Team:Bioriidl Somaiya/Results
Ideation
Our journey began in January of 2019, when the iGEM meetings began. People from a number of fields aggregated to come and speak about their ideas and prototypes of the experiment to be carried out. The idea kept coming aplenty but the decision of choosing the best was the hardest one ever!
- But after so much of the discussion and plotting, we finalized on going forward with a project which respected our fundamentals and would improve the lives and time of many. We needed to now focus on the most primary level in almost all our experiments. The autoclave. It is certain that autoclave usage is time consuming and very taxing. It needs inspection, tests and along with this you also have to wait for the media plates to dry! To battle this we figured out a solution, after tons and tons of experimentation.
The Solution
Media generally used in labs is the nutrient , MacConkey, LB, Sabouraud's media ( broth and agar). For effective and quick sterilization of the desired media, the following conditions were tested and proved - The heating of the media! The most crucial part of our project. Media needs to be heated to high temperatures of approximately 105°C to achieve killing of bacteria, spores and fungi. For getting this, we used our nichrome wire, wound around a basic dilution tube containing media or water which is later tested using a thermometer for checking rate of increase in temperature. We found that the media heated up in approximately 1 - 2 minutes, which was a very good result, compared to the 2.5 hours wasted normally in an autoclave. For preservation of heat and consistent spread, we used the oil surrounds which were not as efficient as expected. The amount of reduction of cells were seen when we used yeast cells for control and heating this way showed a huge decrease in cell count
UV C is known for it microbicidal properties, which were employed here to kill any remaining bacteria or contaminants. The media is passed through the heating coils at high temperatures and then held in the UV chamber with the UV tube for approximately 5 minutes, which reduces count of bacteria further ( UVC causes dimerisation of thymines, the accumulation of which prevents the binding of polymerase due to which replication is inhibited and hence the cell is led to death)
These parameters would handle most of the contaminant non- specifically leading to reduction of count of cells in the media. For the biological part, we targeted Nisin A, for which we had reference of proof from an inhouse researcher. We obtained our Nisin from him and added 2ml of 2% Nisin to prewarmed 10 ml of media. The media was left to activate for 10 minutes and then the liquid was passed through the sterilization unit ( heater plus UV chamber). The results showed completely sterile medium. Since the enzymes are harmful for organisms, we needed to test the final medium for viability for growth of further organisms. The enzyme is heated in the sterilization unit and hence is degraded due to protein denaturation. This was tested by growing of other organisms on the final media. The results showed healthy, normal growth of bacteria. The gene hence selected was that of Nisin A, Subtilosin-A and Sporulation Killing Factor (SKF). Subtilosin- A and SKF were tested similarly and obtained from inhouse researchers. Testing prototype- The final model shows entry of media mix ( non sterile media plus enzyme mix) through the entry cone and its passage through the heating coils ( nichrome wire complex) for a duration of 2 minutes leading to finally, the UV chamber for holding ( 6 minutes) and sterilization.
