Writing DNA with GenoCAD

Abstract

Chemical synthesis of custom DNA made to order calls for software streamlining the design of synthetic DNA sequences. GenoCAD (www.genocad.org) is a free web-based application to design protein expression vectors, artificial gene networks and other genetic constructs composed of multiple functional blocks called genetic parts. By capturing design strategies in grammatical models of DNA sequences, GenoCAD guides the user through the design process. By successively clicking on icons representing structural features or actual genetic parts, complex constructs composed of dozens of functional blocks can be designed in a matter of minutes. GenoCAD automatically derives the construct sequence from its comprehensive libraries of genetic parts. Upon completion of the design process, users can download the sequence for synthesis or further analysis. Users who elect to create a personal account on the system can customize their workspace by creating their own parts libraries, adding new parts to the libraries, or reusing designs to quickly generate sets of related constructs.

Figure 1.

The GenoCAD parts library browser. Parts are associated with individual libraries, each of which is associated with a specific grammar. Users select which parts library they view through choice of a grammar and specific library in drop down boxes on the page. The part category ‘Gene’ is displayed in this figure along with the icon that represents genes in the designs. By clicking on the icon, the list of genes expands, allowing the user to see the available choices in the library. Selecting the link to ‘View Sequence’ for any part opens a small window containing the sequence of the individual part.

Figure 2.

The design interface showing the structure of a genetic toggle switch. The interface has drop down boxes at the top to select the grammar and parts library that will be used in the design. The history panel allows users to select one of the steps in the design process and see the structure of the design at that step. Users are permitted to go back to any step and redesign from that point. The design is presented in the main panel of the page, and icons for each part and the abbreviated parts categories are shown at the top of the design. Choices for each part are shown underneath the part icon. The inset shows the final design for this construct after specific choices (terminals) are selected for each part category.

Figure 3.

A progression through the design of a bistable toggle switch. The starting symbol, S, (1) is where each design begins, and it is transformed into a transcription cassette, CAS, (2). Since the toggle switch contains two transcription cassettes, the single cassette is doubled (3). The design we are following has the cassettes oriented in opposite directions, and we achieve this by transforming the left cassette to the tpc- option, and the right to the tcp+ option (4), which contain a promoter, cistron, and terminator, but in opposite orientations. The right cassette is meant to express a transcriptional repressor and reporter gene in a bicistronic manner, so the cistron is doubled by selecting the 2cis+ option (5). Each cistron is then decomposed to a RBS and gene (6), with the RBS and gene in the reverse orientation in the left cassette. Selection of the specific promoters, RBSs, genes, and terminators produces a final construct that is associated with a DNA sequence (7).

Figure 4.

Creating a custom parts library. Users who create an account at the website are able to create their own parts libraries, and are then able to add custom parts to these libraries. Through the library creation interface, users select the grammar that their library will belong to, provide a name for their library, and can enter a description. Parts can be added to a new library from other libraries of the same grammar, which are loaded in the lower left box on the web page. All parts or a select subset of parts, from the existing library can be copied into or removed from the new library using the orange ‘add’ and ‘remove’ buttons.

Figure 5.

Interface to add a new part. Users that have created a custom library are able to add and save parts that can be used in their designs. The categories of parts permissible in designs are defined in the grammar, so the grammar must be chosen first through a drop down menu. A second drop down, then allows users to choose which part category the new part will belong to; in this case a new terminator is being created. The sequence and description of the part are entered in text boxes, and the library(s) to which the part will be added must be checked.

Figure 6.

GenoCAD data model. Each grammar encompasses a set of rules by which constructs can be designed. The grammar also defines the categories of parts that are available to design the constructs. For each grammar there is a collection of public parts (solid, blue rectangle), which constitute a publicly available parts library (dashed, blue rectangle). ‘User Libraries’ can be created from any subset of the public parts, and this library can be supplemented with user-created parts (solid, red rectangle). Two user libraries (dashed, red and dashed, green rectangles) are shown here that contain different subsets of public and user-created parts. User library 2 contains all user-created parts. When a design is created, all the parts to complete the design must be contained within a single library.