Team:Stony Brook/HP/Silver

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Human Practices

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Insight on the application of a blood test using the GPC1 detection system

Our iGEM team members met with an internal medicine specialist, Bibi Zainul, MD., of Stony Brook Hospital, and discussed our project idea as well as its future applications. We spoke about the viability of using our yeast as a non-invasive early-detection tool by performing periodic blood tests on patients who are more susceptible to pancreatic cancer. She made us aware that the tools we would require to perform such a test, such as a centrifuge and UV lights, are not typically available in most private practice offices and only in some hospitals, which is a concern we would need to address in a future application of our project. She also offered us advice on how to move forward and redesign the application of our project. From her advice, we decided that it would be best to use our project as a lab test that a doctor could order and could be performed off-premises. This affects our project in the following ways:

• Despite going through an intermediate service, reproducible yeast could still be a cheap alternative to current methods of early detection. However, there is the concern that making the test through a third party could increase costs.
• Alternatively, we could optimize our test to make it more hospital-friendly, by selling our modified yeast sensors with a UV light and mini-centrifuge in a comprehensive kit that private practices and hospitals can purchase and have in their offices.

Help with the Design of an HBsAg Expression System

Throughout the summer, Emmanuel Asare, Ph.D., in the Stony Brook University Department of Molecular Genetics and Microbiology, helped our team with the design of a workable system for expressing HIV-B antigen that would be secreted from yeast cells. We needed to develop a construct that would allow us to couple the production of the surface antigen with the production of a visible marker for easy recognition of production, and so decided initially to build a composite part using promoter regions in front of each of the coding sequences. Dr. Asare introduced our team to the adaptable functionality of the common picornaviridae 2A peptide, which is used by viruses to easily cleave proteins without the ribosome needing to be removed form the plus-strand RNA. Ribosomes scanning over 2A during the process of elongation will cleave nascent peptides between a glycine and incoming proline amino acid, which they cannot bind together. As a result, two distinct proteins will be created. We were able to insert 2A between our marker, amilCP (a blue chromoprotein) and the coding sequence for the HIV-B surface antigen in our construct. We placed the 2A and HBsAg coding sequence downstream from the amilCP coding sequence. This eliminated the need for separate regulatory units that could otherwise have affected expression levels. A 2A-based system ensures protein production in a definitive 1:1 ratio for scanning ribosomes.