Team:Stony Brook/Notebook

From 2014.igem.org

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         <label for="ac-1">June</label>
         <label for="ac-1">June</label>
         <article class="ac-large">
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           <h4>Week 1</h4>
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           <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 />
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         <label for="ac-2">July</label>
         <label for="ac-2">July</label>
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         <article class="ac-largeish">
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           <h4>Week 1</h4>
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           <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>
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         <label for="ac-3">August</label>
         <label for="ac-3">August</label>
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          <h4>Week 1</h4>
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        <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>
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         <label for="ac-4">September</label>
         <label for="ac-4">September</label>
         <article class="ac-med">
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          <h4>Week 1:</h4>
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        <br/><br/> <h4>Week 1:</h4>
           <p>Experimental:</p>
           <p>Experimental:</p>
           <ul><li>Expression of melittin</li>
           <ul><li>Expression of melittin</li>

Revision as of 23:32, 17 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

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Notebook

Outreach

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! (Picture from Week 7/2)
  • 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! (picture)


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

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