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The ConcertTM Gel Extraction System was used according to the manufacturer’s instructions (Invitrogen) for separation of a particular PCR product from a mix. The fragment of interest was excised from the gel, the gel slice was cut into small pieces and placed in a microfuge tube. To dissolve the gel slice, 30 μL of gel solubilisation buffer (L1) was added for every 10 μg of gel slice and the tube incubated at 50o_{C for at least}

Table 2.2 PCR primers used in this investigation

Primer name Orientation Sequence (5’-3’) Tm (oC)* Genbank Reference

IR1 Forward GGGGGAACCGCAGAATTC 66.6 Z00027

Mer1 Forward GGAAAATAAAGCACGCTAAGGC 65.0

Mer3 Forward GCATGGCAGGCGCACACC 69.5

Mer4 Forward CGGATGCTGCCATAGGGC 68.2

Mer6 Reverse CATGGTGAACTCCGATCAG 61.8

Mer7 Forward GTCACGCTGTCCGTACCG 68.2 Z00027

PDUMER

Mer9 Reverse CCTAGATGACATGGTCTGC 64.6 PDUMER

Mer10 Reverse CATGGCAGACTCTCCGCG 68.5 Z00027

Mer16 Reverse TGGCGAGGCGGATGTAACG 71.5

Mer19 Forward AATCACCGGCATGACTTGC 66.1 Z00027

AP000342

Mer22 Reverse TGTTGAAGGTCTGCGCCG 68.8 Z00027

Mer23 Forward GCTCGCCCCATATATTTTAG 59.9 PDUMER

Mer24 Reverse GACACTGACGATCGCCAAT 63.8

Mer25 Forward GGCTCTTGATGCCGGGG 69.3 Z00027

Mer27 Forward CTGATGACACGCATTGCCGA 70.7 AP000342

AF120959

Mer28 Reverse CGGCGACACGAAGTCCAG 71.5 AP000342

AF120959

Mer29 Reverse AGSGCAACCTTGACGTGCA 66.4 Z00027

AP000342

Mer32 Reverse ATGCCTTCGTACTTGGCGTG 67.1

Mer33 Forward CACGCCAAGTACGAAGGCAT 67.1

Mer34 Reverse ACGGTCGCCACTTGCGGAT 72.3

Mer35 Forward TGATCGGAGTTCACCATG 60.7 Z00027

Mer36 Reverse GCAAGTCATGCCGGTGATT 66.1 Z00027

AP000342

MerR1 Forward AGGCATAGCCGAACCTGC 65.3 Z00027

MerR2 Reverse GAGAACCTGACCATGGC 60.7

MerT1 Forward TCTGAACCAAAACCGGGC 67.0

MerT2 Reverse GGCATGACGTAGGGAAATC 61.9

MerP1 Forward CTGTTTGCCTCCCTCGC 64.7

MerP2 Reverse TGCTTGACGCTGGACGG 67.8

MercP Forward CCCGATCACWGTCAAGMAVGC 64.4 NM1MER MercA Reverse CGCTCGATCAGCCGWGACVYG 69.5

Pbr8 Forward ATCGGGGAGGCGCCAGAAT 72.1 X71400 Pbr9 Reverse CGCCAGTCGCGAGATGA 67.9 Pbr10 Forward AGGACAGCTTCGCCTTCA 63.8 Pbr11 Reverse CCTTGTTAGCCAGACCT 56.7 Pbr12 Forward TGAGGTACGCGGTCAGTT 62.0 Pbr13 Reverse CTGCGTCTCCTTTCGATT 60.5 Pbr14 Forward TTGTCTTGCGTGGCGAGA 67.1

79

Table 2.2 continued

Primer name Orientation Sequence (5’-3’) Tm (oC)* Genbank Reference Pbr15 Reverse TGCCCGGTGGTGACCAT 69.1 X71400 Pbr16 Forward CAACAGCCCTTCTTGTTC 58.3 Pbr17 Reverse GAGCCAGTACACGACCT 55.9 Pbr18 Forward AGTTCAATCTGGTGCAGC 58.9 Pbr19 Reverse GATCCGCGCCAATGTTGA 69.5

Cad1 Forward GAATGAAGATGGGATGATAA 56.3 PI25CADA

Cad2 Reverse GATTCGCTAGTTTTTCAGGA 58.5

Cad3 Forward GCCCTAGCACATAAGAAAG 56.5

Cad4 Reverse CAGCAACCAAGGCTACAA 60.0

Cad5 Forward CGAAGTATTTGCAGGTACG 58.3

Cad6 Reverse CCCATATCGGAAAGAATCG 61.8

PahAa1 Forward TCACCCGGCGCGMATCRTCAA 73.5 AF036940 AF039533

PahAa2 Reverse CCGCTGGGATAGAASGCATC 66.5

PahAc1 Forward GGGCTGACSCAAAARCACCT 63.0 AF036940 PSENAPDOXA AF039533 AF004283 AF252550 PSEORF1

PahAc2 Reverse CTGTTGTTCGGGAAAACGGTG 68.4

PahAd1 Forward CCACGACGCCGAAGAGTTTC 69.1 PSENAPDOXA

AF039533 PSEORF1

PahAd2 Reverse AGAAGACATCGACTTGATTGCC 64.3

PahC1 Forward CATGGGCATCTCGGTCAAGG 70.0 AF036940

AF039533 PSEORF1

PahC2 Reverse TCAATGAGCCAGCCGGAAGG 71.7

PahE1 Forward GATGCTTCTGAYTGGCGCAG 55.0 AF036940

AF039533 PSEORF1

PahE2 Reverse AACTCCGAAAAGTCGCCACG 54.0

PahF1 Forward AAGCACCCYGTCAGTGGYGAG 66.6 AF036940 AF039533 PSEORF1 AF252550

PahF2 Reverse TTGCCGCAGACCAGCGGATA 56.0

27f (16S) Forward AAGAGTTTGATCCTGGCTCA 62.0 AFARGSSA

1387r (16S) Reverse ACGGGCGGTGTGTACAAGAC 62.0

SP6 Forward ATTTAGGTGACAGTATAGAATAC 50.22

T7 Reverse GTAATACGACTCACTATAGGGC 56.02

*calculated Tm values: provided by Sigma Genosys with the manufacture of primers (http://www.sigma- genosys.com/calc/DNACalc.asp)

Figure 2.5 Location of primers designed to amplify the genes of the pbr operon. This figure indicates the location of and expected size of amplified products for each primer pair. These primers were designed based on the pbr operon of C. metallidurans CH34 (X71400

Figure 2.6 Location of primers designed to amplify the cad operon. This figure indicates the location of and expected size of amplified products for each primer pair. These primers were designed based on the cadCA genes of pI258 (PI25CADA).

Figure 2.7 Location of primers designed to amplify the nahAa and nagAa genes. Primers were designed based on the alignment between the nagAa gene of Ralstonia sp. U2 (AF036940) and the nahAa gene of P. stutzeri (AF039533). Indicated on either side of the gene of interest are the remaining genes in the applicable operon and the total bp flanking this gene within the operon.

Figure 2.8 Location of primers designed to amplify the nahAc, nagAc, ndoAc and pahAc genes. Primers were designed based on the alignment between the nagAc gene of Ralstonia sp. U2 (AF036940), nahAc of P. stutzeri (AF039533), nahAc of P. putida (PSENAPDOXA), ndoC2 of P. fluorescens (AF004283), pahAc of C. testosteroni (AF252550) and pahA3 of P. aeruginosa (PSEORF1). Indicated on either side of the gene of interest are the remaining genes in the applicable operon and the total bp flanking this gene within the operon.

Figure 2.9 Location of primers designed to amplify the nahAd, nagAd and pahA4 genes. Primers were designed based on the alignment between the nahAd gene of P. putida (PSENAPDOXA), the nagAd gene of P. stutzeri (AF039533) and the pahA4 gene of P. aeruginosa (PSEORF1). Indicated on either side of the gene of interest are the remaining genes in the applicable operon and the total bp flanking this gene within the operon.

Figure 2.10 Location of primers designed to amplify the nahC, nagC and pahC genes. Primers were designed based on the alignment between the nagC gene of Ralstonia sp. U2 (AF036940), the, the nahC gene of P. stutzeri (AF039533), and the pahC gene of P. aeruginosa (PSEORF1). Indicated on either side of the gene of interest are the remaining genes in the applicable operon and the total bp flanking this gene within the operon.

Figure 2.11 Location of primers designed to amplify the nahE, nagE and pahE genes. Primers were designed based on the alignment between the nagE gene of Ralstonia sp. U2 (AF036940), the, the nahE gene of P. stutzeri (AF039533), and the pahE gene of P. aeruginosa (PSEORF1). Indicated on either side of the gene of interest are the remaining genes in the applicable operon and the total bp flanking this gene within the operon.

Figure 2.12 Location of primers designed to amplify the nahF, nagF and pahF genes. Primers were designed based on the alignment between the nagF gene of Ralstonia sp. U2 (AF036940), the, the nahF gene of P. stutzeri (AF039533), the pahF gene of P. aeruginosa (PSEORF1) and the pahB gene of C. testosteroni (AF252550). Indicated on either side of the gene of interest are the remaining genes in the applicable operon and the total bp flanking this gene within the operon.

15 minutes, with vortexing every 3 minutes. A further 5 minute incubation was performed once the gel slice had melted.. The spin cartridge (containing a silica matrix) was placed in a 2 mL wash tube (provided with the kit) and the gel solution pipettedinto the cartridge, which was then centrifuged at 12000 rpm for 1 minute. The flow-through liquid was discarded, 500 μL of gel solubilisation buffer was added to the cartridge and incubated for 1 minute at room temperature prior to centrifugation at 12000 rpm for 1 minute. The flow-through liquid was again discarded and 700 μL of wash buffer was added to the cartridge and incubated at room temperature for 5 minutes, then centrifuged for 1 minute. The flow-through was discarded and the cartridge centrifuged again to remove residual wash buffer. The spin cartridge was then placed in a 1.5 mL recovery tube and 50 μL of warm TE buffer was loaded onto the center of the cartridge for elution of the matrix-bound DNA. After incubation for 1 minute at room temperature, the purified DNA was eluted in TE buffer was collected by centrifugation of the cartridge at 12000 rpm for 2 minutes. The size of the purified PCR product was then confirmed by agarose gel electrophoresis.