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 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.

How does it work?

Besides, 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

Constitutive Promoter (BBa_J23119A) A promoter refers to the part of DNA that initiates the gene transcription. A constitutive promoter refers to a promoter that is continuously working. In the specific case of our Aroma module, it will help the bacteria to continuously transcribe the WinterGreen (BSMT1) gene in order to allow the bacteria to continuously produce the aroma. This promoter has a length of 35bp.

RNA Thermometer (BBa_K115017) This RBS (Ribosome Binding Site) is classified as a RNA thermometer, used for temperature post-transcriptional regulation (thermo sensor), and is designed to initiate translation around 32°C. The thermo sensor is repressed when the temperature is below 32°C, it does not translate because the mRNA takes a secondary structure just as the image below and cannot enter the ribosome for translation; when the temperatures reaches a range between 32°C and 37°C, the thermo sensor is activated and the mRNA unfolds into a straight structure that now allows access to the ribosome to start the production of the protein.

The following image (Figure 2) shows the secondary structure of the part after ligation to a protein coding part, as predicted by RNAfold. Notice that the 3' end, including the scar and the start codon, does not belong to the part. The blue region is the Shine Dalgarno (SD) sequence which is the ribosome binding site (TUDelft, 2008).

In the Aroma module, the thermo sensor will give us control over the production of the WinterGreen aroma. This RNA thermometer has a length of 83bp.

WinterGreen Odor Generator (BSMT1) (BBa_J45004) The CDS used in the Aroma module produces a transferase to convert salicylic acid into methyl salicylate, which main characteristic is the WinterGreen odor. The wintergreen odor generator requires of 2mM of salicylic acid to produce methyl salicylate. BSMT1 (WinterGreen Odor Generator original name) has a length of 1074bp.

This CDS works encoding SAM benzoic acid/salicylic acid carboxyl methyltransferase I, derived from BSMT1 from Petunia x hybrida. BSMT1 catalyzes the conversion of salicylic acid to methyl salicylate. Methyl benzoate has a floral smell and methyl salicylate has a wintergreen smell. (MIT, 2006).

Terminator (BBa_B1002) Part made of 6bp, responsible for transcription stop. The terminator stops the production of methyl salicylate.

These 4 genetic parts form the Aroma device of the project(see Figure 4).

Other teams that use RNA thermometer and WinterGreen (BSMT1)

RNA thermometer

·           TUDelft 2008: Temperature-sensing bacteria that changes color at different temperatures; as a temperature reporter system in large-scale fermentations, or as a temperature-inducible protein production system.

·           VictoriaBC 2009: NAND logic gate using the ribo-key/ribo-lock system designed by the 2006 Berkeley 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 the 2008 TUDelft 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)

·           iGEM 2006_MIT: 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.

References

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; American Society of Plant Biologists.

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

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

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

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

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.

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