125mL | Part No. #931147 250mL | Part No. #931144 500mL | Part No. #931141 2.5L | Part No. #931136-B

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Sam Ellis 1, Mark Ultsch 2 , Sandrine Dessoy 3

Thomson Instrument Company is not affiliated with GSK, Genentech (a

fully owned Roche company), Pfizer Inc, Biosilta's Enbase media, Life

Technologies and its Magic Media , Kuhner AG, and New Brunswick Scientific.

For reprints contact folks@htslabs.com

Thomson Instrument Companywww.htslabs.com

800.541.4792

760.757.8080

folks@htslabs.com

#899414

#899415

ULTRA YIELD FLASK™ (SIMPLE DISPOSABLE BIOREACTOR) & ECONOMICAL PARALLEL PROTEIN EXPRESSION SCREENING: SCALE-UP IN E.COLI, YEAST, AND MICROBEST

Ultra Yield Flasks (patented) are a disposable high aeration shake flask or bioreactor . Ultra Yield Flasks achieve high aeration with a 6 baffle design that has been optimized for fermentation for e.coli, Pichia, Pombe, Scizomyces, and other microbes. They provide good airlift with high speed shaking giving an increase of 10 fold oxygenation, and when combined with nutrient enriched media and pH balance, can increase yieldsignificantly. This, combined with the simple AirOTop seal which is made from airporous material, allows for a disposable great aeration system. No cutting of aluminum foil, and exposure to cross contamination with used flask plugs. Ultra Yield Flasks allow for scalable results from the 125mL, 250mL, and 500mL, up to the 2.5L .

How does the yield increase with Ultra Yield Flasks?The Ultra Yield Flasks work to grow more biomass or cells acting as a disposable bioreactor.This leads to an increased yield in protein, DNA, etc. With an increase in density there comes an increase in yield. Cells in Ultra Yield Flasks can be grown for more than 24 hours, because you are not oxygen limited. Cells do not go from logphase to stationary phase but as shown by data (GSK Data ) to a transition phase. This transition stage allows for continued growth without anerobic conditions that use the cells' own nutrients to continue its living, thus without stressing the system. pH maintence is extremely impor-tant in this so buffering with a non-temperaturedependant buffer like MOPS makes a large difference in cell growth with the Ultra Yield Flasks.

Ultra Yield Flasks allow for optimum growth in shake flasks, and allow for scaleable results from the 125mL flask-2.5L flask. In studies, and testing we have found that Ultra Yield Flasks result in consistent standardized results.What ratio can of liquid media can we have in the Ultra Yield Flasks for e.coli and microbes? What are the recommended speeds for e.coli and microbes ?

Media ratio depends on the cells being grown. We are going to give a general rule for e.coli. E.coli use a 40% fill volume. In the 2.5L some people put 500mL for maximum OD, and others use 1L. They use 3-4 flasks to replace a small scale fermentor.

Recommended speeds:

Abstract

Introduction

Media (mL)/Flask40-50mL/flask

75-110mL/flask

125-200mL/flask

500mL (optimum)

1L

Description125mL Ultra Yield Flasks 50/case -- Sterile

250mL Ultra Yield Flasks 50/case -- Sterile

500mL Ultra Yield Flasks 50/case -- Sterile

6/case 2.5L Ultra Yield Flasks -- Sterile

6/case 2.5L (Fermentor Mode)-- Sterile

Shaker Speed300-350 RPM

300-350 RPM

300-350 RPM

300-400 RPM

250 RPM

Part #931147Please cover the flask with AirOtop seals 899415.

931144Please cover the flask with AirOtop seals 899415.

931141Please cover the flask with AirOtop seals 899415.

931136-BPlease cover the flask with AirOtop seals 899414.

931136-BPlease cover the flask with AirOtop seals 899414.

Results

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 101 A1 NI2 A1 I3 A2 NI4 A2 I5 B2 NI6 B2 I7 B4 NI8 B4 I

9 Fcg NI10 Fcg I

*9&10 BL21In Ultra Yield

1 2 3 4 5 6 7 8

Standard 2l Shake Flask w/ 1liter LB

1 2 3 4 5 6 7 8 9 10

Ultra Yield 2.5l Flask w/ 1liter Mod. TB Standard 2L Shake Flask w/ 1liter LB Anti-His Western. Ultra Yield 2.5L Flask w/ 1liter Mod. TB Anti-His Western

Genentech, Inc

Thomson Instrument Company | www.htslabs.com | 800.541.4792 | 760.757.8080 | folks@htslabs.com

GSK

Pfizer

heure culture

0 2 4 6 8 10 12 14 16 18 20 22 24

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10111213141516171819202122232425

pH

4.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.88.0

Acét

ate

(g/l)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0

heure culture

0 2 4 6 8 10 12 14 16 18 20 22 24

Do

0123456789

10111213141516171819202122232425

pH

4.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.88.0

Acét

ate

(g/l)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0

heure culture

0 2 4 6 8 10 12 14 16 18 20 22 24

Do

0123456789

10111213141516171819202122232425

pH

4.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.88.0

Acét

ate

(g/l)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0

heure culture

0 2 4 6 8 10 12 14 16 18 20 22 24

Do

0123456789

10111213141516171819202122232425

pH

4.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.88.0

Acét

ate

(g/l)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0

E.coli : Growth profiles with classical glass flask (2 L, no baffle), 400 mL

Thomson medium, 400 rpm orbital shaker

E.coli : Growth profiles with Thomson flask (2.5L Ultra Yield Flask, baffles),

500 mL Thomson medium, 400 rpm orbital shaker

E.coli : Growth profiles with classical glass flask (2 L, no baffle), 400 mL

medium, 400 rpm orbital shaker

E.coli : Growth profiles with Thomson flask (2.5L Ultra Yield Flask, baffles),

500 mL medium, 400 rpm orbital shaker

610% Yield Increase*

Effect of flask design on E. coli culture growth and production of

recombinant protein. (A) The effect of flask type and growth medium

on the observed optical densities of the cultures at A600 following

overnight protein expression. Cultures carried out in Fernbach flasks

using either LB medium or TB medium are shown, respectively, at the

front (green) and in the center (orange). Cultures grown in Ultra

YieldTM flasks in TB medium are shown at the back (blue). See Table 1

for a list of the recombinant proteins overexpressed by the various

clones. (B) The yields of expressed soluble protein (determined by

protein assay and recorded as mg protein per liter of expression

culture) from the IMAC columns following purification of the twelve

polyHis-tagged recombinant proteins, each expressed under the

three conditions described in Panel A. Clone locations in Panel B are

the same as those in Panel A

*Economical parallel protein expression screening and scale-up in Escherichia coli.

Journal of Structural and Functional Genomics2006 Jun;7(2):101-8. Epub 2006 Dec 23.

A

B

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/L)

25

20

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300

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1 2 3 4 5 6 7 8 9 10 11 12Clone Number

1 2 3 4 5 6 7 8 9 10 11 12Clone Number

O.D. pH Acetate g/L

Thomson Instrument Company | www.htslabs.com | 800.541.4792 | 760.757.8080 | folks@htslabs.com

Ultra Yield Flasks offer a simple alternative to shake flasks, and complex fermentation devices for minimal investment. Thomson Instrument Company with our collaborators of Genentech, GSK, and Pfizer have seen the simple device Ultra Yield Flasks turn mediocre or poor expres-sion into results that are increased up to and beyond 610% as Pfizer reported over 12 constructs. Ultra Yield Flasks can be used for multiple different proteins, antibodies, FABS, MABS, DNA, and different microbial systems, etc.

The data presented in this poster conclusively shows the successful functionality of this device. This is therefore a very simple, inexpensive and unique solution to complex challenges presented by today's ever-changing world of microbial growth.

Fully automated fermentor systems available in the market today range from $15,000 to $90,000 dollars. However, our simple Flask solution can slash that price to a few dollars which fits in any big or small research budget. Apart from the obvious fiscal advantage, the other advantages include the ease of use, the fitting in standard laboratory shaker systems, and the simplicity of construction. Without the need for expensive excess media, time, chromatography columns. It is our hope that Ultra Yield Flasks will become the flask of choice in all biologists’ labs.

What media do we put in the Ultra Yield Flasks? Recommended media is a enriched media with pH balance around 7.4 . TB, EnBase, Magic Media are all good examples of highly enriched medias. The pH balance is most useful with buffers that contain phosphate since these are not temperature dependant. Personally we prefer MOPS at 100 milimolar with any of these medias.

Are the Ultra Yield flasks disposable or re-usable?The Ultra Yield flasks are priced to be a disposable bioreactor or disposable shake flask. Ultra Yield Flasks are reusable, but the cost of the flasks is significantly cheaper than the autoclaving. With labor being around $100.00/per hour it does not make sense to reuse the product? This is why we made the Ultra Yield Flasks a few dollars. If you are to reuse the flasks we would suggest 5 times before any drop off in density, as we know plastic can become brittle over time. Some people have used the flasks multiple times, but remember a little detergent in the flasks in hurting an experiment is more expensive than just using a new one.

Myths and legends of growing cultures ?1. LB is the least healthy growth media. Ultra Yield Flasks are best when using an enriched media.

2. Growing cultures for more than 18 hours will damage my culture. What damages the growth and causes anaerobic growth is the insufficient amount of oxygen, and drop in pH when using normal shake Flasks. Ultra Yield Flasks give sufficient aeration for 7 days worth of growth at 20*C, and 50 hours at 37*C.

What is missing with LB and minimal media? LB or minimal media is like feeding someone bread and water. One needs to get ruffiage, and nutrients and vitamins. This is true with other living organisms as microbes they need a balanced diet to grow strong.

Media choices and recommendations for end users?Different types exist, and by no means is this an endorsement of one over another; TB, Magic Media, Enbase, etc.

What systems are people using with Ultra Yield Flasks?People use the shake flasks, on New Brunswick, ATR, Kuhner, and every shaking platform available.

Conclusion

Materials and Methods

Sam Ellis 1, Mark Ultsch 2 , Sandrine Dessoy 3

1. Thomson Instrument Company E-mail:folks@htslabs.com

Ph:760-757-8080 or 800-541-4792 www.htslabs.com

2. Genentech 1 DNA Way South San Francisco, CA 94080

3. GSK Biologicals Rue de I'Institut 89 Rixensart 1330 Belgium

For reprints contact folks@htslabs.comThomson Instrument Company is not affiliated with GSK, Genentech (a

fully owned Roche company), Pfizer Inc, Biosilta's Enbase media, Life

Technologies and its Magic Media , Kuhner AG, and New Brunswick Scientific.

Thomson Instrument Company | www.htslabs.com | 800.541.4792 | 760.757.8080 | folks@htslabs.com