Transformation of Thermus Species by Natural Competence    

Materials and Reagents

1. 12 ml sterile plastic tubes
   
2. 15 ml sterile plastic tubes
   
3. Sterile plastic Petri dish plates (standard size; 100 x15 mm)
   
4. Sterile tips for micropippettes, 5-200 µl (for instance, Daslab, catalog number: 162001 )
   
5. Sterile tips for micropippettes, 100-1,000 µl (for instance, Daslab, catalog number: 162222 )
   
6. Sterile tips for micropippettes, 10 µl (for instance, Metler Toledo, catalog number: 17004280 )
   
7. Recipient Thermus strain, for instance, HB27 wild type (HB27/ATCC BAA-163 /DSM 7039)^{#
   #}Note: Other Thermus thermophilus strains such as NAR1 and HB8, have been defined as naturally competent, thus, harboring the competence proteins required for transformation (César et al., 2011). All Thermus spp. strains referred here are available at the DSMZ collection.
   
8. Kanamycin sulphate (Sigma-Aldrich, catalog number: K1377-5G )
   
9. Tryptone (Conda, catalog number: 1612 )
   
10. Yeast extract (Conda, catalog number: 1702 )
    
11. Sodium chloride (NaCl) (EMD Millipore, catalog number: 1.06404.1000 )
    
12. Agar (grade A) (Conda, catalog number: 1800 )
    
13. Thermus water (carbonated-rich mineral water)
    
14. DNA template: purified plasmids or genomic DNA** (harbouring the thermostable kat cassette)
    **Note: DNA quantity depends on the topology of the DNA to be transformed and the competence of the recipient strain.
    
15. TB liquid medium (see Recipes)
    
16. TB agar plates (see Recipes)
    

Equipment

1. 50 ml Erlenmeyer flask
   
2. Rotary shaker incubator reaching 70 °C (150 rpm) (Thermo Scientific, Thermo Scientific^TM, catalog number: SHKE 420HP )
   
3. Glass beads or a spreader
   
4. Autoclave device (CertoClav, model: EL 18L )
   
5. Spectrophotometer (Hitachi, model: U-2000 )
   
6. Pipette P2, 0.2-2 μl (Gilson, Pipetman classic^TM, catalog number: F144801 )
   
7. Pipette P20, 2-20 μl (Gilson, Pipetman classic^TM, catalog number: F123600 )
   
8. Pipette P100, 20-100 μl (Gilson, Pipetman classic^TM, catalog number: F123615 )
   
9. Pipette P200, 50-200 μl (Gilson, Pipetman classic^TM, catalog number: F123601 )
   
10. 70 °C heater (Gemini, model: Memmert UE400 )
    
11. Humid chambers
    Note: Tupperware devices are preferentially used. Humidified plastic bags are also valid. Addition of a paper towel, soaked on Thermus water and laid on the bottom of the chamber would enhance humidity maintenance. Ventilation should be avoided in order to conserve humidity. An example is provided in the photographs beneath (Figure 1).
    
    
    Figure 1. Humid chambers. Descriptive photographs of the humid chambers used to incubate Thermus plates. Place a piece of paper soaked with Thermus water and a bottle cap filled with the same water on the bottom of the chambers to maintain high humidity.

Procedure

1. Culture preparation: growth of the subject strain to be transformed (see Figure 2).
1. Inoculate 50 μl of saturated liquid growth culture (optical density at 600 nm [OD₆₀₀] aprox. 2) in 10 ml TB medium in a 50-ml Erlenmeyer or inoculate the culture from the glycerol stock or from a frozen pellet. In the latter cases, one additional day growth would be required to reach optimal culture fitness.
   
2. Incubate overnight the culture at 65 °C in a shaker incubator at 150 rpm.
   
3. Dilute the overgrown culture in 10 ml fresh TB medium to OD₆₀₀ = 0.05 in a 50-ml Erlenmeyer.
   Notes:
1. If the culture has not reached saturation after overnight growth, postposition of the experiment is highly recommended.
   
2. If the culture has not grown optimally overnight and the experiment should be performed, a starting OD₆₀₀ of 0.07-0.08 is possible.
   
4. Incubate in a shaker incubator at 65 °C until an OD₆₀₀ of 0.3-0.4 is reached.
   Note: An OD₆₀₀ close to 0.3 is recommended.
   
   
   Figure 2. Schematic representation of the Thermus transformation procedure. Plate shows a dilution of a transformation of 100 ng of a linearized plasmid harbouring a kat cassette flanked by two genomic arms for double recombination in HB27 strain.
   
   
2. DNA addition
   
1. Add 0.01-1 μg of DNA (genomic ~0.01 μg; plasmids ~0.1-0.25 μg; PCR products ~1 μg) in a sterile 12 ml plastic tube. Tubes can be pre-heated at 65 °C.
   Notes:
1. Genomic DNA (carrying a selectable marker integrated in the genome) should not be added in higher quantities to avoid recombination farther than the target.
   
2. DNA samples should be purified following standard protocols and eluted in water or TE.
   
3. Pre-heating of the tubes keeps the temperature of the growing culture stable.
   
2. Add 500 μl of the exponentially growing culture (see Figure 2) to the DNA.
   
3. As a control, prepare 15 ml plastic tube without DNA, and add 500 μl of the exponentially growing culture to this tube.
   
3. DNA transformation
   
1. Incubate each tube in a shaker incubator at 65 °C for 4 h.
   
2. Plate 50 μl and 400 μl of the cells on selective agar medium, spread by using glass beads or a spreader.
   Notes:
   
1. Volume plated depends on the competence efficiency of the employed Thermus strain. Previous assays plating serial dilution spots can shed light on the appropriate volume to plate.
   
2. Plate the control tube like the others, in addition to plating on a non-selective agar medium, as it will work as the reference of viable cells.
   
3. Incubate plates in humid chambers for 48 h at 65 °C or until colonies become visible.
   Notes:
   
1. Ordinarily, incubation temperature is set according to optimal growth of the strain but it should be adapted to the limitations of the selectable marker employed in the transformations too (for instance, hph5 conferring hygromycin resistance cannot be exposed to temperatures higher than 60 °C whereas kanamycin is selectable in temperatures higher than 70 °C).
   
2. Transformation yields highly depend on the DNA template employed and the strain being transformed. For instance, HB27 transformed with 100 ng of plasmidic DNA shows rates of 10-3 transformants per viable cell.

Data analysis

1. For transformation assays, a set of statistical tests should be performed to compare and analyse the transfer frequencies obtained.
   
2. Transfer frequencies are calculated as the number of CFU transformants divided by the number of CFU of viable cells. To examine differences among transfer frequencies, Student’s t-test and Mann-Whitney U-tests may be used. When comparing transformability of a strain testing several DNA templates, examination of the differences among and within various DNA templates transfer frequencies can be addressed by one-way ANOVA. If different strains are tested, Wilcoxon-tests may be used to evaluate differences among their transfer frequencies. Simple linear regression test can be implemented for modelling the relationship between the DNA template and the frequency of transfer. Post-hoc Tukey and Bonferroni tests should be applied when convenient. 
   
3. At least, 3 independent experiments, should be performed for reliable and reproducible data.
   

Notes

1. This transformation protocol was optimized for Thermus thermophilus HB27, which shows the highest natural competence efficiency within the genus and the highest DNA uptake rates. Other strains with less competence capability should be treated with larger quantities of DNA and/or longer incubation periods.
   
2. In case transformation has been performed to introduce a mutation, transformants should be re-streaked on selective plates and further grown in liquid under each particular condition to ensure a stable mutation.
   
3. DNA amount required varies from 10 ng when employing genomic DNA to 1,000 ng when employing PCR products. On average, replicative plasmids are employed at 100-250 ng.
   
4. Regardless the selective marker employed, post-incubation times are at least 3 to 4 h at 65 °C and 150 rpm. If DNA is added at the start of the exponential phase (OD₆₀₀ = 0.2), times delays up to 5 h incubation.
   

Recipes

1. TB liquid medium
   0.8 % tryptone (w/v)
   0.4 % yeast extract (w/v)
   50 mM NaCl, pH 7.5
   Thermus water (carbonated-rich mineral water)*
   pH may be adjusted by the addition of NaOH
   
2. TB agar plates
   0.8% tryptone (w/v)
   0.4% yeast extract (w/v)
   50 mM NaCl, pH 7.5
   1.8% agar (grade A) (w/v)
   Thermus water (carbonated-rich mineral water)*
   

*Note: The analytical composition of the spring mineral water employed, per mg/ml, is listed beneath (Table 1). Other mineral waters easily purchasable such as Evian water or a laboratory solution resembling this recipe, can be employed.

Table 1. Mineral composition of the spring water from Jaraba Spring in Zaragoza (Spain)

Acknowledgments

This work has been funded by Grants BIO2013-44963-R, and FP7-PEOPLE-2012-IAPP No. 324439. A. Blesa held an FPI contract from the Spanish Ministry of Economy and Competitivity.

References

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2. Brouns, S. J., Wu, H., Akerboom, J., Turnbull, A. P., de Vos, W. M. and van der Oost, J. (2005). Engineering a selectable marker for hyperthermophiles. J Biol Chem 280(12): 11422-11431.
   
3. César, C. E., Alvarez, L., Bricio, C., van Heerden, E., Littauer, D. and Berenguer, J. (2011). Unconventional lateral gene transfer in extreme thermophilic bacteria. Int Microbiol 14(4): 187-199.
   
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6. Lasa, I., Caston, J. R., Fernandez-Herrero, L. A., de Pedro, M. A. and Berenguer, J. (1992). Insertional mutagenesis in the extreme thermophilic eubacteria Thermus thermophilus HB8. Mol Microbiol 6(11): 1555-1564.
   
7. Nakamura, A., Takakura, Y., Kobayashi, H. and Hoshino, T. (2005). In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus. J Biosci Bioeng 100(2): 158-163.
   
8. Schwarzenlander, C. and Averhoff, B. (2006). Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27. FEBS J 273(18): 4210-4218.