Competent Cells. Update 2015/16 CLONING & PROTEIN EXPRESSION

Transcription

1 Ces Update 2015/16 CLONING & PROTEIN EXPRESSION

2 STRAIN PROPERTIES & FORMATS Strain Properties There are many properties to consider when choosing a strain for your experiments. Requirements such as high-quaity pasmid preparations, bue/white screening, cytopasmic disufide bond formation, reguated protein expression and fast coony growth necessitate specific strain choices. The foowing seection chart highights the characteristics of NEB s strains to hep seect the optima strain for a particuar experiment. Coning Strain Properties STRAIN PROPERTIES TRANSFORMATION EFFICIENCY (cfu/µg) (1) STRAIN T1 PHAGE BLUE/WHITE RESISTANT SCREENING aci q ysy COLONIES VISIBLE AFTER 6.5 HRS. ENDO I DEFICIENT (2) PROTEASE DEFICIENT (3) F RecA T7 RNA POLYMERASE CYTOPLASMIC DISULFIDE BOND FORMATION (4) DRUG RESISTANCE (5) NEB Turbo 1 3 x 10 9 K12 nit NEB 5-apha 1 3 x 10 9 K12 none NEB 5-apha F I q 1 3 x 10 9 K12 tet NEB 10-beta 1 3 x 10 9 K12 str dam /dcm 1 3 x 10 6 K12 cam, str, nit NEB Stabe 1 3 x 10 9 K12 tet, str Protein Expression Strain Properties NEB Express x 10 9 B nit NEB Express I q x 10 9 B cam, nit T7 Express x 10 9 B nit T7 Express ysy x 10 9 B cam, nit T7 Express ysy/i q x 10 9 B cam, nit T7 Express Crysta x 10 9 B nit SHuffe Express 1 x 10 7 B spec SHuffe T7 Express SHuffe T7 Express ysy 1 x 10 7 B spec (6) 1 x 10 7 B cam, spec (6) SHuffe T7 1 x 10 6 K12 str, spec BL x 10 7 B BL21(DE3) 1 5 x 10 7 B Lemo21(DE3) 1 3 x 10 7 B cam NiCo21(DE3) 1 5 x 10 7 B (1) TE are given high-quaity for high efficiency chemicay competent strains. TE for eectrocompetent strains are 1-4 x cfu/µg. TE for subconing strains are >1 x 10 6 cfu/µg. (2) Important for high-quaity pasmid preparation. (3) Lacks Lon and OmpT protease activity. (4) Constitutivey expresses a chromosoma copy of the disufide bond isomerase DsbC. (5) nit = nitrofurantoin, tet = tetracycine, cam = choramphenico, str = streptomycin, spec = spectinomycin (6) Resistance to ow eves of streptomycin may be observed. 2

3 STRAIN PROPERTIES & FORMATS Convenient Formats NEB competent ce strains are avaiabe in various formats for your convenience. Most are avaiabe as 50 µ singe-use transformation tubes and many are offered in arger, 200 µ tubes for mutipe simutaneous reactions. Our most popuar coning strains are avaiabe as eectrocompetent ces. SOC outgrowth media and contro pasmids are provided with many of these formats. For even greater vaue, NEB 5-apha is aso avaiabe in a ower efficiency, subconing format, as we as a 96-we pate format. 96-we pate formats are aso avaiabe for other NEB strains upon request. For inquiries regarding buk saes or custom packaging options incuding 96-we pate format, pease contact NEBsoutions@neb.com. TOOLS & RESOURCES Visit to find: Tips for enhancing transformation efficiencies Troubeshooting transformation reactions Product comparisons Coning Formats FORMATS 50 µ SINGLE-USE (H-FORMAT) 200 µ TUBES (I-FORMAT) ELECTROCOMPETENT (K-FORMAT) SUBCLONING (F-FORMAT) 96-WELL PLATE (P-FORMAT) SOC & CONTROL PLASMID INCLUDED NEB Turbo NEB 5-apha NEB 5-apha F I q NEB 10-beta dam /dcm NEB Stabe Protein Expression Formats NEB Express NEB Express I q T7 Express T7 Express ysy T7 Express ysy/i q T7 Express Crysta SHuffe Express SHuffe T7 Express SHuffe T7 Express ysy SHuffe T7 BL21 BL21(DE3) Lemo21(DE3) (1) NiCo21(DE3) (1) Rhamnose soution is provided instead of SOC, contro pasmid is incuded. 3

4 CLONING STRAINS CHARACTERISTICS Coning Strains NEB s growing ine of competent ces incudes severa popuar strains for coning and protein expression, in addition to strains with unique properties incuding fast coony growth, tight contro of expression and disufide bond formation. Our coning strains incude derivatives of the industry standards, DH5α and DH10B. NEB Turbo is unique to NEB and aows coony growth after 6.5 hours. NEB s dam /dcm strain enabes Dam and Dcm methyation-free pasmid growth. NEB Stabe is recommended in a difficut coning experiments. Our ces are a extensivey tested for phage resistance, antibiotic resistance and sensitivity, bue/white screening and transformation efficiency. High efficiency, 5 minute transformation and eectroporation protocos are provided, when appicabe. ADVANTAGES Compatibe with NEBuider HiFi DNA Assemby and Gibson Assemby reactions, as we as igation reactions Strains for coning toxic genes Free of anima products T1 phage resistance (fhua2) Media and contro pasmid incuded A variety of convenient formats Buk saes avaiabe for custom packaging FEATURES NEB 5-apha (NEB #C2987) NEB Turbo (NEB #C2984) NEB 5-apha F I q (NEB #C2992) NEB 10-beta (NEB #C3019) dam /dcm (NEB #C2925) NEB Stabe (NEB #C3040) Versatie Fast growth (< 8 hours) Featured Onine Toos Toxic gene coning Large pasmid/bac coning Dam/Dcm-free pasmid growth Retrovira/entivira vector coning FORMATS Chemicay competent Eectrocompetent Subconing 96-we pate format The Toos & Resources tab, accessibe on our homepage, contains a seection of interactive technica toos. These toos can aso be accessed directy in the footer of every web page. Competitor Cross-Reference Too Use this too to seect another company's competent ce product and find out which NEB strain is compatibe. Choose either the product name or cataog number from the avaiabe seection, and this too wi identify the recommended NEB product, highight its advantages, and provide a ink for ordering the product. NEBconer NEBioCacuator Use this too to find the right products and protocos for each step (digestion, end modification, igation and transformation) of your next traditiona coning experiment. Aso, find other reevant toos and resources to enabe protoco optimization. NEBioCacuator is a coection of cacuators and converters that are usefu in panning bench experiments in moecuar bioogy aboratories. 4

5 PROTEIN STRAINS CHARACTERISTICS Protein Expression Strains NEB aso offers a wide variety of competent ce strains idea for many protein expression appications. These strains address the needs of protein expression contro, toxic protein expression, cytopasmic disufide bond formation, difficut targets and crystaography. NEB Express, T7 Express and SHuffe strains are avaiabe with varying eves of expression contro. Ony NEB offers the exceptiona contro of expression from the ysy gene that reduces basa expression from T7 strains without inhibiting IPTG-induced expression. Lemo21(DE3) features tunabe for difficut targets such as membrane proteins and proteins prone to insoube expression. Our NiCo21(DE3) strain is designed for the expression and purification of His-tagged proteins. Each strain is provided with a detaied protoco for optima expression. ADVANTAGES Deficient in proteases Lon and OmpT Free of anima products T1 phage resistance (fhua2) Media and contro pasmid incuded A variety of convenient formats Buk saes avaiabe for custom packaging STRAIN CHARACTERISTICS PROTEASE DEFICIENT B STRAINS BL21 BL21(DE3) Lemo21(DE3) NiCo21(DE3) NEB Express NEB Express I q T7 Express T7 Express ysy T7 Express ysy/i q T7 Express Crysta SHuffe Express SHuffe T7 Express SHuffe T7 Express ysy Routine non- Tunabe Expression of difficut targets incuding membrane proteins, toxic proteins and proteins prone to insoube expression Expression and purification of His-tagged proteins Versatie non- strain Contro of IPTG induced expression from Pac, Ptac, Ptrc and T5/ac Most popuar strain Better reduction of basa expression Lowest basa expression SeMet abeing of protein for crystaography Enhanced capabiity to correcty fod proteins with mutipe disufide bonds in the cytopasm Enhanced capabiity to correcty fod proteins with mutipe disufide bonds in the cytopasm Tighty controed expression of toxic proteins Enhanced capabiity to correcty fod proteins with mutipe disufide bonds in the cytopasm K12 STRAINS SHuffe T7 Enhanced capabiity to correcty fod proteins with mutipe disufide bonds in the cytopasm 5

6 DIFFICULT TARGETS Protein Expression Strains for Difficut Targets Expression of Toxic Proteins of recombinant protein is often improved by the co-expression of T7 ysozyme which binds and inhibits T7 RNA poymerase function unti the point of induction. NEB has constructed unique T7 Express derivatives with a singe copy of a T7 ysozyme gene (ysy) or a singe copy of ysy and aci q genes on a mini-f pasmid, which is maintained without antibiotic seection. This enhancement is unique to NEB strains and makes them ess susceptibe to ysis during protein overexpression. ysy ensures compete repression of in the absence of inducer moecue. Yet, is activated within 30 minutes after induction. Our ysy strains offer maxima contro of T7-mediated toxic protein expression. Proteins with Mutipe Disufide Bonds SHuffe strains from NEB are engineered strains capabe of expressing proteins with increased disufide bond compexity in the cytopasm. SHuffe strains express the disufide bond isomerase DsbC within the cytopasm. DsbC isomerizes mis-oxidized substrates into their correcty foded state greaty enhancing the fideity of disufide bond formation. Cytopasmic expression aso resuts in significanty higher protein yieds of disufide bonded proteins when compared to peripasmic expression. SHuffe strains are sensitive to kan, amp, tet and in most cases, cam which makes them abe to express proteins from a wide variety of expression vectors offering greater versatiity in experimenta design. PfCHT1 chitinase activity assayed from crude ysates Difficut to Express Proteins Lemo21(DE3) is a tunabe strain designed for the expression of chaenging proteins. A derivative of BL21(DE3), Lemo21(DE3) offers the host features of this popuar expression strain, with the added benefit of being abe to contro expression eves by varying the eve of T7 ysozyme (ysy), the natura inhibitor of T7 RNA Poymerase. The fine contro of expression makes Lemo21(DE3) idea for membrane proteins, toxic proteins, secreted proteins and proteins prone to insoube expression. Overnight expression of a membrane protein PhoA fusion 10,000 6,000 Improved transformation of toxic cones with strains from NEB 8,000 5,000 Percent Transformed (%) Reative Activity 6,000 4,000 2,000 PhoA Activity (units) 4,000 3,000 2,000 1,000 0 BL21 (DE3) T7 Express ysy ysy/ q Transformed Strain 0 Origami (DE3) SHuffe T7 SHuffe T7 Express ,000 µm rhamnose A pasmid and the same pasmid containing a gene encoding a toxic mammaian protein were transformed into each host. Comparison of the reative transformation efficiencies demonstrates that the T7 Express hosts provide the eves of contro necessary for transformation of potentiay toxic cones. BL21(DE3) coud not be transformed with the toxic cone. Pasmodium faciparum chitinase (PfCHT1) with three cysteines was expressed from a pasmid under the reguation of a T7 promoter. After induction, ces were harvested and crude ce ysates were prepared. PfCHT1 was assayed using a chromogenic substrate (CaBioChem #474550) and standardized to protein concentration using Bradford reagent. Lemo21(DE3) System enabes simpe, rapid optimization of membrane protein expression. 6

7 TRANSFORMATION EFFICIENCY Enhancing Transformation Efficiency Transformation efficiency is defined as the number of coony forming units (cfu) that woud be produced by transforming 1 µg of pasmid into a given voume of competent ces. However, 1 µg of pasmid is rarey transformed. Instead, efficiency is routiney cacuated by transforming 100 pg 1 ng of highy purified supercoied pasmid under idea conditions. Transformation Efficiency (TE) is cacuated as: TE = Coonies/µg/Diution. Efficiency cacuations can be used to compare ces or igations. Our recommended protocos and tips are presented here to hep you achieve maximum resuts. Transformation Tips Thawing Ces are best thawed on ice DNA shoud be added as soon as the ast trace of ice in the tube disappears Ces can be thawed by hand, but warming above 0 C decreases efficiency Incubation of DNA with Ces on Ice Incubate on ice for 30 minutes. Expect a 2-fod oss in TE for every 10 minutes this step is shortened. Heat Shock Both temperature and time are specific to the transformation voume and vesse. Typicay, 30 seconds at 42 C is recommended. Outgrowth Outgrowth at 37 C for 1 hour is best for ce recovery and for expression of antibiotic resistance. Expect a 2-fod oss in TE for every 15 minutes this step is shortened. SOC gives 2-fod higher TE than LB medium Incubation with shaking or rotation resuts in 2-fod higher TE Pating Seection pates can be used warm or cod, wet or dry with no significant effects on TE Warm, dry pates are easier to spread and aow for the most rapid coony formation DNA DNA shoud be purified and resuspended in water or TE Buffer Up to 10 µ of DNA from a igation mix can be used with ony a 2-fod oss of efficiency Purification by either a spin coumn or pheno/choroform extraction and ethano precipitation is idea The optima amount of DNA is ower than commony recognized. Using cean, supercoied puc19, the efficiency of transformation is highest in the 100 pg 1 ng range. However, the tota coonies which can be obtained from a singe transformation reaction increase up to about 100 ng. DNA Contaminants to Avoid CONTAMINANT Detergents REMOVAL METHOD Ethano precipitate Pheno Extract with choroform and ethano precipitate Ethano or Isopropano Dry peet before resuspending PEG Coumn purify or pheno/ choroform extract and ethano precipitate DNA binding proteins (e.g., igase) Coumn purify or pheno/ choroform extract and ethano precipitate RECOMMENDED PROTOCOLS HIGH EFFICIENCY TRANSFORMATION PROTOCOL 1. Thaw ces on ice for 10 minutes 2. Add 1 pg 100 ng of pasmid DNA (1 5 µ) to ces and mix without vortexing 3. Pace on ice for 30 minutes 4. Heat shock at 42 C for seconds or according to recommendations 5. Pace on ice for 5 minutes 6. Add 950 µ of room temperature SOC 7. Pace at 37 C for 60 minutes. Shake vigorousy (250 rpm) or rotate. 8. Mix ces without vortexing and perform severa 10-fod seria diutions in SOC 9. Spread µ of each diution onto pre-warmed seection pates and incubate overnight at 37 C (30 C for SHuffe strains) or according to recommendations 5 MINUTE TRANSFORMATION PROTOCOL (10% efficiency compared to above protoco) 1. Thaw ces in your hand 2. Add 1 pg 100 ng of pasmid DNA (1 5 µ) to ces and mix without vortexing 3. Pace on ice for 2 minutes 4. Heat shock at 42 C for 30 seconds or according to recommendations 5. Pace on ice for 2 minutes 6. Add 950 µ of room temperature SOC. Immediatey spread µ onto a seection pate and incubate overnight at 37 C. (30 C for SHuffe strains) NOTE: Seection using antibiotics other than ampiciin may require some outgrowth prior to pating. For tips on protein expression with T7 Express Strains, pease visit: 7

8 Ordering Information Coning Strains PRODUCT NEB # SIZE NEB Turbo C2984H/I 20 x 0.05 m / 6 x 0.2 m NEB Turbo Eectrocompetent C2986K 6 x 0.1 m NEB 10-beta (High Efficiency) C3019H/I 20 x 0.05 m / 6 x 0.2 m NEB 10-beta Eectrocompetent C3020K 6 x 0.1 m NEB 5-apha (High Efficiency) C2987H/I/P 20 x 0.05 m / 6 x 0.2 m/ 1 x 96-we pate NEB 5-apha (Subconing Efficiency) C2988J 6 x 0.4 m NEB 5-apha Eectrocompetent C2989K 6 x 0.1 m NEB 5-apha F I q (High Efficiency) C2992H/I 20 x 0.05 m / 6 x 0.2 m dam /dcm C2925H/I 20 x 0.05 m / 6 x 0.2 m NEB Stabe (High Efficiency) C3040H/I 20 x 0.05 m / 6 x 0.2 m Component Sod Separatey: SOC Outgrowth Medium B9020S 4 x 25 m Protein Expression Strains PRODUCT NEB # SIZE NEB Express (High Efficiency) C2523H/I 20 x 0.05 m / 6 x 0.2 m NEB Express I q (High Efficiency) C3037I 6 x 0.2 m T7 Express (High Efficiency) C2566H/I 20 x 0.05 m / 6 x 0.2 m T7 Express ysy (High Efficiency) C3010I 6 x 0.2 m T7 Express ysy/i q (High Efficiency) C3013I 6 x 0.2 m T7 Express Crysta (High Efficiency) C3022I 6 x 0.2 m SHuffe T7 C3026H 6 x 0.05 m SHuffe Express C3028H 6 x 0.05 m SHuffe T7 Express C3029H 6 x 0.05 m SHuffe T7 Express ysy C3030H 6 x 0.05 m BL21 C2530H 20 x 0.05 m BL21(DE3) C2527H/I 20 x 0.05 m / 6 x 0.2 m Lemo21(DE3) C2528H 6 x 0.05 m NiCo21(DE3) C2529H 20 x 0.05 m Component Sod Separatey: SOC Outgrowth Medium B9020S 4 x 25 m Note: Store Ces at 80 C. Once thawed, do not refreeze. Storage at 20 C wi resut in a significant decrease in transformation efficiency. Ces ose efficiency whenever they are warmed above 80 C, even if they do not thaw. NEBIOCALCULATOR, NEBUILDER, NEBCLONER, NEW ENGLAND BIOLABS, NEB and SHUFFLE are registered trademarks of New Engand Bioabs, Inc. GIBSON ASSEMBLY is a registered trademark of Synthetic Genomics, Inc. DH5 and DH10B are trademarks of Invitrogen Corporation. LEMO21(DE3) SYSTEM is a trademark of Xbrane Biosciences AB. For icensing information, visit ISO 9001 Registered Quaity Management ISO Registered Environmenta Management ISO Registered Medica Devices New Engand Bioabs, Inc., 240 County Road, Ipswich, MA Teephone: (978) To Free: (USA Orders) To Free: (USA Tech) Fax: (978) e-mai: info@neb.com CEL Version 7.0 7/15