Objectives
- To determine the steps to prepare a bioreactor for fermentation.
- To prepare the media required for seed culture fermentation and scale-up fermentation.
- To prepare seed culture required for scale-up fermentation.
The steps below outline what was done by our TSO to prepare the various equipment required to carry out our fermentation.
Day 1
1) First we have to ensure the fermenter is clean and sterile. The whole fermenter was placed into a autoclave machine at 121 degree Celsius for 20minutes. However before doing so, we have to remove all the probes except the temperature probe as it would not be damaged during the autoclaving process. All silicon tubing should be clamped except those for exhaust filter and the female STT coupling of the sampling unit. Aluminum foil is then used to cover all filters and sockets to prevent condensation as these sites.
2) Certain probes require extra steps before they can be placed into the fermenter for use, such probes include the pH electrode probe, which require calibration using specific buffer solutions.
3) The pO2 probe on the other hand is required to be polarized for at least 6 hours, and later calibrated by aerating the probe with nitrogen.
4) When both probes and fermenter are ready, we can install the probes on the fermenter.
Certain probes such as the foam and level probe can be adjusted according to experimental needs.
The probes are also plugged into the machine at their specific jacks
Other accessories are added onto the fermenter, such as exhaust condensers, air inlet and exhaust filters and manual sampler unit.The needs for such accessories are,
6) The additional reagent lines are then connected at this stage. Such addition reagents include, base (sodium hydroxide), acid (sulphuric acid) and anti-foam. The lines should pass through the peristaltic pumps, at the correction location. The use of such pumps aaaaaaallows fluid to be pumped slowly into the fermenter.
7) The picture below shows the peristaltic pumps. Selecting the Auto function allows the computer to control the amount being pumped which allows the computer to control the environment. Selecting the Manual function which is basically an “ON” switch, causes the respective reagent to be continuously pumped into the fermenter. Selecting the Off function which is basically an “OFF” switch deactivates the pump.
8) Now all the equipment is ready and the parameters that you wish to monitor can be set via the computer’s control panel.
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Part II-Media Preparation Procedure
The steps below outline what we have done to prepare the media for the seed culture and the scale-up fermentation during our practical.
Day 1
1) 2 Litres of Luria-Bertani Medium were needed for the experiment (100ml for the shake flask and 1.9 Litres for the fermenter)
2) We used a pre-prepared Luria-Bertani Powder.
3) The content of the powder are as follows:
4) 50 grams of the powder were used to make the 2 liters of Luria-Bertani Medium.
5) 2 portions of 25g of powder was weighed and placed into a 2 liter bottle.
6) The bottle was topped up till the 2 liter mark with distilled water.
8) Since powder took a while to dissolved, many people took turns to shake the mixture.
9) There was no need to adjust the pH of the Luria-Bertani Medium as the powder already adjusted the pH to 7.5. Also due to the fact that we were culturing bacteria cells (E. coli) which are more resistant to pH deviations from the optimum level.
10) The bottle was then labeled.
12) The shake flask was labeled.
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The steps below shows what we did during the 3 days to prepare a seed culture for the fermenter via scale-up fermentation.
Day 1
1) A cryovial of pGLO transformed E. coli was obtained from the -80oC freezer and thawed. Luria-Bertani Agar plates with ampicillin and arabinose were also obtained.
2) Using a disposable inoculating loop, the pGLO transformed E. coli were streaked onto the Luria-Bertani Medium with ampicillin 100ug/ml and arabinose 0.2% to obtain single colonies.
3) The bench was then swapped clean with 70% ethanol solution to sterilize it.
4) The Bunsen flame was ignited to ensure a sterile environment
5) The streaking can then begin.
6) The plate was labelled and left to incubate for 24 hours
Day 2
7) The agar plate which was plated the previous day was obtained. It was placed under UV light to view for any pGLO transformed E. coli growth.
However as you can see above, there was no cell growth; hence we were unable to use the plate to culture cells into the shake flask. There are many possible reasons which could account for the absence of cell growth. The E. coli cells provided to us may have not survived the freezing and thawing process or the culture in the vial was not mixed before streaking.
Luckily our dear TSO, Yong Hao had prepared a plate prepared for us in-case things like this happened.
In the image above you can see, his plate grew very well.
8) Now using disposable inoculation loop.
9) One of the group members took a big loop full of bacteria and inoculated it into the shake flask.
10) Note the Bunsen burner is also ignited, as to prevent contamination of the shake flask.
11) The shake flask was now left to incubate for 24 hours in a 32oC incubator.
12) The shake flask medium is made hence to be used for inoculation of the fermenter for a scale up fermentation.
Day 3
13) We obtained our shake flask from the incubator
As you can see from the image above, there was cell growth, as the medium was no longer clear when we first inoculated.
However based on his experience, Yong Hao suggested we used his flask which he probably had left to incubate for a longer time for inoculating the fermenter. This is because the cell growth in our shake flask was rather low; hence it might not be enough to do a proper inoculation on the fermenter.
14) Before inoculating the fermenter, Yong Hao assisted in adding ampicillin and arabinose into the Luria-Bertani Medium which he has transferred into the fermenter.
15) The ampicillin was added to prevent any unwanted organism or contaminants from growing in the fermenter, while arabinose was added to induce the production of GFP.
16) The culture from the shake flask was pumped into the fermenter for inoculation, which Ms Ang assisted us in.
Note the colour difference between the fermenter medium before and after inoculation
The fermenter was left to run on its own, while the computer and probes constantly measured any changes in the pH, Foam, temperature and dissolved oxygen level, and try to keep them constant.
The green fluorescent protein or GFP which was first isolated from Aequorea Victoria (jellyfish species), is able to give off green colour fluorescent light when illuminated with ultraviolet light (UV).
GFP from Aequorea Victoria has an excitation peaks at wavelengths of 395nm and 475nm where the GFP absorbs light causing its electrons to become excited and emit green light at 509nm.
GFP can be used in applications such as biomarkers or bioreporters where the GFP gene could be incorporated into genes encoding for proteins and be translated out together with the protein of interest. This allows the location and the movement of the protein to be detected. This can serve to be a useful tool as it allows us to determine what are the protein’s target site in the body or when they are made. Another advantage that GFP possess is that unlike other small fluorescent molecules used previously, GFPs are less harmful to living cells when illuminated. Hence GFP is a valuable marker of gene expression.
This blog is created by a group of year 2 students from Nanyang Polytechnic's Diploma in Molecular Biotechnology.
