Team:IISER-Tirupati India/Notebook


Ovi-Cloak

SCROLL

Wetlab Calender

Week 1 (1 Jan 2021 -3 Jan 2021) :

A literature review of the female reproductive tract, found that hZP2 protein mutation in females result in infertility, literature review of the microbiome of the reproductive tract 

Week 2 (4 Jan, 2021 -10 Jan 2021): 

A literature review of the targets of the ovum, ruled out producing non-specific enzymes from the bacteria because of the potential harm 

Week 3 (11 Jan 2021 - 17 Jan 2021) : 

Learning about iGEM parts, kits and registry to get an idea of availablity of resources. 

Categorized the species of bacteria in both fallopian tubes, found ovastacin protein (ASTL gene).

Week 4 (18 Jan 2021 - 24 Jan 2021) : 

Studied more on Lactobacillus, met with experts in our institute to discuss the idea, looked into lex A system for progesterone sensing, completely ruled out production of NAG enzyme by bacteria 

Week 5 (24 Jan 2021 - 31 Jan 2021) : 

A literature review about the immune system of the reproductive system, started review on a kill switch for reversibility.

Week 1 (1 Feb 2021 - 7 Feb 2021 ):

Found more about iGEM competition deliverables and had a meeting with our PIs. Literature review on Zona hardening 

Week 2 ( 8 Feb 2021 - 14 Feb 2021):

 A literature review on the hZP2 cleavage model by ovastacin and learnt more about the structure of ovastacin 

Week 3 (15 Feb 2021 - 21 Feb 2021):

Searched for a commensal for implementation of our idea and an appropriate model organism to use as chassis by looking into secretion systems, looked into progesterone sensing by the SRTF1 . 


Week 4 (22 Feb 2021 - 28 Feb 2021):

Finalised model organism as em>Bacillus subtilis for proof of concept and Lactobacillus acidophilus as the commensal of reproductive tract to modify, finalized SRTF1 for progesterone inducible system

Week 1 (1 March 2021 -7 March 2021): 

Looked at the HGT aspects of the genetically modified bacteria through literature review and found out more about the safety aspects. 

Week 2 (7 March 2021 - 14 March 2021): 

Finalized the different modules of our project, started in-silico work for proof of concept. Looked at different competition deliverables from the wet lab point of view and planned how to achieve them. 

Week 3 (15 March 2021 - 21 March 2021) : 

Looked into more toxin-antitoxin systems for the kill switch in bacteria, started designing preliminary experiments for the proof of concept 

Week 4 (22 March 2021 - 31 March 2021) :

Met Prof Satish K Gupta for getting feedback on our idea, agreement to procure clones of Human ZP2 in E.coli DH5α from his lab at NII, Delhi. Continued work on the toxin-antitoxin system and started looking for more models for the hormonal sensing system

Week 1 (1 April 2021- 3 April 2021) :

Procured clones of hZP2 from Prof Satish K Gupta and shipped them to our lab at IISER Tirupati. Continued the work on designing experiments for the proof of concept of our project. 

Week 2 (4 April 2021 - 10 April 2021): 

Decided to work with yeast as a substitute organism too and started designing experiments for yeast species. Started looking at resources to procure strains of Bacillus subtilis.

Week 3 (11 April 2021 -17 April 2021): 

Meeting with experts working on yeast systems from our institute, started looking for protocols for different experiments, realized the limitations of progesterone inducible systems, and started looking for alternative hormone sensing models in prokaryotes. 

Week 4 (18 April 2021 - 30 April 2021) : 

Realized the sensitivity issue of the estrogen sensing system in prokaryotes 

Week 1 (1 May 2021 - 8 May 2021) : 

Worked on finding alternatives for the progesterone inducible system, literature review on kill switches resumed and protocol review process started 

Week 2 (9 May 2021 - 15 May 2021):

End Semester exams 

Week 3 (16 May 2021- 22 May 2021) : 

Started looking into growth kinetics for reducing ovastacin production time, Heterologous gene expression in Bacillus subtilis. Ruled out the pH, temperature based kill switches and started to look into light inducible Dnase toxins for kill switch and biosafety. And found yqcG and bovine Dnase 1. 

Week 4 (23 May 2021- 31 May 2021) : 

Yeast experimental workflow finalized and started to list out the experiment requirements. Started looking into the ytvA system for biosafety and Decided which promoters to take for experiments, looked for finalized parts in the iGEM parts registry.

Week 1 ( 1 June 2021 - 5 June 2021) : 

Started making the Bacillus subtilis workflow, finalized the experiments, Calculated d-score for terminator efficiency, and selected terminator for making improvement to part. 

Week 2 ( 6 June 2021 - 12 June 2021) : 

Worked on Genetic circuits for all the experiments, finalized the protocols for genome integration in Bacillus subtilis. 

Week 3 (13 June 2021 - 19 June 2021) : 

Started looking for protocols of protein purification in the yeast and Bacillus subtilis and discussed them with various experts from our institute. Found out the reagents required for carrying out the experiments. 

Week 4 (20 June 2021 - 30 June 2021):

Met with Dr Sabari Sankar Thirupathy from IISER Thiruvananthapuram to discuss Bacillus subtilis experiments and had an agreement with him to ship the clones to our laboratory at IISER Tirupati. Did an in-depth analysis of transformation and protein purification protocols for Bacillus subtilis and looked for reagents for the same. 

Week 1 (1 July 2021 - 10 July 2021) :

Discussions on experiments and protocols continued and sent a list of required reagents and consumables to our laboratory and placed initial orders for reagents and consumables. 

Week 2 (11 July 2021 - 17 July 2021) : 

Talked with PhD students and seniors on performing the experiments upon return to campus, ordered gene fragments from IDT for Bacillus subtilis, and ordered yeast gene fragments from Twist Bioscience 

Week 3 (18 July 2021 - 24 July 2021) : 

Returned to IISER Tirupati campus and Quarantined for a week. Compiled final protocols for the experiments and placed more orders of consumables for experiments. 

Week 4 (25 July - 31 July 2021) : 

We received our iGEM distribution kit and NEB molecular biology kits. We presented the wet lab plan to our guide PhDs and Professors. Had our lab safety training to start the experiments. Prepared experimental things to start our initial experimental journey.

Week 1 (1 Aug 2021 - 7 Aug 2021): 

Started our experiments by culturing the E. coli containing the plasmid pDR111 and pDR110 and purified them. Thereafter we did restriction digestion of purified plasmids with BamHI-hf and EcoRI-hf. Performed a growth curve of E Coli DH5α and Bacillus subtilis 168 in LB media and performed Saccharomyces cerevisiae strain S288C in YPD media.

Week 2 (8 Aug 2021 - 14 Aug 2021) : 

Received orders from Himedia. Did Miniprep for the plasmid pDR111 stock preparation. Prepared the stock solutions for the competency of E Coli DH5α and made E Coli DH5α cells competent followed by storing them at -80⁰C. Performed gram staining to check the bacterial contamination on the plates. Had a demo session for Agarose Gel preparation with our seniors. 

Week 3 (15 Aug 2021 - 21 Aug 2021) : 

Transformed the E Coli DH5α cells with pDR111 to check the quality of the prepared competent cells. Received IDT primers and stock solutions were prepared for SDS PAGE. Worked on making the lab setting more inclusive through LabEyes. Performed Restriction digestion on the plasmid pDR111 with BamHI- hf, and EcoRI- hf. 

Week 4 (22 Aug 2021 - 31 Aug 2021) : 

Performed sequential digestion with EcoRI and BamHI and performed gel extraction with the digested vector. Received Twist order for the yeast DNA fragments. Streaked the E. Coli DH5α with hZP2 clones and expressed the hZP2 protein by IPTG induction. Stored the extracted protein in -20⁰C. Received the pRS424, pRS426, pRS425 yeast shuttle vector plasmids from Dr Vijayalakshmi Subramanian lab.

Week 1 ( 1 Sept 2021 - 11 Sept 2021): 

Did protein purification and ran SDS PAGE multiple times for standardization of ZP2 clones with different IPTG concentrations (0.5mM and 1mM). Started working on oligonucleotides received from TWIST ( for golden gate of ZP2- Ovastacin cassette) and did not get satisfactory results. Performed miniprep with the pRS426 plasmid (for S Cerevisiae vector)and pRSETb plasmid (for ZP2 in E. Coli ). Purified the pRS426 plasmid and transformed the E Coli DH5α with pRSETb and got positive results. Prepared stock solutions for Bacillus subtilis transformation and competency.

 

Week 2 (12 Sept 2021 - 18 Sept 2021) : 

Transformed the Bacillus subtilis and observed the growth of cells on plates within 9hrs. Prepared competent cells of E. Coli BL21 strain and transformed them with pRSETb for ZP2 expression. Golden gate trials for ZP2- Ovastacin cassette was unsuccessful. Ran SDS for purified hZP2. 

 

Week 3 (19 Sept 2021 - 25 Sept 2021) : 

Did IMAC(Ni-NTA) for hZP2 protein purification and Western Blot. Started golden gate trials with spacer cassette for terminator check and SRTF1- P22 combined cassette. Did transformation of the ligated product of ZP2- Ovastacin cassette with pRS426 plasmid in NEB 10β cells and got positive results. 

 

Week 4 (26 Sept 2021 - 30 Sept 2021) : 

Mid sem exams 

Week 1 (1 Oct 2021 - 9 Oct 2021): 

Performed golden gate with spacer cassette for terminator check. Mini-prep for the pDR111 and pRS426 plasmids. Did restriction digestion of these plasmids and dephosphorylated them to make them compatible vectors. Did colony PCR with the vector with spacer cassette for terminator check and results were inconsistent. Results with gradient PCR for spacer cassette for terminator check were also not satisfactory. 

 

Week 2 (10 Oct 2021 - 16 Oct 2021) : 

Standardization of PCR with different primers and at different temperatures. Performed golden gate assembly with all the remaining cassettes that we have designed. i.e. ovastacin cassette, BBa_B0010 terminator cassette , BBa_K3889070 terminator cassette, SRTF1- P22 combined cassette. Performed transformation with all the cassettes to the NEB 10β cells and still results were not good. Decided to go with colony PCR, did patching, and performed colony PCR to find out the desired transformant by performing gel electrophoresis. Got the positive result for the BBa_B0010 terminator cassette and did a mini-prep with that to transform it into Bacillus subtilis for further experimentation. 

 

Week 3 (17 Oct 2021 - 23 Oct 2021) : 

BBa_B0010 terminator cassette with pDR111 plasmid transformed into Bacillus subtilis and performed the fluorescence microscopy to check fluorescence (sfGFP and mCherry expression) in bacterial cells. Patched the colonies for spacer cassette for terminator check, BBa_K3889070 terminator cassette, and SRTF1- P22 combined cassette and performed colony PCR and got the positive clones for all cassettes. Inoculated the colonies with clones and performed miniprep and restriction digestion with BsaI-HFv2 for the vector linearization. Transformed the Bacillus subtilis with these different cassettes. We performed the platereader assay with spacer cassette for terminator check and different concentrations of progesterone to check SRTF1- P22 combined cassette.  

Protocols

  1. Growth Curve
    1. Bacillus subtilis 168

      Click here on button to download the PDF file.

    2. E. coli DH5α strain

      Click here on button to download the PDF file.

    3. Yeast (Saccharomyces Cerevisiae strain S288C)

      Click here on button to download the PDF file.

  2. Culture preparation (Streaking, Spreading, Inoculation, Antibotic stock solution preparation)

    Click here on button to download the PDF file.

  3. Revival from glycerol stock and Preparation of glycerol stock

    Click here on button to download the PDF file.

  1. Oligonucleotides Resuspension

    Click here on button to download the PDF file.

  2. Plasmid purification (Miniprep)

    Click here on button to download the PDF file.

  3. Nanodrop

    Click here on button to download the PDF file.

  4. Agarose gel electrophoresis

    Click here on button to download the PDF file.

  5. Agarose Gel Extraction

    Click here on button to download the PDF file.

  6. Restriction digestion

    Click here on button to download the PDF file.

  7. PCR amplification

    Click here on button to download the PDF file.

  8. PCR cleanup

    Click here on button to download the PDF file.

  9. Golden Gate assembly

    Click here on button to download the PDF file.

  10. SpeedVac

    Click here on button to download the PDF file.

  11. Ligation

    Click here on button to download the PDF file.

  12. Patching and Colony PCR

    Click here on button to download the PDF file.

  13. Replica plating and Starch test

    Click here on button to download the PDF file.

  14. Competency and Transformation
    1. E. coli

      Click here on button to download the PDF file.

    2. Bacillus subtilis 168

      Click here on button to download the PDF file.

    3. Saccharomyces Cerevisiae strain S288C

      Click here on button to download the PDF file.

  1. Protein induction

    Click here on button to download the PDF file.

  2. Ni-NTA protein purification

    Click here on button to download the PDF file.

  3. Cell Lysis (with sonication)
    • E. coli BL21 strain

      Click here on button to download the PDF file.

  4. SDS-PAGE

    Click here on button to download the PDF file.

  5. Western blotting
    • E. coli BL21 strain - ZP2 analysis

      Click here on button to download the PDF file.

Our Sponsors
Follow Us