Our objective is to express and purify modified CCMV coat proteins, and to encapsulate HRP within the cavity. Enzymatic activity assays will be carried out, and nanoparticle synthesis will be done varying conditions, such as silver nitrate concentration, temperature and pH.
Modified CCMV coat protein production and purification
HRP encapsulation and Ag-NP synthesis
Nanoparticle analysis to evaluate size distribution and optical properties
A modified gene with a negatively-charged N-terminus was synthesized by Genscript, along with an RBS and IPTG-induced promoter. A double terminator was also included. The cassette is to be transformed into calcium competent E. coli DH5-α.
IPTG is to be used to induce gene expression after the cell culture surpasses a certain OD, indicating sufficient growth. Cells are then separated from the media by a series of centrifugation cycles. A dialysis is to be carried out to separate most of the cell debris left behind and keep proteins, which are then taken to an ultracentrifuge. The resultant purified protein is then kept in an appropriate buffer.
VLPs are to be reassembled in a solution containing HRP, encapsulating the enzyme. Colorimetric assays using TMB with hydrogen peroxide will be used to demonstrate the activity of the enzyme within the viral capsid. Silver nitrate, a precursor for nanoparticle synthesis, will be added to a solution containing the VLPs, and with the addition of the substrate, produce Ag-NPs. Synthesized particles are to be analyzed by transmission electron microscopy and dynamic light scattering.
All material used in the experimental part of the project are not pathogenic or hazardous. According to NIH Guidelines for Research Involving Recombinant DNA Molecules, (May 1999) the classification of biohazardous materials can be divided into four groups. Of these, CCMV corresponds to the first, not being associated with any health risk in humans. No threat against plants could occur because of the lack of the viral genome.
Note: Because of the late delivery of the synthetic gene, this part of the project was not completed in time to be included in this year’s wiki.