One Shot BL21(DE3) - Thermo Fisher Scientific
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1. We recommend using the BL21 DES3 strain for expression of non toxic heterologous genes BL21 DE3 pLysS and BL21 DE3 pLysE are also suitable hosts for expressing non toxic genes however recombinant proteins are generally expressed to higher levels in BL21 DE3 than in BL21 DE3 pLysS or BL21 DE3 pLysE If you are expressing a toxic gene please see below The IPTG inducible lacUV5 promoter controls expression of the T7 polymerase gene in BL21 DE3 BL21 DE3 pLysS and BL21 DE3 pLysE strains Because of the extremely high activity of T7 RNA polymerase some basal level expression of the gene of interest may occur in uninduced cells This creates problems in cases where the gene of interest is toxic to bacterial cells In these cases expression of the toxic gene under uninduced conditions leads to selection of cells that express the lowest levels of the toxic gene These cells are often unable to express high levels of the gene of interest upon IPTG induction of the T7 polymerase For expression of toxic genes we recommend using BL21 DE3 pLysS or BL21 DE3 pLysE These two strains produce T7 lysozyme which helps to reduce basal levels of T7 RNA polymerase Although levels are reduced the cells may still contain a small amount of T7 RNA polymerase See page 9 for precautions to address this issue Continued on next page Expression Guidelines Continued Expression Guidelines The guidelines below assume that expression of your gene2. System 14 Use of the One Shot BL21 DE3 BL21 DE3 pLysS and BL21 DE3 pLysE E coli strains is covered under the licenses detailed below The One Shot BL21 DE3 BL21 DE3 pLysS and BL21 DE3 pLysE E coli strains are genetically modified and carry a chromosomal insertion of a cassette containing the T7 RNA polymerase T7 RNAP gene As a condition of sale this product must be in accordance with all applicable local legislation and guidelines including EC Directive 90 219 EEC on the contained use of genetically modified organisms The composition and or use of this product may beclaimed in U S Patent No 5 693 489 licensed to Life Technologies Corporation by Brookhaven Science Associates LLC The T7 expression system is based on technology developed at Brookhaven National Laboratory under contract with the U S Department of Energy and is the subject of patents and patent applications assigned to Brookhaven Science Associates LLC BSA By provisions of the Distribution License Agreement granted to Invitrogen covering said patents and patent applications Invitrogen grants you a non exclusive sub license under patents assigned to BSA for the use of this technology including the enclosed materials based upon the following conditions 1 these materials are to be used for non commercial research purposes only A separate license under patents owned by BSA is required for any commercial use including the use of these materials fo
3. is not toxic to E coli If you are working with a toxic gene see Precautions page 9 before beginning Pick 34 transformants for overnight culture in 5 mL LB medium containing antibiotic to select for your expression plasmid If using BL21 DE3 pLysS or BL21 DE3 pLysE add 34 ug mL chloramphenicol to select for pLysS or pLysE Grow overnight at 37 C with shaking to saturation ODso 22 Use the overnight cultures to inoculate fresh LB medium containing antibiotic to an ODsoo of 0 05 0 1 1 50 dilution of the overnight culture This dilution allows the cells to quickly return to logarithmic growth and reach the appropriate cell density Use a volume appropriate for taking time points if desired Note If you are using BL21 DE3 pLysS or BL21 DE3 pLysE you may choose not to include chloramphenicol in these cultures Generally the cells will not lose the pLysS or pLysE plasmid during the limited number of cell doublings that occur in the growth and induction stages Use the remainder of each overnight culture to create glycerol stocks Once you have identified the clone that best expresses your protein you can use the glycerol stock to perform additional expression experiments Grow the cultures until they reach mid log phase ODsoo 0 4 2 to 3 hours Induce the cultures by adding IPTG to a final concentration of 0 5 mM and culture for an additional 2 3 hours You may also take time points to analyze for optimal expression of
4. expression using BL21 DE3 BL21 DE3 pLysS or BL21 DE3 pLysE cells please see pages 7 10 e Plasmid DNA ready for transformation e 42 C water bath e 37 C shaking and non shaking incubator e Ice bucket with ice e Spectrophotometer to measure optical density of the cell cultures e Microcentrifuge tube rack optional e Prepare LB agar plates containing the appropriate concentration of antibiotic for plasmid selection If you are using BL21 DE3 pLysS or BL21 DE3 pLysE add 34 ug mL chloramphenicol for pLysS or pLysE selection e Equilibrate a water bath to 42 C e Warm the vial of SOC medium to room temperature e Place the plates in a 37 C incubator to remove excess moisture use two plates for each transformation Continued on next page Basic Transformation Procedure Continued Basic Transformation Procedure 10 Thaw one vial of One Shot cells on ice per transformation Add 5 10 ng of DNA in a volume of 1 5 uL to the cells and mix by tapping gently Do not mix cells by pipetting Incubate the vial s on ice for 30 minutes Heat shock the cells by incubating the vial s for exactly 30 seconds in the 42 C water bath Do not mix or shake Remove the vial s from the 42 C bath and quickly place on ice Add 250 pL of pre warmed SOC medium to the vial s SOC is a rich medium use proper sterile technique to avoid contamination Secure the vial s in a microcentrifuge rack with tape Place th
5. ar plates containing 50 ug mL ampicillin Equilibrate a water bath to 42 C Warm the vial of SOC medium to room temperature Place the plates in a 37 C incubator to remove excess moisture use two plates for each transformation Follow the transformation protocol on page 6 to transform pUC19 into BL21 DE3 BL21 DE3 pLysS or BL21 DE3 pLysE Use the specific modifications below Transform cells with 1 uL 10 pg of pUC19 Plate 50 uL each onto two LB plates containing 50 ug mL ampicillin Calculate the transformation efficiency as transformants per 1 ug of plasmid see below The cells should have an efficiency of either e 10 transformants ug of supercoiled plasmid for BL21 DE3 pLysE OR e 10 transformants ug of supercoiled plasmid for BL21 DE3 or BL21 DE3 pLysS Calculation Use the formula below to calculate transformation efficiency Hof colonies x 10 pg x 300 uL transformed transformants cells 10 pg ug X uL plated ug plasmid transformed DNA DNA 11 Technical Support Web Resources Contact Us Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Tel 1 760 603 7200 Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes SDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional produc
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7. e rack in a shaking incubator and shake the vial s at 37 C for 1 hour at 225 rpm Plate two different volumes of the transformation reaction onto LB plates containing the appropriate antibiotic for plasmid selection Include 34 ug mL chloramphenicol if using BL21 DE3 pLysS or BL21 DE3 pLysE cells Select two volumes ranging from 20 200 uL to ensure well spaced colonies on at least one plate The remaining transformation reaction may be stored at 4 C and plated out the next day if needed Invert the plates and incubate at 37 C overnight Select transformants from the plates and culture as described on page 8 Note Clones may exhibit differences in expression of heterologous genes We recommend choosing 3 4 transformants when characterizing clones for protein expression Expression Guidelines Introduction NANENY Y oy Ya o 00 o je gt Expression of Non Toxic Genes T7 RNA Polymerase and Toxic Genes If you have an expression protocol for the plasmid that you are working with we recommend that you use your own protocol This section provides some general guide lines for the use of T7 RNA polymerase based expression plasmids in BL21 DE3 BL21 DE3 pLysS or BL21 DE3 pLysE cells Transform your expression plasmid into a strain that does not bear the gene for T7 RNA polymerase i e TOP10 DH5a and maintain your construct in this strain Use BL21 DE3 BL21 DE3 pLysS or BL21 DE3 pLysE for expression only
8. invitrogen by technologies One Shot BL21 DE3 One Shot BL21 DE3 pLysS One Shot BL21 DE3 pLysE Competent Cells Catalog nos C6000 03 C6565 03 C6060 10 and C6060 03 Rev Date 2 June 2010 Manual part no 28 0182 MAN0000662 ii Table of Contents About the Kit dd 1 Basic Transformation Procedure ueenesesssenenssesnenennenesennenesennenensenennnenannen 5 Expression Guidelines emitan laico else anne ie adi 7 Testing Transformation Efficiency eeessssesenenenenenennenennnnenen nen 11 Techical SUpport ei 822 en ue AASE 12 Purchaser Notifica ici dal SE VE S SSi 14 iii iv About the Kit Available Kits The table below lists the kits covered by this manual The contents of each kit are listed below Item Transformation Efficiency Catalog no BL21 DE3 1 x 108 cfu ug C6000 03 BL21 DE3 pLysS 1 x 108 cfu ug C6060 10 1 x 108 cfu ug C6060 03 BL21 DE3 pLysE 1 x 10 cfu ug C6565 03 Shipping Each One Shot kit is shipped on dry ice Upon receipt store Storage at 80 C Kit Contents Item SOC Medium store at room temperature or 4 C The table below describes the items included in each of the One Shot chemically competent E coli kits described above Store at 80 C Amount 2 Tryptone 0 5 Yeast 6mL Extract 10 mM NaCl 2 5 mM KCI 10 mM MgCl 10 mM MgSO 20 mM glucose Chemically competent cells pUC19 Control DNA 11 x 50 uL 10 reaction ki
9. on mixture to select for individual clones See next page for details Continued on next page Expression Guidelines Continued Transformation Expression Protocol for Toxic Genes 10 This protocol may be used with either BL21 DE3 pLysS or BL21 DE3 pLysE cells Please note that other protocols are possible depending on your needs Transformation T Follow the basic transformation protocol on page 6 through Step 7 After growing the transformation reaction in SOC for 1 hour page 6 Step 7 add the entire transformation reaction 300 uL to 50 200 mL of LB medium pre warmed to 37 C containing the appropriate selective antibiotic for your expression plasmid and 34 ug mL chloramphenicol Induction 3 Incubate the vial s with shaking at 37 C until the cells reach mid log phase ODeo 0 3 Note Doubling times may vary 30 to 90 minutes depending on the protein expressed Add IPTG to a final concentration of 0 5 1 mM and grow for 2 3 more hours You make take time points if desired Harvest cells by centrifugation and use immediately for analysis or store the cell pellet at 80 C Testing Transformation Efficiency Introduction Before Starting Transformation To test the transformation efficiency of the competent cells contained in the One Shot kit use the supercoiled pUC19 plasmid supplied with the kit as described below An extra vial of cells is included for this purpose Prepare LB ag
10. r research purposes or production purposes by any commercial entity Information about commercial license may be obtained from The Office of Technology Transfer Brookhaven National Laboratory Bldg 475D P O Box 5000 Upton New York 11973 5000 Phone 516 344 7134 2 No materials that contain the cloned copy of the T7 gene 1 the gene for T7 RNA polymerase may be distributed further to third parties outside of your laboratory unless the recipient receives a copy of this sub license and agrees to be bound by its terms This limitation applies to strains BL21 DE3 BL21 DE3 pLysS and BL21 DE3 pLysE CE6 BL21 SI Competent Cells and any derivatives that are made of them You may refuse this sub license by returning this product unused in which case Invitrogen accept return of the product with a full refund By keeping or using this product you agree to be bound by the terms of this license 2010 Life Technologies Corporation All rights reserved The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners invitrogen by Lite technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
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12. tended Use One Shot BL21 DE3 BL21 DE3 pLysS and BL21 DE3 pLysE cells are qualified based on the following criteria 50 uL of competent cells are transformed with 10 pg of supercoiled pUC19 plasmid DNA Transformed cultures are plated on LB plates containing 50 ug mL ampicillin and the transformation efficiency is calculated Test transformations are performed in triplicate Transformation efficiency should be e gt 1x 108 cfu ug DNA for BL21 DE3 cells e gt 1x 108 cfu ug DNA for BL21 DE3 pLysS cells e gt 1 x10 cfu ug DNA for BL21 DE3 pLysE cells Untransformed cells are plated on e LB plates containing 50 ug mL ampicillin to verify the absence of ampicillin resistant contamination e LB plates as a lawn to verify the absence of phage contamination e LB plates containing 34 ug mL chloramphenicol for selection of pLysS or pLysE for BL21 DE3 pLysS or BL21 DE3 pLysE respectively For research use only Not intended for human or animal diagnostic or therapeutic uses Basic Transformation Procedure Introduction Materials Supplied by the User Before Starting A basic transformation protocol for BL21 DE3 BL21 DE3 pLysS and BL21 DE3 pLysE cells is provided below Proceed directly to expression using your own protocol once you have selected transformants Please note that BL21 DE3 BL21 DE3 pLysS and BL21 DE3 pLysE are designed to be used for expression not cloning or subcloning If you have questions about
13. ts OR 21 x 50 pL 20 reaction kits 10 pg pL in 5 mM Tris HCl 50 uL 0 5 mM EDTA pH 8 Continued on next page About the Kit Continued Genotypes pLysS and pLysE BL21 DE3 F ompT hsdSp rg mp gal dem DE3 BL21 DE3 pLysS F ompT hsdSp rg mp gal dem DE3 pLysS Cam BL21 DE3 pLysE F ompT hsdSp rp mp gal dem DE3 pLysE Cam The DE3 designation indicate the strains contain the DE3 lysogen that carries the gene for T7 RNA polymerase under control of the lacUV5 promoter IPTG is required to induce expression of the T7 RNA polymerase The three strains are E coli B r strains and do not contain the lon protease They are also deficient in the outer membrane protease OmpT The lack of two key proteases reduces degradation of heterologous proteins expressed in the strains BL21 DE3 pLysS and BL21 DE3 pLysE carry the pLysS and pLysE plasmids respectively which produce T7 lysozyme see below The BL21 DE3 strain does not carry a plasmid expressing T7 lysozyme The pLysS plasmid carried by the BL21 DE3 pLysS strain produces T7 lysozyme to reduce basal level expression of the gene of interest pLysS confers resistance to chloramphenicol Cam and contains the p15A origin This origin allows pLysS to be compatible with plasmids containing the ColE1 or pMB1 origin i e pUC or pBR322 derived plasmids The pLysE plasmid carried by the BL21 DE3 pLysE strain produces higher amounts of T7 lysoz
14. yme than pLysS to further reduce basal level expression of the gene of interest Like pLysS pLysE confers resistance to chloramphenicol Cam and contains the p15A origin for compatibility with plasmids containing the ColE1 or pMB1 origin Continued on next page About the Kit Continued Expression of Heterologous Genes General Handling Important The BL21 DE3 strain is suitable for use in expressing non toxic heterologous genes Recombinant proteins are generally expressed at higher levels in BL21 DE3 cells than in BL21 DE3 pLysS or BL21 DE3 pLysE but basal expression of heterologous genes is significantly higher in BL21 DES3 cells than in BL21 DE3 pLysS or BL21 DE3 pLysE cells as well The BL21 DE3 pLysS and BL21 DE3 pLysE strains produce T7 lysozyme to reduce basal expression of heterologous genes These two strains are recommended for use when expressing toxic genes Be extremely gentle when working with competent cells Competent cells are highly sensitive to changes in temperature and mechanical lysis caused by pipetting Transformation should be started immediately after thawing the cells on ice Mix the transformation reaction by swirling or tapping the tube gently not by pipetting BL21 DE3 BL21 DE3 pLysS and BL21 DE3 pLysE One Shot cells require IPTG to induce expression of the T7 RNA polymerase from the lacUV5 promoter Continued on next page About the Kit Continued Product Specifications In
15. your protein Analyze clones by western blot or enzymatic assay to determine which clone best expresses your protein of interest Use the glycerol stock created from this clone for expression experiments If you find that expression levels in subsequent inductions decrease or you find that you lose your plasmid your protein may be toxic to E coli see page 9 for additional information Continued on next page Expression Guidelines Continued Precautions Review the guidelines below when basal level expression of a gene of interest is toxic These guidelines assume that the T7 expression plasmid has been correctly designed and created Use BL21 DE3 pLysS or BL21 DE3 pLysE strains for expression experiments Both strains produce T7 lysozyme to inhibit the action of T7 RNA polymerase and reduce basal level expression of the gene of interest Choose BL21 DE3 pLysE when the tightest regulation of expression is required Propagate and maintain your expression plasmid in a strain that does not contain T7 RNA polymerase i e TOP10 DH5a etc Perform a fresh transformation of BL21 DE3 pLysS or BL21 DE3 pLysE cells before each induction experiment Minimize the amount of time that the cells bearing the gene of interest are cultured before IPTG induction Following transformation of BL21 DE3 pLysS or BL21 DE3 pLysE cells grow cells in SOC medium for 1 hour and go directly to protein expression Do not plate the transformati
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