SelProm: A Queryable and Predictive Expression Vector Selection Tool for Escherichia coli

Abstract

The rapid prototyping and optimization of plasmid-based recombinant gene expression is one of the key steps in the development of bioengineered bacterial systems. Often, multiple genes or gene modules need to be coexpressed, and for this purpose compatible, inducible plasmid systems have been developed. However, inducible expression systems are not favored in industrial processes, due to their prohibitive cost, and consequently the conversion to constitutive expression systems is often desired. Here we present a set of constitutive-expression plasmids for this purpose, which were benchmarked using fluorescent reporter genes. To further facilitate the conversion between inducible and constitutive expression systems, we developed SelProm, a design tool that serves as a parts repository of plasmid expression strength and predicts portability rules between constitutive and inducible plasmids through model comparison and machine learning. The SelProm tool is freely available at .

Context‐dependency of synthetic minimal promoters in driving gene expression: a case study

“…The lack of robustness for synthetic promoters would fail their applications. For instance, when used to express the isobutanol synthetic pathway, the J23100, which is characterized to be stronger than P L lacO 1 (Jervis et al ., ), can only produce 0.1 g l −1 isobutanol, which is significantly lower than that of the P L lacO 1 and the natural promoter rrnB p1 (Fig. S1).…”

“…Therefore, the predefined performance of these synthetic promoters may not be reproduced once their sequence contexts are varied in actual situations, and discrepancies among different studies have been witnessed. For example, the J23105 promoter has shown transcriptional activities 95% lower than the commonly used promoter P L lacO 1 (Qi et al, 2012), or comparable to the P L lacO 1 (Jervis et al, 2019). Ruling out the interferences from factors such as the copy number, the type of the reporter protein and the stage of the cell cycle, difference in the upstream sequences among these studies is a non-negligible factor that jeopardizes the robustness of the synthetic promoters.…”

“…These promoters have the potential to be independent of the endogenous transcription regulation and function more robustly than those naturally derived ones (Gilman and Love, 2016;Portela et al, 2017). Some synthetic promoters such as the J231 family in the Registry of Standard Biological Parts have gained wide application in gene circuit construction (Qi et al, 2012), tool development (Jervis et al, 2019), chemical production (Meng et al, 2016;Choi et al, 2017;Zhou et al, 2017) and the characterization of biological systems (Pasotti et al, 2012). However, inconsistencies in their transcriptional strength and responses to environmental signals are often encountered (Pasotti et al, 2012;Kosuri et al, 2013;Zucca et al, 2015;Jervis et al, 2019), for which promoters exhibiting high activities in certain conditions might become the weak ones under another context.…”

“…Some synthetic promoters such as the J231 family in the Registry of Standard Biological Parts have gained wide application in gene circuit construction (Qi et al, 2012), tool development (Jervis et al, 2019), chemical production (Meng et al, 2016;Choi et al, 2017;Zhou et al, 2017) and the characterization of biological systems (Pasotti et al, 2012). However, inconsistencies in their transcriptional strength and responses to environmental signals are often encountered (Pasotti et al, 2012;Kosuri et al, 2013;Zucca et al, 2015;Jervis et al, 2019), for which promoters exhibiting high activities in certain conditions might become the weak ones under another context. The lack of robustness on the performance of synthetic promoters can be partially attributed to the host genetic background, the gene copy number and the environmental factors, while the potential influence of the upstream sequence on the promoter strength has not yet been widely noticed.…”

“…Most synthetic promoters contain only the core promoter elements, which are the r factor binding sites and the spacer (Jervis et al, 2019). The r factor binding sites are usually the derivatives of the naturally defined -10 and -35 boxes, while the spacer is usually a 17-bp random fragment placed in between the two binding sites.…”