Extended Data Fig. 2: Δrpi is a rubisco-dependent E. coli strain with a growth rate that correlates to rubisco flux.
a) Schematic of the Δrpi strain of rubisco-dependent E. coli. PRK and rubisco compensate for the deletion of RPI and rescue growth. b) Growth rates and yields across a titration of rubisco induction by [IPTG]. (N = 4) c) Growth rates and yields across a titration of [CO2]. Yields were calculated up to 40 h. (N = 4) d) A heatmap of growth rates across a two-dimensional titration of CO2 and IPTG. e) Growth rates and yields across a titration of [O2]. Yields were calculated between 15 and 40 h. The BW25113 contained the same plasmid as Δrpi but with GFP in place of rubisco. Growth rates could not be calculated for the control due to non-exponential growth behavior. (N = 6) f) Immunoblots for soluble rubisco with DnaK as a loading control. Left half is wild-type R. rubrum rubisco, right half is the higher-expressing I164T mutant. Samples are of Δrpi cells grown in selection media (see Methods) with different concentrations of IPTG. g) Growth rates of Δrpi cells expressing either WT or I164T rubisco grown in selection media with different concentrations of IPTG. (N = 4) h) Ratio of band intensities from f as a function of IPTG concentration. i) A panel of mutants from the literature and their associated kcat measurements normalised to WT. The WT value is ≈11/s. j) Growth curves of Δrpi expressing the mutants from i. Colouring in i and j is on the same scale and reflects kcat values from the literature. k) Growth rate values calculated from the curves in j, plotted against the normalised kcat values. l) Raw barcode-averaged mutant enrichment values for the same mutants as in k measured in one nanopore sequencing experiment. Error bars in b, c, g and e determined from the SEM of at least four replicates. Error bars in k determined as standard deviations of three or more replicates. Error bars in l determined as standard deviations of three different barcodes (N = 3) for each mutant. Errors in literature values are shown from studies where they were reported.
