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Lactobacillus transformation-PDF

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Lactobacillus transformation-PDF

Overview

Electrotransformation procedure for Lactobacillus acidophilus, Lactobacillus brevis, and Lactobacillus helveticus.

Procedure

Prepare Electrocompetent cells

  1. Use overnight culture (106 CFU/mL) to inoculate 100 mL MRS containing 1% glycine.
  2. Grow until OD660 = 0.2-0.3.
  3. Place culture into two 50mL centrifuge tubes and chill on ice for 10 mins.
  4. Wash 2X with cold washing buffer (5 mM NaH2PO4, 1mM MgCl2, pH 7.4):
    1. Centrifuge for 5min at 4000g.
    2. Resuspend in an unspecified amount of washing buffer.
    3. Repeat.
  5. Concentrate in electroporation-buffer (1mM sucrose, 3mM MgCl2, pH 7.4):
    1. Centrifuge for 5min at 4000g
    2. Resuspend in an unspecified amount of electroporation buffer.
  6. Keep on ice and transform cells within 30 minutes.

Electroporation

  1. Add 1μL (25 ng/μL) of plasmid DNA to 50 ul (108 CFU/ml) of ice-cold cell suspension.
  2. Electroporate at 12.5 kV/cm (pulse number = 10, puse interval = 500 ms).
  3. Dilute electroporated cells to 1ml in MRS broth and incubate at 37°CC for 3 hours.
  4. Plate bacteria onto MRS agar plates with the appropriate antibiotic.
  5. Incubate under anaerobic conditions.

References

  • Kim, Y.H., Han, K.S., Oh, s., You, S. and S.H. Kim (2005) “Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation.” Journal of Applied Microbiology 99: 167–174

 

Lactobacillus planarum miniprep-PDF

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Lactobacillus planarum miniprep-PDF

Overview

Method to miniprep plasmid DNA from Lactobacillus plantarum

Procedure

  1. Supplement Qiagen P1 buffer with 5mg/ml lysozyme, 15U/ml mutanolysin, 100ug/ml RNase.
  2. incubate at 37°C for 15-30 minutes.
  3. Add Qiagen P2 buffer and continue miniprep as usual.
  4. Verify plasmid existence by sequencing or running digests on gel

References

Relevant papers and books

Mathiesen, G., H. M. Namlos, et al. (2004). “Use of bacteriocin promoters for gene expression in Lactobacillus plantarum C11.” Journal of Applied Microbiology 96(4): 819-827.

 

Electro-transformation of Lactobacillus spp-PDF

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Overview

While there is a wide variety of methods used for the electrotransformation of Lactobacillus spp., they all share a typical format.

  • Dilution: This determines the amount of overnight culture to add to new growth media
  • Growth: These are the conditions used to grow the cells on the day you plan to transform.
  • Washing: Cells are repeatedly centrifuged and resuspended in a solution of some type.
  • Buffer: This is what is used for the final resuspension and electroporation.
  • Concentration: This determines how much Electroporation buffer to add relative to the volume of the growth media.
  • Voltage: How hard to shock em.
  • Recovery: What to do with your cells after the shock.

This page will list many of these protocols in a standard format so that you can compare them and choose the one that works for you. In some cases an apples to apples comparison may not be acceptable (e.g. the differences in electroporators can be dramatic) so be sure to check the actual paper.

Procedures

Josson (1989)

  • Species = L. plantarum, L. casei.
  • Dilution = 1/50
  • Growth = MRS + 20mM DL threonine, 37°C, OD600=0.5-1.0
  • Wash = 2x w/ water (RT°C)
  • Buffer = 30% PEG 1000
  • Concentration = 10X cell pellet volume
  • Voltage = 8,500 V/cm
  • Recovery = 30min ice / 2hrs MRS

Bringel (1990)

  • Species = L. plantarum
  • Dilution = to OD600=0.05
  • Growth = MRS + 1% Glycine, 0.75M Sorbitol, 30°C, Shake vigorously, OD600=0.3 (~4 hours)
  • Wash = 3x w/ water (RT°C)
  • Buffer = 30% PEG
  • Concentration = 1/125
  • Voltage = 12,500 V/cm
  • Recovery = 30min ice / 3hrs MRS

Posno (1991)

  • Species = L. casei, L. pentosus, L. plantarum, L. acidophilus, L. fermentum, and L. brevis
  • Dilution = 1/50
  • Growth = MRS + 1% Glycine, 37°C, 3-4 hours
  • Wash = 5mM NaH2PO4 1mM MgCl2 (Ice Cold)
  • Buffer = 0.3M Sucrose 5mM NaH2PO4 1mM MgCl2 (Ice Cold)
  • Concentration = 1/100
  • Voltage = 7,000 V/cm
  • Recovery = 90min MRS

Aukrust (1992)

  • Species = L. plantarum, L. sake
  • Dilution = 1/50
  • Growth = 1% Glycine, 30°C, OD600.=0.6
  • Wash = 1mM MgCl2 (RT°C)
  • Buffer = 30% PEG 1500
  • Concentration = 1/100
  • Voltage = 7,500 V/cm
  • Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl2

Wei (1995)

  • Species = L. fermentum
  • Dilution = 1/50
  • Growth = 1% Glycine, 37°C
  • Wash = 5mM NaH2PO4 1mM MgCl2 (0°C)
  • Buffer = 0.9M Sucrose 3mM MgCl2
  • Concentration = 1/100
  • Voltage = 12,500 V/cm
  • Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl2

Berthier (1996)

  • Species = Lb. sake
  • Dilution = unspecified
  • Growth = MRS, 30°C
  • Wash = 10mM MgCl2 (0°C)
  • Buffer = 0.5M Sucrose, 10%Glycerol
  • Concentration = 1/200
  • Voltage = 7,000 V/cm
  • Recovery = 2hrs MRS, 80mM MgCl2

Thompson (1996)

  • Species = Lb. plantarum
  • Dilution = 1/20
  • Growth = MRS, 6% Glycine, 37°C
  • Wash = 2X water, 1X 50mM EDTA, 2X 0.3M Sucrose ALL ICE COLD
  • Buffer = 0.3M Sucrose
  • Concentration = 1/50
  • Voltage = 7,500 V/cm
  • Recovery = 2hrs MRS

Serror (2002)

  • Species = L. delbrueckii
  • Dilution = Unpecified
  • Growth = MRS, 42°C to OD600 = 1.7
  • Wash = 3X ice-cold EB buffer (1 mM MgCl2, 5mM KH2PO4)
  • Buffer = 0.4M Sucrose, 1mM MgCl2, 5mM KH2PO4; PH: 6)
  • Concentration = 1/30
  • Heat-Shock = 45°C for 20 mins, then ice for 10 mins
  • Voltage = 5,000V/cm
  • Recovery = 3hrs Milk Medium ((0.2 M sucrose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25 mM MgCl2) 37°C

Alegre (2004)

  • Species = L. plantarum
  • Dilution = 1/10
  • Growth = LAB, 37°C, MRS, 30°C
  • Wash = 2X 10mL 10 mM chilled MgCl2, 1X 10mL chilled 0.5M Sucrose, 10% Glycerol
  • Buffer = 0.5M Sucrose, 10% Glycerol
  • Concentration = 1/30
  • Voltage = 13,000 V/cm
  • Recovery = 2hrs MRS, 80mM MgCl2

Kim (2005)

  • Species = L. acidophilus, L. helveticus, L. brevis
  • Dilution = 1/50
  • Growth = 1% Glycine, 37°C, OD600 = 0.2-0.3
  • Wash = Ice 10mins, 2x w/ 5mM NaH2PO4 1mM MgCl2 (0°C)
  • Buffer = 1M Sucrose 3mM MgCl2
  • Concentration = 1/100
  • Voltage = 12,500 V/cm
  • Recovery = 2hrs MRS 0.5M Sucrose, 0.1M MgCl2

Mason (2005)

  • Species = Lb. casei, Lb. crispatus, Lb. delbreuckii, Lb. plantarum, Lb. salivarius
  • Dilution = 1/6
  • Growth = MRS, 8% Glycine, 90mins, 37°C
  • Wash = 2X water; 1X 50mM EDTA; 2X 0.3M Sucrose. ALL ICE COLD
  • Buffer = 0.3M Sucrose
  • Concentration = 1/120
  • Voltage = 7,500 V/cm
  • Recovery = 2hrs MRS

Speer (2012)

  • Species = L. plantarum
  • Dilution = 1/50
  • Growth = 2% Glycine, MRS, 3 hrs, 37°C, shaking
  • Wash = 2X water; 1X 50mM EDTA; 2X E Buffer. ALL ICE COLD
  • Buffer = 0.5M Sucrose, 10%Glycerol
  • Concentration = 1/100
  • Voltage = 12,000 V/cm
  • Recovery = 2hrs MRS 30°C

Notes

  1. Centrifugation at 4000 rpm for 2 minutes was sufficient to pellet the competent cells to give a clear supernatant.
  2. Most of these papers use the Bio-Rad Gene Pulser and have time constants in the range of 9-10 ms with the lower voltages (7-9kV/cm). If you’re using a preset electroporator (like the Eppendorf 2510) you should try a higher voltage (~1200) as your time constand will be in thee range of 5-6 ms. If you’re using PEG in your electroporation buffer expect your time constant to be significantly lower.
  3. If you get ZERO TRANSFORMANTS after trying multiple methods, chances are that your plasmid is incompatible with the strain you’re transforming. This page exists because this happened to me; and after I had tried ALL the protocols listed here, I got a new plasmid and it worked beautifully — Mike.

References

  • Alegre, M.T, Rodrıguez, M.C., and J.M. Mesas. (2004) “Transformation of Lactobacillus plantarum by electroporation with in vitro modified plasmid DNA.” FEMS Microbiology Letters 241: 73-77)
  • Aukrust, T. and H. Blom (1992). “Transformation of Lactobacillus strains used in meat and vegetable fermentaions.” Food Research International 25(4): 253-261.
  • Berthier, F., M. Zagorec, et al. (1996). “Efficient transformation of Lactobacillus sake by electroporation.” Microbiology-Uk 142: 1273-1279.
  • Bringel, F. and J.C. Hubert. (1990) “Optimized transformation by electroporation of Lactobacillus plantarum strains with plasmid vectors” Applied Microbiology and Biotechnology 33:664-670
  • Josson, K., T. Scheirlinck, et al. (1989). “Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii.” Plasmid 21(1): 9-20.
  • Kim, Y.H., Han, K.S., Oh, s., You, S. and S.H. Kim (2005) “Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation.” Journal of Applied Microbiology 99: 167–174
  • Mason, C. K., M. A. Collins, et al. (2005). “Modified electroporation protocol for Lactobacilli isolated frorn the chicken crop facilitates transformation and the use of a genetic tool.” Journal of Microbiological Methods 60(3): 353-363.
  • Posno, M., R. J. Leer, et al. (1991). “Incompatibility of Lactobacillus vectors with replicons derived from small cryptic plasmids and segregational instability of the introduced vectors.” Applied and Environmental Microbiology 57(6): 1822-1828.
  • Serror, P., Sasaki, T., Ehrlich, S.D. and Emmanuelle Maguin. (2002) “Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids.” Applied and Environmental Microbiology, p.46–52
  • Speer, M.A. and T.L. Richard (2012) Will Soon Exist.
  • Thompson, K. and M. A. Collins (1996). “Improvement in electroporation efficiency for Lactobacillus plantarum by the inclusion of high concentrations of glycine in the growth medium.” Journal of Microbiological Methods 26(1-2): 73-79.
  • Wei, M. Q., C. M. Rush, et al. (1995). “An improved method for the transformation of Lactobacillus strains using electroporation.” Journal of Microbiological Methods 21(1): 97-109.

 

In vitro modification of DNA for L. plantarum-PDF

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Overview

The following is a procedure for the in vitro modification of DNA before electrotransformation into Lactobacillus plantarum developed by Alegre et al. The inability to recover successful transformants in many lactic acid bacteria including Lactobacillus plantarum is most likely the result of active host restriction mechanisms. This method was originally developed for Saccharopolyspora spinosa in an attempt to circumvent the active restriction-modification of the host bacterium. See notes for an alternative method.

Materials

  • AEBSF Stock Solution (1mM) (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride)
  • S-adenosylmethionine Stock Solution (0.8mM)
  • 10 mg ml BSA
  • Filtered glycerol
  • Prepared Plasmid DNA
  • Wash Buffer (15mL)
    • 21mg monopotassium phosphate (10mM)
    • 44mg EDTA (10mM)
    • 44mg NaCl (50mM)
    • 12mL Deionized Water
    • 3mL AEBSF Stock Solution (0.2mM)
    • Store at 4°C
  • TNE buffer (5mL)
    • 30mg Tris (50mM)
    • 15mg NaCl (50mM)
    • 15mg EDTA (10mM)

Procedure

Preparation of the Extract

1. Grow up 45 ml of L. plantarum cells in MRS overnight and wait until OD600 is between 1.5 and 2.0.
2. Pellet cells at maximum speed until supernatant is clear (∼4 mins @ 5000g).
3. Resuspend pellet in 10 ml of wash buffer and centrifuge again.
4. Resuspend in 2 ml of wash buffer and put cells on ice.

    • Keep cells chilled (on ice) during the remainder of the procedure

5. Sonicate cells at 12 pulses of 30s with 60s intervals, using a micro tip at 60W.
6. Pellet cells at maximum speed ensuring cells are still cold (i.e. use a prechilled refrigerated centrifuge).
7. Carefully decant the cell extract, isolating only the liquid remains (approximately 1.5ml).
8. Add 1.5mL 100% glycerol and 30μL BSA solution (10mg/mL) to the decanted cell extract.
9. Separate the extract into 25μL aliquots and store at -20°C until use.

DNA Modification

1. Add the following to a 25μL aliquot of cell extract:

    • 50μL TNE Buffer
    • 10μL of S-adenosylmethionine Stock Solution
    • 1μL BSA (10mg/ml)
    • 10μL of plasmid DNA.

2. Incubate the mixture at 30°C for 16 hours.
3. Extract the mixture with a phenol/chloroform extraction.
4. Precipitate using ethanol.

Notes

All questions, input and feedback are welcome!

  • AEBSF should be handled in a fume hood with lab coat, safety gloves and eye protection.
  • AEBSF is a much safer alternative to PMSF that is soluble in water and has a very similar specificity to PMSF as a serine protease inhibitor. It also goes by the name Pefabloc SC.
  • There is a helpful protocol for phenol extraction posted and a protocol for ethanol precipitation posted.
  • An alternative to this protocol is to use a lab strain of Lactococcus lactis (we use strain MG1363) as a shuttle species. The procedure takes just as much linear time, but much less actual time; and is much easier. The process goes as follows.
  1. Miniprep the desired shuttle vector from E. coli.
  2. Electroporate into L. lactis electro-comptent cells at 10,000kv/cm.
  3. Let cells recover in 25ml GM17 media for one hour.
  4. Add the appropriate antibiotic to the media.
  5. Grow overnight at 30°C.
  6. Miniprep L. lactis culture.
  7. Transform L. plantarum electro-competent cells at 10,000kv/cm.
  8. Smile because you didn’t have to buy any extra reagents or work with the loud-ass sonicator!

References

  1. Alegre et al. (FEMS Microbiology Letters 241 (2004), 73-77)
  2. Matsushima et al. (Microbiology 140 (1994), 139-143)

 

Lactobacillus transformation-PDF

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Overview

This is basically a combination of the version of the Berthier (1996) protocol and the Mason (2005) protocol. This protocol is designed for use with Lactobacillus plantarum and an Eppendorf 2510 electroporator. For other protocols check out the Lactobacillus transformation archive.

Materials

  • MRS media
  • Glycine
  • Millipore water
  • 50mM EDTA solution
  • Electroporation Buffer (0.5M Sucrose, 10% glycerol)

Procedure

  1. Grow L. PlantarumInoculate 5ml MRS medium with L. plantarum freezer stock.
    1. Grow overnight at 30°C without shaking.
  2. Make 100ml 2.5% glycine:
    1. Add 1.25g glycine to two flasks of 50ml MRS medium.
    2. Shake to dissolve.
  3. Grow cells:
    1. Add 1 ml overnight culture to each flask (1:50 dilution).
    2. Culture for ~3 hours at 37°C with shaking.
  4. Wash 2x with ice-cold DI water:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 25ml ice-cold DI water.
    3. Repeat.
  5. Treat with ice-cold EDTA:
    1. Centrifuge for 5min at 4000g
    2. Resuspend in 5ml 50mM EDTA.
    3. Incubate on ice for 5 minutes
    4. Add 25ml ice-cold DI water.
  6. Wash with ice-cold DI water:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 25ml ice-cold DI water.
  7. Wash 2x with Electroporation Buffer:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 25ml ice-cold 0.5M Sucrose, 10% glycerol.
    3. Repeat.
  8. Concentrate cells:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 0.8ml ice-cold 0.5M Sucrose, 10% glycerol.
  9. Divide cells:
    1. Aliquot 90μL of cell concentrate into ~20 microcentrifuge tubes.
    2. Keep on ice until use (within the next two hours).
  10. Add DNA:
    1. Add 10μL of plasmid DNA to the 90μL of cell concentrate.
    2. Keep on ice for 5 minutes.
    3. Put 1mm cuvettes on ice too.
  11. Electroporate:
    1. Pipette the cell/DNA mixture into the cuvette
    2. Electrporate at 1200 volts
    3. The time constant should be ~5.0
  12. Recovery:
    1. Immediately transfer the electroporated cells to 900μL of MRS medium.
    2. Incubate at 30°C for 2-3 hours.
  13. Select:
    1. Plate on medium with the appropriate antibiotic
    2. Incubate at 30°C for two days.
    3. Pick colony

Notes

  • All of the liquid reagents/buffers should be autoclaved before use.
  • Work fast and keep the tubes on ice if you’re going to pause for any reason.
  • This protocol works well for chromosomal insertion
  • Notice the high amount of DNA added to the competent cells (10%)
  • While glycerol in the electroporation buffer is typically used to make frozen competent cells, this is not advised and will lead to decreased competency. Instead, the glycerol addition is strictly to increase to competency of the cells. The reason behind this correlation is not understood.

 

 

Lactobacillus chromosomal integration-PDF

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Overview

This procedure is used to integrate a desired DNA cassette into the chromosome of Lactobacillus plantarum at specific locations. This protocol can also be modified to perform gene knockouts and other chrosomal modifications. The two plasmids used for this procedure are pGIP73 and pP7B6 which integrate into the conjugated bile-acid hydrolase (cbh) sequence and the P7B6 prophage sequence respectively. These plasmids are non-replicative in Lactobacillus spp. and operate based on homologous recombination between the plasmid and the chromosome. The desired cassette is inserted in a unique XbaI site in the middle of both the CBH and P7B6 sequences.

Materials

  • Integration plasmid DNA (either pGIP73 or pP7B6)
  • XbaI
  • Antarctic Phosphatase
  • MRS media
  • Erythromycin
  • Lactobacillus plantarum
  • E. coli

Procedure

Preparing the Plasmid

1. Culture the desired plasmid in E. coli in SOB with erythromycin ad a concentration of 300μg/mL.
2. Miniprep the plasmid and digest with XbaI.
3. After two hours digest add Antarctic phosphatase (and the requisite buffer) and digest for one additional hour.

  • Phosphatasing the digest will help prevent self ligation.

4. Digest your desired insert to form compatible ends with XbaI (a SpeI + XbaI double digest works here).

  • Gel extracting the insert will increase ligation efficiency.

5. Ligate and transform into E. coli.
6. Sequence or colony PCR to check successful insertion.

Performing the Integration

1. Transform the Lactobacillus cells according to one of these protocols.
2. After the specificed recovery time, pipette 100μL of the recovered cells into a culture tube of 5ml MRS broth (erythromycin at 1μg/mL) and grow overnight.
3. The next day plate 20μL of this solution onto MRS plates containing 5μg/mL erythromycin to get colonies.

  • Alternately you can just add these 20μL to a tube containing 5ml MRS supplemented with 2.5μg/mL erythromycin.

4. Screen for your gene of interest to ensure successful integration.

  • A plate screen is really good here.

If you don’t want a food grade microorganism then you can stop here.

If you want to remove the erythromycin resistance then continue.

 

5. Grow your integrated culture overnight in 5ml of MRS broth with NO ERYTHROMYCIN!!!
6. Plate a dilution (try 1 to 100,000) of this culture onto MRS plates with NO ERYTHROMYCIN!!!
7. Select ~10 colonies and spot plate them onto two plates: one containing erythromycin and one not.

  • Be sure to label the spots to be sure which colony corresponds to which.

8. Select the colonies that grow on the MRS but not on the MRS+Erythromycin.
9. Screen these colonies for your insert activity.

  • ~50% of colonies will have lost your insert.

Notes

Be sure to do a MIC experiment on your erythromycin every time you get a new batch. The potency of various products can be startlingly different.

References

  • Hols, P., T. Ferain, et al. (1994). “Use of homologous expression secretion signals and vector-free stable chromosomal integration in engineering of Lactobacillus plantarum for alpha amylase and levanase expression.” Applied and Environmental Microbiology 60(5): 1401-1413.
  • Rossi, F., A. Capodaglio, et al. (2008). “Genetic modification of Lactobacillus plantarum by heterologous gene integration in a not functional region of the chromosome.” Applied Microbiology and Biotechnology 80(1): 79-86.

 

Mesoplasma florum:Genomic DNA-PDF

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Mesoplasma florum:Genomic DNA-PDF

Materials

  • ATCC 1161 culture medium
  • TE
  • 10% SDS solution
  • Proteinase-K 20 mg/ml solution
  • 5 M NaCl solution
  • CTAB / NaCl solution
    • CTAB 10%, NaCl 700 mM
    • dissolve 4.1 g NaCl in 80 ml water, and slowly mix and add 10 g CTAB (hexadecyltrimethylammonium bromide) heating to 65° if necessary. Bring the solution to 100 ml.
  • chloroform/isoamyl alcohol 24:1
  • phenol/chloroform/isoamyl alcohol 25:24:1
  • Novagen Pellet Paint NF
  • isopropanol
  • 70% ethanol

CTAB Protocol

  • Culture 10 ml of medium 1161 infected with Mesoplasma florum in 15 ml centrifuge tubes at 30° without shaking.
  • Transfer 2x 1.8 ml into two 2 ml tubes. Spin down at 17000g for 2 minutes and retain the pellet.
  • Resuspend the pellet by vortexing first, then adding 567 μl of TE.
  • Add 3 μl of proteinase-K 20 mg/ml and 30 μl of SDS 10% solution, mix, and incubate at 37° for 1 hour
  • Add 100 μl of 5 M NaCl and mix (critical before adding CTAB)
  • Add 80 μl of CTAB / NaCl solution, mix
  • Incubate 10 minutes at 65°
  • Add an equal volume (800 μl) of chloroform/isoamyl alcohol 24:1, vortex, and spin down at 17000g for 2 minutes
  • Set up fresh 2 ml tubes with 800 μl of phenol/chloroform/isoamyl alcohol 25:24:1, transfer the supernatant, and mix carefully.
  • Spin down at 17000g for 2 minutes
  • Set up fresh 2 ml tubes with 800 μl of chloroform/isoamyl alcohol 24:1, transfer the supernatant, and mix carefully.
  • Spin down at 17000g for 2 minutes
  • Set up fresh 1.5 ml tubes loaded with 1 μl Novagen Pellet Paint NF and transfer the supernatant into those tubes.
  • Add 0.6 volumes of isopropanol and chill in the -80 freezer for 30 minutes
  • Spin down at 17000g for 20 minutes and remove the supernatent carefully retaining the blue pellet.
  • Fill the tube with 70% ethanol to wash, and spin down at 17000g for 2 minutes.
  • Remove the ethanol carefully leaving the blue pellet. This is best done by using a 1 ml pipet to remove the bulk of the ethanol, then a 10 μl tip to remove the remainder. Spinning the tube down after most of the ethanol is removed will assist in removing the last 10-20 μl.
  • Let the tube air dry for 1/2 hour until the ethanol odor is no longer present.
  • Redissolve the DNA pellet in 100 μl TE (this will take an hour or so).
  • Spin down the tube briefly and measure OD and 260/280 ratios.
  • Expect around 500 ng/μl concentration (total 50 μg).

A critical element is the NaCl concentration before adding CTAB. With < 500 mM NaCl, DNA precipitates. Above that point, polysaccharides and proteins precipitate, but the DNA stays in the solution.

 

Reference

  • PMID 7433111
  • This protocol follows closely the bacterial genomic DNA protocol from Current Protocols section 2.4, contributed by Kate Wilson.

 

Qiagen Genomic Tip protocol

  • Grow 40 ml cultures of the Mesoplasma species at 30°C in 1161 medium.
  • Pellet cells at 5000 x g for 10 minutes
  • Resuspend the pellet in 11 ml of buffer B1 (with RNAse)
  • Add 500 μl of proteinase-K solution, 20 mg/ml (10 mg)
  • incubate at 37°C for at least 30 minutes
  • Add 4 ml of buffer B2 and mix or vortex briefly
  • Incubate at 50°C for 30 minutes; the lysate should become clear
  • If necessary, pellet particulates
  • Save a 300 μl sample 1
  • Equilibrate a genomic tip 500/G with 10 ml of buffer QBT and allow flow through
  • Vortex the sample for 10-20 seconds to reduce viscosity
  • Sample should flow through, or can be diluted with buffer QBT before loading
  • limit flow to 20-40 drops/min under pressure
  • save a 1200 μl sample of the flow through — sample 2
  • Wash the column with 15 ml of buffer QC twice or more
  • save a 600 μl sample of the flow through — sample 3
  • Elute with 15 ml of buffer QF prewarmed to 50°C
  • save a 600 μl sample of the elution — sample 4
  • precipitate DNA by adding 10.5 ml (0.7 volumes) of isopropanol
  • invert the tube several times and recover by spooling on a glass rod or
  • Centrifuge at 5000 x g for 15 minutes at 4°C, wash with 70% ethanol
  • dissolve in 100-200 μl of TE pH 8.0 on a shaker at 55°C or overnight
  • For diagnosis of problems:
    • precipitate the DNA from the samples 1-4 taken above with 0.7 volumes of isopropanol
    • wash with 70% ethanol
    • Resuspend in 20 μl TE
    • Run 10 μl on a 0.5% agarose gel

Mesoplasma florum:Electroporation-PDF

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Mesoplasma florum:Electroporation-PDF

Materials

  • overnight culture of Mesoplasma florum in ATCC 1161 medium
    • preparing 10 ml cultures with 1, 10, 100, and 1000 μl of infected cultures assures that one of these will be ready the next day at the correct OD level. Select cultures which are just changing color.
  • chilled mycoplasma electroporation buffer
    • 8 mM HEPES pH 7.4
    • 272 mM sucrose (93.1 g/l)
  • chilled 1 mm electroporation cuvettes
  • 10 ml chilled ATCC 1161 medium
  • Selective medium or plates
    • Tetracycline resistance from TetM is higher than 200 μg/ml
    • Untransformed cells grow poorly at 4 μg/ml
    • Tet selective plates are at 15 μg/ml

Protocol

  • chill the centrifuge to 4°
  • spin down 10 ml of overnight culture at 8000g for 5 minutes, remove the supernatent
  • resuspend the pellet in the remaining liquid by vigorous vortexing
  • add 10 ml of chilled electroporation buffer and mix
  • spin down again, remove supernatent
  • resuspend the pellet in the remaining liquid
  • add 10 ml of chilled electroporation buffer
  • spin down again, remove supernatent
  • resuspend the pellet in the remaining liquid, bring the total volume to 1200 μl with EPB
  • Freeze 400 μl aliquots at -80 indefinitely or use immediately
  • Add 16 μl of transposome or plasmid DNA for transformation
  • Mix and transfer to four chilled 1 mm gap electroporation cuvettes
  • Pulse the cuvette at 1.5 KV
  • Add ml of chilled 1161 medium immediately, mix with the pipet, cover
  • Place the cuvettes into the 30° incubator for outgrowth for 50 minutes
    • cells can be held at 4° following outgrowth
    • dilute 1 μl of the culture into 1 ml of 1161 medium, vortex, and dilute again into 1 ml. Plate 200 μl on a nonselective 1161 plate for counting untransformed CFUs.
  • Plate 360 μl aliquots on three selective plates
  • grow plates for 1.5 – 2 days at 30° for colonies

Notes

9/6/07: Arcing at 1.8 and 1.6 KV; possibly we need a second wash and/or some additional delay in the wash to remove more salt. Gel loading tips do not work. Plating 125 ul seems to work well. Tested outgrowth at 2 ug/ml, 4 ug/ml, 8 ug/ml

Mesoplasma florum:Inverse PCR Transposon location-PDF

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Mesoplasma florum:Inverse PCR Transposon location-PDF

Choice of Enzyme

  • Hutchison used DraI as a cutter, but this is a blunt cutter, making religation difficult
  • MboI cuts at GATC sites, and is insensitive to 5-me dCTP methylation (sensitive to methylation of A)
  • MboI cutting frequency calculation: p(cut) = (.13)(.37)(.37)(.13) = .00231
  • Expected fragment length = 1/ .00231 = 432 bp + length from primer site to end of transposon
  • Expected PCR fragment length is twice this length, or about 1Kbp

Materials

  • Genomic DNA from single colony transposon insertion event
  • MboI
  • NEB buffer 3 10x
  • T4 DNA ligase buffer 10x
  • T4 DNA ligase
  • PCR supermix
  • M13forward(-47) primer
  • T7 universal primer
  • ME primer
  • E-Gel 0.8%

Restriction digest of 500 ng of genomic DNA with MboI

  • Mix 0.5 μl (approx) genomic DNA in TE
  • 5 μl NEB buffer 3
  • 1 μl MboI
  • 43.5 μl water
  • incubate at 37° for 30 minutes
  • heat kill at 65° for 20 minutes

Ligation of cut ends at 5 ng/μl concentration (Hutchison99)

  • 5 μl digested DNA
  • 1 μl T4 DNA ligase buffer
  • 0.2 μl T4 DNA ligase
  • 3.8 μl water
  • incubate 10 minutes at room temperature

Transposon detection PCR reaction 10 μl test volume

  • 0.5 μl ligated DNA
  • 0.3 μl ME primer

9.2 μl PCR supermix High Fidelity

Inverse PCR for transposon location identification

  • 0.5 μl ligated DNA
  • 0.15 μl M13forward(-47) primer
  • 0.15 μl T7 universal primer
  • 9.2 μl PCR supermix high fidelity
  • Cycle 5 minutes at 95° initial denturation
  • 40 cycles of
    • 94° 30 seconds
    • 55° 30 seconds
    • 64° 3 minutes
  • 10 minutes 64° final extension

Detect with E-Gel 0.8%

Inverse PCR for sequencing transposon location

  • 5 μl ligated DNA
  • 1.5 μl M13forward(-47) primer
  • 1.5 μl T7 universal primer
  • 92 μl PCR supermix high fidelity
  • Cycle as above
  • Prepare 1% agarose prep gel
  • add 20 μl of loading dye
  • Run gel
  • Cut the band
  • Prepare DNA the gel with Qiaex II kit
  • Quantitate DNA
  • Sequence with both M13forward(-47) primer and T7 universal primer

Sequence analysis

  • locate the MboI cut site (GATC) in the sequencing results
  • locate the end of the transposon sequence (ME end, reverse complemented here, agatgtgtataagagacag)
  • Identify the duplicated 9bp insertion site surrounding the insertion event
  • Locate the sequence from the ME end to the GATC cut site on the Mesoplasma florum genome
  • The sequence from the M13F(-47) and T7 Universal primers should be adjacent, and oriented in opposite directions on the genome

References

Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650

Mesoplasma florum:Transposome construction-PDF

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Transpose DNA construction from plasmid cutting

  • Cut the transposon out of the plasmid containing PvuII enzyme, which cuts at the correct location at the mosaic end.
  • Cut with an enzyme that produces shorter fragments from the remaining plasmid backbone to make gel purification easier. Sau3AI is a good enzyme.
    • Use NEB Buffer 1 with BSA which is good for Sau3AI and PvuII.
  • TT01 transposon is 2478 bp long
  • Reaction
    • 20 μg of plasmid DNA from a maxiprep
    • 10 μl NEB Buffer 1
    • 1 μl 100x BSA
    • 3 μl PvuII
    • 1 μl Sau3AI
    • QS water to 100 μl
    • Heat 37° 1 hour
    • Heat kill the enzymes for 20 minutes at 65°
    • Add 20 μl loading dye
    • Load and run on a prep gel
    • Cut the band at 2478 bp from the gel
    • Weigh the cut band
    • Add 3x volume Qiagen QX1 buffer
    • Add 30 μl Qiaex II suspension
    • Heat at 50° while vortexing until completely dissolved
    • Spin, discard, and resuspend in 1 ml QX1 buffer
    • Spin, discard, and resuspend in 1 ml PE buffer
    • Spin, discard, and resuspend in 1 ml PE buffer
    • Spin, discard, spin again, and remove remaining PE buffer with 10 μl tip
    • Dry at 50° for 15 minutes until the Qiaex II turns white
    • Resuspend in 30 μl TE
    • Spin, remove supernatant to a fresh tube with a 10 μl tip
    • Add 10 μl TE to the Qiaex II suspension, resuspend, spin
    • Remove supernatant with a 10 μl tip
    • Spin down the fresh tube to pellet any remaining Qiaex II suspension and transfer supernatant to a screw to the vial
    • Measure concentration and label the tube

Transpose DNA construction using PCR and cutting

  • PCR with ext-ME primer using Phusion master mix. Final volume 200 μl
    • 100 μl 2x Phusion master mix
    • 6 μl ext-ME primer (30 PM/μl) (gt ttc ttc agc TGT ctc tea tac aca act)
    • 1 μl transposon template (10 ng/μl)
    • 93 μl water
    • Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m
  • Add 2x 500 μl Qiagen buffer PB
  • Bind to Qiaquick column 2x 600 μl, flowing past the column twice
  • Wash once with 500 μl buffer PB
  • Wash 2x with 750μl buffer PE, and spin at high speed for 5 minutes after emptying.
  • Elute with 2x 50 μl buffer EB into a clean tube.
  • Add 10 μl NEB buffer 2
  • Add 37 μl water
  • Add 3 μl PvuII, mix
  • Incubate at 37° for 1 hour
  • Add 300 μl Qiagen buffer ERC
  • Bind to a Qiagen Minelute column by flowing 2x through the column
  • Wash 2x with 750 μl buffer PE
  • Spin dry
  • Elute with 20 μl TE
  • Measure the concentration of the resulting DNA

Transpose DNA construction using PCR

  • PCR with ME0 primer using Phusion master mix. Final volume 200 μl, split 2x 100μl
    • 100 μl 2x Phusion master mix
    • 6 μl ME0 primer (30 PM/μl) (phos- c tgt ctc tea tac aca act)
    • 1 μl transposon template (10 ng/μl)
    • 93 μl water
    • Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m
  • Add 2x 500 μl Qiagen buffer PB
  • Bind to standard Qiagen column, flowing past the column twice
  • Wash once with 500 μl buffer PB
  • Wash 2x with 750μl buffer PE, spin after emptying.
  • Elute with 2x 50 μl TE
  • vacuum evaporate, resuspend in 20 μl TE
  • Measure the concentration of the resulting DNA, expect 500 ng/μl
  • Dilute to 100 ng/μl
  • Dilute 1 μl into 20 μl, and run a gel to verify the correct size (TT01 is 2478 bp)

Final transposome construction

  • 1 μL transposon DNA, with phosphorylated ME ends, 100 ng/μL
  • 2 μL EZ-Transposase (Epicentre)
  • 1 μL glycerol
  • hold at RT for 1/2 hour
  • reported to age at 4C overnight to provide higher efficiency
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