Complete vessel with head plate
Tubing should be attached with zip ties and male or female luer fi ttings to connect addition or harvest tubing. Make sure that the seal between the head plate and the vessel is suffi ciently greased. The head plate is secured by four gnarled headed bolts. These should be tightened slowly, two at a time (bolts that are opposite each other) until tight. This allows for a uniform connection of the head plate seal to the glass vessel and will give the best possible seal. DO and pH probes are inserted into and secured to the head plate. The vessel should contain freshly made media prior to sterilization plus selective antibiotic. The vessel is now ready to be sterilized by following a liquid sterilization protocol. Make sure all openings to the inside of the vessel are covered with aluminium foil.
Tubing for peristaltic pumps with correct luer fi tting
This tubing should not be one continuous tube but should contain three pieces; two longer pieces to attach one to a reservoir bottle and the other to the addition tube on the head plate, and one shorter piece (6 to 8 inches in length) that will be fed through the peristaltic pump head. All ends will have connectible luer fi ttings. The reason for this is that during the course of the fermentation, the peristaltic pump will pull the tubing into the pump head and if there is not a luer fi tting there, it will continue to pull the tubing. This does two things; it gives an incorrect feed rate and it will also increase the chance for binding up the tubing in the pump head and essentially eliminate the fl ow rate as well.
Sparge tubing going from control unit to head plate
The air should be fi ltered pre- vessel with an autoclavable 0.2 μ m disk fi lter, which is installed between the control unit air outlet and the vessel. As a side note, some fi lter units are uni- directional, so keep this in mind when you set up the sparge. It is important to note that all tubing, fi lters and reservoir bottles with tubing and fi ttings should also be autoclaved, dry cycle. Autoclave bags are available and come in a variety of sizes for tubing and fi lters.
Growth media
Typical growth media for an E. coli fermentation is made up of yeast extract, tryptone or soytone, glucose and NaCl.
The addition of dextrose is also used for fed- batch fermentations to get the culture adapted to this carbon source, since this is what you are going to be feeding it with
later in the fermentation. The ratios can vary depending on the user but a general recipe for this 2XYS media is as follows (per liter):
■ sodium chloride (NaCl) – 5 g
■ yeast extract – 10 g
■ soy hydrolysate – 16 g
■ glucose – 10 g
■ pH to 7.4 using NaOH
Mix with 700 ml of sterile water until dissolved and bring to 1 liter volume using a graduated cylinder.
Glucose feed
The feed solution is an important aspect of the fermentation in terms of its formulation and rate at which it is fed to the culture (see Introduction). In order to keep the growing culture satisfi ed with the availability of micronutrients and a continuous source of amino acids, the feed usually contains yeast extract or a more defi ned source of macro and micro nutrients. For ease of use, this carbon feed will be made up of 2XYS (see growth media above) with NaH 2 PO4 (monobasic) at 20 g per liter. This addition of phosphate is crucial to the continued growth of the culture. As stated in the Introduction, the lack of phosphate can negatively affect the growth and production of the recombinant culture. The recipe is as follows (per liter):
■ sodium chloride (NaCl) – 5.0 g
■ yeast extract – 10.0 g
■ soy hydrolysate – 16.0 g
■ glucose – 400 g
■ sodium phosphate (monobasic) – 20.0 g
Add all reagents to 600 ml of sterile water in a 1000 ml beaker and mix until dissolved. To dissolve in less time you may heat the sample. Be careful, because too much heat will start to caramelize the glucose. When dissolved, bring to 1 liter in a 1 liter graduated cylinder. Transfer this to a 1 liter Corning screw cap pyrex bottle by fi ltering through a VacuCap 90 step fi lter (0.8−0.2 μ m) from Pall. Do not autoclave, due to the caramelization effect of the glucose. Under sterile conditions, add a vented addition cap (New Brunswick Scientifi c) to the feed bottle. The cap should have already been prepared by adding tubing and luer fi ttings to the vent ports and reservoir port. The vent ports are connected to 0.2 μ m sterile Acrodisc syringe fi lters (Pall), while the reservoir port is connected to one of the peristaltic pump addition lines. To ensure how much feed you are adding over time, the feed reservoir can be set on a balance that has previously been zeroed. Once the feed line has been primed, the weight can be recorded and this will give confi rmation of the feed rate. This should be checked once or twice during the feed to compare with the calculated feed rate of the peristaltic pump.
Prior to fermentation, the pump tubing should be set up in the peristaltic pump and the feed rates should be determined for each pump used. Each pump will have a setting option accessible through the control unit that will either be in % cycle or ml/min (given the inside diameter of the tubing). If it is % cycle, as is the case with the older New Brunswick Scientifi c bioreactors, you will have to manually determine fl ow rate (ml/min) by setting a % cycle and then collecting liquid in a graduated cylinder for a given time frame. The fl ow rate will not necessarily be linear, so fl ow rates should be determined at different % cycle settings.
10% antifoam
A substance called anti- foam is added to the growing culture to prevent foaming. Foaming is described as the persistent creation of bubbles by agitation and/or excessive aeration.
The creation of excessive foam can compromise the growth of the culture, create contamination issues, and reduce recombinant protein product yield. Antifoams are typically made from silicone- based polymers, non- silicone organics or a mixture of both types. These are generally not added to the culture as a concentrate but as a 10 to 30% solution. In this way, they can be added using a peristaltic pump and you have a lot less chance of adding too much and effecting the culture growth. Too much antifoam added to a culture will effectively kill the culture by interfering with membrane function(s). In small volume fermentations, such as 2 L cultures, this over addition can happen more easily.
Conversely, it was found in the recombinant yeast Pichia, that if the correct amount of antifoam is added, it can have a positive effect on total cell mass of the culture or increased yield in the recombinant protein produced, depending on which antifoam was used [82]. It is not known whether these positive effects can be observed in E. coli fermentations but it may be worth investigating. What has been established by individual researchers or companies, such as Invitrogen or New Brunswick Scientifi c (NBS), is a list of antifoams that are recommended for use in their respective fermentative systems. For E. coli , Antifoam 204 (Sigma-Aldrich) has been recommended by NBS. The 10% Antifoam 204 recipe is as follows:
1. Add 180 ml of sterile water to a graduated cylinder. Add a stir bar and start mixing on a stir plate.
2. Add 20 ml of Antifoam 204 to the graduated cylinder and mix until homogeneous.
3. Pour into a 250 ml autoclavable bottle and autoclave using standard sterilization protocol for liquids.
5 M acid and 5 M base for pH control
For pH control it is necessary to use a concentrated form of acid and base. In E. coli fermentations, the acid and base most commonly used are H 2 SO 4 and NH 4 OH respectively.
Phosphoric acid can be used in place of sulfuric acid but the total organic phosphate added must be monitored carefully.
Since organic phosphate is essential in controlling cellular metabolism, the addition of too much phosphate can effect cell growth and recombinant protein production. Conversely, it can be used carefully as a source of organic phosphate to help in the maintenance of the growth of the growing culture.
To obviate the use of phosphoric acid and use sulfuric acid in its stead, NaH 2 PO 4 can be added to the glucose feed in order to supply the culture with PO 4 . The base, NH 4 OH, is used instead of NaOH because it can be a source of ammonia that is also essential for cell growth. There is no need to autoclave since both are relatively self- sterilizing. The recipe for both acid and base are:
■ 5M H 2 SO 4 :
1. To a 500 ml beaker add 361 ml of sterile water. With a stir bar in place put the beaker into a bucket of ice, low enough in the bucket so that the stir bar can stir when placed on a stir plate.
2. When the water is well mixed, add 139 ml of concentrated H 2 SO 4 (18 M) slowly to the water.
This will cause an exothermic reaction and generate a large amount of heat. Until it cools in the ice, this solution will be very hot 5M H 2 SO 4 , so be careful!
■ 5M NH 4 OH:
1. It is easiest to buy a 5 M solution of this from a good vendor such as JT Baker.
1 M IPTG stock solution in sterile water
Seventy percent IPA is used for sterilization of the fi tting ends prior to connection of the addition tubing from the reservoirs to the vessel. Add:
1. 700 ml of IPA to a graduated cylinder;
2. add 300 of sterile water and mix thoroughly;
3. transfer to spray bottle(s).