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Supplemental Information

The target sequence generated by primers F-upstream and R-upstream, F-gfp and R-gfp (or R-gfp-truncated)

Keys: Lower case only: corresponds to a region upstream of hoxF up to the translational stop codon of the previous ORF (encodes putative transposase). All transcriptional control elements are located within this region. Caps: gfp sequence from a pGLO vector but translational stop changed to TGA to correspond with the most common translational stop (UGA) found in C. necator. Lower case italics: a portion of the region post hypF2 that includes transcriptional stops. Underlined are the PstI restriction sites.

DOI: 10.7287/peerj.preprints.2120v1/supp-1

Amplifications and ligations of the SH operon elements and the gfp gene

(A) 1.5% agarose gel of PCR amplicons from transcriptional control and stop elements of the soluble hydrogenase operon of C. necatorH16 and a gfp gene in pGLO vector (primary amplification). Lane 1: PCR product from pGLO template generated by primers F-gfp and R-gfp (784 bp), Lane 2: 100bp DNA Ladder (Promega), Lane 3: PCR product from C. necatorH16 chromosomal DNA template generated by F-upstream and R-upstream primers (353 bp). (B) Ligation of primary PCR amplicons and secondary amplification. On the left side is 1% agarose gel of the amplified desired ligation product (secondary amplification). Lane 1: 1 kb DNA Ladder (Promega), Lane 2: An expected 1137 bp PCR product from the ligated DNA fragments (template) and using the F-upstream and R-gfp-truncated primers (relevant band indicated within the red box). On the right side depicted are possible ligations between primary PCR amplicons. A N-Terminal PCR product contains an upstream region of hoxF (grey), whereas a C-Terminal product contains a gfp sequence (green) followed by a downstream region of hypF2 (tan). Phosphorylated ends are shown in purple and PstI sites are in orange. Three possible ligations are i) between two N-terminal products, ii) between an N-terminal product and a C-terminal product (the target insert DNA) and iii) between two C-terminal products.

DOI: 10.7287/peerj.preprints.2120v1/supp-2

Agarose gel visualizations of gene products of cloning steps

(A) 1% agarose gel of the colony PCR product generated from a JM109 transformant harbouring the pGEM-SH::gfp vector. Lane 1: 1 kb DNA Ladder, Lane 2: a 1137 bp PCR product generated from a white colony after transformation (within the red box). (B) 1% agarose gel of the digested fragments. Lane 1: 1 kb DNA Ladder, Lane 2: The PstI-digested pGEM-SH::gfp vector is separated into an approximately 3 kb pGEM-T Easy vector and a 1.1 kb insert fragment of SH operon elements fused to gfp (within the red box), Lane 3: The PstI-digested pJQ200mp18 vector of 5.5 kb (within the blue box) and Lane 4: undigested pGEM-SH::gfp vector. (C) 1% agarose gel of the digested pJQ200mp18-SH::gfp vector. Lane 1: 1 kb DNA Ladder, Lane 2: the 1137 bp insert fragment released from the PstI-digested pJQ200mp18-SH::gfp vector isolated from a white colony after transformation (within the red box). (D) 1% agarose gel of the colony PCR product generated from a transformant harbouring the pJQ200mp18-SH::gfp vector in E. coli S17-1 cells. Lane 1: 1 kb DNA Ladder, Lane 2: the 1137 bp PCR product generated from a white colony after transformation (within the red box). (E) 1% agarose gel of the colony PCR product generated from a transconjugant C. necator H16::gfp cell. Lane 1: the 800 bp PCR product generated from a transconjugant colony after conjugation (within the red box), Lane 2: 1 kb DNA Ladder. (F) 1% agarose gel of amplicons generated from C. necator H16::gfp cells. Lane 1: the 800 bp PCR product generated from a transconjugant with primers F-gfp and R-recombination (within the red box), Lane 2: the 1.14 kb PCR product generated from a transconjugant with primers F-upstream and R-gfp-truncated (within the blue box), Lane 3: 1 kb DNA Ladder.

DOI: 10.7287/peerj.preprints.2120v1/supp-3

Fluorescence of purified and cellular recombinant GFP

Emission spectrum of extracted protein (507nm), at different excitation wavelengths, with maxima observed at 392 and 475 nm, is shown to coincide with that of native GFP.

DOI: 10.7287/peerj.preprints.2120v1/supp-4

Additional Information

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Bat-Erdene Jugder conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Jeffrey Welch conceived and designed the experiments, analyzed the data, wrote the paper, reviewed drafts of the paper.

Nady Braidy performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Christopher P Marquis conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.

Data Deposition

The following information was supplied regarding data availability:

Sequence information supplied as supplementary files.

Funding

Bat-Erdene Jugder received support from AusAID for the provision of an Australian Development Scholarship. Nady Braidy is the recipient of an Alzheimer's Australia Viertel Foundation Postdoctoral Research Fellowship and a National Health and Medical Research Early Career Research Fellowship at the University of New South Wales. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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