Team:OUC-China/Description
1. Where we start
In synthetic biology, microorganisms with modified metabolic pathways are employed as a reaction vessel to synthesis natural or unnatural products. So it involves the introduction of several genes encoding the enzymes of a metabolic pathway . Indeed, pathway optimization requires to adjust expression of multiple genes at appropriately balanced levels for higher production , for example the synthesis of poly(3-hydroxybutyrate) and Mevalonate . As is done in the prokaryotes, grouping a cluster of genes into a single polycistron is a convenient means for regulating genes simultaneously. Thus, for the sake of tuning the expressions of genes within polycistrons, we aim to develop a tightly regulated, predictable components –stem loop to realize cistron concerto.
2. Look closely to the existing strategies
Several prominent strategies have been employed to regulate differential expression of serial genes such as copy number, promoters and RBS . Compared to their obvious advantages, they are limited by high-flux design. When traditional cloning is utilized, constructing libraries of hundreds of configurations of pathway genes with varying copy number, promoter, and RBS strengths is a daunting and time consuming task even for small pathways . Ergo, we are inspired to have a nice try about rational design of one regulated element to reduce the number of trials and transform it into a user-friendly kit. With brainstorms and information collection, we turn to focus on the prior design of the DNA sequences to work on the post- transcriptional level for it can directly determine the relative levels of gene expression.
3. Inspiration from Nature
In C. cellulolyticum, within an polycistron encoding cellulosome, stem-loop structures associated with those intergenic post-transcriptional processed sites located at 3’ termini of highly transcribed genes exhibit folding free energy negatively correlated with transcript-abundance ratio of flanking genes. . Thus we consider the possibility of stem loops inserted in the intergenic region for tuning expression in E.coli which has been widely utilized as an engineered strain.
Fortunately, Keasling has identified that stem loops function well in the post-transcriptional process in E.coli . And he confirmed that TIGR(tunable intergenic regions) sequences generally had a stronger influence on the expression of the gene 3’to the TIGR than on the gene 5’ to the TIGR.
Thus, we decided to develop stem loops within intergenic region as a regulatory elements to coordinate expressions.
4. Our story
Khosla, C. & Keasling, J.D. Metabolic engineering for drug discovery and development. Nat. Rev. Drug Discov. 2, 1019–1025 (2003).
Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J.D. & Keasling, J.D. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21, 796–802 (2003).
Li T, Ye J, Shen R, et al. Semi-rational approach for ultra-high poly (3-hydroxybutyrate) accumulation in Escherichia coli by combining one-step library construction and high-throughput screening[J]. ACS synthetic biology, 2016.
Dudley Q M, Anderson K C, Jewett M C. Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate Synthesis[J]. ACS Synthetic Biology, 2016.
Song C W, Lee J, Ko Y S, et al. Metabolic engineering of Escherichia coli for the production of 3-aminopropionic acid[J]. Metabolic engineering, 2015, 30: 121-129.
Jones, J.A., O.D. Toparlak, and M.A. Koffas, Metabolic pathway balancing and its role in the production of biofuels and chemicals. Curr Opin Biotechnol, 2015. 33: p. 52-9
Xu C, Huang R, Teng L, et al. Cellulosome stoichiometry in Clostridium cellulolyticum is regulated by selective RNA processing and stabilization[J]. Nature communications, 2015, 6.
Smolke C D, Keasling J D. Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon[J]. Biotechnology and bioengineering, 2002, 80(7): 762-776.
Cistrons Concerto
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