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Revision as of 10:26, 16 October 2018
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Experiment Overview
In wet lab section, we developed the detection system to identify which type the patient is infected with.
Pseudovirus Production
The pseudovirus production phase consists of the following parts: construction, transfection and collection of pseudoviruses.
- Construction
- For the pseudovirus production, we prepared structural gene, and non-structural gene with fluorescence protein gene.
About structural gene, capsid (C), membrane (prM) and envelope (E) are necessary for the formation of viral structure. We prepared pCAG-C and pCAG-prM-E for Serotype I to IV.
About non-structural gene with fluorescence protein gene, we prepared EGFP-FMDV2a, DsRed-Express-FMDV2a, ZsYellow-FMDV2a and AmCyan-FMDV2a, and inserted each of them to non-structural genes of dengue virus. - Transfection and collection of pseudoviruses
- We introduced pCAG-C, pCAG-prM-E and FP-FMDV2a (FP: Fluorescence Protein) into HEK293T cells. After cell culture for about 2 days, we collect the supernatant that contains pseudoviruses produced from HEK293T cells.
Pseudovirus Infection
After collecting pseudoviruses from HEK293T cells, we added them to Vero cells. When they can successfully infect the cells, the enclosed fluorescence protein gene is coded and we tried to estimate the amount by measuring the fluorescence intensity.
Since we split up the structural region and non-structural region, the pseudoviruses produced in our system can infect host cells only once. Thus, it will not spread out of the system and will not be harmful.
Parts Validation
To achieve parts validation in iGEM sliver medal criteria, we registered three parts: DENV2 C, EGFP-FMDV2a and DsRed-Express-FMDV2a. Capsid protein (C) is necessary for the formation of pseudovirus structure in the system.
EGFP-FMDV2a or DsRed-Express-FMDV2a is packed in the pseudovirus and codes fluorescence protein after the virus infects host cells.
