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Eric R.  Geertsma

Bert Poolman

High-throughput cloning and expression in _Lactococcus lactis_

We have developed a generic method for high-throughput cloning in bacteria less amenable to conventional DNA manipulations. The method employs ligation-independent cloning in an intermediary _Escherichia coli_ vector, which is rapidly converted via vector backbone exchange \(VBEx) into a bona fide, organism-specific plasmid ready for high-efficiency transformation. Here, we describe the VBEx procedure for _Lactococcus lactis_.


The procedure will yield _L. lactis_ expression vectors from which the gene of interest can be expressed with a cleavable decaHis-tag or alternative tags at either the N- or C-terminus. Simultaneously, _E. coli_ expression vectors holding similar tags can be created.

Plasmid isolation kit \(e.g., Wizard&#xAE; Plus SV Minipreps DNA Purification System, Promega)


T10E0.2 \(10 mM Tris, 0.2 mM EDTA, pH 7.5)


Na-acetate, 3 M, pH 5.3


MilliQ


Ethanol, 96%, ice-cold  


Ethanol, 70%, ice-cold  


Phenol:chloroform \(50/50 v/v)


Chloroform


Tubes \(1.5 ml, PCR tubes)


DpnI \(Roche)


SwaI + 10x SwaI restriction buffer \(Roche), or its isoschizomer SmiI + restriction buffer \(Fermentas). 


Note: digestion with SmiI is done at 30&#xB0;C and not at 25&#xB0;C.


SfiI + 10x SfiI restriction buffer \(Fermentas)


Phusion DNA polymerase + 5x Phusion HF buffer and 5x Phusion GC buffer \(Finnzymes)


dNTP solution \(10 mM each; Roche)


Primers \(desalted, HPLC purification is not necessarily)


TAE, 1x buffer: 980 ml milliQ + 20 ml 50X TAE buffer 


TAE, 50x buffer: 242 gr Tris-base, 57.1 ml acetic acid, 100 ml 0.5 M EDTA, pH 8, add water to 1000 ml


Agarose


Gel purification kit \(e.g., GFX PCR DNA &amp; Gel Band Purification Kit, GE)


dCTP, 25 mM


dGTP, 25 mM


T4 DNA polymerase + 5x T4 DNA polymerase buffer \(Roche)


T4 DNA ligase + 10x T4 DNA ligase buffer \(Roche)


Na2-ATP, 150 mM, pH 7 \(set pH with NaOH)


E. coli MC1061, calcium-competent


L. lactis NZ9000, electrocompetent \(see protocol below)


Luria Broth \(LB), LB agar \(1.5%)


M17, M17 agar \(1.5%) \(Difco)


Glucose, 20% \(w/v)


Sucrose, 2M


Ampicillin, 100 mg/ml in milliQ


Chloramphenicol, 5 mg/ml in EtOH


pRExLIC vectors or derivatives \(see Table 1)


pERL plasmid

Table centrifuge


Small-volume spectrophotometer \(Nanodrop, NanoDrop Technologies)


Freezer \(-80&#xB0;C)

**Step 1**. 


Purify the pRExLIC vector from an E. coli culture using a plasmid isolation kit. Take 20 &#x3BC;g plasmid or more and adjust the volume to 300 &#x3BC;l with milliQ. Add 300 &#x3BC;l phenol:chloroform and vortex the sample until an emulsion forms. Wait for 2 min and vortex again. Centrifuge the tube for 2 min at 16,100g and transfer the upper aqueous phase to a fresh tube without disturbing the interface. Add an equal volume of chloroform to the aqueous phase, vortex and centrifuge for 2 min at 16,100g. Transfer the upper aqueous phase to a fresh tube. Add 0.1 volume 3 M Na-acetate, pH 5.3, and 2.5 volumes ice-cold 96% ethanol and then vortex the sample. Store the sample at -80&#xB0;C for 30 min in a cooling block or use dry ice. Centrifuge for 15 min at 16,100g at 4&#xB0;C. Remove the supernatant without disturbing the pellet and slowly add 1 ml ice-cold 70% ethanol. Centrifuge for 5 min at 16,100g at room temperature. Remove the supernatant without disturbing the pellet and speedvac for 5 min to remove the last traces of ethanol. Take care not to dry the DNA too long \(5 min maximally). Dissolve the DNA pellet in 30 &#x3BC;l T10E0.2 by extensively rinsing the walls of the tube with buffer. Determine the DNA concentration and purity using a spectrophotometer. A 260/280 ratio close to 1.8 indicates a pure DNA sample. The purified plasmid can be stored at 4&#xB0;C.


The pERL plasmid is isolated from a L. lactis culture using similar procedures as described above, with the following modification: Preceding the cell lysis, cells are resuspended in 10 mM Tris-HCl, pH 8.1, 20% \(w/v) sucrose, 10 mM EDTA, 50 mM NaCl plus 10 mg/ml lysozyme and incubated for 15 min at 50&#xB0;C.





**Step 2**.


Set up a digestion with 5 &#x3BC;g of the purified pRExLIC vector, 2.5 &#x3BC;l \(25U) SwaI, 5 &#x3BC;l 10x SwaI-buffer and adjust the volume to 50 &#x3BC;l. Incubate overnight at 25&#xB0;C in an incubator. Next, analyse the full sample on a TAE-gel using a comb with wide wells \(~10 mm). Recover the linearized vector using a gel purification kit, elute in 50 &#x3BC;l T10E0.2. Remove residual agarose and column material by centrifugation for 5 min at 16,100g and transfer the supernatant to a fresh cup. Determine the DNA concentration and purity using a spectrophotometer. A 260/280 ratio close to 1.8 indicates a pure DNA sample. The SwaI-digested vector is stored at 4&#xB0;C.





**Step 3**.


Take 200 ng of SwaI-digested vector and adjust the volume to 10 &#x3BC;l with milliQ. Add 1.5 &#x3BC;l 25 mM dCTP and 3 &#x3BC;l 5x T4 DNA polymerase buffer. Add 0.5 &#x3BC;l \(0.5U) T4 DNA polymerase, vortex shortly and incubate the sample at 20&#xB0;C for 30 min. Heat inactivate the T4 DNA polymerase by incubating at 75&#xB0;C for 20 min. The material can be stored at 4&#xB0;C for several months.





**Step 4**.


Design gene-specific primers extended at the 5&#x2019; side with LIC-specific extensions tails \(see Table 2). Do not include the start and stop codons in the gene-specific part of the primer, these are already present in the LIC specific tails. The annealing temperature of the gene-specific part should be sufficiently high \(preferably above 55&#xB0;C) to allow specific amplification by PCR. Furthermore, the primer should be without hairpin loops that could interfere with the PCR. No modifications to the LIC-specific extensions should be made. Total primer length should be kept below 50 residues to decrease synthesis-costs and assure a sufficient quality.





**Step 5**.


Amplify the gene of interest from genomic or plasmid DNA using Phusion DNA polymerase \(Finnzymes). Set up a 25 &#x3BC;l reaction with 200 &#x3BC;M of each dNTP, 0.5 &#x3BC;M of each primer, 5 &#x3BC;l 5x Phusion HF buffer, template DNA \(0.1-10 ng for plasmid DNA, 25-250 ng for genomic DNA), 0.25 &#x3BC;l \(0.5 U) Phusion DNA polymerase. Pre-heat the block of the PCR machine to 98&#xB0;C before adding the Phusion DNA polymerase to the mixture and placing the cup in the PCR machine.


Cycle as follows: 30 sec at 98&#xB0;C; 15 cycles of 10 sec at 98&#xB0;C, 20 sec at 60&#xB0;C, 15 or 30 sec per kb for plasmid or genomic DNA, respectively. Decrease the annealing temperature every cycle by 0.5&#xB0;C. Next, perform 15 cycles of 10 sec at 98&#xB0;C, 20 sec at 52.5&#xB0;C, 15 or 30 sec per kb for plasmid or genomic DNA, respectively; end with a final extension of 5 min at 72&#xB0;C and hold the sample at 4&#xB0;C. 


If plasmid DNA isolated from dam+ hosts is used as template, it is recommended to add 0.5 &#x3BC;l \(5U) DpnI to the sample and incubate 30 min at 37&#xB0;C. DpnI will digest Dam-methylated template and thereby reduce the number of background-colonies after transformation. Note: not all hosts perform Dam-methylation.





**Step 6**.


Analyse the full sample on a TAE-gel and recover the PCR-product using a gel purification kit, elute the DNA in 30 &#x3BC;l T10E0.2. Remove residual agarose and column material by centrifugation for 5 min at 16,100g and transfer the supernatant to a fresh cup. Determine the concentration and purity using a spectrophotometer. The sample is stored at 4&#xB0;C.





**Step 7**.


Take the molar equivalent to 200 ng vector of insert. The amount of insert to be used can be calculated as follows: ng insert = \(200 ng * insert size in bp) / vector size in bp. Adjust the volume to 10 &#x3BC;l with milliQ. Add 1.5 &#x3BC;l 25 mM dGTP and 3 &#x3BC;l 5x T4 buffer. Add 0.5 &#x3BC;l \(0.5 U) T4 DNA polymerase, vortex shortly and incubate the sample at 20&#xB0;C for 30 min. Heat inactivate the T4 DNA polymerase by incubating at 75&#xB0;C for 20 min. The material can be stored at 4&#xB0;C for several months.





**Step 8**.


Add 1 &#x3BC;l of vector \(step 3) to 3 &#x3BC;l of insert \(step 7) and incubate for 5 min at room temperature. Transform the material to ~75 &#x3BC;l chemically competent _E. coli_ MC1061 according to standard protocols. Plate 0.1 and 0.9 volume of transformed cells on LB agar supplemented with 100 &#x3BC;g/ml ampicillin and incubate overnight at 37&#xB0;C. Analyse the colonies by colony PCR and isolate plasmid using a plasmid isolation kit. No additional purification of the plasmid is needed. Sequence the insert. 


Note: inserts amplified with primers with nLIC-extensions \(Table 1) are compatible with all plasmids holding an nLIC-cassette \(Table 2). For example, using one nLIC-insert, both plasmids pREnLIC-insert and pBADnLIC-insert can be created. The pBAD-derived vectors can immediately be used for expression analysis in E. coli using the AraC/PBAD expression-system \(Guzman _et al_, 1995) and should not be submitted to step 9. Similar, all inserts amplified with primers with cLIC-extensions \(Table 1) are compatible with all plasmids holding a cLIC-cassette \(that are: pREcLIC, pREcLIC-GFP, pBADcLIC, pBADcLIC-GFP; Table 2). The preparation of pBADxLIC vectors is identical to the preparation of the pRExLIC vectors described in step 1-3.





**Step 9**.


Mix approximately 125 ng of the pERL plasmid \(isolated in step 1) and approximately 125 ng of a pRExLIC-derived vector containing the insert \(isolated in step 8). Adjust the volume to 10 &#x3BC;l by adding 1 &#x3BC;l 10x SfiI-buffer, 0.5 &#x3BC;l \(5U) SfiI \(Fermentas) and sufficient milliQ. Use a PCR machine with heated lid to incubate the sample for 80 min at 50&#xB0;C and 20 min at 80&#xB0;C to inactivate SfiI. After cooling to room temperature, start the ligation by the addition of 1.5 &#x3BC;l 8 mM Na2-ATP, pH 7, and 0.5 &#x3BC;l \(0.5U) T4 DNA ligase. Incubate the sample for 1 hr at 20&#xB0;C and, subsequently, 20 min at 65&#xB0;C to heat inactivate the T4 DNA ligase. Transform 2 &#x3BC;l of sample to 30 &#x3BC;l electrocompetent L. lactis NZ9000 \(see protocol below) and plate aliquots on M17 plates supplemented with 0.5% \(w/v) glucose, 0.5 M sucrose and 5 &#x3BC;g/ml chloramphenicol. Seal the plates with parafilm and incubate at 30&#xB0;C until colonies appear \(~18 hrs).


The pNZxLIC vectors derived in this step can immediately be used for expression analysis in L. lactis using the nisin-controlled expression-system \(for details, see Kunji _et al_, 2003 and de Ruyter _et al_, 1996)








**Electrotransformation of L. lactis**. 


Preparation of electrocompetent L. lactis NZ9000 was essentially done as described \(Holo and Nes, 1989; Wells _et al_, 1993), but with some critical modifications. Briefly: Streak a frozen stock of L. lactis NZ9000 on M17 agar supplemented with 0.5% \(w/v) glucose and incubate overnight at 30&#xB0;C. Use a single colony to inoculate 5 ml M17 supplemented with 0.5% \(w/v) glucose. Grow at 30&#xB0;C for 6h. Use this culture to inoculate 50 ml M17 supplemented with 0.5% \(w/v) glucose plus 1% \(w/v) glycine and incubate overnight. Note: glycine can be autoclaved with the medium, sucrose and glucose need to be added seperately. Use the 50 ml culture to inoculate 400 ml M17 supplemented with 0.5% \(w/v) glucose, 0.5 M sucrose and 2% \(w/v) glycine and continue cultivation until OD600 = 0.5. Harvest the cells by centrifugation at 5000g for 15 min at 4&#xB0;C. Wash the cells with 1 volume ice-cold solution A \(0.5 M sucrose and 10% \(v/v) glycerol, prepared in milliQ) and centrifuge at 5000g for 15 min at 4&#xB0;C. Next, resuspend the cells in 0.5 volume ice-cold solution A supplemented with 50 mM Na-EDTA, pH 7.5, and 0.25 volume solution A, and incubate for 15 min on ice before centrifugation at 5000g for 15 min at 4&#xB0;C. Finally, resuspend the cells in 0.01 volume ice-cold solution A. Aliquots of 40 &#x3BC;l are flash-frozen in liquid nitrogen and stored at -80&#xB0;C until use.


 For electroporation, thaw cells on ice, combine them with the plasmid DNA, and transfer the sample to an ice-cooled electroporation cuvet \(2 mm electrode gap). Expose cells to a single electrical pulse with a field strength of 2 kV, capacitance of 25 &#x3BC;F and resistance of 200 Ohm. Immediately following discharge, mix the cells with 1 ml ice-cold M17 supplemented with 0.5% \(w/v) glucose, 0.5 M sucrose, 20 mM MgCl2 plus 2 mM CaCl2, and leave them on ice for 10 min. Subsequently, incubate the cells at 30&#xB0;C for 2 hrs and plate aliquots on M17 agar supplemented with 0.5% \(w/v) glucose, 0.5 M sucrose and 5 &#x3BC;g/ml chloramphenicol. Seal the plates with parafilm and incubate overnight at 30&#xB0;C.

The full procedure can be performed in 4 days.

**Step 1**. Although the additional phenol:chloroform purification step might seem over-cautious, we find this step essential for high-efficiency digestion with SwaI.





**Step 3**. More extensive treatment with T4 DNA polymerase resulting from higher incubation temperatures, prolonged incubation or higher concentrations of T4 DNA polymerase will decrease the efficiency of the procedure dramatically.





**Step 7**. More extensive treatment with T4 DNA polymerase resulting from higher incubation temperatures, prolonged incubation or higher concentrations of T4 DNA polymerase will decrease the efficiency of the procedure dramatically.

**Step 6**. If the PCR was not successful, we refer to the manufacturer&#x2019;s protocol for optimization. For complex DNA \(e.g., genomic DNA or GC-rich DNA), the use of 5x Phusion GC buffer should be considered.

The ligation-independent cloning procedure described should yield transformation efficiencies of approximately 10,000 CFU/&#x3BC;g DNA of which at least 90% will contain the insert. The VBEx procedure will yield transformation efficiencies of approximately 1,000,000 CFU/&#x3BC;g DNA; all the L. lactis transformants will contain the correct insert.

L. M. Guzman, D. Belin, M. J. Carson _et al_., _J Bacteriol_ **177** \(14), 4121 \(1995).





H. Holo and I. F. Nes, _Appl Environ Microbiol_ **55** \(12), 3119 \(1989).





J. M. Wells, P. W. Wilson, and R. W. Le Page, _J Appl Bacteriol_ **74** \(6), 629 \(1993)





E. R. Kunji, D. J. Slotboom, and B. Poolman, _Biochim Biophys Acta_ **1610** \(1), 97 \(2003)





P. G. de Ruyter, O. P. Kuipers, and W. M. de Vos, _Appl Environ Microbiol_ **62** \(10), 3662 \(1996).

Method Article

Eric R. Geertsma, Bert Poolman

Eric R. Geertsma

University of Groningen, Biochemistry department

Bert Poolman

University of Groningen, Biochemistry department

DOI:

10.1038/nprot.2007.290

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High-throughput cloning and expression in recalcitrant bacteria

by Eric R Geertsma & Bert Poolman

Nature (04 July, 2007)

Method Article

Eric R. Geertsma, Bert Poolman

Eric R. Geertsma

University of Groningen, Biochemistry department

Bert Poolman

University of Groningen, Biochemistry department

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DOI:

10.1038/nprot.2007.290

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Associated Publications

High-throughput cloning and expression in recalcitrant bacteria

by Eric R Geertsma & Bert Poolman

Nature (04 July, 2007)

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