1.Introduction
The study of mycobacterial genomes has exploded during the last 10 yr. Initially, no systems were available for the direct manipulation of mycobacterial genes in mycobacteria, so Escherichia coli was used as the primary cloning host. Several genomic libraries were created
(1-5) in E.coli. Although these proved useful for the identification of many protein antigens (6), the use of E.coli as a cloning host has several limitations. It is now known that many mycobacterial promoters do not function at all in E.coli; therefore, it is difficult to study the expression and control of mycobacterial genes in such a host. In addition, certain posttranslational modifications of proteins do not take place in E.coli and therefore the antigenicity and properties of proteins expressed in E.coli may differ (7-9).
The development of electroporation techniques for use with both fast-and slow-growing mycobacterial species has provided a means for the genetic manipulation of mycobacteria directly. Many types of DNA have been introduced into various mycobacterial species using this technology, including plasmids, cosmids, Integrating vectors, and transposon-delivery vectors. The use of high-efficiency electroporation in conjunction with these vector systems has enabled many studies to be performed; for example, investigations into mycobacterial-gene expression (10), the production of recombinant BCG vaccines (11), and the production of transposon mutant libraries (12).
The fast-growing Mycobacterium smegmatis is now commonly used as a cloning host for the study of genes from pathogenic mycobacteria. Although M.smegmatis mc26, taken from ATCC607, showed poor transformation efficiencies by electroporation, a mutant strain designated mc2155 that shows transformation efficiencies of 104-105 per μg of DNA (13) was isolated. Using plasmid and phage DNAs, evidence has also been provided that M.smegmatis mc2155 does not possess a restriction and modification system (13). This is an obvious advantage because it means that exogenous DNAs will not be degraded upon entry into the cell.
Although M.smegmatis has been the most widely-used species, many other species of mycobacteria have been transformed using electroporatrion, including Mycobacterium bovis BCG (14-17) Mycobacterium vaccae (l8-20), Mycobacterium phlei (21), Mycobacterium w (19), Mycobacterium fortuitum(17,21), Mycobacterium aurum(22,23), Mycobacterium intracellulare (24), and Mycobacterium parafortuitum(25). The efficiencies for electroporation vary among different mycobacterial species (Table 1). Other factors that can influence the efficiency are the DNA used and the selectable marker carried on the transforming DNA. The differences observed in efficiencies found for different markers may reflect the stabilities of the gene products in the mycobacterial cell. In addition, certain mycobacterial-plasmid replicons may not function in particular species, for example, pAL5000-based vectors have been unable to successfully transform M.intracellulare(24).
Several vector systems have been developed for use in mycobacteria, including plasmids, integrating vectors, and transposon-delivery systems. Vectors for use in mycobacteria must carry suitable selectable markers; the choice of selection markers is critical and is dependent on the particular species of mycobacteria being used. Table 2 shows selection markers that have been successfully used for various species. Most slow-growers possess only one ribosomal RNA (rDNA)operon; this unusual situation means that resistance to agents such as kanamycin can easily be acquired by mutation in the rDNA operon itself (26), which is not likely to occur where there is more than one operon. Most of the fast-growers contain two rDNA operons, and therefore have a much lower rate of spontaneous resistance to kanamycin. For M. smegmatis, kanamycin resistance has been widely used (27), because the commonly used mc2155 strain is sensitive to kanamycin at low concentrations and the rate of spontaneous mutation to kanamycin resistance for mc2l55 is 10-7 to 10-9 (27,28). Kanamycin resistance has also been used as a selectable marker in M. aurum, M. parafortuitum, Mycobacterium tuberculosis, and M. bovis BCG (15,22,25,29,30).
An alternative to kanamycin resistance, is hygromycin resistance, which has been used in both M. smegmatis and BCG 16. Vectors carrying kanamycin resistance have been unable to transform at least two species that are potential vaccine candidates, M. w and M. vaccae (19). Hygromycin resistance has been successfully used as a selectable marker in these species (19), and more recently apramycin resistance has been used (31).
It has been reported that tetracycline can be used in M. smegmatis(32); however, this antibiotic is not suitable for slow-growing mycobacteria because tetracycline is unstable over the time required for culture (3-6 wk). Ampicillin resistance is not suitable for use in mycobacteria because they are naturally resistant to β-lactams. Chloramphenicol resistance cannot be used for direct selection owing to the high rate of spontaneous mutations (10-4 to 10-5) (13), although it has been used in conjunction with other antibiotic-resistance genes (13,21).
Genes conferring resistance to sulfonamides(32), gentamicin(33), or streptomycin(25) may also be used for selection, although it may not be advisable to introduce such antibiotic resistances into pathogenic mycobacteria because of biohazard considerations, nor would they be appropriate for use in live recombinant vaccines owing to the possibility of transfer to other pathogenic bacteria. (Streptomycin, sulfonamides, and kanamycin may all be used in treatment of infections caused by pathogenic mycobacteria.) Therefore nonantibiotic resistance markers are required for use, such as the L5-phage superimmunity gene, which confers resistance to mycobacteriophage L5 infection(34) or mercury-resistance genes (35).
2. Materials
2.1. Growth of Mycobacteria and E. coli
1 Lowenstein Jensen (LJ) slopes (Difco, West Molsey, Surrey, UK): store at 40C
2 Middlebrook 7H9 broth (Difco) dissolve in deionized water at 4.7 g per 900 mL and autoclave.
3 Middlebrook ADC enrichment (Difco), containmg bovine albumin fraction V, dextrose, catalase, and NaCl- store at 40C (see Note 3)
4 Middlebrook OADC enrichment (as ADC with oleic acid, see Note 3), (Difco), store at 40C
5 Tween-80 (Sigma, Poole, Dorset, UK) prepare as a 20% v/v stock, filter sterilize through a 0.2-μm membrane and store at 40C (see Note 5)
6 For fast-growing species such as M. smegmatis, media should be prepared using 7H9 broth supplemented with 10% v/v ADC and 0.05% v/v Tween-80.
7 For slow-growing species such as M. tuberculosis or M. bovis BCG, media should be prepared using 7H9 broth supplemented with 10% v/v OADC and 0.05% v/v Tween-80.
8 2M glycine (Analar grade, Sigma), autoclave (see Note 7).
9 LB (Luria-Bertani) broth (Difco) for culture of E. coli should be prepared at 25 g/L and autoclaved. Agar for plates should be added at 15 g/L prior to autoclavmg.
2.2. Preparation of Electrocompetent Cells
1 10% v/v glycerol; sterilize by autoclaving.
2 Electroporation cuvets, 0.2-cm gap electrodes (see Note 12)
3 Electroporation apparatus with pulse controller (see Notes 11 and 12)
4 DNA in solution (see Notes 10 and 13), this should be free from salts, enzymes and other substances. In order to clean up DNA, it can be ethanol precipitated and thoroughly washed with 70% ethanol (this will also remove excess salts) The concentration of DNA should be approximately 0.2-1 mg/mL
5 Lemco broth. 10 g/L peptone, 5 g/L Lemco powder, 5 g/L NaCl, autoclave and supplement with 0.05% v/v final concentration Tween-80. For plates, Tween-80 should be omitted, and agar should be added to 1.5% final concentration prior to autoclaving
2.3. Selection of Transformants
1 Middlebrook 7H10 agar, 19 g agar base per 900 mL, autoclave.
2 Middlebrook ADC or OADC enrichment (see Subheading 2.1. and Note 3)
3 Cycloheximide (Sigma) stock solution of 20 mg/mL in water; store at -20℃(see Note 16)
4 For plates, Middlebrook 7H10 agar should be supplemented with 10% v/v ADC enrichment (fast-growing species) or 10% v/v OADC enrichment (slow-growing species) plus selection antibiotic. Plates for slow-growing species must be poured thick (i.e., 90 mm diameter plates with 40 mL agar) to prevent drying out during the long incubation period required for growth, cycloheximide should be added to a final concentration of 100 μg/mL (see Note 16)
5 Kanamycin sulphate (Sigma), 50 mg/mL stock (filter sterilize), store at -20℃
6 Hygromycin B (Boehringer Mannheim, Lewes, East Sussex, UK), obtained as a 50 mg/mL stock in phosphate buffered salme; store at 4!ˉC in the dark 7 Selection plates for kanamycm resistance should contain 10-30 pg/mL kanamycm, and for hygromycm resistance, 50-100 μg/mL (see Table 3 for other antibiotics)
2.4. Plasmid Preparation
1 GET buffer; 50 mM glucose, 10 mM EDTA, pH 8.0, 25 mM Tris-HCl, pH 8.0, filter-sterilize through 0.2-μm membrane. EDTA should be prepared as a 0.5M solution and buffered to pH 8.0 with NaOH-EDTA only dissolves fully at pH 8.0-before diluting to final concentration. Lysozyme (Sigma) should be added to 10 mg/mL before use.
2 Alkaline SDS solution; 0.2M NaOH, 1% sodium dodecyl sulphate (SDS) SDS should be prepared as a 20% solution and autoclaved
3 3M Na acetate, pH to 4.8 with glacial acetic acid and sterilize by autoclaving
2.5. Preparation of Electrocompetent E. coli
10 mM Tris-HCl, pH 7.5, 1 mM MgCl2; Tris buffer should be prepared as a 1M stock, buffered with HCl and autoclaved. MgCl2 should be prepared as a 1M stock; filter-sterilize through a 0.2-um membrane. Dilute with sterile distilled water to achieve final concentration. Use LB broth and LB agar plates containing suitable antibiotic for selection of transformants (see Subheading 2.1.)
3. Methods
Caution: Because some mycobacterial species are pathogenic to humans, appropriate containment facilities for each species should be used for all procedures (see Note 1).
3. 1. Mycobacterial Culture
Mycobacteria should be maintained in the laboratory by regular subculture on LJ slopes. Inocula for overnight or small cultures can be taken directly off the slope (see Note 2).
3. 1. 1 Fast-Growing Species
Inoculate 5 mL Lemco broth with a loopful of mycobacteria; the cells can be dispersed using a vortex (see Notes 2 and 5). Incubate at 37℃ with shaking (100 rpm) overnight.
Inoculate large scale culture (100-500 mL in 250-1000 mL conical flask) with a 1/100 dilution of the overnight culture and continue incubation at 37℃ with shaking until OD600 = 0.8-1.0(usually between 16-24 h; see Note 5).
3.1.2. Slow-Growing Species
1 Inoculate 5 mL 7H9 broth (containing OADC and Tween-80) with a loopful of mycobacteria, vortex to disperse cells, and incubate at 37℃ with shaking (100 rpm) for 10-15 d (see Note 2).
2 Use this culture to inoculate a further 10 mL of broth and Incubate for 10-15 d.
3 Inoculate a large-scale culture 100-500 mL with 10 mL of culture and continue incubation at 37℃ with shaking until OD600 is 0.5-1.0, usually between 14-28 d incubation (see Notes 5 and 6).
4 Optional: add 0.1 volumes 2M glycine (final concentration is 15% w/v) 24 h before harvesting cells (see Note 7).
3.2. Preparation of Electrocompetent Cells
1 Incubate cells on ice for 1.5 h (see Notes 8 and 13) before harvesting by centrifugation at 3000g for 10 min. This improves transformation efficiencies fourfold.
2 Wash cells three times in ice-cold 10% glycerol. Reduce the volume each time; e.g., for 100mL, wash one, 25 mL; wash two, 10 mL; and wash three, 5 mL. Finally, resuspend in 1/100 to 1/500 original culture volume of ice-cold 10% glycerol (see Note 9).
3 At this stage, cells may frozen in a dry ice/ethanol bath and stored in aliquots at-70℃ for future use, Cells frozen in this way should be thawed on ice and used as required (see Note 9)
3.3. Electroporation
1 Add approx 1 μg salt-free DNA (no more than 5 μL volume; see Notes 10 and 13) to 0.4 mL mycobacterial suspension and leave on ice for 10 min (see Note 9) Transfer to a 0.2 cm electrode-gap electroporation cuvet (see Note 9) The cuvet should be chilled on ice before use (see Note 13)
2 Place cuvet in electroporation chamber and subject to one single pulse of 25 kV, 25 μF, with the pulse-controller resistance set at 1000 Ω resistance (see Notes 9 and 11-13).
3 Put cuvet back on ice for 10 min, transfer cell suspension to a sterile universal bottle, add 5mL Lemco broth, and incubate at 37℃ for 2 h for fast-growers (see Note 14); add 5 mL 7H9 (plus OADC and Tween) and incubate at 37℃ for at least 3 h for slow-growers. This step allows expression of any antibiotic-resistance gene carried on the DNA (see Notes 14 and 18).
4 Harvest cells by centrifugation at 3000g for 10 min and plate out suitable dilutions (to give 30-300 colonies per plate) on 7HlO agar plus ADC or OADC enrichment and appropriate antibiotic (see Notes 15 and 16).
5 Incubate plates at 37℃ until colonies become visible, this will take 3-7 d for fast-growers and 2-4 wk for slow-growers (see Table 4 for species requirements) When using slow-growing species, the plates must be sealed with parafilm to prevent desiccation (see Note 16). 
3.4. Growth of Mycobacterial Transformants
3.4.1. Fast-Growing Species
Inoculate 5 mL Lemco broth plus selection antibiotic with transformant colonies. Incubate at 37℃ with shaking (100 rpm) for 2-3 d.
3.4.2. Slow-Growing Species
Inoculate 5 mL 7H9 (plus OADC and Tween) containing appropriate selection antibiotic with transformant colonies. Incubate at 37℃ with slow shakmg (100 rpm) for 2 wk.
3.5. Plasmid Preparation from Mycobacteria
1 Harvest 1.5 mL and resuspend in 100 μL GET containing 10 mg/mL lysozyme and incubate at 37℃ for 24 h.
2 Add 200 μL alkaline SDS, incubate at 37℃ for 1 h
3 Add 150 μL 3M Na acetate, pH 4.8, incubate on ice for 1 h.
4 Spin 15 mm in microcentrifuge at 10,000g and recover supernatant
5 Precipitate plasmid DNA with 800 μL absolute ethanol overnight at -20℃ recover by 15 mm spin in a microcentrifuge at 10,000g, and redissolve pellet in 40 μL TE.
6 Plasmid DNA can be used to retransform E. coli for larger-scale plasmid preparation, for direct visualization of plasmid DNA, 20 μL can be electrophoresed on a 0.8% agarose gel (see Note 17) See Chapter 4 for preparation of plasmids/cosmids for direct analysis.
3.6. Electroduction Between Mycobacteria and E. coli
3.6.1. Preparation of Electrocompetent E. coli
1 Inoculate 100 mL of LB broth with a loopful of E. coli, incubate at 37℃ with shaking (250 rpm) until OD660 = 0.6
2 Incubate cells on ice for 10 min
3 Harvest cells at 3000g for 10 mm and wash in 10 mM Tris-HCl, pH 7.5, 1 mM MgCl2
4 Resuspend cells in same buffer at 1o9 cells/ml (OD660 = 20), roughly 1/30 of original volume
3.6.2. Electroduction of E. coli
1 Pick a transformed mycobacterial colony from selection plate, patch onto a new plate and mix the remainder of the colony with 20 μL ice-cold glycerol (see Note 17).
2 Vortex and leave on ice for 10 min.
3 Add to 0.4 mL E. coli electrocompetent cells, leave on ice for 10 min
4 Place in electroporation chamber and give a single pulse of 2.5 kV, 4.5 μF (no pulse controller required)
5 Transfer cells to sterile universal, add 4 mL LB and incubate for 1 h at 37℃
6 Plate on LB agar containing appropriate antibiotic for 12-16 h.
7 Slow-growing species such as M. bovis BCG should yield 10-100 E. coli colonies, M.smegmatis should yield 1o4 E. coli transformants (see Note 17).
4. Notes
1 Pathogenic mycobacteria represent an important biohazard, therefore, all culture and genetic manipulation must be carried out in appropriate containment facilities inside a Class I safety cabinet. In most countries, genetic manipulation involvmg pathogenic mycobacteria or their DNA must be met with approval by the relevant authorities. In any case, risk assessment must form the first part of any experiment with pathogenic mycobacteria. A list of mycobacterial species and the type of containment required should be consulted prior to use.
2 Mycobacteria are relatively slow-growing organisms; the fast-growing species have a generation time of 2-3 h and the slow-growing species of around 20 h. This often leads to a problem with contamination of cultures because many common contaminants have a much quicker doubling time and will rapidly outgrow mycobacteria. It is extremely important to maintain a good aseptic technique, especially with slow-growers. It is often wise to set up duplicate cultures in case one becomes contaminated. Cultures should be checked for purity using acid-fast staining at all stages (36)
3 Both ADC and OADC supplements are extremely heat-labile and should only be added to 7H10 or 7H9 media after cooling. M. bovis BCG can be grown in medium supplemented with ADC rather than OADC, but growth is slower. Growth of mycobacterial cultures is enhanced by the provision of up to 10% CO2 in the an above the medium.
4. Mycobacteria have chemically-resistant cell walls that are difficult to lyse, thus they are able to survive high voltages even when pulses have long time constants. Several factors, outlined below, affect the efficiency of transformation; these include the growth phase of cells when harvested, electroporation media, and the field strength and time constant of the delivered pulse
5 Growth of cells: mycobacterial cells, particularly M. tuberculosis, have a tendency to clump together in culture, this is owing to the thick waxy nature of the mycobacterial coat. The addition of Tween-80, a nonionic detergent, to media reduces the amount of clumping and provides a more homogenous suspension of cells. The medium used for growth of mycobacteria for electroporation is not important and a variety of different recipes are used, the most common being Middlebrook 7H10. In general, mycobacterial cultures should be removed from the incubator when in the logarithmic phase of growth. In contrast to E. coli, the cells can be harvested at any point from the early to late log phase (A600 of 0.2-10)
6 Because mycobacteria have the ability to remain dormant for long periods of time, cultures may contain many cells that are not in an active phase of growth. The maintenance of slow-growing species in a mid-log phase of growth by regular dilution of cultures over several months improves the efficiency of transformation (probably by increasing the number of cells actively growing) (37)
7 For slow-growing species such as BCG, the addition of glycine (to a final concentration of 15%) to young growing cultures can improve transformation efficiencies (37,38) Glycine affects the cell wall of mycobacteria and presumably makes DNA entry easier. Ideally, glycine should be added 1-2 days prior to harvesting. The best efficiencies are obtained with younger cultures of 4-7 d (15).
8 Preincubation on ice. Once cultures have reached the required stage of growth, they should be removed from the incubator and incubated on ice for 1.5 h prior to harvesting. This results in a fourfold increase in transformation efficiency (39). Longer incubations on ice result in reduced efficiency, probably owing to increased cell lysis. This may also increase the possibility of arcing during pulse delivery. Recently, it has been reported that incubation of slow-growers at room temperature and the use of reagents warmed to 37℃ during the preparation of competent cells results in improved transformation efficiencies, whereas the preparation of M. smegmatis competent cells was more efficient at 0℃ (40)
9 Pulse delivery it is important to have an even cell suspension for electroporation because any clumping of cells will lead to arcing and a reduced transformation efficiency. During the standing time on ice prior to pulse delivery, the cells may settle in the tube and it is necessary to redistribute them using a pipet or a vortex immediately prior to the high-voltage pulse. This step serves to resuspend the cells and to ensure thorough mixing of the DNA. In addition, care must be taken to ensure that no bubbles are introduced between the two electrodes of the cuvet imediately prior to the high-voltage pulse. This step serves to resuspend the cells and to ensure thorough mixing of the DNA In addition, care must be taken to ensure that no bubbles are mtroduced between the two electrodes of the cuvet. It is recommended that competent cells that have been thawed from frozen should be harvested and resuspended in fresh 10% glycerol prior to use
10 DNA concentratron: The efficiency of electroporation depends on the choice of DNA for transformation; some vectors have been unable to transform particular mycobacterial species and the efficiency often depends on the choice of selectable marker (see Table 2). The efficiency of transformation is not affected by the amount of DNA added; addition of 0 5-500 ng DNA produces the same efficiency, and as much as 5 μg can be used. However, the volume of DNA used is critical; for small volumes of cell suspensions, the addition of a large amount of DNA in water will alter the conductivity of the suspension. Therefore, it is important that not more than 5 μL of DNA solution are added to the cell suspension
11 Pulse conditions. the use of a pulse controller in addition to the actual electroporation apparatus allows control over the parallel resistance and therefore the time constant; higher parallel resistance produces a longer time constant. Observations have shown that the optimum time constant is 15-25 ms (1000 ohms resistance) (39). The use of 0.2-cm electrode gap cuvets, as opposed to the 0.4-cm gap cuvets originally used, results in a higher field strength (39). The electroporation medium also has an effect on the time constant. Use of diluted glycerol provides a high-resistance medium, allowing longer time constants to be achieved
12 There are many different electroporation devices available commercially, we use the BioRad Gene Pulser, but any apparatus that can deliver high-voltage pulses can be used. There are also different makes of cuvets available, although the gap or path length may be the same, the maximum volume of the cell suspension can vary from 50 μL to 400 μL. The volume of cell suspension used does not seem to affect the efficiency (16), so it does not matter which cuvets (and therefore what volume of cells) are used.
13 Arcing the use of the pulse-controller apparatus serves to reduce the probability of arcing when using high-voltages applied to high-resistance media, although it may still occur. Factors that cause arcing include the presence of lysed cells in the sample, salts in the DNA solution, and electroporation with cuvets that have not been chilled on ice. These factors can be minimized by ensuring that during preparation of electrocompetent cells, the preincubation on ice is no longer than 1.5 h. Also, ensure that the cuvets are chilled on ice before use, make sure that the outside of the cuvet is dry before placing in the pulse chamber, the cuvet slide may also be chilled. Always ensure that the DNA for transformation is free from salts and other containnants; ethanol precipitation and washing with 70% ethanol can be used to clean up DNA, which should preferably be dissolved in sterile deionized, distilled water. The settings for the pulse are important as well. Increasing the parallel resistance to ∞Ω increases the possibility of arcing, therefore a setting of 1000 Q produces more consistent results. In some cases, arcing may be violent enough to blow the lid off the electroporation cuvet, dispersing the cell suspension over the inside of the electroporation chamber (thereby creating aerosols). When working with pathogenic organisms, it is imperative that the pulse is delivered with the electroporation chamber placed inside a Class I safety cabinet and that appropriate disinfectants (freshly diluted 2% Hycolin) are at hand. For nonpathogenic species, the pulse can be delivered on the bench, but Hycolin should be available to deal with any spills
14 The dilution of cells immediately after the pulse is important. Cells should be diluted at least 1o-fold and incubated for several h prior to plating. Omission of this step leads to greatly reduced efficiencies (41). Presumably, the dilution allows better recovery from the pulse and therefore greater survival of transformants. Slow-growers should be incubated for 3-16 h before plating out
15 The problem of clumping is also important when plating out cells after electroporation. It is important to ensure that resistant colonies have arisen from single cells, so the cells must be thoroughly resuspended before plating. For particularly "sticky" cells, the suspension may be passed through a 23-gage needle several times prior to plating. Caution: This must not be attempted with pathogenic mycobacteria owing to the risk of needle-stick injuries. Appropriate dilutions may also help to alleviate this problem by thinning out the suspension. It is important to dilute cells prior to plating because the cell suspensions used for electroporation are very concentrated. If the cells are not diluted, then it may be very difficult to visualize true resistant colonies against a background lawn of sensitive cells. This is owing to aggregation, which protects some cells from the effects of the antibiotic
16 Because slow-growing organisms take up to 4 wk to form colonies from single cells, it is important to pour plates thickly and to wrap them securely in parafilm to prevent drying out during the incubation period. Cycloheximide can be added to plates at 100 μg/mL to prevent fungal contamnation, which is possible during such long incubation times. If fungal contamination is recurrent, it may be better to use Lemco-agar plates with cycloheximide, because 7H10 agar appears to interfere slightly with the action of cycloheximide (L. Brooks, personal communication). The long incubation period also means that antibiotic-containing plates should be freshly poured for each experiment, because this will minimize the loss of antibiotic activity
17 Several methods for the preparation of plasmid DNA from mycobacterial species exist(39,42) Plasmids can be isolated from mycobacteria for direct analysis (see Chapter 4). However, owing to the low-copy-number of most mycobacterial vectors, the yield may still be too low for subsequent analysis. Therefore it is often advisable to retransform plasmlds isolated from mycobacteria into E. coli (using a recA deletion strain such as TG2 or JM109) and prepare plasmid DNA from the more amenable species. Because all mycobacterial species used in electroporation are wild-type with respect to recA, recombmation and rearrangements of plasmid DNA can and do occur, therefore, it is important to check the identity of all DNAs introduced into mycobacteria. An alternative method is to use direct electroduction from mycobacteria to E. coli (43); this involves taking a mycobacterial colony, resuspending it in small amount of glycerol, and mixing with electrocompetent E. coli. The mixture is then pulsed using conditions suitable for E. coli electroporation, and E. coli transformants are selected for on media containing the relevant antibiotic. E. coli transformants appear after 16 h, which is much sooner than the appearance of the mycobacterial colonies (2-3 d). This method is much quicker, and may be more convenient for slow-growing species, where the growth of mycobacterial transformants for plasmid preparation may take 2-4 wk. It is important that not too much of the agar is carried over in this step because this will result in arcing. Transferring too many mycobacterial cells should also be avoided because this can give problems with background growth.
18 Spontaneous kanamycin resistance can often be a problem with slow-growers, owing to mutations to the rRNA operon, of which slow-growing species possess only one. Therefore, it is always necessary to perform a control electroporation with no DNA to check for the frequency of such mutants. The problem is usually less pronounced with fast-growers which possess two rRNA operons. Problems of spontaneous resistance will apply to all antibiotics that act on rRNA(e.g., streptomycin).
19 We have found that the use of hygromycin at 50 μg/mL leads to a high level of background growth resulting in a lawn. Therefore we recommend using 100 μg/mL as selection for mycobacteria. Some strains of E. coli require as much as 250 μg/mL for successful selection
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