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.
How is the Aroma module composed?

http://2014hs.igem.org/wiki/images/0/
06/AromaHoja.png

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).

Figure 1A. Aroma Module

http://2014hs.igem.org/wiki/imag
es/5/56/AromaFigure1Aa.png

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. The RNA thermometer was also selected as a regulator in
order to continue the CIDEB UANL 2013 work with it.

How does it work?

Figure 1B. Production of Wintergreen Odor

http://2014hs.igem.org/wiki/images/6/6e/Aroma
Figure1Bb.png

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

http://2014hs.igem.org/wiki/images/d/da/AromaPartsChart-.png

Full Device:
These 4 genetic parts form the Aroma device of the project (see Figure 4). The full
devices length is 1,251 bp (including restriction sites).

Figure 4. Aroma Device

http://2014hs.igem.org/wiki/images/
3/3a/AromaFigure4.png

Other teams that used 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.

Figure 5. RNA Thermometer circuit, excerpted from TUDelft 2008 team

http://2014hs.igem.org/wiki/images/e/e0/AromaF
igure5.png

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.

Figure 6. Circuit from iGEM CIDEB UANL 2013 team

http://2014hs.igem.org/wiki/images/3/3f/Aro
maFigure6.jpg

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 (Wintergreen Genetating Device) did not
produce methyl salicylate when salicylic acid was added to the medium, while
E. coli with the WGD did produce methyl salicylate when salicylic acid was
added to the medium.

Figure 7. Wintergreen odor enzyme (BSMT1) generator circuit by MIT 2006.

http://2014hs.igem.org/wiki/images/9/98/AromaFigu
re7.jpg

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:
http://2006.igem.org/wiki/index.php/MIT_2006.
TUDelft iGEM Team. (2008). TUDelft 2008. Retrieved on March 31th, 2014.
From: http://2008.igem.org/Team:TUDelft.
VictoriaBC. (2009). VictoriaBC 2009. Retrieved on March 31th, 2014. From:
http://2009.igem.org/Team:VictoriaBC.
iGEM CIDEB Team. (2013). iGEM CIDEB UANL 2013. Retrieved on March
31th, 2014. http://2013hs.igem.org/Team:CIDEB-UANL_Mexico/Project.