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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:
ahem.. according to the prac manual, we're suppose to set up the bioreactor as well. but.. it's so nice of the lab tech to set it up for us. think maybe they were afraid that we wld mess up the whole bioreactor?
anyway. this is what we HAVE to do if we were to set up the bioreactor by ourselves.
first, we must calibrate the pH electrode by using the standard buffer solution, meaning we must measure the optimun pH levels with 2 buffers, a pH7.0 buffer and either an acid or a base buffer, depending on the range of pH that is to be measured.
then have to install the pH, pO2, foam and level probe...
the height of the foam and level probe must be adjusted if necessary. After connecting the 4 probes, more connections.. the weak acid, base and antifoam..
other accessories (air inket, exhaust filter, cooling jacket sampling unit etc) for the 'pretty' bioreactor must also be connected before using it. After dressing up, the bioreactor is ready for sterilization. Those equipments that are heat sensitive are being wrapped up in aluminium foil..
the pO2 probe must be polarized then calibrate after sterilizing. polarization means putting the probe in an O2 environment to make sure it adapt to the O2 in the bioreactor. this will ensure that the probe will read and measure the amount of O2 at a high rate when the probe is polar. and the last connection is made with the peristaltic pumps, a positive displacement pump used for pumping fluids contained within a flexible tube fitted inside a circular pump casing..