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F1 Karen (Dim Sum Dolly) | M1 Lucas (EMO boy) |
F2 Delia (Chua Hong Hong) | M2 Alvin (Guest-Of-Honor) |
F3 Miki (pied piper) | M3 Zhao Zhi (Mr. BOO) |
F4 Flavia (Ms. pokey) | M4 Mr. Tung Zhi Kang (Kun-jun virus) |
F5 Siti (sweet lil thing) | |
F6 Sajini (she smells nice) | |
F7 Faith (she gives us hope) |
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 temperature used to cultivate E.coli must be regulated. at a high temperature, they will be kill off as their enzymes are denatured (this will be a waste of our efforts). if they are growing at a much slower rate, the operating cost in cultivating E. coli will increase.
E. coli prefers to grow at a pH range of 4 to 8. as they are living organism, they produce wastes and require oxygen. the wastes that these organisms produced are very acidic (ouch~! i doubt ours are the same.) if the solution is not neutralize, or maintain at the optimum pH, the environment will not be conducive for their survival. if the pH is kept under the optimum pH over a period, they can only survive for a few hours to a day.
dissolved oxygen is crucial for their survival. without oxygen, they will not be able to carry out their biological processes and multiply in numbers. if so, due to the insufficient oxygen, they will die (and yes, just like any other living organism!!!). without having enough cells to replace the dead cells, soon, there will be no more live cells.
♥ flavia & faith.