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

 
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<h2>Sept 17</h2>
 +
<ul>
 +
<li>First meeting of Team:IIT-Madras for iGEM 2015 in our UG lab.</li>
 +
<li>Ideation begins.&#9786;</li>
 +
</ul>
 +
 +
 +
<h2>Feb15</h2>
 +
<ul>
 +
<li>Engaged in talk with the mentors about the project, Dr. Nitish Mahapatra and Dr. Kartik Raman.</li>
 +
<li>Introducing few changes in the project.</li>
 +
</ul>
 +
  
 
<h2>May 18-24</h2>
 
<h2>May 18-24</h2>
 
<ul>
 
<ul>
−
<li>Cleaned fridge and transferred materials.</li>
+
<li>Project being finalized.</li>
−
<li>Circuit finalised toggle switch/ mutual depressor switch using TAL repressor.</li>
+
<li>A sketch of proposed work being ready.</li>
 +
</ul>
  
−
<br></br>
 
  
 
<h2>May 25-31</h2>
 
<h2>May 25-31</h2>
−
<ol>
+
<ul>
 
<li>Inventory of all supplies is to be done.</li>
 
<li>Inventory of all supplies is to be done.</li>
−
<li>Alyteserin was chosen as the other protein APO has no defined structure. ZDOCK and online tools were used to generate a peptide that binds to hydrophobic surface. Possible peptides: IAGYAEEILEHVIAE (aka naly)</li>
+
<li>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.</li>
 +
<li>The pdb structure of Alyteserin-1a was generated in pymol, while introducing two mutations D4E and S23N in the pdb structure of Alyteserin-1c.
 +
<li>The structural features of Alyteserin-1a was analyzed carefully to design a novel peptide which could interact with it.</li>
 +
<li>Pymol and Pepstr, an online tool, were used to generate a large number of peptide pdb structures of size 10-18 amino acid.</li>
 +
<li>A software, ZDOCK, was used to assess the docking parameters of  Alyteserin-1a and novel peptide.</li>
 +
<li>Best peforming peptide was chosen to test it's functionality in molecular dynamic simulation.</li>
 +
</ul>
  
−
<!--
 
−
--------------------------------------------------------
 
  
−
Week 3
+
<h2>June 1-7</h2>
−
-> GROMACS simulations started
+
<ul>
−
-> Made a catalog of all available materials
+
<li>Molecul Dynamic Simulation started.</li>
−
-> Lacto Bacilus strains are available NZ9000 and MG1363
+
<li>Made a catalog of all available materials.</li>
−
-> Simulations finished the proteins were found to interact favourably  
+
<li><em>Lactococcus lactis</em> strains, NZ9000 and MG1363, were collected from Prof. KBR's lab.</li>
−
-> Heidelberg 2008 Auto Inducer 2 produced by LuxS sensed by LuxQ
+
<li>MD Simulations finished the proteins were found to interact favourably.</li>  
−
-> Reference:Quorum Sensing AI@ in campylobacter Orla Cloak, Barbara Solow doi:10.1128
+
</ul>
−
Can we do the whole circuit using just Lux components??!!
+
  
−
--------------------------------------------------------
 
  
−
Week 4
+
<h2>June 8-14</h2>
−
 
+
<ul>
−
->Circuit will be composed of purely Lux components.
+
<li>Started working on the design of genetic circuit.</li>
−
-> MD Simulations were done with ionic solution of protein complex. MD Simultions showed that naly  
+
<li>One more MD Simulation was performed with the ionic solution of protein complex. MD Simultions showed that naly  
−
interacts favorably with Alytesterin forming a cavity of hydrophobic residues
+
interacts favorably with Alyteserin forming a cavity of hydrophobic residues.</li>
−
-> Sender is finalised to be E.Coli DH5Alpha with LuxP-PFS gene
+
<li>Sender is finalised to be <em>E.Coli</em> DH5α, which would constitutively synthesize the AI-2 signaling molecules.</li>
−
Receiver is L.lactis NZ9000/MG1316 with LUXPQOU coupled with Lux R through sRNA's qrr1-5 and HFQ
+
<li>Receiver is finalised to be <em>Lactococcus lactis</em> NZ9000, which would sense the AI-2 signaling molecules and would behave in the desired way.</li>
 +
</ul>
 
   
 
   
−
--------------------------------------------------------
 
  
−
Week 5
+
<h2>June 15-21</h2>
 +
<ul>
 +
<li>Sequences were finalised for qr1-5 and HFQ.</li>
 +
<li>Request to iGEM HQ to order extra parts for LuxS, LUXPQUO, Sigma 54, L. lactis constitutive promoter and HFQ.</li>
 +
<li>MD simulation job in which both peptide were dis-oriented at intial condition was submitted.</li>
 +
<li>Prepared SOC stock, stored at 4C for autoclaving the next day.</li>
 +
<li>LB broth, and LB agar was prepared.
 +
<li>Use of usp45 secretion tag for the secretion of both peptides Alyteserin-1a and NAly.</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>
 +
</ul>
  
−
-> Sequences were finalised for qr1-5 and HFQ.
 
−
Email sent to iGEM HQ requesting extra parts for LuxS, LUXPQUO, Sigma 64, constitutive promoter and HFQ.
 
−
MD Simulations on dis-oriented peptides job was submitted.
 
−
Victoria provided us with L.lactis, restriction enzymes and buffers.
 
−
Enzymes and buffers were stored at -20C yellow box labelled master mix.
 
−
Strains(2 of each strain) were stored in Abrar's box at -80C.
 
−
-> 11 Jun:Prepared SOC stock, stored in freezer for autoclaving the next day.
 
−
-> 12 Jun:Made LB broth and LB agar. Plated one petri plate, made stock of 1M 45ml CaCl2.
 
−
Alyteserin was found to have a localization signal attached to it in form of USP45. The same sequence was then attached to NAly.
 
−
-> 13 Jun: We found some inconsistencies in the promoter sequences of LuxCDABE and expS and
 
−
their role when bound to LuxR. It should work, but in vitro experiments carried out earlier
 
−
showed that the promoters did not function as expected. Refer to this link for luxPQ which
 
−
was found to have a signalling region at the beginning http://www.ncbi.nlm.nih.gov/nuccore/16082689?report=fasta.
 
−
The stop codon for LuxP and the start codon for LuxQ overlap by one amino acid.
 
−
For inoculation, first the bacteria on the plate were inoculated in a test tube of LB broth and
 
−
kept overnight. Then bacteria from that LB broth are to be again taken and grown till OD of
 
−
around 0.4 is reached. Negative control was also prepared, where the broth contained antibiotic.
 
  
−
-------------------------------------------------------------
+
<h2>June 22-28</h2>
 +
<ul>
 +
<li>We started preparation of fresh 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>
  
−
Week 6
 
−
15, 16 Jun: We performed competent cell preparation and transformation over two days. The first batch showed no colonies. This could be because after heat shock and addition of broth, the tubes were inoculated for only 40 miinutes. According to iGEM protocol they must be inoculated for atleast 2 hours. None of the transformed bacteria showed growth on 16th Jun. On 16th, this was attempted again following iGEM protocol.
 
−
19 Jun: After analysing the results of our previous experiments, we decided that the competent cells prepared did not have hig transformation efficiency. We started preparation of new competent cells today afresh. Instead of SOC media, we used only LB broth throughout. SOC media might have degraded after autoclaving, so it is better to avoid it. Maybe next time we can use SOB media?
 
−
21 Jun: Agar Stabs for 5 plasmids arrived. We streaked them on agar plates for plasmid isolation the next day. Also, RBS and promoter did not give good results. The DNA had dried up from the wells. Also, a new terminator that we cloned gave only 2 colonies. Adding salts to the competent cells before transformation helped in improving transformation efficiency.
 
  
−
------------------------------------------------------------
+
<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>
  
−
Week 7
+
<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>
  
−
22 Jun: We performed plasmid isolation from bacteria received through agar stabs. We made one error in the procedure. GPS needs to be mixed well before it is used, because it is a suspension.
+
<h2>Aug 17-23</h2>
−
23-27 Jun: We took a break from wet-lab work to focus on primer and sequence design for the plasmids we needed to order from IDT.
+
<ul>
 +
<li>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. </li>
 +
<li>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. </li>
 +
<li>Started site-directed-mutagenesis for BBa_K218006. No success.</li>
 +
</ul>
  
−
-----------------------------------------------------------
+
<h2>Aug 24-30</h2>
−
 
+
<ul>
−
Week 8
+
<li>SDM was repeated 3 times on various possible parameters. Still no success. We gave up on this.</li>
−
 
+
<li>2-3 time cloning experiments were performed. No success.</li>
−
29-30 Jun : We performed transformation for Biobricks [promoter, terminator], we failed as chemically competent cells were older than a week. We decides to make Ultra-competent cells.  
+
</ul>
−
1 Jul : Qrr1-3 and Qrr4-5 were reported to have problems by IDT guys. We made changes and made them as Qrr1, Qrr2, Qrr3, Qrr4, Qrr5.
+
−
2 Jul : Order for all gBlocks were confirmed and placed. Inoculation of a single colony to 5ml tubes [with antibiotic and without antibiotic].The bigger shaker in UG lab were occupied, so we couldn't go ahead with this. We booked the shaker for next two days.
+
−
3 Jul : Agian, we incoulated the colony into two test tubes (with Antibiotic and without antibiotic), due to unknown error we did not get any growth.
+
−
4 Jul : Agian, we incoulated the colony into three test tubes (with Antibiotic (Ampicillin)+ old LB broth and without antibiotic (old LB broth ans new LB broth)), no growth. Cells on plate are not viable. Streak a new E. coli DH5alpha plate.
+
  
−
--------------------------------------------------------------
+
<h2>Sept 1-16</h2>
−
 
+
−
5 Jul - 25 Jul
+
−
Plasmid isolation was performed 3 times(!).
+
−
More or less no work was done.
+
−
Work on the wiki has started.
+
−
 
+
−
----------------------------------------------------------------
+
−
 
+
−
10th August
+
−
Plasmid Minipreps were run again, however the quantity of plasmid was found to be too low. We will run again taking larger quantity of cells in falcon tubes.
+
−
IDT delivery delayed, should come by today.
+
−
 
+
−
-----------------------------------------------------------------
+
−
 
+
−
29-30 Aug
+
−
 
+
−
We tried SDM with PQOU plasmid, unfortunately no amplification was observed. When checking the primers, we discovered that two primers were in low concentration. However, even with the other primers we obtained no amplification. We can only infer that the sequence for the part is wrong, as the primers should work otherwise.
+
−
 
+
−
3A assembly was attempted for the first time with a sample of QRR1 and some PSB1K3.
+
−
 
+
−
<p> Document the dates you worked on your project.</p>
+
−
 
+
−
<h5>What should this page have?</h5>
+
 
<ul>
 
<ul>
−
<li>Chronological notes of what your team is doing.</li>
+
<li>3A assembly for Biobrick BBa_K622000 and BBa_K1033225</li>
−
<li> Brief descriptions of daily important events.</li>
+
−
<li>Pictures of your progress. </li>
+
−
<li>Mention who participated in what task.</li>
+
 
</ul>
 
</ul>
  
  
−
<h4>Inspiration</h4>
 
−
<p>You can see what others teams have done to organize their notes:</p>
 
−
 
−
<ul>
 
−
<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
 
−
<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
 
−
<li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li>
 
−
<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
 
−
</ul>
 
−
-->
 
−
</div>
 
 
</html>
 
</html>

Latest revision as of 14:55, 18 September 2015

Sept 17

  • First meeting of Team:IIT-Madras for iGEM 2015 in our UG lab.
  • Ideation begins.☺

Feb15

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

May 18-24

  • Project being finalized.
  • A sketch of proposed work being ready.

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.
  • Lactococcus lactis 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 DH5α, 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 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.

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. :(

Aug 17-23

  • 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.

Aug 24-30

  • 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.

Sept 1-16

  • 3A assembly for Biobrick BBa_K622000 and BBa_K1033225