Team:IIT Madras/Notebook

Sept 17

Engaged in talk with the mentors about the project, Dr. Nitish Mahapatra and Dr. Kartik Raman.
Introducing few changes in the project.

Inventory of all supplies is to be done.
Alyteserin-1a was chosen to test our model as the structural feature, mechanism of action and other relevent details of anit-microbial peptide Alyteserin-1c, which has two mutations (D4E, N23S), were available in the literature.
The pdb structure of Alyteserin-1a was generated in pymol, while introducing two mutations D4E and S23N in the pdb structure of Alyteserin-1c.
The structural features of Alyteserin-1a was analyzed carefully to design a novel peptide which could interact with it.
Pymol and Pepstr, an online tool, were used to generate a large number of peptide pdb structures of size 10-18 amino acid.
A software, ZDOCK, was used to assess the docking parameters of Alyteserin-1a and novel peptide.
Best peforming peptide was chosen to test it's functionality in molecular dynamic simulation.

Molecul Dynamic Simulation started.
Made a catalog of all available materials.
Lactococcus lactis strains, NZ9000 and MG1363, were collected from Prof. KBR's lab.
MD Simulations finished the proteins were found to interact favourably.

Started working on the design of genetic circuit.
One more MD Simulation was performed with the ionic solution of protein complex. MD Simultions showed that naly interacts favorably with Alyteserin forming a cavity of hydrophobic residues.
Sender is finalised to be E.Coli DH5Î±, which would constitutively synthesize the AI-2 signaling molecules.
Receiver is finalised to be Lactococcus lactis NZ9000, which would sense the AI-2 signaling molecules and would behave in the desired way.

Sequences were finalised for qr1-5 and HFQ.
Request to iGEM HQ to order extra parts for LuxS, LUXPQUO, Sigma 54, L. lactis constitutive promoter and HFQ.
MD simulation job in which both peptide were dis-oriented at intial condition was submitted.
Prepared SOC stock, stored at 4C for autoclaving the next day.
LB broth, and LB agar was prepared.
Use of usp45 secretion tag for the secretion of both peptides Alyteserin-1a and NAly.
In biobrick BBa_K218006, the stop codon for LuxP and the start codon for LuxQ overlap by one base pair.
Chemical competent cell preparation and transformation over two days. The first transformed batch showed no colonies.

We started preparation of fresh competent cells. Instead of SOC media, we used only LB broth throughout.
Agar Stabs for 5 plasmids arrived. We streaked them on agar plates for plasmid isolation on the next day.
We checked the transformation efficiency using the transformation efficiency kit provided by iGEM. Transformation failed again.
First plasmid isolation failed with known errors.

The sequence of all parts and primers to fix the problem in biobrick BBa_K218006 via site-directed mutagenesis, were designed and ordered to IDT.
Preparation of ultra-competent cells. Tranformation efficiency was found out to be overwhelming.

Plasmid isolation was performed for transformed colonies and other biobrickes which were ordered from iGEM.
Received the gBlocks and primers from IDT. This delay took a lot from us. :(

Started cloning the gBlocks from IDT. But failed. No overhangs at the 5' and 3' end of the gBlocks therefore we can not use our two RE site, EcoRI and PstI. A big mistake.
We wished to use the other two REs, XbaI and SpeI, but they would result in self ligation of plasmid backbone and formation of scar at the insert plus backbokne region. We relied on luck.
Started site-directed-mutagenesis for BBa_K218006. No success.

SDM was repeated 3 times on various possible parameters. Still no success. We gave up on this.
2-3 time cloning experiments were performed. No success.