Abstract
Cupriavidus necator H16 can convert CO2 into industrial chemicals and fuels. To facilitate its engineering, we designed, built and tested the pMTL70000 modular plasmids comprising standardised Cupriavidus and E. coli replicons, selectable markers and application specific modules. Plasmids were characterised in terms of transmissibility, stability, copy number and compatibility.
Highlights
Cupriavidus necator, is a nonpathogenic, Gram-negative, aerobic chemolithoautotroph
Plasmid stability was assessed by estimating the number of cells retaining resistance to plasmid-encoded chloram phenicol after one to nine, 24-h serial passages at 30 ◦C in 0.4% (w/v) sodium gluconate-minimal medium (SG-MM) (Schlegel et al, 1961) lacking antibiotic supplementation
Plasmids based on the Pseudomonas pVS1 replicon were found to most stable (Fig. 2), with more than 97% of the cells maintaining pMTL75111 over the nine days compared to only 9% and 74% in the case of pMTL72111 and
Summary
Cupriavidus necator (formerly Ralstonia eutropha), is a nonpathogenic, Gram-negative, aerobic chemolithoautotroph. The suitability of the various vectors and their replicons for engi neering of C. necator H16 was tested as detailed in the Supplementary information, with respect to segregational stability, plasmid copy number and compatibility. Plasmids based on the Pseudomonas pVS1 replicon were found to most stable (Fig. 2), with more than 97% of the cells maintaining pMTL75111 over the nine days compared to only 9% and 74% in the case of pMTL72111 (pMOL28) and
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