5.11.1 Purification of the plasmid pIJ86 carrying mmr gene
100 µl of E. coli TOP10 (carrying the DNA construct pIJ86/mmr) from a glycerol stock was added to 5ml of LB medium containing 50 µg/ml apramycin and was incubated at 37 oC, 180 rpm overnight. Plasmid DNA was purified from the cells and the presence of the methylenomyin resistance gene mmr was confirmed by PCR amplification using mmr-fwd and mmr-rev primers provided in Table 5.6 and the components and conditions outlined below. The PCR product was analysed by
Table 5.8: PCR mixture for amplifying mmr
Component of PCR Volume (µl) Purified plasmid DNA 2.0
Buffer with MgCl2 5.0
dNTPs (10mM) 1.0
mmr-fwd (10µM) 1.5
mmr-rev (10µM) 1.5
Taq DNA polymerase 0.5
DMSO 5.0
Distilled water 33.5
TOTAL 50.0
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Initial denaturation 94 oC 5 min Denaturation 94 oC 1 min
Annealing 55 oC 1 min 33x Extension 72 oC 1.5 min
Final extension 72 oC 10 min End and storage 4 oC
agarose gel electrophoresis, which showed a single band corresponding to the 1.4 kb mmr. The construct was kept at – 20 oC pending further use.
5.11.2 Transformation of E. coli ET12567/pUZ8002
Purified plasmid pIJ86/mmr (2 µl) was added to 100 µl of electrocompetent E. coli ET12567/pUZ8002 cells and mixed gently before being transferred to an electroporation cuvette where 1.8 V was applied. 1 ml of ice-cold liquid LB medium was added and the mixture was incubated at 37 oC, 180 rpm for 1 hour. It was thereafter spread over solid LB plates containing apramycin (50 µg/ml), kanamycin (50 µg/ml) and chloramphenicol (25 µg/ml). The plates were incubated at 37 oC for 18 hours to generate individual colonies.
Transformation of the E. coli ET12567/pUZ8002 and E. coli TOP10 by the cosmid C73_787/mmyR::apr and its various mutated derivatives were also carried out in a similar manner. The selective plates however contained only apramycin in the case of TOP10 transformation.
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5.11.3 Conjugation of E. coli ET12567/pUZ8002 with Streptomyces species
The procedure described here was used to conjugate ET12567/pUZ8002 (ET) carrying pIJ86/mmr (from section 5.11.2) with S. coelicolor strains W110, M145 and
S. albus J1074, and also for the conjugation of the ET carrying the cosmid
C73_787/mmyR::apr or any of its mutated derivatives with S. coelicolor, S. albus or
S. lividans as required.
Single colonies of the E. coli ET12567/pUZ8002 carrying the required construct was inoculated into 5 ml LB medium containing kanamycin, chloramphenicol and apramycin. The culture was incubated overnight at 37 oC, and 100 µl was added to
10 ml LB containing the antibiotics and incubated for 5 hours (O.D600 approximately
0.4). The cells were collected by centrifuging at 3000 rpm for 10 minutes, washed twice with liquid LB medium containing no antibiotics, and suspended in 400 µl of LB medium.
400µl of liquid LB medium was added to a 50 µl spore stock of each of the
Streptomyces strains from -80 oC glycerol stocks. The cells were then heat-shocked at
55 oC for 10 minutes and were immediately transferred to an ice bath for 5 minutes. They were then mixed with the fresh cells of E. coli ET12567/pUZ8002 carrying the required construct. The mixture was centrifuged for 2 minutes at 10,000 rpm, half of the supernatant discarded, and the cells were resuspended in the remaining liquid. The resulting suspension was spread over three 25 ml solid SFM plates containing 100µl of 2.5 M MgCl2 and incubated overnight at 30 oC. The plates were overlayed in the
morning with 1ml of sterile water containing 25 µl of apramycin and 20 µl of nalidixic acid. The plates were then incubated at 30 oC for an additional 5 days.
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Single colonies of the resulting Streptomyces transconjugants were picked and cultured on SFM plates containing apramycin and nalidixic acid. The inclusion of nalidixic acid ensured any residual E. coli that might be picked with the Streptomyces colonies were all eliminated. The plates were then incubated for 7 days and the spore stocks were generated. Strains W110 and M145 carrying the pIJ86/mmr construct were named W301 and W302 respectively. Table 5.3 provides the names of
Streptomyces transconjugants carrying the cosmid C73_787 or its derivatives.
Integration of the cosmid into these Streptomyces species was confirmed by LC-MS analysis of the resulting transconjugants which showed that they produced methylenomycin antibiotics and/or the methylenomycin furans relative to their respective wild type strains. Genetic confirmation was obtained for strains W301 and W302 as detailed in section 5.11.4.
5.11.4Colony PCR to confirm the presence of mmr in W301 and W302
W301 and W302 strains were inoculated on SFM agar plates and were incubated at 30 oC for 4 days. Wild type S. coelicolor M145 was also grown in parallel under the same conditions. Colonies of each of the strains were lysed using the following procedure: 1 loop of the spores was scrapped from the plates and 30 µl of fast screening lysis buffer was added. The mixture was incubated at 37 oC for 2 hours before adjusting to pH 8 with 1M HCl. The content was cooled on ice for 10 minutes and centrifuged at 13,200 rpm for 10 minutes. 20 µl of the resulting supernatant was diluted to 100 µl with sterile distilled water. 2 µl of this solution was analysed by PCR using the same primers and conditions outlined in section 5.11.1. The PCR products were analysed by agarose gel electrophoresis.
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