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.

Design:

The images used in both of the games were the following:

(1)   Laboratory warning:

(2)   Project related:

Pictures:

Results:

The shape of the game and its size contributed to its attractiveness for the students and people in general. They would came closer to it just for curiosity and when the team mentioned them its purpose and explained the rules to them, they would become interested in participating actively, as well as asking questions about the signs and the project. This incremented their knowledge about both aspects included in the memory game.

All of the students had fun and learned something new about safety and the team after playing this game.