Synthetic biology (Oxford, England)最新文献

{"title":"Mouse chromosomes get supersized but find their limits.","authors":"David M Truong","doi":"10.1093/synbio/ysac024","DOIUrl":"https://doi.org/10.1093/synbio/ysac024","url":null,"abstract":"© The Author(s) 2022. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Humans diverged from primates when an ancestral chromosomal fusion—the event when two chromosomes join together to form one—gave us 23 instead of 24 sets of chromosomes. In muntjac deer, small deer native to South and Southeast Asia, chromosome fusions occurred so often that Indian muntjacs have only 3 chromosomes, whereas Chinese muntjacs have 23 (1). Fusions matter not only during the evolution of species but can also cause diseases such as cancer or Down’s syndrome. While fusions occur often in nature, engineering events like these on purpose have been difficult to do. The field of synthetic genomics attempts feats like this, along with building new designer chromosomes for applications in medicine, agriculture and industrial processing. Completely synthetic genomes have been built for bacteria (2), as well as for yeast (2). Additionally, it was shown that all 16 yeast chromosomes can be fused into one single chromosome 12 megabases long (3). Besides these achievements, it remained an open question what the actual size limit of a single chromosome would be, for example, whether the 100–200 megabase mammalian chromosomes could be fused and whether changes like these would persist through multiple generations. Answers could be used to model speciation and human diseases, as well as biologically ‘contain’ engineered organisms from natural populations. In a groundbreaking new study (4), researchers from the Chinese Academy of Sciences have generated the largest designed fusion chromosomes so far reported in mice as their research model. To technically achieve this, they used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to make targeted DNA breaks. These breaks would induce recombination—a natural repair process of the cell—thereby fusing the two largest mouse chromosomes 1 and 2 into a single one, in two different orientations, followed by fusing medium size chromosomes 4 and 5. The longest of these chromosomes was 377 megabases long and functional. In addition, they accomplished all this engineering in haploid mouse embryonic stem cells (i.e. cells with only one set of chromosomes), showing the potential to make mice easier to engineer using haploid cells in a Petri dish. Although the authors could generate heterozygous embryos with the largest fused chromosomes, one fused orientation was lethal to the developing embryo, while embryos of the other orientation grew to adulthood. Yet, the resultant mice could not breed homozygous offspring. Surprisingly, the 308 megabase medium-sized fused chromosome mice coul","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysac024"},"PeriodicalIF":0.0,"publicationDate":"2022-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/19/9b/ysac024.PMC9659764.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40687767","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"basicsynbio and the BASIC SEVA collection: software and vectors for an established DNA assembly method.","authors":"Matthew C Haines,&nbsp;Benedict Carling,&nbsp;James Marshall,&nbsp;Vasily A Shenshin,&nbsp;Geoff S Baldwin,&nbsp;Paul Freemont,&nbsp;Marko Storch","doi":"10.1093/synbio/ysac023","DOIUrl":"https://doi.org/10.1093/synbio/ysac023","url":null,"abstract":"<p><p>Standardized deoxyribonucleic acid (DNA) assembly methods utilizing modular components provide a powerful framework to explore designs and iterate through Design-Build-Test-Learn cycles. Biopart Assembly Standard for Idempotent Cloning (BASIC) DNA assembly uses modular parts and linkers, is highly accurate, easy to automate, free for academic and commercial use and enables hierarchical assemblies through an idempotent format. These features enable applications including pathway engineering, ribosome binding site (RBS) tuning, fusion protein engineering and multiplexed guide ribonucleic acid (RNA) expression. In this work, we present basicsynbio, open-source software encompassing a Web App (https://basicsynbio.web.app/) and Python Package (https://github.com/LondonBiofoundry/basicsynbio), enabling BASIC construct design via simple drag-and-drop operations or programmatically. With basicsynbio, users can access commonly used BASIC parts and linkers while designing new parts and assemblies with exception handling for common errors. Users can export sequence data and create instructions for manual or acoustic liquid-handling platforms. Instruction generation relies on the BasicBuild Open Standard, which is parsed for bespoke workflows and is serializable in JavaScript Object Notation for transfer and storage. We demonstrate basicsynbio, assembling 30 vectors using sequences including modules from the Standard European Vector Architecture (SEVA). The BASIC SEVA vector collection is compatible with BASIC and Golden Gate using BsaI. Vectors contain one of six antibiotic resistance markers and five origins of replication from different compatibility groups. The collection is available via Addgene under an OpenMTA agreement. Furthermore, vector sequences are available from within the basicsynbio application programming interface with other collections of parts and linkers, providing a powerful environment for designing assemblies for bioengineering applications. <b>Graphical Abstract</b>.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysac023"},"PeriodicalIF":0.0,"publicationDate":"2022-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9664905/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40687691","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Could a simple model of COVID-19 infections be the key to designing better virus-based therapies?","authors":"Connor R King","doi":"10.1093/synbio/ysac019","DOIUrl":"https://doi.org/10.1093/synbio/ysac019","url":null,"abstract":"© The Author(s) 2022. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Since the emergence of COVID-19, the spotlight on viruses has become negative. However, viruses are not only our enemies, due to their ability to deliver DNA and RNA into cells, viruses can also be repurposed as therapeutics. This ability is already used to treat genetic diseases and cancer (2, 4). However, an important factor that is often overlooked when developing virus-based therapies is the fact that each patient’s immune system might respond differently to the treatment which can determine its effectiveness (3). Recently, the Ke Lab at Los Alamos National Laboratory developed a simplistic model of the immune response to SARS-CoV-2 infection that can explain why some individuals have severe symptoms while others quickly resolve the infection (1). The simplicity of this model suggests that one day it could not only help understand viral infections but help improve virus-based therapies. Current designs for virus-based therapeutics mainly focused on the delivery system itself. Much like if you were designing a system to deliver medicine to houses, you might first want to optimize the delivery vehicle to be used for delivering the medicine and identify routes for the delivery. Now imagine that this system was developed just around the delivery of the medication itself without consideration of what might happen when the medicine is delivered. If you deliver this medicine to a house that is, much like a cell, unaware of why the medicine is coming, the recipient may dispose of the said medication. Since the injection of DNA or RNA from a virus into a cell is typically associated with a disease, it is reasonable to assume that the cell has processes at hand that interfere with virus-based treatments. The model from the Ke lab might help therapy-designers to predict and mitigate these processes just as the model is able to explain why some people get severe COVID-19 and others do not. The focus of the model is the immune response generated by the molecular footprint left by the viral disease. This footprint results from the viral infection itself but is also generated by cells being damaged by the immune system. Both contribute to sustaining an active immune response in patients. The model simplifies many of the processes that generate and remove this footprint in order to reduce complexity. Their approach to making the model simple can be compared to trying to plan out how long it takes to drive from San Francisco to Los Angeles. There are many factors that contribute to how quickly one can drive—traffic and headwind—but one could p","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysac019"},"PeriodicalIF":0.0,"publicationDate":"2022-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/07/5c/ysac019.PMC9518665.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40390049","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A toolkit for enhanced reproducibility of RNASeq analysis for synthetic biologists.","authors":"Benjamin J Garcia,&nbsp;Joshua Urrutia,&nbsp;George Zheng,&nbsp;Diveena Becker,&nbsp;Carolyn Corbet,&nbsp;Paul Maschhoff,&nbsp;Alexander Cristofaro,&nbsp;Niall Gaffney,&nbsp;Matthew Vaughn,&nbsp;Uma Saxena,&nbsp;Yi-Pei Chen,&nbsp;D Benjamin Gordon,&nbsp;Mohammed Eslami","doi":"10.1093/synbio/ysac012","DOIUrl":"https://doi.org/10.1093/synbio/ysac012","url":null,"abstract":"<p><p>Sequencing technologies, in particular RNASeq, have become critical tools in the design, build, test and learn cycle of synthetic biology. They provide a better understanding of synthetic designs, and they help identify ways to improve and select designs. While these data are beneficial to design, their collection and analysis is a complex, multistep process that has implications on both discovery and reproducibility of experiments. Additionally, tool parameters, experimental metadata, normalization of data and standardization of file formats present challenges that are computationally intensive. This calls for high-throughput pipelines expressly designed to handle the combinatorial and longitudinal nature of synthetic biology. In this paper, we present a pipeline to maximize the analytical reproducibility of RNASeq for synthetic biologists. We also explore the impact of reproducibility on the validation of machine learning models. We present the design of a pipeline that combines traditional RNASeq data processing tools with structured metadata tracking to allow for the exploration of the combinatorial design in a high-throughput and reproducible manner. We then demonstrate utility via two different experiments: a control comparison experiment and a machine learning model experiment. The first experiment compares datasets collected from identical biological controls across multiple days for two different organisms. It shows that a reproducible experimental protocol for one organism does not guarantee reproducibility in another. The second experiment quantifies the differences in experimental runs from multiple perspectives. It shows that the lack of reproducibility from these different perspectives can place an upper bound on the validation of machine learning models trained on RNASeq data. Graphical Abstract.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysac012"},"PeriodicalIF":0.0,"publicationDate":"2022-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/4f/a8/ysac012.PMC9408027.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33444691","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A universal approach to gene expression engineering.","authors":"Rahmi Lale,&nbsp;Lisa Tietze,&nbsp;Maxime Fages-Lartaud,&nbsp;Jenny Nesje,&nbsp;Ingerid Onsager,&nbsp;Kerstin Engelhardt,&nbsp;Che Fai Alex Wong,&nbsp;Madina Akan,&nbsp;Niklas Hummel,&nbsp;Jörn Kalinowski,&nbsp;Christian Rückert,&nbsp;Martin Frank Hohmann-Marriott","doi":"10.1093/synbio/ysac017","DOIUrl":"https://doi.org/10.1093/synbio/ysac017","url":null,"abstract":"<p><p>In this study, we provide a universal approach to Gene Expression Engineering (GeneEE) for creating artificial expression systems. GeneEE leads to the generation of artificial 5^' regulatory sequences (ARES) consisting of promoters and 5^' untranslated regions. The ARES lead to the successful recruitment of RNA polymerase, related sigma factors and ribosomal proteins that result in a wide range of expression levels. We also demonstrate that by engaging native transcription regulators, GeneEE can be used to generate inducible promoters. To showcase the universality of the approach, we demonstrate that 200-nucleotide (nt)-long DNA with random composition can be used to generate functional expression systems in six bacterial species, <i>Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Thermus thermophilus, Streptomyces albus</i> and <i>Streptomyces lividans</i>, and the eukaryote yeast <i>Saccharomyces cerevisiae</i>.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysac017"},"PeriodicalIF":0.0,"publicationDate":"2022-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f4/c8/ysac017.PMC9534286.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33497337","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluating the persistence and stability of a DNA-barcoded microbial system in a mock home environment.","authors":"Nathan D McDonald,&nbsp;Katherine A Rhea,&nbsp;John P Davies,&nbsp;Julie L Zacharko,&nbsp;Kimberly L Berk,&nbsp;Patricia E Buckley","doi":"10.1093/synbio/ysac016","DOIUrl":"https://doi.org/10.1093/synbio/ysac016","url":null,"abstract":"<p><p>Recent advancements in engineered microbial systems capable of deployment in complex environments have enabled the creation of unique signatures for environmental forensics operations. These microbial systems must be robust, able to thrive in specific environments of interest and contain molecular signatures, enabling the detection of the community across conditions. Furthermore, these systems must balance biocontainment concerns with the stability and persistence required for environmental forensics. Here we evaluate the stability and persistence of a recently described microbial system composed of germination-deficient <i>Bacillus subtilis</i> and <i>Saccharomyces cerevisiae</i> spores containing nonredundant DNA barcodes in a controlled simulated home environment. These spore-based microbial communities were found to be persistent in the simulated environment across 30-day periods and across multiple surface types. To improve the repeatability and reproducibility in detecting the DNA barcodes, we evaluated several spore lysis and sampling processes paired with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -CRISPR-associated proteins (Cas) detection (Sherlock). Finally, having optimized the detectability of the spores, we demonstrate that we can detect the spores transferring across multiple material types. Together, we further demonstrate the utility of a recently described microbial forensics system and highlight the importance of independent validation and verification of synthetic biology tools and applications. Graphical Abstract.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysac016"},"PeriodicalIF":0.0,"publicationDate":"2022-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9423098/pdf/ysac016.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40335395","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multicolor plate reader fluorescence calibration.","authors":"Jacob Beal,&nbsp;Cheryl A Telmer,&nbsp;Alejandro Vignoni,&nbsp;Yadira Boada,&nbsp;Geoff S Baldwin,&nbsp;Liam Hallett,&nbsp;Taeyang Lee,&nbsp;Vinoo Selvarajah,&nbsp;Sonja Billerbeck,&nbsp;Bradley Brown,&nbsp;Guo-Nan Cai,&nbsp;Liang Cai,&nbsp;Edward Eisenstein,&nbsp;Daisuke Kiga,&nbsp;David Ross,&nbsp;Nina Alperovich,&nbsp;Noah Sprent,&nbsp;Jaclyn Thompson,&nbsp;Eric M Young,&nbsp;Drew Endy,&nbsp;Traci Haddock-Angelli","doi":"10.1093/synbio/ysac010","DOIUrl":"https://doi.org/10.1093/synbio/ysac010","url":null,"abstract":"<p><p>Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysac010"},"PeriodicalIF":0.0,"publicationDate":"2022-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/71/0a/ysac010.PMC9357555.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40696991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Variability in cell-free expression reactions can impact qualitative genetic circuit characterization.","authors":"Katherine A Rhea,&nbsp;Nathan D McDonald,&nbsp;Stephanie D Cole,&nbsp;Vincent Noireaux,&nbsp;Matthew W Lux,&nbsp;Patricia E Buckley","doi":"10.1093/synbio/ysac011","DOIUrl":"https://doi.org/10.1093/synbio/ysac011","url":null,"abstract":"<p><p>Cell-free expression systems provide a suite of tools that are used in applications from sensing to biomanufacturing. One of these applications is genetic circuit prototyping, where the lack of cloning is required and a high degree of control over reaction components and conditions enables rapid testing of design candidates. Many studies have shown utility in the approach for characterizing genetic regulation elements, simple genetic circuit motifs, protein variants or metabolic pathways. However, variability in cell-free expression systems is a known challenge, whether between individuals, laboratories, instruments, or batches of materials. While the issue of variability has begun to be quantified and explored, little effort has been put into understanding the implications of this variability. For genetic circuit prototyping, it is unclear when and how significantly variability in reaction activity will impact qualitative assessments of genetic components, e.g. relative activity between promoters. Here, we explore this question by assessing DNA titrations of seven genetic circuits of increasing complexity using reaction conditions that ostensibly follow the same protocol but vary by person, instrument and material batch. Although the raw activities vary widely between the conditions, by normalizing within each circuit across conditions, reasonably consistent qualitative performance emerges for the simpler circuits. For the most complex case involving expression of three proteins, we observe a departure from this qualitative consistency, offering a provisional cautionary line where normal variability may disrupt reliable reuse of prototyping results. Our results also suggest that a previously described closed loop controller circuit may help to mitigate such variability, encouraging further work to design systems that are robust to variability. Graphical Abstract.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysac011"},"PeriodicalIF":0.0,"publicationDate":"2022-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9365049/pdf/ysac011.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40413432","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Signal amplification of <i>araC pBAD</i> using a standardized translation initiation region.","authors":"Patrick J Shilling,&nbsp;Diana Khananisho,&nbsp;Alister J Cumming,&nbsp;Bill Söderström,&nbsp;Daniel O Daley","doi":"10.1093/synbio/ysac009","DOIUrl":"https://doi.org/10.1093/synbio/ysac009","url":null,"abstract":"<p><p><i>araC pBAD</i> is a genetic fragment that regulates the expression of the <i>araBAD</i> operon in bacteria, which is required for the metabolism of L-arabinose. It is widely used in bioengineering applications because it can drive regulatable and titratable expression of genes and genetic pathways in microbial cell factories. A notable limitation of <i>araC pBAD</i> is that it generates a low signal when induced with high concentrations of L-arabinose (the maximum ON state). Herein we have amplified the maximum ON state of <i>araC pBAD</i> by coupling it to a synthetically evolved translation initiation region (<i>TIR^EVOL</i> ). The coupling maintains regulatable and titratable expression from <i>araC pBAD</i> and yet increases the maximal ON state by >5-fold. The general principle demonstrated in the study can be applied to amplify the signal from similar genetic modules. Graphical Abstract.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysac009"},"PeriodicalIF":0.0,"publicationDate":"2022-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/88/e2/ysac009.PMC9316229.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40556576","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform.","authors":"Andrei Sakai,&nbsp;Christopher R Deich,&nbsp;Frank H T Nelissen,&nbsp;Aafke J Jonker,&nbsp;Daniela M de C Bittencourt,&nbsp;Christopher P Kempes,&nbsp;Kim S Wise,&nbsp;Hans A Heus,&nbsp;Wilhelm T S Huck,&nbsp;Katarzyna P Adamala,&nbsp;John I Glass","doi":"10.1093/synbio/ysac008","DOIUrl":"https://doi.org/10.1093/synbio/ysac008","url":null,"abstract":"<p><p>Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a <i>Mycoplasma</i> bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative <i>Mycoplasma capricolum</i> (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of <i>Escherichia coli</i>. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of <i>in vitro</i> transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease (RNase) activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNase activity yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned <i>E. coli</i> cell-free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNase activity. However, it is not clear which gene encodes the RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Graphical Abstract.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysac008"},"PeriodicalIF":0.0,"publicationDate":"2022-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9239315/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40573536","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}