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Step 1: Cloning the Target Gene into pNZ8048 and Transformation into L. lactis

Step 1: Cloning the Target Gene into pNZ8048 and Transformation into L. lactis

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90



Martin S. King et al.



Table 3 The PCR program

Step



Time



Temp. (°C)



Initial denaturation/activation



2 min



95



Denaturation



20 s



95



AnnealingNB1



20 s



60



40 s



70



10 min



70



Elongation



NB2



Final elongation



We use 30 cycles of denaturation, annealing, and elongation.

NB1

Annealing temperature chosen according to the lowest Tm of the primers and the kit manufacturers

recommendation.

NB2

Length of elongation was chosen according to the size of the expected PCR product and kit

manufacturers recommendation.



3. Follow the Qiagen PCR purification kit manual; elute plasmid with

30 μL EB buffer prewarmed to 60 °C for 5 min

4. Run a 1% agarose gel to confirm PCR amplification has been successful

using standard protocols

Restriction digestion

1. Set up the following restriction digestion reaction of the PCR-amplified

gene product (Table 4)

2. Incubate at 37 °C for 90 min to ensure complete digestion of the insert

(this digestion reaction can be left overnight without any observable star

activity), and inactivate the restriction enzymes by incubation at 80 °C

for 10 min



Table 4 Reaction mixture for restriction digests of the PCR-amplified gene insert

Reagent

Volumes (μL)



CutSmart buffer

DpnI-treated PCR product



5

30



NcoI (10,000 μ/mL)



2



XbaI (20,000 μ/mL)



1



Ultrapure water



12



Final volume



50



Membrane Protein Expression in Lactococcus lactis



91



3. Follow the Qiagen PCR purification kit manual; elute plasmid with

30 μL EB buffer prewarmed to 60 °C for 5 min, and determine the

DNA concentration using the nanodrop. Dilute the digested PCRamplified gene insert to 50 ng/μL with buffer EB

Preparation of pNZ vector

1. Inoculate 100 mL M17 (supplemented with 5 μg/mL chloramphenicol

and 1% glucose) with L. lactis transformed with the pNZ8048 vector;

grow overnight at 30 °C without shaking

2. Centrifuge (4000 Â g, 10 min, 4 °C) and discard the supernatant

3. Dissolve lysozyme (to 10 mg/mL) in buffer P1, and resuspend the pellet;

heat at 50 °C for 20 min, removing the tubes and vortexing them every

5 min to ensure a homogenous solution; isolate plasmid DNA using the

miniprep kit (following the manufacturer’s instructions)

Restriction digestion

1. Set up the following restriction digestion reaction of the pNZ vector

(Table 5)

2. Incubate at 37 °C for 90 min to ensure complete digestion of the inserts

(this digestion reaction can be left overnight without any observable star

activity), and inactivate the restriction enzymes by incubation at 80 °C

for 10 min

3. Run the digested pNZ8048 on a 1% agarose gel following standard protocols; excise the top band, which represents the digested plasmid, with a

sharp scalpel. Follow the QIAquick gel extraction kit protocol; elute

plasmid with 30 μL EB buffer prewarmed to 60 °C for 5 min, and determine the DNA concentration using the nanodrop. Dilute the digested

plasmid DNA to 50 ng/μL with buffer EB

Table 5 Reaction mixture for restriction digest of the vector

Reagent



CutSmart buffer

pNZ8048 plasmid



Volumes (μL)



5

30



NcoI (10,000 μ/mL)



2



XbaI (20,000 μ/mL)



1



Ultrapure water



12



Final volume



50



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Martin S. King et al.



Table 6 Reaction mixture for the ligation of the carrier genes into yeast vectors

Reagent

Volumes



T4 DNA ligase buffer



2 μL



Vector (for 4000 bp vector)



50 ng DNA (% 1 μL)



Insert (for 1000 bp insert)



50 ng DNA (% 1 μL)



T4 DNA ligase



1 μL



Ultrapure water



15 μL



Final volume



20 μL



The calculations shown are for a gene product of 1000 bp; the amount of insert DNA needed is calculated

from Eq. 1.



Ligation and pellet paint coprecipitation

1. Set up the ligation reaction at a 3:1 insert:vector molar ratio; use 50 ng of

vector and calculate the amount of insert using Eq. (1) and Table 6:

Amount of insert ngị ẳ 3 insert length=vector lengthị

amount of vector ðngÞ



(1)



2. Incubate at room temperature for 1 h; inactivate the enzyme by incubation at 65 °C for 10 min

3. Add 3 μL pellet paint to each ligation mix, followed by 0.1 vol. 3 M

sodium acetate, pH 5.2 (typically 2.5 μL)

4. Add 2 vol. of 100% ethanol (typically 45 μL), vortex briefly, and incubate at room temperature for 5 min

5. Centrifuge (14,000 Â g, 10 min, room temperature); a small pink pellet

should be visible

6. Carefully remove the supernatant; add 300 μL 70% ethanol, vortex

briefly, and centrifuge (14,000 Â g, 10 min, room temperature)

7. Carefully remove the supernatant; add 300 μL 100% ethanol, vortex

briefly, and centrifuge (14,000 Â g, 10 min, room temperature)

8. Carefully remove the supernatant and dry at 90 °C for 20 min

9. Resuspend the pellet in 10 μL ultrapure sterile water; store at À20 °C for

long-term storage, or place on ice for transformation

Preparation of electrocompetent L. lactis

1. Inoculate L. lactis strain NZ9000 from a frozen glycerol stock into 50 mL

SM17 media supplemented with 0.8% glucose and 1.5% glycine; incubate overnight at 30 °C without aeration

2. Add the overnight culture to 1 L SM17 media supplemented with 0.8%

glucose and 1.5% glycine to give an initial starting OD600 of 0.1, and



Membrane Protein Expression in Lactococcus lactis



93



incubate at 30 °C; measure the OD600 every hour until it reaches 0.5–0.7

(this should take about 4 h)

3. Centrifuge (7500 Â g, 10 min, 4 °C); discard the supernatant, and

resuspend gently the pellet in 150 mL wash solution

4. Repeat the wash step three times in total. Resuspend the cells to a final

volume of 3 mL and aliquot into 110 μL portions; store at À80 °C

Transformation into electrocompetent L. lactis cells

1. Prechill electrophoretic cuvettes on ice; thaw competent cells

2. Transfer 50 μL of cells into the cuvette; add the entire ligation product

(after the pellet paint precipitation) and mix gently by pipetting

3. Prepare 2-mL sterile Eppendorf tubes with 2 mL recovery media

4. On the GenePulser II

– Voltage ¼ 2.5 kV

– Capacitance ¼ 25 μF

– Low range resistance ¼ 200 Ω

– High range resistance ¼ infinite

5. Place the prepared electrophoretic cuvettes into the electroporation

chamber and pulse the cells; immediately add 1 mL L. lactis recovery

media to the cuvette and transfer back into 2-mL Eppendorf tubes

6. Leave cells to recover at 30 °C for 2–3 h (without shaking)

7. Centrifuge cells (14,000 Â g, 2 min, room temperature); remove all

supernatant apart from final 150 μL; resuspend the cells in remaining

buffer, and plate out 100 μL cells onto prewarmed SM17 plates

supplemented with 0.5% glucose and 5 μg/mL chloramphenicol, and

incubate at 30 °C for 3 days

8. Pick individual colonies, and inoculate M17 media supplemented with

1% glucose and 5 μg/mL chloramphenicol. Incubate overnight at 30 °C

without shaking

9. Using sterile technique, mix 500 μL 30% glycerol and 500 μL L. lactis

overnight culture in a sterile 1.5-mL Eppendorf tube. Mix by inverting

12 times, and store at À80 °C

10. Centrifuge the rest of the culture (4000 Â g, 10 min, 4 °C) and discard

the supernatant

11. Dissolve lysozyme (to 10 mg/mL) in buffer P1, and resuspend the

pellet; heat at 50 °C for 20 min, removing the tubes and vortexing

them every 5 min to ensure a homogenous solution; isolate plasmid

DNA using the Qiagen miniprep kit (following the manufacturer’s

instructions)

12. Confirm transformation has been successful by DNA sequencing



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Martin S. King et al.



5.2 Tips

– DpnI digests methylated template DNA

– Usually, a 3:1 ratio (insert to vector) is recommended for a ligation. For

L. lactis vectors, this ratio can be increased from 5:1 to 10:1

– L. lactis transformation is comparably inefficient compared to E. coli;

typically, we use 50 ng plasmid per transformation

– Incubation with lysozyme facilitates lysis of the bacterial cell wall, which

is required for the miniprep procedure to work; otherwise no DNA will

be isolated

– Cut off the end of pipette tips when resuspending electrocompetent

L. lactis to reduce shearing forces

– Pellet paint coprecipitation removes salt from the ligation product; excess

salt during electroporation causes sparking, reducing the efficiency of

transformation



6. STEP 2: GROWTH OF L. LACTIS, EXPRESSION OF THE

TARGET PROTEIN AND PREPARATION OF MEMBRANE

VESICLES

6.1 Overview

The protocol described below is optimized for the expression of members of

the mitochondrial carrier family in L. lactis, but this is a good starting point

for the expression of other membrane proteins. Still it would be good to

optimize parameters, such as induction time and nisin A concentration

for each construct.

Growth of L. lactis and the Expression of the Target Protein

1. Remove the L. lactis glycerol stock from the À80 °C freezer; keep on

dry ice and ensure the sample does not thaw. Scrape the frozen cell stock

with a sterile cocktail stick and inoculate 50 mL M17 media supplemented with 1% glucose and 5 μg/mL chloramphenicol; swirl, and

incubate the culture overnight at 30 °C overnight (without shaking)

2. Measure OD600 of the overnight culture; inoculate 500 mL prewarmed

M17 media supplemented with 1% glucose and 5 μg/mL chloramphenicol with the overnight culture, aiming for a starting OD600 of 0.1

3. Incubate at 30 °C without shaking; measure OD600 every 45 min to

construct growth curve (the doubling time is approximately 45 min)

4. When OD600 reaches $0.5, add nisin A (100 μL/L; which is a 1:10,000

dilution) and swirl immediately; return the flasks to the 30 °C incubator

(without shaking) for a further 3 h



Membrane Protein Expression in Lactococcus lactis



95



5. Centrifuge (6000 Â g, 10 min, 4 °C) and discard the supernatant.

Resuspend the pellet in TBS and centrifuge (6000 Â g, 10 min, 4 °C).

Resuspend the pellet in TBS, aliquot the sample into 15-mL falcon

tubes, and centrifuge (4000 Â g, 10 min, 4 °C). Discard the supernatant,

snap-freeze thoroughly in liquid nitrogen, and store at À80 °C

6. If using whole cells for functional assays, it is imperative that the frozen

cells are “snap-thawed” to prevent cell lysis. Add 2 mL TBS to the frozen

pellet, and resuspend by gentle pipetting. To aid thawing, place the falcon tube in a beaker of warm water during resuspension; place the

thawed cells on ice

Isolation of L. lactis membrane vesicles

1. Thaw the frozen cells, and lyse by mechanical disruption with one pass at

30 kpsi through a cell disruptor. We use a Constant Systems Cell

disrupter.

2. Centrifuge the disrupted sample at low speed (11,000 Â g, 15 min, 4 °C) to

remove whole cells and debris; decant the supernatant into ultracentrifuge

tubes, and centrifuge (200,000 Â g, 1 h, 4 °C) to collect the membranes.

3. Resuspend the pellet in TBS and centrifuge (200,000 Â g, 1 h, 4 °C) to

wash the membranes.

4. Resuspend the pellet in TBS and homogenize; determine the concentration using the BCA assay, and adjust the concentration to 5 mg/mL;

snap-freeze and store in liquid nitrogen.

Analysis

Determine expression using SDS-PAGE and Western blot analysis

according to standard protocols. For highly expressed proteins, a band

may be seen on a Coomassie-stained SDS-PAGE gel; for lowerexpressed proteins, Western blot analysis will be necessary to confirm

expression.



6.2 Tips

– After the addition of nisin A, swirl immediately to promote global induction and to prevent localized lysis, as nisin A inserts into the membrane to

form pores.

– Using the correct procedures (as detailed above), whole cells can be frozen and stored at À80 °C without adverse effects.

– The one-shot disruption system of Constant Systems is the most suitable

system to disrupt the lactococcal cell wall as the yield of crude membranes

improves more than fivefold when compared to that obtained by lysozyme

and French press treatment (Frelet-Barrand, Boutigny, Kunji, et al., 2010).



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