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

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<h2>May 18-24</h2>
 
<h2>May 18-24</h2>
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<h2>May 25-31</h2>
 
<h2>May 25-31</h2>
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<h2>June 8-14</h2>
 
<h2>June 8-14</h2>
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<h2>June 15-21</h2>
 
<h2>June 15-21</h2>
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<li>In biobrick BBa_K218006, the stop codon for LuxP and the start codon for LuxQ overlap by one base pair.</li>
 
<li>In biobrick BBa_K218006, the stop codon for LuxP and the start codon for LuxQ overlap by one base pair.</li>
 
<li>Chemical competent cell preparation and transformation over two days. The first transformed batch showed no colonies.</li>
 
<li>Chemical competent cell preparation and transformation over two days. The first transformed batch showed no colonies.</li>
 +
</ul>
 +
 +
<br></br>
 +
 +
<h2>June 22-28</h2>
 +
<ul>
 +
<li>We started preparation of afresh competent cells. Instead of SOC media, we used only LB broth throughout.</li>
 +
<li>Agar Stabs for 5 plasmids arrived. We streaked them on agar plates for plasmid isolation on the next day.</li>
 +
<li>We checked the transformation efficiency using the transformation efficiency kit provided by iGEM. Transformation failed again.</li>
 +
<li>First plasmid isolation failed with known errors.</li>
 +
</ul>
 +
 +
<br></br>
 +
 +
<h2>June 29- July 5</h2>
 +
<ul>
 +
<li>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.</li>
 +
<li>Preparation of ultra-competent cells. Tranformation efficiency was found out to be overwhelming.</li>
 +
</ul>
 +
 +
<br></br>
 +
 +
<h2>Aug 10-16</h2>
 +
<ul>
 +
<li>Plasmid isolation was performed for transformed colonies and other biobrickes which were ordered from iGEM.</li>
 +
<li>Received the gBlocks and primers from IDT. This delay took a lot from us. :( </li>
 
</ul>
 
</ul>

Revision as of 16:44, 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.


June 22-28

  • We started preparation of afresh 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.


June 29- July 5

  • 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.


Aug 10-16

  • 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. :(