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Expression of Recombinant Genes in the Yeast Pichia pastoris 重组基因在毕赤酵母中的表达
Current Protocols Essential Laboratory Techniques Pub Date : 2018-09-11 DOI: 10.1002/cpet.25
James M. Cregg, Ilya Tolstorukov, Anasua Kusari, Anthony Jay Sunga, Knut Madden, Thomas Chappell
{"title":"Expression of Recombinant Genes in the Yeast Pichia pastoris","authors":"James M. Cregg,&nbsp;Ilya Tolstorukov,&nbsp;Anasua Kusari,&nbsp;Anthony Jay Sunga,&nbsp;Knut Madden,&nbsp;Thomas Chappell","doi":"10.1002/cpet.25","DOIUrl":"10.1002/cpet.25","url":null,"abstract":"<p>The synthesis of specific recombinant proteins using single-celled organisms from bacteria to mammalian tissue culture cells has become a major source of biopharmaceutical products for the industry and a source of a wide variety of proteins for academic research. A range of organisms are utilized for this purpose. One of the newest and most promising of these is the yeast <i>Pichia pastoris</i>. This article provides detailed basic protocols for the expression of heterologous genes and the synthesis of recombinant proteins utilizing this yeast. Specifically provided are protocols for the insertion of foreign vector DNAs into the yeast by electroporation, amplification of vector sequences by the post-translational vector amplification (PTVA) method, and growth and expression of foreign genes in shake flask cultures. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.25","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42748509","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Overview of Protein Expression Vectors for E. coli 大肠杆菌蛋白表达载体综述
Current Protocols Essential Laboratory Techniques Pub Date : 2018-08-21 DOI: 10.1002/cpet.23
Paul D. Riggs
{"title":"Overview of Protein Expression Vectors for E. coli","authors":"Paul D. Riggs","doi":"10.1002/cpet.23","DOIUrl":"10.1002/cpet.23","url":null,"abstract":"<p><i>Escherichia</i> <i>coli</i> is often the first host chosen for recombinant protein production, due to its low cost, ease of use, and the wide range of molecular tools available. After the gene of interest is cloned, the first task of the researcher is to choose a suitable protein expression vector for protein production. The number of possible vectors is vast, but a good background of the advantages and disadvantages of the various types of vectors can help in designing a successful strategy. This unit provides an introduction to the elements that make up an expression vector and guidance in applying those elements to the production of a particular protein. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.23","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47404846","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 6
Zebrafish: Development of a Vertebrate Model Organism 斑马鱼:一种脊椎动物模式生物的发育
Current Protocols Essential Laboratory Techniques Pub Date : 2018-05-04 DOI: 10.1002/cpet.19
Jason R. Meyers
{"title":"Zebrafish: Development of a Vertebrate Model Organism","authors":"Jason R. Meyers","doi":"10.1002/cpet.19","DOIUrl":"10.1002/cpet.19","url":null,"abstract":"<p>Zebrafish (<i>Danio rerio</i>) are a small-bodied tropical, freshwater fish species originally from South Asia. The ease of care, year-round prolific breeding, and transparent, external development have made these fish a popular model vertebrate for many fields of biology. Over the past 40 years, the development of methods for genetic and developmental manipulation have driven a rapid rise in their use in research. This review provides an orientation to the zebrafish as a model system, from its ecology and life history to its early use in research and adoption as a key genetic model for vertebrate development. Its current uses and key techniques that highlight future areas of development of the system are also discussed. Finally, an introduction to the essentials of zebrafish use and care are presented, highlighting the ease of adoption of this system for teaching and research use. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.19","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"50803764","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 107
Radiation Safety and Measurement 辐射安全与测量
Current Protocols Essential Laboratory Techniques Pub Date : 2018-04-24 DOI: 10.1002/cpet.22
Jill Meisenhelder, Steve Bursik
{"title":"Radiation Safety and Measurement","authors":"Jill Meisenhelder,&nbsp;Steve Bursik","doi":"10.1002/cpet.22","DOIUrl":"10.1002/cpet.22","url":null,"abstract":"<p>Scientific knowledge has been considerably advanced by the use of biochemical molecules which incorporate radioisotopes at specific sites. This unit provides an overview of the use of radioisotopes in the biochemical laboratory. It begins with a discussion of the principles of radioactivity in order to provide the reader/user with knowledge on which to base a common sense approach to the safe use of isotopes. The characteristics of isotopes most commonly used in a biochemical laboratory are then detailed, as well as the safety precautions and monitoring methods peculiar to each one. Detection and imaging methods used in experimental analysis are reviewed. Finally, an outline of an orderly, proper response to a spill of radioactive material is presented. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.22","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47283902","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Preparing and Presenting a Poster 准备和展示海报
Current Protocols Essential Laboratory Techniques Pub Date : 2018-04-24 DOI: 10.1002/cpet.18
Sharon E. Torigoe
{"title":"Preparing and Presenting a Poster","authors":"Sharon E. Torigoe","doi":"10.1002/cpet.18","DOIUrl":"10.1002/cpet.18","url":null,"abstract":"<p>A poster is one method of informing the world about results from a research project. Whether serving as an aid for a presentation or standing alone, it should provide information about a project. Both substantive and stylistic qualities should contribute to effectively communicating one's conclusions. This article gives general guidelines and suggestions for poster content, layout, and design to assist in preparing an effective poster. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.18","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44932982","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Saccharomyces cerevisiae: A Unicellular Model Genetic Organism of Enduring Importance 酿酒酵母：一种具有持久重要性的单细胞模式遗传生物
Current Protocols Essential Laboratory Techniques Pub Date : 2018-04-24 DOI: 10.1002/cpet.21
Pamela K. Hanson
{"title":"Saccharomyces cerevisiae: A Unicellular Model Genetic Organism of Enduring Importance","authors":"Pamela K. Hanson","doi":"10.1002/cpet.21","DOIUrl":"10.1002/cpet.21","url":null,"abstract":"<p>Though perhaps best known for its roles in baking and brewing, the budding yeast <i>Saccharomyces cerevisiae</i> is found in a wide range of habitats and has been adapted for use in the laboratory. <i>S. cerevisiae</i>’s reputation as a powerful genetic model system stems in part from its remarkably efficient homologous recombination, which allows researchers to readily modify yeast genes. This genetic tractability also contributed to yeast's selection as the system of choice for Nobel laureates who studied everything from the cell cycle to membrane trafficking. As the first eukaryote to have its genome fully sequenced, <i>S. cerevisiae</i> has long been at the leading edge of genomic-scale research, including microarrays, systematic gene deletion, and more recently, construction of a fully synthetic eukaryotic genome. A dedicated, well-curated database, and a wide range of commercially available collections make it easy for new researchers to adopt this system to answer fundamentally important questions in eukaryotic cell biology. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.21","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42387049","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 12
How to Write a Research Manuscript 如何写研究稿件
Current Protocols Essential Laboratory Techniques Pub Date : 2018-04-24 DOI: 10.1002/cpet.20
Deborah J. Frank
{"title":"How to Write a Research Manuscript","authors":"Deborah J. Frank","doi":"10.1002/cpet.20","DOIUrl":"10.1002/cpet.20","url":null,"abstract":"<p>This article provides a step-by-step guide to help you turn your high-quality data into a high-quality manuscript for publication in a scientific journal. It covers all aspects of the writing process, including: choosing a journal to which to submit your paper, writing each section, formulating your “story,” making figures, soliciting constructive criticism, and navigating the review process. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.20","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45027040","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Preparing and Presenting a Talk 准备和演讲
Current Protocols Essential Laboratory Techniques Pub Date : 2018-02-13 DOI: 10.1002/cpet.13
Michael More, Christine Sjolander, Stephen Chang
{"title":"Preparing and Presenting a Talk","authors":"Michael More,&nbsp;Christine Sjolander,&nbsp;Stephen Chang","doi":"10.1002/cpet.13","DOIUrl":"10.1002/cpet.13","url":null,"abstract":"<p>How information is presented can have as much impact as the information itself. In order to ensure that you're getting your message across to the audience, you need to prepare your talk with as much care as you designed your research. This unit outlines information on how to prepare a great talk and deliver it for maximum effectiveness. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.13","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49544761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Practical Considerations When Altering Digital Images 修改数字图像时的实际考虑
Current Protocols Essential Laboratory Techniques Pub Date : 2018-02-13 DOI: 10.1002/cpet.11
Jerry Sedgewick
{"title":"Practical Considerations When Altering Digital Images","authors":"Jerry Sedgewick","doi":"10.1002/cpet.11","DOIUrl":"10.1002/cpet.11","url":null,"abstract":"<p>Worldwide, Adobe Photoshop is the primary means in research for altering images. Training on the use of this powerful tool most often is peer to peer within a laboratory, or it is self-taught through trial and error. As a result, varying amounts of visual data can be removed unwittingly; contrast- and brightness-enhancement of images can be done without adjusting color and grayscale levels to conform to the output (which includes publication, projection, Web placement, and inclusion into Microsoft Word and Adobe Acrobat); images can be unethically altered; and pixelation can be introduced. Correct methods for use of Adobe Photoshop are outlined in this unit so that these errors can be avoided. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.11","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45279061","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Dictyostelium discoideum: A Model System for Cell and Developmental Biology 盘状盘基骨菌:细胞与发育生物学的模式系统
Current Protocols Essential Laboratory Techniques Pub Date : 2017-11-09 DOI: 10.1002/cpet.15
Sabateeshan Mathavarajah, Ana Flores, Robert J. Huber
{"title":"Dictyostelium discoideum: A Model System for Cell and Developmental Biology","authors":"Sabateeshan Mathavarajah,&nbsp;Ana Flores,&nbsp;Robert J. Huber","doi":"10.1002/cpet.15","DOIUrl":"10.1002/cpet.15","url":null,"abstract":"<p>The social amoeba <i>Dictyostelium discoideum</i> has long served as a model system for studying fundamental processes in cell and developmental biology. This eukaryotic microbe is also recognized as a model organism for biomedical and human disease research since the genome encodes homologs of genes linked to human disease, such as those linked to cancer and neurodegeneration. <i>Dictyostelium</i> has a unique life cycle composed of a unicellular growth phase and a multicellular developmental phase that is induced by starvation. During its life cycle, <i>Dictyostelium</i> undergoes conserved cellular processes including, but not limited to, cell proliferation, phagocytosis, intercellular signaling, cell adhesion and motility, chemotaxis, and cell differentiation. The history of the organism, the resources available to researchers in the community, and the diverse ways that <i>Dictyostelium</i> is used in the contemporary research lab are discussed. © 2017 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":500994,"journal":{"name":"Current Protocols Essential Laboratory Techniques","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-11-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpet.15","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45192512","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 20
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