Team:SVCE CHENNAI/Noot

Notebooks

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January 2016

Week 1: 4^th Jan to 10^th Jan

Selections were done to recruit team members of iGEM 2k16.

Week 2: 11^th Jan to 17^th Jan

Organized our first team meet up and the freshers were addressed about iGEM competition and its values.

Week 3: 18^th Jan to 24^th Jan

The team brainstormed ideas of various disciplines and raised funds for the competition.

Week 4: 25^th Jan to 31^st Jan

Decanting work was done and the lab was setup with necessary chemicals and equipment.

February to April 2016

Brainstorming and fund raising

May 2016

Week 1: 2^nd May to 8^th May

•   The team was divided to do human practices and lab work.

Human Practices


Prepared questionnaires and finalized Google forms for urban and rural population.
Conducted a survey in a village named Kunthambakkam.

Lab

Mother plate of E.coli DH5α was prepared.
Antibiotic stocks and buffers required for molecular biology works were prepared.
Protocol optimization for competent cells was carried out.

Week 2: 9^th May to 15^th May

Human Practices


Questionnaire was prepared for biotech students.
A survey was done among urban people and educated them genetically modified organisms.

Lab

The transformation didn’t yield a fruitful result first time due to handling errors.
The competent cells and transformation was repeated using both CaCl₂ protocol and CCMB80 buffer protocol for the following parts:

•    Lambda pR promoter
•    RNA thermometer
•    Anderson strong promoter+ strong RBS
•    Double terminator
•    cjBlue
•    tsPurple

Colonies were observed in control plates of CaCl₂ protocol.
Trouble shooting was done and the procedure was repeated. It was found that visible transformants were seen using CaCl₂ protocol.
Plasmid isolation and colony PCR was done to confirm the transformants.
Staining was done to check contamination in the transformed colonies. It was found that it was contaminated by cocci .
PCR amplification of tsPurple and λ pR promoter was done.

Week 3: 15^th May to 21^st May:

University exams were held and so no work was carried out.

Week 4: 21^th May to 30^th May:

University exams were held and so no work was carried out.

June 2016

Week 1: 1^st June to 7^th June:

University exams were held and so no work was carried out.

Week 2: 8^th June to 15^th June:

University exams were held and so no work was carried out.

Week 3: 15^th June to 26^th June

•   The team was divided to do human practices and lab work.

Human Practices


Created awareness about genetically modified organisms (GMO’s) for various village people.
A list of college and schools was made to educate students and required processes were carried out.
We approached various diary industries requesting appointment for a presentation about our project.
We spoke to Prof. Iyappan(from SRM University) regarding temperature sensitive systems and, following his suggestions, we decided to use RNA thermometer along with λ pR promoter.

Lab

Transformation was carried out for ligated λ pR product.
Again witnessed problem in transformation due to contamination. Troubleshooting was done to check for contamination during competent cell preparation by staining buffers.
Checked for contamination in DH5α culture by streaking them in McConkey plates. There was no sign of contaminants in the plate.
Freshly prepared new CaCl₂ and CCMB80 buffers and competent cell efficiency was tested. It was found to be efficient at 20pg and 50pg.

Week 4: 27^th June to 3^rd July

Human Practices

Contacted teams on twitter to talk about collaboration. Got positive response from 7 teams.
Ethical survey form was prepared.
A google form for world survey was prepared.
Visited Pasky Diary industry and opinion of industry people was noted.
A skype call was made to Purdue industry and Collaboration oppurtunities were discussed.

Lab

PCR amplification of RNA thermometer was done.
Plasmid isolation of RFP transformants was done and a positive result was seen.
Transformation was carried out for the following parts.
The competent cells and transformation was repeated using both CaCl₂ protocol and CCMB80 buffer protocol for the following parts:

•    GFP
•    Double terminator
•    Anderson strong promoter
•    cjBlue
•    RNA thermometer

PCR amplification of λ pR promoter and tsPurple was carried out and observed negative result.
Transformation of λ pR promoter and tsPurple was carried out.

July 2016

Week 1: 4^th July to 10^th July

Lab

Plasmid isolation of ligated λ pR promoter along with tsPurple was done with internal and positive control. Visible band was observed only for RFP plasmid.
Plasmid isolation of cjBlue, GFP, RNA thermometer and RFP was carried out and size confirmation was done.
PCR amplification of GFP and cjBlue was done.

Lab

We took a list of various milk industries and contacted their administrative heads to hold a discussion regarding survey and presentations.We explained our idea and we got suggestions on how the project could be improved.

Week 2: 11^th July to 17^th July

Lab

PCR amplification was found to be positive for phusion polymerase while showed negative result for taq polymerase.
Restriction of cjBlue, RNA thermometer and RFP was done and size confirmation was carried out.
RNA thermometer+cjBlue and RNA thermometer+GFP were kept for ligation and observed negative results. It was kept for overnight incubation but the ligation didn’t happen.

Week 3: 18^th July to 24^th July

Lab

Restriction of RNA at E and S site didn’t happen due to buffer incompatibility.
To clear the doubt on restriction enzymes each enzymes were individually tested by linearizing GFP plasmid. It was concluded that restriction enzymes works well.
Tried concentrating tango buffer from 1X to 2X and restriction was carried out resulting in negative result.
Finally used NEB 2.1 buffer for restriction at E and S Sites. It was observed that restriction occured using this common buffer.

Human Practices

Collaboration works were initiated.
We contacted various Indian and international teams- IISC Banglore, IIT Madras, iGEM_Westminster2016, Tec Chihuahua 2016, iGEM Tel Hai 2016, Edinburgh iGEM OG 16, Newcastle iGEM, Virginia iGEM, XMU-China iGEM, Trinity iGEM, iGEM DTU Denmark and established collaboration relations.

Week 4: 25^th July to 31^st July

Lab

AMP construct g blocks was checked by doing PCR amplification and distinct band was found by using 30 cycles in amplification. Competent cells stock was tested for efficiency but in vain.
AMP cassette was restricted at X and P site and ligated with Chloramphenicol backbone. The size confirmation wasn’t proper. RNA thermometer+cjBlue blue plasmid was isolated from the transformed colonies .
Protocol was optimized for ligation using restricted GFP plasmid at E and P site at varying time periods.( 10 min, 45 min, 2 hours, and overnight). It was found that ligation happened at 10 minutes.

Human Practices

We contacted all 14 food and nutrition teams through social networking sites for conducting international iGEM meet up, but we couldn't hold the meet. We conducted the Indian iGEM Meetup on 23^rdJuly.

August 2016

Week 1: 1^stAugust to 7^th August

Lab

•    Transformation of parts on Cam+ plates
  K314103 (1638 bp) - Lac inducible cassette
  K608351 (948 bp) - λ pR promoter
  K1357008 (845 bp) - tsPurple
  Positive control - RFP
  No growth was observed in the plates except for the negative control.
•    Plasmid isolation of ligated plasmid - RNA thermometer+ cjBlue+ backbone with Kanamycin resistance. On performing AGE, a single distinct band of size greater than 10kb was observed.
•    To confirm the size of the above plasmid, the plasmid was linearised by single digestion (EcoRI) but couldn't observe the right size for the plasmid.
•    Restriction digestion of RNA thermometer and cjBlue performed followed by a one hour and also a 4 hour ligation so as to optimize the ligation protocol. Didn't get the size required on performing AGE.
•    Optimisation of restriction digestion using Cj blue performed.
•    Preparation of subtilis seed stock
•    g blocks order placed
•    PCR of RNA thermometer - correct result observed
•    PCR of g block - WB1 and lac cassette performed
•    λ pR promoter + tsPurple + backbone with Kanamycin resistance. Plasmid was transformed on Kan+ plates. Colonies were observed on the plate. However, similar ones were observed on the negative control plate too hence it was concluded that the plates had been contaminated.
•    Streaking of B.subtilis on McConkey Agar Plate to confirm its identity
•    Restriction of PCR products - RNA thermometer and cjBlue followed by a 10 minute ligation in 1:1 ratio.

Human Practices

•    Human Practices Presentation at Anna University, Chennai
•    Presentation was done at Kwality Industry and we documented their opinions.
  Presentations were done at Sri Sankara College of Arts and Science and Sri Ramachandra University.

Week 2 : 8^thAugust to 15^th August

Lab

•    Ligation of RNA thermometer + cjBlue with backbone having Kanamycin resistance in 1:2 ratio. Faint band was observed at 3kb. The obtained product was transformed . No transformants were observed and cell growth was observed in the negative control plate.
•    PCR of cjBlue and lac cassette was performed
•    Restriction digestion of RNA thermometer and cjBlue parts
•    Ligation of restricted parts – RNA thermometer, cjBlue and backbone with Chloramphenicol resistance. Obtained product was transformed. No colonies observed on the plates except for contamination.
•    PCR of restricted lac cassette part
•    Restriction of WB1 and ligation of restricted lac cassette with WB1. No band was observed on performing AGE

Human Practices

•    Compilation of milk industries reports was done.
  We acquired support from Paskey Dairy industry administrative head and they decided to fund our project.
  We spoke to polymer chemists regarding improving our membrane material.

Week 3: 15^thAugust to 22^nd August

Lab

•    Restriction of WB1, lac cassette and d.Terminator parts.
•    Restriction of RNA thermometer, cjBlue and double terminator parts.
•    Ligation of RNA thermometer and cjBlue restricted parts in 1:1 ratio. No ligation has occurred.
•    Ligation of RNA thermometer, cjBlue and backbone with Chloramphenicol resistance. Band was observed near 3kb for 10 minutes ligation and near 800bp for 1 hour ligation.
•    Ligation of lac cassette and WB1 performed twice . No band was observed.

Human Practices

•    Had talks with few food scientists regarding our project.
  After doing a lot of background research based on the tips given by the polymer chemists, we came up with polycaprolactone(PCL) as a possible alternative to the inner membrane material(PVC).

Week 4: 23^rdAugust to 31^st August

Lab

•    Antibiotic broth dilution tests were performed to check the activity of the antibiotics. Growth of cells was observed even in the highest concentration of antibiotic containing test tube. So we concluded that the antibiotic being used so far has lost its activity.
•    Triple Iron Sugar test was performed to confirm the identity of B.subtilis culture we had.
•    Sporulation studies began . Starting with streaking of B.subtilis on AK Agar plate . Spores were observed on visualizing the cells on compound microscope after staining.
•    Ligation of ligated lac cassette+WB1 with double terminator for 10 minutes was performed. No band observed.
•    Restriction digestion of lac cassette and WB1 parts
•    Ligation of the above restricted parts in 1:1 ratio. A smear was observed on the gel.
•    Ligation of RNA thermometer, cjBlue and backbone with Chloramphenicol resistance in 1:3 ratio for 10 minutes. Band obtained near 3kb
•    PCR of ligated product.
•    Transformation of ligated products – lac cassette+WB1+double terminator+ backbone with Chloramphenicol resistance and RNA thermometer+cjBlue+backbone with Chloramphenicol resistance. Positive results were observed.

Human Practices

•    Collaboration with UPO Sevilla Team. GMO survey was shared among the Indian population and a report was made based on the results.
•    Industrial visit to Kwality Food Industry

September 2016

Week 1: 1^stSeptember to 7^th September

Lab

•    PCR and restriction digestion WB2 g blocks
•    Ligation of lac cassette and WB2 in 1:2 ratio.
•    PCR of Anderson promoter part using the Master Mix of NEB Kit
•    Plasmid isolation from the transformed plate of RNA thermometer+cjBlue+backbone with Chloramphenicol resistance. A light band was observed around 3kb and a high intensity band around 2 kb observed. Couldn’t clearly conclude the presence of the plasmid.
•    PCR of the above plasmid performed

Human Practices

•    Compilation of school reports and village reports.

Week 2: 8^thSeptember to 14^th September

Lab

•    Transformation of lac cassette+WB1+backbone with Chloramphenicol resistance and lac cassette+WB2+backbone with Chloramphenicol resistance. No transformants growth observed. No significant growth of transformants was observed.
•    Linearisation of RNA thermometer+cjBlue+backbone with Chloramphenicol resistance by single digestion . Band was observed at 2kb
•    Ligation of RNA thermometer+cjBlue with backbone with Chloramphenicol resistance and double terminator. Band observed between 2kb and 3kb size.
•    Ligation of lac cassette, WB2 and backbone with Chloramphenicol resistance
•    Transformation of the above 2 ligated products . Control plate had contamination.
•    PCR of the ligated product – RNA thermometer+double terminator+backbone with Chloramphenicol resistance
•    Restriction digestion of WB2, GFP and double terminator parts

Human Practices

•    Spread the knowledge of SynBio and educated students about our project in the National Workshop on Genetic Engineering organized by SVCE, Chennai

Week 3: 15^thSeptember to 22^nd September

Lab

•    Ligation of RNA thermometer+cjBlue with double terminator and lac cassette with WB2.
•    PCR of g blocks – λ pR construct
•    Restriction digestion of double terminator part
•    Ligation of restricted g block λ pR and double terminator in 1:3 ratio.

Human Practices

•    Lab troubleshooting form drafted and shared with other iGEM Teams via Twitter and Facebook

Week 4: 23^rdSeptember to 30^th September

Lab

•    Restriction of λ pR PCR product
•    Ligation of the restricted λ pR with backbone with Chloramphenicol resistance. No visible band was observed.
•    PCR of λ pR g blocks
•    Restriction digestion of λ pR PCR product and backbone with Chloramphenicol resistance.

Human Practices

•    Project presentation at Sri Ramachandra University, Chennai
•    iGEM Stall at OMICS 16', SVCE
•    Compilation of reports for schools, colleges, collaboration, scholar interviews and their proofreading was done.

October 2016

Week 1: 1^st October to 7^th October:

Lab

•   Performed sporulation studies of Bacillus subtilis.
  Optimization of ligation protocol.

Human Practices

•   Compilation of reports and surveys.
  Designing of Poster

Week 2: 8^st October to 16^th October:

Lab

•   Judging form was filled.
  Transformed our parts for validation.
  Compiled the lab results.

Human Practices

•   Designing and editing of the Poster.
  Wiki page updated.

Sponsors

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igemsvce@gmail.com

Sri Venkateswara College of Engineering
Tamil Nadu, India

    

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