Alexander Ditzel, Fanglong Zhao, Xue Gao, George N Phillips
Natural products are a valuable source of pharmaceuticals, providing a majority of the small molecule drugs in use today. However, their production through organic synthesis or in heterologous hosts can be difficult and time-consuming. Therefore, to allow for easier screening and production of natural products, we demonstrated the use of a cell-free protein synthesis (CFPS) system to partially assemble natural products in vitro using SAM-dependent methyltransferase enzyme reactions. The tea caffeine synthase TCS1 was utilized to synthesize caffeine within a CFPS system. Cell-free systems also provide the benefit of allowing the use of substrates that would normally be toxic in a cellular environment to synthesize novel products. However, TCS1 is unable to utilize a compound like AdoEt as a cofactor to create ethylated caffeine analogs. The automation and reduced metabolic engineering requirements of CFPS systems, in combination with other synthesis methods, may enable the more efficient generation of new compounds.
{"title":"Utilizing a Cell-free Protein Synthesis Platform for the Biosynthesis of a Natural Product, Caffeine","authors":"Alexander Ditzel, Fanglong Zhao, Xue Gao, George N Phillips","doi":"10.1093/synbio/ysad017","DOIUrl":"https://doi.org/10.1093/synbio/ysad017","url":null,"abstract":"\u0000 Natural products are a valuable source of pharmaceuticals, providing a majority of the small molecule drugs in use today. However, their production through organic synthesis or in heterologous hosts can be difficult and time-consuming. Therefore, to allow for easier screening and production of natural products, we demonstrated the use of a cell-free protein synthesis (CFPS) system to partially assemble natural products in vitro using SAM-dependent methyltransferase enzyme reactions. The tea caffeine synthase TCS1 was utilized to synthesize caffeine within a CFPS system. Cell-free systems also provide the benefit of allowing the use of substrates that would normally be toxic in a cellular environment to synthesize novel products. However, TCS1 is unable to utilize a compound like AdoEt as a cofactor to create ethylated caffeine analogs. The automation and reduced metabolic engineering requirements of CFPS systems, in combination with other synthesis methods, may enable the more efficient generation of new compounds.","PeriodicalId":22158,"journal":{"name":"Synthetic Biology","volume":"76 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2023-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138945344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Matthew W Lux, Elizabeth A Strychalski, Gary J Vora
Abstract Reproducibility has been identified as an outstanding challenge in science, and the field of synthetic biology is no exception. Meeting this challenge is critical to allow the transformative technological capabilities emerging from this field to reach their full potential to benefit the society. We discuss the current state of reproducibility in synthetic biology and how improvements can address some of the central shortcomings in the field. We argue that the successful adoption of reproducibility as a routine aspect of research and development requires commitment spanning researchers and relevant institutions via education, incentivization and investment in related infrastructure. The urgency of this topic pervades synthetic biology as it strives to advance fundamental insights and unlock new capabilities for safe, secure and scalable applications of biotechnology. Graphical Abstract
{"title":"Advancing reproducibility can ease the ‘hard truths’ of synthetic biology","authors":"Matthew W Lux, Elizabeth A Strychalski, Gary J Vora","doi":"10.1093/synbio/ysad014","DOIUrl":"https://doi.org/10.1093/synbio/ysad014","url":null,"abstract":"Abstract Reproducibility has been identified as an outstanding challenge in science, and the field of synthetic biology is no exception. Meeting this challenge is critical to allow the transformative technological capabilities emerging from this field to reach their full potential to benefit the society. We discuss the current state of reproducibility in synthetic biology and how improvements can address some of the central shortcomings in the field. We argue that the successful adoption of reproducibility as a routine aspect of research and development requires commitment spanning researchers and relevant institutions via education, incentivization and investment in related infrastructure. The urgency of this topic pervades synthetic biology as it strives to advance fundamental insights and unlock new capabilities for safe, secure and scalable applications of biotechnology. Graphical Abstract","PeriodicalId":22158,"journal":{"name":"Synthetic Biology","volume":"27 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135611266","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Abstract Recombineering is an important tool in gene editing, enabling fast, precise and highly specific in vivo modification of microbial genomes. Oligonucleotide-mediated recombineering via the in vivo production of single-stranded DNA can overcome the limitations of traditional recombineering methods that rely on the exogenous delivery of editing templates. By modifying a previously reported plasmid-based system for fully in vivo single-stranded DNA recombineering, we demonstrate iterative editing of independent loci by utilizing a temperature-sensitive origin of replication for easy curing of the editing plasmid from recombinant cells. Optimization of the promoters driving the expression of the system’s functional components, combined with targeted counterselection against unedited cells with Cas9 nuclease, enabled editing efficiencies of 90–100%. The addition of a dominant-negative mutL allele to the system allowed single-nucleotide edits that were otherwise unachievable due to mismatch repair. Finally, we tested alternative recombinases and found that efficiency significantly increased for some targets. Requiring only a single cloning step for retargeting, our system provides an easy-to-use method for rapid, efficient construction of desired mutants. Graphical Abstract
{"title":"Efficient and iterative retron-mediated in vivo recombineering in Escherichia coli","authors":"A. Ellington, Christopher R. Reisch","doi":"10.1093/synbio/ysac007","DOIUrl":"https://doi.org/10.1093/synbio/ysac007","url":null,"abstract":"Abstract Recombineering is an important tool in gene editing, enabling fast, precise and highly specific in vivo modification of microbial genomes. Oligonucleotide-mediated recombineering via the in vivo production of single-stranded DNA can overcome the limitations of traditional recombineering methods that rely on the exogenous delivery of editing templates. By modifying a previously reported plasmid-based system for fully in vivo single-stranded DNA recombineering, we demonstrate iterative editing of independent loci by utilizing a temperature-sensitive origin of replication for easy curing of the editing plasmid from recombinant cells. Optimization of the promoters driving the expression of the system’s functional components, combined with targeted counterselection against unedited cells with Cas9 nuclease, enabled editing efficiencies of 90–100%. The addition of a dominant-negative mutL allele to the system allowed single-nucleotide edits that were otherwise unachievable due to mismatch repair. Finally, we tested alternative recombinases and found that efficiency significantly increased for some targets. Requiring only a single cloning step for retargeting, our system provides an easy-to-use method for rapid, efficient construction of desired mutants. Graphical Abstract","PeriodicalId":22158,"journal":{"name":"Synthetic Biology","volume":"23 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2022-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74123254","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
P. Salerno, Matthew W. Leckenby, Bruce Humphrey, Rocky M. Cranenburgh
Abstract Antibiotic resistance genes are widely used to select bacteria transformed with plasmids and to prevent plasmid loss from cultures, yet antibiotics represent contaminants in the biopharmaceutical manufacturing process, and retaining antibiotic resistance genes in vaccines and biological therapies is discouraged by regulatory agencies. To overcome these limitations, we have developed X-mark™, a novel technology that leverages Xer recombination to generate selectable marker gene-free plasmids for downstream therapeutic applications. Using this technique, X-mark plasmids with antibiotic resistance genes flanked by XerC/D target sites are generated in Escherichia coli cytosol aminopeptidase (E. coli pepA) mutants, which are deficient in Xer recombination on plasmids, and subsequently transformed into enteric bacteria with a functional Xer system. This results in rapid deletion of the resistance gene at high resolution (100%) and stable replication of resolved plasmids for more than 40 generations in the absence of antibiotic selective pressure. This technology is effective in both Escherichia coli and Salmonella enterica bacteria due to the high degree of homology between accessory sequences, including strains that have been developed as oral vaccines for clinical use. X-mark effectively eliminates any regulatory and safety concerns around antibiotic resistance carryover in biopharmaceutical products, such as vaccines and therapeutic proteins. Graphical Abstract
摘要:抗生素耐药基因被广泛用于选择用质粒转化的细菌和防止质粒从培养中丢失，然而抗生素在生物制药生产过程中是污染物，在疫苗和生物疗法中保留抗生素耐药基因是监管机构不鼓励的。为了克服这些限制，我们开发了X-mark™，这是一种利用Xer重组产生可选择的无标记基因质粒的新技术，用于下游治疗应用。利用该技术，在大肠杆菌胞浆氨基肽酶(e.c oli pepA)突变体中产生带有XerC/D靶位的抗生素耐药基因的x标记质粒，并将其转化为具有功能Xer系统的肠道细菌。这导致在没有抗生素选择压力的情况下，以高分辨率(100%)快速删除抗性基因，并稳定地复制已分解的质粒超过40代。这项技术对大肠杆菌和肠沙门氏菌都有效，因为附属序列之间的高度同源性，包括已开发为临床使用的口服疫苗的菌株。X-mark有效地消除了对生物制药产品(如疫苗和治疗性蛋白质)中抗生素耐药性遗留的任何监管和安全担忧。图形抽象
{"title":"Xer recombination for the automatic deletion of selectable marker genes from plasmids in enteric bacteria","authors":"P. Salerno, Matthew W. Leckenby, Bruce Humphrey, Rocky M. Cranenburgh","doi":"10.1093/synbio/ysac005","DOIUrl":"https://doi.org/10.1093/synbio/ysac005","url":null,"abstract":"Abstract Antibiotic resistance genes are widely used to select bacteria transformed with plasmids and to prevent plasmid loss from cultures, yet antibiotics represent contaminants in the biopharmaceutical manufacturing process, and retaining antibiotic resistance genes in vaccines and biological therapies is discouraged by regulatory agencies. To overcome these limitations, we have developed X-mark™, a novel technology that leverages Xer recombination to generate selectable marker gene-free plasmids for downstream therapeutic applications. Using this technique, X-mark plasmids with antibiotic resistance genes flanked by XerC/D target sites are generated in Escherichia coli cytosol aminopeptidase (E. coli pepA) mutants, which are deficient in Xer recombination on plasmids, and subsequently transformed into enteric bacteria with a functional Xer system. This results in rapid deletion of the resistance gene at high resolution (100%) and stable replication of resolved plasmids for more than 40 generations in the absence of antibiotic selective pressure. This technology is effective in both Escherichia coli and Salmonella enterica bacteria due to the high degree of homology between accessory sequences, including strains that have been developed as oral vaccines for clinical use. X-mark effectively eliminates any regulatory and safety concerns around antibiotic resistance carryover in biopharmaceutical products, such as vaccines and therapeutic proteins. Graphical Abstract","PeriodicalId":22158,"journal":{"name":"Synthetic Biology","volume":"24 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2022-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91366580","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Abstract Advances in synthetic biology have allowed the generation of strains of bacteria that are genetically altered to have specific therapeutic benefits. These synthetic biotics, also widely referred to as engineered living therapeutics, have tremendous potential as a new therapeutic modality, and several have advanced into the clinic and human testing. This review outlines some of the unique attributes of synthetic biotics as well as some of the challenges in their development as prescription products. Regulatory considerations are discussed, and a case study of a program that has advanced into Phase 2 testing is provided: SYNB1618 for the treatment of PKU.
{"title":"Development of synthetic biotics as treatment for human diseases","authors":"A. Brennan","doi":"10.1093/synbio/ysac001","DOIUrl":"https://doi.org/10.1093/synbio/ysac001","url":null,"abstract":"Abstract Advances in synthetic biology have allowed the generation of strains of bacteria that are genetically altered to have specific therapeutic benefits. These synthetic biotics, also widely referred to as engineered living therapeutics, have tremendous potential as a new therapeutic modality, and several have advanced into the clinic and human testing. This review outlines some of the unique attributes of synthetic biotics as well as some of the challenges in their development as prescription products. Regulatory considerations are discussed, and a case study of a program that has advanced into Phase 2 testing is provided: SYNB1618 for the treatment of PKU.","PeriodicalId":22158,"journal":{"name":"Synthetic Biology","volume":"60 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2022-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86028452","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
N. Dang, Alissa Lance-Byrne, Angela Tung, Xiaoge Guo, Ryan J. Cecchi, Joanna Buchthal, Alejandro Chavez, N. C. Yeo
CRISPR/Cas9 has revolutionized the field of genome engineering. Yet, as the CRISPR toolbox has rapidly expanded, there remains a need for a comprehensive library of CRISPR/Cas9 reagents that allow users to perform complex cellular and genetic manipulations without requiring labor-intensive generation of reagents to meet each user’s unique experimental circumstances. Here we described the creation and validation of a pNAX CRISPR library consisting of 72 different Cas9 and gRNA expression plasmids to allow for efficient multiplex gene editing, activation, and repression in mammalian cells. The toolkit plasmids, which are piggyBac or lentiviral based, provide the means for reliable and rapid delivery of Cas9/gRNA through either transient transfection or stable integration. Using the toolkit, we demonstrate the ease with which users can perform single or multiplex gene editing and modulate the expression of both coding and non-coding genes. We also highlight the use of the comprehensive toolkit to perform combinatorial gene knockout to identify factors that regulate homologous recombination, along with investigating the regulatory role of a 68-kb intronic region associated with human disease.
{"title":"Generation and Application of a Versatile CRISPR Toolkit for Mammalian Cell Engineering","authors":"N. Dang, Alissa Lance-Byrne, Angela Tung, Xiaoge Guo, Ryan J. Cecchi, Joanna Buchthal, Alejandro Chavez, N. C. Yeo","doi":"10.1093/synbio/ysab033","DOIUrl":"https://doi.org/10.1093/synbio/ysab033","url":null,"abstract":"\u0000 CRISPR/Cas9 has revolutionized the field of genome engineering. Yet, as the CRISPR toolbox has rapidly expanded, there remains a need for a comprehensive library of CRISPR/Cas9 reagents that allow users to perform complex cellular and genetic manipulations without requiring labor-intensive generation of reagents to meet each user’s unique experimental circumstances. Here we described the creation and validation of a pNAX CRISPR library consisting of 72 different Cas9 and gRNA expression plasmids to allow for efficient multiplex gene editing, activation, and repression in mammalian cells. The toolkit plasmids, which are piggyBac or lentiviral based, provide the means for reliable and rapid delivery of Cas9/gRNA through either transient transfection or stable integration. Using the toolkit, we demonstrate the ease with which users can perform single or multiplex gene editing and modulate the expression of both coding and non-coding genes. We also highlight the use of the comprehensive toolkit to perform combinatorial gene knockout to identify factors that regulate homologous recombination, along with investigating the regulatory role of a 68-kb intronic region associated with human disease.","PeriodicalId":22158,"journal":{"name":"Synthetic Biology","volume":"1 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2021-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83629927","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Laboratory automation and mathematical optimisation are key to improving the efficiency of synthetic biology research. While there are algorithms optimising the construct designs and synthesis strategies for DNA assembly, the optimisation of how DNA assembly reaction mixes are prepared remains largely unexplored. Here, we focus on reducing the pipette tip consumption of a liquid-handling robot as it delivers DNA parts across a multi-well plate where several constructs are being assembled in parallel. We propose a linear programming formulation of this problem based on the capacitated vehicle routing problem, along with an algorithm which applies a linear programming solver to our formulation, hence providing a strategy to prepare a given set of DNA assembly mixes using fewer pipette tips. The algorithm performed well in randomly generated and real-life scenarios concerning several modular DNA assembly standards, proving capable of reducing the pipette tip consumption by up to 61% in large-scale cases. Combining automatic process optimisation and robotic liquid-handling, our strategy promises to greatly improve the efficiency of DNA assembly, either used alone or in combination with other algorithmic methods.
{"title":"A linear programming-based strategy to save pipette tips in automated DNA assembly","authors":"Kirill Sechkar, Z. Tuza, G. Stan","doi":"10.1093/synbio/ysac004","DOIUrl":"https://doi.org/10.1093/synbio/ysac004","url":null,"abstract":"Laboratory automation and mathematical optimisation are key to improving the efficiency of synthetic biology research. While there are algorithms optimising the construct designs and synthesis strategies for DNA assembly, the optimisation of how DNA assembly reaction mixes are prepared remains largely unexplored. Here, we focus on reducing the pipette tip consumption of a liquid-handling robot as it delivers DNA parts across a multi-well plate where several constructs are being assembled in parallel. We propose a linear programming formulation of this problem based on the capacitated vehicle routing problem, along with an algorithm which applies a linear programming solver to our formulation, hence providing a strategy to prepare a given set of DNA assembly mixes using fewer pipette tips. The algorithm performed well in randomly generated and real-life scenarios concerning several modular DNA assembly standards, proving capable of reducing the pipette tip consumption by up to 61% in large-scale cases. Combining automatic process optimisation and robotic liquid-handling, our strategy promises to greatly improve the efficiency of DNA assembly, either used alone or in combination with other algorithmic methods.","PeriodicalId":22158,"journal":{"name":"Synthetic Biology","volume":"51 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2021-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84976747","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jing Wui Yeoh, Neil Swainston, Peter Vegh, Valentin Zulkower, Pablo Carbonell, M. B. Holowko, Gopal Peddinti, C. Poh
Abstract Advances in hardware automation in synthetic biology laboratories are not yet fully matched by those of their software counterparts. Such automated laboratories, now commonly called biofoundries, require software solutions that would help with many specialized tasks such as batch DNA design, sample and data tracking, and data analysis, among others. Typically, many of the challenges facing biofoundries are shared, yet there is frequent wheel-reinvention where many labs develop similar software solutions in parallel. In this article, we present the first attempt at creating a standardized, open-source Python package. A number of tools will be integrated and developed that we envisage will become the obvious starting point for software development projects within biofoundries globally. Specifically, we describe the current state of available software, present usage scenarios and case studies for common problems, and finally describe plans for future development. SynBiopython is publicly available at the following address: http://synbiopython.org.
{"title":"SynBiopython: an open-source software library for Synthetic Biology","authors":"Jing Wui Yeoh, Neil Swainston, Peter Vegh, Valentin Zulkower, Pablo Carbonell, M. B. Holowko, Gopal Peddinti, C. Poh","doi":"10.1093/synbio/ysab001","DOIUrl":"https://doi.org/10.1093/synbio/ysab001","url":null,"abstract":"Abstract Advances in hardware automation in synthetic biology laboratories are not yet fully matched by those of their software counterparts. Such automated laboratories, now commonly called biofoundries, require software solutions that would help with many specialized tasks such as batch DNA design, sample and data tracking, and data analysis, among others. Typically, many of the challenges facing biofoundries are shared, yet there is frequent wheel-reinvention where many labs develop similar software solutions in parallel. In this article, we present the first attempt at creating a standardized, open-source Python package. A number of tools will be integrated and developed that we envisage will become the obvious starting point for software development projects within biofoundries globally. Specifically, we describe the current state of available software, present usage scenarios and case studies for common problems, and finally describe plans for future development. SynBiopython is publicly available at the following address: http://synbiopython.org.","PeriodicalId":22158,"journal":{"name":"Synthetic Biology","volume":"1 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90839804","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Alejandro Arce-Rodríguez, Ilaria Benedetti, Rafael Silva-Rocha, V. de Lorenzo
Although inducible heterologous expression systems have been available since the birth of recombinant DNA technology, the diversity of devices and genetic architectures of the corresponding vectors have often resulted in a lack of reproducibility and interoperability. In an effort to increase predictability of expression of genes of interest in a variety of possible bacterial hosts we propose a composition standard for debugging and reassembling all regulatory parts that participate in the performance of such devices. As a case study we address the n-octane and dicyclopropyl ketone (DCPK)-inducible PalkB promoter of the alkane biodegradation pOCT plasmid of Pseudomonas putida. The standardized expression module consisted of an edited alkS regulatory gene that is divergently expressed and separated of PalkB by a synthetic DNA buffer sequence. The native DNA sequence of the structural alkS gene was modified to alleviate the catabolite repression exerted by some carbon and nitrogen sources through the Crc/Hfq complex of some hosts. The PalkB promoter along with the alkS variants were then formatted as SEVA (Standard European Vector Architecture) cargoes and their activity parameters in P. putida determined with GFP and luminiscent reporters. The thereby refactored system showed improvements in various features desirable in conditional expression modules: inducibility, capacity, noise reduction and on/off ratio. When applied to other promoter/regulator pairs, the compositional standard thereby implemented in the AlkS/PalkB module will enable more complex genetic programming in non-model bacteria.
{"title":"Standardization of inducer-activated broad host range expression modules: debugging and refactoring an alkane-responsive AlkS/PalkB device","authors":"Alejandro Arce-Rodríguez, Ilaria Benedetti, Rafael Silva-Rocha, V. de Lorenzo","doi":"10.1093/synbio/ysab030","DOIUrl":"https://doi.org/10.1093/synbio/ysab030","url":null,"abstract":"Although inducible heterologous expression systems have been available since the birth of recombinant DNA technology, the diversity of devices and genetic architectures of the corresponding vectors have often resulted in a lack of reproducibility and interoperability. In an effort to increase predictability of expression of genes of interest in a variety of possible bacterial hosts we propose a composition standard for debugging and reassembling all regulatory parts that participate in the performance of such devices. As a case study we address the n-octane and dicyclopropyl ketone (DCPK)-inducible PalkB promoter of the alkane biodegradation pOCT plasmid of Pseudomonas putida. The standardized expression module consisted of an edited alkS regulatory gene that is divergently expressed and separated of PalkB by a synthetic DNA buffer sequence. The native DNA sequence of the structural alkS gene was modified to alleviate the catabolite repression exerted by some carbon and nitrogen sources through the Crc/Hfq complex of some hosts. The PalkB promoter along with the alkS variants were then formatted as SEVA (Standard European Vector Architecture) cargoes and their activity parameters in P. putida determined with GFP and luminiscent reporters. The thereby refactored system showed improvements in various features desirable in conditional expression modules: inducibility, capacity, noise reduction and on/off ratio. When applied to other promoter/regulator pairs, the compositional standard thereby implemented in the AlkS/PalkB module will enable more complex genetic programming in non-model bacteria.","PeriodicalId":22158,"journal":{"name":"Synthetic Biology","volume":"18 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2020-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85708124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Nymul E. Khan, Enoch Yeung, Yuliya Farris, S. Fansler, Hans C. Bernstein
Modern microbial biodesign relies on the principle that well-characterized genetic parts can be reused and reconfigured for different functions. However, this paradigm has only been successful in a limited set of hosts, mostly comprised from common lab strains of Escherichia coli. It is clear that new applications such as chemical sensing and event logging in complex environments will benefit from new host chassis. This study quantitatively compared how the same chemical event logger performed across four strains and three different microbial species. An integrase-based sensor and memory device was operated by two representative soil Pseudomonads—Pseudomonas fluorescens SBW25 and Pseudomonas putida DSM 291. Quantitative comparisons were made between these two non-traditional hosts and two benchmark E. coli chassis including the probiotic Nissle 1917 and common cloning strain DH5α. The performance of sensor and memory components changed according to each host, such that a clear chassis effect was observed and quantified. These results were obtained via fluorescence from reporter proteins that were transcriptionally fused to the integrase and downstream recombinant region and via data-driven kinetic models. The Pseudomonads proved to be acceptable chassis for the operation of this event logger, which outperformed the common E. coli DH5α in many ways. This study advances an emerging frontier in synthetic biology that aims to build broad-host-range devices and understand the context by which different species can execute programmable genetic operations.
{"title":"A broad-host-range event detector: expanding and quantifying performance between Escherichia coli and Pseudomonas species","authors":"Nymul E. Khan, Enoch Yeung, Yuliya Farris, S. Fansler, Hans C. Bernstein","doi":"10.1093/synbio/ysaa002","DOIUrl":"https://doi.org/10.1093/synbio/ysaa002","url":null,"abstract":"Modern microbial biodesign relies on the principle that well-characterized genetic parts can be reused and reconfigured for different functions. However, this paradigm has only been successful in a limited set of hosts, mostly comprised from common lab strains of Escherichia coli. It is clear that new applications such as chemical sensing and event logging in complex environments will benefit from new host chassis. This study quantitatively compared how the same chemical event logger performed across four strains and three different microbial species. An integrase-based sensor and memory device was operated by two representative soil Pseudomonads—Pseudomonas fluorescens SBW25 and Pseudomonas putida DSM 291. Quantitative comparisons were made between these two non-traditional hosts and two benchmark E. coli chassis including the probiotic Nissle 1917 and common cloning strain DH5α. The performance of sensor and memory components changed according to each host, such that a clear chassis effect was observed and quantified. These results were obtained via fluorescence from reporter proteins that were transcriptionally fused to the integrase and downstream recombinant region and via data-driven kinetic models. The Pseudomonads proved to be acceptable chassis for the operation of this event logger, which outperformed the common E. coli DH5α in many ways. This study advances an emerging frontier in synthetic biology that aims to build broad-host-range devices and understand the context by which different species can execute programmable genetic operations.","PeriodicalId":22158,"journal":{"name":"Synthetic Biology","volume":"24 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85385553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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