Team:Mingdao/Design

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• About Us
• Team Members
• Attribution

• Motivation
• Design
• Experiment
• Proof
• Demo
• Result
• Notebook

• Basic Parts
• Composite Parts
• Parts Collection

• Introduction
• Principal
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• Future
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• Lab Work
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Sobriety test conducted by an alcohol breathalyzer met some problems as mentioned previously. Blood glucose meter is a smart design to measure the glucose concentration in the blood. We’d like to apply this technique to innovate IGEM BLOOD ALCOHOL METER (iMeter).

BLOOD GLUCOSE METER

Blood glucose meter (BGM) is a common commercialized household health care appliance. It is small, hand-held, portable. It is “smart” with an easy-to-use operation interface. Data processing is quick, accurate and reliable. You can obtain the measures within 5-8 seconds.

PRINCIPLE OF BGM

The BGM is an electronic device built with electrodes covered by the enzyme of glucose oxidase (GOX), which catalyzes the oxidation of glucose, and converts it to gluconic acid and hydrogen peroxide (H2O2). When H2O2 meets electrode (e.g., Ag/AgCl), the electrons are generated and give a strength of current which can be measured in amperometry.

GLUCOSE OXIDASE (GOX)

 

 

 

 

 

Glucose oxidase (GOX) is an oxido-reductase existing in several insects and fungi. The redox reaction performed by GOX can generate hydrogen peroxide as antibacterial materials when glucose and oxygen are present. GOX extracted from Aspergillus niger is widely used in glucose-related diagnostics and biotechnologies including biosensors and food industries. In the redox reaction, GOX requires FAD as electron mediators and finally transfer electrons to oxygen to generate hydrogen peroxide with the reduction potential of -97 ± 3 mV at pH 7.4 (Anal Chem. 2014).

ALCOHOL OXIDASE (AOX)

 

 

 

 

 

Alcohol oxidase (AOX) is also an oxido-reductase that catalyzes the oxidation of alcohol and produce aldehyde and hydrogen peroxide. The substrates of AOX are primary alcohols including methanol and some short-chain alcohols such as ethanol. AOX is present in the methylotrophic yeast like Pichia pastoris, which has two genes, AOX1, AOX2 under a strong methanol inducible promoter. The yeast uses AOX genes to metabolize methanol as a carbon and energy source. The AOX enzymes were used in biosensors (Biosens Bioelectron. 2005) but with a limited application because its electrochemical properties including reduction potential are not well studied.

DISPLAYING AOX ENZYME ON THE CELL SURFACE OF E. COLI

Protein induction and purification may cost much time up to 12 hours and need tricky technical skills. To simplify the procedure of AOX enzyme application, we obtained the idea from the project of an iGEM Team – NCTU_Formosa in 2015. A protein can be displayed on the cell surface of E. coli by fusing to Lpp-OmpA. Lipoprotein (Lpp) is a major outer membrane of E. coli which interacts with the peptidoglycan to maintain the structure and function of cell membrane. Another transmembrane protein called outer membrane protein A (OmpA) is involved in bacterial conjugation and phage infection. A study showed that Lpp-OmpA hybrid can direct the heterologous protein GFP to the external surface of E. coli (Enzyme Microb Technol. 2001). Bacillus lipase (J Microbiol. 2014) and Fungi xylanase (Curr Microbiol. 2015) were demonstrated to be displayed on the cell surface of E. coli and maintained the functional enzyme activities. We’d like to display the AOX enzyme on bacterial surface by fusing with Lpp-OmpA and apply to the electrochemical analyzer by depositing on the test strip.

DEVELOPING IGEM BLOOD ALCOHOL METER (iMeter)

In order to replace GOX with bacteria displaying AOX on the cell surface to the sample test strips and apply it to the electrochemical analyzer, we’ve conducted the following experiments to demonstrate it works.

GENE CLONING

AOX genes synthesized by IDT and Lpp-OmpA got from NCTU-Formosa were amplified by PCR followed by restriction enzyme (RE) digestion, and finally cloned onto pSB1C3 based on the biobrick standard assembly rule. The recombinant vectors carrying the AOX genes were checked by colony PCR and RE, and further confirmed by sequencing.

PROTEIN EXPRESSION

GFP reporter gene was expressed and the fluorescence level was assayed to test our gene expression system.

PROTEIN ANALYSIS

Coomassie blue staining was performed on SDS-PAGE to check the expression of AOX gene.

ENZYME ACTIVITY ASSAY

H2O2 production assay was conducted as AOX enzyme activity assay, demonstrating that AOX catalyzed the oxidation of alcohol and generated a product of hydrogen peroxide.

MODELING

An electrochemical simulator was utilized to study the electrochemical properties of AOX enzyme. The experiment was performed by a collaboration with the biotech company, BIONIME CORP. who is a worldwide leader in blood glucose meter innovation.

This year, we’ve done all the experiment to prove the function of AOX and demonstrated the electrochemical activity of AOX with alcohol. Now we’re going to develop IGEM BLOOD ALCOHOL METER (iMeter) by creating an alcohol test strip to apply in an electrochemical analyzer (i.e., blood glucose meter).