O M G O M Vs! Outer Membrane Vesicles Spherical proteoliposomes - - PowerPoint PPT Presentation

O M G O M Vs!

• 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

Outer Membrane Vesicles

• OMVs form when portions of cell membrane

bulge, pinch off, and release

OMV Production

• A deletion in the tolRA genes will weaken the

cell membrane, increasing vesiculation

Hypervesiculation

• 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

ClyA

• Vaccination
• Drug delivery
• Single ClyA fusion

constructs

– Fluorescent Proteins – Single-chain antibody fragment

Applications

• 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

Advantages

• Create and co-transform multiple ClyA-

fluorescent protein fusions

• Quantify relative levels of expression

Objectives

Measure relative expression levels of multiple ClyA fusions

Objective

Create ClyA Fusion Proteins

• E. coli

budding OMVs ClyA fusion proteins expressed on surface of OMVs Co-transform with fusions and express

Create Fluorescent Protein Biobricks

Test Fluorescent Protein Devices

Create and Test ClyA-FP devices

Measure relative expression levels of multiple ClyA fusions

Objective Create ClyA Biobrick Co-transform and Measure relative levels

• f expression

• 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

Fluorescent Protein Biobricks

Create RFC Honeydew, Strawberry and Tangerine Biobricks

Create Fluorescent Protein Biobricks

Test Fluorescent Protein Devices

Create and Test ClyA-FP devices

Measure relative expression levels of multiple ClyA fusions

Objective Create ClyA Biobrick Co-transform and Measure relative levels

• f expression

• 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

Test Florescent Protein Devices

Create RFC 10 Honeydew, Strawberry and Tangerine Biobricks

Create Fluorescent Protein Biobricks

Characterize New Fluorescent Protein Biobricks

Test Fluorescent Protein Devices

Create and Test ClyA-FP devices

Measure relative expression levels of multiple ClyA fusions

Objective Create ClyA Biobrick Co-transform and Measure relative levels

• f expression

• Use RFC 25 (Freiburg Fusion) standard

T G C C G C C G A C G G

Part A Part B Scar Site

XbaI NgoMIV EcoRI SpeI PstI AgeI

Create ClyA Biobrick

Create RFC 10 Honeydew, Strawberry and Tangerine Biobricks

Create Fluorescent Protein Biobricks

Characterize New Fluorescent Protein Biobricks

Test Fluorescent Protein Devices

Create and Test ClyA-FP devices

Measure relative expression levels of multiple ClyA fusions

Objective

Create RFC 25 C-Terminal ClyA Fusion

Create ClyA Biobrick Co-transform and Measure relative levels

• f expression

• Ligate the new FPs and pTet Promoter/RBS

to ClyA

• Induce the cells
• Obtain the vesicles by centrifuging cultures
• Measure fluorescence of OMVs in plate reader
• Use previous fluorescence results to compare

Create and Test ClyA-Fluorescent Protein Devices

RBS ClyA FP

Create RFC 10 Honeydew, Strawberry and Tangerine Biobricks

Create Fluorescent Protein Biobricks

Characterize New Fluorescent Protein Biobricks

Test Fluorescent Protein Devices

Measure fluorescence levels of OMVs using plate reader

Create and Test ClyA-FP devices

Measure relative expression levels of multiple ClyA fusions

Objective

Create RFC 25 C-Terminal ClyA Fusion

Create ClyA Biobrick Co-transform and Measure relative levels

• f 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

Measure relative levels of expression

1 2 3 550 575 600 625 650 675 700

Percent Increase Wavelength [nm]

mStrawberry Reporter Shows Characteristic Emission Spectrum

Accomplishments BBa_K399000 BBa_K399001 BBa_K399002

mHoneydew mStrawberry mTangerine

Create Fluorescent Protein Biobricks

Test Fluorescent Protein Devices

Create and Test ClyA-FP devices Create ClyA Biobrick Co-transform and Measure relative levels

• f expression

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

Future Work

• 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: CURIE

Outreach: CURIE

Outreach: CIBT

• Unique one-week experience for high school biology teachers
• Presentation about synthetic biology
• ApE (A Plasmid Editor) software and gel electrophoresis

Outreach: CIBT

Funding

• College of Engineering
• Weill Institute
• CALS

Advisers

• Dr. Mathew DeLisa
• Dr. Xiling Shen
• Pengcheng Bu
• Sean O’Brien

Thank You!