thermometer, also called ribo-switch; 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).

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 for Union module, because when performing the filtration by silica, WinterGreen can demonstrate the presence of bacteria in the beads. The team added the RNA thermometer for regulating the production of the aroma in the project. Another reason for selecting the RNA thermometer as a regulator was to continue the CIDEB UANL 2013 work with it.

How does it work?

This module has a constitutive promoter which 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.

Parts of the module

Full Device:

These 4 genetic parts form the Aroma device of the project (see Figure 3). The full device's length is 1,251bp (including restriction sites).

Other teams that use RNA thermometer and WinterGreen (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.

·           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 (BSMT1)

·          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.

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