Benedetti, I. | bioRxiv Science

bioRxiv2021

Parameterization of regulatory nodes for engineering broad host range heterologous gene expression

By building on the SEVA (Standard European Vector Architecture) format we have refactored a number of regulatory nodes recruited from both Gram-negative and Gram-positive bacteria for rigorously comparing and parameterizing five expression devices that respond to diverse and unrelated chemical inducers, i.e. LacIq-Ptrc, XylS-Pm, AlkS-PalkB, CprK-PDB3 and ChnR-PchnB. These were assembled as cargoes following the SEVA standard within exactly the same vector backbone and bearing the different functional segments arrayed in an invariable DNA scaffold. Their performance in an Escherichia coli strain of reference was then analyzed through the readout a fluorescence reporter gene that contained strictly identical translation signal elements in all cases and in the same DNA context. This study allowed us to describe and compare the cognate expression systems with unprecedented quantitative detail. The systems under scrutiny diverged considerably in their capacity, expression noise, inducibility and OFF/ON ratios. These features, along with the absence of physiological effects caused by the inducers and the lack of cross-regulation offer a panoply of choices to potential users and help interoperability of the specific constructs.

synthetic biology↗

bioRxiv2020

Standardization of inducer-activated broad host range expression modules: Debugging and refactoring an alkane-responsive AlkS/PalkB device

Although inducible heterologous expression systems have been available since the birth of recombinant DNA technology, the diversity of devices and genetic architectures of the corresponding vectors have often resulted in a lack of reproducibility and interoperability. In an effort to increase predictability of expression of genes of interest in a variety of possible bacterial hosts we propose a composition standard for debugging and reassembling all regulatory parts that participate in the performance of such devices. As a case study we address the n-octane and dicyclopropyl ketone (DCPK)-inducible PalkB promoter of the alkane biodegradation pOCT plasmid of Pseudomonas putida. The standardized expression module consisted of an edited alkS regulatory gene that is divergently expressed and separated of PalkB by a synthetic DNA buffer sequence. The native DNA sequence of the structural alkS gene was modified to alleviate the catabolite repression exerted by some carbon and nitrogen sources through the Crc/Hfq complex of some hosts. The PalkB promoter along with the alkS variants were then formatted as SEVA (Standard European Vector Architecture) cargoes and their activity parameters in P. putida determined with GFP and luminiscent reporters. The thereby refactored system showed improvements in various features desirable in conditional expression modules: inducibility, capacity, noise reduction and on/off ratio. When applied to other promoter/regulator pairs, the compositional standard thereby implemented in the AlkS/PalkB module will enable more complex genetic programming in non-model bacteria.

synthetic biology↗