Composite

Part:BBa_K4347010

Designed by: Victor Di Donato, Nicoletta de Maat   Group: iGEM22_Queens_Canada   (2022-07-07)

Bst fusion with Sac7e codon optimized for E.coli

This fusion protien was designed by linking the N-terminus of wildtype Bst polymerase with thermostable DNA binding protien Sac7e using a flexible (GGGGS)4 linker to increase polymerase processivity in LAMP reaction.

Usage and Biology

Wildtype bst polymerase Large Fragment is a family I DNA polymerase derived from the thermophilic bacterium Geobacillus stearothermophilus. Bst polymerase Large Fragment is notable for its strong strand displacement activity and thermal stability [1]. Bst also contains a 5' to 3' DNA polymerase activity but lacks 3' to 5' exonuclease activity[2]. These unique features allow Bst polymerase to facilitate isothermal amplification techniques such as LAMP and rt-LAMP.

Bst polymerase derived from thermophilic bacterium Geobacillus stearothermophilus used in LAMP modelled on PyMol.

Sac7e is part of the 7 kDa DNA-binding family and is a highly thermostable and pH resistant protien that aids in the binding of double stranded DNA. Sac7e is thermally stable to 85.5°C and compared to other similar proteins, Sac7e showed the highest affinity for dsDNA (KD = 11 μM), with binding sites ~ 6-8 bases per protein[3].

DNA binding protien "Sac7e" modelled in Pymol.

Enhanced processivity

The end goal of fusing Bst polymerase to double stranded binding protien Sac7e is to increase the polymerases processivity in the LAMP reaction. This fusion was based off of previous work from Yang et, al (2004) where the structural homologue of Bst was fused with a structural homologue of Sac7e to produce a more processive and efficient polymerase in the PCR reaction[4]. Sac7e sharply kinks the double DNA helix upon binding into the minor groove[5] upstream of the DNA polymerase. This will keep the polymerase attached to the DNA template longer, thus yield more amplification product.

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


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]


References


1. Ignatov, K. B., Barsova, E. V., Fradkov, A. F., Blagodatskikh, K. A., Kramarova, T. V., & Kramarov, V. M. (2014). A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification. BioTechniques, 57(2), 81–87. https://doi.org/10.2144/000114198

2. Aliotta JM, Pelletier JJ, Ware JL, Moran LS, Benner JS, Kong H (1996). Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. (5-6):185-95. PMID: 8740835

3. 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

4. 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

5. Robinson, H., Gao, Y.-G., McCrary, B. S., Edmondson, S. P., Shriver, J. W., & Wang, A. H.-J. (1998). The hyperthermophile chromosomal protein sac7d sharply kinks DNA. Nature, 392(6672), 202–205. https://doi.org/10.1038/32455

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