Obtaining a Panel of Cascade Promoter-5′-UTR Complexes in Escherichia coli

Abstract

A promoter is one of the most important and basic tools used to achieve diverse synthetic biology goals. Escherichia coli is one of the most commonly used model organisms in synthetic biology to produce useful target products and establish complicated regulation networks. During the fine-tuning of metabolic or regulation networks, the limited number of well-characterized inducible promoters has made implementing complicated strategies difficult. In this study, 104 native promoter-5'-UTR complexes (PUTR) from E. coli were screened and characterized based on a series of RNA-seq data. The strength of the 104 PUTRs varied from 0.007% to 4630% of that of the P promoter in the transcriptional level and from 0.1% to 137% in the translational level. To further upregulate gene expression, a series of combinatorial PUTRs and cascade PUTRs were constructed by integrating strong transcriptional promoters with strong translational 5'-UTRs. Finally, two combinatorial PUTRs (P-UTR and P-UTR) and two cascade PUTRs (PUTR-PUTR and PUTR-PUTR) were identified as having the highest activity, with expression outputs of 170%, 137%, 409%, and 203% of that of the P promoter, respectively. These engineered PUTRs are stable for the expression of different genes, such as the red fluorescence protein gene and the β-galactosidase gene. These results show that the PUTRs characterized and constructed in this study may be useful as a plug-and-play synthetic biology toolbox to achieve complicated metabolic engineering goals in fine-tuning metabolic networks to produce target products.

A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

“…(e) Promoter strengths at the transcriptional level with methyl laurate as the sole carbon source. The promoter strength was defined as described previously (S. H.Zhou et al, ). The data represent the mean ± standard deviations of biological triplicates [Color figure can be viewed at wileyonlinelibrary.com]…”

“…Cellular fluorescence (485 nm excitation, 510 nm emission) and density (OD 600 ) were measured using the Cytation TM 5 cell imaging multi‐mode reader system (BioTek, Winooski, VT). The promoter strength was defined as the fluorescence (in relative fluorescence units) divided by the cell density (OD 600 ; S. H. Zhou et al, ).…”

Fine‐tuning the (2S)‐naringenin synthetic pathway using an iterative high‐throughput balancing strategy

“…Based on our previous study (J. Wu, Zhou et al, ), the optimal expression ratio of (2 S )‐naringenin biosynthetic genes are Tctal : Pc4cl : Phchs : Mschi = 1:1:10:10. Hence, in the first round of screening, 16 promoters (PUTR ssrA , PUTR dnaKJ , PUTR rplNXE , PUTR grpE , PUTR alsRBACE , PUTR trxA , PUTR rrnG , PUTR sdhCDAB , PUTR rrnH , PUTR yccFS , PUTR yibN , PUTR infC‐rplT , PUTR racR , PUTR talB , PUTR rpmH , and PUTR ssrA ‐PUTR infC‐rplT ) with an activity levels from 0.049‐ to 46.3‐fold of that of the P BAD promoter (S. Zhou, Ding et al, ) were used to regulate the expression of Tctal and Pc4cl genes. Fifteen promoters (PUTR ssrA ‐PUTR infC‐rplT , P ssrA ‐UTR rpsT , P grpE ‐UTR rpsT , P dnaKJ ‐UTR rpsT , PUTR alsRBACE ‐PUTR infC‐rplT , PUTR rpsU , PUTR rrnA , PUTR glpD , PUTR ssrS , PUTR accBC , PUTR mdh , PUTR rplNXE , PUTR trxA , PUTR sdhCDAB , and PUTR rrnG ) with activity levels from 0.44‐ to 46.3‐fold of that of the P BAD promoter (S. Zhou, Ding et al, ) were used to regulate the expression of Phchs and Mschi genes.…”

“…However, such optimization approaches are often limited by a lack of genetic tools, such as promoters and plasmids. More specifically, a key element in balancing metabolic fluxes is the availability of promoters with gradient strengths (S. Zhou, Ding et al, ; S. Zhou, Du et al, ). In addition, testing such promoters in multigene pathways results in an astronomical number of combinations.…”

Construction of Synthetic Promoters by Assembling the Sigma Factor Binding −35 and −10 Boxes

“…In the past decade, many well‐characterized promoters have been utilized for building biosensors, gene circuits, as well as constructing biosynthetic pathways for chemicals, biofuels, and natural products . There are many strong promoters with a wide range of potential applications have been characterized . However, most natural promoters have long sequences and unidentified regulatory elements that may interfere with gene expression.…”