In the event that your workflow requires you to optimize your sequence for recombinant expression in other organisms, Benchling supports codon optimization.
This article discusses how to use the codon optimization tool for both back translated AA sequences and for DNA sequences.
Access the codon optimization tool
The codon optimization tool can be accessed through the back translation tool for AA sequences as well as through the Analyze menu for a selected region of a DNA sequence (as shown in the image below).
Note: you will not be able to access the codon optimization tool in a DNA sequence unless the highlighted region is a multiple of three.
Once you create the back translation or codon optimization analysis for your sequence, the Codon optimization tab will open for that sequence (shown in the image below). In this tab, you will be able to set and adjust a number of parameters.
Codon optimization parameters
The table below outlines the parameters that you are able to set for codon optimization:
| Organism | Allows you to specify the target organism the sequence will be expressed in. The algorithm will create a sequence whose codons match the codon frequencies of the specified organism |
| Method | Allows you to specify how you want the algorithm to generate your new sequence’s codon usage |
| GC Content | Allows you to select a range of desired GC content. The algorithm will attempt the create a sequence that matches the specified range |
| mRNA uridine depletion | Allows you to select for mRNA uridine depletion in an attempt to help reduce the immunogenicity of RNA |
| Hairpin parameters | Allows you to define the stem size and window that you want the algorithm to avoid hairpins for |
| Restriction sites to avoid | Allows you to select restriction enzyme cut sites you want to avoid creating recognition sites for in your new DNA sequence |
| Patterns to reduce | Allows you to designate a DNA pattern using AGTC bases that you would like to see less of in the optimized sequence |
| Restriction sites to protect* | Allows you to select which restriction enzyme cut sites you want to ensure still exist in your new DNA sequence |
| Protected regions* | Allows you select regions that will not be modified by the algorithm |
*this is only an option if you access the codon optimization tool from a DNA sequence
Generate a codon optimized sequence
In order to generate a codon optimized sequence, whether through back translation or from a DNA sequence, follow the steps below which outline how you set parameters in the Codon optimization tab. You may find yourself setting some or all of these parameters.
- Use the Organism dropdown menu to select your target organism
- Click on the box for the codon usage method you’d like the algorithm to use
- Use the GC content dropdown menu to select the range you’d like to target
- Note: if you select custom range, a text box will appear where you will need to set the minimum and maximum range yourself
- Use the checkbox to toggle mRNA uridine depletion off if needed
- Use the checkbox to toggle hairpin parameters on if needed
- Note: if you toggle the checkbox on, you will have the option to adjust the stem size and window parameters manually, or use the default values that Benchling uses
- If you want to avoid adding cut sites to your sequence, click the + Add cut site to avoid button and choose an enzyme from the list. Repeat as many times as needed to add multiple cut sites
- If you used a DNA sequence for optimization and you'd like to preserve cut sites in your sequence, click into the Preserve Cut Sites tab and then click the + Add cut site to preserve and choose an enzyme from the list. Repeat as many times as needed to add multiple cut sites
- If you used a DNA sequence for optimization and you'd like to protect regions in your sequence, click into the Protected Regions tab, highlight a region of interest in the original sequence, and then click the + Add selected region. Repeat as many times as needed to add multiple regions
- Note: you may find it easier to use the the split workspace view so that you can highlight the region to preserve in one window and click the Add button in the other
- If you want to reduce nucleotide patterns in the back-translated sequence, click into the Patterns to Reduce tab, then click the + Add pattern to reduce button and enter a DNA pattern using ATGC bases only in the text box
- Once you’ve set all the relevant parameters, click Preview optimization
- Review the optimization summary and then click Save as new sequence
- Note: you can click Back to return to the parameter settings page if you want to make any adjustments before you save the sequence
- Select the project and folder that you want to save the sequence in and click Select