Part:BBa_K4347010:Design

Bst fusion with Sac7e codon optimized for E.coli

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 5
  Illegal XhoI site found at 209
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

When compared to other protiens from the 7 kDa DNA-binding family, Sac7e showed the highest affinity for dsDNA (KD = 11 μM), with binding sites ~ 6-8 bases per protein^[1]. It also has a thermostability of 85.5°C which is well above the optimal 65°C for LAMP making it a reliable candidate as our fusion protien.

Once we were established Sac7e to the the fusion protien of choice, we conducted an AlphaFold analysis in combination with Pymol to predict and model a fused protien consisting of Sac7e fused via N-terminal to Bst polymerase. The linker of choice was (GGGGS)₄ as glycine linkers are very flexible and limit strain between each component.

Wildtype Bst polymerase (green) fused with Sac7e (red) using (GGGGS)₄ flexible linker (blue).

Considerations

The idea of this fusion protien was inspired by previous work from Yang et, al (2004) who fused Bst homologue Taq polymerase to a Sac7e homologue Sso7d to improve Taq polymerase proccessivity in PCR amplification^[2]. It was found that the fusion protien had a processivity score of 0.98 equating to an average primer extension length of 51 nt compared to a non-fused Taq polymerase, which had a processivity score of 0.64 equating to an average primer extension length of 2.9 nt. The catalytic activity of the fusion polymerase was not affected compared to the wild-type Taq as the k_cat values of nucleotide incorporation were nearly identical. It was also found that the fusion polymerase had ~4 to 8-fold decrease K _{m (DNA)} values than the origional enzyme, suggesting a stabilizing effect of the fusion with DNA template.

Source

• Sac7e: https://parts.igem.org/Part:BBa_K4347006
• (GGGGS)₄ linker: https://parts.igem.org/Part:BBa_K3117004
• Bst: https://parts.igem.org/Part:BBa_K3790000

References

1. Kalichuk, V., Béhar, G., Renodon-Cornière, A., Danovski, G., Obal, G., Barbet, J., Mouratou, B., & Pecorari, F. (2016). The archaeal “7 KDA DNA-binding” proteins: Extended characterization of an old gifted family. Scientific Reports, 6(1). https://doi.org/10.1038/srep37274

2. Wang, Y. (2004). A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Research, 32(3), 1197–1207. https://doi.org/10.1093/nar/gkh271