When the bacteria are growing under non-limiting conditions, growth is exponential and consequently the culture will soon run into oxygen limitation. High glucose concentration also causes overflow metabolism, which in E. coli means accumulation of acetate. Both oxygen limitation and acetate accumulation will limit the growth to relatively low cell densities (usually less than OD 10), and also reduce the capacity of the cells to produce recombinant protein. In addition, exponential growth is coupled with very fast synthesis of the recombinant product, which often exceeds the capacity of the cell’s folding machinery and results in product aggregation. When the growth rate is controlled by slow glucose feeding, these problems can be avoided and the cells can grow at a linear rate to higher densities (up to OD 70-80 in EnBase). The slower rate of protein synthesis facilitates higher fraction of the product to fold into native, soluble state.

What is the difference between the liquid medium EnBase Flo and the tabletised medium EnPresso?

Both media compositions are based on mineral salt defined medium for high cell density cultivation and enable controlled glucose delivery by addition of a defined amount of EnZ I´m. The difference between both media can be found in the polysaccharide being used as base substrate leading to different optimal enzyme concentrations for the cultivations.

What is the difference between the three liquid media EnBase Flo, EnBase Flo Min and EnBase Flo Max?

All three media contain small amounts of complex additives to overcome the long lag phase of bacteria during their adaption to the EnBase medium. EnBase Flo Min contains only 20% complex additives from EnBase Flo. Lower concentrations of complex compounds have been seen to be beneficial for production of certain soluble protein. EnBase Flo Max can support the growth of mutants and contains 200% complex additives compared to EnBase Flo.

Does the medium tablet from Enpresso contain nitrogen source?

Yes, the tablet contains substrates with ammonia.

Compatibility of EnBase with my constructs and downstream processes

Is the use of the Enbase system restricted to any strains?

There might be a problem with expression systems, where bacteria secrete proteases into the medium. These proteases could interrupt the cultivation system by the degradation of the enzyme in the medium being responsible for glucose delivery.

What inducers can be used with EnBase? Can I use autoinduction?

EnBase media are compatible with different induction systems. IPTG, lactose, arabinose, rhamnose and other inducers can all be used in EnBase cultivations. It is also possible to combine autoinduction strategy with EnBase by adding autoinducer (lactose or rhamnose) to the culture from the start. However, in most cases the best results are probably achieved by not adding inducer to the culture until after overnight incubation.

Can I use my standard pre-culture method to inoculate an EnBase culture? I would like to prepare the pre-culture in a same way I am doing with LB cultures: growing an over-night culture at 37^oC.

For best possible results, an actively growing culture should be used for inoculation. A certain amount of fresh, living cells used as inoculum is a requirement for a successful EnBase cultivation. Over-night cultures contain mainly cells from the stationary phase. Using only a limited amount of living cells as starter culture will cause glucose accumulation in the EnBase culture and will end up in very poor growth. Alternatively harvested cells from an agar plate grown over night can be used to inoculate EnBase cultures. Please check our website for further details.

Is EnBase compatible with my purification system?

Expression in EnBase can be combined with virtually any purification tag. In purification applications it is recommended that the cell pellet is washed before lysis to get rid of medium components. This is essential for successful MBP-tag purification after expression in EnBase. For other tags the washing step is usually not absolutely necessary, but may improve recovery of the product. Washing of the pellet can be easily done as follows: spin the cells down and discard the supernatant, then wash the cells by resuspending in 0.9% NaCl solution, spin down again and discard the supernatant. Repeat the wash for three times to and then proceed with your protocol for cell lysis/product extraction.

Have EnBase media been tested for production of heterologous proteins using Fungi as the expression host? Specifically Aspergillus niger?

No, fungi have not been used previously. However, a large number of different yeasts, which have similar requirements, have be cultivated using the EnBase Flo Yeast Medium. We would be happy to provide a sample and let you try this for your organism.

EnZ I'm

If I accidentally stored the EnZ I’m and Booster at room temperature for a couple of weeks, can they still be used?

According to our quality control tests a few weeks storage at RT does not cause any detectable change in the performance of either EnZ I’m or Booster, and they can be used without any concern. For periods longer than three to four weeks storage at 4 ^oC is recommended.

Does the enzyme EnZ I´m need to remain on ice during inoculation or induction step?

No, during the pipetting step no cooling is needed. The enzyme is very stabile and should be stored at 4-8°C for longterm storage.

Do I need to optimize the concentration of EnZ I´m for my specific expression system?

BioSilta has optimized the concentrations for typical shaking conditions (amplitude and shaking speed) and commonly used strains (e.g. E. coli BL21 or Bacillus subtilis). In case of non-optimal shaking conditions, other cultivation formats or special strains further optimizations by the customers are beneficial. Recommended test ranges are: 20%, 50%, 100% and 300% of recommended concentration. The better the oxygenation and the higher the specific growth rate of the strain, the higher enzyme concentration will be optimal for good results.

How can I dilute EnZ I´m? I need a lower concentration for suitable pipetting step.

You can dilute the enzyme with sterile water short before usage. This diluted solution is not recommended for storage.

Cultivation temperature

Is EnBase suitable for low cultivation temperatures?

EnBase media has been shown to perform well at cultivation temperatures between 15 to 42 ^oC.

How should I adapt the EnBase protocol concerning enzyme concentration when performing a temperature shift from 30 °C to 18 °C after induction?

First one general comment to the temperature shift: The effect of getting reduced amount of aggregated protein by setting a lower induction temperature to slow down the cell growth can be obtained by simply using EnBase. In EnBase cultivations the cell growth rate is reduced by controlled glucose delivery. We have even seen with Fab production in E. coli that yield at 18 °C was lower than at 30 °C. That´s why we recommend to test also temperature of 25 °C or 30 °C with the normal protocol. If you like to test lower temperatures after induction (15-20 °C) we recommend to test in parallel 50% enzyme concentration described in the manual. But probably the lower glucose consumption rate at lower temperature compensates for the lower enzyme activity and no changes in enzyme concentration is needed.

Do I need to extend the cultivation time when using lower temperature than 30 °C?

It depends on the expression system. If the pH stays stable after 24 h induction, longer expression time might result in higher protein yield. Comparison of different harvest time up to 48 hours might be useful.

Cultivation vessels, shaking speeds and scale-up

What kind of cultivation vessel should I use with EnBase medium?

You can use ordinary Erlenmeyer shake flasks, the high-aeration Ultra Yield Flasks, other types of shake flasks and various sizes of multiwell plates. Also disposable bioreactors can be used with EnBase. Please note that in shake flasks the broth volume should not exceed 10% (20% in Ultra Yield Flasks) of the flask volume (e.g. 50 ml broth in a 500 ml flask) to ensure sufficient aeration. As flask closures we highly recommend the AirOtop airporous membrane seals or other oxygen-permeable closures that allow for high aeration Cotton plugs, foil caps and the like are not recommended due to poor oxygen permeability.

Does EnBase work well in the 96-deepwell plates?

Cultivations done in a 96-deepwell plate are generally very challenging, as the oxygenation is very poor in this format. The success of cultivation with EnBase medium is strongly dependent on the available oxygen, which is determined by the ratio of the liquid surface and the cultivation volume, the shaking speed, the shaking amplitude, the filling volume and the shape of the well. A table showing the oxygen transfer rates in microwell plates in combination with Sandwich covers is published on the homepage of the System Duetz (http://www.enzyscreen.com/1551010.htm). On this website, the oxygen transfer rate was measured when changing the cultivation conditions from a shaker with 25 mm amplitude and 1000 µL cultivation volume to a shaker with 50 mm amplitude and 500 µL cultures; the oxygen transfer rate was more than 12-fold higher. EnBase Flo is suitable for cultivation in 96-deepwell plates. Using E. coli BL21 cultures with 750 µL an optical density of 45 was obtained with a shaker having an amplitude of 50mm, but only OD=25 with an amplitude of 25mm. Higher cell densities are possible when only 500 µl is used.

In our shaker the so-called “diameter orbit” is 1.9 cm. Does it mean the same as the amplitude? And how can this influence the results?

The diameter orbit is the same like amplitude. We have seen some limitations when using 50 mm amplitude due to lower oxygenation. But 1.9 cm is close to 2.5 cm and we recommend enzyme concentrations as described for 2.5 cm amplitude. Best indication for optimal dosing besides cell growth and protein production is the measurement of pH. If pH drops to values lower than 6.5 enzyme concentration should be reduced (e.g. 50% of recommended amount), as this might be a sign of glucose accumulation.

We have only shakers in the lab available, which can be set to a maximum of 200 rpm. Will the protocol work with the lower shaking speed than recommended?

The enzyme doses needs to be adapted, as less oxygen is available. The lower cell growth rate results in lower amount of needed glucose. Reduce the enzyme concentration about 30-50% and do some parallel tests to find the optimum.

Can I scale-up the the experiment to achieve similar results as in the small scale screening experiments?

Generally fed-batch conditions will be provided with EnBase media in all cultivation formats. However, the cell growth and protein production depends on available oxygen. The better oxygen transfer can be ensured, e.g. by Ultra Yield Flasks and gas-permeable AirOTop seals, the higher the productivity. The different oxygen conditions require different optimal EnZ I´m concentrations. BioSilta has listed the optimal EnZ I´m concentrations for the most common shaking conditions. (Check the corresponding datasheet and the webpage for the complete protocol.)

Ultra Yield Flasks and AirOtop Seals

Are the Ultra Yield Flasks autoclavable?

Yes, they can be reused for sterile cultivation by autoclavation.

Antifoam and EnBase cultivations

Do we know if antifoam is needed with the Yeast cultures in UYF?

Cultivation in Ultra Yield flasks can cause foaming, and increased protein content due to cell death and secrated proteins can cause foaming. Antifoam would be a good additive as a precaution, unless it becomes too expensive.

Troubleshooting

When using EnPresso the final cell density of my culture was OD600=5. How can I improve the cellular growth?

There are several critical points you should remember:

take cells from exponential growth phase as inoculum (cells after 6 h growth)
take at least 1:100 dilution from pre-culture, the ideal start OD of your main culture should be about OD600=0.15
add the needed amino acids, in case the slow growth is caused due to auxotrophic properties
add the booster tablet after overnight cultivation (OD600 needs to be at least 5)
ensure very good oxygenation:

by breathable membrane as cover
by high shaking speed (250 rpm)
by filling volume of max 10%

How to face the problem of poor bacterial growth caused by oxygen limitation when using EnPresso?

Providing optimal shaking conditions for the culture oxygen limitations can be partly overcome by adding additional trace elements like Selenium, Nickel and Molybdenium to the medium. This might be an option to activate the anaerobic metabolism and avoid for example the accumulation of formate. (see: Soini et al., High cell density media for Escherichia coli are generally designed for aerobic cultivations – consequences for large-scale bioprocesses and shake flask cultures. Microb. Cell Fact. 2008, 7:26)

There is some turbidity in the EnBase Flo medium bottle. What is it and can I still use the medium?

Slight precipitation of the EnBase substrate may occur during prolonged storage, but this should not cause any detectable change in the performance of the medium. If needed, the precipitates can be removed by filtration through a sterile 0.2-0.45 µm filter, but for usual microbial cultivation and protein expression applications this is not necessary and the product can be used without precautions.

More questions?

Frequently Asked Questions (FAQs)

Latest News/Press Releases

Latest Publications

Where to Find Us

Technology

Products

Applications

Support/Publications

Ordering

About BioSilta