Description
Origin
According to the updated WHO database, more than 80% of people around the world are exposed to air quality levels that exceed the allowed limits and a not so surprising fact is that low income cities (nearly 98%) are impacted the most with such bad quality air stemming from the increasing levels of noxious pollutants in the environment. As the air quality declines, cases for stroke, heart diseases, lung cancer and chronic respiratory problems all shoot up. We have targeted major pollutants which have probably taken many lives after being well above their acceptable limits, namely xylene and acetaldehyde, whose levels drastically increase inside closed vehicles due to enormous aerosol accumulation. So, the major concern for us (as it has been for 2 decades) is to find a way to curb this problem. Referring to previous iGEM projects, we found that teams from many countries have worked in this field and designed circuits mainly to detect the levels of harmful contaminants. Some of them have been successful in their approach but they had to compromise on either of the two factors: sensitivity and efficiency. Our approach lies not only in sensing the levels of these pollutants, but also in finding a way to reduce their levels, which has been depicted in our circuit designs.
Circuit Design
The quest began with the search for ways to curb the problem to capture as well as detection of noxious gases and harmful chemicals. The task was to make a circuit with feedback positive and negative loops for increasing the sensitivity and specificity of detection. We came up with a novel circuit that can be modified easily for detection of various pollutants. Some pollutants activate a promoter whereas others repress a promoter. For eg: CO activates the COOA promoter whereas Acetaldehyde represses XylR promoter. The circuit we have designed can be used for both kinds of pollutants.
Genetic Design and Clonning Strategies
Cloning of module 1 in pZS21MCS:
For cloning of construct 1, Ptet- RBS1- T7 RNA polymerase –terminator 1-terminator 2 and construct 2, PT7-RBS2- Chromoprotein II- RBS3-tetR-terminator3. Different parts like Ptet, PT7, RBS, T7 RNA polymerase, Chromoprotein II, tetR and terminator parts are taken from BioBricks library and are amplified by PCR. Different amplified PCR products are fused together through splice overlap extension PCR.
Cloning of Module 2 in pACYC177 :
For cloning of construct 1 of module 2, Pmar-RBS4- ToxR-terminator 4 and construct 2, Pctx-RBS5- chromoprotein I- RBS6-TetR- terminator5. Different parts like ToxR and chromoprotein I are PCR amplified from BioBricks library. Other parts like- Pmar is amplified from E. coli genome (BW25113), and terminator 4 is cloned from plasmid (modified pAH125). Parts are amplified by PCR and different parts are fused together by splice overlap extension PCR.
Co-culturing
Co-culturing is a technique of culturing or growing two or more than two different populations of cells in the same culture plate. It is a useful way to understand the interaction between two different types of cells, making it advantageous for studies of cell-cell communication.
Here in we have the plan to co-culture our engineered strain of E.coli (which can independently detect and capture each of the noxious gases and harmful chemicals) with naturally occurring microbes which can consume these harmful gases and chemicals through their evolved metabolic pathways. This ensures the existence of a sink for these pollutants, and thereby the longevity of the synthetic microbes.
Growth on Paper
