SpECTRAŽ S. pombe Expression Systemtools.thermofisher.com/.../sfs/manuals/spectrasystem_man.pdfS. pombe host strain, expression of the PCR product can be regulated using thiamine

SpECTRA S. pombe Expression System Version D October 2, 2002 25-0403

SpECTRA S. pombe Expression System Rapid cloning of Taq polymerase-amplified PCR products into vectors for thiamine-regulated expression in S. pombe

Catalog no. K180-01

A Limited Label License covers this product (see Purchaser Notification). By use of this product, you accept the terms and conditions of the Limited Label License.

www.invitrogen.com [email protected]

http://www.invitrogen.com/

iii

Table of Contents

Table of Contents.................................................................................................................................................. iii Important Information ............................................................................................................................................v Accessory Products................................................................................................................................................ix

Introduction ..................................................................................................................1 Overview ................................................................................................................................................................1

Methods ........................................................................................................................6 PCR Primer Design.................................................................................................................................................6 Producing PCR Products ......................................................................................................................................10 TOPO® Cloning and Transformation ...................................................................................................................11 Optimizing the TOPO® Cloning Reaction............................................................................................................16 Yeast Transformation ...........................................................................................................................................17 Expression and Purification of the PCR Product..................................................................................................21

Appendix.....................................................................................................................25 pNMT TOPO® Control Reactions ........................................................................................................................25 Purifying PCR Products........................................................................................................................................28 Addition of 3´ A-Overhangs Post-Amplification .................................................................................................30 Lithium Acetate Transformation Protocol ............................................................................................................31 Electroporation Transformation Protocol .............................................................................................................32 pNMT-TOPO® Vectors ........................................................................................................................................33 pNMT/CAT Vectors.............................................................................................................................................35 Recipes..................................................................................................................................................................36 Technical Service .................................................................................................................................................40 Purchaser Notification ..........................................................................................................................................42 Product Qualification............................................................................................................................................43 References ............................................................................................................................................................46

v

Important Information

Shipping/Storage The components in the SpECTRA S. pombe Expression System are supplied in 4 boxes

and are shipped as described below. For a description of the reagents in each box, see below and pages vi-vii. Upon receipt, store each box as indicated below.

Box Contents Shipping Storage Temp 1 pNMT TOPO TA Cloning® Reagents Dry ice -20°C 2 & 3 One Shot® TOP10 Chemically

Competent E. coli Dry ice -80°C

4 SpECTRA S. pombe Accessory Kit Room temperature Room temperature and +4°C (see page vii for details)

pNMT TOPO TA Cloning® Reagents

The pNMT TOPO TA Cloning® reagents (Box 1) are listed below. Note that the user must supply Taq polymerase. Store Box 1 at -20°C.

Item Concentration Amount

pNMT1-TOPO® vector, linearized 10 ng/µl plasmid DNA in: 50% glycerol 50 mM Tris-HCl, pH 7.4 (at 25°C) 1 mM EDTA 2 mM DTT 0.1% Triton X-100 100 µg/ml BSA 30 µM phenol red

10 µl

pNMT41-TOPO® vector, linearized Same as above 10 µl pNMT81-TOPO® vector, linearized Same as above 10 µl 10X PCR Buffer 100 mM Tris-HCl, pH 8.3 (at

42°C) 500 mM KCl 25 mM MgCl2 0.01% gelatin

100 µl

dNTP Mix 12.5 mM dATP 12.5 mM dCTP 12.5 mM dGTP 12.5 mM dTTP in water (pH 8)

10 µl

Salt Solution 1.2 M NaCl 0.06 M MgCl2

50 µl

Sterile Water -- 1 ml

continued on next page

vi

Important Information, continued

pNMT TOPO TA Cloning® Reagents, continued

Item Concentration AmountNMT pombe Forward Sequencing Primer

Lyophilized in TE buffer, pH 8 2 µg

URA4 pombe Reverse Sequencing Primer

Lyophilized in TE buffer, pH 8 2 µg

pNMT1/CAT Expression Control Plasmid

0.5 µg/µl in TE buffer, pH 8 10 µl





Control PCR Primers 0.1 µg/µl each in TE Buffer, pH 8 10 µl Control PCR Template 0.05 µg/µl in TE Buffer, pH 8 10 µl

One Shot® TOP10 Reagents

The table below describes the items included in each One Shot® TOP10 Chemically Competent E. coli kit (Boxes 2 and 3). The cells are supplied at a transformation efficiency of at least 1 x 109 cfu/µg DNA. Store Boxes 2 and 3 at -80°C.

Item Composition Amount

SOC Medium (may be stored at room temperature or +4°C)

2% Tryptone 0.5% Yeast Extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO4 20 mM glucose

6 ml

TOP10 cells -- 21 x 50 µl pUC19 Control DNA 10 pg/µl in 5 mM Tris-HCl,

0.5 mM EDTA, pH 8 50 µl


vii


SpECTRA S. pombe Accessory Kit

The table below describes the items included in the SpECTRA S. pombe Accessory Kit (Box 4). The amount of powdered EMM S. pombe Expression Medium provided is sufficient to prepare more than 7.5 liters of medium. Store the TCP1 stab and the thiamine at +4°C. Store the powdered EMM S. pombe Expression Medium at room temperature. For long-term storage, store the EMM S. pombe Expression Medium at +4°C.

Item Composition Amount

EMM S. pombe Expression Medium

See below 250 g

TCP1 S. pombe Cells YPD agar 1 stab 1000X Thiamine 10 mM Thiamine 8 ml

Composition of EMM

The EMM S. pombe Expression Medium provided in the kit is supplied as a powdered stock that can be dissolved in deionized, distilled water to prepare liquid medium. The table below lists the composition and final concentration of one liter of liquid EMM Expression Medium. For instructions to prepare EMM Expression Medium, see page 19.

Final Concentration Amount/liter

111 mM Glucose 93.5 mM NH4Cl 14.7 mM KH phthalate 15.5 mM Na2HPO4 5.2 mM MgCl2·6H2O 13.4 mM KCl 281.7 µM Na2SO4 100 µM CaCl2·2H2O 81.2 µM nicotinic acid 55.5 µM myo-inositol 4.2 µM D-pantothenic acid·½Ca 4.8 µM citric acid·H2O 8.1 µM H3BO3 1.4 µM ZnSO4·7H2O 2.4 µM MnSO4·H2O 739.9 nM FeCl3·6H2O 661.4 nM NaMoO4·2H2O 602.4 nM KI 160.2 nM CuSO4·5H2O 40.9 nM D-biotin

20 g 5 g 3 g 2.2 g 1.066 g 1 g 40 mg 14.7 mg 10 mg 10 mg 1 mg 1 mg 500 µg 400 µg 400 µg 200 µg 160 µg 100 µg 40 µg 10 µg


viii


Primer Sequences The table below lists the sequence and pmoles for the primers included in the pNMT

TOPO® TA Expression Kits.

Primer Sequence pMoles SuppliedNMT pombe Forward

5´-TTTCAATCTCATTCTCACTTTCTGA-3´ 267

URA4 pombe Reverse

5´-ACAAGGCATCGACTTTTTCAATA-3´ 286

Genotype of TOP10 Cells

F- mcrA ∆(mrr-hsdRMS-mcrBC) Φ80lacZ∆M15 ∆lacΧ74 recA1 deoR araD139 ∆(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG

ix

Accessory Products

Introduction The products listed in this section are intended for use with the pNMT TOPO® TA

Expression Kits. For more information, refer to our World Wide Web site (www.invitrogen.com) or call Technical Service (see page 40).

Products Available Separately

Some of the reagents included in the pNMT TOPO® TA Expression Kits as well as other products that may be used for expression in S. pombe are available separately from Invitrogen. Ordering information is provided below.

Product Amount Catalog no. 10 reactions C4040-10 20 reactions C4040-03

One Shot® Kit (TOP10 Chemically Competent Cells)

40 reactions C4040-06 One Shot® Kit 10 reactions C4040-50 (TOP10 Electrocompetent Cells) 20 reactions C4040-52 EMM S. pombe Expression Medium* 250 g, powder Q800-01 pNMT1 TOPO® TA Expression Kit 20 reactions K181-01 pNMT41 TOPO® TA Expression Kit 20 reactions K182-01 pNMT81 TOPO® TA Expression Kit 20 reactions K183-01 S.c. EasyComp Kit 6 x 20 transformations K5050-01

*Sufficient to prepare more than 7.5 liters of EMM Expression Medium

Detection of Recombinant Proteins

Expression of your recombinant fusion protein can be detected using an antibody to the appropriate epitope. The table below describes the antibodies available for detection of C-terminal fusion proteins expressed using one of the pNMT-TOPO® vectors. Horseradish peroxidase (HRP) and alkaline phosphatase (AP)-conjugated antibodies allow one-step detection using colorimetric or chemiluminescent detection methods. Fifty microliters of each antibody is supplied which is sufficient for 25 westerns.

Product Epitope Catalog no.

Anti-V5 Antibody R960-25 Anti-V5-HRP Antibody R961-25 Anti-V5-AP Antibody

Detects 14 amino acid epitope derived from the P and V proteins of the paramyxovirus, SV5 (Southern et al., 1991) GKPIPNPLLGLDST

R962-25

Anti-His (C-term) Antibody R930-25 Anti-His(C-term)-HRP Antibody R931-25 Anti-His(C-term)-AP Antibody

Detects the C-terminal polyhistidine (6xHis) tag (requires the free carboxyl group for detection (Lindner et al., 1997) HHHHHH-COOH

R932-25



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Accessory Products, continued

Purification of Recombinant Protein

The metal binding domain encoded by the polyhistidine tag allows simple, easy purification of your recombinant protein by Immobilized Metal Affinity Chromatography (IMAC) using Invitrogen's ProBond Resin (see below). To purify proteins expressed from one of the pNMT-TOPO® vectors, the ProBond Purification System or the ProBond resin in bulk are available separately. See the table below for ordering information.

Product Quantity Catalog no. 50 ml R801-01 ProBond Metal-Binding Resin

(precharged resin provided as a 50% slurry in 20% ethanol)

150 ml R801-15

ProBond Purification System 6 purifications K850-01 ProBond Purification System with Anti-V5-HRP Antibody

1 kit K854-01

ProBond Purification System with Anti-His(C-term)-HRP Antibody

1 kit K853-01

Purification Columns (10 ml polypropylene columns)

50 R640-50

1

Introduction

Overview

Introduction The SpECTRA S. pombe Expression System uses TOPO® Cloning technology to

provide a highly efficient, rapid cloning strategy for the direct insertion of Taq polymerase-amplified PCR products into a series of plasmid vectors for regulated expression of the gene of interest in the fission yeast, Schizosaccharomyces pombe (S. pombe). TOPO® Cloning requires no ligase, post-PCR procedures, or PCR primers containing special, additional sequences. Once cloned, analyzed, and transformed into an S. pombe host strain, expression of the PCR product can be regulated using thiamine. When used together, the three vectors allow expression of your protein of interest over a greater than 10,000-fold range in S. pombe.

pNMT-TOPO® Vectors

The pNMT1-TOPO®, pNMT41-TOPO®, and pNMT81-TOPO® vectors are 6.1 kb expression vectors designed to facilitate rapid cloning and thiamine-regulated expression of PCR products in S. pombe. The pNMT-TOPO® vectors contain the following elements: • The nmt1 promoter for thiamine-regulated expression of the gene of interest in

S. pombe cells (Maundrell, 1990). Each vector contains a distinct form of the nmt1 promoter differing only in the TATA box region (see page 3 for details) (Basi et al., 1993).

• TOPO® Cloning site for rapid and efficient cloning of Taq-amplified PCR products (see the next page for more information)

• C-terminal peptide containing the V5 epitope and a polyhistidine (6xHis) tag for detection and purification of recombinant protein (optional)

• S. pombe ars1 origin of replication for non-integrative, high-copy maintenance of the plasmid in S. pombe cells (Heyer et al., 1986)

• S. cerevisiae LEU2 auxotrophic marker for selection of yeast transformants (Andreadis et al., 1984)

• Ampicillin resistance gene for selection in E. coli Three control plasmids (pNMT1/CAT, pNMT41/CAT, or pNMT81/CAT) are included in the kit for use as positive controls for transformation and expression in S. pombe cells.


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Overview, continued

How TOPO® Cloning Works

The plasmid vectors, pNMT1-TOPO®, pNMT41-TOPO®, and pNMT81-TOPO®, are supplied linearized with: • Single 3´ thymidine (T) overhangs for TA Cloning® • Topoisomerase covalently bound to the vector (this is referred to as activated vector) Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in this kit has single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector. Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5′-CCCTT in one strand (Shuman, 1991). The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3′ phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I. The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5′ hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase (Shuman, 1994). TOPO® Cloning exploits this reaction to efficiently clone PCR products (see below).

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Overview, continued

nmt1 Promoter and Thiamine Regulation

In S. pombe, the no message in thiamine (nmt1) gene encodes a 39 kDa protein that is both regulated by thiamine and thought to be involved in its biosynthesis (Maundrell, 1990). The nmt1 gene and its promoter have been well-studied and found to exhibit the following characteristics: • In minimal medium, the nmt1 gene is highly transcribed (Maundrell, 1990). • In minimal medium supplemented with thiamine, expression of the nmt1 gene is

repressed (Maundrell, 1990). • Complete repression of transcription occurs when thiamine is added to a final

concentration of 0.5 µM or greater (Maundrell, 1990). • Addition of thiamine to the culture medium results in the disappearance of message

within 3 hours (Maundrell, 1990). • Removal of thiamine results in approximately 100-200-fold induction of expression

from the nmt1 promoter. • Removal of thiamine results in detectable message after 10 hours and maximal, steady-

state levels after 16 hours (Maundrell, 1990). The SpECTRA S. pombe Expression System uses the nmt1 promoter to impart thiamine-regulation to a heterologous gene of interest. In the system, expression of your gene of interest is repressed in the presence of thiamine and induced in the absence of thiamine. In general, the kinetics of heterologous gene expression are similar to those described above with the native nmt1 gene.

TATA Box Mutations in the nmt1 Promoter

Detailed analysis of the nmt1 promoter has demonstrated that it contains a canonical TATA box element located 25 base pairs upstream of the transcriptional start site (Maundrell, 1990) (see the diagram on page 7). Further studies have shown that stepwise truncation of the TATA box leads to a progressive decrease in the strength of the nmt1 promoter (Basi et al., 1993). Both the induced level and the basal, repressed level of the nmt1 gene (and heterologous genes) are reduced (Basi et al., 1993). The pNMT1-TOPO®, pNMT41-TOPO®, and pNMT81-TOPO® vectors contain the wild-type nmt1 promoter and two TATA box mutants, respectively (see figure below). TOPO® Cloning your gene of interest into pNMT1-TOPO®, pNMT41-TOPO®, and pNMT81-TOPO® allows the user to vary the range of thiamine-regulated expression of the recombinant protein of interest more than 10,000-fold.

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Overview, continued

An Example of Thiamine-Regulated Expression

In the experiment below, one microgram of pNMT1/CAT, pNMT41/CAT, and pNMT81/CAT (included with the kit) were used to transform 1 x 108 S. pombe cells. The transformation mixtures were plated on EMM + T agar plates and 3 transformants containing the correct constructs were screened. Starter cultures were prepared by inoculating each transformant into 50 ml of EMM + T medium. Cells were grown at 30°C with shaking for 24 hours. After 24 hours, the starter cultures were washed twice with 50 ml of EMM and resuspended in 50 ml of EMM. 500 µl aliquots of each starter culture were inoculated into 2 flasks of 100 ml EMM. One of the two experimental cultures was supplemented with 10 µM thiamine. Cultures were incubated with shaking at 30°C for 18 hours and cells were collected for analysis. Cell extracts were prepared by glass bead breakage and quantitative ELISA assays performed using the Roche Sandwich ELISA Kit following the manufacturers instructions. The results depicted below demonstrate the distinct induction profile and expression level obtained from each vector. Note that both the basal and induced levels of transcription vary with each vector.

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Sample 1: pNMT1/CAT, induced Sample 2: pNMT1/CAT, uninduced Sample 3: pNMT41/CAT, induced Sample 4: pNMT41/CAT, uninduced Sample 5: pNMT81/CAT, induced Sample 6: pNMT81/CAT, uninduced


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Overview, continued

Experimental Outline

The table below describes the general steps needed to clone and express your gene of interest. For more details, refer to the pages indicated.

Step Action Page 1 Design PCR primers to clone your gene of interest into one of the pNMT-

TOPO® vectors in frame with the C-terminal peptide containing the V5 epitope and the polyhistidine (6xHis) tag (if desired). Consult the diagrams on pages 7-9 to help you design your PCR primers.

6-9

2 Produce your PCR product. 10 3 TOPO® Clone your PCR product into the appropriate pNMT-TOPO®

vector and transform into One Shot® TOP10 E. coli. Select transformants on LB plates containing 50-100 µg/ml ampicillin.

11-13

4 Analyze your transformants for the presence and orientation of insert by restriction enzyme digestion or PCR.

14

5 Select a transformant with the correct restriction pattern and sequence it to confirm that your gene is in the correct orientation, cloned in frame with the C-terminal peptide, and is free from PCR-induced mutations.

14

6 Prepare purified plasmid DNA and transform into competent S. pombe cells. Maintain transformants in EMM Expression Medium containing thiamine.

17-20

7 Remove thiamine to induce expression of the gene of interest. 21-228 Assay for expression of your recombinant protein. 23-249 Purify recombinant protein, if desired. 24

6

Methods

PCR Primer Design

Introduction It is important to properly design your PCR primers to ensure that you obtain the

recombinant protein you need for your studies. Use the information below and the diagram on page 7 to design your PCR primers. Remember that your PCR product will have single 3´ adenine overhangs.

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The pNMT1-TOPO®, pNMT41-TOPO®, and pNMT81-TOPO® vectors differ from one another only in the sequence of the TATA box region of the nmt1 promoter. All other sequences, including the region surrounding the TOPO® Cloning site and the C-terminal peptide are identical among the 3 vectors. If you wish to TOPO® Clone your PCR product into all 3 pNMT-TOPO® vectors, you only need to design your PCR primers once. After producing your PCR product, you may TOPO® Clone the same PCR product into all 3 vectors.

General Molecular Biology Techniques

For help with E. coli transformations, restriction enzyme analysis, DNA sequencing, and DNA biochemistry, refer to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et al., 1994).

Do not add 5´ phosphates to your primers for PCR. The PCR product synthesized will not ligate into the pNMT-TOPO® vector.

Translation Initiation

Your PCR product must contain an ATG start codon for proper initiation of translation. You may want to include the ATG within the context of a Kozak translation initiation sequence (Kozak, 1987; Kozak, 1991; Kozak, 1990). If so, you may need to design a Kozak sequence into your forward PCR primer. An example of a Kozak consensus sequence is provided below. Note that other sequences are possible, but the G or A at position 3 and the G at position +4 are the most critical for function (shown in bold). The ATG start codon is shown underlined.

(G/A)NNATGG

Fusion to the C-terminal Peptide

If you wish to include the C-terminal peptide for detection with either the V5 or His(C-term) antibodies or purification using the polyhistidine (6xHis) tag, you must design your reverse PCR primer to remove the native stop codon and maintain the frame through the DNA encoding the C-terminal peptide. If you do not wish to include the C-terminal peptide, include the native stop codon in the reverse PCR primer or design the primer to anneal downstream of the native stop codon. Note: Cloning efficiencies may vary depending on the 5′ nucleotide sequence of your primer (see page 27). Use the diagrams on pages 7-9 to design your PCR primers. Once you have designed your PCR primers, proceed to page 10.


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PCR Primer Design, continued

TOPO® Cloning Site for pNMT1-TOPO®

The diagram below is supplied to help you design appropriate PCR primers to correctly clone and express your PCR product using pNMT1-TOPO®. Restriction sites are labeled to indicate the actual cleavage site. The vector is supplied linearized between base pair 1150 and 1151. This is the TOPO® Cloning site. The complete sequence of pNMT1-TOPO® is available for downloading from our World Wide Web site (www.invitrogen.com) or from Technical Service (page 40). For a map and a description of the features of pNMT1-TOPO®, refer to the Appendix, pages 33-34.

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Producing PCR Products

Introduction Once you have decided on a PCR strategy and have synthesized the primers you are

ready to produce your PCR product.

Materials Supplied by the User

You will need the following reagents and equipment. • Taq polymerase • Thermocycler • DNA template and primers for PCR product

Polymerase Mixtures

If you wish to use a mixture containing Taq polymerase and a proofreading polymerase, Taq must be used in excess of a 10:1 ratio to ensure the presence of 3´ A-overhangs on the PCR product (i.e. Expand or eLONGase). If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only, you can add 3′ A-overhangs using the method on page 30.

Producing PCR Products

1. Set up the following 50 µl PCR reaction. Use less DNA if you are using a plasmid for template and more DNA if you are using genomic DNA as a template. Use the cycling parameters suitable for your primers and template. Be sure to include a 7 to 30 minute extension at 72°C after the last cycle to ensure that all PCR products are full length and 3´ adenylated. DNA Template 10-100 ng 10X PCR Buffer 5 µl 50 mM dNTP Mix 0.5 µl Primers (0.1-0.2 µg each) 1 µM each Sterile water add to a final volume of 49 µl Taq Polymerase (1 unit/µl) 1 µl Total Volume 50 µl

2. Check the PCR product by agarose gel electrophoresis. You should see a single, discrete band. If you do not see a single band, please refer to the Note below.

If you do not obtain a single, discrete band from your PCR, you may gel-purify your fragment before TOPO® Cloning into pNMT1-TOPO®, pNMT41-TOPO®, or pNMT81-TOPO® (see pages 28-29). Take special care to avoid sources of nuclease contamination and long exposure to UV light. Alternatively, you may optimize your PCR to eliminate multiple bands and smearing (Innis et al., 1990). The PCR Optimizer Kit (Catalog no. K1220-01) from Invitrogen can help you optimize your PCR. Call Technical Service for more information (page 40).

11

TOPO® Cloning and Transformation

Introduction TOPO® Cloning technology allows you to produce your PCR products, ligate them into

pNMT1-TOPO®, pNMT41-TOPO®, or pNMT81-TOPO®, and transform the recombinant vector into TOP10 E. coli in one day. It is important to have everything you need set up and ready to use to ensure that you obtain the best possible results. If this is the first time you have TOPO® Cloned, perform the control reactions on pages 25-26 in parallel with your samples. If you have previously TOPO® Cloned, read the Note below.

Recent experiments at Invitrogen have demonstrated that inclusion of salt (200 mM NaCl, 10 mM MgCl2) in the TOPO® Cloning reaction results in the following: • A 2- to 3-fold increase in the number of transformants. • Allows for longer incubation times (up to 30 minutes). Longer incubation times can

result in an increase in the number of transformants obtained. Including salt in the TOPO® Cloning reaction prevents topoisomerase I from rebinding and potentially nicking the DNA after ligating the PCR product and dissociating from the DNA. The result is more intact molecules leading to higher transformation efficiencies. If you do not include salt in the TOPO® Cloning reaction, the number of transformants obtained generally decreases as the incubation time increases beyond 5 minutes.

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Because of the above results, we recommend adding salt to the TOPO® Cloning reaction. A stock salt solution is provided in the kit for this purpose. Note that the amount of salt added to the TOPO® Cloning reaction varies depending on whether you plan to transform chemically competent cells (provided) or electrocompetent cells (see below). For this reason two different TOPO® Cloning reactions are provided to help you obtain the best possible results. Read the following information carefully.

Chemically Competent E. coli

For TOPO® Cloning and transformation into chemically competent E. coli, adding sodium chloride and magnesium chloride to a final concentration of 200 mM NaCl, 10 mM MgCl2 in the TOPO® Cloning reaction increases the number of colonies obtained. A Salt Solution (1.2 M NaCl; 0.06 M MgCl2) is provided to adjust the TOPO® Cloning reaction to the recommended concentration of NaCl and MgCl2.

Electrocompetent E. coli

For TOPO® Cloning and transformation of electrocompetent E. coli, salt must also be included in the TOPO® Cloning reaction, but the amount of salt must be reduced to 50 mM NaCl, 2.5 mM MgCl2 to prevent arcing when electroporating. The Salt Solution provided in the kit must be diluted 4-fold to prepare a 300 mM NaCl, 15 mM MgCl2 solution for convenient addition to the TOPO® Cloning reaction (see next page).


12

TOPO® Cloning and Transformation, continued

Materials Supplied by the User

In addition to general microbiological supplies (i.e. plates, spreaders), you will need the following reagents and equipment. • 42°C water bath (or electroporator with cuvettes, if using electrocompetent cells) • LB plates containing 50-100 µg/ml ampicillin (two for each transformation) • Reagents and equipment for agarose gel electrophoresis • 37°C shaking and non-shaking incubator

There is no blue-white screening for the presence of inserts. Individual recombinant plasmids need to be analyzed by restriction analysis, sequencing, or PCR for the presence and orientation of insert. Sequencing primers included in each kit can be used to sequence across an insert in the multiple cloning site to confirm orientation and reading frame.

Preparation for Transformation

For each transformation, you will need one vial of competent cells and two selective plates. • Equilibrate a water bath to 42°C (for chemical transformation) or set up your

electroporator if you are using electrocompetent E. coli. • For electroporation, dilute a small portion of the Salt Solution 4-fold to prepare Dilute

Salt Solution (e.g. add 5 µl of the Salt Solution to 15 µl sterile water) • Warm the vial of SOC medium from Box 2 (or Box 3) to room temperature. • Warm selective plates at 37°C for 30 minutes. • Thaw on ice 1 vial of One Shot® cells for each transformation.

Setting Up the TOPO® Cloning Reaction

The table below describes how to set up your TOPO® Cloning reaction (6 µl) for eventual transformation into either chemically competent One Shot® TOP10 E. coli (provided) or electrocompetent E. coli. Additional information on optimizing the TOPO® Cloning reaction for your needs can be found on page 16. Note: The red or yellow color of the TOPO® vector solution is normal and is used to visualize the solution.

Reagent* Chemically Competent E. coli Electrocompetent E. coli

Fresh PCR product 0.5 to 4 µl 0.5 to 4 µl Salt Solution 1 µl --

Dilute Salt Solution (1:4) -- 1 µl Sterile Water add to a final volume of 5 µl add to a final volume of 5 µl TOPO® vector 1 µl 1 µl

*Store all reagents at -20°C when finished. Salt solutions and water can be stored at room temperature or +4°C.


13


Performing the TOPO® Cloning Reaction

1. Mix reaction gently and incubate for 5 minutes at room temperature (22-23°C). Note: For most applications, 5 minutes will yield plenty of colonies for analysis. Depending on your needs, the length of the TOPO® Cloning reaction can be varied from 30 seconds to 30 minutes. For routine subcloning of PCR products, 30 seconds may be sufficient. For large PCR products (> 1 kb) or if you are TOPO® Cloning a pool of PCR products, increasing the reaction time will yield more colonies.

2. Place the reaction on ice and proceed to One Shot® TOP10 Chemical Transformation (below) or Transformation by Electroporation (below). Note: You may store the TOPO® Cloning reaction at -20°C overnight.

One Shot® TOP10 Chemical Transformation

1. Add 2 µl of the TOPO® Cloning reaction from Step 2 above into a vial of One Shot® TOP10 Chemically Competent E. coli and mix gently. Do not mix by pipetting up and down.

2. Incubate on ice for 5 to 30 minutes. Note: Longer incubations on ice seem to have a minimal effect on transformation efficiency. The length of the incubation is at the users discretion (see above).

3. Heat-shock the cells for 30 seconds at 42°C without shaking. 4. Immediately transfer the tubes to ice. 5. Add 250 µl of room temperature SOC medium. 6. Cap the tube tightly and shake the tube horizontally (200 rpm) at 37°C for 1 hour. 7. Spread 25-200 µl from each transformation on a prewarmed selective plate and

incubate overnight at 37°C. We recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies.

8. An efficient TOPO® Cloning reaction will produce hundreds of colonies. Pick ~10 colonies for analysis (see Analysis of Positive Clones, next page).

Transformation by Electroporation

1. Add 2 µl of the TOPO® Cloning reaction into a 0.1 cm cuvette containing 50 µl of electrocompetent E. coli and mix gently. Do not mix by pipetting up and down. Avoid formation of bubbles.

2. Electroporate your samples using your own protocol and your electroporator. Note: If you have problems with arcing, see next page.

3. Immediately add 250 µl of room temperature SOC medium. 4. Transfer the solution to a 15 ml snap-cap tube (i.e. Falcon) and shake for at least

1 hour at 37°C to allow expression of the antibiotic resistance gene. 5. Spread 10-200 µl from each transformation on a prewarmed selective plate and

incubate overnight at 37°C. To ensure even spreading of small volumes, add 20 µl of SOC. We recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies.

6. An efficient TOPO® Cloning reaction will produce hundreds of colonies. Pick ~10 colonies for analysis (see Analysis of Positive Clones, next page).


14


Addition of the Dilute Salt Solution in the TOPO® Cloning Reaction brings the final concentration of NaCl and MgCl2 in the TOPO® Cloning reaction to 50 mM and 2.5 mM, respectively. To prevent arcing of your samples during electroporation, the volume of cells should be between 50 and 80 µl (0.1 cm cuvettes) or 100 to 200 µl (0.2 cm cuvettes). If you experience arcing during transformation, try one of the following suggestions: • Reduce the voltage normally used to charge your electroporator by 10% • Reduce the pulse length by reducing the load resistance to 100 ohms • Ethanol-precipitate the TOPO® Cloning reaction and resuspend in water prior to

electroporation

Analysis of Positive Clones

1. Pick 10 colonies and culture them overnight in 2-5 ml LB or SOB medium containing 50-100 µg/ml ampicillin.

2. Isolate plasmid DNA using your method of choice. If you need ultra-pure plasmid DNA for automated or manual sequencing, we recommend the S.N.A.P. MiniPrep Kit (Catalog no. K1900-01) or the S.N.A.P. MidiPrep Kit (Catalog no. K1910-01).

3. Analyze the plasmids for the presence and orientation of insert by restriction analysis or sequencing. The NMT pombe Forward and URA4 pombe Reverse sequencing primers are included in the kit to help you sequence your insert. Refer to the diagrams on pages 7-9 for the sequence surrounding the TOPO® Cloning site. Note: Resuspend each primer in 20 µl of sterile water to prepare a 0.1 µg/µl stock solution.

4. If you need help with setting up restriction enzyme digests or DNA sequencing, refer to general molecular biology texts (Ausubel et al., 1994; Sambrook et al., 1989).

Alternative Method of Analysis

You may wish to use PCR to directly analyze positive transformants. For PCR primers, use a combination of one of the primers included with the kit and a primer that binds within your insert. You will have to determine the amplification conditions. If this is the first time you have used this technique, we recommend that you perform restriction analysis in parallel to confirm that PCR gives you the correct result. Artifacts may be obtained because of mispriming or contaminating template. Please note that this method will allow you to check for both the presence of cloned PCR product and the orientation of the insert. The following protocol is provided for your convenience. Other protocols are suitable. 1. Prepare a PCR cocktail consisting of PCR buffer, dNTPs, primers, and Taq

polymerase. Use a 20 µl reaction volume. Multiply by the number of colonies to be analyzed (e.g. 10).

2. Pick 10 colonies and resuspend them individually in 20 µl of the PCR cocktail. Don't forget to make a patch plate to preserve the colonies for further analysis.

3. Incubate the reaction for 10 minutes at 94°C to lyse the cells and inactivate nucleases.

4. Amplify for 20 to 30 cycles using the appropriate conditions (see text above). 5. For the final extension, incubate at 72°C for 10 minutes. Hold at +4°C. 6. Visualize by agarose gel electrophoresis.


15


��

If you have problems obtaining transformants or the correct insert, perform the control reactions described on page 25-26. These reactions will help you troubleshoot your experiment.

Preparing a Glycerol Stock

Once you have identified the correct clone, be sure to isolate a single colony and prepare a glycerol stock for long term storage. We recommend that you also store the purified plasmid DNA at -20°C. 1. Streak the original colony on LB plates containing 50-100 µg/ml ampicillin. 2. Isolate a single colony and inoculate into 1-2 ml of LB containing 50-100 µg/ml

ampicillin. 3. Grow the culture to mid-log phase (OD600 = 0.5-0.7). 4. Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a cryovial. 5. Store at -80°C.

16

Optimizing the TOPO® Cloning Reaction

Introduction The information below will help you optimize the TOPO® Cloning reaction for your

particular needs.

Faster Subcloning The high efficiency of TOPO® Cloning technology allows you to streamline the cloning

process. If you routinely clone PCR products and wish to speed up the process, consider the following: • Incubate the TOPO® Cloning reaction for only 30 seconds instead of 5 minutes.

You may not obtain the highest number of colonies, but with the high efficiency of TOPO® Cloning, most of the transformants will contain your insert.

• After adding 2 µl of the TOPO® Cloning reaction to chemically competent cells, incubate on ice for only 5 minutes. Increasing the incubation time to 30 minutes does not significantly improve transformation efficiency.

More Transformants

If you are TOPO® Cloning large PCR products, toxic genes, or cloning a pool of PCR products, you may need more transformants to obtain the clones you want. To increase the number of colonies: • Incubate the salt-supplemented TOPO® Cloning reaction for 20 to 30 minutes

instead of 5 minutes. Note: Increasing the incubation time of the salt-supplemented TOPO® Cloning reaction allows more molecules to ligate, increasing the transformation efficiency. Addition of salt appears to prevent topoisomerase from rebinding and nicking the DNA after it has ligated the PCR product and dissociated from the DNA.

Cloning Dilute PCR Products

To clone dilute PCR products, you may: • Increase the amount of the PCR product • Incubate the TOPO® Cloning reaction for 20 to 30 minutes • Concentrate the PCR product by precipitation

17

Yeast Transformation

Introduction Once you have obtained your pNMT-TOPO® expression construct, you are ready to

transform and express your protein of interest in S. pombe. General guidelines for transformation and expression are provided below. We recommend that you include a positive control (see below) for transformation and expression to help you evaluate your results.

Basic Yeast Molecular Biology Techniques

We recommend that the user be familiar with basic yeast molecular biology and microbiological techniques. For a general reference source, refer to the Guide to Yeast Genetics and Molecular Biology, pages 795-823 (Guthrie and Fink, 1991).

Plasmid Preparation

You may use any method of choice to prepare purified plasmid DNA for small-scale yeast transformation. Standard protocols may be found in Current Protocols in Molecular Biology (Ausubel et al., 1994) or Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989). We recommend isolating plasmid DNA using the S.N.A.P. MiniPrep Kit (10-15 µg DNA, Catalog no. K1900-01), the S.N.A.P. MidiPrep Kit (10-200 µg DNA, Catalog no. K1910-01), or CsCl gradient centrifugation.

Yeast Host Strain The TCP1 S. pombe strain is included with the kit for use as a host for your pNMT1-

TOPO®, pNMT41-TOPO®, and pNMT81-TOPO® constructs. The TCP1 strain contains a leu1 mutation resulting in leucine auxotrophy. Note that prior to introduction of the pNMT-TOPO® vectors, the strain must be maintained on plates containing leucine. Genotype: h, leu1-32 Note: The TCP1 strain may also be obtained from the American Type Culture Collection (ATCC Catalog no. 38399). For more information about this strain, refer to the ATCC Web site at www.atcc.org.

Initiating TCP1 Culture

To initiate a culture of TCP1 from the stab provided with the kit, streak a small amount from the stab on a YPD plate (see Appendix for recipe, page 38) and incubate at 30°C. Once growth is established, you may check the phenotype of the strain by streaking a single colony on a PDM minimal plate supplemented with leucine. TCP1 will not grow in PDM minimal medium deficient in leucine. Be sure to make glycerol stocks of the strain. Store glycerol stocks at -80°C. If you plan to use the strain directly from plates, be sure that the plates are less than 4 days old.

Positive Control The pNMT1/CAT, pNMT41/CAT, and pNMT81/CAT vectors are provided with the

SpECTRA S. pombe Expression System as positive controls for yeast transformation and expression from pNMT1-TOPO®, pNMT41-TOPO®, and pNMT81-TOPO®, respectively. Each vector allows thiamine-regulated expression of a chloramphenicol acetyltransferase (CAT) protein fused to the C-terminal peptide containing the V5 epitope and the polyhistidine (6xHis) tag. For a detailed map and a description of the features of the vectors, see page 35.


http://www.atcc.org/

18

Yeast Transformation, continued

Assay for CAT Protein

The CAT fusion protein expressed from the pNMT1/CAT, pNMT41/CAT, and pNMT81/CAT control plasmids is approximately 32 kDa in size. You may assay for CAT expression by ELISA assay, western blot analysis, fluorometric assay, or radioactive assay (Ausubel et al., 1994; Neumann et al., 1987). If you wish to detect CAT protein using western blot analysis, you may use the following antibodies available from Invitrogen: • Anti-CAT Antiserum (Catalog no. R902-25) • Anti-V5 Antibodies (see page ix for more information) • Anti-His(C-term) Antibodies (see page ix for more information) Other kits to assay for CAT protein are suitable.

Medium Requirements

The table below lists the types of media required for transformation and expression experiments. We generally use: • pombe Dropout Medium (PDM) lacking leucine after transformation to select for

the pNMT-TOPO® expression construct. PDM contains thiamine, therefore, it is not suitable for expression experiments with the pNMT-TOPO® vectors. See page 37 for a recipe.

• Edinburgh Minimal Medium (EMM) for expression experiments. EMM is a defined, minimal medium that does not contain thiamine. EMM must be supplemented with 10 µM thiamine (EMM + T) to repress transcription from the nmt1 promoter. Note: You may use EMM + T for both transformation and expression experiments, if desired.

Medium Use for....

PDM leucine (L) Selecting pNMT-TOPO® transformants

EMM + 10 µM thiamine (T) Selecting pNMT-TOPO® transformants and starting cultures for expression experiments

EMM Inducing expression of the gene of interest from pNMT-TOPO®

Note that since PDM is a richer medium, S. pombe cells will grow faster in PDM than in EMM.

EMM Expression Medium

Expression of your recombinant fusion protein from the pNMT-TOPO® vectors in S. pombe requires a specialized medium, EMM Expression Medium. As mentioned above, EMM is a defined, minimal medium that does not contain thiamine. A powdered stock of EMM S. pombe Expression Medium is provided in the kit to allow you to prepare more than 7.5 liters of medium. The powder must be dissolved into deionized water prior to use. Instructions are provided on the next page to prepare liquid medium and agar plates. Additional EMM Expression Medium may be obtained separately from Invitrogen (see page ix for ordering information).


19


Preparation of EMM Expression Medium

Follow the instructions below to prepare 1 liter of EMM Expression Medium. If you need more or less medium, adjust the volumes accordingly. Note that the EMM powdered stock and liquid medium may be slightly yellow in color. 1. Dissolve 32 g of the powdered stock of EMM in 900 ml of deionized, distilled

water. Note: If you are making supplemented medium, add the appropriate amount of each amino acid. If you wish to add thiamine (T), use the 10 mM stock of thiamine included with the kit and add to a final concentration of 10 µM (e.g. 1 ml of a 10 mM stock solution to 1 liter of EMM).

2. Bring the volume up to 1 liter with deionized, distilled water. 3. Filter-sterilize. 4. Store at room temperature or at +4ºC. EMM Expression Medium may be stored at

room temperature for up to 1 month and at +4ºC for up to 6 months, if uncontaminated.

Note: We do not recommend autoclaving EMM Expression Medium. EMM contains glucose and autoclaving will cause the glucose to caramelize and the medium to darken in color.

Preparation of EMM Agar Plates

To prepare EMM agar plates, we generally prepare a 2X stock of EMM and dilute with melted agar prior to pouring plates. A procedure is provided below to prepare 1 liter of EMM containing agar. 1. Dissolve 32 g of the powdered stock of EMM in 400 ml of deionized, distilled water. 2. Bring the volume up to 500 ml with deionized, distilled water. 3. Filter-sterilize and store at room temperature until use. 4. To prepare agar plates, dilute the 2X EMM into an equal volume of a 4% sterile

solution of melted agar (e.g. 500 ml of 2X EMM plus 500 ml of 4% melted agar). 5. Allow the agar to cool to 50ºC.

Note: If you wish to add thiamine, use the 10 mM stock of thiamine included with the kit and add to a final concentration of 10 µM (e.g. 1 ml of a 10 mM stock solution to 1 liter of EMM).

6. Pour plates and allow to harden. Invert the plates and store at +4ºC. Plates are stable for up to 6 months, if uncontaminated.

Reagents for Yeast Transformation

Many protocols are suitable for preparing competent TCP1 S. pombe cells. The S. c. EasyComp Kit from Invitrogen (Catalog no. K5050-01) provides a quick and easy method for preparing competent yeast cells that can be used immediately. Transformation efficiency is guaranteed at greater than 103 transformants per µg DNA. Two small-scale S. pombe transformation protocols are included in the Appendix (see pages 31 and 32) for your convenience. You may use either the lithium acetate procedure or electroporation to transform your pNMT-TOPO® expression construct into the TCP1 S. pombe strain. In each case, transformation efficiency should be greater than 103 transformants per µg DNA.


20


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We have found that frozen competent S. pombe cells prepared with the S.c. EasyComp Kit transform poorly; therefore, we recommend using competent cells that have been freshly prepared for transformation.

Yeast Transformation

Use one of the methods described on the previous page (or one of your own choosing) to transform your pNMT-TOPO® expression construct into the TCP1 strain. We recommend that you include the appropriate control vector (see page 35) as a positive control for expression and a sample with no DNA as a negative control for transformation. Select for transformants on PDM L or EMM + T agar plates. Transformants should exhibit leucine prototrophy. One you have identified a transformant, be sure to purify the colony and make a glycerol stock for long-term storage.

Maintaining Transformants

We generally maintain S. pombe cells containing the pNMT-TOPO® construct in EMM + T. Remember that the medium must contain thiamine to repress transcription of your gene from pNMT-TOPO®.

21

Expression and Purification of the PCR Product

Introduction Once you have transformed the pNMT-TOPO® construct into S. pombe cells, you will

remove the thiamine from the growth medium to induce expression of the gene of interest. To induce expression of the gene of interest, S. pombe cells containing the pNMT-TOPO® construct are switched from EMM + T to EMM (medium lacking thiamine). To detect the protein encoded by your PCR product, you may use a functional assay or Western blot analysis if you have the appropriate antibodies (see below). If you have expressed your PCR product as a fusion to the C-terminal peptide, you may purify the recombinant fusion protein using metal ion chromatography. General guidelines are provided in this section for expression, detection, and purification. We recommend that you read through this section before beginning.

Materials to Have on Hand

You should have the following materials on hand before beginning: • EMM + T (see page 19 for a recipe) • EMM (see page 19 for a recipe) • Sterile 250 ml flasks • Table top centrifuge • Sterile microcentrifuge tubes • Microcentrifuge • 1X TE + 100 mM NaCl (see page 39 for a recipe)


22

Expression and Purification of the PCR Product, continued

Inducing Expression of Recombinant Protein

To induce expression of your protein of interest from pNMT1-TOPO®, pNMT41-TOPO®, and pNMT81-TOPO®, you will switch cells from EMM + T to EMM. Generally, recombinant protein may be detected in as little as 10 hours after removal of thiamine from the medium, with maximal expression levels achieved by 16 hours after removal of thiamine. Expression conditions may vary depending on the nature of your gene of interest, therefore, you may want to perform time course experiments to optimize expression conditions for your protein of interest. Follow the protocol below to induce expression of your recombinant protein. 1. Inoculate a single colony of TCP1 containing your pNMT1-TOPO®, pNMT41-

TOPO®, or pNMT81-TOPO® construct into 50 ml of EMM + T. Grow overnight at 30°C with shaking.

2. Pellet the cells at 1500 x g for 5 minutes at room temperature. Discard the supernatant.

3. To remove any residual thiamine, resuspend the cells in 50 ml of EMM and centrifuge at 1500 x g for 5 minutes at room temperature. Repeat the wash once more.

4. Resuspend the cells in 50 ml of EMM. Inoculate 500 µl aliquots of starter culture in parallel into 2 flasks of 100 ml EMM. Supplement one of the two experimental cultures with 10 µµM thiamine (this will represent the uninduced culture).

5. Incubate at 30°C with shaking for 18 hours. Collect cells for analysis. Note: If you are performing a time course experiment, you may harvest an aliquot of cells at various time points after inoculation into EMM (e.g. 0, 6, 12, 18, 24 hours after addition of cells to EMM).

6. Centrifuge the cells at 1500 x g for 5 minutes at +4°C. 7. Resuspend the cells in 10 ml of 1X TE + 100 mM NaCl. Centrifuge the cells at

1500 x g for 5 minutes at +4°C. 8. Resuspend the cell pellet in 1 ml of 1X TE + 100 mM NaCl. 9. Transfer cells to a sterile microcentrifuge tube. Centrifuge samples for 2 minutes at

top speed in the microcentrifuge. 10. Remove the supernatant. 11. Store the cell pellets at -80°C until ready to use. Proceed to the next page to prepare

cell lysates to detect your recombinant protein.

Detection of Recombinant Fusion Proteins

To detect expression of your recombinant fusion protein by western blot analysis, you may use the Anti-V5 Antibodies or the Anti-His(C-term) Antibodies available from Invitrogen (see page ix for ordering information) or an antibody to your protein of interest. In addition, the Positope Control Protein (Catalog no. R900-50) is available from Invitrogen for use as a positive control for detection of fusion proteins containing a V5 epitope or a polyhistidine (6xHis) tag. The ready-to-use WesternBreeze® Chromogenic Kits and WesternBreeze® Chemiluminescent Kits are available from Invitrogen to facilitate detection of antibodies by colorimetric or chemiluminescent methods. For more information, refer to our World Wide Web site (www.invitrogen.com) or call Technical Service (see page 40).



23


Preparing Cell Lysates

A general protocol for small-scale preparation of cell lysates using acid-washed glass bead breakage is provided below for your convenience. Other protocols are suitable. Materials Needed: • 1X TE + 100 mM NaCl (see page 39 for a recipe) • Acid-washed glass beads (0.4-0.6 mm size; Sigma, Catalog no. G8772) • Bead beater (Biospec Mini 8 Beadbeater; Biospec Products, Bartlesville, OK) • Sterile microcentrifuge tubes Protocol: 1. You may prepare cell lysates from either frozen cells or fresh cells. Resuspend the

cell pellets from Step 11, page 22 in 500 µl of 1X TE + 100 mM NaCl. 2. Add 400 µl of acid-washed glass beads. 3. Break cells at top speed for 45 seconds in a bead beater (e.g. Biospec Mini 8

Beadbeater). Place tubes on ice for 5 minutes. Repeat 5 times. 4. Centrifuge in a microcentrifuge for 2 minutes at maximum speed. 5. Remove supernatant and transfer to a fresh microcentrifuge tube. Assay the lysate for

protein concentration using BSA as a standard. 6. Store the extracts at -20°C until use. For long-term storage, store the extracts at -

80°C. 7. To perform SDS-PAGE analysis, remove the appropriate amount of extract and add

SDS-PAGE sample buffer to a final concentration of 1X. Boil the sample for 5 minutes, if needed.

8. Load samples onto an SDS-PAGE gel (see below) and electrophorese. Use the appropriate percentage of acrylamide to resolve your recombinant fusion protein.

The C-terminal peptide containing the V5 epitope and the polyhistidine region will add approximately 3.6 kDa to your protein.

Polyacrylamide Gel Electrophoresis

To facilitate separation of your recombinant fusion protein by polyacrylamide gel electrophoresis, a wide range of pre-cast NuPAGE® and Novex® Tris-Glycine polyacrylamide gels and electrophoresis apparatus are available from Invitrogen. The NuPAGE® Gel System avoids the protein modifications associated with Laemmli-type SDS-PAGE, ensuring optimal separation for protein analysis. In addition, Invitrogen also carries a large selection of molecular weight protein standards and staining kits to facilitate visualization of your recombinant protein. For more information about the appropriate gels, standards, and stains to use to visualize your recombinant protein, refer to our World Wide Web site (www.invitrogen.com) or call Technical Service (see page 40).



24


Scale-up of Expression for Purification

Once you have determined the optimal conditions for expression of your recombinant protein, you may scale-up expression to purify the recombinant fusion protein. If you plan to use ProBond resin to purify your recombinant fusion protein. see the Note below. If you are using another metal-chelating resin. refer to the manufacturers instructions to prepare the cells.

When purifying recombinant fusion proteins using ProBond resin, note that the largest culture volume that can be used with the 2 ml columns included in the ProBond Purification System is generally 50 ml of cells. If you need to purify recombinant fusion protein from larger culture volumes, you may need more ProBond resin (e.g. bulk ProBond resin). See page x for ordering information.

Purification of Recombinant Protein

For help with purification of your recombinant fusion protein, refer to the ProBond Purification System manual for details about sample preparation for chromatography. If you are using another type of resin, please refer to the manufacturers recommendations for sample preparation.

25

Appendix

pNMT TOPO® Control Reactions

Introduction If you have trouble obtaining transformants or vector containing insert, please perform

the following control reactions to help troubleshoot your experiment. Performing the control reactions involves producing a control PCR product containing the lac promoter and the LacZα fragment using the reagents included in the kit. Successful TOPO® Cloning of the control PCR product in either direction will yield blue colonies on LB agar plates containing antibiotic and X-gal.

Before Starting Be sure to prepare LB plates containing 50-100 µg/ml ampicillin and X-gal (see page 36

for recipe) before performing the control reaction:

Producing Control PCR Product

1. To produce the 500 bp control PCR product containing the lac promoter and LacZα, set up the following 50 µl PCR: Control DNA Template (50 ng) 1 µl 10X PCR Buffer 5 µl 50 mM dNTPs 0.5 µl Control PCR Primers (0.1 µg/µl each) 1 µl Sterile Water 41.5 µl Taq Polymerase (1 unit/µl) 1 µl Total Volume 50 µl

2. Overlay with 70 µl (1 drop) of mineral oil, if required. 3. Amplify using the following cycling parameters:

Step Time Temperature Cycles Initial Denaturation 2 minutes 94°C 1X Denaturation 1 minute 94°C Annealing 1 minute 60°C 25X Extension 1 minute 72°C Final Extension 7 minutes 72°C 1X

4. Remove 10 µl from the reaction and analyze by agarose gel electrophoresis. A discrete 500 bp band should be visible. Proceed to the Control TOPO® Cloning Reactions, next page.


26

pNMT TOPO® Control Reactions, continued

Control TOPO® Cloning Reactions

Using the control PCR product produced on the previous page and the TOPO® vector, set up two 6 µl TOPO® Cloning reactions as described below. 1. Set up control TOPO® Cloning reactions:

Reagent "Vector Only" "Vector + PCR Insert"Sterile Water 4 µl 3 µl Salt Solution or Dilute Salt Solution 1 µl 1 µl Control PCR Product -- 1 µl TOPO® vector 1 µl 1 µl

2. Incubate at room temperature for 5 minutes and place on ice. 3. Transform 2 µl of each reaction into separate vials of One Shot® TOP10 cells

(page 13). 4. Spread 25-100 µl of each transformation mix onto LB plates containing 50-100 µg/ml

ampicillin and X-Gal (see page 36). Be sure to plate two different volumes to ensure that at least one plate has well-spaced colonies. For plating small volumes, add 20 µl of SOC to allow even spreading.

5. Incubate overnight at 37°C.

Analysis of Results

Hundreds of colonies from the vector + PCR insert reaction should be produced. Greater than 85% of these will be blue. The vector only plate should yield very few colonies (

27

pNMT TOPO® Control Reactions, continued

Factors Affecting Cloning Efficiency

Note that lower cloning efficiencies will result from the following variables. Most of these are easily correctable, but if you are cloning large inserts, you may not obtain the expected 85% (or more) cloning efficiency.

Variable Solution

pH>9 in PCR amplification reaction Check the pH of the PCR amplification reaction and adjust with 1 M Tris-HCl, pH 8.

Incomplete extension during PCR Be sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need a longer extension time.

Cloning large inserts (>3 kb) Increase amount of insert. Or gel-purify as described on pages 28-29.

Excess (or overly dilute) PCR product Reduce (or concentrate) the amount of PCR product. Please note that you may add up to 4 µl of your PCR to the TOPO® Cloning reaction (page 12).

Cloning blunt-ended fragments Add 3´ A-overhangs by incubating with Taq polymerase (page 30).

PCR cloning artifacts ("false positives") TOPO® Cloning is very efficient for small fragments (< 100 bp) present in certain PCR reactions. Gel-purify your PCR product (page 28) or optimize your PCR. If your template DNA carries an ampicillin marker, carryover into the TOPO® Cloning reaction from the PCR may lead to false positives. Linearize the template DNA prior to PCR to eliminate carryover.

PCR product does not contain sufficient 3´ A-overhangs even though you used Taq polymerase

Taq polymerase is less efficient at adding a nontemplate 3´ A next to another A. Taq is most efficient at adding a nontemplate 3´ A next to a C. You may have to redesign your primers so that they contain a 5´ G instead of a 5´ T (Brownstein et al., 1996).

28

Purifying PCR Products

Introduction Smearing, multiple banding, primer-dimer artifacts, or large PCR products (>3 kb) may

necessitate gel purification. If you intend to purify your PCR product, be extremely careful to remove all sources of nuclease contamination. There are many protocols to isolate DNA fragments or remove oligonucleotides. Refer to Current Protocols in Molecular Biology, Unit 2.6 (Ausubel et al., 1994) for the most common protocols. Three simple protocols are provided below.

Note that cloning efficiency may decrease with purification of the PCR product. You may wish to optimize your PCR to produce a single band (see Producing PCR Products, page 10).

Using the S.N.A.P. Gel Purification Kit

The S.N.A.P. Gel Purification Kit (Catalog no. K1999-25) allows you to rapidly purify PCR products from regular agarose gels. 1. Electrophorese amplification reaction on a 1 to 5% regular TAE agarose gel. Note:

Do not use TBE to prepare agarose gels. Borate interferes with the NaI step, below. 2. Cut out the gel slice containing the PCR product and melt it at 65°C in 2 volumes of

6 M NaI. 3. Add 1.5 volumes Binding Buffer. 4. Load solution (no more than 1 ml at a time) from Step 3 onto a S.N.A.P. column.

Centrifuge 1 minute at 3000 x g in a microcentrifuge and discard the flow-through. 5. If you have solution remaining from Step 3, repeat Step 4. 6. Add 900 µl of the Final Wash Buffer. 7. Centrifuge 1 minute at 3000 x g in a microcentrifuge and discard the flow-through. 8. Elute the purified PCR product in 40 µl of TE or sterile water. Use 4 µl for the

TOPO® Cloning reaction and proceed as described on page 12.

Quick S.N.A.P. Method

An even easier method is to simply cut out the gel slice containing your PCR product, place it on top of the S.N.A.P. column bed, and centrifuge at full speed for 10 seconds. Use 1-2 µl of the flow-through in the TOPO® Cloning reaction (page 12). Be sure to make the gel slice as small as possible for best results.


29

Purifying PCR Products, continued

Low-Melt Agarose Method

If you prefer to use low-melt agarose, use the procedure below. Note that gel purification will result in a dilution of your PCR product and a potential loss of cloning efficiency. 1. Electrophorese as much as possible of your PCR reaction on a low-melt agarose gel

(0.8 to 1.2%) in TAE buffer. 2. Visualize the band of interest and excise the band. 3. Place the gel slice in a microcentrifuge tube and incubate the tube at 65°C until the

gel slice melts. 4. Place the tube at 37°C to keep the agarose melted. 5. Add 4 µl of the melted agarose containing your PCR product to the TOPO® Cloning

reaction as described on page 12. 6. Incubate the TOPO® Cloning reaction at 37°C for 5 to 10 minutes. This is to keep

the agarose melted. 7. Transform 2 to 4 µl directly into chemically competent One Shot® TOP10 cells using

the method on page 13.

30

Addition of 3´ A-Overhangs Post-Amplification

Introduction Direct cloning of DNA amplified by Vent® or Pfu polymerases into TOPO® Cloning

vectors is often difficult because of very low cloning efficiencies. These low efficiencies are caused by the 3´ to 5´ exonuclease activity, which removes the 3´ A-overhangs necessary for TOPO® Cloning. Invitrogen has developed a simple method to clone these blunt-ended fragments.

Before Starting You will need the following items:

• Taq polymerase • A heat block equilibrated to 72°C • Phenol-chloroform (optional) • 3 M sodium acetate (optional) • 100% ethanol (optional) • 80% ethanol (optional) • TE buffer (optional)

Procedure This is just one method for adding 3´ adenines. Other protocols may be suitable.

1. After amplification with Vent® or Pfu polymerase, place vials on ice and add 0.7-1 unit of Taq polymerase per tube. Mix well. It is not necessary to change the buffer.

2. Incubate at 72°C for 8-10 minutes (do not cycle). 3. Place the vials on ice. The DNA amplification product is now ready for ligation into

the appropriate pNMT-TOPO® vector. Note: If you plan to store your sample(s) overnight before proceeding with TOPO® Cloning, you may want to extract your sample(s) with phenol-chloroform to remove the polymerases. After phenol-chloroform extraction, precipitate the DNA with ethanol and resuspend the DNA in TE buffer to the starting volume of the amplification reaction.

You may also gel-purify your PCR product after amplification with Vent® or Pfu (see previous page). After purification, add Taq polymerase buffer, dATP, and 0.5 unit of Taq polymerase and incubate 10-15 minutes at 72°C. Use 4 µl in the TOPO® Cloning reaction.

Vent® is a registered trademark of New England Biolabs.

31

Lithium Acetate Transformation Protocol

Introduction A small-scale protocol to transform S. pombe cells using lithium acetate is provided

below. The protocol has been modified from a protocol originally described by Okazaki et al., 1990. Other protocols are suitable.

Materials Needed Be sure to have the following reagents on hand before starting.

• EMM + LT liquid medium (see recipe, page 19) • 0.5X YPD • 1X TE (see recipe, page 38) • 100 mM LiAc, pH 4.9 (see recipe, page 39) • 100 mM LiAc/50% PEG-3350 (see recipe, page 39) • pNMT-TOPO® vector construct (or other plasmid DNA to be transformed) • PDM L or EMM + T selective plates and medium

Protocol 1. Inoculate 10 ml of EMM + LT medium with a colony of your S. pombe strain and

shake overnight at 30°C. 2. Determine the OD600 of your overnight culture. Dilute culture to an OD600 of 0.4 in

50 ml of EMM + LT medium and grow an additional 2-4 hours. 3. Pellet the cells at 1500 x g for 5 minutes at +4°C. Wash the cells once with 0.5

culture volumes of 100 mM LiAc. 4. Pellet the cells at 1500 x g for 5 minutes at +4°C. Estimate the cell pellet volume and

resuspend the cells in 4X pellet volume of 100 mM LiAc. 5. Incubate the cells at room temperature for 60-120 minutes. 6. For each transformation, mix 1 µg of plasmid DNA with 100 µl of cell suspension

from Step 5. 7. Add 290 µl of 100 mM LiAc/50% PEG-3350 and mix gently. 8. Incubate the DNA/cell solution at room temperature for 60 minutes. 9. Heat shock for 15 minutes at 42°C. 10. Centrifuge in a microcentrifuge for 10 seconds and remove supernatant. 11. Resuspend the cell pellet in 10 ml of 0.5X YPD and incubate with shaking at 30°C

for 60-90 minutes. 12. Pellet the cells at 1500 x g for 5 minutes at +4°C. Wash the cells once with 5 ml of

selective medium (PDM L or EMM + T) and re-pellet. 13. Resuspend the cell pellet in 50-100 µl of selective medium and plate on the

appropriate selective plate (PDM L or EMM + T).

32

Electroporation Transformation Protocol

Introduction A protocol to transform S. pombe cells by electroporation is provided below. For more

details, please refer to the original published reference (Prentice, 1992). Other protocols are suitable.

Materials Needed Be sure to have the following reagents on hand before starting.

• EMM + LT liquid medium (see recipe, page 19) • 1 M sorbitol (see recipe, page 39) • 0.2 cm electroporation cuvettes • Electroporator • pNMT-TOPO® vector construct (or other plasmid DNA to be transformed) • PDM L or EMM + T selective plates and medium

Protocol 1. Inoculate 10 ml of EMM + LT medium with a colony of your S. pombe strain and

shake overnight at 30°C. 2. Determine the OD600 of your overnight culture. Dilute culture to an OD600 of 0.4 in

50 ml of EMM + LT medium and grow an additional 2-4 hours. 3. Pellet the cells at 1500 x g for 5 minutes at +4°C and wash the cells once with 0.5

culture volumes of ice-cold sterile water. 4. Pellet the cells at 1500 x g for 5 minutes at +4°C and wash the cells twice with 0.5

culture volumes each of ice-cold 1 M sorbitol. 5. Pellet the cells at 1500 x g for 5 minutes at +4°C. Estimate the cell pellet volume and

resuspend the cells in 4X pellet volume of ice-cold 1 M sorbitol. 6. For each transformation, transfer 100 µl of cell suspension from Step 5 to a pre-

chilled 0.2 cm electroporation cuvette. Add 1 µg of plasmid DNA and vortex to mix. 7. Incubate the DNA/cell solution on ice for 5-10 minutes. 8. Vortex the cells and electroporate the sample using your own protocol and your

electroporator. The settings used to electroporate S. cerevisiae cells are generally suitable for S. pombe.

9. Immediately after pulsing, remove the cell suspension to a pre-chilled 15 ml conical tube. Use 1 ml of ice-cold 1 M sorbitol to rinse the cuvette and transfer to the same pre-chilled, conical tube.

10. Remove a 50-100 µl aliquot and plate on the appropriate selective plate (PDM L or EMM + T).

33

pNMT-TOPO® Vectors

Map The figure below summarizes the features of the pNMT1-TOPO®, pNMT41-TOPO®,

and pNMT81-TOPO® vectors. Note that, except for the differences in the TATA box region, all other sequence among the three vectors is identical. Each vector is supplied linearized at the TOPO® Cloning site. The complete sequences of pNMT1-TOPO®, pNMT41-TOPO®, and pNMT81-TOPO® are available for downloading from our Web site (www.invitrogen.com) or from Technical Service (see page 40).

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34

pNMT-TOPO® Vectors, continued

Features of the pNMT-TOPO® Vectors

pNMT1-TOPO®, pNMT41-TOPO®, and pNMT81-TOPO® are 6.1 kb vectors that inducibly express your gene of interest under the control of the wild-type S. pombe nmt1 promoter, a four bp TATA box deletion mutant nmt1 promoter, and a seven bp TATA box deletion mutant nmt1 promoter, respectively. The table below describes the relevant features of the pNMT-TOPO® vectors. All features have been functionally tested.

Feature Benefit S. pombe nmt1 promoter Permits high-level, thiamine-regulated expression of

your gene of interest (Maundrell, 1990) TATA box region of the nmt1 promoter • wild-type (pNMT1-TOPO®) • 4 bp deletion (pNMT41-TOPO®) • 7 bp deletion (pNMT81-TOPO®)

Alters transcriptional efficiency of the nmt1 promoter to allow varying levels of thiamine-regulated gene expression (Basi et al., 1993)

NMT Forward priming site Allows sequencing in the sense orientation TOPO® Cloning site Allows insertion of your PCR product V5 epitope (Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr)

Permits detection of your recombinant protein with the Anti-V5 Antibodies available from Invitrogen (Southern et al., 1991)

Polyhistidine (6xHis) tag Permits purification of your recombinant protein on metal-chelating resin such as ProBond In addition, the C-terminal 6xHis tag is the epitope for the Anti-His(C-term) antibodies available from Invitrogen (Lindner et al., 1997)

S. pombe ura4 transcription termination sequence

Permits efficient transcription termination and poly-adenylation of mRNA

URA4 Reverse priming site Permits sequencing of the non-coding strand S. pombe ars1 origin Allows high-copy, non-integrative replication and

maintenance in S. pombe (Heyer et al., 1986) SV40 early promoter Allows expression of the LEU2 gene S. cerevisiae LEU2 gene Permits auxotrophic selection of the plasmid in leu1

yeast hosts (Andreadis et al., 1984) nmt1/2 transcription termination sequence

Allows efficient transcription termination and polyadenylation of mRNA

Ampicillin resistance gene (β-lactamase)

Permits selection of transformants in E. coli

pUC origin Allows high-copy number replication and growth in E. coli

35

pNMT/CAT Vectors

Description pNMT1/CAT, pNMT41/CAT, and pNMT81/CAT are 6.7 kb control vectors expressing

chloramphenicol acetyltransferase (CAT). The CAT gene was PCR-amplified and TOPO® Cloned into pNMT1-TOPO®, pNMT41-TOPO®, or pNMT81-TOPO® in frame with the C-terminal peptide containing the V5 epitope and the polyhistidine (6xHis) tag. The CAT protein expressed from each pNMT/CAT vector is approximately 32 kDa in size.

Map The figure below summarizes the features of the pNMT/CAT vectors. The complete

nucleotide sequence for pNMT1/CAT, pNMT41/CAT, and pNMT81/CAT are available for downloading from our World Wide Web site (www.invitrogen.com) or by contacting Technical Service (see page 40).

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Documents

SpECTRAŽ S. pombe Expression Systemtools.thermofisher.com/.../sfs/manuals/spectrasystem_man.pdfS. pombe host strain, expression of the PCR product can be regulated using thiamine