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
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