Biology subjects

Simmons, T. R.

Publications and source records attributed to Simmons, T. R..

3 recordsLinked to original sources

bioRxiv2024

Concentration dependent CsrA regulation of the uxuB transcript leads to development of post-transcriptional BANDPASS Filter

Post-transcriptional control systems offer new avenues to design synthetic circuits that offer reduced burden and less synthetic regulatory components compared to transcriptionally based tools. Herein, we repurpose a newly identified post-transcriptional interaction between the uxuB leader sequence and the E. coli CsrA regulatory protein to design a biological post-transcriptional BANDPASS filter. In this work, we characterize the uxuB mRNA as heterogenous target of the Carbon Storage Regulatory A (CsrA) protein, where the protein can both activate and repress uxuB activity depending on its intracellular concentration. We leverage this interaction to implement a novel strategy of regulation within the 5UTR of an mRNA. Specifically, we report a hierarchical binding strategy that may be leveraged by CsrA within uxuB to result in a dose- dependent response in regulatory outcomes. In our semi-synthetic circuit, the uxuB mRNA leader sequence is used as a scaffold that is fused to a gene of interest, which allows the circuit to transition between ON/OFF states based on a range of free native concentration of the CsrA regulatory protein. Notably, this system exerts regulation comparable to previously developed transcriptional BANDPASS filters while reducing the number of circuit components and can be used in concert with additional controlled circuits to achieve complex multi-signal control. We anticipate that future characterization of native regulatory RNA-protein systems will allow for development of more complex RNP-based circuits for synthetic biology applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/615595v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@107348dorg.highwire.dtl.DTLVardef@b4be0org.highwire.dtl.DTLVardef@1a272a3org.highwire.dtl.DTLVardef@1443c3_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Rewiring native post-transcriptional carbon regulators to build multi-layered genetic circuits and optimize engineered microbes for bioproduction

As the bioprocessing and synthetic biology spaces rapidly expand, post-transcriptional regulation is emerging as driver for maximizing signal response rate and for minimizing cost per protein within cells. Designing robust post-transcriptional control systems that have precise tunability and can achieve diverse regulatory outcomes are paramount to advance the field. Herein, we develop a new approach for engineered post-transcriptional control in bacteria by rewiring a native regulatory system in Escherichia coli, the Carbon Storage Regulatory (Csr) Network, to create tunable, complex genetic circuits. First, by co-opting native components of the Csr Network to regulate translation of a target mRNA transcript, we establish a Csr-regulated Buffer Gate. Next, by rationally engineering the interactions between our synthetic construct and the native components of the Csr Network, we expand our original design into a genetic toolbox of 12 Buffer Gates that achieve precise tunability across a 10-fold range of target gene expression. Subsequently, to further regulatory capabilities using this approach, we develop a Csr-regulated NOT Gate through engineering a Csr-activated sequence into our synthetic constructs. We then build upon the Csr Buffer and Not Gates to create post-transcriptional dual input Boolean OR, NOR, AND and NAND Logic Gates, as well as a genetic pulse circuit. As a third step, we demonstrate portability of our Csr-regulated Buffer Gates into three industrially relevant bacteria by recapitulating Buffer Gate activity simply by leveraging the conserved homologous Csr Network in each species. Lastly, as a demonstration of downstream application, we apply our system to a proof-of-concept synthetic mevalonate pathway. Using our engineered constructs, we optimize mevalonate production in E. coli resulting in a three-fold increase in production relative to a transcriptionally controlled mevalonate pathway. As a whole, we establish a novel approach to rewire post-transcriptional regulatory networks for complex bacterial computation that can be utilized for efficient bioproduction in engineered microbes.

bioengineering↗

bioRxiv2023

CsrA Shows Selective Regulation of sRNA-mRNA Networks

Post-transcriptional regulation, by small RNAs (sRNAs) as well as the global Carbon Storage Regulator A (CsrA) protein, play critical roles in bacterial metabolic control and stress responses. The CsrA protein affects selective sRNA-mRNA networks, in addition to regulating transcription factors and sigma factors, providing additional avenues of cross talk between other stress-response regulators. Here, we expand the known set of sRNA-CsrA interactions and study their regulatory effects. In vitro binding assays confirm novel CsrA interactions with ten sRNAs, many of which are previously recognized as key regulatory nodes. Of those 10 sRNA, we identify that McaS, FnrS, SgrS, MicL, and Spot42 interact with CsrA in vivo. We find that the presence of CsrA impacts the downstream regulation of mRNA targets of the respective sRNA. In vivo evidence supports enhanced CsrA McaS-csgD mRNA repression and showcase CsrA-dependent repression of the fucP mRNA via the Spot42 sRNA. We additionally identify SgrS and FnrS as potential new sRNA sponges of CsrA. Overall, our results further support the expanding impact of the Csr system on cellular physiology via CsrA impact on the regulatory roles of these sRNAs.

molecular biology↗