Team:Stony Brook/Notebook

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<h1 >WELCOME TO iGEM 2014! </h1>
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<p>Your team has been approved and you are ready to start the iGEM season!
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<br>On this page you can document your project, introduce your team members, document your progress <br> and share your iGEM experience with the rest of the world! </p>
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<p style="color:#E7E7E7"> <a href="https://2014.igem.org/wiki/index.php?title=Team:Stony_Brook/Notebook&action=edit"style="color:#FFFFFF"> Click here  to edit this page!</a> </p>
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<tr><td colspan="3"> <h3>Notebook</h3></td></tr>
 
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<p>You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well. </p>
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<div id="yellow_header"> <a href="http://www.stonybrook.edu/">Stony Brook University</a></div>
 +
<div id="blue_header"> <a href="https://2014.igem.org/Team:Stony_Brook"><img src="https://static.igem.org/mediawiki/2014/1/1e/Stony_Brook_Apis-biotics_header.png"/ id="apis-biotics"/></a>
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    <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook"><img src="https://static.igem.org/mediawiki/2014/7/78/Stony_Brook_NavIcon2_Home.png"/></a>
 +
      <p> Home </p>
 +
    </div>
 +
    <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook/Team"><img src="https://static.igem.org/mediawiki/2014/6/66/Stony_Brook_NavIcon2_Team.png"/></a>
 +
      <p> Team </p>
 +
    </div>
 +
    <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook/Project"><img src="https://static.igem.org/mediawiki/2014/9/99/Stony_Brook_NavIcon2_Project.png"/></a>
 +
      <p> Project </p>
 +
    </div>
 +
    <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook/Notebook"><img src="https://static.igem.org/mediawiki/2014/5/57/Stony_Brook_NavIcon2_Notebook.png"/></a>
 +
      <p> Notebook </p>
 +
    </div>
 +
<div id="yellow_navigation">
 +
  <a href="https://2014.igem.org/Team:Stony_Brook/Results"> <img src="https://static.igem.org/mediawiki/2014/0/04/Stony_Brook_NavIcon2_Results.png"/></a>
 +
      <p>Results</p>
 +
</div>
 +
    <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook/Outreach"><img src="https://static.igem.org/mediawiki/2014/8/80/Stony_Brook_NavIcon2_Outreach.png"/></a>
 +
      <p> Outreach </p>
 +
    </div>
 +
<div id="yellow_navigation">
 +
  <a href="https://2014.igem.org/Team:Stony_Brook/Safety"> <img src="https://static.igem.org/mediawiki/2014/0/07/Stony_Brook_NavIcon2_Safety.png"/></a>
 +
      <p>Safety</p>
 +
</div>
 +
    <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook/Attributions"><img src="https://static.igem.org/mediawiki/2014/0/03/Stony_Brook_NavIcon2_Acknowledgements.png"/></a>
 +
      <p> Attributions </p>
 +
    </div>
 +
  </div>
 +
</div>
 +
<div id="project">
 +
  <p>Notebook</p>
 +
</div>
 +
 
 +
<div class="picture"><img src="https://static.igem.org/mediawiki/2014/7/78/Stony_Brook_BetterLabSpace.jpg"/></div>
 +
 
 +
</div>
 +
 
 +
<p class="glossary"><a href="https://2014.igem.org/Team:Stony_Brook/MaterialsMethods">Click here to see our procedures</a></p>
 +
 
 +
<div id="contents"> <section class="ac-container">
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    <section class="ac-container">
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    <div>
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        <input id="ac-1" name="accordion-1" type="checkbox" />
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        <label for="ac-1">June</label>
 +
        <article class="ac-large">
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          <br/><br/><h4>Week 1</h4>
 +
          <p>Literary research: </p>
 +
          <p>We looked into SUMO protein, a protective group that we could use to stabilize the melittin peptide. We also looked into mechanisms to express or release melittin:<br />
 +
          <ul><li> Using a signalling sequence, signal peptides and pathways (OmpA, SecB, IgA beta)in order to secrete melittin</li>
 +
            <li>Lysing our cells with a killswitch and then using FLAG tags, affinity tags to purify melittin from lysate</li>
 +
            <li>Proteases to cleave off a protecting group.</li></ul></p>   
 +
<p> We also looked into understanding biobricking assembly standards</p>
 +
 
 +
          <h4>Week 2</h4>
 +
          <p> Literary research:</p>
 +
        <ul> <li>Prepromelittin nucleotide sequence was found.</li>
 +
        <li> Decided on a fusion protein with melittin and GST tag</li>
 +
        <li>Reached out to Genomics Core Facility on Stony Brook University Campus to talk with Dr. John Schwedes about RNA extraction and Dr. Dmitri Gnatenko about primer design.</li>
 +
        <li>Searching for plasmids: met with Yelena from Molecular Cloning Facility to discuss appropriate plasmids for our experiment and search through their plasmid library</li></ul>
 +
          <p>Experimental:</p>
 +
          <ul><li>Competent E.coli cell line made. </li>
 +
          <li> Visited local beehive to obtain Apis mellifera samples; dissected our bees and extracted their RNA  </li>
 +
            <li>Transformed RhlR biobrick</li></ul>
 +
          <p> Outreach:</p>
 +
          <p>We presented on synthetic biology at four classes at a local high school, East Sachem High School!</p>
 +
 
 +
  <h4> Week 3</h4>
 +
<p>Literary research:</p>
 +
<ul><li> Looking into P. syringae as a model for P. aeruginosa?</li>
 +
<li> Optimal expression conditions</li>
 +
<li> Looking into TEV protease site to cleave off GST protein</li>
 +
<li>Detection methods</li></ul>
 +
<p>Experimental:</p>
 +
<ul><li> Designed primers for melittin amplification and tagged melittin</li>
 +
<li>Successful RNA extraction!</li>
 +
<li>Obtained plasmid from our advisor, Dr. Czaplinski</li>
 +
<li>Designed RhlR circuit and obtained signaling compound C4HSL</li>
 +
<li>Constitutive promoter BBa_J23101 and J23102 transformed for later fluorescence testing</li></ul>
 +
 
 +
<h4>Week 4</h4>
 +
<p>Experimental:</p>
 +
<ul><li>Synthesized cDNA library</li>
 +
<li> Designed and ordered Biobrick melittin primers</li>
 +
<li>mCherry and fluorescence testing</li></ul>
 +
  <p>Outreach:</p>
 +
<ul><li>Looked into doing an iGEM survey with other schools with the Stony Brook Institutional Review Board </li>
 +
  <li> Attended iGEM High School Jamboree!</li></ul>
 +
        </article>
 +
    </div>
 +
    <div>
 +
        <input id="ac-2" name="accordion-1" type="checkbox">
 +
        <label for="ac-2">July</label>
 +
        <article class="ac-largeish">
 +
          <br/><br/><h4>Week 1</h4>
 +
          <p>Experimental:</p>
 +
          <ul><li> Ligated and transformed for GST-melittin fusion protein</li>
 +
<li>Induced plasmid with IPTG and checked with SDS page gel- no protein detected </li>
 +
<li>Redid PCR and digest</li>
 +
          <li>Sequenced RhlR/Rhl circuit samples</li></ul>
 +
 
 +
          <h4>Week 2</h4>
 +
          <p>Experimental:</p>
 +
          <ul><li>Prepared insert for our melittin plasmid- used PCR to amplify melittin, insert TEV cleavage site, then digested insert, and purified</li>
 +
<li>Digested RhlR/ rhl plasmid, phosphatased, purified, ligated, and transformed. Cultured overnight, miniprepped and ran on gel to check results.</li>
 +
          <li>RhlR + Promoter miniprep, sent for sequencing</li></ul>
 +
<p>Outreach</p>
 +
          <p>Presentated at Stony Brook University Biotechnology Camp for high school students!</p>
 +
          <h4>Week 3</h4>
 +
          <p>Experimental:</p>
 +
          <ul><li>Digest check, sequencing results for RhlR/Rhl</li>
 +
          <li>Screened colonies for melittin </li>
 +
            <li>Began PCR from beginning to amplify prepromelittin</li>
 +
add-on PCR, inserted into duet plasmid</li></ul>
 +
          <p> Mathematical modeling:</p>
 +
          <p>Researching articles for rates of RhlR degradation</p>
 +
<p>Comparison of our model with arbitrary values to experimental model for fluorescence</p>
 +
 
 +
 
 +
          <h4>Week 4</h4>
 +
          <p>Experimental:</p>
 +
          <ul><li>PCR amplification of prepromelittin from cDNA, addition of TEV site to melittin gene</li>
 +
            <li>Ligation of TEV-melittin into GST plasmid</li>
 +
            <li>Transformation of E.coli with ligation</li>
 +
            <li>Checked successful transformation by digesting transformed plasmid and viewing diegest on gel to cross-check with expected band size-70% success! </li>
 +
          <li>Samples from transformations sent for senquencing</li>
 +
          <li>N17+RhlR ligation into duet plasmid troubleshooting</li></ul>
 +
          <p> Mathematical modeling:</p>
 +
          <ul><li>Rates for C4HSL and RhlR degradation, dimerization</li>
 +
          <li>Preliminary mathematical models created!</li></ul>
 +
     
 +
        </article>
 +
    </div>
 +
  <div>
 +
        <input id="ac-3" name="accordion-1" type="checkbox">
 +
        <label for="ac-3">August</label>
 +
        <article class="ac-small">
 +
        <br/><br/> <h4>Week 1</h4>
 +
          <p>Experimental:</p>
 +
          <ul><li>Results from sequencing: 3/4 samples were in frame; 1 had a missene mutation</li>
 +
          <li>Inducing of cultures of GST plasmid (control) and GST+TEV+melittin with IPTG</li>
 +
        <li> Attempted to checked protein expression by SDS-page</li>
 +
          <li>Co-transformations of RhlR plasmids</li></ul>
 +
          <h4>Week 2</h4>
 +
          <ul><li>Expression of melittin confirmed by SDS-PAGE!</li>
 +
          <li>Completion of Rhl circuit</li>
 +
          <li>Samples sent in for sequencing</li>
 +
          </ul>
 +
          <h4>Week 3</h4>
 +
          <p>School starts! </p>
 +
        </article>
 +
    </div>
 +
<div>
 +
        <input id="ac-4" name="accordion-1" type="checkbox">
 +
        <label for="ac-4">September</label>
 +
        <article class="ac-med">
 +
        <br/><br/> <h4>Week 1:</h4>
 +
          <p>Experimental:</p>
 +
          <ul><li>Expression of melittin</li>
 +
          <li>Checked protein expression by SDS-PAGE</li>
 +
          <li>Ligation of Rhl into mCherry plasmid</li>
 +
          <li> Double digestion to verify that ligation was successful</li></ul>
 +
          <p> Mathematical Modeling</p>
 +
          <ul><li>Adjusting model for protein expression</li>
 +
          <li>Visualization of melittin expression using MATLAB</li></ul>
 +
<p>Outreach:</p>
 +
          <ul><li>Feature in Stony Brook Scholars Newsletter</li>
 +
          <li>Stony Brook involvement fair!</li></ul>
 +
 
 +
<h4>Week 2:</h4>
 +
          <p>Experimental:</p><ul>
 +
          <li>Measuring of fluroresence expression using TECAN </li>
 +
          <li>Found that Rhl is a leaky promoter, performed experiments to validate our concerns</li></ul>
 +
 
 +
          <h4>Week 3</h4>
 +
          <ul><li>Culturing of cells with GST+TEV+Melittin</li>
 +
          <li>Checking protein expression by SDS-PAGE</li>
 +
        <li> Biobricking</li></ul>
 +
        </article>
 +
    </div>
 +
</section> 
 +
</div>
 +
</body>
</html>
</html>

Latest revision as of 03:39, 18 October 2014

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> Stony Brook iGEM Team Page

Home

Team

Project

Notebook

Results

Outreach

Safety

Attributions

Notebook

Click here to see our procedures



Week 1

Literary research:

We looked into SUMO protein, a protective group that we could use to stabilize the melittin peptide. We also looked into mechanisms to express or release melittin:

  • Using a signalling sequence, signal peptides and pathways (OmpA, SecB, IgA beta)in order to secrete melittin
  • Lysing our cells with a killswitch and then using FLAG tags, affinity tags to purify melittin from lysate
  • Proteases to cleave off a protecting group.

We also looked into understanding biobricking assembly standards

Week 2

Literary research:

  • Prepromelittin nucleotide sequence was found.
  • Decided on a fusion protein with melittin and GST tag
  • Reached out to Genomics Core Facility on Stony Brook University Campus to talk with Dr. John Schwedes about RNA extraction and Dr. Dmitri Gnatenko about primer design.
  • Searching for plasmids: met with Yelena from Molecular Cloning Facility to discuss appropriate plasmids for our experiment and search through their plasmid library

Experimental:

  • Competent E.coli cell line made.
  • Visited local beehive to obtain Apis mellifera samples; dissected our bees and extracted their RNA
  • Transformed RhlR biobrick

Outreach:

We presented on synthetic biology at four classes at a local high school, East Sachem High School!

Week 3

Literary research:

  • Looking into P. syringae as a model for P. aeruginosa?
  • Optimal expression conditions
  • Looking into TEV protease site to cleave off GST protein
  • Detection methods

Experimental:

  • Designed primers for melittin amplification and tagged melittin
  • Successful RNA extraction!
  • Obtained plasmid from our advisor, Dr. Czaplinski
  • Designed RhlR circuit and obtained signaling compound C4HSL
  • Constitutive promoter BBa_J23101 and J23102 transformed for later fluorescence testing

Week 4

Experimental:

  • Synthesized cDNA library
  • Designed and ordered Biobrick melittin primers
  • mCherry and fluorescence testing

Outreach:

  • Looked into doing an iGEM survey with other schools with the Stony Brook Institutional Review Board
  • Attended iGEM High School Jamboree!


Week 1

Experimental:

  • Ligated and transformed for GST-melittin fusion protein
  • Induced plasmid with IPTG and checked with SDS page gel- no protein detected
  • Redid PCR and digest
  • Sequenced RhlR/Rhl circuit samples

Week 2

Experimental:

  • Prepared insert for our melittin plasmid- used PCR to amplify melittin, insert TEV cleavage site, then digested insert, and purified
  • Digested RhlR/ rhl plasmid, phosphatased, purified, ligated, and transformed. Cultured overnight, miniprepped and ran on gel to check results.
  • RhlR + Promoter miniprep, sent for sequencing

Outreach

Presentated at Stony Brook University Biotechnology Camp for high school students!

Week 3

Experimental:

  • Digest check, sequencing results for RhlR/Rhl
  • Screened colonies for melittin
  • Began PCR from beginning to amplify prepromelittin
    • add-on PCR, inserted into duet plasmid

Mathematical modeling:

Researching articles for rates of RhlR degradation

Comparison of our model with arbitrary values to experimental model for fluorescence

Week 4

Experimental:

  • PCR amplification of prepromelittin from cDNA, addition of TEV site to melittin gene
  • Ligation of TEV-melittin into GST plasmid
  • Transformation of E.coli with ligation
  • Checked successful transformation by digesting transformed plasmid and viewing diegest on gel to cross-check with expected band size-70% success!
  • Samples from transformations sent for senquencing
  • N17+RhlR ligation into duet plasmid troubleshooting

Mathematical modeling:

  • Rates for C4HSL and RhlR degradation, dimerization
  • Preliminary mathematical models created!


Week 1

Experimental:

  • Results from sequencing: 3/4 samples were in frame; 1 had a missene mutation
  • Inducing of cultures of GST plasmid (control) and GST+TEV+melittin with IPTG
  • Attempted to checked protein expression by SDS-page
  • Co-transformations of RhlR plasmids

Week 2

  • Expression of melittin confirmed by SDS-PAGE!
  • Completion of Rhl circuit
  • Samples sent in for sequencing

Week 3

School starts!



Week 1:

Experimental:

  • Expression of melittin
  • Checked protein expression by SDS-PAGE
  • Ligation of Rhl into mCherry plasmid
  • Double digestion to verify that ligation was successful

Mathematical Modeling

  • Adjusting model for protein expression
  • Visualization of melittin expression using MATLAB

Outreach:

  • Feature in Stony Brook Scholars Newsletter
  • Stony Brook involvement fair!

Week 2:

Experimental:

  • Measuring of fluroresence expression using TECAN
  • Found that Rhl is a leaky promoter, performed experiments to validate our concerns

Week 3

  • Culturing of cells with GST+TEV+Melittin
  • Checking protein expression by SDS-PAGE
  • Biobricking

Retrieved from "http://2014.igem.org/Team:Stony_Brook/Notebook"