Team:ICT-Mumbai/Notebook
Week 1: Ideation
The first few weeks we ideated upon our problem statement and discussed ways to tackle them. After long discussions and much debate, we finally decided to work upon the issue that we think is quite relevant not only in our country but also in a lot of developing and developed countries, the real reason why public toilets are frowned upon and not used, despite new government policies that encourage building and usage of public toilets. For our inspiration and project summary see ‘project abstract’.
Week 2: Ground Reality
During this week, we decided to approach different NGO’s and organizations that are involved in promoting and providing better sanitation. Sulabh International is an India-based NGO that promotes and provides better sanitation by building public pay and use toilets and community toilets to name a few. To understand and evaluate the feasibility of our idea and to understand the ground reality we decided to meet with the XYZ of Sulabh Internation. A detailed transcript of our conversation with him can be found at our ‘human practices’ tab.
Week 3: We step into the lab!
Week 3, and we step into the lab!
During the first week in the lab, we learnt about the techniques and the principles behind each one of them. It marked our first foray into the world of synthetic biology and it was great fun learning and performing these experiments.
1.Wet lab begins! We learn about different kinds of growth media, composition and their significance.
2.Introduction to agarose gel electrophoresis and DNA quantification methods.
Additionally, we learnt the precautions to follow while handling ethidium bromide containing gels and its disposal procedures.
3.We also kicked off our very first step towards establishing our proof of concept – extracting genomic DNA from Escherichia. coli, BW25113. We checked the size of the extracted product by agarose gel electrophoresis.
5ul and 10ul of the extracted products were loaded in lanes 1,3 and 2,4 respectively.
Week 4:The idea!
Glutamine synthetase (glnA), smaller and bigger subunits of glutamate synthetase (gltD and gltB respectively) were our genes of primary interest. All the three genes of interest were amplified from E. coli , BW25113 genomic DNA using polymerase chain reaction (PCR).PCR products were loaded and run on 1% agarose gel to check for the correct sizes of these three genes ( glnA:1640bp, gltB:4462bp, gltD:1420bp ). Among the three, we were successful in obtaining two genes - glnA and gltD. We repeated PCR for gltB for the second time by adding DMSO. This time too we could not see bands of correct size. We again carried out PCR using different annealing temperatures and the product obtained with 62°C as annealing temperature gave good bands. This was further gel purified and used as a template to carry out another PCR for the same gene. We could not see bands of correct size again.
Week 5: What next?
We anyway proceeded with cloning glnA in pSD113 (Deb et. al) and gltD in pUC19 cloning vector.Step 1: RE digestion of glnA(insert) and pSD113(vector) with NdeI and HindIII RE digestion of gltD(insert) and pUC19(vector) with SmaI and KpnI
