1. Copyright OpenHelix. No use or reproduction without express written consent1

2. GenoCAD Advanced Topics Computer-Assisted Design Software for Synthetic Biology Materials prepared by: Mary E. Mangan PhD www.openhelix.com/genocad2 Version 1

3. Copyright OpenHelix. No use or reproduction without express written consent3 GenoCAD Advanced Agenda Introduction and Review New Library with Advanced Searching Advanced Design Copying and Reworking Export GenBank format  other tools Grammar from Scratch Modifying (Editing) Grammar Summary Exercises GenoCAD: http://www.genocad.org/http://www.genocad.org/

4. Introduction to GenoCAD Computer-assisted design software for synthetic biology Copyright OpenHelix. No use or reproduction without express written consent4 genocad.org

5. Register and Log In at GenoCAD Completed first tutorial Features we explore here require saving to your workspace Copyright OpenHelix. No use or reproduction without express written consent5 http://dx.doi.org/10.6084/m9.figshare.153827 figshare.com GenoCAD

6. Conceptual Framework for GenoCAD Aspects of DNA function explained with language metaphors: transcription, translation, code GenoCAD lets you develop a language for DNA designs Copyright OpenHelix. No use or reproduction without express written consent6

7. 7 GenoCAD Advanced Agenda GenoCAD: http://www.genocad.org/http://www.genocad.org/ Introduction and Review New Library with Advanced Searching Advanced Design Copying and Reworking Export GenBank format  other tools Grammar from Scratch Modifying (Editing) Grammar Summary Exercises

8. New Project Library Best practice: New Library for each project Locate and reuse parts Continue to use Training Set Grammar for now Copyright OpenHelix. No use or reproduction without express written consent8 click

9. Add New Library in Training Set In the Training Set, create a “new library” Name it “Fluors” Copyright OpenHelix. No use or reproduction without express written consent9 search

10. Search for Project Parts Locate specific parts quickly with a search Advanced searches offer precision Build queries with multiple options Copyright OpenHelix. No use or reproduction without express written consent10 Advanced Value box search options: use text words from name, description, etc use DNA sequence: aggggtgct employ wildcards: *aggggtgct* choose “phrase” or “exclude” options

11. Advanced Search Example Copyright OpenHelix. No use or reproduction without express written consent11 Set field to: Part Name Set value to: *fp Set field to: Category Set value to: pro* Choose MAY include *fp Query components will appear here pro* and or click

12. Save a Search Save searches that you might use often Copyright OpenHelix. No use or reproduction without express written consent12 click

13. Saved Searches and My Cart Load and run the saved search Highlight the “Training Set” category Then select the five parts shown Click to add to My Cart Copyright OpenHelix. No use or reproduction without express written consent13 click

14. My Cart My Cart has my selections Copyright OpenHelix. No use or reproduction without express written consent14 click Remove any unneeded items

15. Add More Parts We need other parts from Training grammar Find T7 terminator, and an RBS Key point: parts need to be compatible with the grammar you intend to use Choose ones compatible with Training Grammar Copyright OpenHelix. No use or reproduction without express written consent15 click Build a query Part name: T7 Category: RBS Join with “MAY Include”

16. Return to My Cart Cart now has the items we need to design Select all, copy to Fluors library Copyright OpenHelix. No use or reproduction without express written consent16 click

17. Examine the Fluors Library Access the libraries tab Click the Fluors item All the parts are available Next let’s design some color constructs Copyright OpenHelix. No use or reproduction without express written consent17 Next

18. Copyright OpenHelix. No use or reproduction without express written consent18 GenoCAD Advanced Agenda GenoCAD: http://www.genocad.org/http://www.genocad.org/ Introduction and Review New Library with Advanced Searching Advanced Design Copying and Reworking Export GenBank format  other tools Grammar from Scratch Modifying (Editing) Grammar Summary Exercises

19. Design New Fluors Constructs Use the Training Set Grammar + Fluors Library This time let’s reverse the orientation, use PCT- Copyright OpenHelix. No use or reproduction without express written consent19 click designs click

20. Quickly Clone a Construct From Designs list you can quickly “clone” copies Click to “clone” the RFP construct Copyright OpenHelix. No use or reproduction without express written consent20 Click Clone Click View

21. Work with a Cloned Design You can quickly swap out pieces Generate similar constructs easily Copyright OpenHelix. No use or reproduction without express written consent21 click designs

22. Copyright OpenHelix. No use or reproduction without express written consent22 GenoCAD Advanced Agenda GenoCAD: http://www.genocad.org/http://www.genocad.org/ Introduction and Review New Library with Advanced Searching Advanced Design Copying and Reworking Export GenBank format  other tools Grammar from Scratch Modifying (Editing) Grammar Summary Exercises

23. Exporting GenBank Format Categories may be mapped to GenBank features Can use this to visualize features in GenBank-compliant tools Illustrated with VectorEditor Demo web tool Copyright OpenHelix. No use or reproduction without express written consent23 bit.ly/GBtable code.google.com/p/vectoreditor/

24. Obtain YFP GenBank Format File Obtain the YFP design from My Designs Click “View” to access GenBank output Copyright OpenHelix. No use or reproduction without express written consent24 Click View

25. Exporting with GenBank Features Select the GenBank button Obtain your text file Examine the Features area Copyright OpenHelix. No use or reproduction without express written consent25

26. Upload the YFP File Access VectorEditor http://bit.ly/VectorEditorhttp://bit.ly/VectorEditor Import the YFP construct file View the features Copyright OpenHelix. No use or reproduction without express written consent26 orientation

27. Copyright OpenHelix. No use or reproduction without express written consent27 GenoCAD Advanced Agenda GenoCAD: http://www.genocad.org/http://www.genocad.org/ Introduction and Review New Library with Advanced Searching Advanced Design Copying and Reworking Export GenBank format  other tools Grammar from Scratch Modifying (Editing) Grammar Summary Exercises

28. Grammar from Scratch Can create a new grammar to suit project needs Use them to create a construct with “Design” options Copyright OpenHelix. No use or reproduction without express written consent28 Credit: bit.ly/chlplst

29. A Blank Grammar Template Name and describe a grammar, choose an icon set Brand new grammar is created Copyright OpenHelix. No use or reproduction without express written consent29 click

30. Build a Grammar The new grammar has only default categories Just add new categories and rules Copyright OpenHelix. No use or reproduction without express written consent30

31. Copy a Grammar Copyright OpenHelix. No use or reproduction without express written consent31 Another way to create a new grammar: copy one It will then become editable to modify for your needs

32. Export a Grammar Copyright OpenHelix. No use or reproduction without express written consent32 Another way to work with a grammar: export Any grammar can also be exported and shared

33. Copyright OpenHelix. No use or reproduction without express written consent33 GenoCAD Advanced Agenda GenoCAD: http://www.genocad.org/http://www.genocad.org/ Introduction and Review New Library with Advanced Searching Advanced Design Copying and Reworking Export GenBank format  other tools Grammar from Scratch Modifying (Editing) Grammar Summary Exercises

34. Modifying Grammars Grammars establish the expression strategy Subject–Verb–Object Promoter–Cistron–Terminator Copyright OpenHelix. No use or reproduction without express written consent34

35. Manage Grammar Access the Grammars tab Select Training Set grammar Click “Manage Grammar” to examine rules Copyright OpenHelix. No use or reproduction without express written consent35 select

36. Grammars Copyright OpenHelix. No use or reproduction without express written consent36 Categories: parts characteristics Rules: specify the number, order, orientation, etc click

37. Conceptual Illustration of Rules Rewritable category items transform to another rule Parent  child Child  child Terminal category has no child rules Copyright OpenHelix. No use or reproduction without express written consent37 rewrites S (start) is the parent CAS (cassette) is a child CAS (cassette) is a parent rewrites CIS (cistron) is a parent GEN (gene) is terminal RBS (binding site) is terminal RBGN (rbs + gene) is a child parent parent + child term p + c

38. Grammars Copyright OpenHelix. No use or reproduction without express written consent38 click Categories: edit characteristics Rules: edit, remove or add rules click

39. Adding Custom Grammar Elements Add new grammar items useful for your projects A commonly used strategy: protein tags We’ll employ a his-tag, poly-histidine stretch Copyright OpenHelix. No use or reproduction without express written consent39 http://en.wikipedia.org/wiki/File:His-tag.png http://en.wikipedia.org/wiki/File:His-tag-primers.png

40. Add Category Add a new category to the Training Grammar Histidine tags Letter: HisTag GenBank Qualifier: misc_binding Icon: protein-stability- element.png Copyright OpenHelix. No use or reproduction without express written consent40

41. Add Rules to a Grammar Cistron with histidine tags need rules: CIS  RBS GEN HIS One tag at the end of a cistron (histag-C) Copyright OpenHelix. No use or reproduction without express written consent41 C-histag

42. New Rule is Available, Old Rules can be edited Cistron-using designs can now have his-tag HisTag is now a terminal category because it is used Copyright OpenHelix. No use or reproduction without express written consent42 Click Edit

43. Rule Editing has Consequences Change to rbgn rule affects existing constructs View the “Copy of RFP construct” Copyright OpenHelix. No use or reproduction without express written consent43 Examine designs click

44. Validation Status Return to the Design list Note the change in status Examine the status key Re-work the design by clicking the steps again until complete Copyright OpenHelix. No use or reproduction without express written consent44 Examine designs click

45. Copyright OpenHelix. No use or reproduction without express written consent45 GenoCAD Advanced Agenda GenoCAD: http://www.genocad.org/http://www.genocad.org/ Introduction and Review New Library with Advanced Searching Advanced Design Copying and Reworking Export GenBank format  other tools Grammar from Scratch Modifying (Editing) Grammar Summary Exercises

46. Copyright OpenHelix. No use or reproduction without express written consent46 GenoCAD Summary GenoCAD is a toolbox for synthetic biology Parts, grammars, designs, and simulations

47. Copyright OpenHelix. No use or reproduction without express written consent47 GenoCAD Advanced Agenda GenoCAD: http://www.genocad.org/http://www.genocad.org/ Introduction and Review New Library with Advanced Searching Advanced Design Copying and Reworking Export GenBank format  other tools Grammar from Scratch Modifying (Editing) Grammar Summary Exercises

48. Copyright OpenHelix. No use or reproduction without express written consent48