TITLE: DIVA Design instructions SOP (User&#39;s section)
AUTHORS: Henrique De Paoli, Christopher J Petzold

[DESCRIPTION]
This is a "how to" step-by-step instruction for creating Projects and Designs in DIVA for automated design of oligos, plasmids and build instructions. User can either build plasmids on their own or submit the final Design for review/PI approval and third party (e.g., DIVA team) construction pipeline. DIVA also allows for tracking Design progress and is linked to ICE (a repository for parts, plasmids and strains used as Design input and/or construction output in DIVA).

[BEFORE_START]
1. Define backbone(s), insert(s), and assembly strategy.
   a.Guided by Target/Host expert (e.g., expression of a gene cluster) and/or Learn (e.g., design of experiments), compile all ICE entries (i.e., DNA parts linked to Strains with samples) and/or annotated files of sequence-verified DNA/protein sequences (e.g., genbank) to be used.
        i.Synthetic fragments will not require physical template pre-available.
        ii.Synthetic fragments must be ≤5kb and verified for synthesis constraints (e.g., https://boost.jgi.doe.gov/index.html)
        iii.Optional: Codon juggle, polish and/or partition synthetic fragments
2. Resource Crop data and Relevant milestone (contact PI for reference)
3. You will be asked to select a Project from a self-complete list. If you don’t have a Project, create one first. Although Projects can be added later to each Design from the Design page, you must have a Project linked to your Design to submit it for review.

[STEPS]
SECTION: DIVA access
1. Go to DIVA server at https://diva.agilebiofoundry.org/  and login by using your LBNL LDAP/affiliate username and password.

 

SECTION: Define Project
2. Click the New Project button.

  
 


SECTION: Define Project
2.1. Input a Project Name, Description and define the DIVA Team who will review your design. 

 

 

SECTION: Define Project
2.2. Add your Principal Investigator/Supervisor’s Name, who is responsible for approving your expenses, then save the project.
SECTION: Create Designs
3. Click the New Design button.




  

SECTION: Create Designs
4. Input Design Name, Description(with Crop data, e.g., 11-2-TRY_M1, and Relevant milestone, e.g., FY21Q4_DBTL_AS2) and Project. Hit Create (it will take you directly to Device Editor).

  

SECTION: Create Designs
5. Input desired sequences in each cell using ‘right click’ and collecting from the appropriate source (e.g., Search for Part, Fig.5B; insert from ICE, Fig.5C-D, e.g., ABF_009895 ). Use left-most column for plasmid backbone. 

 


  
  
  
  

SECTION: Create Designs
6. Define Part Source, Name (with ‘_marker’ and ‘_enzyme’, e.g., bb_tDNA_kan and ABF_009842_EcoRI_amp), and Start BP and End BP. If using gDNA, use ‘_gDNA’ instead of ‘_marker’. 


  

SECTION: Create Designs
8. Under the Part tab, define Forced Assembly Strategy for each part individually (if left blank, PCR will be automatically adopted by j5). Avoid PCRs larger than 5.1kb.
- Optional: define Eugene rules.


    

SECTION: Create Designs
9. Under the Collection tab, define Columns names (double click on e.g., ‘No_name_0’). Also, define combinatorial (set by default) vs ‘one per row’ (must have all rows filled to be toggled).
a. Optional: select cells and insert empty part as needed
b. Optional: edit DNA assembly directives and parameters
c. Optional: define Column Direction (must toggle in the Device Editor area, not in the table).


  

SECTION: Run j5
10. Press the J5 (green) button to open the ‘j5 controls’ and select Preview assembly (this is simply putting sequences together). Inspect output visually for desired output (e.g., number of variants) and adjust as needed.

  

SECTION: Run j5
11. While in the pop-up section, select the Advanced j5 tab for more options.
a. Optional: Edit j5 parameters
b. Define master lists

  

SECTION: Run j5
12. Select desired assembly from the drop-down menu.

You must select SLIC/Gibson/CPEC or Golden Gate (i.e., non-mock assembly; checks for assembly feasibility, designs oligos and output warnings and Build instructions) to be able to submit design for review. 

Select the  'Run j5' button.

 

SECTION: Run j5
13. Download results and open the combinatorial file (e.g., pmas00001_combinatorial.csv, or simply pmas00001.csv if making just one construct). Must be free of critical warnings (e.g., mispriming, incompatibilities between assembly pieces, homologous sequence repeat, etc).
Examples of “non-critical” warnings:
- Primer3 could not find a reasonable set of primers.
- The master plasmids/oligos list file is empty.

  
  
  

SECTION: Run j5
14. If no changes are needed, you can close the Advanced j5 Controls pop up and select the link Design Details (top right corner) to go to the main Design page.

  

SECTION: Submit for DIVA Review
15. You can now Submit for DIVA Review. Pay attention to prompted messages and (un)check boxes accordingly.

  
  

SECTION: Submit for DIVA Review
16. Wait for Reviewer feedback. Address Reviewer requests (if any) and re-submit for Review, or solicit PI budgetary approval (Submit to PI button).
- PI was defined while creating the Project you linked to the Design.


 

SECTION: Create Designs
7. Add columns and rows as needed for the remaining parts (e.g., Insert 1 ABF_009896, Insert 2 ABF_009569).