Team:CIDEB-UANL Mexico/project aroma

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Description Capture Module Aroma Module Resistance Module Union Module Parts
iGEM CIDEB 2014 - Project

Aroma Module

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.


Figure 1. Production of Wintergreen Odor

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.

Figure 2. The device originally thought, and the 2 modules derived from it.


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


IMAGE

CODE

DESCRIPTION

 

BBa_J23119A

 

In the specific case of our aroma module, it will help the bacteria to continuously transcribe the WinterGreen gene in order to allow the bacteria to continuously produce the aroma. This promoter has a length of 35bp.

BBa_K115017

A RNA thermometer, used for temperature post-transcriptional regulation (thermo sensor), and is designed to initiate transcription around 32°C.

BBa_K1255001

 

Produces a transferase to convert salicylic acid into methyl salicylate (WinterGreen odor). The wintergreen odor generator requires of 2mM of salicylic acid to produce methyl salicylate. BSMT1 (BBa_J45004), WinterGreen Odor Generator original name, was created by MIT 2006. This year, the team optimized the sequence for Escherichia coli. The new biobrick has a length of 1,074bp.

BBa_B1002

 

Part made of 6bp, responsible for transcription stop. The terminator stops the production of methyl salicylate.


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

Team

Part Used

Use

TuDelft 2008 :

RNA themometer

 

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

 

ITB_Indonesia 2013 :

 

pUV and mCherry (BBa_J06702) to detect DNA damage.

 

Colombia_Israel 2007 :

 

Expression of the UV promoter in presence of UV irradiation light lead to the expression of the EYFP reporter.

Team
Part used
Description
TU Delft 2008RNA themometerTemperature-sensing bacteria that changescolor at different temperatures; as a temperature reporter system inlarge-scale fermentations, or as a temperature-inducible protein productionsystem.
Victoria BC 2009RNA themometerNAND 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.
CIDEB UANL 2013RNA themometerProduction 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.
MIT 2006BSMT1This 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.

Project Zoom in


Bibliography/References

● Huang, H. (2006, August 30). Part:BBa_B1002. Retrieved August 30, 2014, from http://parts.igem.org/wiki/index.php?title=Part:BBa_B1002.

● iGEM2006_Berkeley. (2006). Part:BBa_J23100. Retrieved August 30, 2014, from http://parts.igem.org/Part:BBa_J23100.

● iGEM2006_MIT. (2006). Part:BBa_J45004. Retrieved August 30, 2014, from http://parts.igem.org/Part:BBa_J45004.

● iGEM CIDEB Team. (2013). iGEM CIDEB UANL 2013. Retrieved on March 31th, 2014. https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/Project.

● iGEM08_TUDelft. (2008). Part:BBa_K115017. Retrieved August 30, 2014, from http://parts.igem.org/Part:BBa_K115017.

● MIT IGEM Team. (2006). MIT 2006. Retrieved on March 31th, 2014, from: https://2006.igem.org/wiki/index.php/MIT_2006.

● TUDelft iGEM Team. (2008). TUDelft 2008. Retrieved on March 31th, 2014, from: https://2008.igem.org/Team:TUDelft.

● VictoriaBC. (2009). VictoriaBC 2009. Retrieved on March 31th, 2014, from: https://2009.igem.org/Team:VictoriaBC.

● Zubieta, Chole et al. (2003). Structural Basis for Substrate Recognition in the Salicylic Acid Carboxyl Methyltransferase Family. Manuscript submitted for publication. Retrieved from www.plantcell.org.


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