Team:IIT Madras/Notebook

Sept 17

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.

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 DH5Alpha, 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.