Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/10—Preservation of living parts
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/10—Preservation of living parts
  • A01N1/16—Physical preservation processes
  • A01N1/162—Temperature processes, e.g. following predefined temperature changes over time
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/04—Preserving or maintaining viable microorganisms
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/848—Escherichia
  • Y10S435/849—Escherichia coli

Definitions

• This invention relates to a method for producing cells which are competent for transformation and which may be stably stored for extended periods of time at various temperatures.
• the method involves growing cells in a growth conducive medium, rendering said cells competent, and lyophilizing said competent cells.
• the invention further relates to competent cells produced by such a method, to methods of transforming said cells with a DNA molecule, and to a method of producing a desired protein or polypeptide from said transformed cells.
• E. coli E. coli
• these factors include the addition of other cations such as Mg, Mn, or Rb to the Ca-treated cells as well as the prolonged incubation of the cells in CaCl 2 .
• the efficiency of transformation of E. coli cells is substantially enhanced by the method described by Hanahan (JMB (1983), hereinafter referred to as "Hanahan (1983)").
• Hanahan (1983) the cells are grown at 37°C in the presence of 20 mM Mg.
• Plasmid DNA is combined with the cells at 0°C in the presence ofMn, Ca, Rb or K, dimethylsulfoxide (DMSO), dithiothreitol (DTT) and hexamine cobalt chloride.
• Competent cells of several strains of Escheric a coli (E. coli) prepared by the latter method have transformation efficiencies of from 1 to 5 x 10 8 transformants/ ⁇ g plasmid DNA.
• frozen competent cells prepared by methods such as those summarized above have transformation efficiencies of about 1 x 10 8 transformants/ ⁇ g plasmid DNA. These competent cells can be stored at -80°C for several months without significant loss of transformation efficiency. However, cells prepared by the methods outlined above are extremely unstable when stored at temperatures higher than -80°C ⁇ e.g., -20°C). Stable storage of competent cells at higher temperatures is highly desirable, since many research labs do not have access to -80°C freezers.
• the present invention relates to a method which allows competent cells to be lyophilized and stored for extended periods of time at various temperatures (- 80°C to room temperature) without appreciably losing transformation efficiency.
• the method of the invention provides competent cells which do not require specialized storage conditions (e.g., extremely low temperatures) to maintain the transformation efficiency of such cells.
• the method of the invention comprises growing the cells in a growth conducive medium, rendering said cells competent, and lyophilizing said competent cells.
• the cells are rendered competent by incubating the cells in a competence buffer, preferably at a low temperature (e.g., 0°C to 4°C).
• the cells are then lyophilized in the presence of a cryoprotectant.
• the invention further relates to competent cells produced by such a method.
• the cells are Escherichia cells, most preferably E. coli.
• the E. coli cells are selected from the group consisting of DH5 ⁇ and DH10B.
• the invention relates to a method for transforming the lyophilized competent cells produced by the above-summarized method with a DNA molecule.
• the invention also relates to the transformed cell produced by this method.
• the invention in another embodiment, relates to a method of producing a desired protein by first obtaining a lyophilized competent cell produced by the above method, transforming the lyophilized competent cell with a DNA molecule capable of expressing the desired protein, and then culturing the transformed cell under conditions sufficient to produced the desired protein.
• the invention also relates to a protein produced by this method.
• Figure 1 is a graph showing the transformation efficiency of competent E. coli DH5 ⁇ that had been lyophilized and stored at -20°C in NUNC cryovials, where the cryoprotectant used was either trehalose or sucrose.
• Figure 2 is a graph showing the cell viability of competent E. coli DH5 ⁇ that had been lyophilized and stored at -20°C in NUNC cryovials, where the cryoprotectant used was either trehalose or sucrose.
• Figure 3 is a graph showing the transformation efficiency of competent E. coli DH5 ⁇ that had been lyophilized and stored at -20°C, where the cryoprotectant was trehalose, and the product was vacuum sealed in glass vials.
• Figure 4A is a graph showing the transformation efficiency of competent E. coli DH5 ⁇ that had been lyophilized and stored at -20°C, where the cryoprotectant was trehalose, and the product was vacuum sealed in glass vials.
• Figure 4A is a graph showing the transformation efficiency of competent
• FIG. 4B is a graph showing the transformation efficiency of competent E. coli DH5 ⁇ that had been lyophilized in NUNC cryovials, where the cells were first frozen in liquid nitrogen, lyophilized, and then stored at -20°C for varying periods of time.
• Figure 5 is a graph showing the cell viability of competent E.
• coli DH5 ⁇ that had been lyophilized in NUNC cryovials and stored at -20°C for varying periods of time, where lyophilization and storage at -20°C is preceded by either freezing at -80°C for 16 hours, or freezing in liquid nitrogen.
• Figure 6 is a graph showing transformation efficiency of competent E. coli DH5 ⁇ that had been lyophilized according to Example 3.
• Figure 7 is a graph showing the cell viability of competent E. coli DH5 that had been lyophilized according to Example 3.
• Figure 8 is a graph showing the transformation efficiency of competent E. coli DH10B that had been lyophilized in glass vials which were vacuum sealed, and stored at -20°C, where the cryoprotectant used was either trehalose or sucrose.
• DNA Molecule Any DNA molecule, of any size, from any source, including DNA from viral, prokaryotic, and eukaryotic organisms.
• the DNA molecule may be in any form, including, but not limited to, linear or circular, and single or double stranded.
• Non-limiting examples of DNA molecules include plasmids, vectors, and expression vectors.
• Cloning vector A plasmid, phage DNA, a cosmid, or other DNA molecule which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of restriction endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a DNA fragment may be spliced in order to bring about its replication and cloning.
• the cloning vector may further contain a marker suitable for use in the identification of cells transformed with the cloning vector. Markers, for example, provide tetracycline resistance or ampicillin resistance.
• Expression vector A vector similar to a cloning vector but which is capable of expressing a gene which has been cloned into it, after transformation into a host.
• the cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.
• competent cells which can be "stably stored” are able to withstand storage for extended periods of time at a suitable temperature, without appreciably losing their transformation efficiency.
• the competent cells maintain about 40% to 100% preferably 60% to 100%, more preferably 70% to 100%, and most preferably about 80% to 100% of their original transformation efficiency (just after lyophilization) during a storage period of 30 days, preferably 60 days, more preferably 90 days, and most preferably 120 days, at a temperature of -20°C.
• Suitable storage temperatures vary from about room temperature to about -180°C.
• the storage temperature ranges from about 4°C to about -80°C, more preferably from about -20°C to about -80°C.
• the cells are stored at about -20°C.
• the storage period or time may range from about 0 days to about 150 days, preferably from about 240 days to about 365 days, and more preferably from about 365 days to about 450 days, although longer storage times may be used at temperatures of about -20°C and below.
• Competent cells Cells having the ability to take up and establish an exogenous DNA molecule. Substantially pure. As used herein means that the desired purified protein is free from contaminating cellular components which would be associated with the protein in nature. "Substantially pure” does not indicate that the protein must be completely free of all contaminants.
• the method of the invention provides for the production of cells which are competent for transformation and which may be stably stored for extended periods at various temperatures. Both gram negative and gram positive prokaryotic cells (e.g., bacteria) can be used in accordance with the invention.
• prokaryotic cells include, but are not limited to, Escherichia sp., Klebsiella sp., Salmonella sp., Bacillus sp., Streptomyces sp., Streptococcus sp., Shigella sp.,
• Staphylococcus sp. and Pseudomonas sp.
• species within each aforementioned genus include Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, Salmonella typhimurium, Streptomyces aureus, Streptococcus utans, Streptococcus pneumoniae, and Pseudomonas syringae .
• the cells which are rendered competent by the claimed method are Escherichia, most preferably E. coli.
• E. coli strains which can be made competent by the claimed methods include DH5, DH5c , DH10, DH10B, HB101, RR1, JV30, DH11S, DM1, DH10B/p3, DH5 ⁇ MCR, DH5 ⁇ 5'IQ, DH5 ⁇ 5', SCSI, Stab2, DH12S, DH5 -E, DH10BAC,
• the cells to be made competent are grown in a growth conducive medium. Any medium capable of supporting growth of the cells to be made competent can be used.
• Such media include but are not limited to Luria Broth, Luria Broth supplemented with 20 mM MgCl 2 , 0.001% thiamine, and 0.2% glucose; SOB medium containing 0.001% PPG (recipe given below); and 15/10 medium (recipe given below).
• Other suitable media will be readily recognized by one of skill in the art.
• the incubation temperatures for growing the cells may vary from about 10° to about 42°C. Preferably, the temperatures range from about 12° to about 37°C, more preferably from about 15°C to about 32°C, and most preferably from about 20° to about 25°C. In a preferred aspect of the invention, the cells are grown at about 23 °C.
• growth conditions and culture age can affect both the viability and the transformation efficiency of cells following cryopreservation.
• Cells grown in shake flask culture are generally more resistant to the stress of freeze-drying than are static broth cultures.
• the age of a culture also effects the ability of the culture to survive freeze-drying.
• cells harvested in late log or early stationary growth exhibit the greatest resistance to freeze-drying.
• the cells are grown in shake flasks, although other means of growth may be used including fermentators.
• Shake flasks used in the invention can be of any size and any type.
• baffled 2.8 L liter shake flasks are used for this processing.
• Incubation times will vary according to the conditions used (temperature, medium, aeration, etc.) and the cell type. Aeration in flasks varies according to the rotation per minute (rpm) used, with higher rpms resulting in higher aeration. Flasks are typically shaken at 100-500 rpms, preferably 200-400 rpms, and most preferably 200-300 rpms, although one of ordinary skill in the art may determine other preferred ranges. Cells are typically grown for a time and under conditions sufficient to reach an optical density (OD) at 550 nm between 0.1 to 2.0.
• OD optical density
• the OD ranges from 0.1 to 1.0, more preferably from 0.3 to 0.8, more preferably from 0.5 to 0.8, even more preferably from 0.5 to 0.7, still more preferably from 0.6 to 0.8, and most preferably from 0.66 to 0.75.
• the cells may be collected for further processing.
• the cells can again be reinoculated and the growth process repeated until a culture of sufficient optical density is obtained
• the cells may optionally be chilled (e.g., 0° to 4°C for 5 minutes to 2 hours) Collection of the cells may be accomplished by centrifuging the cells to obtain a cell pellet
• Collection may also be accomplished by concentrating the cells and then centrifuging the concentrated cultures to obtain a cell pellet
• Methods of concentrating the cells include, but are not limited to, dewatering the culture, filtering, or subjecting the culture to size exclusion chromatography, e.g. using CentriconTM columns (Amicon Corp , Lexington, MA)
• competence buffer is any solution that enables cells to take up and establish exogenous DNA.
• competence buffers include 50 mM CaCl 2 , 10 mM Tris/HCl (Maniatis, T et al., Molecular Cloning A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY (1989)), 0 1 M MOPS (pH
• the cells suspended in the competence buffer are incubated for a sufficient time and at a temperature sufficient to make the cells competent to DNA uptake
• the cells are incubated at low temperature (0 to 4°C) for 0 to 3 hours, more preferably 5 min. to 1 hr., and most preferably 5 min. to 30 min.
• a cryoprotectant may be added directly to the cell suspension.
• the cells are collected and then resuspended in a cryoprotectant.
• the concentration of the cryoprotectant will vary depending on the cell type, buffers used, the type of cryoprotectant and other factors. Optimal conditions can be determined by one skilled in the art without undue experimentation.
• Cryoprotectants provide protection of the cells during the freezing process by depressing the freezing point, minimizing the effect of solution changes external to the cell, penetrating the cell to protect against solute concentration effects, and/or shifting the optimum cooling rate to lower values (F.
• cryoprotectant must not be toxic to the cells. Further information on the preservation of living cells by freeze drying may be found in Simione, 1992.
• cryoprotectant and combination thereof may be used in accordance with the invention.
• the type and amount used may vary depending on the cell type and conditions used.
• Cryoprotectants that can be used in the present invention include, but are not limited to, carbohydrates and carbohydrate derivatives such as trehalose, sucrose, lactose, maltose, mannitol, galactose, ribose, fructose, xylose, mannose, dextrose, glucose, and sorbitol, and polymers such as polyethyleneamine, polyvinylpyrrolidone (PVP), ficoll, etc.
• PVP polyvinylpyrrolidone
• cryoprotectants which can be used in accordance with the invention, such as acacia gum, albumin, gelatin, and sugar alcohols, will be readily recognized by one skilled in the art.
• the cell suspension may be aliquoted into containers to be used for lyophilization and storage, such as chilled cryovials, e.g., NUNC tubes (Gibco BRL, Gaithersburg, MD, Cat. No. 366656), or glass vials (Wheaton, Millville, N.J.)
• chilled cryovials e.g., NUNC tubes (Gibco BRL, Gaithersburg, MD, Cat. No. 366656), or glass vials (Wheaton, Millville, N.J.)
• the cells Prior to lyophilization, the cells are frozen at about -20°C to about -180°C, preferably at about -80°C to about -180°C, most preferably about -80°C Methods of freezing a sample to a temperature from about -80° to about -180°C are well known in the art.
• lyophilization is a process by which ice and/or moisture is removed from frozen cells by sublimation under vacuum at low, subzero temperatures (e.g. , -40° to -50°C). Any residual moisture associated with the "dried" preparation is then removed by gradually raising the temperature, resulting in evaporation.
• lyophilization comprises subjecting frozen cells to a vacuum under conditions sufficient to substantially remove moisture and/or ice from said cells (also referred to herein as substantially dried cells).
• the substantially dried cells may then be stored at various temperatures (room temperature to about -180°C, preferably about 4°C to about -80°C, more preferably about -20°C to about -80°C, and most preferably about -20°C).
• One such process for lyophilizing cells comprises the steps of
• the vacuum is less than about 100 ⁇ m, and the cells are dried by:
• the cell container may then be sealed and stored for extended time at various temperatures.
• the viable cell count of competent cells produced by the method of the invention will remain at greater than about 1 x 10 7 cells/ml, preferably greater than about 1 x 10 8 cells/ml, and more preferably greater than about 1 x 10 9 cells/ml when stored at -20°C for any time period from about 0 days to about 450 days, preferably from about 240 days to about 365 days, and more preferably from about 365 days to about 450 days.
• These cells will retain a transformation efficiency of at least about 1 x 10 5 , preferably at least about 1 x 10 6 , more preferably at least about 1 x 10 7 , still more preferably at least about 1 x 10 8 and most preferably at least about 1 x 10 9 transformants per microgram of DNA (T/ ⁇ g).
• Suitable storage temperatures vary from about room temperature to about -180°C.
• the storage temperature ranges from about 4°C to about -80°C, more preferably from about -20°C to about -80°C.
• the cells are stored at about -20°C.
• the storage period or time may range from about 0 days to about 45 days, preferably from about 0 days to about 90 days, still more preferably from about 0 days to about 150 days, yet more preferably from about 240 days to about 365 days, and still more preferably from about 365 days to about 450 days, although longer storage times may be used at temperatures of about -20°C and below.
• Competent cells produced by the method of the invention may be stored at -20°C for at least one year while retaining substantially their transformation efficiency.
• Substantial retention of transformation efficiency means that after lyophilization, the cells will have a transformation efficiency after storage that is about 40% to 100%, preferably at about 60% to 100%, more preferably about 70% to 100% and most preferably about 80% to 100% of the transformation efficiency of the cells immediately after lyophilization.
• the lyophilized competent cell may be any gram positive or gram negative bacteria including, but not limited to, Escherichia, Klebsiella, Salmonella, Bacillus, Streptomyces, Streptococcus, and Pseudomonas.
• gram negative prokaryotic cells are transformed according to the method of the invention, more preferably Escherichia, and most preferably E. coli.
• any DNA molecule e.g. vectors, plasmids, phagemids, expression vectors, etc.
• the cells are mixed with the DNA molecule in the presence of a competence buffer.
• the competence buffer may be added to the lyophilized competent cells prior to adding the DNA molecule or the DNA molecule and competence buffer may be added simultaneously to the lyophilized competent cells.
• any solution may be used to rehydrate and mix the competent cells with the DNA molecule. Such solutions include water, saline, or any suitable buffer.
• the transformed cells may be grown in a growth conducive medium.
• a growth conducive medium contains an antibiotic to assist in selection of transformed cells.
• the DNA molecule to be transformed may contain a selective marker (e.g. an antibiotic resistance gene), allowing selection of transformed cells when the corresponding antibiotic is used in the medium.
• the invention also concerns a method of producing a desired protein by transforming a lyophilized competent cell with a DNA molecule encoding said desired protein.
• the invention concerns a method of producing a desired protein comprising obtaining a lyophilized competent cell produced according to the invention, transforming said cell with a DNA molecule capable of expressing said desired protein, and culturing said transformed cell under conditions sufficient to produce said desired protein.
• Cells which can be used according to this aspect of the invention including both gram negative and gram positive bacteria, preferably Escherichia, and most preferably E. coli.
• the cells are transformed by mixing the cells with a DNA molecule and incubating the mixture under conditions sufficient to transform said cell with said DNA molecule.
• This mixing step may be accomplished in any solution.
• the lyophilized competent cells are rehydrated in a competence buffer prior to adding the DNA molecule, or the lyophilized competent cells are simultaneously mixed with the DNA molecule and the competence buffer.
• Transformed cells may be selected according to techniques well known in the art including, for example, selection for marker genes on the DNA molecule (e.g. antibiotic resistance genes).
• the cell may then be cultured according to well known techniques in a growth conducive medium. Upon culturing the cell under appropriate conditions, the cell is capable of producing the desired protein.
• the desired protein may then be isolated, and a substantially pure protein obtained by well known protein purification techniques.
• a seed stock of E. coli DH5 ⁇ cells (Gibco BRL, Gaithersburg, MD) was prepared as follows: DH5 ⁇ cells were streaked on an LB plate (32 g Gibco BRL LB agar (Life Technologies, Inc., Gaithersburg, MD) per liter distilled water) and the plate was incubated for 36 hours at 23°C. Several colonies were picked into a 500 ml non-baffled shake flask containing 25 ml SOB medium (2% Bacto tryptone, 0.5% Bacto yeast extract 10 mMNaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 ).
• the flask was shaken at 23 °C, 275 rpm, for several hours and the optical density at 550 nm is followed.
• 10 ml of the cells were mixed with 10 ml of SOB:glycerol 60:40 (60 ml SOB, 40 ml glycerol, (Gibco BRL) in a 50 ml conical tube.
• the cells were mixed using a vortex mixer and were allowed to remain for 10 min. on ice. 1 ml aliquots were dispensed into NUNC cryovials (Catalog No. 366656) (Life Technologies, Inc.,
• a seed of DH5 ⁇ cells stored at -80°C was thawed on ice for 10 min. 0.450 ml of the thawed seed was inoculated into 1500 ml of SOB medium containing 0.001% PPG in a 2.8 L Fernbach flask. The flask was shaken at 23°C
• the cells were chilled on ice for 15 min. and collected by centrifugation using Corning 250 ml bottles with 250 ml of cell culture/bottle. The cells were centrifuged in a GS3 rotor at 4000 rpm 4°C for 10 minutes in a Sorvall RC2B centrifuge.
• Each cell pellet was resuspended in 75 ml of cold CCMB80 buffer (10 mM potassium acetate pH 7.0, 80 mM CaCl 2 -H 2 O, 20 mM MnCl 2 -4H 2 O, 10 mM MgCl 2 -6H 2 O, 10% glycerol adjusted to pH 6.4 with 0.1 N HC1).
• the cells were kept on ice for 20 min.
• the cell pellet was collected at 4°C by centrifugation and each cell pellet was resuspended in 16 ml of either 9% aqueous trehalose
• the cells were removed from the lyophilizer and taken to a 4°C cold room where the caps were tightened The cells were then placed in a foil pouch containing desiccant and the pouches were placed in a -20°C freezer for storage.
• the vials were removed from the -20°C freezer and placed on wet ice for 6 minutes.
• the cells were rehydrated in 400 ⁇ l of a 1: 1 mixture of CCMB80 buffer Vv'ithout glycerol (10 M potassium acetate pH 7.0, 80 mM CaCl 2 -2H 2 O, 20 mM
• Figure 1 indicates that DH5 ⁇ cells lyophilized in NUNC cryovials using either sucrose or trehalose as the cryoprotectant retained a transformation efficiency of >1 0 X 10 7 T/ug when stored for at least 12 months at about -20°C
• Figure 2 indicates that the viable cell count of the cells stored in NUNC cryovials at about -20°C remained at approximately 1 0 X 10 9 cells/ml for at least 12 months
• Figure 3 indicates that the cells lyophilized using trehalose in glass vials which are vacuum sealed retain a transformation efficiency of >1 O 10 8 T/ ⁇ g after storage at -20°C for 12 months
• Example 2 The following example was carried out essentially as Example 1 with the following exceptions
• the DH5 ⁇ seed stored at -80°C was thawed and 600 ⁇ l of the seed was inoculated into 1 7 L of SOB medium containing 0 001% PPG in a 2 8 L Fernbach flask
• the flask was shaken for 18 hours at 23 °C, 275 rpm
• 175 ml of the culture was inoculated into 1 7 L of SOB medium containing 0 001% PPG in a 2 8 L Fernbach flask
• the flask was shaken for approximately 4 hours at 23°C, 275 rpm
• 2 7 ml of the culture was oculated into 2 Fernbach flasks each containing 1 7 L SOB medium and 0 001% PPG
• the flasks were shaken at 23°C, 275 rpm, for approximately 22 hours
• the cultures were harvested and processed as in Example 1, with the exception that the cells were not chilled prior to collection by centrifugation 200 ml of cells were then centrifuged at about 4°C as described in Example 1 and the cell pellet was resuspended in 60 ml cold CCMB80 buffer. The cells were placed on ice for 20 minutes and again collected by centrifugation at about 4°C. The cell pellet was resuspended in 25.6 ml of cold 12% aqueous sucrose and the cells were placed on ice for 2 hours. 250 ⁇ l of the cells were vialed after a 2 hour incubation on ice into chilled NUNC cryovials. The cells were frozen by placing the vials in a -80°C freezer for 16 hours or were frozen by immersion in a liquid nitrogen bath. The cells were lyophilized as described in Example 1.
• Example 2 The following example was carried out essentially as in Example 2 with the following exceptions. Two DH5oc seeds were thawed and 800 ⁇ l was inoculated into two 2.8 L Fernbach flasks, each containing 1.7 L of SOB medium and
• the flasks were shaken at 23°C, 275 rpm, for approximately 19 hours.
• the optical density of the two flasks were 0.3918 and 0.3578, respectively.
• the cells were collected by centrifugation at 4°C. Each cell pellet was resuspended in 10 ml of room temperature SOB medium containing 0 001% PPG and were pooled (total of 120 ml). A 1 100 dilution of the cells had an optical density of 0.0754, indicating that the cell density was 7.54.
• Twenty ml of the cells were inoculated into 6 Fernbach flasks, each containing 1.7 L SOB medium and 0.001% PPG.
• the flasks were shaken at 23°C, 275 rpm, for 6.5 hours, at which time the optical densities of the flasks were 0.705, 0.783, 0.701, 0.749, 0.704, and 0.702.
• the flasks were chilled on ice for 15 minutes.
• Ten liters of the cells were concentrated in the 4°C cold room by dewatering to 3 L in the following manner.
• the peristaltic pump used was a Masterflex model 7529-30 (Cole Palmer) and the column used was a Microgon column # M22M-300-01N The column was connected to the pump and rinsed once with 2 liters of autoclaved deionized water.
• the pump was turned on and 2 liters of 0.37% bleach was circulated through the system for 20 minutes
• the system was then rinsed with 10 liters of autoclaved deionized water. 10 liters of the cell suspension was poured into the reservoir.
• the cell suspension was then circulated through the system with the pump speed set at 3 5 to reduce the volume to 3 liters
• the process required approximately 25 minutes (approximately 280 ml/minute)
• the 3 liters of concentrated cell suspension was recovered from the reservoir by pumping the cell suspension out of the reservoir.
• Figure 6 indicates that the cells can be stored at -20°C for at least 4 months and maintain a transformation efficiency greater than 1 x 10 7 T/ug.
• Figure 7 indicates that the cell viability remains at greater than 1 x 10 9 cells/ml after storage for 4 months at -20°C.
• Example 2 was performed essentially as Example 1 with the following exceptions.
• E. coli strain DH10B was streaked from a master seed stored at -80°C onto an LB plate containing 100 ⁇ g/ml streptomycin. The plate was incubated for 24 hours at 23°C. A single colony was picked from the plate and inoculated into a flask containing 250 ml of 15/10 medium (1.0% Bacto tryptone,
• 550 nm was initially 0.1. The flask was shaken until the optical density was 0.7, and the cells were then collected by centrifugation at 4°C. The cells were processed as in Example 1. After resuspension in the cryoprotectant, 1 ml of the cell suspension was aliquoted into sterile 5 ml glass vials (Wheaton) and the cells were frozen for 5 minutes in a dry ice ethanol bath. Rubber stoppers were fitted loosely on the vials and the vials were placed in the lyophilizer. The cells were lyophilized according to the program described in Example 1 except that the vials were sealed under vacuum.

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• 1998
  • 1998-02-11 AT AT98906440T patent/ATE294229T1/de not_active IP Right Cessation
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  • 1998-02-11 WO PCT/US1998/002880 patent/WO1998035018A1/en not_active Ceased
  • 1998-02-11 JP JP53508298A patent/JP2001512310A/ja not_active Ceased
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