Universal Self Amplifying (USA) Biosensor Clemson University iGEM - - PowerPoint PPT Presentation

Universal Self Amplifying (USA) Biosensor

Clemson University iGEM 2013

Outline

• Introduction
• USA Biosensor System
• Results
• Future work
• Human practices
• Questions

Foodborne Pathogens

• Center for Disease Control (CDC)

estimates that each year roughly 1 in 6 Americans (or 48 million people) gets sick, 128,000 are hospitalized, and 3,000 die of foodborne diseases.

• Department of Defense (DoD)

food safety and QA action levels require zero tolerance for the following pathogens: Esherichia coli O157:H7, Salmonella, Staphylococcus, and Listeria monocytogenes

*2011 estimates of foodborne illness in the united states. (2011, April 15). Retrieved from http://www.cdc.gov/features/ dsfoodborneestimates/

Foodborne Pathogens

• Some possible pathogens are allowed low levels of CFU/g

such as some species of E. coli, which has a limit of 100 CFU/g

• Limitation with PCR detection

– Not able to differentiate between live or dead cells

• Current methods (Biological or Immunoassay) suffer

– Limited detection usually between 1,000-10,000 CFU/g. Most DoD food safety limits of microorganisms like species of E. coli (100 CFU/g) fall bellow this detection range. – Require sample enrichment in order to detect – Only qualitative not quantitative

Our Project:

Universal Self Amplifying (USA) Biosensor

• USA System to optimize pathogen detection:

– Increased pathogen specificity – No pathogen enrichment – Detection of lower CFU per ml or g – Viable cells only – High sample volume capacity – Simple step detection

luxR luxI gfp

LuxR

LuxI

rfp luxI

LuxI

AHL GFP

RFP AHL

AHL

AHL

PCat PLux PLac

RBS RBS RBS RBS SAM

Universal Self-Amplifying Biosensor

SAM

Model Pathogen

RBS

DT DT DT

LuxR LuxR

USA Biosensor System

Results

BioBrick Submitted

• MP grown on minimal medium supplemented with glucose or

lactose to test effect upon lac promoter

UB & USA-B Response to AHL

• Increased GFP emission over time as expected

UB & USA-B Response to Model Pathogen

• Increased GFP emission over time as expected
• The higher concentrations of the AHL-producing K1090000

resulted in higher GFP signal

UB & USA-B With All Three

• Excess AHL produced the highest GFP fluorescence as

expected

• However, non-AHL producing model pathogen and AHL-

producing model pathogen produced almost the same results: as the bacteria grew, the GFP fluorescence increased

Conclusions

• All four constructs on

a Petri plate: MP, RFP, UB, and USA-B

• USA-B has

constitutive GFP expression regardless

• f AHL presence

Future Work

Biosensor

• Further develop enzymatic construct
• Improve promoter stringency
• Determine optimal detection rate
• Evaluate efficiency of multiple copies of

regulator, co-inducer, and reporter

• Develop unique (synthetic) signaling

system

Future Work

Pathogen--Signal Construct

• Replace AHL with unique signal under pathogen-

specific promoter Phage

• Construction of lysogenic bacterial phage

– Specific to pathogen

Human Practices

Clemson Elementary School

• two 4th grade classes
• “bad” bacteria vs. “good” bacteria
• Very curious and imaginative
• Will return and play games

Human Practices

Pendleton High School

• Spoke to 3 classes of juniors and seniors
• Had good discussions about the ethics of genetic

engineering

• Science teachers wanted us to return to teach as a

part of general biology

Human Practices

Student Survey

• Surveyed the students

before and after the presentation

• Had a sample size of 44

students

• Used yes/no responses

and 7-point scales for qualitative responses

Clemson University – iGEM Survey: Before

• 1. Have you ever heard of Synthetic Biology?

Yes No

• 2. What is your opinion on genetic engineering?

• 3. What is your opinion on genetically modified organisms?

• 4. Does it change your opinion when they are called GMOs?

Yes No

• 5. If you could modify a bacterium to perform a task or solve a problem what

would the organism be able to do? A. Degrade pollutants B. Improve food safety C. Detect pathogens D. Produce energy useable by humans E. All of the above F. None of the above G. Other H. If other, please specify below:

Human Practices

1 2 3 4 5 6 7

genetic engineering GMOs

Average Opinion Before and After

before after

Clemson iGEM Team 2013

Questions?