Saccharomyces cerevisiae can serve as a key production platform for biofuels, nutraceuticals, industrial compounds, and therapeutic proteins. Over the recent years, synthetic biology tools and ...libraries have expanded in yeast to provide newfound control over regulation and synthetic circuits. This review provides an update on the status of the synthetic biology toolbox in yeast for use as a cell factory. Specifically, we discuss the impact of plasmid selection and composition, promoter, terminator, transcription factor, and aptamer selection. In doing so, we highlight documented interactions between these components, current states of development, and applications that demonstrate the utility of these parts with a particular focus on synthetic gene expression control.
In this review, the authors highlight the ways in which the yeast synthetic biology toolbox continues to expand in recent years.
Next-generation sequencing (NGS) technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the ...biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. Escherichia coli DNA polymerase I proteolytic (Klenow) fragment was originally utilized in Sanger's dideoxy chain-terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today's standard capillary electrophoresis (CE) and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ϕ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ϕ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies.
The unicellular cyanobacterium Synechocystis sp. PCC 6803 has been widely used as a photoautotrophic host for synthetic biology studies. However, as a green chassis to capture CO
for biotechnological ...applications, the genetic toolbox for Synechocystis 6803 is still a limited factor.
We systematically characterized endogenous genetic elements of Synechocystis 6803, including promoters, ribosome binding sites, transcription terminators, and plasmids. Expression from twelve native promoters was compared by measuring fluorescence from the reporter protein EYFP in an identical setup, exhibiting an 8000-fold range of promoter activities. Moreover, we measured the strength of twenty native ribosome binding sites and eight native terminators, indicating their influence on the expression of the reporter genes. In addition, two shuttle vectors, pCA-UC118 and pCB-SC101, capable of replication in both Synechocystis 6803 and E. coli were constructed. Expression of reporter proteins were significantly enhanced in cells containing these new plasmids, thus providing superior gene expression platforms in this cyanobacterium.
The results of this study provide useful and well characterized native tools for bioengineering work in the model cyanobacterium Synechocystis 6803.
In bacterial genomes, there are two mechanisms to terminate the DNA transcription: the "intrinsic" or Rho-independent termination and the Rho-dependent termination. Intrinsic terminators are ...characterized by a RNA hairpin followed by a run of 6-8 U residues relatively easy to identify using one of the numerous available prediction programs. In contrast, Rho-dependent termination is mediated by the Rho protein factor that, firstly, binds to ribosome-free mRNA in a site characterized by a C > G content and then reaches the RNA polymerase to induce its release. Conversely on intrinsic terminators, the computational prediction of Rho-dependent terminators in prokaryotes is a very difficult problem because the sequence features required for the function of Rho are complex and poorly defined. This is the reason why it still does not exist an exhaustive Rho-dependent terminators prediction program.
In this study we introduce RhoTermPredict, the first published algorithm for an exhaustive Rho-dependent terminators prediction in bacterial genomes. RhoTermPredict identifies these elements based on a previously proposed consensus motif common to all Rho-dependent transcription terminators. It essentially searches for a 78 nt long RUT site characterized by a C > G content and with regularly spaced C residues, followed by a putative pause site for the RNA polymerase. We tested RhoTermPredict performances by using available genomic and transcriptomic data of the microorganism Escherichia coli K-12, both in limited-length sequences and in the whole-genome, and available genomic sequences from Bacillus subtilis 168 and Salmonella enterica LT2 genomes. We also estimated the overlap between the predictions of RhoTermPredict and those obtained by the predictor of intrinsic terminators ARNold webtool. Our results demonstrated that RhoTermPredict is a very performing algorithm both for limited-length sequences (F
-score obtained about 0.7) and for a genome-wide analysis. Furthermore the degree of overlap with ARNold predictions was very low.
Our analysis shows that RhoTermPredict is a powerful tool for Rho-dependent terminators search in the three analyzed genomes and could fill this gap in computational genomics. We conclude that RhoTermPredict could be used in combination with an intrinsic terminators predictor in order to predict all the transcription terminators in bacterial genomes.
Next-generation sequencing platforms Mardis, Elaine R
Annual review of analytical chemistry (Palo Alto, Calif.),
06/2013, Letnik:
6
Journal Article
Recenzirano
Automated DNA sequencing instruments embody an elegant interplay among chemistry, engineering, software, and molecular biology and have built upon Sanger's founding discovery of dideoxynucleotide ...sequencing to perform once-unfathomable tasks. Combined with innovative physical mapping approaches that helped to establish long-range relationships between cloned stretches of genomic DNA, fluorescent DNA sequencers produced reference genome sequences for model organisms and for the reference human genome. New types of sequencing instruments that permit amazing acceleration of data-collection rates for DNA sequencing have been developed. The ability to generate genome-scale data sets is now transforming the nature of biological inquiry. Here, I provide an historical perspective of the field, focusing on the fundamental developments that predated the advent of next-generation sequencing instruments and providing information about how these instruments work, their application to biological research, and the newest types of sequencers that can extract data from single DNA molecules.
Detailed transcription maps of bacteriophages are not usually explored, limiting our understanding of molecular phage biology and restricting their exploitation and engineering. The ONT-cappable-seq ...method described here brings phage transcriptomics to the accessible nanopore sequencing platform and provides an affordable and more detailed overview of transcriptional features compared to traditional RNA-seq experiments. With ONT-cappable-seq, primary transcripts are specifically capped, enriched, and prepared for long-read sequencing on the nanopore sequencing platform. This enables end-to-end sequencing of unprocessed transcripts covering both phage and host genome, thus providing insight on their operons. The subsequent analysis pipeline makes it possible to rapidly identify the most important transcriptional features such as transcription start and stop sites. The obtained data can thus provide a comprehensive overview of the transcription by your phage of interest.
Genetic studies in Arabidopsis established FLOWERING LOCUS T (FT) as a key flower-promoting gene in photoperiodic systems. Grafting experiments established unequivocal one-to-one relations between ...SINGLE FLOWER TRUSS (SFT), a tomato homolog of FT, and the hypothetical florigen, in all flowering plants. Additional studies of SFT and SELF PRUNING (SP, homolog of TFL1), two antagonistic genes regulating the architecture of the sympodial shoot system, have suggested that transition to flowering in the day-neutral and perennial tomato is synonymous with "termination." Dosage manipulation of its endogenous and mobile, graft-transmissible levels demonstrated that florigen regulates termination and transition to flowering in an SP-dependent manner and, by the same token, that high florigen levels induce growth arrest and termination in meristems across the tomato shoot system. It was thus proposed that growth balances, and consequently the patterning of the shoot systems in all plants, are mediated by endogenous, meristem-specific dynamic SFT/SP ratios and that shifts to termination by changing SFT/SP ratios are triggered by the imported florigen, the mobile form of SFT. Florigen is a universal plant growth hormone inherently checked by a complementary antagonistic systemic system. Thus, an examination of the endogenous functions of FT-like genes, or of the systemic roles of the mobile florigen in any plant species, that fails to pay careful attention to the balancing antagonistic systems, or to consider its functions in day-neutral or perennial plants, would be incomplete.
The bacteriophage T7 expression system is one of the most prominent transcription systems used in biotechnology and molecular-level research. However, T7 RNA polymerase is prone to read-through ...transcription due to its high processivity. As a consequence, enforcing efficient transcriptional termination is difficult. The termination hairpin found natively in the T7 genome is adapted to be inefficient, exhibiting 62% termination efficiency in vivo and even lower efficiency in vitro. In this study, we engineered a series of sequences that outperform the efficiency of the native terminator hairpin. By embedding a previously discovered 8-nucleotide T7 polymerase pause sequence within a synthetic hairpin sequence, we observed in vivo termination efficiency of 91%; by joining 2 short sequences into a tandem 2-hairpin structure, termination efficiency was increased to 98% in vivo and 91% in vitro. This study also tests the ability of these engineered sequences to terminate transcription of the Escherichia coli RNA polymerase. Two out of 3 of the most successful T7 polymerase terminators also facilitated termination of the bacterial polymerase with around 99% efficiency.
The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered ...by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.
•A 0.8 kb centromere-like sequence was identified from the I. orientalis genome and shown to improve plasmid stability.•A set of constitutive promoters and terminators was discovered and characterized under different culture conditions.•An efficient in vivo DNA assembly method was developed for plasmid and pathway assembly in I. orientalis.
•ALD6 overexpression strains were constructed by a scarless replacement method.•Improved acidity and a reduction in higher alcohols production were achieved.•Ethanol production was regained by ...increasing the inoculation amount of yeast.
Low-alcohol Huangjiu (LAH), which contains reduced contents of ethanol and higher alcohols, is prepared by diluting original Huangjiu that has a high ethanol content, which leads to a weakened flavor (i.e., acidity). To increase acidity and reduce higher alcohols level in LAH, the gene ALD6 encoding aldehyde dehydrogenase was expressed in yeast HJ-1 under the control of the pPGK1 promoter and terminators with varying activities (tGIC1, tPGK1 and tCPS1) by scarless replacement at BAT2 locus, yielding the engineered strains HJΔB-AG, HJΔB-AP, and HJΔB-AC. The acetate concentration produced by HJΔB-AG, HJΔB-AP, and HJΔB-AC was 1.26-, 1.84-, and 2.51-fold of that of HJ-1, respectively. Furthermore, the concentration of higher alcohols produced by HJΔB-AG, HJΔB-AP, and HJΔB-AC decreased by 39.91%, 45.55%, and 52.80%, respectively. This study resulted in the creation of promising recombinant yeast strains and introduced a method that can be used for the high-quality production of LAH by acid-producing Saccharomyces cerevisiae.
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