| 8th June |
12 new lab members were inducted into the iGEM IITM Team |
| 14th June |
"First lab team meet New members were briefed about directed evolution and iDEC" |
| 15th June |
Work was initiated to ideate on a project for iDEC. One avenue was to integrate directed evolution with an ongoing project on bacterial cellulose. The other avenue was to come with a new project entirely. |
| 16th June |
Resources regarding directed evolution and iDEC were shared with the team |
| 21st June |
"Review Meet – 1 Ideas for projects were presented by lab members and discussed" |
| 25th June |
"Review Meet – 2 Members presented their final project ideas" |
| 27th June |
"Faculty Advisor Meet We presented a set of ideas for the competition and our advisor critically assessed them, helping us choose the idea for our final abstract and detail it." |
| 28th June |
"Review Meet - 3 Discussed Faculty Advisor’s feedback with team members and solidified our project idea with slight modifications" |
| 29th June |
"Project Abstract Meet Divided work amongst ourselves to cover various sections of the project abstract required for the submission" |
| 30th June |
Finalization of Project Abstract |
| 1st July |
Team Meet - Submission of project abstract |
| July Week-2 |
"Team Meet Coming up with a convenient team structure to enable different sub-groups working on different verticals under the project" |
| 14th July |
Discussed project experimental design with sub-groups designated to work on different steps of the experimental design |
| 18th July |
"Updates Meet Brief updates regarding research on topics under experimental design" |
| 21st July |
Detailed updates regarding experimental design including working with yeast, mutagenesis/mutant library generation, recombination techniques to form varied mutant library, yeast cloning, screening of mutants, making cells stable in hypoxic regions and unstable in other regions |
| July Week-4 |
Start of new semester - break |
| 5th - 6th Aug |
"Wet Lab Intro session for new team members Cultured and plated bacteria" |
| 8th Aug |
Further Literature Review and obtaining protocols for the experimental design from isolation of gene to methods of mutagenesis like ePCR, discussion on using E.coli as expression system for yCD |
| 8th August |
Initial efforts were made to decide on possible computational methods that can be used to enhance the project. This mainly involved literature review. |
| 9th August |
An overview report on already available computational tools for directed evolution was presented. This included tools like SCHEMA, MAP, HotSpot Wizard, PEDEL and B-FITTER |
| 19th August |
The tools discussed earlier were explored further. HotSpot Wizard was run using default parameters to get a general idea on the mutational hotspots present in yeast cytosine deaminase. |
| 28th August |
Software for site-directed mutagenesis and random mutagenesis were discussed. The applicability of the FynchBio eVOLVER framework for high-throughput yeast continuous culture in the lab was also looked at. |
| 4th September |
"CFI Review Meet Current progress on computational work was presented to the Head of the Centre for Innovation, IIT Madras" |
| 11th September |
Protein charged domain prediction, the use of Rosetta and the in silico simulation of protein stability in different pH environments was discussed |
| 13th September |
A meeting with Dr. Athi N. Naganathan, a faculty member at IITM working on protein biophysics proved fruitful. We were introduced to his software pStab, which was designed to predict stable protein mutants at different pH, based on electrostatic frustration. |
| 14th September |
Based on Dr. Naganathan’s suggestions, pStab was used with wild-type yeast cytosine deaminase as input. Residue-residue interactions were tracked with PropKa. Wild-type stability at pH 5 was found. |
| 17th September |
The Protein Data Bank structure of yCD was completed using WHAT IF. |
| 18th September |
Looked at the principles and protocols of site specific mutagenesis in order to validate the results we derive computationally |
| 21st September |
pStab was run again using the completed structure. Results were similar to those obtained with the raw pdb file. It was decided that we would continue working with just the raw file instead of the WHAT IF completed structure. |
| 23rd September |
"Structural Analysis of Cytosine Deaminase Detailed reading and inferencing based on the reaction mechanism and zinc ion participation in the ligand- binding site interaction. Changes in the same due to reduction in pH were hypothesized" |
| 24th September |
Based on the mutant index vs mutability prediction by pStab, mutants were created using PyMOL. These mutants were then put through pStab to determine their electrostatic stability at pH 5. Mutants with interesting characteristics were chosen for further analysis. |
| 25th September |
The team began a formal drafting of the project report. |