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Replication of M13 single-stranded DNA bearing a sitespecific ethenocytosine lesion by Escherichia coil cell extracts

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cell Research 1997年第1期7卷 1-12页

作者： WANGGE,PAULMDUNMAN,MZAFRIHUMAYUN DepartmentofMicrobiologyandMolecularGenetics!UMDNewJerseyMedicalSchool185 SouthOrangeAvenue MSBF607Newark NJ07103-2714 USA DepartmentofMicrobiologyandMolecularGenetics!UMDNewJerseyMedicalSchool185 SouthOrangeAvenue MSBF607Newark NJ07103-2714 USA Departmen

Previous investigation on the mutagenic effects of 3,N4-Ethenocytosine (εC), a nonpairing DNA lesion,revealed the existence of a novel SOS-independent inducible mutagenic mechanism in E. coli termed UVM for UV modulation of mutagenesis. To investigate whether UVM is mediated by an alteration of DNA replication, we have set up an in vitro replication system ill which phage M13 viral single-stranded DNA bearing a single site-specific (εC) residue is replicated by soluble protein extracts from E. coli cells. Replication products were analyzed by agarose gel electrophoresis and the frequency of translesion synthesis was determined by restriction endonuclease analyses. Our data indicate that DNA replication is strongly inhibited by εC, but that translesion DNA synthesis does occur in about 14% of the replicated DNA molecules. These results are very similar to those observed previously in vivo, and suggest that this experimental system may be suitable for evaluating alterations in DNA replication in UVM-induced cells.

关键词： Ethenocytosine M13 in vitro replication cell extract

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Methods to reduce variability in E. Coli-based cell-free protein expression experiments

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Synthetic and Systems Biotechnology 2019年第4期4卷 204-211页

作者： Jared L.Dopp Yeong Ran Jo Nigel F.Reuel 不详

cell-free protein synthesis(CFPS)is an established biotechnology tool that has shown great utility in many applications such as prototyping proteins,building genetic circuits,designing biosensors,and expressing cytotoxic *** CFPS has been widely deployed,the many,varied methods presented in the literature can be challenging for new users to *** our experience and others who newly enter the field,one of the most frustrating aspects of applying CFPS as a laboratory can be the large levels of variability that are present within experimental *** we provide a retrospective summary of CFPS methods that reduce variability *** methods include optimized extract preparation,fully solubilizing the master mix components,and careful mixing of the *** have reduced our coefficient of variation from 97.3%to 1.2%.Moreover,these methods allow complete novices(*** rotation undergraduate students)to provide data that is comparable to experienced users,thus allowing broader participation in this exciting research area.

关键词： cell-free protein synthesis CFPS cell extract In vitro protein synthesis In vitro transcription-translation cell-free synthetic biology

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Main advances in the Escherichia coli-based cell-free protein synthesis systems

Main advances in the Escherichia coli-based cell-free protei...

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山东省药学会2010年生化与生物技术药物学术研讨会

作者：廉倩倩王凤山 Institute of Biochemical and Biotechnological Drugs School of Pharmaceutical SciencesShandong University

＜正＞cell-free protein synthesis plays an important part as a basal method for many technologies in life science and the cell-free protein synthesis systems have been developed greatly over the past several ***,we mainly review the main advances in cell extract preparation,energy regeneration system,reaction formats of the Escherichia coli-based cell-free protein synthesis systems.

关键词： cell-free protein synthesis(CFPS) system Escherichia coli cell extract energy regeneration system reaction formats

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