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A step-by-step introduction to rule-based design of synthetic genetic constructs using GenoCAD

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GenoCAD is an open source web-based system that provides a streamlined, rule-driven process for designing genetic sequences. GenoCAD provides a graphical interface that allows users to design sequences consistent with formalized design strategies
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  C H A P T E R E I G H T  A Step-by-Step Introduction toRule-Based Design of SyntheticGenetic Constructs Using GenoCAD Mandy L. Wilson, Russell Hertzberg, Laura Adam,  and   Jean Peccoud Contents 1 . Introduction  1742 . Overview of GenoCAD  1753 . Requesting an Account on GenoCAD.org  1764 . Browsing the Parts Catalog  1765 . Searching for Parts  1786 . Using My Cart to Create Libraries  1797 . My Libraries  1818 . My Parts  1839 . Designing Sequences  18310 . Installing GenoCAD  18511 . Anticipated Evolutions  186 Acknowledgments  187 References  188  Abstract GenoCAD is an open source web-based system that provides a streamlined,rule-driven process for designing genetic sequences. GenoCAD provides agraphical interface that allows users to design sequences consistent withformalized design strategies specific to a domain, organization, or project.Design strategies include limited sets of user-defined parts and rules indicatinghow these parts are to be combined in genetic constructs. In addition toreducing design time to minutes, GenoCAD improves the quality and reliabilityof the finished sequence by ensuring that the designs follow established rulesof sequence construction. GenoCAD.org is a publicly available instance of GenoCAD that can be found at www.genocad.org. The source code and latestbuild are available from SourceForge to allow advanced users to install andcustomize GenoCAD for their unique needs. Methods in Enzymology,  Volume 498  # 2011 Elsevier Inc.ISSN 0076-6879, DOI: 10.1016/B978-0-12-385120-8.00008-5 All rights reserved.Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, USA 173  This chapter focuses primarily on how the GenoCAD tools can be used toorganize genetic parts into customized personal libraries, then how theselibraries can be used to design sequences. In addition, GenoCAD’s parts man-agement system and search capabilities are described in detail. Instructions areprovided for installing a local instance of GenoCAD on a server. Some of thefuture enhancements of this rapidly evolving suite of applications are brieflydescribed. 1. Introduction The vision of rationally designing synthetic biological systems hasproved more elusive than anticipated (Kwok, 2010). The complexity of artificial gene networks has not made significant progress since 2006(Purnick and Weiss, 2009), which may indicate that the  ad hoc   processesused to develop proof-of-concept systems do not scale up well. The fieldstill lacks a suitable framework to design more complex systems. Severalauthors have proposed to approach DNA sequences as a language toprogram biological systems (Clancy and Voigt, 2010; Goler   et al.,  2008).This idea may provide the foundation upon which it will be possible todevelop computer-assisted design software applications for synthetic biol-ogy. A fast growing ecology of software tools to assist synthetic biologists inthe development of new biological systems has been reviewed recently(Marchisio and Stelling, 2009). Gene Designer (Villalobos  et al.,  2006) is astand-alone application with smooth graphical editor allowing users to dragand drop genetic parts into a larger DNA sequence. TinkerCell is another desktop application allowing users to design genetic constructs from stan-dard parts and simulate the dynamics of the gene network they encode(Chandran  et al.,  2009). SynBIOSS is a web-based alternative to TinkerCell(Hill  et al.,  2008; Weeding  et al.,  2010). GEC (Pedersen and Philipps, 2009) and Clotho (www.clothocad.org) are programming environments specifi-cally designed for synthetic biology.Like TinkerCell or Gene Designer, GenoCAD has a graphical user interface accessible to users without any programming experience. Insteadof being a stand-alone application, GenoCAD is a database-driven web-based application (Czar   et al.,  2009). Like Clotho and GEC, GenoCADrelies on a solid foundation derived from the theory of computer languages(Cai  et al.,  2007, 2009). GenoCAD is an open source application distributedunder the Apache software license. An instance of GenoCAD is available atwww.genocad.org and is referred to as GenoCAD.org in this chapter todifferentiate it from the GenoCAD software itself. 174  Mandy L. Wilson  et al.  2. Overview of GenoCAD Before building sequences in GenoCAD, it is helpful to understandthe overall structure of the application and how the various pieces fittogether to provide the user with a safe and streamlined design experience.DNA sequences are made up of smaller standardized genetic DNAsegments such as promoters, transcription terminators, genes, proteindomains, and others. Within GenoCAD, these segments are referred to as“parts.” GenoCAD.org has a library with thousands of distinct basic parts(Cai  et al.,  2010; Peccoud  et al.,  2008). Users are not limited to the partsincluded in the global GenoCAD database. They can add new sequences intheir personal workspace without having to make them available to other GenoCAD users. Design strategies composed of rules describing how partscan be combined are called grammars in GenoCAD.The concept of a Design Strategy within GenoCAD is similar to the rolea grammar plays within language. A writer may use a series of words thatinclude a subject, a predicate, indirect objects, and prepositions, but if theyfail to use the prescribed grammar for the language in question, the wordsmay not come together to form a meaningful sentence. Design strategies inGenoCAD work much the same way. A design strategy uses rules to definewhich classes of parts, called categories, can be used to design a DNAsequence, and in what order they may appear. For example, the design of an  E. coli   gene expression cassette requires—at minimum—a Promoter, aRibosome Binding Site (RBS), a Gene, and a Terminator. In GenoCAD,Promoters, RBS, Genes, and Terminators are categories, and the rules thatensure parts from each of the categories above are included define the  E. coli  design strategy. The design strategy ensures that categories may only be usedin the appropriate order, so, in the example above, the RBS and Gene canonly be inserted between a Promoter and Terminator. The rules of thedesign strategy also prevents parts from categories external to the  E. coli  design strategy from being included in the design.Design strategies are currently coded within the GenoCAD database.That’s where personal libraries come in. Libraries are named lists of partsthat include only the parts the user wants to have available when creatingDNA sequences for a specific project. Libraries are always design strategy-specific (e.g., an  E. coli   library may not contain Yeast parts), and work inconjunction with their design strategies to prevent user error during thedesign of a sequence. Personal libraries in GenoCAD can contain a combi-nation of user-defined and global parts. GenoCAD Designs are DNAsequences that have been constructed using design strategies, libraries, andparts. Step by Step Introduction to GenoCAD  175  3. Requesting an Account on GenoCAD.org When accessing GenoCAD.org for the first time, the first page pre-sented is the Parts tab. Although most of the available features of Geno-CAD.org may be viewed without logging in, many of them are disabled or have limited functionality for the unauthenticated user. To take full advan-tage of the features GenoCAD.org has to offer, a user account is required.The link to apply for an account is located on the Log in tab. After loading the Log In page, the applicant would then click on the link, “Don’thave an account? Request one.” At this point, the Application for Accountpage is loaded in the browser (Fig. 8.1).The use of GenoCAD.org is free. However, in order to minimize therisk that this resource may be used to develop biological weapons, applica-tions for account are reviewed to verify that the applicants can be identifiedand have legitimate needs to use the GenoCAD.org Web site. Moreinformation on the guidelines GenoCAD.org uses for this validation isavailable from the GenoCAD Privacy Policy referenced at the top of theApplication for Account page. Accordingly, the more information theapplicant provides on the application, the more quickly his account canbe validated; if the request can be verified from the information submittedin the initial application, the turn-around time for approval is less than48 hours. Once the account is approved, the applicant receives an e-mailthat includes a link to log into the system. Users can change their personalinformation or password by clicking on the My Profile link available in thesubmenu at the top of most of the pages in the system.This user registration process is specific to GenoCAD.org because theresource is publicly available. Organizations installing GenoCAD on their own servers could most probably link GenoCAD to an existing user directory (LDAP, Active Directory). 4. Browsing the Parts Catalog When logging into GenoCAD, the Parts tab, or parts listing, is thedefault page (Fig. 8.2). The tabs along the top of the page guide the user todifferent features of the GenoCAD application, while the navigationalmenu on the left side of the screen contains functionality pertaining onlyto the Parts tab. The default navigational tab selected is Public Libraries.GenoCAD.org has thousands of public, or global, parts spread across four design strategies and a number of public libraries that users may choose fromin developing their own personal libraries. GenoCAD offers two differentoptions to aid users in finding parts. 176  Mandy L. Wilson  et al.  Below the Public Libraries tab, there is a collapsible hierarchical menuthat is made up of Design Strategies, Libraries, and Categories, althoughinitially only design strategies (often called grammars) and libraries aredisplayed. In order to see which categories are represented beneath a specificlibrary, the user can use the mouse to click on the small arrow next to thelibrary in question to expand the list of categories below the selected library.Alternately, theExpandAllbutton expandsthe menuto showthecategoriesbeneath all of the libraries, and the Collapse All button collapses the treestructure back to the design strategy/library levels.To view the parts available under a given library or category, the user can click on the library or category name in the hierarchical menu on the left.The parts that fall under the selected library or category are displayed on the Figure 8 . 1  Application for Account. In order totake fulladvantage of the site features,users ofGenoCAD.org mustrequest an account. The identity ofthe applicant isverifiedbefore the account is approved. Users are encouraged to fill out the application ascompletely as possible to expedite the review process. Step by Step Introduction to GenoCAD  177
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