Biology subjects

Jewett, M. C.

Publications and source records attributed to Jewett, M. C..

6 recordsLinked to original sources

bioRxiv2018

Deconstructing cell-free extract preparation for in vitro activation of transcriptional genetic circuitry

Recent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits designed as biosensors. Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the method by which an extract is prepared affects its functionality. Specifically, a key goal toward developing cell-free biosensors based on native genetic regulators is activating the transcriptional machinery present in bacterial extracts for protein synthesis. However, protein yields from genes transcribed in vitro by the native Escherichia coli RNA polymerase are quite low in conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial {sigma}70 promoters is constrained by the rate of transcription in crude extracts and that processing the extract with a ribosomal run-off reaction and subsequent dialysis can alleviate this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture, but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing and will encourage the further proliferation of cell-free gene expression technology for new applications.

synthetic biology↗

bioRxiv2018

Selection removes Shine-Dalgarno-like sequences from within protein coding genes

The Shine-Dalgarno (SD) sequence motif facilitates translation initiation and is frequently found upstream of bacterial start codons. However, thousands of instances of this motif occur throughout the middle of protein coding genes in a typical bacterial genome. Here, we use comparative evolutionary analysis to test whether SD sequences located within genes are functionally constrained. We measure the conservation of SD sequences across Gammaproteobacteria, and find that they are significantly less conserved than expected. Further, the strongest SD sequences are the least conserved whereas we find evidence of conservation for the weakest possible SD sequences given amino acid constraints. Our findings indicate that most SD sequences within genes are likely to be deleterious and removed via selection. To illustrate the origin of these deleterious costs, we show that ATG start codons are significantly depleted downstream of SD sequences within genes, highlighting the potential for these sequences to promote erroneous translation initiation.

evolutionary biology↗

bioRxiv2017

Computational Design of Asymmetric Three-dimensional RNA Structures and Machines.

The emerging field of RNA nanotechnology seeks to create nanoscale 3D machines by repurposing natural RNA modules, but successes have been limited to symmetric assemblies of single repeating motifs. We present RNAMake, a suite that automates design of RNA molecules with complex 3D folds. We first challenged RNAMake with the paradigmatic problem of aligning a tetraloop and sequence-distal receptor, previously only solved via symmetry. Single-nucleotide-resolution chemical mapping, native gel electrophoresis, and solution x-ray scattering confirmed that 11 of the 16 miniTTR designs successfully achieved clothespin-like folds. A 2.55 [A] diffraction-resolution crystal structure of one design verified formation of the target asymmetric nanostructure, with large sections achieving near-atomic accuracy (< 2.0 [A]). Finally, RNAMake designed asymmetric segments to tether the 16S and 23S rRNAs together into a synthetic singlestranded ribosome that remains uncleaved by ribonucleases and supports life in Escherichia coli, a challenge previously requiring several rounds of trial-and-error.

bioengineering↗

bioRxiv2017

Diversity of translation initiation mechanisms across bacterial species is driven by environmental conditions and growth demands

The Shine-Dalgarno (SD) sequence is often found upstream of protein coding genes across the bacterial kingdom, where it enhances start codon recognition via hybridization to the anti-SD (aSD) sequence on the small ribosomal subunit. Despite widespread conservation of the aSD sequence, the proportion of SD-led genes within a genome varies widely across species, and the evolutionary pressures shaping this variation remain largely unknown. Here, we conduct a phylogenetically-informed analysis and show that species capable of rapid growth have a significantly higher proportion of SD-led genes in their genome, suggesting a role for SD sequences in meeting the protein production demands of rapidly growing species. Further, we show that utilization of the SD sequence mechanism co-varies with: i) genomic traits that are indicative of efficient translation, and ii) optimal growth temperatures. In contrast to prior surveys, our results demonstrate that variation in translation initiation mechanisms across genomes is largely predictable, and that SD sequence utilization is part of a larger suite of translation-associated traits whose diversity is driven by the differential growth strategies of individual species.

systems biology↗

bioRxiv2017

A pipeline for interrogating and engineering single-subunit oligosaccharyltransferases

Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of post-translational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3. Given their relative simplicity, these single-subunit OSTs (ssOSTs) have emerged as important targets for mechanistic dissection of poorly understood aspects of N-glycosylation and at the same time hold great potential for the biosynthesis of custom glycoproteins. To take advantage of this utility, this chapter describes a multipronged approach for studying and engineering ssOSTs that integrates in vivo screening technology with in vitro characterization methods, thereby creating a versatile and readily-adaptable pipeline for virtually any ssOST of interest.\n\nAbbreviations

synthetic biology↗

bioRxiv2017

A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 440 {micro}g/mL for the single-subunit OST enzyme, Protein glycosylation B (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

synthetic biology↗