Team:Stony Brook/Notebook

From 2014.igem.org

(Difference between revisions)
 
(25 intermediate revisions not shown)
Line 5: Line 5:
<title>Stony Brook iGEM Team Page</title>
<title>Stony Brook iGEM Team Page</title>
<style type="text/css">
<style type="text/css">
 +
 +
#contentSub, #footer-box, #catlinks, #search-controls, #p-logo, .printfooter, .firstHeading,.visualClear {display: none;}
 +
a{
a{
outline: 0;
outline: 0;
Line 92: Line 95:
#yellow_navigation_container{
#yellow_navigation_container{
-
width: 405px;
+
width: 600px;
height: 86px;
height: 86px;
position: absolute;
position: absolute;
top: 38%;
top: 38%;
right: 50%;
right: 50%;
-
margin-right: -203px;
+
margin-right: -335px;
}
}
Line 153: Line 156:
top: 300px;
top: 300px;
left: 50%;
left: 50%;
-
margin-left: -334px;
+
margin-left: -670px;
 +
        width: 800px;
}
}
 +
.glossary{
.glossary{
position:absolute;
position:absolute;
Line 239: Line 244:
.ac-container input:checked ~ article.ac-large{
.ac-container input:checked ~ article.ac-large{
-
height: 1200px;
+
height: 1100px;
width: 800px;
width: 800px;
}
}
.ac-container input:checked ~ article.ac-largeish{
.ac-container input:checked ~ article.ac-largeish{
-
height: 1050px;
+
height: 950px;
width: 800px;
width: 800px;
}
}
.ac-container input:checked ~ article.ac-med{
.ac-container input:checked ~ article.ac-med{
-
height: 600px;
+
height: 500px;
width: 800px;
width: 800px;
}
}
.ac-container input:checked ~ article.ac-small{
.ac-container input:checked ~ article.ac-small{
-
height: 500px;
+
height: 300px;
width: 800px;
width: 800px;
}
}
Line 383: Line 388:
<div id="yellow_header"> <a href="http://www.stonybrook.edu/">Stony Brook University</a></div>
<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>
<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>
-
  <div id="yellow_navigation_container">
+
    <div id="yellow_navigation_container">
     <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>
     <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>
       <p> Home </p>
Line 396: Line 401:
       <p> Notebook </p>
       <p> Notebook </p>
     </div>
     </div>
-
     <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook"><img src="https://static.igem.org/mediawiki/2014/8/80/Stony_Brook_NavIcon2_Outreach.png"/></a>
+
<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>
       <p> Outreach </p>
     </div>
     </div>
-
     <div id="yellow_navigation"> <a href="https://2014.igem.org/Team:Stony_Brook"><img src="https://static.igem.org/mediawiki/2014/0/03/Stony_Brook_NavIcon2_Acknowledgements.png"/></a>
+
<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>
       <p> Attributions </p>
     </div>
     </div>
Line 408: Line 421:
</div>
</div>
-
<div class="picture"><img src="https://static.igem.org/mediawiki/2014/0/00/Stony_Brook_LabSpace.png" /></div>
+
<div class="picture"><img src="https://static.igem.org/mediawiki/2014/7/78/Stony_Brook_BetterLabSpace.jpg"/></div>
</div>
</div>
-
<p class="glossary">Click here to see our procedures</p>
+
<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">
<div id="contents"> <section class="ac-container">
Line 420: Line 433:
         <label for="ac-1">June</label>
         <label for="ac-1">June</label>
         <article class="ac-large">
         <article class="ac-large">
-
           <h4>Week 1</h4>
+
           <br/><br/><h4>Week 1</h4>
           <p>Literary research: </p>
           <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 />
           <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 />
Line 449: Line 462:
  <p>Experimental:</p>
  <p>Experimental:</p>
  <ul><li> Designed primers for melittin amplification and tagged melittin</li>
  <ul><li> Designed primers for melittin amplification and tagged melittin</li>
-
<li>Successful RNA extraction! (Picture from Week 7/2)</li>
+
<li>Successful RNA extraction!</li>
<li>Obtained plasmid from our advisor, Dr. Czaplinski</li>
<li>Obtained plasmid from our advisor, Dr. Czaplinski</li>
<li>Designed RhlR circuit and obtained signaling compound C4HSL</li>
<li>Designed RhlR circuit and obtained signaling compound C4HSL</li>
Line 461: Line 474:
   <p>Outreach:</p>
   <p>Outreach:</p>
  <ul><li>Looked into doing an iGEM survey with other schools with the Stony Brook Institutional Review Board </li>
  <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! (picture) </li></ul>
+
   <li> Attended iGEM High School Jamboree!</li></ul>
         </article>
         </article>
     </div>
     </div>
Line 468: Line 481:
         <label for="ac-2">July</label>
         <label for="ac-2">July</label>
         <article class="ac-largeish">
         <article class="ac-largeish">
-
           <h4>Week 1</h4>
+
           <br/><br/><h4>Week 1</h4>
           <p>Experimental:</p>
           <p>Experimental:</p>
           <ul><li> Ligated and transformed for GST-melittin fusion protein</li>
           <ul><li> Ligated and transformed for GST-melittin fusion protein</li>
Line 511: Line 524:
         <label for="ac-3">August</label>
         <label for="ac-3">August</label>
         <article class="ac-small">
         <article class="ac-small">
-
          <h4>Week 1</h4>
+
        <br/><br/> <h4>Week 1</h4>
           <p>Experimental:</p>
           <p>Experimental:</p>
           <ul><li>Results from sequencing: 3/4 samples were in frame; 1 had a missene mutation</li>
           <ul><li>Results from sequencing: 3/4 samples were in frame; 1 had a missene mutation</li>
Line 530: Line 543:
         <label for="ac-4">September</label>
         <label for="ac-4">September</label>
         <article class="ac-med">
         <article class="ac-med">
-
          <h4>Week 1:</h4>
+
        <br/><br/> <h4>Week 1:</h4>
           <p>Experimental:</p>
           <p>Experimental:</p>
           <ul><li>Expression of melittin</li>
           <ul><li>Expression of melittin</li>

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"