View source for Team:IIT Madras/Experiments

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<h2>Getting the structure of Alyteserin-1a</h2>
<p>Alyteserin-1a is a modified antimicrobial peptide which was made by introducing two mutations (D4E & N23S) in Alyteserin-1c peptide. The structure for Alyteserin-1a is not available therefore we obtained the pdb structure for Alyteserin-1c (pdb id : 2L5R) and created the given mutations in pymol. We carefully analyzed the structural feature of Alyteserin-1a peptide to come up with another peptide which could interact with it. We seem to find an interesting pattern which was a hydrophobic groove on one side of the peptide. We targeted this region to neutralize the anit-microbial peptide.</p>

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<h2>Predicting the structure of Naly</h2>
<p>In our project, a key step was to neutralize the antimicrobial peptide, Alyteserin-1a, so that we can create a stress free environment. For this, we designed a novel peptide of smaller length, which can interact favorably with Alyteserin-1a. We used following step to get the best possible peptide :
<ul>
<li> We used pepstr, an online tool, which takes amino-acid sequence as input and gives us possible structures, in pdb form, of the protein.</li>
<li> After getting several possible structures, we used another online tool, ZDOCK, which docks two molecules and give a score, which tells us about that how favorably peptides are interacting.</li>
<li>We screened few peptides from above process and proceeded for MD simulations.</li>
</ul>
  
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<h2>Molecular Dynamics Simulation</h2>
<p>We performed molecular dynamics simulation experiments to confirm that Naly is able to interact with Alyteserin-1a antimicrobial peptide, hence it can neutralize it's activity to kill the pathogemic bacteria. Following are the steps that we followed in this experiment :</p>
<ol>
<li>Convert both the pdb files to .gro files and get the topology files using pdb2gmx tool. Use GROMOS96 53a6 force field.</li>
<li>Change the positions of both the peptides in a way that they are far away and disoriented.
<li>Build the .gro file for the complex (both proteins) by merging the above .gro files. Create the .top file for  complex both peptides.</li>
<li>Define the box for the complex and solvate with water molecules.</li>
<li>Add NA+ and CL- atoms to neutralize the charge of whole system. Create the index.ndx file using make_ndx.</li>
<li>Prepare the .tpr file for energy minimization step.</li>
<li>Steepest descent minimization algorithm was used for a maximum of 50,000 steps.</li>
<li>Position restrain the water molecules in complex.top file.</li>
<li>NVT equilibrate the system. We used leap-frog integrator, lincs constraint_algorithm, Particle Mesh Ewald for electrostatic interactions, position restrain both the peptides, reference temperature 300K with no pressure couppling for 50,000 max steps.</li>  
<li>NPT equilibrate the system with Parrinello-Rahman pressure coupling.</li>
<li>Run the NPT for a total of five times with position constraint [fcx, fcy, fcz] values 1000, 100, 10, 1, in both posre_.itp file. Use the output of one step as the input for the nnext step.</li>
<li>Final production run for 30,000 ps.</li> 

</ol>
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<h2>Analyzing the md simulation</h2>
<p>Once we have run the md simulations, we need to validate the simulation based on several parameters, follow the following protocol to obtain and see the results. </p>
<ol>
<li>Using index.ndx file, remove the water and other atoms i.e. NA and CL from the system.</li>
<li>Use the complex.gro file and .xtc file to see the simulation in vmd.</li>
<li>Using the .edr file calculate different parameters like potential energy, kinetic energy, total energy, radius of gyration etc. to prove your hypothesis. </li>

</ol>



<h2> Preparing Ultra-competent cells</h2>
<p>To get good transformation efficiency from <em>E. coli</em> cells, we prefer ultra-competent cells rather normal chemical competent cells. Following is the procedure :</p>

<ol type="a">
<li>Materials/Buffers: </li>
<ol type="I">
<li>SOB SOLUTION FOR COMPETENT CELL PREPARATION</li>
<ol>
<li>0.5% yeast Extract</li>
<li>2% Tryptone</li>
<li>10mM NaCl</li>
<li>2.5mM KCl</li>
<li>10mM MgCl2</li>
<li>10mM MgSO4.</li>
<li>Dissolve all in nanopure water and autoclave</li>
</ol>
<li>TRANSFORMATION BUFFER FOR COMPETENT CELL PREPARATION</li>
<ol>
<li>10mM PIPES</li>
<li>15mM CaCl2</li>
<li>250mM KCl</li>
<li>Dissolve in nanopure water and adjust pH to 6.7 with KOH or HCl. The add MnCl2 to 55mM and adjust final volume. Sterilize by filtration with 0.45 µm filter. Store at 4C. </li>
</ol>
</ol>

<li>Cells were cultured on 2xYT agar plate overnight at 37C.</li>
<li>10-12 colonies were cultured in 250ml SOB medium.</li>
<li>It was incubated at 37C for 1hour. Then the flasks were transferred to 19C. It was incubated till the OD600 reached 0.5.</li>
<li>Flask was placed on ice for 10min.</li>
<li>The cells were pelleted by spinning at 4000rpm for 10min at 4C.</li>
<li>Cells were resuspended in 80ml ice cold TB(Transformation Buffer) and stored on ice for 10min.</li>
<li>It was centrifuged again at 4000rpm for 10min at 4C.</li>
<li>Pellet was resuspended in 20ml of TB with 1.5ml DMSO.</li>
<li>Final volume was aliquoted into microcentrifuge tubes (100-500µl) and stored at -80C. </li>
</ol>

<p><b>CAUTION!</b></p>
<ul>
<li><b>Caution:</b> The whole procedure after the cells are pelleted out needs to be carried out in ice.</li>
<li><b>Caution:</b> TB buffer is heat sensitive, never take it out of ice.</li>
</ul>




<h2>Mini Prep: DNA Isolation using HiPura Silica Kit</h2>
<ol type="I">
<li>Single isolated colonies were picked and inoculated in 3ml media.</li> 
<li>Overnight incubation was done at 37°C. </li>
<li>1.5ml of culture was taken and centrifuged at 12,000 rpm for 2 min.</li> 
<li>Supernatant was discarded and pellet was kept on Ice. </li>
<li>The pellet was re-suspended with 100µl of chilled TGE buffer (25mM Tris pH 8.0, 50mM Glucose, 10mM EDTA) treated with RNAse (0.25 µl / 200 µl TGE buffer) was added to it. </li>
<li>200µl of Sol II containing 0.2N NaOH and 1% SDS was added and the solution was mixed gently and kept on ice for 3-5 min. </li>
<li>150µl of cold potassium acetate solution (3M Potassium and 5M acetate) was added and the solution was mixed gently and finally kept on ice for 3-5 min. </li>
<li>Mixture was centrifuged at 14,000 rpm for 5 min. </li>
<li>The supernatant was transferred to a clean centrifuge tube.</li> 
<li>3 volumes of Chaotropic Salt Solution was added. </li>
<li>10µl of Glass Powder Suspension was added and then kept for mixing in rotospin for 30 min.</li> 
<li>Centrifugation was done at 14,000 rpm for 2 min and the supernatant was discarded. </li>
<li>The glass powder pellet was re-suspended in 500µl of diluted wash solution (1 volume of wash Solution as provided with the HiPura Silica kit added to 9 volume of Milli-Q and 10 volume of absolute ethanol).</li> 
<li>Centrifugation was done at 12,000 rpm for 2 min and the supernatant was discarded.</li> 
<li>Repeated aboe two steps twice. After the last wash, the supernatant was removed completely and the pellet was air dried for 5 min. </li>
<li>Glass Powder Pellet was re-suspended into 20µl of autoclaved double distilled water (or TE buffer, pH 8.0 for long term storage). </li>
<li>Incubation at 55°C for 20 min was done.</li> 
<li>Centrifugation at 14,000 rpm was done for 2 min and the supernatant was carefully taken out and was stored in -20°C. </li>
<li>Confirmation was done by running 2µl of isolated plasmid DNA on agarose gel against a standard marker.</li>
</ol>

<h2>Restriction Digestion</h2>
<ol type="I">
<li>20µl of restriction digestion reaction was set up on ice. </li>
<li>2µl of NEB buffer 4 (CutSmart) was used for the High Fidelity enzymes PstI and SpeI. </li>
<li>1µg of DNA was added to it. </li>
<li>0.2µl each of restriction enzymes were added to the mixture. </li>
<li>Autoclaved double distilled water was used to make up the volume to 20µl.</li> 
<li>The reaction mixture was kept at 37°C for 8-12h. </li>
<li>The restriction digestion was confirmed by loading on agarose gel against a standard marker.</li>
</ol>

<h2>Elution of Plasmid DNA using HiPura Silica Kit.</h2>
<ol type="I">
<li>Digested sample was run on agarose gel. </li>
<li>The required size band was excised under UV light and the gel pieces were loaded into a vial.</li> 
<li>3 volume of Chaotropic Salt Solution was added to it. </li>
<li>Incubated at 55°C till agarose melted completely. </li>
<li>10µl of Glass Powder Suspension was added to it. </li>
<li>The mixture was mixed and incubated at room temperature for 15-20 min in the rotospin. </li>
<li>The mixture was centrifuged at 12,000 rpm for 2 minutes and the supernatant was discarded.</li> 
<li>500µl of diluted Wash Solution (1 volume of wash Solution as provided with the HiPura Silica kit added to 9 volume of Milli-Q and 10 volume of absolute Ethanol.) was added to the pellet. </li>
<li>Centrifugation was done at 12,000 rpm for 2 min and the supernatant was discarded. </li>
<li>Above both steps were repeated twice. After the last wash, the supernatant was removed completely and the pellet was air dried for 5 min. </li>
<li>Glass Powder Pellet was re-suspended into 20µl of autoclaved double distilled water (or TE buffer, pH 8.0 for long term storage).</li> 
<li>Incubation at 55°C for 20 min was done. </li>
<li>Centrifugation at 14,000 rpm was done for 2 min and the supernatant was carefully taken out and was stored in -20°C. </li> 
<li>Confirmation was done by running 2µl of isolated plasmid DNA on agarose gel against a standard marker.</li>
</ol>


<h2>Ligation</h2>
<ol type="I">
<li>10µl of reaction was set up. </li>
<li>Purified Vector and Insert were taken in the ratio of 1:3.</li> 
<li>1µl of 10X Ligase buffer was added to it. </li>
<li>1µl of T4 Ligase was added to the mixture. </li>
<li>Finally, the volume was made up to 10µl by adding autoclaved double distilled water. </li>
<li>Ligation reaction mixture was kept overnight at gradient temperature over the range of 8-22°C.</li>
</ol>


<h2>3A Assembly</h2>
<ol type="A">
<li>Restriction Digest </li>
<ol type="I">
<li>Mix contains:</li>
<ol>
<li>5 µl NEB buffer 2.</li>
<li>0.5 µl BSA.</li>
<li>1 µl Forward Enzyme.</li>
<li>1 µl Backward Enzyme.</li>
<li>5 µl DNA part.</li>
<li>37.5 µl MilliQ water(DNAase free).</li>
</ol>
<li>Reaction mix is incubated at 37°C for 30 min.</li>
<li>It is then heat inactivated at 80°C for 20 min.</li>

</ol>
<li>Ligation</li>
<ol type="I">
<li>Mix contains:</li>
<ol>
<li>9 µl MilliQ water.</li>
<li>3 µl Upstream Digest.</li>
<li>3 µl Downstream Digest.</li>
<li>2 µl Plasmid backbone.</li>
<li>2 µl Ligase Buffer.</li>
<li>1 µl Ligase.</li>
</ol>
<li>Mix in incubated at Room Temperature for 15 min.</li>
<li>It is then heat inactivated at 65C for 20 min.</li>
</ol>

</ol>


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<h2>Experiments &amp; Protocols</h2>

<p>Describe the experiments, research and protocols you used in your iGEM project.</p>

<h5>What should this page contain?</h5>
<ul>
<li> Protocols </li>
<li> Experiments </li>
<li>Documentation of the development of your project </li>
</ul>



<h4>Inspiration</h4>
<ul>
<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
</ul>
</div>
-->
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