Difference between revisions of "Team:IIT Madras/Notebook"

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<h2>May 25-31</h2>
 
<h2>May 25-31</h2>
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<h2>June 1-7</h2>
 
<h2>June 1-7</h2>

Revision as of 16:16, 12 September 2015

Sept 17

  • First meeting of Team:IIT_Madras for iGEM 2015.
  • Ideation begins.

May 18-24

May 25-31

  • 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.






June 1-7

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

June 8-14

  • 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.

June 15-21

  • 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.