Abstract
Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5′ vs. 3′ untranslated region) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems.
Highlights
Synthetic biology is an interdisciplinary field that relies on biologists, engineers, mathematicians, and others to create novel biological systems by engineering and interchanging genetic parts derived from nature [1,2]
While we observed that these tools were able to modulate gene expression in two model systems, there was a lack of correlation between RNA secondary structure prediction algorithms and the experimental data. These results show that self-cleaving ribozymes combined with upstream competing sequences can modulate gene expression in eukaryotic systems, and that other factors, besides ribozyme self-cleavage and base-pair interactions, influence gene expression
After normalizing the fold-reduction levels by accounting for the loss of gene expression, we observed that some ribozyme constructs reduced gene expression more weakly compared to that data prior to normalization (Fig 2A and 2C, S2 Table)
Summary
Synthetic biology is an interdisciplinary field that relies on biologists, engineers, mathematicians, and others to create novel biological systems by engineering and interchanging genetic parts derived from nature [1,2]. Previous work has shown that these tools have the ability to regulate different steps of gene expression, including transcription [3], mRNA processing and stability [4], translation [5], and protein synthesis/stability [6]. This ability has been useful in the construction of synthetic gene circuits, such as counting devices [7], patterning devices [8], toggle switches [9], and gene oscillators [10], as well as the production of novel drugs, therapeutics, and biofuels.
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