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Team:UI-Indonesia/Description
From 2013.igem.org
Project Blue Ivy
World’s Problem Waiting to be Solved
Tuberculosis is still one of the worst infection-related health problem. It kills approximately 2 million people annually, making it the number one killer of single infection disease. The global epidemic is growing and becoming more dangerous. Today, tuberculosis has infected approximately a third of world’s population, even after WHO declared a global emergency on tuberculosis on 1993.
Where is the problem? Is it that hard to cure Tuberculosis? No!
WHO’s data about Tuberculosis epidemiology shows that 95% cases and 98% deaths from tuberculosis infection occur in poor countries; at the same time, 78% of world’s population dont have access to proper Tuberculosis diagnostic facilities. This clearly tells us that, the real problem in eradicating Tuberculosis is that we don’t have a reliable diagnostic tool for people in poor countries. A mathematical modelling of TB epidemiology shows that if every man and woman in this planet have an access to appropriate diagnostic facility, the prevalence of the disease will decrease 30% globally. Thus, providing a cheap, portable, reliable and easy to use TB diagnostic tool is exactly what we aim to do.
Our diagnostic tool (or biosensor) consist of an antibody fragment as the sensitive element which binds to antigen 85, a specific protein produced by Mycobacterium sp., and split Beta-galactosidase as the reporter.
The Split Beta-galactosidase a.k.a The Reporter
Our system is based on ternary complexation mediated protein complementation principle. To put it in a simpler way, it is two proteins interacting with mediation of another protein and uses a reporter protein thatis split into two fragments, called as split reporter. Each fragment is then fused to one of the interacting protein of interest. These reporter fragment are individually inactive, and will be re-activated when the two fragments interact and re-assemble into a functioning enzyme which can convert substrate into products and gives a unique signal.
Figure 1. Ternary complexation mediated protein complementation (Source: Stains, C.I., et al. (2010), with slight modification)
We choose split Beta-Galactosidase as our reporter protein based on several considerations. First, Broome et al., (2010), have shown that they were able to split the Beta-galactosidase enzyme into two fragments that are capable to re-assemble and shows stable enzymatic activity in bacteria and in mammalian cells1. Hence, we decided to adopt their split reporter system into our biosensor.
Second, The substrate for Beta-Galactosidase, Xgal, is cheaper than the substrate for some other protein that can also functioned as split reporter, for example, luciferin (substrate for luciferase enyzme). This is important, because our goal is to make a cheap detection system to be used in suburban area.
The signal produced by Beta-Galactosidase is visible so that we dont need another device to define the infection. The signal also lasts for a long time so that we can minimize the false negative that is often occurred in short time observation.
The Single Chain Fragment Variable a.k.a The Sensitive Element
The antibody fragment (scFv) used in our detection system is selected based on the previous experiment conducted by Ferrara et al., (2012)2. They select 111 scFvs from the previously created antibody library3 using flow cytometry. From these 111 scFvs they tested 48 of them for their specificity to Ag85, also by flow cytometry, using biotinylated Ag85; myoglobin and streptavidin-Alexa-Fluor-633 were used as negative controls. Also, they were tested to their recognition for the three different Ag85components.From the result, 14 of them were then selected for ELISA test. Three of the candidates were eliminated, leaving 11 remains. The top eight clones were further tested in an 8x8 sandwich assay, in which yeast displaying all eightselected antibodies were used as Ag85 capture reagents, and testedwith all eight scFv-Fc fusions as detection reagents. To fulfill the need of our system, we need to select two scFvs which recognize two different sites on Ag85. Based on the data on the journal, we decided to choose antibody C11 and A2 for our biosensor.
Figure 2. Antibody selection method (Source:Ferrara, F., et al. (2012))
References:
1. Broome, A.M., et al. (2010). Expanding the utility of Beta-galactosidase complementation: pieceby piece. Mol Pharm. 7(1): 60–74. doi:10.1021/mp900188e.
2. Ferrara, F., et al. (2012) Using Phage and Yeast Display to Select Hundreds of Monoclonal Antibodies:Application to Antigen 85, a Tuberculosis Biomarker. PLoS ONE. 7(11):e49535.doi:10.1371/journal.pone.0049535.
3. Sblattero, D., Bradbury, A. (1999). Exploiting recombination in single bacteriato make large phage antibody libraries. Nature America.
4. Stains, C.I., et al. (2010). A General Approach for Receptor and Antibody-TargetedDetection of Native Proteins utilizing Split-LuciferaseReassembly. ACS Chem Biol. 5(10): 943–952. doi:10.1021/cb100143m.
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