Enzymatic Assembly of Small Synthetic Genes with Repetitive Elements.

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences, which results in synthesis failure or delays by DNA synthesis vendors. This represents a major obstacle for the development of synthetic biology since repetitive elements are increasingly being used in the design of genetic circuits and design of biomolecular nanostructures. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden-Gate-compatible overhangs. Then, synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate Assembly. We demonstrate the method by constructing eight challenging genes with repetitive elements, e.g., multiple repeats of RNA aptamers and RNA origami scaffolds with multiple identical aptamers. The genes range in size from 133 to 456 base pairs and are assembled with fidelities of up to 87.5%. The method was developed to facilitate our own specific research but may be of general use for constructing challenging and repetitive genes and, thus, a valuable addition to the molecular cloning toolbox.

Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control.

Designer RNA scaffolds constitute a promising tool for synthetic biology, as they can be genetically expressed to perform specific functions in vivo such as scaffolding enzymatic cascades and regulating gene expression through CRISPR-dCas9 applications. RNA origami is a recently developed RNA design approach that allows construction of large RNA nanostructures that can position aptamer motifs to spatially organize other molecules, including proteins. However, it is still not fully understood how positioning multiple aptamers on a scaffold and the orientation of a scaffold affects functional properties. Here, we investigate fusions of single-guide RNAs and RNA origami scaffolds (termed sgRNAO) capable of recruiting activating domains for control of gene expression in yeast. Using MS2 and PP7 as orthogonal protein-binding aptamers, we observe a gradual increase in transcriptional activation for up to four aptamers. We demonstrate that different aptamer positions on a scaffold and scaffold orientation affect transcriptional activation. Finally, sgRNAOs are used to regulate expression of enzymes of the violacein biosynthesis pathway to control metabolic flux. The integration of RNA origami nanostructures at promoter sites achieved here, can in the future be expanded by the addition of functional motifs such as riboswitches, ribozymes and sensor elements to allow for complex gene regulation.

Synthetic Translational Regulation by Protein-Binding RNA Origami Scaffolds.

Rational design approaches for the regulation of gene expression are expanding the synthetic biology toolbox. However, only a few tools for regulating gene expression at the translational level have been developed. Here, we devise an approach for translational regulation using the MS2 and PP7 aptamer and coat-protein pairs in Escherichia coli. The aptamers are used as operators in transcription units that encode proteins fused to their cognate coat proteins, which leads to self-repression. RNA origami scaffolds that contain up to four aptamers serve as an alternate binder to activate translation. With this system, we demonstrate that the increase in expression of a reporter protein is dependent on both the concentration and number of aptamers on RNA origami scaffolds. We also demonstrate regulation of multiple proteins using a single MS2 coat protein fusion and apply this method to regulate the relative expression of enzymes of the branched pathway for deoxyviolacein biosynthesis.

Synthetic gene regulation for independent external induction of the Saccharomyces cerevisiae pseudohyphal growth phenotype.

Pseudohyphal growth is a multicellular phenotype naturally performed by wild budding yeast cells in response to stress. Unicellular yeast cells undergo gross changes in their gene regulation and elongate to form branched filament structures consisting of connected cells. Here, we construct synthetic gene regulation systems to enable external induction of pseudohyphal growth in Saccharomyces cerevisiae. By controlling the expression of the natural PHD1 and FLO8 genes we are able to trigger pseudohyphal growth in both diploid and haploid yeast, even in different types of rich media. Using this system, we also investigate how members of the BUD gene family control filamentation in haploid cells. Finally, we employ a synthetic genetic timer network to control pseudohyphal growth and further explore the reversibility of differentiation. Our work demonstrates that synthetic regulation can exert control over a complex multigene phenotype and offers opportunities for rationally modifying the resulting multicellular structure.

Construction of hybrid regulated mother-specific yeast promoters for inducible differential gene expression.

Engineered promoters with predefined regulation are a key tool for synthetic biology that enable expression on demand and provide the logic for genetic circuits. To expand the availability of synthetic biology tools for S. cerevisiae yeast, we here used hybrid promoter engineering to construct tightly-controlled, externally-inducible promoters that only express in haploid mother cells that have contributed a daughter cell to the population. This is achieved by combining elements from the native HO promoter and from a TetR-repressible synthetic promoter, with the performance of these promoters characterized by both flow cytometry and microfluidics-based fluorescence microscopy. These new engineered promoters are provided as an enabling tool for future synthetic biology applications that seek to exploit differentiation within a yeast population.

Using Spinach aptamer to correlate mRNA and protein levels in Escherichia coli.

In vivo gene expression measurements have traditionally relied on fluorescent proteins such as green fluorescent protein (GFP) with the help of high-sensitivity equipment such as flow cytometers. However, fluorescent proteins report only on the protein level inside the cell without giving direct information about messenger RNA (mRNA) production. In 2011, an aptamer termed Spinach was presented that acts as an RNA mimic of GFP when produced in Escherichia coli and mammalian cells. It was later shown that coexpression of a red fluorescent protein (mRFP1) and the Spinach aptamer, when included into the same gene expression cassette, could be utilized for parallel in vivo measurements of mRNA and protein production. As accurate characterization of component biological parts is becoming increasingly important for fields such as synthetic biology, Spinach in combination with mRFP1 provide a great tool for the characterization of promoters and ribosome binding sites. In this chapter, we discuss how live-cell imaging and flow cytometry can be used to detect and measure fluorescence produced in E. coli cells by different constructs that contain the Spinach aptamer and the mRFP1 gene.

The spinach RNA aptamer as a characterization tool for synthetic biology.

Characterization of genetic control elements is essential for the predictable engineering of synthetic biology systems. The current standard for in vivo characterization of control elements is through the use of fluorescent reporter proteins such as green fluorescent protein (GFP). Gene expression, however, involves not only protein production but also the production of mRNA. Here, we present the use of the Spinach aptamer sequence, an RNA mimic of GFP, as a tool to characterize mRNA expression in Escherichia coli. We show how the aptamer can be incorporated into gene expression cassettes and how co-expressing it with a red fluorescent protein (mRFP1) allows, for the first time, simultaneous measurement of mRNA and protein levels from engineered constructs. Using flow cytometry, we apply this tool here to evaluate ribosome binding site sequences and promoters and use it to highlight the differences in the temporal behavior of transcription and translation.