PROJECT PROPOSAL PLAN:
Baculovirus is widely used for expressing recombinant proteins. Structural of baculovirus is a rod-shaped nucleocapsid containing genomic DNA. The baculovirus Autographa California multiple nuclear polyhedrosis virus (AcMNPV) used as a vector in many gene therapies studies in the science world. The wild-type AcMNPV does not replicate in mammalian cells but it can transfer then. Due to higher level of gene expression, the baculovirus are active. In insect cell culture it easy to maintain the culture compared to mammalian cells. The infection of Baculovirus can turn off host cell protein expression and impair secretion apparatus of Endoplasmic reticulum (ER)-Golgi pathway proteins. But some strategies have been developed to improve secretion protein. Chaperones proteins are potential on making secrete protein production up to two fold. Secretory alkaline phosphate-EGFP fusion protein (SEFP) as co-expressed with chaperones, calreticulin (CALR) which located in storage compartment associated with ER. Alpha Synuclein (α-syn) and Beta Synuclein (β-syn) is a small protein, α-syn work as a small heat shock protein. Both of them are molecular chaperones.
The aim of this project is to enhance the expression of the SEFP testing chaperones (α-syn, β-syn with Calreticulin as a positive control, and dsRed as a negative control) which may increase and enabled correct folding protein forms. We will test promoters on virus expression levels: gp64 to use if timing of promoter expression/protein production is crucial to expression levels. -------- > more explanation about gp64! How it works?
In order for making Baculovirus based on AcMNPV this project will use Flash BacUltra. It is a new platform technology for the production of recombinant baculoviruses, optimised for producing 100 % recombinant virus by eliminating the need for plaque purification and it also increases protein yield.
The main content:
Firstly, used the plaque assay (describe 4 viruses) to titre viruses (pfu/ml Multiplicity of Infection) and used the alkaline phosphatase activity assay to compare secreted protein in the 4 viruses. After comparing the viruses, continue with the western blot and SDS-PAGE to analyse(marker protein production using anti-GFP antibody), at the end observe the expression in fluorescence microscopy.
The expectation is to see the co-expressed SEFP and chaperones either α-syn or β-syn can deliver enhanced on secretion protein production in BEVS which can improve up to 2.3 fold.
Design Project Proposal:
For this piece of coursework you will prepare a project proposal based on the actual project.
Your full project proposal should be between 2000 and 2500 words and should include:
• a scientific abstract (200 words),
• an abstract for a general audience (200 words),
• an introduction (including aim and objectives) up to 1000 words,
• materials and methods, including a description of the techniques you will use, how your data will be presented and analysed (including appropriate statistical tests),
• references (using Endnote to cite and list references in the bibliography in the Harvard style).