Enter RBS Design Inputs
Example Data Table
These rows show how the scoring model compares different bacterial translation leaders.
| Construct | RBS Sequence | Spacer | Start Codon | Hairpin ΔG | AU % | Score | Band |
|---|---|---|---|---|---|---|---|
| Leader A | AGGAGG | 6 | ATG | -2.5 | 74.0 | 94.40 | Very Strong |
| Leader B | AAGGAA | 7 | GTG | -5.5 | 63.0 | 68.05 | Moderate |
| Leader C | GGAGAT | 5 | ATG | -4.0 | 58.0 | 67.17 | Moderate |
| Leader D | ACCTTA | 10 | TTG | -9.0 | 39.0 | 22.54 | Weak |
Formula Used
This calculator uses a composite heuristic for bacterial RBS screening. It is intended for comparative design work, not as a substitute for full thermodynamic simulation or experimental validation.
Overall Score
RBS Strength = 0.35 × Motif Score + 0.25 × Spacing Score + 0.15 × Start Codon Score + 0.15 × Accessibility Score + 0.10 × AU Context Score
Motif Score
The calculator scans the entered sequence for the best 6 nt window against the consensus AGGAGG. Exact positional matches receive full weight, while purine-preserving mismatches receive partial weight.
Spacing Score
Spacing Score = 100 − [12 × |Spacer − 6|^1.65], clipped to 0–100
A 6 nt spacer is treated as the best default bacterial spacing target.
Start Codon Score
ATG = 100, GTG = 75, TTG = 55, CTG = 40
Accessibility Score
Accessibility Score = 100 + (8 × Hairpin ΔG), clipped to 0–100
More negative local hairpin energies decrease ribosome access near the start region.
Estimated Binding ΔG
Estimated Binding ΔG ≈ −[1.25 × Exact Matches + 0.35 × Purine Partial Matches + 0.02 × GC%]
How to Use This Calculator
- Enter a construct name so the output is easier to identify in exports.
- Paste the bacterial RBS sequence you want to evaluate.
- Set the spacer length between the RBS core and the start codon.
- Select the start codon and enter the local hairpin ΔG.
- Estimate upstream AU-richness, then press the calculate button.
- Review the final score, component scores, interpretation, and the spacer sensitivity graph.
- Download the result as CSV or PDF if you want a record.
FAQs
1) What does RBS strength mean here?
It estimates how favorable a bacterial translation initiation region looks based on motif quality, spacing, codon choice, local folding, and AU-rich context. The score is comparative, not absolute.
2) Is this a full thermodynamic simulator?
No. It is a practical screening model for fast design comparison. Use it to rank candidate leaders, then confirm performance with dedicated tools and wet-lab measurements.
3) Why is spacer length important?
Ribosome positioning depends strongly on the distance between the Shine-Dalgarno region and the start codon. Many bacterial systems perform best near 5 to 8 nucleotides, often close to 6.
4) Why do strong hairpins reduce the score?
Stable secondary structure can hide the RBS or start codon from the ribosome. More negative ΔG values usually mean tighter folding and lower local accessibility.
5) Can GTG or TTG still work well?
Yes. Alternative start codons can still express effectively, especially in optimized sequence contexts. They usually receive lower weighting because initiation is often weaker than with ATG.
6) Should I always maximize the score?
Not always. Very strong initiation can create metabolic burden, misfolding, or poor pathway balance. Target the expression level your design actually needs.
7) Does host choice affect interpretation?
Yes. Ribosome preferences, transcript processing, and cellular physiology differ across organisms. Compare constructs within a similar host context whenever possible.
8) Which inputs matter most for reliable comparison?
Use realistic spacer length, a well-defined local ΔG estimate, and a clean RBS sequence. Comparisons are strongest when all candidates are scored using the same assumptions.