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Contributor/author(s): Sebastian Lim Yang
There is a glitch which involves reverting a completed scenario to free-play mode. This trick is helpful for users who would like to save their achievements in the scenarios to a regular city file. To do this, complete a scenario, then click 'Play another scenario' and then click on the 'X' button in the right hand corner. You can then save the city.
(This trick was first discovered in SimCity 3000 UK Edition, v1.1.)
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Serving San Joaquin County
The Velvet Grill And Creamery in Lodi, CA is your number-one choice for a family-run restaurant and homemade ice cream. We serve all the family favorites, and some of our own, from steak to chicken in a sandwich, from home made ice cream to salads, and much more. Call 209-369-8231 to find out more about our available services:
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Whether you're looking for breakfast on the go, a light and healthy lunch, or a delicious dinner, our restaurant promises to satisfy. The Velvet Grill And Creamery has your table ready now.
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subset: commoncrawl


text:
Bathroom caulking is an important part of any repair or renovation in your bathroom. Moisture will penetrate your bathroom walls and cause mold, mildew and rotting and can lead to expensive damage to your house, unless you take precautions with your shower enclosure and/or bathtub. All points of moisture entry or seepage should be sealed, for example, between the tub and wall, surrounding sinks and faucets, and the area where the toilet touches the floor. Here are some tips to keep in mind while applying bathroom caulking.
1) For a smooth, clean caulk line, use masking tape each side of the seam you are caulking. Leave a gap between the two pieces of tape the size of the bead you want. Put down your bead of caulk. Use your finger to push excess caulk onto the tape. Then remove the masking tape.
2) A sharpened flathead screwdriver works good for removing old caulking if you do not have other tools. Take care not to damage the underlying surface.
3) Silicon caulk is extremely sticky and will not easily clean off your hands, so wear latex gloves when applying silicon caulk.
4) For minute gaps like those around sink faucets, use caulk from squeeze tubes, rather a cartridge.
5). For smoothing caulk, a paint stick makes a handy tool.
6). To unplug a used tube of caulk, cut off the nozzle end off of the tube so that the hole is slightly larger than the ole hole. Then screw a screw with coarse sized threads into the nozzle through the hardened caulk. Now you can use the screw to pull the hardened caulk out of the nozzle. A drywall screw will work great for this.
7). To seal your half-used caulk cartridge, put a 2 inch nail into the nozzle. Wrap up the whole nozzle in Saran Wrap® to keep the caulk in the nozzle from hardening.
8). Never use oil-based caulk around thermal-pane windows. The oils in these kinds of caulking will attack the polymer sealants on the windows, causing the window to fog up and lose it's insulating properties.
9). Avoid the self-adhesive caulking strips sold as replacement for tube caulking around tub enclosures. They are next to useless for sealing against water seepage, and at best will protect the underlying surface against only the most benign humidity.
10). Elastomeric latex caulks require from one to three days to fully cure before painting them. Elastomeric caulks are, as their name would indicate, more elastic than paint that is laid over them. Your paint will not be able to stretch enough to avoid cracking during the caulk's curing and resultant shrinkage.
photo by A. Godber / CreativeCommons


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...will probably be the thing for me for the next couple of day. I have been online now, only about an hour and a half, wrighting one short rant, checking my email, sending out a few emails, and visiting a few select sites, and I am exhausted. Imagine becoming exhausted by that, and of course by my two runs to the throne in the basement, in that short a time. Oh well, once I am feeling better I will be back at it as usual.
All the best,
Glenn B
Fakes, fakes, and more fakes…
8 hours ago


text: It's Officially Unofficially Fall...And That Means Halloween! 09/04/2017 Labor Day weekend is now behind us and that means, unofficially, it's Fall. While many people here in Upstate New York are revealing in apple picking, scarf wearing, and pumpkin spice everything, I get giddy that it's Halloween season. I recognize that Halloween is indeed solely one day on the calendar, but it's sooooo much more than that when you're into costumes, makeup, wigs, and well, the utter ridiculousness. When I do Halloween, I DO Halloween! The light up skulls come out, the caution


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In Chester
Our chefs prepare over 120 authentic dishes from around the world every day and each dish is expertly created in small amounts to ensure they are always fresh.
Our Live Cooking Stations provide fabulous entertainment and aromas while the dishes are cooked in front of you. We provide a casual dining experience like no other, offering a balanced choice of delicious food and exceptional value for money, giving our guests added value at one set price, all within luxurious surroundings. This is the pinnacle of casual dining. Welcome to Panda Mami, where your gastronomic journey begins.


text:
Transformation of pichia methanolica
Methods for transforming Pichia methanolica, and DNA molecules useful in transformation of P. methanolica, are disclosed. P. methanolica cells are exposed, in the presence of DNA molecules, to a pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm and a pulse duration of from 1 to 40 milliseconds, whereby the DNA molecules are introduced into the cells. The DNA molecules may comprise an expression unit that includes a transcription promoter of a P. methanolica gene operably linked to a segment encoding a polypeptide or protein of interest. The DNA molecules may also encode a selectable marker, such as a P. methanolica ADE2 gene. Cells transformed according to the invention may be used in production systems for the preparation of proteins of commercial importance.
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Methylotrophic yeasts are those yeasts that are able to utilize methanol as a sole source of carbon and energy. Species of yeasts that have the biochemical pathways necessary for methanol utilization are classified in four genera, Hansenula, Pichia, Candida, and Torulopsis. These genera are somewhat artificial, having been based on cell morphology and growth characteristics, and do not reflect close genetic relationships (Billon-Grand, Mycotaxon 35:201-204, 1989; Kurtzman, Mycologia 84:72-76, 1992). Furthermore, not all species within these genera are capable of utilizing methanol as a source of carbon and energy. As a consequence of this classification, there are great differences in physiology and metabolism between individual species of a genus.
Methylotrophic yeasts are attractive candidates for use in recombinant protein production systems. Some methylotrophic yeasts have been shown to grow rapidly to high biomass on minimal defined media. Certain genes of methylotrophic yeasts are tightly regulated and highly expressed under induced or de-repressed conditions, suggesting that promoters of these genes might be useful for producing polypeptides of commercial value. See, for example, Faber et al., Yeast 11:1331, 1995; Romanos et al., Yeast 8:423, 1992; and Cregg et al., Bio/Technology 11:905, 1993.
Development of methylotrophic yeasts as hosts for use in recombinant production systems has been slow, due in part to a lack of suitable materials (e.g., promoters, selectable markers, and mutant host cells) and methods (e.g., transformation techniques). The most highly developed methylotrophic host systems utilize Pichia pastoris and Hansenula polymorpha (Faber et al., Curr. Genet. 25:305-310, 1994; Cregg et al., ibid.; Romanos et al., ibid.; U.S. Pat. No. 4,855,242; U.S. Pat. No. 4,857,467; U.S. Pat. No. 4,879,231; and U.S. Pat. No. 4,929,555).
There remains a need in the art for methods of transforming additional species of methylotrophic yeasts and for using the transformed cells to produce polypeptides of economic importance, including industrial enzymes and pharmaceutical proteins. The present invention provides such methods as well as other, related advantages.
The present invention provides methods for introducing DNA molecules into Pichia methanolica cells and cells transformed according to these methods.
Within one aspect of the invention, the methods comprise exposing a Pichia methanolica cell, in the presence of a linear DNA molecule, to an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm and a time constant of from 1 to 40 milliseconds, whereby the DNA molecule is introduced into the cell. Within one embodiment of the invention, the DNA molecule comprises a segment encoding a polypeptide, other than a P. methanolica polypeptide, operably linked to a P. methanolica gene transcription promoter and a P. methanolica gene transcription terminator. Within a preferred embodiment, the transcription promoter is a P. methanolica AUG1 gene promoter, which, within one embodiment, comprises a sequence of nucleotides as shown in SEQ ID NO:2 from nucleotide 24 to nucleotide 1354. The DNA molecule may further comprise a selectable marker gene that complements a mutation in the host cell. Within one embodiment, the selectable marker gene is a P. methanolica gene, such as a P. methanolica ADE2 gene.
Within a second aspect of the invention there are provided methods for transforming P. methanolica with heterologous DNA, comprising exposing a population of P. methanolica cells, in the presence of heterologous linear DNA molecules, to an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm and a time constant of from 1 to 40 milliseconds, whereby the heterologous DNA is introduced into at least a portion of the population, and recovering cells into which the DNA has been introduced. Within one embodiment of the invention, from 10.sup.3 to 10.sup.5 cells are recovered per microgram of heterologous DNA. Within another embodiment, from 0.9.times.10.sup.4 to 1.1.times.10.sup.4 cells are recovered per microgram of heterologous DNA. Within a further embodiment, the methods further comprise the step of recovering integrative transformants from the recovered cells, such as by culturing the cells in a growth medium comprising sorbitol as a carbon source. Within an additional embodiment, the population of cells is in early log phase growth.
Within other aspects, the present invention provides Pichia methanolica cells transformed by the methods disclosed above.
These and other aspects of the invention will become evident upon reference to the following detailed description and the attached drawings.
FIG. 1 illustrates the effects of field strength and pulse duration on electroporation efficiency of P. methanolica.
FIG. 2 is a schematic diagram of a recombination event between plasmid pCZR140 and P. methanolica genomic DNA.
FIG. 3 is a schematic diagram of a recombination event between plasmid pCZR137 and P. methanolica genomic DNA.
Prior to setting forth the invention in more detail, it will be useful to define certain terms used herein:
Early log phase growth--That phase of cellular growth in culture when the cell concentration is from 2.times.10.sup.6 cells/ml to 8.times.10.sup.6 cells/ml.
Heterologous DNA--A DNA molecule, or a population of DNA molecules, that does not exist naturally within a given host cell. DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species so long as that host DNA is combined with non-host DNA. For example, a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promoter is considered to be a heterologous DNA molecule.
Integrative transformants--Cells into which have been introduced heterologous DNA, wherein the heterologous DNA has become integrated into the genomic DNA of the cells.
Linear DNA--DNA molecules having free 5' and 3' ends, that is non-circular DNA molecules. Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption.
Operably linked--The term operably linked indicates that DNA segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
As noted above, the present invention provides methods for introducing DNA into cells of the methylotrophic yeast Pichia methanolica, and for selecting cells into which the DNA has been introduced. Those skilled in the art will recognize that transformation of cells with both homologous DNA (DNA from the host species) and heterologous DNA is a prerequisite to a large number of diverse biological applications. The methods of the present invention are particularly well suited to the preparation of cells transformed with heterologous DNA, which cells can be used for the production of polypeptides and proteins, including polypeptides and proteins of higher organisms, including humans. The present invention further provides for the transformation of Pichia methanolica cells with other DNA molecules, including DNA libraries and synthetic DNA molecules. The invention thus provides techniques that can be used to express genetically diverse libraries to produce products that are screened for novel biological activities, to engineer cells for use as targets for the screening of compound libraries, and to genetically modify cells to enhance their utility within other processes.
Strains of Pichia methanolica are available from the American Type Culture Collection (Rockville, Md.) and other repositories. Within one embodiment of the invention, cells to be transformed with heterologous DNA will have a mutation that can be complemented by a gene (a "selectable marker") on the heterologous DNA molecule. This selectable marker allows the transformed cells to grow under conditions in which untransformed cells cannot multiply ("elective conditions"). The general principles of selection are well known in the art. Commonly used selectable markers are genes that encode enzymes required for the synthesis of amino acids or nucleotides. Cells having mutations in these genes cannot grow in media lacking the specific amino acid or nucleotide unless the mutation is complemented by the selectable marker. Use of such "selective" culture media ensures the stable maintenance of the heterologous DNA within the host cell. A preferred selectable marker of this type for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 220.127.116.11). The ADE2 gene, when transformed into an ade2 host cell, allows the cell to grow in the absence of adenine. The coding strand of a representative P. methanolica ADE2 gene sequence is shown in SEQ ID NO:1. The sequence illustrated includes 1006 nucleotides of 5' non-coding sequence and 442 nucleotides of 3' non-coding sequence, with the initiation ATG codon at nucleotides 1007-1009. Within a preferred embodiment of the invention, a DNA segment comprising nucleotides 407-2851 is used as a selectable marker, although longer or shorter segments could be used as long as the coding portion is operably linked to promoter and terminator sequences. Those skilled in the art will recognize that this and other sequences provided herein represent single alleles of the respective genes, and that allelic variation is expected to exist. Any functional ADE2 allele can be used within the present invention. Other nutritional markers that can be used within the present invention include the P. methanolica ADE1, HIS3, and LEU2 genes, which allow for selection in the absence of adenine, histidine, and leucine, respectively. For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Gene-deficient mutants can be prepared by known methods, such as site-directed mutagenesis. P. methanolica genes can be cloned on the basis of homology with their counterpart Saccharomyces cerevisiae genes. The ADE2 gene disclosed herein was given its designation on the basis of such homology.
Within another embodiment of the invention, a dominant selectable marker is used, thereby obviating the need for mutant host cells. Dominant selectable markers are those that are able to provide a growth advantage to wild-type cells.
Typical dominant selectable markers are genes that provide resistance to antibiotics, such as neomycin-type antibiotics (e.g., G418), hygromycin B, and bleomycin/phleomycin-type antibiotics (e.g., Zeocin.TM.; available from Invitrogen Corporation, San Diego, Calif.). A preferred dominant selectable marker for use in P. methanolica is the Sh bla gene, which inhibits the activity of Zeocin.TM..
Electroporation is used within the present invention to facilitate the introduction of DNA into P. methanolica cells. Electroporation is the process of using a pulsed electric field to transiently permeabilize cell membranes, allowing macromolecules, such as DNA, to pass into cells. Electroporation has been described for use with mammalian (e.g., Neumann et al., EMBO J. 1:841-845, 1982) and fungal (e.g., Meilhoc et al., Bio/Technology 8:223-227, 1990) host cells. However, the actual mechanism by which DNA is transferred into the cells is not well understood. For transformation of P. methanolica, it has been found that electroporation is surprisingly efficient when the cells are exposed to an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm and a time constant (.tau.) of from 1 to 40 milliseconds. The time constant .tau. is defined as the time required for the initial peak voltage V.sub.0 to drop to a value of V.sub.0 /e. The time constant can be calculated as the product of the total resistance and capacitance of the pulse circuit, i.e., .tau.=R.times.C. Typically, resistance and capacitance are either preset or may be selected by the user, depending on the electroporation equipment selected. In any event, the equipment is configured in accordance with the manufacturer's instructions to provide field strength and decay parameters as disclosed above. Electroporation equipment is available from commercial suppliers (e.g., BioRad Laboratories, Hercules, Calif.).
DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide or protein production, the DNA molecules will include, in addition to the selectable marker disclosed above, an expression casette comprising a transcription promoter, a DNA segment (e.g., a cDNA) encoding the polypeptide or protein of interest, and a transcription terminator. These elements are operably linked to provide for transcription of the DNA segment of interest. It is preferred that the promoter and terminator be that of a P. methanolica gene. A preferred promoter is that of a P. methanolica alcohol utilization gene (AUG1), a representative coding strand sequence of which is shown in SEQ ID NO:2. Within SEQ ID NO:2, the initiation ATG codon is at nucleotides 1355-1357. Nucleotides 1-23 of SEQ ID NO:2 are non-AUG1 polylinker sequence. It is particularly preferred to utilize as a promoter a segment comprising nucleotides 24-1354 of SEQ ID NO:2, although additional upstream sequence can be included. P. methanolica contains a second alcohol utilization gene, AUG2, the promoter of which can be used within the present invention. Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. The DNA molecules will further include a selectable marker to allow for identification, selection, and maintenance of transformants. The DNA molecules may further contain additional elements, such an origin of replication and a selectable marker that allow amplification and maintenance of the DNA in an alternate host (e.g., E. coli). To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment, comprising the promoter--gene of interest--terminator plus selectable marker, flanked at both ends by host DNA sequences. This is conveniently accomplished by including 3' untranslated DNA sequence at the downstream end of the expression segment and relying on the promoter sequence at the 5' end. When using linear DNA, the expression segment will be flanked by cleavage sites to allow for linearization of the molecule and separation of the expression segment from other sequences (e.g., a bacterial origin of replication and selectable marker). Preferred such cleavage sites are those that are recognized by restriction endonucleases that cut infrequently within a DNA sequence, such as those that recognize 8-base target sequences (e.g., Not I).
Proteins that can be produced in P. methanolica using the methods of the present invention include proteins of industrial and pharmaceutical interest. Such proteins include enzymes such as lipases, cellulases, and proteases; enzyme inhibitors, including protease inhibitors; growth factors such as platelet derived growth factor, fibroblast growth factors, and epidermal growth factor; cytokines such as erythropoietin and thrombopoietin; and hormones such as insulin, leptin, and glucagon.
For use within the present invention, P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25.degree. C. to 35.degree. C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium is YEPD (Table 1). The cells may be passaged by dilution into fresh culture medium or stored for short periods on plates under refrigeration. For long-term storage, the cells are preferably kept in a 50% glycerol solution at -70.degree. C.
2% D-glucose
2% Bacto.TM. Peptone (Difco Laboratories, Detroit, Mich.)
5 1% Bacto.TM. yeast extract (Difco Laboratories)
0.004% adenine
0.006% L-leucine
0.056% -Ade-Trp-Thr powder
0.67% yeast nitrogen base without amino acids
2% D-glucose
0.5% 200.times. tryptophan, threonine solution
0.056% -Ade-Trp-Thr powder
0.67% yeast nitrogen base without amino acids
2% D-glucose
0.5% 200.times. tryptophan, threonine solution
18.22% D-sorbitol
0.052% -Leu-Trp-Thr powder
0.67% yeast nitrogen base without amino acids
2% D-glucose
0.5% 200.times. tryptophan, threonine solution
0.052% -His-Trp-Thr powder
0.67% yeast nitrogen base without amino acids
2% D-glucose
0.5% 200.times. tryptophan, threonine solution
0.056% -Ura-Trp-Thr powder
0.67% yeast nitrogen base without amino acids
2% D-glucose
0.5% 200.times. tryptophan, threonine solution
Table 1, continued
0.056% -Ura-Trp-Thr powder
0.67% yeast nitrogen base without amino acids
2% D-glucose
0.5% 200.times. tryptophan, threonine solution
18.22% D-sorbitol
-Leu-Trp-Thr powder
powder made by combining 4.0 g adenine, 3.0 g arginine, 5.0 g aspartic acid,
2.0 g histidine, 6.0 g isoleucine, 4.0 g lysine, 2.0 g methionine, 6.0 g
phenylalanine, 5.0 g serine, 5.0 g tyrosine, 4.0 g uracil, and 6.0 g valine (all L-amino acids)
-His-Trp-Thr powder
powder made by combining 4.0 g adenine, 3.0 g arginine, 5.0 g aspartic acid,
6.0 g isoleucine, 8.0 g leucine, 4.0 g lysine, 2.0 g methionine, 6.0 g
phenylalanine, 5.0 g serine, 5.0 g tyrosine, 4.0 g uracil, and 6.0 g valine (all L-amino acids)
-Ura-Trp-Thr powder
powder made by combining 4.0 g adenine, 3.0 g arginine, 5.0 g aspartic acid,
2.0 g histidine, 6.0 g isoleucine, 8.0 g leucine, 4.0 g lysine, 2.0 g methionine,
6.0 g phenylalanine, 5.0 g serine, 5.0 g tyrosine, and 6.0 g valine (all L-amino acids)
-Ade-Trp-Thr powder
powder made by combining 3.0 g arginine, 5.0 g aspartic acid, 2.0 g histidine,
6.0 g isoleucine, 8.0 g leucine, 4.0 g lysine, 2.0 g methionine, 6.0 g
phenylalanine, 5.0 g serine, 5.0 g tyrosine, 4.0 g uracil, and 6.0 g valine (all L-amino acids)
200.times. tryptophan, threonine solution
3.0% L-threonine, 0.8% L-tryptophan in H.sub.2 O
For plates, add 1.8% Bacto.TM. agar (Difco Laboratories)
Electroporation of P. methanolica is preferably carried out on cells in early log phase growth. Cells are streaked to single colonies on solid media, preferably solid YEPD. After about 2 days of growth at 30.degree. C., single colonies from a fresh plate are used to inoculate the desired volume of rich culturc media (e.g., YEPD) to a cell density of about 5-10.times.10.sup.5 cells/ml. Cells are incubated at about 25.degree.-35.degree. C., preferably 30.degree. C., with vigorous shaking, until they are in early log phase. The cells are then harvested, such as by centrifugation at 3000.times.g for 2-3 minutes, and resuspended. Cells are made electrocompetent by reducing disulfide bonds in the cell walls, equilibrating them in an ionic solution that is compatible with the electroporation conditions, and chilling them. Cells are typically made electrocompetent by incubating them in a buffered solution at pH 6-8 containing a reducing agent, such as dithiothreitol (DTT) or .beta.-mercaptoethanol (BME), to reduce cell wall proteins to facilitate subsequent uptake of DNA. A preferred incubation buffer in this regard is a fresh solution of 50 mM potassium phosphate buffer, pH 7.5, containing 25 mM DTT. The cells are incubated in this buffer (typically using one-fifth the original culture volume) at about 30.degree. C. for about 5 to 30 minutes, preferably about 15 minutes. The cells are then harvested and washed in a suitable electroporation buffer, which is used ice-cold. Suitable buffers in this regard include pH 6-8 solutions containing a weak buffer, divalent cations (e.g., Mg.sup.++, Ca.sup.++) and an osmotic stabilizer (e.g., a sugar). After washing, the cells are resuspended in a small volume of the buffer, at which time they are electrocompetent and can be used directly or aliquotted and stored frozen (preferably at -70.degree. C.). A preferred electroporation buffer is STM (270 mM sucrose, 10 mM Tris, pH 7.5, 1 mM MgCl.sub.2). Within a preferred protocol, the cells are subjected to two washes, first in the original culture volume of ice-cold buffer, then in one-half the original volume. Following the second wash, the cells are harvested and resuspended, typically using about 3-5 ml of buffer for an original culture volume of 200 ml.
Electroporation is carried out using a small volume of electrocompetent cells (typically about 100 .mu.l) and up to one-tenth volume of linear DNA molecules. For example, 0.1 ml of cell suspension in a buffer not exceeding 50 mM in ionic strength is combined with 0.1-10 .mu.g of DNA (vol. .ltoreq.10 .mu.l). This mixture is placed in an ice-cold electroporation cuvette and subjected to a pulsed electric field of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant of from 1 to 40 milliseconds, preferably 10-30 milliseconds, more preferably 15-25 milliseconds, most preferably about 20 milliseconds, with exponential decay. The actual equipment settings used to achieve the desired pulse parameters will bc determined by the equipment used. When using a BioRad (Hercules, Calif.) Gene Pulser.TM. electroporator with a 2 mm electroporation cuvette, resistance is set at 600 ohms or greater, preferably "infinite" resistance, and capacitance is set at 25 .mu.F to obtain the desired field characteristics. After being pulsed, the cells are diluted approximately 10.times. into 1 ml of YEPD broth and incubated at 30.degree. C. for one hour.
The cells are then harvested and plated on selective media. Within a preferred embodiment, the cells are washed once with a small volume (equal to the diluted volume of the electroporated cells) of 1.times. yeast nitrogen base (6.7 g/L yeast nitrogen base without amino acids; Difco Laboratories, Detroit, Mich.), and plated on minimal selective media. Cells having an ade2 mutation that have been transformed with an ADE2 selectable marker can be plated on a minimal medium that lacks adenine, such as ADE D (Table 1) or ADE DS (Table 1). In a typical procedure, 250 .mu.l aliqouts of cells are plated on 4 separate ADE D or ADE DS plates to select for Ade.sup.+ cells.
P. methanolica recognizes certain infrequently occuring sequences, termed autonomously replicating sequences (ARS), as origins of DNA replication, and these sequences may fortuitously occur within a DNA molecule used for transformation, allowing the transforming DNA to be maintained extrachromosomally. However, integrative transformants are generally preferred for use in protein production systems. Integrative transformants have a profound growth advantage over ARS transformants on selective media containing sorbitol as a carbon source, thereby providing a method for selecting integrative transformants from among a population of transformed cells. ARS sequences have been found to exist in the ADE2 gene and, possibly, the AUG1 gene of P. methanolica. ade2 host cells of Pichia methanolica transformed with an ADE2 gene can thus become Ade.sup.+ by at least two different modes. The ARS within the ADE2 gene allows unstable extrachromosomal maintenance of the transforming DNA (Hiep et al., Yeast 9:1189-1197, 1993). Colonies of such transformants are characterized by slower growth rates and pink color due to prolific generation of progeny that are Ade.sup.-. Transforming DNA can also integrate into the host genome, giving rise to stable transformants that grow rapidly, are white, and that fail to give rise to detectable numbers of Ade.sup.- progeny. ADE D plates allow the most rapid growth of transformed cells, and unstable and stable transformants grow at roughly the same rates. After 3-5 days of incubation on ADE D plates at 30.degree. C. stable transformant colonies are white and roughly twice the size of unstable, pink transformants. ADE DS plates are more selective for stable transformants, which form large (.apprxeq.5 mm) colonies in 5-7 days, while unstable (ARS-maintained) colonies are much smaller (.apprxeq.1 mm). The more selective ADE DS media is therefore preferred for the identification and selection of stable transformants. For some applications, such as the screening of genetically diverse libraries for rare combinations of genetic elements, it is sometimes desirable to screen large numbers of unstable transformants, which have been observed to outnumber stable transformants by a factor of roughly 100. In such cases, those skilled in the art will recognize the utility of plating transformant cells on less selective media, such as ADE D.
Integrative transformants are preferred for use in protein production processes. Such cells can be propagated without continuous selective pressure because DNA is rarely lost from the genome. Integration of DNA into the host chromosome can be confirmed by Southern blot analysis. Briefly, transformed and untransformed host DNA is digested with restriction endonucleases, separated by electrophoresis, blotted to a support membrane, and probed with appropriate host DNA segments. Differences in the patterns of fragments seen in untransformed and transformed cells are indicative of integrative transformation. Restriction enzymes and probes can be selected to identify transforming DNA segments (e.g., promoter, terminator, heterologous DNA, and selectable marker sequences) from among the genomic fragments.
Differences in expression levels of heterologous proteins can result from such factors as the site of integration and copy number of the expression cassette and differences in promoter activity among individual isolates. It is therefore advantageous to screen a number of isolates for expression level prior to selecting a production strain. A variety of suitable screening methods are available. For example, transformant colonies are grown on plates that are overlayed with membranes (e.g., nitrocellulose) that bind protein. Proteins are released from the cells by secretion or following lysis, and bind to the membrane. Bound protein can then be assayed using known methods, including immunoassays. More accurate analysis of expression levels can be obtained by culturing cells in liquid media and analyzing conditioned media or cell lysates, as appropriate. Methods for concentrating and purifying proteins from media and lysates will be determined in part by the protein of interest. Such methods are readily selected and practiced by the skilled practitioner.
For small-scale protein production (e.g., plate or shake flask production), P. methanolica transformants that carry an expression cassette comprising a methanol-regulated promoter (such as the AUG1 promoter) are grown in the presence of methanol and the absence of interfering amounts of other carbon sources (e.g., glucose). For small-scale experiments, including preliminary screening of expression levels, transformants may be grown at 30.degree. C. on solid media containing, for example, 20 g/L Bacto-agar (Difco), 6.7 g/L yeast nitrogen base without amino acids (Difco), 10 g/L methanol, 0.4 .mu.g/L biotin, and 0.56 g/L of -Ade-Thr-Trp powder. Because methanol is a volatile carbon source it is readily lost on prolonged incubation. A continuous supply of methanol can be provided by placing a solution of 50% methanol in water in the lids of inverted plates, whereby the methanol is transferred to the growing cells by evaporative transfer. In general, not more than 1 mL of methanol is used per 100-mm plate. Slightly larger scale experiments can be carried out using cultures grown in shake flasks. In a typical procedure, cells are cultivated for two days on minimal methanol plates as disclosed above at 30.degree. C., then colonies arc used to inoculate a small volume of minimal methanol media (6.7 g/L yeast nitrogen base without amino acids, 10 g/L methanol, 0.4 .mu.g/L biotin) at a cell density of about 1.times.10.sup.6 cells/ml. Cells are grown at 30.degree. C. Cells growing on methanol have a high oxygen requirement, necessitating vigorous shaking during cultivation. Methanol is replenished daily (typically 1/100 volume of 50% methanol per day).
For production scale culturing, fresh cultures of high producer clones are prepared in shake flasks. The resulting cultures are then used to inoculate culture medium in a fermenter. Typically, a 500 ml culture in YEPD grown at 30.degree. C. for 1-2 days with vigorous agititation is used to inoculate a 5-liter fermenter. The cells are grown in a suitable medium containing salts, glucose, biotin, and trace elements at 28.degree. C., pH 5.0, and >30% dissolved O.sub.2. After the initial charge of glucose is consumed (as indicated by a decrease in oxygen consumption), a glucose/methanol feed is delivered into the vessel to induce production of the protein of interest. Because large-scale fermentation is carried out under conditions of limiting carbon, the presence of glucose in the feed does not repress the methanol-inducible promoter.
The invention is further illustrated by the following non-limiting examples.
Example 1
P. methanolica cells (strain CBS6515 from American Type Culture Collection, Rockville, Md.) were mutagenized by UV exposure. A killing curve was first generated by plating cells onto several plates at approximately 200-250 cells/plate. The plates were then exposed to UV radiation using a G8T5 germicidal lamp (Sylvania) suspended 25 cm from the surfaces of the plates for periods of time as shown in Table 2. The plates were then protected from visible light sources and incubated at 30.degree. C. for two days.
TABLE 2 ______________________________________ Viable Cells Time Plate 1 Plate 2 Average ______________________________________ 0 sec. 225 229 227 1 sec. 200 247 223 2 sec. 176 185 181 4 sec. 149 86 118 8 sec. 20 7 14 16 sec. 0 2 1 ______________________________________
Large-scale mutagenesis was then carried out using a 2-second UV exposure to provide about 20% killing. Cells were plated at approximately 10.sup.4 cells/plate onto eight YEPD plates that were supplemented with 100 mg/L each of uracil, adenine, and leucine, which were added to supplement the growth of potential auxotrophs having the cognate deficiencies. Following UV exposure the plates were wrapped in foil and incubated overnight at 30.degree. C. The following day the colonies on the plates (.about.10.sup.5 total) were resuspended in water and washed once with water. An amount of cell suspension sufficient to give an OD.sub.600 of 0.1-0.2 was used to inoculate 500 ml of minimal broth made with yeast nitrogen base without amino acids or ammonia, supplemented with 1% glucose and 400 .mu.g/L biotin. The culture was placed in a 2.8 L baffled Bell flask and shaken vigorously overnight at 30.degree. C. The following day the cells had reached an OD.sub.600 of .about.1.0-2.0. The cells were pelleted and resuspended in 500 ml of minimal broth supplemented with 5 g/L ammonium sulfate. The cell suspension was placed in a 2.8 L baffled Bell flask and shaken vigorously at 30.degree. C. for 6 hours. 50 ml of the culture was set aside in a 250-ml flask as a control, and to the remainder of the culture was added 1 mg nystatin to select for auxotrophic mutants (Snow, Nature 211:206-207, 1966). The cultures were incubated with shaking for an additional hour. The control and nystatin-treated cells were then harvested by centrifugation and washed with water three times. The washed cells were resuspended to an OD.sub.600 of 1.0 in 50% glycerol and frozen. Titering of nystatin-treated cells versus the control cells for colony forming units revealed that nystatin enrichment had decreased the number of viable cells by a factor of 10.sup.4.
10.sup.-2 dilutions of nystatin-treated cells were plated on 15 YEPD plates. Colonies were replica-plated onto minimal plates (2% agar, 1.times. YNB, 2% glucose, 400 .mu.g/L biotin). The frequency of auxotrophs was about 2-4%. Approximately 180 auxotrophic colonies were picked to YEPD+Ade, Leu, Ura plates and replica-plated to various dropout plates. All of the auxotrophs were Ade.sup.-. Of these, 30 were noticably pink on dropout plates (LEU D, HIS D, etc.; see Table 1). Of the 30 pink mutants, 21 were chosen for further study; the remainder were either leaky for growth on ADE D plates or contaminated with wild-type cells.
The Ade.sup.- mutants were then subjected to complementation analysis and phenotypic testing. To determine the number of loci defined by the mutants, all 21 mutants were mated to a single pink, Ade.sup.- tester strain (strain #2). Mating was carried out by mixing cell suspensions (OD.sub.600 =1) and plating the mixtures in 10 .mu.l aliquots on YEPD plates. The cells were then replicated to SPOR media (0.5% Na acetate, 1% KCl, 1% glucose, 1% agar) and incubated overnight at 30.degree. C. The cells were then replica-plated to ADE D plates for scoring of phenotype. As shown in Table 3, some combinations of mutants failed to give Ade.sup.+ colonies (possibly defining the same genetic locus as in strain #2), while others gave rise to numerous Ade.sup.+ colonies (possibly defining a separate genetic locus). Because mutant #3 gave Ade.sup.+ colonies when mated to #2, complementation testing was repeated with mutant #3. If the group of mutants defined two genetic loci, then all mutants that failed to give Ade.sup.+ colonies when mated to strain #2 should give Ade.sup.+ colonies when mated to #3. Results of the crosses are shown in Table 3.
TABLE 3 ______________________________________ Mutant x Mutant #2 x Mutant #3 ______________________________________ #1 + - #3 + - #10 + - #15 + - #18 + - #24 + - #28 + - #30 + - #2 - + #6 - + #8 - + #9 - + #11 - + #17 - + #19 - + #20 - + #22 - + #27 - + #4 + + #12 + + #16 + + ______________________________________
As shown in Table 3, most mutants fell into one of two groups, consistent with the idea that there are two adenine biosynthetic genes that, when missing, result in pink colonies on limiting adenine media. Three colonies (#4, #12, and #16) may either define a third locus or exhibit intragenic complementation. Two intensely pigmented mutants from each of the two complementation groups (#3 and #10; #6 and #11) were selected for further characterization. Additional analysis indicated that Ade.sup.- was the only auxotrophy present in these strains.
A P. methanolica clone bank was constructed in the vector pRS426, a shuttle vector comprising 2.mu. and S. cerevisiae URA3 sequences, allowing it to be propagated in S. cerevisiae. Genomic DNA was prepared from strain CBS6515 according to standard procedures. Briefly, cells were cultured overnight in rich media, spheroplasted with zymolyase, and lysed with SDS. DNA was precipitated from the lysate with ethanol and extracted with a phenol/chloroform mixture, then precipitated with ammonium acetate and ethanol. Gel electrophoresis of the DNA preparation showed the presence of intact, high molecular weight DNA and appreciable quantities of RNA. The DNA was partially digested with Sau 3A by incubating the DNA in the presence of a dilution series of the enzyme. Samples of the digests were analyzed by electrophoresis to determine the size distribution of fragments. DNA migrating between 4 and 12 kb was cut from the gel and extracted from the gel slice. The size-fractionated DNA was then ligated to pRS426 that had been digested with Bam HI and treated with alkaline phosphatase. Aliquots of the reaction mixture were electroporated in E. coli MC 1061 cells using a BioRad Gene Pulser.TM. device as recommended by the manufacturer.
The genomic library was used to transform S. cerevisiae strain HBY21A (ade2 ura3) by electroporation (Becker and Guarente, Methods Enzymol. 194:182-187, 1991). The cells were resuspended in 1.2M sorbitol, and six 300-.mu.l aliquots were plated onto ADE D, ADE DS, URA D and URA DS plates (Table 1). Plates were incubated at 30.degree. C. for 4-5 days. No Ade.sup.+ colonies were recovered on the ADE D or ADE DS plates. Colonies from the URA D and URA DS plates were replica-plated to ADE D plates, and two closely spaced, white colonies were obtained. These colonies were restreaked and confirmed to be Ura.sup.+ and Ade.sup.+. These two strains, designated Ade1 and Ade6, were streaked onto media containing 5 FOA (5 fluoro orotic acid; Sikorski and Boeke, Methods Enzymol. 194:302-318). Ura.sup.- colonies were obtained, which were found to be Ade.sup.+ upon replica plating. These results indicate that the Ade.sup.+ complementing activity is genetically linked to the plasmid-borne URA3 marker. Plasmids obtained from yeast strains Ade1 and Ade6 appeared to be identical by restriction mapping as described below. These genomic clones were designated pADE 1-1 and pADE 1-6, respectively.
Total DNA was isolated from the HBY21A transformants Ade1 and Ade6 and used to transform E. coli strain MC 1061 to Amp.sup.R. DNA was prepared from 2 Amp.sup.R colonies of Ade1 and 3 Amp.sup.R colonies of Ade6. The DNA was digested with Pst I, Sca I, and Pst I+Sca I and analyzed by gel electrophoresis. All five isolates produced the same restriction pattern.
PCR primers were designed from the published sequence of the P. methanolica ADE2 gene (also known as ADE1; Hiep et al., Yeast 9:1251-1258, 1993). Primer 9080 (SEQ ID NO:3) was designed to prime at bases 406-429 of the ADE2 DNA (SEQ ID NO:1), and primer 9079 (SE ID NO:4) was designed to prime at bases 2852-2829. Both primers included tails to introduce Avr II and Spe I sites at each end of the amplified sequence. The predicted size of the resulting PCR fragment was 2450 bp.
PCR was carried out using plasmid DNA from the five putative ADE2 clones as template DNA. The 100 .mu.l reaction mixtures contained 1.times. Taq PCR buffer (Boehringer Mannheim, Indianapolis, Ind.), 10-100 ng of plasmid DNA, 0.25 mM dNTPs, 100 pmol of each primer, and 1 .mu.l Taq polymerase (Boehringer Mannheim). PCR was run for 30 cycles of 30 seconds at 94.degree. C., 60 seconds at 50.degree. C., and 120 seconds at 72.degree. C. Each of the five putative ADE2 genomic clones yielded a PCR product of the expected size (2.4 kb). Restriction mapping of the DNA fragment from one reaction gave the expected size fragments when digested with Bgl II or Sal I.
The positive PCR reactions were pooled and digested with Spe I. Vector pRS426 was digested with Spe I and treated with calf intestinal phosphatase. Four .mu.l of PCR fragment and 1 .mu.l of vector DNA were combined in a 10 .mu.l reaction mix using conventional ligation conditions. The ligated DNA was analyzed by gel electrophoresis. Spe I digests were analyzed to identify plasmids carrying a subclone of the ADE2 gene within pRS426. The correct plasmid was designated pCZR 118.
Because the ADE2 gene in pCZR118 had been amplified by PCR, it was possible that mutations that disabled the functional character of the gene could have been generated. To test for such mutations, subclones with the desired insert were transformed singly into Saccharomyces cerevisiae strain HBY21A. Cells were made electrocompetent and transformed according to standard procedures. Transformants were plated on URA D and ADE D plates. Three phenotypic groups were identified. Clones 1, 2, 11, and 12 gave robust growth of many transformants on ADE D. The transformation frequency was comparable to the frequency of Ura.sup.+ transformants. Clones 6, 8, 10, and 14 also gave a high efficiency of transformation to both Ura.sup.+ and Ade.sup.+, but the Ade.sup.+ colonies were somewhat smaller than those in the first group. Clone 3 gave many Ura.sup.+ colonies, but no Ade.sup.+ colonies, suggesting it carried a non-functional ade2 mutation. Clones 1, 2, 11, and 12 were pooled.
To identify the P. methanolica ade2 complementation group, two representative mutants from each complementation group (#3 and #10; #6 and #11), which were selected on the basis of deep red pigmentation when grown on limiting adenine, were transformed with the cloned ADE gene. Two hundred ml cultures of early log phase cells were harvested by centrifugation at 3000.times.g for 3 minutes and resuspended in 20 ml of fresh KD buffer (50 mM potassium phosphate buffer, pH 7.5, containing 25 mM DTT). The cells were incubated in this buffer at 30.degree. C. for 15 minutes. The cells were then harvested and resuspended in 200 ml of ice-cold STM (270 mM sucrose, 10 mM Tris, pH 7.5, mM MgCl.sub.2). The cells were harvested and resuspended in 100 ml of ice-cold STM. The cells were again harvested and resuspended in 3-5 ml of ice-cold STM. 100-.mu.l aliquouts of electrocompetent cells from each culture were then mixed with Not I-digested pADE1-1 DNA. The cell/DNA mixture was placed in a 2 mm electroporation cuvette and subjected to a pulsed electric field of 5 kV/cm using a BioRad Gene Pulser.TM. set to 1000 .OMEGA. resistance and capacitance of 25 .mu.F. After being pulsed, the cells were diluted by addition of 1 ml YEPD and incubated at 30.degree. C. for one hour. The cells were then harvested by gentle centrifugation and resuspended in 400 .mu.l minimal selective media lacking adenine (ADE D). The resuspended sarnples were split into 200-.mu.l aliqouts and plated onto ADE D and ADE DS plates. Plates were incubated at 30.degree. C. for 4-5 days. Mutants #6 and #1 gave Ade.sup.+ transformants. No Ade.sup.+ transformants were observed when DNA was omitted, hence the two isolates appeared to define the ade2 complementation group. The ADE2 sequence is shown in SEQ ID NO:1.
Example 2
The P. methanolica clone bank disclosed in Example 1 was used as a source for cloning the Alcohol Utilization Gene (AUG1). The clone bank was stored as independent pools, each representing about 200-250 individual genomic clones. 0.1 .mu.l of "miniprep" DNA from each pool was used as a template in a polymerase chain reaction with PCR primers (8784, SEQ ID NO:5; 8787, SEQ ID NO:6) that were designed from an alignment of conserved sequences in alcohol oxidase genes from Hansenula polymorpha, Candida boidini, and Pichia pastoris. The amplification reaction was run for 30 cycles of 94.degree. C., 30 seconds; 72.degree. C., 60 seconds; followed by a 7 minute incubation at 72.degree. C. One pool (#5) gave a .about.600 bp band. DNA sequencing of this PCR product revealed that it encoded an amino acid sequence with .about.70% sequence identity with the Pichia pastoris alcohol oxidase encoded by the AOX1 gene and about 85% sequence identity with the Hansenula polymorpha alcohol oxidase encoded by the MOX1 gene. The sequence of the cloned AUG1 gene is shown in SEQ ID NO:2.
Sub-pools of pool #5 were analyzed by PCR using the same primers used in the initial amplification. One positive sub-pool was further broken down to identify a positive colony. This positive colony was streaked on plates, and DNA was prepared from individual colonies. Three colonies gave identical patterns after digestion with Cla I.
Restriction mapping of the genomic clone and PCR product revealed that the AUG1 gene lay on a 7.5 kb genomic insert and that sites within the PCR fragment could be uniquely identified within the genomic insert. Because the orientation of the gene within the PCR fragment was known, the latter information provided the approximate location and direction of transcription of the AUG1 gene within the genomic insert. DNA sequencing within this region revealed a gene with very high sequence similarity at the amino acid level to other known alcohol oxidase genes.
Example 3
ade2 mutant P. methanolica cells are transformed by electroporation essentially as disclosed above with an expression vector comprising the AUG1 promoter and terminator, human GAD65 DNA (Karlsen et al., Proc. Natl. Acad. Sci. USA 88:8337-8341, 1991), and ADE2 selectable marker. Colonies are patched to agar minimal methanol plates (10 to 100 colonies per 100-mm plate) containing 20 g/L Bacto.TM.-agar (Difco), 6.7 g/L yeast nitrogen base without amino acids (Difco), 10 g/L methanol, and 0.4 .mu.g/L biotin. The agar is overlayed with nitrocellulose, and the plates are inverted over lids containing 1 ml of 50% methanol in water and incubated for 3 to 5 days at 30.degree. C. The membrane is then transferred to a filter soaked in 0.2M NaOH, 0.1% SDS, 35 mM dithiothreitol to lyse the adhered cells. After 30 minutes, cell debris is rinsed from the filter with distilled water, and the filter is neutralized by rinsing it for 30 minutes in 0.1M acetic acid.
The filters are then assayed for adhered protein. Unoccupied binding sites are blocked by rinsing in TTBS-NFM (20 mM Tris pH 7.4, 0.1% Tween 20, 160 mM NaCl, 5% powdered nonfat milk) for 30 minutes at room temperature. The filters are then transferred to a solution containing GAD6 monoclonal antibody (Chang and Gottlieb, J. Neurosci. 8:2123-2130, 1988), diluted 1:1000 in TTBS-NFM. The filters are incubated in the antibody solution with gentle agitation for at least one hour, then washed with TTBS (20 mM Tris pH 7.4, 0.1% Tween 20, 160 mM NaCl) two times for five minutes each. The filters are then incubated in goat anti-mouse antibody conjugated to horseradish peroxidase (1 .mu.g/ml in TTBS-NFM) for at least one hour, then washed three times, 5 minutes per wash with TTBS. The filters are then exposed to commercially available chemiluminescence reagents (ECL.TM.; Amersham Inc., Arlington Heights, Ill.). Light generated from positive patches is detected on X-ray film.
To more accurately detect the level of GAD.sub.65 expression, candidate clones are cultured in shake flask cultures. Colonies are grown for two days on minimal methanol plates at 30.degree. C. as disclosed above. The colonies are used to inoculate 20 ml of minimal methanol media (6.7 g/L yeast nitrogen base without amino acids, 10 g/L methanol, 0.4 .mu.g/L biotin) at a cell density of 1.times.10.sup.6 cells/ml. The cultures are grown for 1-2 days at 30.degree. C. with vigorous shaking. 0.2 ml of 50% methanol is added to each culture daily. Cells are harvested by centrifugation and suspended in ice-cold lysis buffer (20 mM Tris pH 8.0, 40 mM NaCl, 2 mM PMSF, 1 mM EDTA, 1 .mu.g/ml leupeptin, 1 .mu.g/ml pepstatin, 1 .mu.g/ml aprotinin) at 10 ml final volume per 1 g cell paste. 2.5 ml of the resulting suspension is added to 2.5 ml of 400-600 micron, ice-cold, acid-washed glass beads in a 15-ml vessel, and the mixture is vigorously agitated for one minute, then incubated on ice for 1 minute. The procedure is repeated until the cells have been agitated for a total of five minutes. Large debris and unbroken cells are removed by centrifugation at 1000.times.g for 5 minutes. The clarified lysate is then decanted to a clean container. The cleared lysate is diluted in sample buffer (5% SDS, 8M urea, 100 mM Tris pH 6.8, 10% glycerol, 2 mM EDTA, 0.01% bromphenol glue) and electrophoresed on a 4-20% acrylamide gradient gel (Novex, San Diego, Calif.). Proteins are blotted to nitrocellose and detected with GAD6 antibody as disclosed above.
Clones exhibiting the highest levels of methanol-induced expression of foreign protein in shake flask culture are more extensively analyzed under high cell density fermentation conditions. Cells are first cultivated in 0.5 liter of YEPD broth at 30.degree. C. for 1-2 days with vigorous agitation, then used to inoculate a 5-liter fermentation apparatus (e.g., BioFlow III; New Brunswick Scientific Co., Inc., Edison, N.J.). The fermentation vessel is first charged with mineral salts by the addition of 57.8 g (NH.sub.4).sub.2 SO.sub.4, 68 g KH.sub.2 PO.sub.4, 30.8 g MgSO.sub.4.7H.sub.2 O, 8.6 g CaSO.sub.4.2H.sub.2 O, 2.0 g NaCl, and 10 ml antifoam (PPG). H.sub.2 O is added to bring the volume to 2.5 L, and the solution is autoclaved 40 minutes. After cooling, 350 ml of 50% glucose, 250 ml 10.times. trace elements (Table 4), 25 ml of 200 .mu.g/ml biotin, and 250 ml cell inoculum are added.
TABLE 4 ______________________________________ 10 X trace elements: ______________________________________ FeSO.sub.4.7H.sub.2 O 100 mM 27.8 g/L CuSO.sub.4.5H.sub.2 O 2 mM 0.5 g/L ZnCl.sub.2 8 mM 1.09 g/L MnSO.sub.4.H.sub.2 O 8 mM 1.35 g/L CoCl.sub.2.6H.sub.2 O 2 mM 0.48 g/L Na.sub.2 MoO.sub.4.2H.sub.2 O 1 mM 0.24 g/L H.sub.3 BO.sub.3 8 mM 0.5 g/L KI 0.5 mm 0.08 g/L biotin 5 mg/L thiamine 0.5 g/L ______________________________________ Add 1-2 mls H.sub.2 SO.sub.4 per liter to bring compounds into solution.
The fermentation vessel is set to run at 28.degree. C., pH 5.0, and >30% dissolved O.sub.2. The cells will consume the initial charge of glucose, as indicated by a sharp demand for oxygen during glucose consumption followed by a decrease in oxygen consumption after glucose is exhausted. After exhaustion of the initial glucose charge, a glucose-methanol feed supplemented with NH.sub.4.sup.+ and trace elements is delivered into the vessel at 0.2% (w/v) glucose, 0.2% (w/v) methanol for 5 hours followed by 0.1% (w/v) glucose, 0.4% (w/v) methanol for 25 hours. A total of 550 grams of methanol is supplied through one port of the vessel as pure methanol using an initial delivery rate of 12.5 ml/hr and a final rate of 25 ml/hr. Glucose is supplied through a second port using a 700 ml solution containing 175 grams glucose, 250 ml 10.times. trace elements, and 99 g (NH.sub.4).sub.2 SO.sub.4. Under these conditions the glucose and methanol are simultaneously utilized, with the induction of GAD.sub.65 expression upon commencement of the glucose-methanol feed. Cells from the fermentation vessel are analyzed for GAD.sub.65 expression as described above for shake flask cultures.
Cells are removed from the fermentation vessel at certain time intervals and subsequently analyzed. Little GAD.sub.65 expression is observed during growth on glucose. Exhaustion of glucose leads to low level expression of the GAD.sub.65 protein; expression is enhanced by the addition of MeOH during feeding of the fermentation culture. The addition of methanol has a clear stimulatory effect of the expresion of human GAD.sub.65 driven by the methanol-responsive AUG1 promoter.
Example 4
Transformation conditions were investigated to determine the electric field conditions, DNA topology, and DNA concentration that were optimal for efficient transformation of P. methanolica. All experiments used P. methanolica ade2 strain #11. Competent cells were prepared as previously described. Electroporation was carried out using a BioRad Gene Pulser.TM..
Three field parameters influence transformation efficiency by electroporation: capacitance, field strength, and pulse duration. Field strength is determined by the voltage of the electric pulse, while the pulse duration is determined by the resistance setting of the instrument. Within this set of experiments, a matrix of field strength settings at various resistances was examined. In all experiments, the highest capacitance setting (25 .mu.F) of the instrument was used. 100 .mu.l aliquots of electrocompetent cells were mixed on ice with 10 .mu.l of DNA that contained approximately 1 .mu.g of the ADE2 plasmid pCZR133 that had been linearized with the restriction enzyme Not I. Cells and DNA were transferred to 2 mm electroporation cuvettes (BTX Corp., San Diego, Calif.) and electropulsed at field strengths of 0.5 kV (2.5 kV/cm), 0.75 kV (3.75 kV/cm), 1.0 kV (5.0 kV/cm), 1.25 kV (6.25 kV/cm), and 1.5 kV (7.5 kV/cm). These field strength conditions were examined at various pulse durations. Pulse duration was manipulated by varying the instrument setting resistances to 200 ohms, 600 ohms, or "infinite" ohms. Pulsed cells were suspended in YEPD and incubated at 30.degree. C. for one hour, harvested, resuspended, and plated. Three separate sets of experiments were conducted. In each set, electroporation conditions of 0.75 kV (3.75 kV/cm) at a resistance of "infinite" ohms was found to give a dramatically higher transformation efficiency than other conditions tested (see FIG. 1).
After the optimal pulse conditions were established, the influence of DNA topology on transformation efficiency was investigated. Electrocompetent cells were mixed with 1 .mu.g of uncut, circular pCZR133 or with 1 .mu.g of Not I-digested pCZR133. In three separate experiments, an average of roughly 25 transformants were recovered with circular DNA while linear DNA yielded an average of nearly 1.times.10.sup.4 transformants. These data indicate that linear DNA transforms P. methanolica with much greater efficiency than circular DNA.
Finally, the relationship between DNA concentration and transformation efficiency was investigated. Aliquots of linear pCZR133 DNA (1 ng, 10 ng, 100 ng and 1 .mu.g in 10 .mu.l H.sub.2 O) were mixed with 100 .mu.l electrocompetent cells, and electroporation was carried out at 3.75 kV/cm and "infinite" ohms. The number of transformants varied from about 10 (1 .mu.g DNA) to 10.sup.4 (1 .mu.g DNA) and was found to be proportional to the DNA concentration.
Example 5
Integration of transforming DNA into the genome of P. methanolica was detected by comparison of DNA from wild-type cells and stable, white transformant colonies. Two classes of integrative transformants were identified. In the first, transforming DNA was found to have integrated into a homologous site. In the second class, transforming DNA was found to have replaced the endogenous AUG1 open reading frame. While not wishing to be bound by theory, this second transformant is believed to have arisen by a "transplacement recombination event" (Rothstein, Methods Enzymol. 194:281-301, 1991) whereby the transforming DNA replaces the endogenous DNA via a double recombination event.
P. methanolica ade2 strain #11 was transformed to Ade.sup.+ with Asp I-digested pCZR140, a Bluescript.RTM. (Stratagene Cloning Systems, La Jolla, Calif.)-based vector containing the P. methanolica ADE2 gene and a mutant of AUG1 in which the entire open reading frame between the promoter and terminator regions has been deleted (FIG. 2). Genomic DNA was prepared from wild-type and transformant cells grown for two days on YEPD plates at 30.degree. C. About 100-200 .mu.l of cells was suspended in 1 ml H.sub.2 O then centrifuged in a microcentrifuge for 30 seconds. The cell pellet was recovered and resuspended in 400 .mu.l of SCE+DTT+zymolyase (1.2M sorbitol, 10 mM Na citrate, 10 mM EDTA, 10 mM DTT, 1-2 mg/ml zymolyase 100T) and incubated at 37.degree. C. for 10-15 minutes. 400 .mu.l of 1% SDS was added, and the solution was mixed until clear. 300 .mu.l of 5M potassium acetate, pH 8.9 was added, and the solution was mixed and centrifuged at top speed in a microcentrifuge for five minutes. 750 .mu.l of the supernatant was transferred to a new tube and extracted with an equal volume of phenol/chloroform. 600 .mu.l of the resulting supernatant was recovered, and DNA was precipitated by the addition of 2 volumes of ethanol and centrifugation for 15 minutes in the cold. The DNA pellet was resuspended in 50 ml TE (10 mM Tris pH 8, 1 mM EDTA)+100 .mu.g/ml RNAase for about 1 hour at 65.degree. C. 10-.mu.l DNA samples were digested with Eco RI (5 .mu.l) in a 100 .mu.l reaction volume at 37.degree. C. overnight. DNA was precipitated with ethanol, recovered by centrifugation, and resuspended in 7.5 .mu.l TE+2.5 .mu.l 5.times. loading dye. The entire 10 ml volume was applied to one lane of a 0.7% agarose in 0.5.times. TBE (10.times. TBE is 108 g/L Tris base 7-9, 55 g/L boric acid, 8.3 g/L disodium EDTA) gel. The gel was run at 100 V in 0.5.times.TBE containing ethidium bromide. The gel was photographed, and DNA was electrophoretically transferred to a positively derivatized nylon membrane (Nytran.RTM. N+, Schleicher & Schuell, Keene, N.H.) at 400 mA, 20 mV for 30 minutes. The membrane was then rinsed in 2.times. SSC, blotted onto denaturation solution for five minutes, neutralized in 2.times. SSC, then crosslinked damp in a UV crosslinker (Stratalinker.RTM., Stratagene Cloning Systems) on automatic setting. The blot was hybridized to a PCR-generated AUG1 promoter probe using a commercially available kit (ECL.TM. kit, Amersham Corp., Arlington Heights, Ill.). Results indicated that the transforming DNA altered the structure of the AUG1 promoter DNA, consistant with a homologous integration event (FIG. 2).
In a second experiment, P. methanolica ade 2 strain #11 was transformed to Ade.sup.+ with Not I-digested pCZR137, a vector containing a human GAD65 cDNA between the AUG1 promoter and terminator (FIG. 3). Genomic DNA was prepared as described above from wild-type cells and a stable, white, Ade.sup.+ transformant and digested with Eco RI. The digested DNA was separated by electrophoresis and blotted to a membrane. The blot was probed with a PCR-generated probe corresponding to either the AUG1 open reading frame or the AUG1 promoter. Results demonstrated that the AUG1 open reading frame DNA was absent from the transformant strain, and that the AUG1 promoter region had undergone a significant rearrangement. These results are consistent with a double recombination event (transplacement) between the transforming DNA and the host genome (FIG. 3).
From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.
__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 6 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3077 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CAGCTGCTCTGCTCCTTGATTCGTAATTAATGTTATCCTTTTACTTTGAACTCTTGTCGG60 TCCCCAACAGGGATTCCAATCGGTGCTCAGCGGGATTTCCCATGAGGTTTTTGACAACTT120 TATTGATGCTGCAAAAACTTTTTTAGCCGGGTTTAAGTAACTGGGCAATATTTCCAAAGG180 CTGTGGGCGTTCCACACTCCTTGCTTTTCATAATCTCTGTGTATTGTTTTATTCGCATTT240 TGATTCTCTTATTACCAGTTATGTAGAAAGATCGGCAAACAAAATATCAACTTTTATCTT300 GAACGCTGACCCACGGTTTCAAATAACTATCAGAACTCTATAGCTATAGGGGAAGTTTAC360 TGCTTGCTTAAAGCGGCTAAAAAGTGTTTGGCAAATTAAAAAAGCTGTGACAAGTAGGAA420 CTCCTGTAAAGGGCCGATTCGACTTCGAAAGAGCCTAAAAACAGTGACTATTGGTGACGG480 AAAATTGCTAAAGGAGTACTAGGGCTGTAGTAATAAATAATGGAACAGTGGTACAACAAT540 AAAAGAATGACGCTGTATGTCGTAGCCTGCACGAGTAGCTCAGTGGTAGAGCAGCAGATT600 GCAAATCTGTTGGTCACCGGTTCGATCCGGTCTCGGGCTTCCTTTTTTGCTTTTTCGATA660 TTTGCGGGTAGGAAGCAAGGTCTAGTTTTCGTCGTTTCGGATGGTTTACGAAAGTATCAG720 CCATGAGTGTTTCCCTCTGGCTACCTAATATATTTATTGATCGGTCTCTCATGTGAATGT780 TTCTTTCCAAGTTCGGCTTTCAGCTCGTAAATGTGCAAGAAATATTTGACTCCAGCGACC840 TTTCAGAGTCAAATTAATTTTCGCTAACAATTTGTGTTTTTCTGGAGAAACCTAAAGATT900 TAACTGATAAGTCGAATCAACATCTTTAAATCCTTTAGTTAAGATCTCTGCAGCGGCCAG960 TATTAACCAATAGCATATTCACAGGCATCACATCGGAACATTCAGAATGGACTCGCAAAC1020 TGTCGGGATTTTAGGTGGTGGCCAACTTGGTCGTATGATCGTTGAAGCTGCACACAGATT1080 GAATATCAAAACTGTGATTCTCGAAAATGGAGACCAGGCTCCAGCAAAGCAAATCAACGC1140 TTTAGATGACCATATTGACGGCTCATTCAATGATCCAAAAGCAATTGCCGAATTGGCTGC1200 CAAGTGTGATGTTTTAACCGTTGAGATTGAACATGTTGACACTGATGCGTTGGTTGAAGT1260 TCAAAAGGCAACTGGCATCAAAATCTTCCCATCACCAGAAACTATTTCATTGATCAAAGA1320 TAAATACTTGCAAAAAGAGCATTTGATTAAGAATGGCATTGCTGTTGCCGAATCTTGTAG1380 TGTTGAAAGTAGCGCAGCATCTTTAGAAGAAGTTGGTGCCAAATACGGCTTCCCATACAT1440 GCTAAAATCTAGAACAATGGCCTATGACGGAAGAGGTAATTTTGTTGTCAAAGACAAGTC1500 ATATATACCTGAAGCTTTGAAAGTTTTAGATGACAGGCCGTTATACGCCGAGAAATGGGC1560 TCCATTTTCAAAGGAGTTAGCTGTTATGGTTGTGAGATCAATCGATGGCCAAGTTTATTC1620 CTACCCAACTGTTGAAACCATCCACCAAAACAACATCTGTCACACTGTCTTTGCTCCAGC1680 TAGAGTTAACGATACTGTCCAAAAGAAGGCCCAAATTTTGGCTGACAACGCTGTCAAATC1740 TTTCCCAGGTGCTGGTATCTTTGGTGTTGAAATGTTTTTATTACAAAATGGTGACTTATT1800 AGTCAACGAAATTGCCCCAAGACCTCACAATTCTGGTCACTATACCATCGACGCTTGTGT1860 CACCTCGCAATTTGAAGCTCATGTTAGGGCCATTACTGGTCTACCCATGCCGAAGAACTT1920 CACTTGTTTGTCGACTCCATCTACCCAAGCTATTATGTTGAACGTTTTAGGTGGCGATGA1980 GCAAAACGGTGAGTTCAAGATGTGTAAAAGAGCACTAGAAACTCCTCATGCTTCTGTTTA2040 CTTATACGGTAAGACTACAAGACCAGGCAGAAAAATGGGTCACATTAATATAGTTTCTCA2100 ATCAATGACTGACTGTGAGCGTAGATTACATTACATAGAAGGTACGACTAACAGCATCCC2160 TCTCGAAGAACAGTACACTACAGATTCCATTCCGGGCACTTCAAGCAAGCCATTAGTCGG2220 TGTCATCATGGGTTCCGATTCGGACCTACCAGTCATGTCTCTAGGTTGTAATATATTGAA2280 GCAATTTAACGTTCCATTTGAAGTCACTATCGTTTCCGCTCATAGAACCCCACAAAGAAT2340 GGCCAAGTATGCCATTGATGCTCCAAAGAGAGGGTTGAAGTGCATCATTGCTGGTGCTGG2400 TGGTGCCGCTCATTTACCGGGAATGGTTGCGGCGATGACGCCGCTGCCTGTTATTGGTGT2460 CCCTGTTAAAGGCTCTACTTTGGATGGTGTTGATTCACTACACTCCATCGTTCAAATGCC2520 AAGAGGTATTCCTGTTGCTACTGTGGCTATTAACAATGCTACTAACGCTGCCTTGCTAGC2580 TATCACAATCTTAGGTGCCGGCGATCCAAATACTTGTCTGCAATGGAAGTTTATATGAAC2640 AATATGGAAAATGAAGTTTTGGGCAAGGCTGAAAAATTGGAAAATGGTGGATATGAAGAA2700 TACTTGAGTACATACAAGAAGTAGAACCTTTTATATTTGATATAGTACTTACTCAAAGTC2760 TTAATTGTTCTAACTGTTAATTTCTGCTTTGCATTTCTGAAAAGTTTAAGACAAGAAATC2820 TTGAAATTTCTAGTTGCTCGTAAGAGGAAACTTGCATTCAAATAACATTAACAATAAATG2880 ACAATAATATATTATTTCAACACTGCTATATGGTAGTTTTATAGGTTTGGTTAGGATTTG2940 AGATATTGCTAGCGCTTATCATTATCCTTAATTGTTCATCGACGCAAATCGACGCATTTC3000 CACAAAAATTTTCCGAACCTGTTTTTCACTTCTCCAGATCTTGGTTTAGTATAGCTTTTG3060 ACACCTAATACCTGCAG3077 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3386 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GAATTCCTGCAGCCCGGGGGATCGGGTAGTGGAATGCACGGTTATACCCACTCCAAATAA60 AAGTGTAGTAGCCGGACTGAAAGGTTTTAGGAGTCTGTTTGTTTGTTCATGTGCATCATT120 CCCTAATCTGTTAACAGTCTCGGAGTATACAAAAAAGTAAGTCAAATATCAAGGTGGCCG180 GGGGCAGCATCGAGACTCGAGATGGTACATACTTAAAAGCTGCCATATTGAGGAACTTCA240 AAGTTTTATCTGTTTTTAGAATTAAAAGACGATTGTTGTAACAAAACGTTGTGCCTACAT300 AAACTCAAATTAATGGAAATAGCCTGTTTTGAAAAATACACCTTCTTAAGTACTGACAAA360 GTTTTGTTAAATGACTATCGAACAAGCCATGAAATAGCACATTTCTGCCAGTCACTTTTA420 ACACTTTCCTGCTTGCTGGTTGACTCTCCTCATACAAACACCCAAAAGGGAAACTTTCAG480 TGTGGGGACACTTGACATCTCACATGCACCCCAGATTAATTTCCCCAGACGATGCGGAGA540 CAAGACAAAACAACCCTTTGTCCTGCTCTTTTCTTTCTCACACCGCGTGGGTGTGTGCGC600 AGGCAGGCAGGCAGGCAGCGGGCTGCCTGCCATCTCTAATCGCTGCTCCTCCCCCCTGGC660 TTCAAATAACAGCCTGCTGCTATCTGTGACCAGATTGGGACACCCCCCTCCCCTCCGAAT720 GATCCATCACCTTTTGTCGTACTCCGACAATGATCCTTCCCTGTCATCTTCTGGCAATCA780 GCTCCTTCAATAATTAAATCAAATAAGCATAAATAGTAAAATCGCATACAAACGTCATGA840 AAAGTTTTATCTCTATGGCCAACGGATAGTCTATCTGCTTAATTCCATCCACTTTGGGAA900 CCGCTCTCTCTTTACCCCAGATTCTCAAAGCTAATATCTGCCCCTTGTCTATTGTCCTTT960 CTCCGTGTACAAGCGGAGCTTTTGCCTCCCATCCTCTTGCTTTGTTTCGGTTATTTTTTT1020 TTCTTTTGAAACTCTTGGTCAAATCAAATCAAACAAAACCAAACCTTCTATTCCATCAGA1080 TCAACCTTGTTCAACATTCTATAAATCGATATAAATATAACCTTATCCCTCCCTTGTTTT1140 TTACCAATTAATCAATCTTCAAATTTCAAATATTTTCTACTTGCTTTATTACTCAGTATT1200 AACATTTGTTTAAACCAACTATAACTTTTAACTGGCTTTAGAAGTTTTATTTAACATCAG1260 TTTCAATTTACATCTTTATTTATTAACGAAATCTTTACGAATTAACTCAATCAAAACTTT1320 TACGAAAAAAAAATCTTACTATTAATTTCTCAAAATGGCTATTCCAGATGAATTTGATAT1380 TATTGTTGTCGGTGGTGGTTCCACCGGTTGTGCTCTTGCTGGTAGATTAGGTAACTTGGA1440 CGAAAACGTCACAGTTGCTTTAATCGAAGGTGGTGAAAACAACATCAACAACCCATGGGT1500 TTACTTACCAGGTGTTTATCCAAGAAACATGAGATTAGACTCAAAGACTGCTACTTTTTA1560 CTCTTCAAGACCATCACCACACTTGAACGGTAGAAGAGCTATTGTTCCATGTGCTAACAT1620 CTTGGGTGGTGGTTCTTCCATCAACTTCTTGATGTACACCAGAGCCTCTGCCTCCGATTA1680 CGATGATTGGGAATCTGAAGGTTGGACTACCGATGAATTATTACCACTAATGAAGAAGAT1740 TGAAACTTATCAAAGACCATGTAACAACAGAGAATTGCACGGTTTCGATGGTCCAATTAA1800 GGTTTCATTTGGTAACTATACTTATCCAAACGGTCAAGATTTCATTAGAGCTGCCGAATC1860 TCAAGGTATTCCATTTGTTGATGATGCTGAAGATTTGAAATGTTCCCACGGTGCTGAGCA1920 CTGGTTGAAGTGGATCAACAGAGACTTAGGTAGAAGATCCGATTCTGCTCATGCTTACAT1980 TCACCCAACCATGAGAAACAAGCAAAACTTGTTCTTGATTACTTCCACCAAGTGTGAAAA2040 GATTATCATTGAAAACGGTGTTGCTACTGGTGTTAAGACTGTTCCAATGAAGCCAACTGG2100 TTCTCCAAAGACCCAAGTTGCTAGAACTTTCAAGGCTAGAAAGCAAATTATTGTTTCTTG2160 TGGTACTATCTCATCACCATTAGTTTTGCAAAGATCTGGTATCGGTTCCGCTCACAAGTT2220 GAGACAAGTTGGTATTAAACCAATTGTTGACTTACCAGGTGTTGGTATGAACTTCCAAGA2280 TCACTACTGTTTCTTCACTCCATACCATGTCAAGCCAGATACTCCATCATTCGATGACTT2340 TGTTAGAGGTGATAAAGCTGTTCAAAAATCTGCTTTCGACCAATGGTATGCTAACAAGGA2400 TGGTCCATTAACCACTAATGGTATTGAGGCAGGTGTTAAGATTAGACCAACTGAAGAAGA2460 ATTAGCCACTGCTGATGACGAATTCAGAGCTGCTTATGATGACTACTTTGGTAACAAGCC2520 AGATAAGCCATTAATGCACTACTCTCTAATTTCTGGTTTCTTTGGTGACCACACCAAGAT2580 TCCAAACGGTAAGTACATGTGCATGTTCCACTTCTTGGAATATCCATTCTCCAGAGGTTT2640 CGTTCACGTTGTTTCTCCAAACCCATACGATGCTCCTGACTTTGATCCAGGTTTCATGAA2700 CGATCCAAGAGATATGTGGCCAATGGTTTGGTCTTACAAGAAGTCCAGAGAAACTGCCAG2760 AAGAATGGACTGTTTTGCCGGTGAAGTTACTTCTCACCACCCACACTACCCATACGACTC2820 ACCAGCCAGAGCTGCTGACATGGACTTGGAAACTACTAAAGCTTATGCTGGTCCAGACCA2880 CTTTACTGCTAACTTGTACCACGGTTCATGGACTGTTCCAATTGAAAAGCCAACTCCAAA2940 GAACGCTGCTCACGTTACTTCTAACCAAGTTGAAAAACATCGTGACATCGAATACACCAA3000 GGAGGATGATGCTGCTATCGAAGATTACATCAGAGAACACACTGAAACCACATGGCATTG3060 TCTTGGTACTTGTTCAATGGCTCCAAGAGAAGGTTCTAAGGTTGTCCCAACTGGTGGTGT3120 TGTTGACTCCAGATTAAACGTTTACGGTGTTGAAAAGTTGAAGGTTGCTGATTTATCAAT3180 TTGCCCAGATAATGTTGGTTGTAACACTTACTCTACTGCTTTGTTAATCGGTGAAAAGGC3240 TTCTACCTTAGTTGCTGAAGACTTGGGCTACTCTGGTGATGCTTTGAAGATGACTGTTCC3300 AAACTTCAAATTGGGTACTTATGAAGAAGCTGGTCTAGCTAGATTCTAGGGCTGCCTGTT3360 TGGATATTTTTATAATTTTTGAGAGT3386 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TGATCACCTAGGACTAGTGACAAGTAGGAACTCCTGTA38 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CAGCTGCCTAGGACTAGTTTCCTCTTACGAGCAACTAGA39 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TGGTTGAAGTGGATCAA17 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GTGTGGTCACCGAAGAA17 __________________________________________________________________________
1. A method for introducing DNA into a Pichia methanolica (P. methanolica) cell, comprising exposing said Pichia methanolica cell, in the presence of a linear DNA molecule, to an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm and a time constant of from 15 to 40 milliseconds, whereby said linear DNA molecule is introduced into said cell.
2. The method according to claim 1 wherein said linear DNA molecule comprises a DNA sequence encoding a polypeptide, other than a P. methanolica polypeptide, operably linked to a P. methanolica gene transcription promoter and a P. methanolica gene transcription terminator.
3. The method according to claim 2 wherein said transcription promoter is a P. methanolica AUG1 gene promoter.
4. The method according to claim 3 wherein said AUG1 gene promoter comprises a sequence of nucleotides as shown in SEQ ID NO:2 from nucleotide 24 to nucleotide 1354.
5. The method according to claim 2 wherein said linear DNA molecule further comprises a selectable marker gene that complements a mutation in said cell.
6. The method according to claim 5 wherein said selectable marker gene is a P. methanolica gene.
7. The method according to claim 5 wherein said marker gene is a P. methanolica ADE2 gene.
8. The method according to claim 7 wherein said ADE2 gene comprises a sequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 407 to nucleotide 2851.
9. The method according to claim 1 wherein said linear DNA molecule comprises a selectable marker gene that complements a mutation in said cell.
10. The method according to claim 9 wherein said marker gene is a P. methanolica gene.
11. The method according to claim 9 wherein said marker gene is a P. methanolica ADE2 gene.
12. The method according to claim 11 wherein said ADE2 gene comprises a sequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 407 to nucleotide 2851.
13. The method according to claim 1 wherein said time constant is from 15 to 25 milliseconds.
14. A method for transforming P. methanolica cells with heterologous DNA, comprising exposing a population of said P. methanolica cells, in the presence of heterologous linear DNA molecules, to an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm and a time constant of from 15 to 40 milliseconds, whereby said heterologous DNA is introduced into at least a portion of said population, and recovering cells into which said DNA has been introduced.
15. The method according to claim 14 wherein from 10.sup.3 to 10.sup.5 cells are recovered per microgram of said heterologous linear DNA molecules.
16. The method according to claim 15 wherein from 0.9.times.10.sup.4 to 1.1.times.10.sup.4 cells are recovered per microgram of said heterologous linear DNA molecules.
17. The method according to claim 14 further comprising the step of recovering integrative transformants from said recovered cells.
18. The method according to claim 17 wherein said recovering step comprises culturing said recovered cells in a growth medium comprising sorbitol as a carbon source.
19. The method according to claim 14 wherein said population of cells is in early log phase growth.
20. The method according to claim 14 wherein said time constant is from 15 to 25 milliseconds.
Referenced Cited
U.S. Patent Documents
|November 7, 1989
|Stroman et al.
|March 20, 1990
|April 19, 1994
|February 10, 1998
Other references
- Piredda et al., Yeast 10: 1601-1612, 1994. Jong et al., Soc. Indust. Microbiol. News 46: 199-203, 1996. Meilhoc et al., Bio/Technology 8: 223-227, 1990. Gellissen et al., Tibtech 10: 413-417, 1992. Faber et al., Curr. Genet. 25: 305-310, 1994. Hiep et al., Yeast 9: 1251-1258, 1993. Hiep et al., Yeast 9: 1189-1197, 1993.
Patent History
Type: Grant
Filed: Jul 14, 1997
Date of Patent: Dec 29, 1998
Assignee: ZymoGenetics, Inc. (Seattle, WA)
Inventors: Christopher K. Raymond (Seattle, WA), Susan D. Holderman (Seattle, WA), Erica Vanaja (Seattle, WA)
Primary Examiner: Terry A. McKelvey
Attorney: Gary E. Parker
Application Number: 8/914,066
International Classification: C12N 1563;


text:
Five One Two Counseling & Coaching
Shaina Singh, LCSW, Board Approved Supervisor
Psychotherapist. Life Coach. Dating Coach
About Me
About Shaina
Imagine walking into a room where you are greeted with warmth, compassion, and acceptance. Now imagine sitting in front of someone who listens to you, really gets you and lets you be you. That is my goal as your therapist. I accept you as you are, offer you support and guidance, help you learn tools and offer you hope. In fact, most of my clients say that when they leave the sessions, they start to notice changes and feel hopeful about their life, relationship, and the problems.
I believe that if it's not broken, don't fix it; if it works, do more of it; if it's not working, do something different; small steps can lead to big changes; the solution is not necessarily related to the problem; & the future is both created and negotiable.
I have worked in the behavioral health field for over nine years. In that time, I have provided counseling to couples, families, worked with victims of domestic violence, rape, trauma, homelessness, military individuals, & families, helped people enduring grief, counseled on healthy relationships, and dating dilemmas. I am a poly and kink friendly therapist who will provide you with a safe space along with understanding and compassion as you work together. I am also a proud and committed ALLY to the LGBTQII community and really enjoy working and supporting the community.
Licensed Clinical Social Worker certified by the Texas State Board of Examiners License # 54876
Texas State Board Approved Supervisor for LMSW supervisees
Emotionally Focused Therapy Training and Certification by ICCEFT
Life and Dating Coach certified by Life Coaching Institute of America
Certified Solution Focused Therapist by BRIEF INTERNATIONAL
PreMarital Counseling Educator by SYMBIS (Saving Your Marriage Before It Starts)
Master of Science in Social Work from the University of Texas at Arlington
Bachelor of Arts in Psychology from University of Texas at Tyler
My areas of specialty include relationships, individual therapy, couples therapy, dating coaching, breakup, separation or divorce, sexuality and gender, poly and kink relationships and transgender community.


text:
(CNN) -- As times get tough, people look for change. Not just new presidents and prime ministers, but change they can count on -- or, at least, count. And they're willing to turn over couch cushions to find it.
The piggy bank business is one industry that has seen business boom in the wake of the financial crisis.
As the financial crisis thrust billion-dollar figures onto the front pages, it also has cast a spotlight on how we perceive and use the smallest of currencies -- namely, metal coins.
In Thailand, the Treasury Department has found that coin exchange for paper currency has doubled in the first half of the year. In Argentina, the year began with a drought of coins as people began to hoard pesos.
While most Christmas gift-giving slumped, retailers in the U.S. and the U.K. saw a rise in piggy bank sales -- U.K. retailer Debenhams reported a 150 percent increase in sales during the holiday season.
Priya Raghubir is not surprised. As a researcher at New York University, she studies how coins and small denominations of cash alter people's spending habits -- research inspired partly by her son's delight in getting change after a 7-Eleven purchase on a trip to Hong Kong
"Yet my son refused to cash in a $100 bill he'd been given as a present," she said. She was curious if the same was true for adults.
Her research suggests, indeed, adults spend like children. She found that if two groups are given an equivalent amount of cash -- say a $1 bill versus four quarters, or a $100 bill versus five $20 bills -- the latter group will always spend more cash. Watch why people gravitate toward small change during a recession »
The phenomena crosses borders. When homemakers in China received a week's pay from researchers in large bills vs. small, the ones with smaller bills spent it faster.
The research suggests one way to loosen consumer spending is to increase the supply of small bills or change.
"It suggests that if central banks changed to smaller denominations, people would spend it faster," Raghubir said. Also, if governments gave tax rebates in the form of money orders or traveler's checks that can be cashed at locations other than banks, spending might increase, she added.
There's a lot of spare change out of circulation -- in the United States alone, at least $10 billion sitting unused in piggy banks and coffee cups, according to Coinstar -- a U.S. business which operates and markets change counting machines primarily in the U.S., Canada, the U.K. and Ireland.
Unlike most companies, Coinstar has been able to grow through the credit crisis. "In 2006 we had 13,000 (coin changing) kiosks in place, and in 2008 we had 18,000," said Mike Skinner, president of Coinstar's change business. "We plan to add 1000 or 1500 more this year."
More people are using that change to make purchases at the stores where coin change machines are located -- 55 percent of change ends up being spent immediately on purchases, up nearly 10 percent from 2003, Skinner said. "They tend (to use redeemed change) to upgrade their purchases, like stepping up to a steak rather than a roast, or buying a nicer bottle of wine," he said.
Company research suggests coins are becoming more important to cash-strapped consumers -- a survey late last year found that 42 percent of people aged 18 to 34 are cashing in more change than the previous year. Almost four in 10 survey respondents said they value loose change more than before the credit crisis.
This has all been good business for Coinstar, which saw its stock value jump 70 percent this year, making it one of the better performing stocks this year.
A big reason for Coinstar's success this year came from using revenue built from loose change to buy Redbox, which provides self-service kiosks at stores across the U.S. for $1-a-night rentals on new DVD releases.
The low-cost DVD rental business is doing so well in this economy, it has raised the ire of Hollywood titans -- Redbox filed a lawsuit against Universal, Fox and Warner Bros. (owned by Time Warner, parent company of CNN) after the companies began holding back releases to Redbox out of fears it will further erode new video sales.
Unfortunately, the hunt for change has resulted in some extreme measures. Police in Illinois and Iowa are looking for a Bonnie-and-Clyde pair of bandits caught twice on video tape robbing video game machines at Wal-Marts in north central Illinois, said Detective Jim Merica of the Normal, Ill., police department.
The pair is believed to be responsible for a string of robberies that has netted $10,000 in change from vending machines from June to July. "They hide in plain sight, going into game rooms at times when there are only kids present," he said. "It's a pretty unusual case because of the interstate nature of it."
And in Italy, hard times have brought back the country's most famous coin bandit. For 30 years Roberto Cancelletto was a fixture at Rome's Trevi fountain using a high-powered magnet and fishing reel to nick change from the waters until his arrest in 2002 and a clampdown by Rome police on coin fishing at the fountain. It was reported that Cancelletto made as much as $250,000 a year fishing for change.
This past July, Cancelletto was back at Trevi -- but this time atop the fountain staging a one-man protest until police arrested him. The subject of his protest: He can't find a job, Italian press reported.


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text:
The first Friday of the month is reserved for recipes. You can see additional First Friday Food posts here.
The Reason:
The Journey:
Okay, so I haven't actually made this, which is a first for First Friday Food. So I can't speak for how difficult it is to make, but I can speak for how awesome it is to eat!
The Verdict:
In a day of a lot of really good recipes, this one stood out to me as the best. The bacon really added something; it was neither extraneous nor overpowering. It is sweet, but not too sweet, and it goes perfect for breakfast. There wasn't anybody who didn't like this (even Evie!), in fact, I don't think there was anybody who didn't take seconds (or thirds, or in some cases fourths).
The Recipe:
Adapted from Smittenkitchen.com
- 1 cup Whole wheat flour
- 1 cup all-purpose flour
- 1 Tbsp. baking powder
- 1/4 cup granulated sugar
- 1/2 tsp. salt
- 6 Tbsp cold unsalted butter
- 1/2 to 3/4 cup dried figs (rehydrated)
- 1/2 to 3/4 cup (10 slices) cooked bacon (apple wood smoked)
- 3/4 cup whole milk ricotta
- 1/3 cup heavy cream
- 2 Tbsp. brown sugar
- 1/4 cup maple syrup
- Cook bacon until crispy. Chop into small pieces.
- Prepare figs by chopping them into small pieces (roughly the same size as your bacon pieces). Place the pieces into the bottom of a medium bowl . Cover the fig pieces in boiling water and allow to sit for 10 minutes. Strain fig pieces.
- Preheat oven to 425 degrees.
- Line a large baking sheet with parchment paper.
- In the bottom of a large bowl, whisk the flours, baking powder, sugar, and salt together.
- Cut the butter into small pieces with a knife and work the butter into the flour mixture with your fingertips until the mixture resembles a coarse meal.
- Toss in the figs and bacon and mix.
- Add the ricotta and heavy cream together and stir them in to form a dough with a flexible spatula. Using your hands, gently knead dough into and even mass at the bottom of the bowl.
- Transfer the dough to a slightly floured counter. Lightly flour the top of the dough and pat it into a circle (about 7 in. in diameter) about 1 inch thick. With a large knife, divide the dough into 8 equal pieces. Transfer the scones to the prepared baking sheet with a spatula.
- Bake the scones for 15 mins, until lightly golden at the edges. Cool in a pan for a minute then transfer to a wire rack. Allow them to cool about 5 minutes before glazing.
Maple glaze:
- Combine the maple syrup and brown sugar. Lightly brushed over the baked scones.
Scones are best the day they are baked. However, if you wish to get a lead on them, you can make them, arrange them on the parchment-lined sheet and freeze them. If you are prepping just one day in advance, cover the tray with plastic wrap and bake them the day you need them. If you are prepping them more than one day in advance, once the scones are frozen, transfer the scones to a freezer bag or container. Bring them back to a parchment lined sheet when you are ready to bake them. Do not defrost, just throw the frozen scones in the oven and add 2-3 mins to the baking time.


text:
United Sold; New Focus on Stadium
Saturday, January 6, 2007
A San Francisco real estate developer has partnered with former Duke basketball star Brian Davis and at least two other investors to purchase D.C. United's operating rights, build the team's stadium in Anacostia and develop the land around it, sources said yesterday.
Victor MacFarlane, who heads the largest minority-owned real estate management firm in the country, will lead the group. He will be joined by Davis, a Bladensburg High graduate, and Discovery Communications founder John Hendricks. The group will pay Anschutz Entertainment Group, the MLS team's operator the past five years, an estimated $33 million.
The group is more interested in a proposed 27,000-seat soccer stadium at Poplar Point in Southeast and the mixed-use development that would surround it than it is in the team, said the sources, who refused to be identified because they did not want to upstage the official announcement at a news conference scheduled for Monday.
District Mayor Adrian M. Fenty, Del. Eleanor Holmes Norton (D-D.C.) and two of Fenty's top deputies, City Administrator Dan Tangherlini and Neil O. Albert, deputy mayor for economic development, are scheduled to attend the news conference.
Fenty's team is increasingly leaning toward supporting the new stadium as a way to kick-start development in that area along the Anacostia River, a high-level D.C. government source said.
A further announcement related to the stadium development could come from Fenty in as soon as two weeks, after he has time to work out the specifics of how the project would be financed, the source said. Fenty wants to ensure that a significant amount of private money will be used to build the stadium.
A city planning agency has developed two financing options, one with United controlling the development and paying for the stadium, and one with the city using tax money generated from the site to fund the stadium, but Fenty's aides say they are examining all options.
The soccer facility would be built across the Anacostia from the Washington Nationals' new, publicly funded baseball stadium, scheduled to open in 2008.
United President Kevin Payne, who oversaw the formation of the group and will continue to run the club's front office, said he had no comment on the sale. MacFarlane's spokesman, Doug Holm, did not respond to telephone and e-mail messages.
MLS Commissioner Don Garber, who will also attend Monday's news conference, said through a spokesman that he deferred comment to United officials.
AEG, which also runs MLS teams in Chicago, Los Angeles and Houston, has had United's operating rights on the market for several years. In the summer of 2005, AEG announced a $26 million deal with a group headed by local real estate investors Willi Lauterbach and Tim Kissler, only to see the agreement collapse a few months later amid questions about the new group's financial resources.
Unlike other U.S. professional sports franchises, MLS teams are owned by the league, which awards operating rights to individual groups. AEG, founded by Colorado billionaire Philip Anschutz, took over United's operations five years ago following the disintegration of the club's original investment team, Washington Soccer LP.
MacFarlane, 55, owns 25 percent of the Southeast Federal Center project near the new Nationals stadium. He was involved in a failed attempt by a group of developers to build a mix of condominiums, shops and restaurants, along with parking garages, next to the new baseball stadium. Under that proposal, MacFarlane and local developer Herbert S. Miller had pledged to invest money from the California Public Employees' Retirement System to build the mixed-use development. However, the deal fell through when MacFarlane and Miller failed to reach agreement with District government officials. Miller is not a partner in the soccer deal.
MacFarlane Partners, founded in 1987 in San Francisco, specializes in urban development projects and, as of last year, was managing $2.2 billion in investor equity and $8 billion in properties. MacFarlane, sources said, is the primary financier in the United deal, with Davis, Hendricks and at least one other unidentified investor serving smaller roles.
Davis, 36, graduated from Bladensburg High in 1988 and won NCAA championships in 1991 and '92 at Duke. He and former teammate Christian Laettner are partners in Blue Devil Ventures, a real estate development firm based in Durham, N.C. They have been unsuccessful in attempts to purchase the NBA's Memphis Grizzlies.
Unlike MacFarlane and Davis, Hendricks has a soccer background. In 2001, he founded the Women's United Soccer Association, which folded in 2003. With a new women's league planning to launch within two years, Hendricks could end up overseeing a local team that would share a stadium with United.
Hendricks also helped create the Maryland Soccer Foundation, which runs the Maryland SoccerPlex in Montgomery County.
The deal was brokered by Michael Sears, a local financial advisor and investment manager. Sears is advising the parties on the sale and on developing Poplar Point.
Staff writer Thomas Heath contributed to this report.