iGEM 2014 — Team Stony_Brook

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# Team:Stony Brook/Notebook

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Stony Brook iGEM Team Page

[Stony Brook University](http://www.stonybrook.edu/)

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Home

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Team

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Project

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Notebook

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Results

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Outreach

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Safety

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Attributions

Notebook

![](https://static.igem.org/mediawiki/2014/7/78/Stony_Brook_BetterLabSpace.jpg)

[Click here to see our procedures](https://2014.igem.org/Team%3AStony_Brook/MaterialsMethods)

[ ]
June

#### 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!

[ ]
July

#### 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!

[ ]
August

#### 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!

[ ]
September

#### 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