Synthetic biology (Oxford, England)最新文献

{"title":"GoldBricks: an improved cloning strategy that combines features of Golden Gate and BioBricks for better efficiency and usability.","authors":"Vishalsingh R Chaudhari,&nbsp;Maureen R Hanson","doi":"10.1093/synbio/ysab032","DOIUrl":"https://doi.org/10.1093/synbio/ysab032","url":null,"abstract":"<p><p>With increasing complexity of expression studies and the repertoire of characterized sequences, combinatorial cloning has become a common necessity. Techniques like BioBricks and Golden Gate aim to standardize and speed up the process of cloning large constructs while enabling sharing of resources. The BioBricks format provides a simplified and flexible approach to endless assembly with a compact library and useful intermediates but is a slow process, joining only two parts in a cycle. Golden Gate improves upon the speed with use of Type IIS enzymes and joins several parts in a cycle but requires a larger library of parts and logistical inefficiencies scale up significantly in the multigene format. We present here a method that provides improvement over these techniques by combining their features. By using Type IIS enzymes in a format like BioBricks, we have enabled a faster and efficient assembly with reduced scarring, which performs at a similarly fast pace as Golden Gate, but significantly reduces library size and user input. Additionally, this method enables faster assembly of operon-style constructs, a feature requiring extensive workaround in Golden Gate. Our format allows such inclusions resulting in faster and more efficient assembly.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab032"},"PeriodicalIF":0.0,"publicationDate":"2021-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/eb/9e/ysab032.PMC8578713.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39713192","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":"Data sharing policies: share well and you shall be rewarded.","authors":"Jean Peccoud","doi":"10.1093/synbio/ysab028","DOIUrl":"https://doi.org/10.1093/synbio/ysab028","url":null,"abstract":"<p><p>Sharing research data is an integral part of the scientific publishing process. By sharing data, authors enable their readers to use their results in a way that the textual description of the results does not allow by itself. In order to achieve this objective, data should be shared in a way that makes it as easy as possible for readers to import them in computer software where they can be viewed, manipulated and analyzed. Many authors and reviewers seem to misunderstand the purpose of the data sharing policies developed by journals. Rather than being an administrative burden that authors should comply with to get published, the objective of these policies is to help authors maximize the impact of their work by allowing other members of the scientific community to build upon it. Authors and reviewers need to understand the purpose of data sharing policies to assist editors and publishers in their efforts to ensure that every article published complies with them.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab028"},"PeriodicalIF":0.0,"publicationDate":"2021-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8482415/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39483587","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":"Contemporary biomedical engineering perspective on volitional evolution for human radiotolerance enhancement beyond low-earth orbit.","authors":"Alexander M Borg,&nbsp;John E Baker","doi":"10.1093/synbio/ysab023","DOIUrl":"https://doi.org/10.1093/synbio/ysab023","url":null,"abstract":"<p><p>A primary objective of the National Aeronautics and Space Administration (NASA) is expansion of humankind's presence outside low-Earth orbit, culminating in permanent interplanetary travel and habitation. Having no inherent means of physiological detection or protection against ionizing radiation, humans incur capricious risk when journeying beyond low-Earth orbit for long periods. NASA has made large investments to analyze pathologies from space radiation exposure, emphasizing the importance of characterizing radiation's physiological effects. Because natural evolution would require many generations to confer resistance against space radiation, immediately pragmatic approaches should be considered. Volitional evolution, defined as humans steering their own heredity, may inevitably retrofit the genome to mitigate resultant pathologies from space radiation exposure. Recently, uniquely radioprotective genes have been identified, conferring local or systemic radiotolerance when overexpressed <i>in vitro</i> and <i>in vivo</i>. Aiding in this process, the CRISPR/Cas9 technique is an inexpensive and reproducible instrument capable of making limited additions and deletions to the genome. Although cohorts can be identified and engineered to protect against radiation, alternative and supplemental strategies should be seriously considered. Advanced propulsion and mild synthetic torpor are perhaps the most likely to be integrated. Interfacing artificial intelligence with genetic engineering using predefined boundary conditions may enable the computational modeling of otherwise overly complex biological networks. The ethical context and boundaries of introducing genetically pioneered humans are considered.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab023"},"PeriodicalIF":0.0,"publicationDate":"2021-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8434797/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39436532","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":"Evaluation of inducible promoter-riboswitch constructs for heterologous protein expression in the cyanobacterial species Anabaena sp. PCC 7120.","authors":"Jessee Svoboda,&nbsp;Brenda Cisneros,&nbsp;Benjamin Philmus","doi":"10.1093/synbio/ysab019","DOIUrl":"https://doi.org/10.1093/synbio/ysab019","url":null,"abstract":"Abstract Cyanobacteria are promising chassis for synthetic biology applications due to the fact that they are photosynthetic organisms capable of growing in simple, inexpensive media. Given their slower growth rate than other model organisms such as Escherichia coli and Saccharomyces cerevisiae, there are fewer synthetic biology tools and promoters available for use in model cyanobacteria. Here, we compared a small library of promoter–riboswitch constructs for synthetic biology applications in Anabaena sp. PCC 7120, a model filamentous cyanobacterium. These constructs were designed from six cyanobacterial promoters of various strengths, each paired with one of two theophylline-responsive riboswitches. The promoter–riboswitch pairs were cloned upstream of a chloramphenicol acetyltransferase (cat) gene, and CAT activity was quantified using an in vitro assay. Addition of theophylline to cultures increased the CAT activity in almost all cases, allowing inducible protein production with natively constitutive promoters. We found that riboswitch F tended to have a lower induced and uninduced production compared to riboswitch E for the weak and medium promoters, although the difference was larger for the uninduced production, in accord with previous research. The strong promoters yielded a higher baseline CAT activity than medium strength and weak promoters. In addition, we observed no appreciable difference between CAT activity measured from strong promoters cultured in uninduced and induced conditions. The results of this study add to the genetic toolbox for cyanobacteria and allow future natural product and synthetic biology researchers to choose a construct that fits their needs.","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab019"},"PeriodicalIF":0.0,"publicationDate":"2021-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8546608/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39825393","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":"Automated design and implementation of a NOR gate in Pseudomonas putida.","authors":"Huseyin Tas,&nbsp;Lewis Grozinger,&nbsp;Angel Goñi-Moreno,&nbsp;Victor de Lorenzo","doi":"10.1093/synbio/ysab024","DOIUrl":"https://doi.org/10.1093/synbio/ysab024","url":null,"abstract":"<p><p>Boolean NOR gates have been widely implemented in <i>Escherichia coli</i> as transcriptional regulatory devices for building complex genetic circuits. Yet, their portability to other bacterial hosts/chassis is generally hampered by frequent changes in the parameters of the INPUT/OUTPUT response functions brought about by new genetic and biochemical contexts. Here, we have used the circuit design tool CELLO for assembling a NOR gate in the soil bacterium and the metabolic engineering platform <i>Pseudomonas putida</i> with components tailored for <i>E. coli.</i> To this end, we capitalized on the functional parameters of 20 genetic inverters for each host and the resulting compatibility between NOT pairs. Moreover, we added to the gate library three inducible promoters that are specific to <i>P. putida</i>, thus expanding cross-platform assembly options. While the number of potential connectable inverters decreased drastically when moving the library from <i>E. coli</i> to <i>P. putida</i>, the CELLO software was still able to find an effective NOR gate in the new chassis. The automated generation of the corresponding DNA sequence and <i>in vivo</i> experimental verification accredited that some genetic modules initially optimized for <i>E. coli</i> can indeed be reused to deliver NOR logic in <i>P. putida</i> as well. Furthermore, the results highlight the value of creating host-specific collections of well-characterized regulatory inverters for the quick assembly of genetic circuits to meet complex specifications.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab024"},"PeriodicalIF":0.0,"publicationDate":"2021-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/ff/5e/ysab024.PMC8546601.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39839147","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":"Synthetic biological toggle circuits that respond within seconds and teach us new biology.","authors":"Sonja Billerbeck","doi":"10.1093/synbio/ysab027","DOIUrl":"https://doi.org/10.1093/synbio/ysab027","url":null,"abstract":"Imagine it would take several minutes or even hours for your light bulb to turn on after you hit the switch—not very useful for many daily (and nightly) activities. Light switches are made from the so-called toggle switches; basic and widely used electrical components that provide binary on–off control over electrical circuits, allowing quick decisionmaking and memory. Synthetic biologists have built analogues genetic toggle switches but until now those only responded in time-ranges of minutes to hours, limiting their use in applications that require real-time action. Mishra et al. have recently built a biological bistable toggle switch in yeast that responds within seconds by mimicking nature’s way of rapid response generation (1). Synthetic biologists envision to control cellular behavior by engineering biology in analogy to electrical circuits. Implementing a synthetic biological toggle switch was thus one of the early achievements of the field (2). While, over the last two decades, synthetic biologists have mastered to build toggle switches that respond to various inputs—chemicals, light or temperature—and show high switching robustness (3); one challenge remained: timing! Existing circuits act slow as they rely on transcription and translation for signal progression, resulting in significant delays between input-sensing and toggling into the corresponding response state. Not only Synthetic Biologists but nature itself controls important decisions—such as cell cycle progression, embryonal development or induced cell death—via bistable toggle switches. But nature knows how to act fast: rapid responses are not mediated by genetics but via post-translational protein modifications, such as phosphorylations. Although phosphor-regulation has long been known, it was difficult to engineer and concert into designed behavior. Mishra et al. overcame this hurdle by developing (phosphorylation-required interaction and mediated effect (PRIME) that harnesses the modularity of natural phosphate regulators and allows us to build chimeric proteins that can be combined to ‘phosphor-in phosphorout’ gates. One gate consists of two chimeric proteins that interact in a phosphate-dependent manner: once the upstream protein partner gets activated by a trigger, it binds to and activates or deactivates (phosphorylates or de-phosphorylates) its downstream partner. The downstreampartner then acts as the activator within the next PRIME gate. Using the PRIME gates, the authors build a network of logic gates resulting in a new-to-nature toggle network architecture that could be switched from one state to the other by two different chemical inputs, sorbitol and isopentenyl adenine—two chemicals for which receptors were readily available. As the first test read-out of the system, they used a green fluorescent protein that could be toggled between localization in the cytosol or the nucleus. Eventually, the authors showed to control a complex cellular function, yeast bud format","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab027"},"PeriodicalIF":0.0,"publicationDate":"2021-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8434798/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39436531","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":"Erratum to: A MATLAB toolbox for modeling genetic circuits in cell-free systems.","authors":"Vipul Singhal,&nbsp;Zoltan A Tuza,&nbsp;Zachary Z Sun,&nbsp;Richard M Murray","doi":"10.1093/synbio/ysab016","DOIUrl":"https://doi.org/10.1093/synbio/ysab016","url":null,"abstract":"<p><p>[This corrects the article DOI: 10.1093/synbio/ysab007.].</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab016"},"PeriodicalIF":0.0,"publicationDate":"2021-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/68/7d/ysab016.PMC8379372.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39341295","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":"High-yield 'one-pot' biosynthesis of raspberry ketone, a high-value fine chemical.","authors":"Simon J Moore,&nbsp;Tommaso Tosi,&nbsp;David Bell,&nbsp;Yonek B Hleba,&nbsp;Karen M Polizzi,&nbsp;Paul S Freemont","doi":"10.1093/synbio/ysab021","DOIUrl":"https://doi.org/10.1093/synbio/ysab021","url":null,"abstract":"<p><p>Cell-free extract and purified enzyme-based systems provide an attractive solution to study biosynthetic strategies towards a range of chemicals. 4-(4-hydroxyphenyl)-butan-2-one, also known as raspberry ketone, is the major fragrance component of raspberry fruit and is used as a natural additive in the food and sports industry. Current industrial processing of the natural form of raspberry ketone involves chemical extraction from a yield of ∼1-4 mg kg^-1 of fruit. Due to toxicity, microbial production provides only low yields of up to 5-100 mg L^-1. Herein, we report an efficient cell-free strategy to probe into a synthetic enzyme pathway that converts either L-tyrosine or the precursor, 4-(4-hydroxyphenyl)-buten-2-one, into raspberry ketone at up to 100% conversion. As part of this strategy, it is essential to recycle inexpensive cofactors. Specifically, the final enzyme step in the pathway is catalyzed by raspberry ketone/zingerone synthase (RZS1), an NADPH-dependent double bond reductase. To relax cofactor specificity towards NADH, the preferred cofactor for cell-free biosynthesis, we identify a variant (G191D) with strong activity with NADH. We implement the RZS1 G191D variant within a 'one-pot' cell-free reaction to produce raspberry ketone at high-yield (61 mg L^-1), which provides an alternative route to traditional microbial production. In conclusion, our cell-free strategy complements the growing interest in engineering synthetic enzyme cascades towards industrially relevant value-added chemicals.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab021"},"PeriodicalIF":0.0,"publicationDate":"2021-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8546603/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39825394","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":"Enzyme engineering and <i>in vivo</i> testing of a formate reduction pathway.","authors":"Jue Wang,&nbsp;Karl Anderson,&nbsp;Ellen Yang,&nbsp;Lian He,&nbsp;Mary E Lidstrom","doi":"10.1093/synbio/ysab020","DOIUrl":"https://doi.org/10.1093/synbio/ysab020","url":null,"abstract":"<p><p>Formate is an attractive feedstock for sustainable microbial production of fuels and chemicals, but its potential is limited by the lack of efficient assimilation pathways. The reduction of formate to formaldehyde would allow efficient downstream assimilation, but no efficient enzymes are known for this transformation. To develop a 2-step formate reduction pathway, we screened natural variants of acyl-CoA synthetase (ACS) and acylating aldehyde dehydrogenase (ACDH) for activity on one-carbon substrates and identified active and highly expressed homologs of both enzymes. We then performed directed evolution, increasing ACDH-specific activity by 2.5-fold and ACS lysate activity by 5-fold. To test for the <i>in vivo</i> activity of our pathway, we expressed it in a methylotroph which can natively assimilate formaldehyde. Although the enzymes were active in cell extracts, we could not detect formate assimilation into biomass, indicating that further improvement will be required for formatotrophy. Our work provides a foundation for further development of a versatile pathway for formate assimilation.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab020"},"PeriodicalIF":0.0,"publicationDate":"2021-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8511477/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39519298","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":"Rational gRNA design based on transcription factor binding data.","authors":"David Bergenholm,&nbsp;Yasaman Dabirian,&nbsp;Raphael Ferreira,&nbsp;Verena Siewers,&nbsp;Florian David,&nbsp;Jens Nielsen","doi":"10.1093/synbio/ysab014","DOIUrl":"https://doi.org/10.1093/synbio/ysab014","url":null,"abstract":"<p><p>The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has become a standard tool in many genome engineering endeavors. The endonuclease-deficient version of Cas9 (dCas9) is also a powerful programmable tool for gene regulation. In this study, we made use of <i>Saccharomyces cerevisiae</i> transcription factor (TF) binding data to obtain a better understanding of the interplay between TF binding and binding of dCas9 fused to an activator domain, VPR. More specifically, we targeted dCas9-VPR toward binding sites of Gcr1-Gcr2 and Tye7 present in several promoters of genes encoding enzymes engaged in the central carbon metabolism. From our data, we observed an upregulation of gene expression when dCas9-VPR was targeted next to a TF binding motif, whereas a downregulation or no change was observed when dCas9 was bound on a TF motif. This suggests a steric competition between dCas9 and the specific TF. Integrating TF binding data, therefore, proved to be useful for designing guide RNAs for CRISPR interference or CRISPR activation applications.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab014"},"PeriodicalIF":0.0,"publicationDate":"2021-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/3d/cc/ysab014.PMC8546606.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39825389","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}