O M G O M Vs!
Outer Membrane Vesicles • Spherical proteoliposomes ranging in size from 50-200 nm • Naturally secreted by gram-negative bacteria • Contain phospholipids and periplasmic proteins • Play role in pathogenesis, biofilm formation, quorum signaling, and nutrient acquisition
OMV Production • OMVs form when portions of cell membrane bulge, pinch off, and release
Hypervesiculation • A deletion in the tolRA genes will weaken the cell membrane, increasing vesiculation
ClyA • Cytolysin A (ClyA) is a surface hemolytic protein found in E. coli • OMVs naturally enriched in ClyA • Other proteins can be attached, negating the natural hemolytic activity
Applications • Vaccination • Drug delivery • Single ClyA fusion constructs – Fluorescent Proteins – Single-chain antibody fragment
Advantages • OMVs have great potential as a multifunctional platform – easily produced en masse via simple genetic mutations in E. coli – easily purified by ultracentrifugation – inexpensive to produce – functional expression of foreign proteins on surfaces possible
Objectives • Create and co-transform multiple ClyA- fluorescent protein fusions • Quantify relative levels of expression
Measure relative expression Objective levels of multiple ClyA fusions E. coli budding OMVs Co-transform with fusions and Create ClyA Fusion express Proteins ClyA fusion proteins expressed on surface of OMVs
Measure relative expression Objective levels of multiple ClyA fusions Create Fluorescent Protein Biobricks Test Fluorescent Protein Devices Create ClyA Biobrick Create and Test ClyA-FP devices Co-transform and Measure relative levels of expression
Fluorescent Protein Biobricks • Decided to add more fluorescent proteins to Registry • Strawberry, Honeydew, Tangerine Fluorescent Proteins • Initially created FP Biobricks in the most commonly used RFC 10 format PTet Fluorescent Protein
Measure relative expression Objective levels of multiple ClyA fusions Create RFC Honeydew, Strawberry Create Fluorescent and Tangerine Biobricks Protein Biobricks Test Fluorescent Protein Devices Create ClyA Biobrick Create and Test ClyA-FP devices Co-transform and Measure relative levels of expression
Test Florescent Protein Devices • Ligate FP Biobricks to inducible pTet promoter/RBS construct from kit • Induce and measure expression using a plate reader • Characterize these biobricks so we had a baseline read to compare ClyA-FP fusions against PTet RBS Fluorescent Protein
Measure relative expression Objective levels of multiple ClyA fusions Create RFC 10 Honeydew, Create Fluorescent Strawberry and Tangerine Biobricks Protein Biobricks Characterize New Fluorescent Test Fluorescent Protein Devices Protein Biobricks Create ClyA Biobrick Create and Test ClyA-FP devices Co-transform and Measure relative levels of expression
Create ClyA Biobrick • Use RFC 25 (Freiburg Fusion) standard XbaI NgoMIV EcoRI AgeI SpeI PstI A C C G G C Part A Part B T G G C C G Scar Site
Measure relative expression Objective levels of multiple ClyA fusions Create RFC 10 Honeydew, Create Fluorescent Strawberry and Tangerine Biobricks Protein Biobricks Characterize New Fluorescent Test Fluorescent Protein Devices Protein Biobricks Create RFC 25 C-Terminal ClyA Create ClyA Biobrick Fusion Create and Test ClyA-FP devices Co-transform and Measure relative levels of expression
Create and Test ClyA-Fluorescent Protein Devices • Ligate the new FPs and pTet Promoter/RBS to ClyA RBS ClyA FP • Induce the cells • Obtain the vesicles by centrifuging cultures • Measure fluorescence of OMVs in plate reader • Use previous fluorescence results to compare
Measure relative expression Objective levels of multiple ClyA fusions Create RFC 10 Honeydew, Create Fluorescent Strawberry and Tangerine Biobricks Protein Biobricks Characterize New Fluorescent Test Fluorescent Protein Devices Protein Biobricks Create RFC 25 C-Terminal ClyA Create ClyA Biobrick Fusion Measure fluorescence levels of Create and Test ClyA-FP devices OMVs using plate reader Co-transform and Measure relative levels of expression
Measure relative levels of expression • Co-transform ClyA fusions • Express and ultracentrifuge • Check if functionality of both fusions is preserved • Measure relative amounts of each with a plate reader
Accomplishments Create Fluorescent mStrawberry Reporter Shows Protein Biobricks Characteristic Emission Spectrum BBa_K399000 3 mHoneydew Test Fluorescent Protein Devices Percent Increase 2 BBa_K399001 Create ClyA Biobrick mStrawberry 1 BBa_K399002 Create and Test ClyA-FP devices 0 mTangerine Co-transform and 550 575 600 625 650 675 700 Measure relative levels Wavelength [nm] of expression
Future Work Future Work • Construct and co-transform multiple ClyA-protein fusions • Measure relative expression levels • Make and test a streptavidin construct • Check for attachment of vesicles to biotin
Outreach: CURIE Outreach: CURIE • One week summer program for high school girls who excel in math and science • Presentation on principles of Synthetic Biology and iGEM Jamboree • Lab tour • Gel electrophoresis demo
Outreach: CIBT Outreach: CIBT • Unique one-week experience for high school biology teachers • Presentation about synthetic biology • ApE (A Plasmid Editor) software and gel electrophoresis
Thank You! Funding • College of Engineering • Weill Institute • CALS Advisers • Dr. Mathew DeLisa • Dr. Xiling Shen • Pengcheng Bu • Sean O’Brien
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