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
Step 2: Digested vectors were gel purified and digested inserts were PCR purified. Further, glnA was ligated with pSD113 and gltD with pUC19 in 1:3 molar ratio.Each ligation mix was separately transformed in E. coli DH5α competent cells and plated separately on Luria Bertini (LB) medium + ampicillin agar plates. Few colonies from each plate were patched to create a masterplate and inoculated for plasmid preps and glycerol stocks.
Step 3:We carried out blue-white screening for gltD+pUC19, whereas we screened for glnA by carrying out a restriction digestion.
Week 6: We don't give up!
We repeated PCR for gltB using a different polymerase enzyme - Long PCR Enzyme Mix (Thermo Scientific #K0182).
We checked for the correct band (4462 bp) using agarose gel electrophoresis . Hurray!Positive results finally. gltB was further gel purified and RE digested with SalI and SmaI. pUC19 was digested with BamHI. Digested pUC19 was gel purified and digested gltB was PCR purified and a ligation reaction of both was was set up in 1:3 molar ratio.
We sent out all three genes for sequencing.
Week 7: Interlab study begins!
While we were awaiting our sequencing results, we began our interlab study. All the test devices and controls were reconstituted and transformed from the kitplates. All of them were inoculated for making glycerol stocks the next day. Since our plate reader was under maintenance, we performed the measurements later
Week 8: Making way for proteorhodopsin!
In order to make the GS-GOGAT cycle work, we needed proteorhodopsin (PR) that would produce ATP with the help of the proton gradient. However, this activity of PR is seen only in the presence of retinal. We found both, the gene for PR (BBa_K773002) and a device for retinal biosynthesis (BBa_K1604022) in the provided kitplate. For PR, we decided to use an araC regulated promoter (BBa_R0080) which was also present in the kitplate.
We reconstituted BBa_K773002 (Plate 2, well 1P), BBa_K1604022 (Plate 7, well 17K), BBa_R0080 (Plate 3, well 5G) and transformed in E. coli DH5-α competent cells.
Colonies were found only for BBa_K773002. BBa_K1604022 and BBa_R0080 gave no colonies. We repeated the transformation for these two parts, this time we increased the incubation time for each. We were successful.
Week 9: Eureka moments!
We learned that farnesyl diphosphate (FPP) an intermediate in the carotene biosynthesis pathway is is also the starting molecule for a fragrant compound, limonene. Catalysed by limonene synthase (LIMS), FPP is converted to limonene. To our surprise we found the gene coding for LIMS+rbs (BBa_I74211) is present in the kitplate. We planned to clone this part downstream of BBa_K1604022 so as to obtain limonene. Thus, we reconstituted and transformed this part from well 4I of plate 4 in E. coli DH5-α competent cells. Colonies obtained were inoculated for making glycerol stocks and miniprepped the next day.
Week 10: Gene deletions!
We learned that ammonia assimilation in E. coli is regulated by PII proteins that adenylate glutamine synthetase (GS) under nitrogen rich conditions and render it inactive. It was found that the cell also contains another PII like protein called GlnK.(Ref. 2)
Since we wanted the cells to work under ammonia rich conditions, we had to delete glnB – gene coding for PII, and, glnK – gene coding for glnK. Gene deletions were carried out as per Datsenko and Wanner, 2000 (Ref. 3)
Week 11: Sequencing results arrive
Sequencing results were positive only for glnA. We anyway decided to get biobricked glnA synthesized from IDT. Since the PR in our kitplate is without a rbs, we decided to get that too synthesized from IDT. We sent out the sequences for the same. We decided to have glnA, gltB, gltD along with PR in one plasmid and BBa_K1604022 with BBa_I74211 in another plasmid containing a different ori in the same cell. We planned to carry out a 3A assembly using BBa_1604022, BBa_I74211 and BBa_JO4450.
Week 11: 3A assembly!
Sequencing results were positive only for glnA We carried out a variant of 3A assembly using BBa_1604022 (cut with EcoRI and SpeI), BBa_I74211(cut with XbaI and PstI) and BBa_JO4450 (cut with EcoRI and PstI). Unfortunately, it did not work.
Week 12: Discussions and decisions
After a negative 3A result, we discussed and planned our next plan of action .
Week 13: Examination time!
We could not work this week as our university mid term examinations were scheduled.
