DAY 1! -Experiment 1 and 2

Finally its Week 13 when its our class turn to have our BPT lab. LIKE long man. Quite excited. Firstly, cannot wait to get the experiment started and secondly, cannot wait to finish. Haha.

So, Day 1 of our lab. Firstly, we were split into two groups. One group was sent to the other room to be familiarised with the Bioreactor and its operation and another was to start with the experiment first.

The objectives of the experiment are simple. Firstly, to familiarize with the parts and components of microbial and mammalian bioreactors and secondly, to know the basic operation procedure of a bioreactor.

Since our group, The Buffalo Wings ( name by Fadzly ) was seated nearer to the opposite room, we started with the Familiarisation of the Bioreactor.

We went in and was told to identify and label the different components of bioreactor on our book... but......




















WHAT IS THIS?!

all we ( or at least I ) ever knew of was..























HAHAHAHA.
OKay...

So the group kind of teamed up! and we got certain answers but there were a few unsure ones.. Then, then Ms Ang saves the day!

Slowly, she explains the different parts of the fermentor and their uses.
What we have learnt:


















Labelling of different parts of the fermentor..
and
knowing the functions of these parts..

1. Motor- To turn the impeller
2. Impeller- To mix the media and not allowing the cells to settle down. It is also used to distribute the air.
3. Sparger- To introduce air
4. Baffles- To ensure a better mixing in the fermentor and to prevent whirlpool effect.
5. Inlet Air Filter- To remove contaminants nefore it enters the reactor.
6. Exhaust Air Filter- Not allowing the contaminants or toxins to be released into environtment
7. Condenser- Condense water from the air exiting the reactor
8. Rotameter- Measure the flowrate of gas
9. Pressure Gauge- Measure the pressure of air in fermentor
10. Temperature Probe- Measure temperature in the media
11. Cooling Jacket- Cool or heat the reactor
12. pH probe- Measure pH of the media
13.Dissolved Oxygen Probe- Measure the amount of dissolved oxygen
14. Level Probe- Measure level of media in fermentor
15. Foam Probe- Measure the foam
16. Acid- Sulphuric acid used. Hydrochloric acid not used as it is corrosive.
17. Base- Sodium Hydroxide
18. Antifoam- Reduce amount of foam
19. Sampling Tube- Take sample asceptically from fermentor
20.Control Panel- Control the parameters of the fermentor

After this, we proceeded back into the same room to continue our next experiment which is to Prepare the Media and Seed Culture.

We were briefed on our next instructions and to carry out the experiment. BUT, what is this WHOLE experiment about? Basically, a seed culture which 'contains' Green Fluorescent Protein (GFP) is prepared and scaled up using fermentation.



Green fluorescent protein, GFP, is a spontaneously fluorescent protein isolated from coelenterates, most commonly the Pacific jellyfish, Aequoria victoria. Its role is to transduce, the blue chemiluminescence of another protein, when aequorin interacts with Ca²⁺ ions, inducing a blue glow, by energy transfer into green fluorescent light.

http://www-bioc.rice.edu/Bioch/Phillips/Papers/fig1.jpeg

The overall shape of the protein is made up of eleven strands of b-sheet (green) form the walls of a cylinder. Short segments of a-helices (blue) cap the top and bottom of the 'b-can' and also provide a scaffold for the fluorophore, which is near geometric center of the can. This folding motif, with b-sheet outside and a-helix inside, represents a new class of proteins. Two monomers are associated into a dimer in the crystal and in solution at low ionic strengths.

http://www-bioc.rice.edu/Bioch/Phillips/Papers/fig3.jpeg

Above is a topology diagram of the folding pattern in GFP. The b-sheet strands are shown in light green, a-helices in blue, and connecting loops in yellow. The positions in the sequence that begin and end each major secondary structure element are also given. The anti-parallel strands (except for the interactions between stands 1 and 6) make a tightly formed barrel.

Green fluorescent protein is comprised of 238 amino acids. GFP, has all of its own light handling machinery built in, constructed using only amino acids. It has a special sequence of three amino acids: serine-tyrosine-glycine (sometimes, the serine is replaced by the similar threonine). When the protein chain folds, this short segment is buried deep inside the protein. Then, several chemical transformations occur: the glycine forms a chemical bond with the serine, forming a new closed ring, which then spontaneously dehydrates. Finally, over the course of an hour or so, oxygen from the surrounding environment attacks a bond in the tyrosine, forming a new double bond and creating the fluorescent chromophore. Since GFP makes its own chromophore, it is perfect for genetic engineering.



http://www.apsnet.org/Education/K-12PlantPathways/TeachersGuide/Activities/PlantBiotechnology/text/act4fig.htm


The Aequorea victoria green fluoroscent protein is a 30kDa monomer, cloned in a plasmid which has been inserted within in an EcoRI fragment containing the cDNA sequence of the Aequorea victoria green fluoroscent protein (GFP). The fragment was obtained from (delta)GFP10 by amplification using polymerase chain reaction. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase with primers cleaving at the EcoRI sites and subsequent digestion with EcoRI.


The chemical structure of the chromophore in Aequorea victoria GFP.

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EXPERIMENT TIME!

The experiment started.. with Nurul!

Firstly, the 40g of seed culture was weighed.























After weighing, the culture is poured into a bottle using a funnel.

Done? .. Nope!

Because the weighing scale was too small, the previous step had to be repeated a few times. Again... and again..

Next, 2 litres of LB media is prepared.

2 ladies measuring the volume of water..










































Inoculating asceptically and later, using parafilm to wrap around the plate.


























DONE!

topping up to 2 Litres.. Next, 100mL of the LB medium is transferred into a 500mL shaker flask.

Measuring 100mL of the media using a measuring cylinder..

The seed media!

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Summary of the procedures for Experiment 2!

A. Preparation of Media

1) 2 litres of Lura-Bertani Medium (LB medium) was prepared.
2) 100mL of the medium was transferred into 500mL shaker flask and the remaining 1900mL was used for the bioreactor the next practical.
3) The medium was autoclaved at 121 degrees celsius for 20 minutes.
4) The medium is allowed to cool, followed by adding ampicillin to the final concentration of 100ug/ml to both the seed and fermentation media.
5) The media was kept at 4 degrees celsius till inoculation.

-During practical, our group has only prepared till step 2 where the medium was prepared.


B. Preparation of the Bioreactor

1. The pH electrode was calibrated using a standard buffer solution.
2. The pH probe, pO2 probe, foam and level probe were installed into the top plate.
3. The addtion agent lines for acid, base and antifoam were connected to the fermenter.
4. Other accessories such as exhaust condensers, air inlet and exhaust filters and manual sampler unit were installed.
5. Ensure that the water jacket is filled with water.
6. Sterilization: -All cables were disconnected except the temperature probe. - All silicone tubings were clamped except the exhaust filter and female STT coupling of sampling unit. - All filters and sockets were covered aluminium foil to protect from condensing moisture. - The set up was autoclaved with steam at 121 degrees celsius for 20 minutes.
7. The pO2 electrode was polarised for about 6 hours and the pO2 probe was calibrated by aerating with nitrogen.
8. The addition lines to the peristaltic pumps were connected and the bioreactor was switched to 'auto' or 'manual'.

-The set up of the bioreactor was already done for us during practical by our lovely lab technician!


C. Preparation of Seed culture

1. pGLO transformed E.coli was retrieved from -80 degrees celsius freezer.
2. Streaking of the bacteria was done on a LB/Amp/Ara plate and incubated for 24 hours.

~~The End~~