bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2020.01.26.920280

Robustness testing and scalability of phosphate regulated promoters useful for two-stage autoinduction in E. coli

Moreb, E. A.·Ye, Z.·Efromson, J. P.·Hennigan, J. N.·Menacho-Melgar, R.·Lynch, M. D.
View original source ↗Export metadata ↓

Abstract

A key challenge in synthetic biology is the successful utilization of characterized parts, such as promoters, in different biological contexts. We report the robustness testing of a small library of E. coli PhoB regulated promoters that enable heterologous protein production in two-stage cultures. Expression levels were measured both in a rich Autoinduction Broth as well as a minimal mineral salts media. Media dependent differences were promoter dependent. 4 out of 16 promoters tested were identified to have tightly controlled expression which was also robust to media formulation. Improved promoter robustness led to more predictable scale up and consistent expression in instrumented bioreactors. This subset of PhoB activated promoters, useful for two-stage autoinduction, highlight the impact of the environment on the performance of biological parts, and the importance of robustness testing in synthetic biology. HighlightsO_LICharacterization of the impact of media on promoter activity C_LIO_LIIdentification of promoters robust to environmental variables C_LIO_LIIdentification of promoters whose expression scale from microtiter plates to bioreactors C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/920280v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@94f035org.highwire.dtl.DTLVardef@264edaorg.highwire.dtl.DTLVardef@102ee30org.highwire.dtl.DTLVardef@163d16e_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

Source connections

Explore related subjects

synthetic biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Moreb, E. A., Ye, Z., Efromson, J. P., Hennigan, J. N., Menacho-Melgar, R., Lynch, M. D.. 2020-01-26. Robustness testing and scalability of phosphate regulated promoters useful for two-stage autoinduction in E. coli. https://doi.org/10.1101/2020.01.26.920280

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Creation of genomic chimeras via megabase scale genome expansion

An organism's physiology is controlled by its genomic content. Building genomic chimeras would enable melding of the diverse functions and properties of life. However, prior arts in genome synthesis are limited to reconstituting pre-existing functions within a singular genome rather than combining diverse genomic functions across multiple distinct genomes. Existing methods are also prohibitively expensive, labor intensive, and time consuming, and thus fundamentally not scalable for creating large synthetic chimeric genomes. To address all these limitations, we invented Additive Conjugative-CAST Engineering (ACE) combining conjugation with CRISPR-associated transposition (CAST) to deliver and integrate up to half a genome per step from a donor cell into a precisely defined position in the recipient's genome. We have demonstrated ACE's capability to integrate at least a 2-megabase donor genomic segment in a single step and at least 3 megabase in two consecutive steps. Importantly, we confirmed ACE's generality through the creation of multiple genomic chimeras across species, genus, and at least order barriers. With ACE, we further demonstrate such chimeric organisms, or what we call genome expanded organisms (GEOs), can be stably forged from at least three starting bacterial strains, and can be stably maintained to express all their acquired genomic parts in a single chimera. ACE offers a new paradigm of creating artificial lifeforms to combine desired properties and potentially create novel functions beyond the constraints of nature. ACE also offers a unique perspective to probe unexplored plasticity in genome architecture and expression patterns of novel, massively expanded genomes.

synthetic biology↗
bioRxiv2026

Functional analysis of small-molecule and protein binding domains using monomeric streptavidin-based two-component biosensors

Protein biosensors are important analytical and diagnostic tools that convert molecular recognition into measurable activity. Most common two-component biosensors require two non-competing recognition domains, yet binding affinity and specificity alone do not reliably predict their compatibility with the rest of the biosensor. We report a modular platform that exploits monomeric streptavidin (mSA2) as a surrogate recognition domain, enabling rapid functional evaluation of candidate binders in the context of biosensor scaffold without requiring a second ligand-specific binding protein. We established a chromatography-free protocol for recovering functional mSA2-based biosensor components from Escherichia coli. Using biotinylated small molecules methotrexate and vitamin D3, as well as heart failure protein biomarkers galectin-3 and S100A7 we demonstrate ligand-dependent reporter biosensor activation. The developed platform provides a scalable approach for rapid binder evaluation and biosensor prototyping, streamlining biosensor development using binding domains generated by directed evolution or computational protein design.

synthetic biology↗
bioRxiv2026

Developing synthetic auxin-based trans-kingdom signaling

Synthetic trans-kingdom signaling systems with tunable transduction properties provide a unique avenue to explore how communication between organisms coordinate behaviors without the confounding complexity of natural systems. However, existing trans-kingdom signaling systems suffer from either toxic signal molecules, a need for rare precursors for signal generation, or transduction machinery which is either species-specific or cannot be easily tuned. This limits their capacity to be broadly applied across kingdoms and their utility as a model system to study trans-kingdom interactions. In this work we elucidate the design principles for a trans-kingdom signaling system based on the plant phytohormone, Indole 3-Acetic Acid (auxin), that overcomes these challenges. We demonstrate how auxin biosynthesis can be engineered into a model sender organism, the cyanobacteria Synechococcus elongatus PCC 7942, and how this signal can be tuned with low leak and high dynamic range. We also show these signals can be connected to gene expression using tunable synthetic auxin-responsive signaling systems across multiple kingdoms. We demonstrate that S. elongatus generated auxin signals can tune gene expression in co-cultured Saccharomyces cerevisiae via auxin-induced degradation. Additionally, we show that when the S. elongatus are associated with the roots of the model plant, Arabidopsis thaliana, the auxin they generate can control gene expression via auxin-responsive synthetic transcription factors expressed from the plants genome. Taken together, our results demonstrate the design principles for a tunable auxin-based trans-kingdom signaling system that can be broadly applied across species.

synthetic biology↗