ena-STUDY-KTH, SCIENCE FOR LIFE LABORATORY-18-03-2021-13:15:47:598-28
Investigation of gene fitness in the versatile chemolithoautotroph Cupriavidus necator using a randomly barcoded transposon mutant library
The aim of this study was to understand how autotrophic (CO2-fixing) bacteria balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. To this end, the proteome of the model chemolithoautotroph Cupriavidus necator (formerly Ralstonia eutropha) was studied in different environmental conditions. Gene essentiality and gene fitness was probed using a randomly barcoded transposon mutant library according to the RB-TnSeq workflow (Wetmore et al., mBio, 2015). Transposon insertions were first mapped to genomic locations by isolating and PCR-amplifying genome fragments containing a transposon (fragment). Transposons can then be identified by a 20 nt barcode. In a second step, the pooled mutant library was cultivated in different selective conditions, foe example fructose-limited chemostats with either continuous or pulsed feed. The contribution to strain fitness of each gene was then probed by determining the enrichment or depletion of knockout mutants from the library over time (0, 8, 16 generations).
The aim of this study was to understand how autotrophic (CO2-fixing) bacteria balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. To this end, the proteome of the model chemolithoautotroph Cupriavidus necator (formerly Ralstonia eutropha) was studied in different environmental conditions. Gene essentiality and gene fitness was probed using a randomly barcoded transposon mutant library according to the RB-TnSeq workflow (Wetmore et al., mBio, 2015). Transposon insertions were first mapped to genomic locations by isolating and PCR-amplifying genome fragments containing a transposon (fragment). Transposons can then be identified by a 20 nt barcode. In a second step, the pooled mutant library was cultivated in different selective conditions, foe example fructose-limited chemostats with either continuous or pulsed feed. The contribution to strain fitness of each gene was then probed by determining the enrichment or depletion of knockout mutants from the library over time (0, 8, 16 generations).