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Lucigen Corporation | 2007/2008 Catalog

NEW! OverExpress™ Competent CellsExpress toxic proteins from any organismLucigen’s new OverExpress Electrocompetent and Chemically Competent Cells are unique in their ability to express toxic proteins from all classes of organisms, including eubacteria, yeasts, plants, viruses, and mammals. Proteins that have been difficult or impossible to express in bacteria – including most membrane proteins, some cytoplasmic proteins, and nucleases – are easily expressed in OverExpress Cells.

The OverExpress strains contain genetic mutations phenotypically selected for conferring tolerance to toxic proteins. The strain C41(DE3) was derived from E. coli BL21(DE3). This strain has at least one uncharacterized mutation, which prevents cell death associated with expression of many recombi-nant toxic proteins. The strain C43(DE3) was derived from C41(DE3) by selecting for resistance to expression of a different set of toxic proteins.

As in standard BL21(DE3) cells, the OverExpress are lysogens of λDE3. These strains carry a chromosomal copy of the T7 RNA polymerase gene under the control of the lacUV5 promoter. They are suitable for production of protein from genes cloned into T7-driven expression vectors; e.g., Lucigen’s pSMART®-cDNA vector. OverExpress pLysS strains also carry a chloramphenicol-resistant plasmid that encodes T7 lysozyme, which is a natural inhibitor of T7 RNA polymerase. Cells contain-ing pLysS produce a small amount of T7 lysozyme that suppresses basal expression of T7 RNA poly-merase prior to induction, thus providing additional stability for recombinants encoding particularly toxic proteins. OverExpress Competent Cells are deficient in the lon and ompT proteases, enhancing recombinant protein yields. OverExpress Cells are recA+ and endA+, so they are not recommended for plasmid DNA isolation.

OverExpress Advantages• Exceptional reliability in protein expression. OverExpress Cells greatly increase the probability

of successful expression of cloned genes.

• Successful expression of toxic proteins. BL21(DE3) cells fail to express many proteins due to expression toxicity. Membrane proteins are a particular problem in this regard, yet often these proteins represent important targets in basic research and the development of new diagnostics and pharmaceuticals. OverExpress Competent Cells excel in expressing membrane proteins, as well as many other types of toxic proteins.

• High recombinant yields. At > 1 x 1010 cfu/µg (> 1 x 109 cfu/µg for pLysS), OverExpress Electrocompetent Cells maximize the chance of finding the expressing clone you are looking for. Choose OverExpress Chemically Competent Cells (> 1 x 106 cfu/µg) when maximum recombinant yields are not critical.

• Unbiased expression libraries. OverExpress Electrocompetent Cells increase the complexity of expression libraries, avoiding the problem of lost clones due to expression toxicity. In addition, construction and screening of expression libraries can be done in the same competent cell, saving two days of work.

OverExpress ExamplesWhen transformed with deleterious recombinant clones, OverExpress Competent Cells show uni-formly high expression and robust growth, whereas standard BL21(DE3) cells show poor expression and slow growth (Figure 1). Figure 2 shows transformation effectiveness, tolerance of expression-induced toxicity, and protein expression for T7 expression plasmids coding for a variety of recombi-nant proteins. These results demonstrate that the OverExpress C41(DE3) and C43(DE3) strains are clearly superior to the parental BL21(DE3) in transformation and expression of toxic proteins.

More than 350 publications have validated the effectiveness of OverExpress Competent Cells in ex-pressing many different toxic proteins from a wide variety of organisms. Table 1 (next page) shows representative examples; a more extensive OverExpress bibliography is available on-line at: www.lucigen.com/catalog/overexpress.php.

OverExpress Applications• Toxic protein expression

• Construction of expression libraries (Electrocompetent Cells)

• Routine expression using any E. coli vector with a T7 promoter

Figure 1. Toxic protein expression examples. Green Fluorescent Protein (top) or Red Fluorescence Inducing Protein (bottom) were expressed from a T7 promoter construct that was transformed into C41, BL21, or C43 competent cells spread on IPTG plates to induce protein expression.

C41 BL21 C43

Figure 2. Comparison of OverExpress C41(DE3) and C43(DE3) cells with the parental strain BL21(DE3) in transformation and expression of heterologous proteins.*a Transformation Success Rate corresponds to the number

of colonies on LB+ampicillin agar without induction.b Post-expression Viability corresponds to the number of

colonies on LB+ampicillin+IPTG agar (i.e., with induction).c Protein Expression corresponds to observation of a heter-

ologous protein by SDS-PAGE after induction with IPTG.

* L. Dumon-Seignovert, G. Cariot, and L. Vuillard (2004). Protein Expression and Purification 37, 203. Data used with permission.

BL21(DE3)C41(DE3)C43(DE3)

Transformation Success Ratea

Post-expression Viabilityb

Protein Expressionc

100

80

60

40

20

0

Section 5 Protein Expression

Lucigen Corporation | Advanced Products for Molecular Biology

73

Table 1. Selected published examples of toxic proteins successfully expressed in OverExpress C41 or C43 cells.

Protein Type Organism Strain

Accelerated cell death 1 (ACD1) Arabidopsis thaliana C43(DE3)

AcpM (malonyl acyl carrier protein) Mycobacterium tuberculosis C41(DE3)

AcrA-AcrB-TolC multidrug efflux pump Membrane Enterobacter aerogenes C43(DE3)

ADP/ATP translocase Membrane Bovine C43(DE3)

AHSP (alpha-haemoglobin stabilizing protein) Human C41(DE3)

AIDA-ß domain Membrane Escherichia coli C41(DE3)

AKR1C (aldo-keto reductase 1C) Human C41(DE3)

ATP/ADP translocase Membrane Rickettsia prowazekii C41(DE3)

ATPase (V-ATPase subunit C) Membrane Saccharomyces cerevisiae C41(DE3)

BCR-ABL oncogenic protein Human C41(DE3)

BcrC Bacillus subtilis C41(DE3)

BmrA ATP Binding Cassette transporter Membrane Escherichia coli C41(DE3)

BRCT domain of 53BP1 Human C41(DE3)

C5 methyltransferase M.HaeIII Haemophilus influenzae C41(DE3)

Cytochrome P450 CYP79B2 Membrane Arabidopsis thaliana C43(DE3)

DNA polymerase Bacteriophage T5 C43(DE3)

Dystrophin 226 Rat C41(DE3)

EmrA (membrane fusion protein) Membrane Escherichia coli C41(DE3)

Estrogen receptor-related receptors Human C41(DE3)

FtsH (Zn2+-metalloprotease) Membrane Mycobacterium smegmatis C41(DE3)

Glucocorticoid receptor ligand-binding domain Human C41(DE3)

growth hormones gfGH-I /-II Goldfish C41(DE3)

Heptad repeats HR1 & HR2 PPR virus C41(DE3)

IntI1 integrase Transposon Tn21 C41(DE3)

KMCP1 (kidney mitochondrial carrier protein-1) Membrane Mouse C41(DE3)

LH2 (light harvesting complex 2) Membrane Pea C41(DE3)

M2 proton channel Membrane Influenza A virus C41(DE3)

NA+/glucose cotransporter (hSGLT1) Membrane Human C41(DE3)

NS3 serine protease Dengue virus Type 2 C41(DE3)

Nsp9 protein SARS coronavirus C41(DE3)

Orange fluorescent protein Cnidaria tube anemone Cerianthus sp. C41(DE3)

p53 Human C41(DE3)

Rop1 (antisense RNA-binding protein) Escherichia coli C41(DE3)

TAT-Bc1-2 delta loop protein Membrane Rat C43(DE3)pLysS

terpene synthases/cyclases Rice (Oryza sativa) C41(DE3)

TnI (troponin inhibitory subunit) Chicken C41(DE3)

Tocopherol cyclase Zea mays C43(DE3)

Ubiquitin E3 ligase MDM2 Human C41(DE3)

UCP1 (uncoupling protein 1) Membrane Mouse C41(DE3)

UCP1 anion carrier Membrane Rat C41(DE3)

UDP-N-acetylglucosamine acyltransferase Helicobacter pylori C41(DE3)

YibK Membrane Haemophilus influenzae C41(DE3)

YvcC, a multidrug ATP-binding cassette transporter Membrane Bacillus subtilis C41(DE3)

KasA (beta-ketoacyl-ACP synthase) Membrane Mycobacterium tuberculosis C41(DE3)pLysS

(See www.lucigen.com/catalog/overexpress.php for a complete list including references.)



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Lucigen Corporation | 2007/2008 Catalog

OverExpress™ Genotypes• OverExpress C41(DE3): F – ompT hsdSB (rB

- mB-) gal dcm (DE3)

• OverExpress C41(DE3)pLysS: F – ompT hsdSB (rB- mB

-) gal dcm (DE3) pLysS (CmR)

• OverExpress C43(DE3): F – ompT hsdSB (rB- mB

-) gal dcm (DE3)

• OverExpress C43(DE3)pLysS: F – ompT hsdSB (rB- mB

-) gal dcm (DE3) pLysS (CmR)

C41(DE3) and C43(DE3) can be differentiated from each other and from BL21(DE3) by transforma-tion with a plasmid verification vector, pAVD10. pAVD10 contains the uncF gene (encoding the beta subunit of E. coli ATPase) under the control of the T7 promoter. This plasmid is unconditionally lethal to BL21(DE3). It is lethal to C41(DE3) only upon induction with IPTG, but it is tolerated by C43(DE3) regardless of induction. pAVD10 is provided with OverExpress Cells.

OverExpress UseWhich OverExpress cell strain should I use?It is difficult to predict which of the four OverExpress strains – C41(DE3), C43(DE3), C41(DE3)pLysS, or C43(DE3)pLysS – will work best in expressing a given protein. We recommend initially using an OverExpress ComboPack™, which contains 3 reactions each of the four OverExpress competent cell strains, to determine which one is best for your application.

OverExpress AvailabilityOverExpress Competent Cells are now available exclusively from Lucigen.

IMPORTANT NOTEOverExpress Cells are licensed exclusively to Lucigen Corporation by Imaxio S.A. (Imaxio) under US Pat. 6,361,966 and others. Purchase of OverExpress Cells is accompanied by a non-exclusive, non-transferable license for research use only by nonprofit and for-profit organizations. Commercial use (i.e., to produce a product or service for sale) requires a separate license available from Imaxio.

For More Information…• Publications referencing OverExpress Cells. A bibliography is available on-line at:

www.lucigen.com/catalog/overexpress.php

• High efficiency expression of toxic proteins. eLucidations, Vol. 6. Available on-line at: www.lucigen.com/catalog/eLucidations.php

• OverExpress protocols. OverExpress Competent Cell manuals can be accessed on-line at: www.lucigen.com/catalog/manuals.php



75Lucigen Corporation | Advanced Products for Molecular Biology

ORDER INFORMATIONEach OverExpress Kit contains: the indicated OverExpress Electrocompetent or Chemically Competent Cells in SOLO packaging (1 transformation per tube), Expression Recovery Medium (lactose minus), pUC19 Positive Control Plasmid, pAVD10 Verification Plasmid, and complete protocols. ComboPacks contain 3 reactions each of C41(DE3), C43(DE3), C41(DE)pLysS, and C43(DE3)pLysS, as either electro-competent or chemically competent cells, as indicated. Expression Recovery Medium is also available separately.

OverExpress™ Competent Cells Cat. No. Size Price

Electrocompetent Cells

OverExpress C41(DE3) Cells (>1 x 1010 cfu/µg)

60341-1 12 reactions (SOLOs) $205


OverExpress C41(DE3)pLysS Cells (>1 x 109 cfu/µg)

60343-1 60343-2

12 reactions (SOLOs) 24 reactions (SOLOs)

$205 $364


60345-1 60345-2


$205 $364


60347-160347-2


$205 $364

OverExpress ElectroComboPack (3 reactions each of the above 4 strains)


Chemically Competent Cells


60442-1 60442-2


$173 $307


60444-1 60444-2


$173 $307


60446-1 60446-2


$173 $307


60448-1 60448-2


$173 $307

OverExpress ChemComboPack (3 reactions each of the above 4 strains)


Expression Recovery Medium

Expression Recovery Medium (lactose minus)

80030-1 8 x 12 ml $79


ORDER INFORMATIONEach OverExpress Kit contains: the indicated OverExpress Electrocompetent or Chemically Competent Cells in SOLO packaging (1 transformation per tube), Expression Recovery Medium (lactose minus), pUC19 Positive Control Plasmid, pAVD10 Verification Plasmid, and complete protocols. ComboPacks contain 3 reactions each of C41(DE3), C43(DE3), C41(DE)pLysS, and C43(DE3)pLysS, as either electro-competent or chemically competent cells, as indicated. Expression Recovery Medium is also available separately.

OverExpress™ Competent Cells Cat. No. Size

Electrocompetent Cells


60341-1 12 reactions (SOLOs)



60343-1 60343-2



60345-1 60345-2



60347-160347-2


OverExpress ElectroComboPack (3 reactions each of the above 4 strains)




60442-1 60442-2



60444-1 60444-2



60446-1 60446-2



60448-1 60448-2


OverExpress ChemComboPack (3 reactions each of the above 4 strains)


Expression Recovery Medium

Expression Recovery Medium (lactose minus)

80030-1 8 x 12 ml

OverExpress™


IMPORTANT! -86°C Storage Required

Immediately Upon Receipt

Lucigen® Corporation Advanced Products for Molecular Biology

2120 W. Greenview Drive Middleton, WI 53562

Toll Free (888) 575-9695 Phone (608) 831-9011 FAX (608) 831-9012

www.lucigen.com

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE MA031 v1.0

OverExpress™ Chemically Competent Cells Notice of Limited Label License, Copyright, Patents, Warranties, Disclaimers and Trademarks Copyright© 2001-2004 by Lucigen Corp. All rights reserved. UltraCloneTM, CloneDirectTM, pcrSMARTTM, pEZTM, pEZSeqTM, cSMARTTM, PCR-SMARTTM, CopyRightTM and ReplicatorTM are trademarks of Lucigen Corp. Lucigen®, CloneSmart®, ClonePlex®, DNATerminator®, E. cloni®, PCRTerminator® and pSMART® are registered trademarks of Lucigen Corp. OverExpress™ is a trademark of Imaxio, S.A. (Saint-Beauzire, France). HydroShear® and GeneMachines® are registered trademarks of Genomics Solutions (Ann Arbor, MI).

Lucigen’s products are sold for research use only and are not to be used in humans or for medical diagnostics. Lucigen’s liability with respect to any CloneSmart product is limited to the replacement of the product. No other warranties of any kind, expressed or implied, including without limitation, any implied fitness for any particular use, are provided by Lucigen. Lucigen is not liable for any direct, indirect, incidental or consequential damages arising out of or in connection with the use or inability to use any of its CloneSmart products.

Limited Label License

OverExpress products are sold under an exclusive license issued to Lucigen Corporation (“Lucigen”) by Imaxio, S.A. (“Imaxio”). The OverExpress technology, including evolved expression strain(s) C41(DE3), C43(DE3) and the pLysS versions (all hereinafter referred to as “MATERIALS”), is proprietary to Imaxio and Lucigen. Purchase of the MATERIALS is subject to the following Terms and Conditions: 1. Purchase of the MATERIALS is accompanied by a non-exclusive, non-transferable license to use said MATERIALS for the purchaser’s internal research purposes only. The Purchaser will use the MATERIALS in accordance with all applicable laws, regulations and governmental guidelines. The Purchaser agrees not to distribute the MATERIALS to any third parties, including recipient(s) in different department(s)/division(s) within the Purchaser’s organization. 2. The MATERIALS, including their use and all parts, derivatives and progeny thereof and information related thereto, are the subject of U.S. Patent No. 6,361,966, AU Patent No. 716694, and other pending patent applications, and will remain the property of Imaxio. 3. In accordance with scientific custom, all written or oral public disclosures concerning the Purchaser’s research using the MATERIALS shall include a reference as is appropriate to either U.S. Patent No. 6,361,966 and/or AU Patent No. 716694 and/or the following publication: Bruno Miroux and John E. Walker. Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. Journal of Molecular Biology (1996) Volume 260, pages 289–298. 4. The Purchaser agrees to make no commercial use of the MATERIALS. Commercial use means use of the MATERIALS to produce a product, which product will be sold or used commercially. For the avoidance of doubt, commercial uses include any activity for which consideration is received, for example, resale of the MATERIALS, use of the MATERIALS to provide services or in manufacturing, or use of the MATERIALS for diagnostic purposes. Such commercial uses require a Commercial Product License. Consequently, internal research purposes are all uses of the MATERIALS that are not commercial uses. Information about the Terms and Conditions of Commercial Product License can be obtained from IMAXIO Business Development Director, Biopôle Clermont-Limagne, 63360 Saint Beauzire, France. Fax: + 33 473 644 393 E-mail: [email protected]. 5. PLEASE NOTE: The MATERIALS contain the T7 RNA polymerase gene. The use of the T7 expression system, developed at Brookhaven National Laboratory under contract with the U.S. Department of Energy and comprising bacteria, bacteriophages and plasmids containing the T7 RNA polymerase gene, is the subject of patent applications assigned to Brookhaven Science Associates LLC. MATERIALS are sold under a license issued to Lucigen Corporation by Brookhaven Science Associates, LLC for noncommercial research purposes only. A separate license is required for any commercial use, INCLUDING THE USE OF MATERIALS FOR RESEARCH PURPOSES OR PRODUCTION PURPOSES BY ANY COMMERCIAL ENTITY. Information about commercial licenses may be obtained from the Office of Intellectual Property and Sponsored Research, Brookhaven National Laboratory, Bldg. 475D, P. O. Box 5000, Upton, New York 11973-5000, telephone (631)-344-7134. In using this product, you agree that MATERIALS or its derivitives containing the cloned gene for T7 RNA Polymerase may not be distributed further to third parties outside of your laboratory, unless the recipient receives a copy of this limited label license and agrees to be bound by its terms.

If the Purchaser is not willing to accept these use limitations, Lucigen Corporation is willing to accept return of the product for a full refund. For information on obtaining a license, contact Lucigen Corporation, 2120 W. Greenview Dr., Middleton, WI 53562. Email: [email protected]. Phone: 608-831-9011. Fax 608-831-9012

Lucigen® Corporation 2 (888) 575-9695 MA031 v1.0 www.lucigen.com

mailto:[email protected]

OverExpress™ Chemically Competent Cells

Contents Components & Storage Conditions ..................................................................................................... 3

OverExpress Chemically Competent Cells.......................................................................................... 4

Transformation Protocol ...................................................................................................................... 5

Strain Verification Protocol .................................................................................................................. 6

Sample Induction Protocol .................................................................................................................. 6

Media Recipes..................................................................................................................................... 7

Related Products................................................................................................................................. 7

References.......................................................................................................................................... 8

Components & Storage Conditions Four strains of Lucigen’s OverExpress Chemically Competent Cells are available: C41(DE3), C43(DE3), C41(DE3) pLysS, and C43(DE3)pLysS. The cells are shipped on dry ice in one container, along with supercoiled control pUC19 DNA at 1 ng/μl, supercoiled control plasmid pAVD10 at 5 ng/µl, and Expression Recovery Medium. C41(DE3), C43 (DE3), C41(DE3)pLysS, and C43(DE3)pLysS SOLOs are packaged in 25 μl aliquots, sufficient for one transformation per tube. Please refer to the table below for materials and catalog numbers All OverExpress Chemically Competent Cells require storage at –86o C.

OverExpress Chemically Competent Cells

STRAIN Efficiency (cfu/µg pUC19) Transformations Catalog # Storage

OverExpress C41(DE3) SOLOs

> 1 x 106 12 ( 12 x 50 µl) 24 ( 24 x 50 µl)

60442-1 60442-2

-86°C

OverExpress C41(DE3) pLysS SOLOs

> 1 x 106 12 ( 12 x 50 µl) 24 ( 24 x 50 µl)

60444-1 60444-2

-86°C

OverExpress C43(DE3) SOLOs

> 1 x 106 12 ( 12 x 50 µl) 24 ( 24 x 50 µl)

60446-1 60446-2

-86°C

OverExpress C43(DE3) pLysS SOLOs

> 1 x 106 12 ( 12 x 50 µl) 24 ( 24 x 50 µl)

60448-1 60448-2

-86°C

OverExpress ComboPack (3 reactions of each of the above)

> 1 x 106 12 ( 12 x 50 µl) 60452-1 -86°C

Expression Recovery Medium* (lactose-free)

12 ( 1 x 12 ml) 24 ( 2 x 12 ml) 96 ( 8 x 12 ml)

---- ----

80030-1

-20 to -86°C

Supercoiled pAVD10 DNA (5 ng/ μl)

10 (1 x 10 µl) ---- -20 to -86°C

Supercoiled pUC19 DNA (1 ng/µl)

10 (1 x 10 µl) ---- -20 to -86°C



OverExpress Electroporation Competent Cells

OverExpress C41 (DE3), C41 (DE3) pLysS, C43 (DE3), and C43 (DE3) pLysS Chemically Competent Cells are E. coli strains that are effective in expressing toxic proteins from all classes of organisms, including bacteria, yeast, plant, viruses, and mammals. These new OverExpress strains contain genetic mutations phenotypically selected for conferring tolerance to toxic proteins (1-5). The strain C41(DE3) was derived from BL21(DE3). This strain has at least one uncharacterized mutation, which prevents cell death associated with expression of many recombinant toxic proteins. The strain C43(DE3) was derived from C41(DE3) by selecting for resistance to a different toxic protein. It can express a different set of toxic proteins than C41(DE3). As in standard BL21(DE 3) strains, OverExpress C41(DE3), C41(DE3)pLysS, C43(DE3), and C43 (DE3)pLysS are lysogens of λDE3. These strains carry a chromosomal copy of the T7 RNA polymerase gene under the control of the lacUV5 promoter. These strains are suitable for production of protein from target genes cloned into T7-driven expression vectors. OverExpress C41(DE3), C41(DE3) pLysS, C43(DE3), and C43(DE3)pLysS are also deficient in the lon and ompT proteases. OverExpress C41(DE3)pLysS and C43(DE3)pLysS carry a chloramphenicol resistant plasmid that expresses a small amount of T7 lysozyme, which is a natural inhibitor of T7 RNA polymerase. These strains are used to suppress basal expression of T7 RNA polymerase prior to induction, thus stabilizing recombinants encoding particularly toxic proteins. Genotypes OverExpress C41(DE3)

F – ompT hsdSB (rB B- mB

-) gal dcm (DE3)

OverExpress C41(DE3)pLysS F – ompT hsdSB (rB B

- mB-) gal dcm (DE3) pLysS (Cm ) R

OverExpress C43(DE3)

F – ompT hsdSB (rB B- mB

-) gal dcm (DE3) OverExpress C43(DE3)pLysS F – ompT hsdSB (rB B

- mB-) gal dcm (DE3) pLysS (Cm ) R

As a control for transformation, OverExpress Chemically Competent Cells are provided with supercoiled pUC19 DNA at a concentration of 1 ng/μl. Dilute the plasmid 1:100 in dH2O, and use 1 μl for transformation. As a control for differentiating C41(DE3) and C43(DE3) strains from each other and from BL21 (DE3), OverExpress Chemically Competent cells are provided with the plasmid vector pAVD10 at a concentration of 5 ng/µl. Use 1μl for transformation.



Preparation for Transformation

OverExpress Chemically Competent Cells are provided in aliquots of 50 µl sufficient for one transformation reaction. Transformation is performed by heat shock at 42oC, followed by incubation on ice. To ensure successful transformation results, the following precautions must be taken:

• For best results, use a minimum of 1 µl of miniprep DNA (10-50 ng) for transforming OverExpress

Chemically Competent Cells. • The cells must be completely thawed on ice before use. • For highest transformation efficiency, use the provided Expression Recovery Medium to

resuspend the cells after transformation. Use of TB or other media may result in lower transformation efficiencies and induction of protein expression.

Transformation Protocol 1. Remove OverExpress cells from the -86°C freezer and thaw completely on wet ice (10-15 minutes).

2. Add 1 µl of miniprep DNA sample to the 50 µl of cells on ice. Stir briefly with a pipet tip; do not pipet up and down to mix, which can introduce air bubbles and warm the cells.

3 . Incubate on ice for 30 minutes.

4. Heat-shock cells by placing them in a 42oC water bath for 45 seconds.

5. Return the cells to ice for 2 minutes.

6. Add 950 µl of room-temperature Expression Recovery Medium to the cells in the culture tube.

7 . Place the tubes in a shaking incubator at 250 rpm for 1 hour at 37oC.

8 . Plate up to 100 µl of transformed cells on LB agar plates containing the appropriate antibiotic.

9. Incubate the plates overnight at 37°C. 10. Transformed clones can be further grown in LB or any other lactose free medium.



Strain Verification Protocol The vector pAVD10 is provided with OverExpress Chemically Competent Cells to verify the identity of the cells. This vector encodes a protein that is toxic to BL21(DE3) cells, even at a very low level of expression. C41 (DE3) cells tolerate basal expression of the protein, but not induced expression. C43(DE) cells are viable even at high levels of expression.

1. Transform the competent cell sample with 1 µl (5 ng) of pAVD10, using the protocol described

above.

2. Plate 100 µl of the transformation reaction onto an LB+ ampicillin plate and 100 µl onto an LB + ampicillin + ITPG plate.

3. Incubate the plates overnight at 37°C.

4. Observe the growth of colonies on each plate.

Expected Results:

BL 21(DE3) C41(DE3) C43(DE3) LB+Amp No Colonies Colonies Colonies

LB+Amp+IPTG No Colonies No Colonies Colonies Sample Induction Protocol

1. Inoculate a single colony from a freshly streaked plate into 5 ml of LB medium containing the appropriate antibiotic for the plasmid and host strain.

2. Incubate with shaking at 37°C overnight. To minimize the amount of expression of the target protein prior to induction, add glucose to the growth medium at a concentration of 0.2% (w/v).

3. Inoculate 50 ml of LB medium containing the appropriate antibiotic with 0.5 ml of the overnight culture prepared in step 2.

4. Incubate with shaking at 37°C until the OD600 reaches 0.8-1.

5. Add IPTG to a final concentration of 1 mM (Prepare a 1 M solution of IPTG by dissolving 2.38 g of IPTG in water and adjust the final volume to 10 ml. Filter sterilize before use). Optimal time for induction of the target protein may vary from 2-16 hours, depending on the protein.

6. Incubate at 37°C for 3-4 hours. To determine the optimal time for induction of the target protein, it is recommended that a time course experiment be performed varying the induction from 2-16 hours.

7. Place the culture on ice for 10 minutes. Harvest cells by centrifugation at 5,000 x g for 10 minutes at 4°C.

8. Remove the supernatant and store the cell pellet at -20°C (storage at lower temperatures is also acceptable).



Media Recipes

LB Culture Medium for Growth of Transformants

Per liter: 5 g yeast extract 8 g tryptone 5 g NaCl

Add all components to deionized water. Adjust pH to 7.0 with NaOH. Autoclave and cool to 55°C.

LB Agar Plates Per liter: 5 g yeast extract 8 g tryptone 5 g NaCl 15 g agar

Add deionized water to 1 liter. Adjust pH to 7.0 with NaOH. Autoclave. Cool to 55°C and add the appropriate filter-sterilized antibiotic (e.g., 30-50 mg kanamycin for kanamycin-resistant transformants; 50-100 mg ampicillin or carbenicillin for ampicillin-resistant transformants).

For blue/white screening, add 3 ml 100mM IPTG and 10 ml 2% X-gal to the molten agar at 55°C before pouring.

Pour approximately 25 ml per petri plate.

Related Lucigen Products • E. cloni® EXPRESS BL21(DE3) Chemically Competent Cells • CloneSmart® Blunt Cloning Kit • DNATerminator® End Repair Kit • PCRTerminator® End Repair Kit • UltraClone™ DNA Ligation & Transformation Kit • CloneDirect™ Rapid Ligation Kit • PCR-SMART™ Cloning Kit • ClonePlex® Library Construction Kit • pEZSeq™ Blunt Cloning Kit • cSMART™ cDNA Cloning Kit • E. cloni® 10G Chemically Competent Cells



References 1. B. Miroux and J.E. Walker (1996). Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol. 260, 289-298. 2. L. Dumon-Seignovert, G. Cariot, and L. Vuillard (2004). The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3). Protein Expression and Purification 37, 203-206. Data used with permission. 3. U.S. Pat. No. 6,361,966; PCT/GB97/01879; and additional patents pending or issued to Avidis S.A. or Imaxio S.A. Purchase of these products is accompanied by a limited use license for research purposes only. A separate license is required for commercial use. Contact Imaxio S.A. (phone: +33 4 73 64 42 71; fax: + 33 4 73 64 43 93) for details. See the Limited Label License terms on p. 2 of this manual. 4. BL21(DE3) cells and derivatives, including C41(DE3) and C43(DE3) strains, are patented. Purchase of these products is accompanied by a limited use license for noncommercial research purposes only. A separate license is required for any commercial use, including the use of these materials for research purposes or production purposes by any commercial entity. Information about commercial licenses may be obtained from the Office of Intellectual Property and Sponsored Research, Brookhaven National Laboratory, Bldg. 475D, P.O. Box 5000, Upton, New York 11973-5000, telephone (631) 344-7134. See the Limited Label License terms on p. 2 of this manual. 5. F.W. Studier (2005). Protein production by auto-induction in high-density shaking cultures. Protein Expression and Purification 41, 207-234.


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