pLaq U e Out ! in vivo imaging and breakdown of atherosclerotic - - PowerPoint PPT Presentation

pLaqUe Out!

in vivo imaging and breakdown of atherosclerotic plaque

NTU-SINGAPORE iGEM ’09

Atherosclerosis

• Kills millions yearly
• Hard to diagnose early
• Medications don’t cure,
• nly relieve symptoms
• Major surgery

as last resort

• What if plaque breakdown

is possible in vivo?

• Non-invasive
• Painless

Anatomy of a plaque site

Reduced lumen area means faster flow rate Abundant p-selectin expressed on damaged endothelium Blood [NO] levels fall drastically at plaque site Lipid core consists of cholesteryl esters Hard to diagnose

What if?

Engineering a solution

• Detect falling levels of [NO].
• Identify the plaque site.
• Break down the lipid core

enzymatically

• Bonus! Dilate the vessel

locally with NO.

Sensing device Release devices Dilation device

Sensing device is active in high [NO]. Sensing device shuts down in low [NO]. Release & Dilation devices activate. In the vicinity of plaque, [NO] falls. Catch-bond mechanism attaches system to site.

THE GOAL

It actively represses the other constructs.

Degradation device releases enzyme to attack lipid core. Degrading enzyme breaks down cholesterol esters in plaque. Imaging device releases an imaging protein in vivo.

Dilation device

Plaque site is visually identified.

Imaging Degradation

Dilation device releases enzyme to produce NO after programmed delay. As [NO] increases, Sensing device re-activates, shutting down system.

Sensing device Release devices Dilation device

Our Genetic Circuit

Prototype Specifications

LuxR-AHL NO NO TetR TetR

Dilation device

Imaging Degradation Sensing device CHE IFP

Modeling Profile

Simulink circuit representation for modeling

Time-variant model activity

Low [NO] Cholesteryl esterase Infrared fluorescent protein (IFP)

pLaqUe Out!

Low [TetR] Low [TetR]

NO-sensing device Cholesteryl esters degradation device Infrared imaging device

NO-sensing device :

• E. coli K12 genome

NO-sensing construct pNO RBS TetR Term TetR NO Conversion to a standard biobrick by synthesis PCR For enhanced pNO activity *Tucker, N. P., B. D. Autreaux, et al. (2006). "Mechanism of transcriptional regulation by Escherichia coli nitric oxide sensor NorR." Biochemical Society Transactions 34(1): 191 - 194.

NorR expressing construct

J23119 B0034 NorR B0015

Double digestion results. Length

• f system construct corresponds to

expected size ~ 1750 bp

• ve

Ctrl

42 deg C 37 deg

C

30 deg C

NO-sensing promoter (pNO) is

pNO RBS GFP Term Response of pNO to increasing [SNP]

↑Fluorescence with ↑ [SNP]

Test Construct

[SNP] (mM) Time (min) GFP Fluorescence (AU)

Low [NO] Cholesteryl esterase Infrared fluorescent protein (IFP)

pLaqUe Out!

Low [TetR] Low [TetR]

NO-sensing device Infrared imaging device Cholesteryl esters degradation device

Cholesteryl esters degradation device produces cholesteryl esterase in absence of

CHE LuxI Low [TetR]

Cholesteryl esters degradation device breaks down cholesteryl esters

pLacI RBS CHE Term

Test Construct

CHE

Cholesteryl esters degraded!! Fluorescence (AU)

Time (min)

Extent of cholesteryl esters degradation with controls

Untransformed Cells Transformed Cells w induction Background Tansformed Cells w/o induction

Low [NO] Cholesteryl esterase Infrared fluorescent protein (IFP)

pLaqUe Out!

Low [TetR] Low [TetR]

NO-sensing device Cholesteryl esters degradation device Infrared imaging device

In vivo IR imaging device: IFP is a novel reporter protein

• Discovered by Roger

Tsien’s lab at UCSD.

• Excitation (684 nm) and

emission (708nm) do not interfere with blood constituents

• Biliverdin is essential

for IFP to fluoresce Infrared signals in mice

Images by Shu Xiaokun * S. Xiaokun, A. Royant, M.Z. Lin, T.A. Aguilera, V. Lev-Ram, P .A. Steinbach and R.Y Tsien, “Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome” Science, vol 324, 804, 2009.

IR imaging device produces IFP & HO-1

HO-1 IFP

IFP device emits an infrared signal

Test Construct

Emission wavelength (nm) Intensity (AU)

IR signals at different temperatures

IFP is pH dependent

Infrared emission intensity vs pH pH

IFP intensity (AU)

IFP is concentration and temperature dependent

Concentratio n Temperature IR fluorescence intensity (AU) IFP fluorescence vs Temp and Concentration

Human Practices - DIYBio

45% “Neutral” 37% “No”

19% “Yes”

Should DIYBio be banned? Can DIYBio help people engage in science?

61% “Yes”

37% “No” 25% “Neutral”

516 respondents from 11 countries!

Human Practices - DIYBio

Majority of our respondents are willing to accept DIYBio if:

• There is a biosafety management
• Bio-ethics are being addressed

“Have fun but act RESPONSIBLY!”

Our Accomplishments

 Submitted and verified 17 parts.  Characterized 3 working new parts  Characterized 1 existing part  Improved 2 existing parts  We helped 3 teams with their iGEM projects!  We looked into the fledgling hobbyist phenomenon DIYbio for Human Practices Advance

Our Team

Advisors

• Asst. Prof. Lee, Kijoon
• Dr. Ling, Hua

Saeidi Nazanin Nguyen Xuan Hung Wong Choon Kit Sponsors Genentech Singapore Johnson & Johnson Singapore 1st Base Lee Foundation NTU Student Affairs Office NTU Alumni Affairs Office TWC/BIO Pte Ltd Administrative School of Chemical & Biomedical Engineering

• General Office
• Technical Staff
• Ms. Yeo, Kah-Yan (Laboratory Executive)

Tsien’s Lab at UCSD DIYBio Community Jankowski, Tito (DIYbiologist) Cowell, Mackenzie (Founder) Bobe, Jason (Founder)

Our heartfelt thanks to:

pLaqUe Out!

in vivo imaging and breakdown of atherosclerotic plaque