5.3 Isolation of Genomic DNA from the strains of M elsdenii
5.3.1 PCR cloning and sequencing of gene coding for Short-Chain Acyl-CoA
5.3.1.3 SCAD gene sequence analysis
Purified PCR amplification products of the SCAD gene for the strain ATCC 25940, ME5 and ME7 of M. elsdenii are shown in Figure 5.6 were used as a template for cyclo-sequencing reactions. The primers used for sequencing were MEF–4 (forward) and MER–7 (reverse) for 5’–3’ and 3’–5’ direction respectively. The nucleotide sequence of 1.55 kb fragment of three strains was determined using the ABI-PRISM Dye Terminator Cycle Sequencing Kit in an automated DNA sequencer as described in section 2.6.12. After sequence assembly (Figure 5.7), the completed sequence of the SCAD gene (1.55 kb) of the three M. elsdenii strains was found to represent the whole gene. A sequence similarity search based on the NCBI database was performed using ANGIS and BLASTN analysis, which showed that the SCAD gene was identical to the published sequence for M. elsdenii Accession number L04528. Figure 5.7 shows the multiple sequences alignment of the SCAD gene sequences of strains ATCC 25940, ME5, ME7 and Accession no. L04528, which was performed using a multiple comparison program Clustal.
Figure 5.7: Multiple sequence alignment of the Short-chain Acyl-CoA dehydrogenase (SCAD) gene sequence of M. elsdenii strains ATCC 25940, ME5, ME7 and database sequence L04528 using a multiple comparison program (Clustal).
10 20 30 40
1 ATGGATTTTA ACTTAACAGA TATTCAACAG GACTTCTTAA L04528
1 ATGGATTTTA ACTTAACAGA TATTCAACAG GACTTCTTAA ME7
1 ATGGATTTTA ACTTAACAGA TATTCAACAG GACTTCTTAA ATCC 25940
1 ATGGATTTTA ACTTAACAGA TATTCAACAG GACTTCTTAA ME5
50 60 70 80
41 AACTCGCTCA TGATTTCGGC GAAAAGAAAT TAGCACCGAC L04528
41 AACTCGCTCA TGATTTCGGC GAAAAGAAAT TAGCACCGAC ME7
41 AACTCGCTCA TGATTTCGGC GAAAAGAAAT TAGCACCGAC ATCC 25940
41 AACTCGCTCA TGATTTCGGC GAAAAGAAAT TAGCACCGAC ME5
90 100 110 120
81 CGTTACGGAA CGCGACCACA AAGGTATTTA TGACAAAGAA L04528
81 CGTTACGGAA CGCGACCACA AAGGTATTTA TGACAAAGAA ME7
81 CGTTACGGAA CGCGACCACA AAGGTATTTA TGACAAAGAA ATCC 25940
81 CGTTACGGAA CGCGACCACA AAGGTATTTA TGACAAAGAA ME5
130 140 150 160
121 CTCATCGACG AATTGCTCAG CCTCGGTATT ACCGGCGCTT L04528
121 CTCATCGACG AATTGCTCAG CCTCGGTATT ACCGGCGCTT ME7
121 CTCATCGACG AATTGCTCAG CCTCGGTATT ACCGGCGCTT ATCC 25940
121 CTCATCGACG AATTGCTCAG CCTCGGTATT ACCGGCGCTT ME5
170 180 190 200
161 ACTTCGAAGA AAAATACGGC GGTTCCGGCG ATGACGGCGG L04528
161 ACTTCGAAGA AAAATACGGC GGTTCCGGCG ATGACGGCGG ME7
161 ACTTCGAAGA AAAATACGGC GGTTCCGGCG ATGACGGCGG ATCC 25940
161 ACTTCGAAGA AAAATACGGC GGTTCCGGCG ATGACGGCGG ME5
210 220 230 240
201 CGACGTTTTG AGCTACATCC TCGCTGTTGA AGAATTGGCT L04528
201 CGACGTTTTG AGCTACATCC TCGCTGTTGA AGAATTGGCT ME7
201 CGACGTTTTG AGCTACATCC TCGCTGTTGA AGAATTGGCT ATCC 25940
250 260 270 280
241 AAATACGACG CTGGTGTTGC TATCACCTTG TCGGCAACGG L04528
241 AAATACGACG CTGGTGTTGC TATCACCTTG TCGGCAACGG ME7
241 AAATACGACG CTGGTGTTGC TATCACCTTG TCGGCAACGG ATCC 25940
241 AAATACGACG CTGGTGTTGC TATCACCTTG TCGGCAACGG ME5
290 300 310 320
281 TTTCCCTTTG CGCTAACCCG ATTTGGCAGT TCGGTACAGA L04528
281 TTTCCCTTTG CGCTAACCCG ATTTGGCAGT TCGGTACAGA ME7
281 TTTCCCTTTG CGCTAACCCG ATTTGGCAGT TCGGTACAGA ATCC 25940
281 TTTCCCTTTG CGCTAACCCG ATTTGGCAGT TCGGTACAGA ME5
330 340 350 360
321 AGCTCAGAAA GAAAAATTCC TCGTTCCTTT GGTTGAAGGC L04528
321 AGCTCAGAAA GAAAAATTCC TCGTTCCTTT GGTTGAAGGC ME7
321 AGCTCAGAAA GAAAAATTCC TCGTTCCTTT GGTTGAAGGC ATCC 25940
321 AGCTCAGAAA GAAAAATTCC TCGTTCCTTT GGTTGAAGGC ME5
370 380 390 400
361 ACTAAACTCG GCGCTTTCGG CTTGACCGAA CCGAACGCAG L04528
361 ACTAAACTCG GCGCTTTCGG CTTGACCGAA CCGAACGCAG ME7
361 ACTAAACTCG GCGCTTTCGG CTTGACCGAA CCGAACGCAG ATCC 25940
361 ACTAAACTCG GCGCTTTCGG CTTGACCGAA CCGAACGCAG ME5
410 420 430 440
401 GTACTGATGC TTCCGGCCAG CAGACCATTG CTACGAAGAA L04528
401 GTACTGATGC TTCCGGCCAG CAGACCATTG CTACGAAGAA ME7
401 GTACTGATGC TTCCGGCCAG CAGACCATTG CTACGAAGAA ATCC 25940
401 GTACTGATGC TTCCGGCCAG CAGACCATTG CTACGAAGAA ME5
450 460 470 480
441 CGATGACGGC ACTTACACGT TGAACGGCTC CAAGATCTTC L04528
441 CGATGACGGC ACTTACACGT TGAACGGCTC CAAGATCTTC ME7
441 CGATGACGGC ACTTACACGT TGAACGGCTC CAAGATCTTC ATCC 25940
441 CGATGACGGC ACTTACACGT TGAACGGCTC CAAGATCTTC ME5
490 500 510 520
481 ATCACCAACG GCGGCGCTGC TGACATCGAC ATTGTCTTCG L04528
481 ATCACCAACG GCGGCGCTGC TGACATCGAC ATTGTCTTCG ME7
481 ATCACCAACG GCGGCGCTGC TGACATCGAC ATTGTCTTCG ATCC 25940
481 ATCACCAACG GCGGCGCTGC TGACATCGAC ATTGTCTTCG ME5
530 540 550 560
521 CTATGACCGA TAAGAGCAAA GGCAACCACG GCATTACAGC L04528
521 CTATGACCGA TAAGAGCAAA GGCAACCACG GCATTACAGC ME7
521 CTATGACCGA TAAGAGCAAA GGCAACCACG GCATTACAGC ATCC 25940
521 CTATGACCGA TAAGAGCAAA GGCAACCACG GCATTACAGC ME5
570 580 590 600
561 CTTCATCCTC GAAGACGGTA CTCCGGGCTT TACTTACGGC L04528
561 CTTCATCCTC GAAGACGGTA CTCCGGGCTT TACTTACGGC ME7
561 CTTCATCCTC GAAGACGGTA CTCCGGGCTT TACTTACGGC ATCC 25940
610 620 630 640
601 AAGAAAGAAG ACAAGATGGG CATCCATACT TCGCAGACCA L04528
601 AAGAAAGAAG ACAAGATGGG CATCCATACT TCGCAGACCA ME7
601 AAGAAAGAAG ACAAGATGGG CATCCATACT TCGCAGACCA ATCC 25940
601 AAGAAAGAAG ACAAGATGGG CATCCATACT TCGCAGACCA ME5
650 660 670 680
641 TGGAACTCGT ATTCCAGGAC GTCAAAGTTC CGGCTGAAAA L04528
641 TGGAACTCGT ATTCCAGGAC GTCAAAGTTC CGGCTGAAAA ME7
641 TGGAACTCGT ATTCCAGGAC GTCAAAGTTC CGGCTGAAAA ATCC 25940
641 TGGAACTCGT ATTCCAGGAC GTCAAAGTTC CGGCTGAAAA ME5
690 700 710 720
681 CATGCTCGGC GAAAGAAGGCA AAGGCTTCAA GATTGCTATG L04528
681 CATGCTCGGC GAAAGAAGGCA AAGGCTTCAA GATTGCTATG ME7
681 CATGCTCGGC GAAAGAAGGCA AAGGCTTCAA GATTGCTATG ATCC 25940
681 CATGCTCGGC GAAAGAAGGCA AAGGCTTCAA GATTGCTATG ME5
730 740 750 760
721 ATGACCTTGG ACGGCGGCCG TATCGGCGTT GCTGCTCAGG L04528
721 ATGACCTTGG ACGGCGGCCG TATCGGCGTT GCTGCTCAGG ME7
721 ATGACCTTGG ACGGCGGCCG TATCGGCGTT GCTGCTCAGG ATCC 25940
721 ATGACCTTGG ACGGCGGCCG TATCGGCGTT GCTGCTCAGG ME5
770 780 790 800
761 CTCTCGGCAT TGCAGAAGCT GCTTTGGCAG ATGCTGTTGA L04528
761 CTCTCGGCAT TGCAGAAGCT GCTTTGGCAG ATGCTGTTGA ME7
761 CTCTCGGCAT TGCAGAAGCT GCTTTGGCAG ATGCTGTTGA ATCC 25940
761 CTCTCGGCAT TGCAGAAGCT GCTTTGGCAG ATGCTGTTGA ME5
810 820 830 840
801 ATACTCCAAA CAGCGTGTAC AGTTCGGCAA ACCGCTCTGC L04528
801 ATACTCCAAA CAGCGTGTAC AGTTCGGCAA ACCGCTCTGC ME7
801 ATACTCCAAA CAGCGTGTAC AGTTCGGCAA ACCGCTCTGC ATCC 25940
801 ATACTCCAAA CAGCGTGTAC AGTTCGGCAA ACCGCTCTGC ME5
850 860 870 880
841 AAATTCCAGT CCATTTCCTT CAAACTGGCT GACATGAAGA L04528
841 AAATTCCAGT CCATTTCCTT CAAACTGGCT GACATGAAGA ME7
841 AAATTCCAGT CCATTTCCTT CAAACTGGCT GACATGAAGA ATCC 25940
841 AAATTCCAGT CCATTTCCTT CAAACTGGCT GACATGAAGA ME5
890 900 910 920
881 TGCAGATCGA AGCTGCTCGT AACCTCGTTT ACAAAGCTGC L04528
881 TGCAGATCGA AGCTGCTCGT AACCTCGTTT ACAAAGCTGC ME7
881 TGCAGATCGA AGCTGCTCGT AACCTCGTTT ACAAAGCTGC ATCC 25940
881 TGCAGATCGA AGCTGCTCGT AACCTCGTTT ACAAAGCTGC ME5
930 940 950 960
921 TTGCAAGAAA CAGGAAGGCA AACCCTTCAC CGTTGACGCT L04528
921 TTGCAAGAAA CAGGAAGGCA AACCCTTCAC CGTTGACGCT ME7
921 TTGCAAGAAA CAGGAAGGCA AACCCTTCAC CGTTGACGCT ATCC 25940
970 980 990 1000
961 GCTATCGCAA AACGCGTTGC TTCCGACGTC GCTATGCGCG L04528
961 GCTATCGCAA AACGCGTTGC TTCCGACGTC GCTATGCGCG ME7
961 GCTATCGCAA AACGCGTTGC TTCCGACGTC GCTATGCGCG ATCC 25940
961 GCTATCGCAA AACGCGTTGC TTCCGACGTC GCTATGCGCG ME5
1010 1020 1030 1040
1001 TAACGACCGA AGCTGTCCAG ATCTTCGGCG GCTATGGCTA L04528
1001 TAACGACCGA AGCTGTCCAG ATCTTCGGCG GCTATGGCTA ME7
1001 TAACGACCGA AGCTGTCCAG ATCTTCGGCG GCTATGGCTA ATCC 25940
1001 TAACGACCGA AGCTGTCCAG ATCTTCGGCG GCTATGGCTA ME5
1050 1060 1070 1080
1041 CAGCGAAGAA TATCCGGTTG CTCGTCACAT GCGCGATGCT L04528
1041 CAGCGAAGAA TATCCGGTTG CTCGTCACAT GCGCGATGCT ME7
1041 CAGCGAAGAA TATCCGGTTG CTCGTCACAT GCGCGATGCT ATCC 25940
1041 CAGCGAAGAA TATCCGGTTG CTCGTCACAT GCGCGATGCT ME5
1090 1100 1110 1120
1081 AAGATTACTC AGATCTACGA AGGCACGAAC GAAGTTCAGC L04528
1081 AAGATTACTC AGATCTACGA AGGCACGAAC GAAGTTCAGC ME7
1081 AAGATTACTC AGATCTACGA AGGCACGAAC GAAGTTCAGC ATCC 25940
1081 AAGATTACTC AGATCTACGA AGGCACGAAC GAAGTTCAGC ME5
1130 1140 1150 1160
1121 TCATGGTTAC AGGCGGTGCT CTGTTAAGAT AA L04528
1121 TCATGGTTAC AGGCGGTGCT CTGTTAAGAT AATTGAAGTT ME7
1121 TCATGGTTAC AGGCGGTGCT CTGTTAAGAT AATTGAAGTT ATCC 25940
1121 TCATGGTTAC AGGCGGTGCT CTGTTAAGAT AATTGAAGTT ME5
1170 1180 1190 1200
1161 L04528
1161 TATGCTCGGG CCTGGCCCTT TGCTGGGCCC GTTACATAAA ME7
1161 TATGCTCGGG CCTGGCCCTT TGCTGGGCCC GTTACATAAA ATCC 25940
1161 TATGCTCGGG CCTGGCCCTT TGCTGGGCCC GTTACATAAA ME5
1210 1220
1201 L04528
1201 AAAAGATTTT AGGAGGCAAAA ME7
1201 AAAAGATTTT AGGAGGCAAAA ATCC 25940
1201 AAAAGATTTT AGGAGGCAAAA ME5