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The protein that is responsible for the fluorescent glow in the mouse is called Green Fluorescent Protein, also known as GFP. in this experiment, we will be scaling up colonies of E.coli that is been inserted with GFP.
What is GFP?
GFP is a protein that has a unique β-barrel structure that consists of 11 β-strands + 1 α-helix + chromophore running through the middle. GFP is one of the most commonly used bioreporter of a gene-expression or gene product at the molecular level.
Though GFP was first isolated and purified in the 1960s by Osamu Shimomura from Aequorea victoria, a type of jellyfish., the true potential of GFP was not comprehended until in 1987 when Douglas Prasher first thought of the idea of using GFP as a ‘tag’ that reports whenever a particular protein was being produced by a cell. Since a protein molecule was extremely small to be observed unless under an electron microscope, why not attach a marker so that it can be easily detected even by the naked eye?
So HOW GFP is inserted into the nucleotide sequence and expressed, for example in the case of this experiment, an E.coli??
A simplify diagram of a nucleotide sequence
A simplify diagram of a GFP inserted nucleotide sequence
Overall Objectives
The main objective of the whole practical is … *drum roll*… To be able to get an A+++ for our report!!!!!! Hahaha just kidding, well that’s about 80% of the whole objective. The other 20% is to learn and experience the idea of scaling up the growth of E.coli inserted with GFP in a step-wise manner from a seed culture all the way to a bioreactor. At the end of the experiment, we also aim to be able to:
The following table shows the functions each parts of the bioreactor.
after that, we realised that the mammalian cell bioreactor is actually slightly different frm the microbial bioreactor that we are using. the mammalian cell bioreactor uses modified impeller (marine propeller type) and modified sparger for gentle aeration, whereas the microbial bioreactor uses flat-blade propeller. the mammalian cell bioreactor does not have the baffles which reduces the shear force that is crucial for the mammalian cells as they are very delicate.
that's all for now. shall post up some more info abt wat we did for the preparation of that stinky LB broth and streaking of the agar plates. (: