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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:
with the computer control system, the conditions of the fermentor at different time of the day are collected and stored also known as data logging. the system also helps perform calculations based on the mathematical models of the process also known as data analysis. the system also process control and optimization. it sends signals to pumps, valves, switches and alarms to adjust the conditions when no one is there to manually check. to sum up, IT JUST MAKE LIFE EASIER FOR US~! (how great with machines arnd??) oh yes, and we rmbed to take out 10ml medium before inoculation which is for our _____. (in case if u didnt know we meant BLANK!)
let's recap on what we have done for the last 2 days:
seed culture from the streak plate was transferred to the shake flask --> magnified from the shake flask (E.coli cells were PROLIFERATING!!)
dont you wanna know what we did to them?!
okay. we inoculated 100ml of seed culture from the shake flask to the fermentor, this is in line with increasing the yield of desired protein (the GFP), in other words, we were gradually scaling up the fermentation process. how interesting!
steak plate --> shake flask --> fermentor
samples were drawn to monitor cell growth and product formation hourly, and we divided ourselves into pairs. (how wise were we??! (: )
each pair assigned to each hour to take the sample, at the same time computer data logging was set up by our lab tech so we could monitor the fermentor parameters.
to be continued...
♥ flavia & faith.