Coding

Part:BBa_K3682001:Design

Designed by: Yen-Syun,Huang   Group: iGEM20_NYMU-Taipei   (2020-10-15)


pepP sequence


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 260
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1243
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We are looking to clone the pepP sequence from Escherichia coli strain K12 using PCR. The annealing strand of primers will be: 5'atgagtgagatatccc3'(forward) and 5'tcattgctttctcgca3'(reverse). The suspension strand of the primers will depend on how you are going to anneal your parts. Notice: This sequence involves a start codon and a stop codon, so one should delete the stop codon sequence of the reverse primer if there are other coding sequences behind pepP sequence.

Fig1. pepP structure from uniport

Source

We cloned it from E.coli K12 strain genomic DNA

References

1. Weaver, J., Watts, T., Li, P., and Rye, H. S. (2014). Structural basis of substrate selectivity of E. coli prolidase. PLOS ONE 9:e111531. doi: 10.1371/journal.pone.0111531

2. Jeyakanthan, J., Takada, K., Sawano, M., Ogasahara, K., Mizutani, H., Kunishima, N., et al. (2009). Crystal structural and functional analysis of the putative dipeptidase from Pyrococcus horikoshii OT3. J. Biophys. 2009:434038. doi: 10.1155/2009/434038

3. Graham, S. C., Bond, C. S., Freeman, H. C., and Guss, J. M. (2005). Structural and functional implications of metal ion selection in aminopeptidase P, a metalloprotease with a dinuclear metal center. Biochemistry 44, 13820–13836. doi: 10.1021/bi0512849

4. Wilce, M., Bond, C., Dixon, N., Freeman, H., Guss, J., Lilley, P., et al. (1998). Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 95, 3472–3477. doi: 10.1073/pnas.95.7.3472

5. Graham, S. C., Bond, C. S., Freeman, H. C., and Guss, J. M. (2005). Structural and functional implications of metal ion selection in aminopeptidase P, a metalloprotease with a dinuclear metal center. Biochemistry 44, 13820–13836. doi: 10.1021/bi0512849

6. Liu, J. W., Hadler, K. S., Schenk, G., and Ollis, D. (2007). Using directed evolution to improve the solubility of the C-terminal domain of Escherichia coli aminopeptidase P. FEBS J. 274, 4742–4751. doi: 10.1111/j.1742-4658.2007.06022.x

https://febs.onlinelibrary.wiley.com/doi/full/10.1111/j.1742-4658.2007.06022.x

7. https://www.uniprot.org/uniprot/P15034