Improved dynamic range of a rhamnose-inducible promoter for gene expression in Burkholderia

A diverse genetic toolkit is critical for understanding bacterial physiology and genotype-phenotype relationships. Inducible promoter systems are an integral part of this toolkit. In Burkholderia and related species, the L-rhamnose-inducible promoter is among the first choices due to its tight control and the lack of viable alternatives. To improve upon its maximum activity and dynamic range, we explored the effect of promoter system modifications in B. cenocepacia with a LacZ-based reporter. By combining the bacteriophage T7 gene 10 stem loop and engineered rhaI transcription factor-binding sites, we obtained a rhamnose-inducible system with a 6.5-fold and 3.0-fold increase in maximum activity and dynamic range, respectively, compared to the native promoter. We then added the modified promoter system to pSCrhaB2 and pSC201, common genetic tools used for plasmid-based and chromosome-based gene expression, respectively, in Burkholderia, creating pSCrhaB2plus and pSC201plus. We demonstrated the utility of pSCrhaB2plus for gene expression in B. thailandensis , B. multivorans and B. vietnamiensis and used pSC201plus to control highly expressed essential genes from the chromosome of B. cenocepacia . The utility of the modified system was demonstrated as we recovered viable mutants to control ftsZ , rpoBC , and rpsF , whereas the unmodified promoter was unable to control rpsF . The modified expression system allowed control of an essential gene depletion phenotype at lower levels of L-rhamnose, the inducer. pSCRhaB2plus and pSC201plus are expected to be valuable additions to the genetic toolkit for Burkholderia and related species. Importance Species of Burkholderia are dually recognized as being of attractive biotechnological potential but also opportunistic pathogens for immunocompromised individuals. Understanding the genotype-phenotype relationship is critical for synthetic biology approaches in Burkholderia to disentangle pathogenic from beneficial traits. A diverse genetic toolkit, including inducible promoters, is the foundation for these investigations. We thus sought to improve on the commonly used rhamnose-inducible promoter system. Our modifications resulted in both higher levels of heterologous protein expression and broader control over highly-expressed essential genes in B. cenocepacia . The significance of our work is in expanding the genetic toolkit to enable more comprehensive studies into Burkholderia and related bacteria.

Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

ABSTRACTPrecisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

Upstream development of Escherichia coli fermentation process with PhoA promoter using design of experiments (DoE)

In this work, a fed-batch fermentation development was performed with recombinant E. coli carrying the PhoA promoter system. The phosphate concentrations tested for this PhoA strain, 2.79 mM to 86.4 mM, were beyond the concentrations previously evaluated for cell growth and product titer. The results from the scouting work was used for design of experiments (DoE) where a range of phosphate levels from 27.1 mM to 86.4 mM was simultaneously evaluated with temperature, pH and DO set points. Definitive screening was used to evaluate these parameters simultaneously and the results indicate that fermentation temperature and phosphate content are the major contributors of product titer. The other factors tested such as pH had a minimal effect and DO had no impact on product titer.

Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.