Open Access Highly Accessed Research article

Genetic changes during a laboratory adaptive evolution process that allowed fast growth in glucose to an Escherichia coli strain lacking the major glucose transport system

César Aguilar1, Adelfo Escalante1*, Noemí Flores1, Ramón de Anda1, Fernando Riveros-McKay12, Guillermo Gosset1, Enrique Morett1 and Francisco Bolívar1

Author Affiliations

1 Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología. Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, 62210, México

2 Winter Genomics, México D.F. 07300, Cuernavaca, Morelos, 62210, México

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BMC Genomics 2012, 13:385  doi:10.1186/1471-2164-13-385

Published: 10 August 2012

Additional files

Additional file 1Additional file 1:

Table S1. Mutations in coding regions detected by Roche NimbleGen Inc. This table includes the data provided by Roche NimbleGen Inc. (RN) for the whole genome sequence analysis of the evolved PB12 strain. Section A lists 26 (21+5) non-synonymous point mutations in structural genes that accordingly to RN changed the coding regions of these genes. In fact only in 21 of these genes detected also by Winter Genomics Inc (WG), the mutations occurred (Table 1A). Section B presents the list of the 12 genes included in a large deletion in this strain. This list also includes the genes deleted in the parental PB11 strain, for the construction of this derivative lacking PTS (Figure 2). The table also includes (Section C), the list of the 20 genes in which synonymous point mutations occurred accordingly to RN. Those 16 in common with WG are in bold letters.

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Additional file 2:

Table S2. Mutations in coding regions detected by Winter Genomics Inc. This table lists the data provided by Winter Genomics Inc. (WG) obtained for the whole genome sequence of the PB12 strain in comparison to the parental strains JM101 and PB11. Importantly, PB11 strain was sequenced by WG and the same nucleotide sequences as in the parental strain JM101 were determined (data not shown). Therefore all the point mutations detected in PB12 by RN and WG appeared during the laboratory evolution process. Section A includes a list of 27 genes (21+6) in which, accordingly to this company non-synonymous point mutations occurred changing the coding regions in structural genes. 21 of these genes were also detected by RN (Table 1A). The table also includes (Section B) the list of 18 genes in which synonymous mutations also occurred, accordingly to WG. Those 16 in common with RN are in bold letters.

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Additional file 3:

Figure S1. Nucleotide sequence of the chromosomal genes fusion that occurred in the evolved PB12 strain. This figure includes the nucleotide sequence of the genomic region where the deletion occurred (Figures 3 and 4) between the ptsP and the galR genes in the PB12 strain.

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Additional file 4:

Table S3. Oligonucleotides employed in this study. This table lists the oligonucleotides utilized in this work. Section A shows the oligonucleotides used for DNA sequencing with the Sanger method, including those for the confirmation of the deletion that occurred in the PB12 strain (Figures 2, 3 and 4), the reported mutations provided by RN and WG (Table 1 and Tables S1 and S2 in Additional file 1 and Additional file 2) and the ones employed for gene disruption confirmation. Section B lists the oligos utilized for gene disruption with the Datsenko-Wanner methodology [46]. Section C lists the oligonucleotides utilized for RT-qPCR analysis not previously reported. The sequences of the oligos utilized for the remaining genes listed in Table 3, have been previously published [9-11] (see Materials and Methods).

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Figure S2. Amplification curves for the ihfB gene in different strains. This figure shows the positions of the amplification curves for the ihfB gene (Section A) and the Ct values of this gene (Section B), in the different strains employed in this study. As can be seen, all the amplification curves of the ihfB gene that has been used as the reference gene for the determination of the RT-qPCR levels, have very similar values. The values presented in the table are from three different fermentations (F1, F2 y F3) for each utilized strain. Since all the values included in section B are very similar, only one third of them are presented (labeled with an asterisk *) in section A. These results demonstrate that the same reproducible expression levels were obtained for the ihfB gene in all strains. This is the most important characteristic that a reference gene should have, in agreement to the MIQE guidelines [59-61]. These results corroborate the stability of the expression of the reference ihfB gene in these strains in the utilized conditions. (EPS 308 kb)

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