Composite

Part:BBa_K5129002:Design

Designed by: Ansal' Diassova, Adil Ispambetov, Anastassiya Tyazhelova, Rauan Muratuly, Tomiris Kudassova, Makpal Akishova, Nuriya Nurlankyzy, Yernur Kenzhegazin, Artyom German, Abylaikhan Seraliyev, Temirlan Karat   Group: iGEM24_NU-Kazakhstan   (2024-10-01)


L-lactate inducible production of anticancer peptide PNC27 (ALPaGA promoter)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 166
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 249
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

  1. ALPaGA promoter - BBa_K4244000
    1. No scar between ALPaGA and SP3
  2. Spacer 3- BBa_K3700001
    1. No scar between SP3 and RBS
  3. RBS - BBa_B0033
    1. TACTAG 6bp scar
  4. NSP4-PNC-27 composite part - BBa_K5129001
    1. TACTAGAG 8bp BioBrick scar
  5. Terminator - BBa_B1006

ALPaGA was chosen to express the NSP-PNC-27 in high lactate concentration - see more in Main Page Spacers are needed to create primers with spacer region to prevent from nonspecific binding if repetitive regions in plasmid are present. BBa_K3700001 was used Weaker RBS BBa_B0033 was used to exclude recombination, since BBa_B0034 was already used in Kill Switch System Terminator BBa_B1006 was also used to prevent recombination, since more commonly used terminator BBa_B0015 was included in Kill Switch system.

As for the construction of the plasmid, the genetic sequence of PNC-27 was not available on open sources prior to our work. Therefore, the sequence had to be computed for the creation of the PNC-27 coding part. To do so, we utilized amino acid alignments of the peptide to p53 and CPP obtained for the visualization. Afterward, appropriate amino acid fragments of both components of PNC-27 were aligned to the mRNA sequences that code for these amino acids in native proteins from which the synthetic construct was made. BLAST alignment was used for this analysis as well. Then, reverse DNA sequences coding these mRNA fragments were manually computed, which was followed by codon optimization and insertion of the obtained parts into the plasmid designed for this project through SnapGene. Hence, we were able to compute a new part coding PNC-27 anticancer peptide having only its amino acid sequence. Sequence for the NSP4 protein was fused to to the initial sequence of PNC-27 sequence. There are no scars or additioanl sequences between NSP4 and PNC-27

Source

ALPaGA is the promoter, reconstructed from the backbone of existing lldPRD operon of Escherichia coli, involved in l-lactate metabolism, in previous works[1].

BBa_K3700001 was designed by R2oDNA web application in previous work to create a useful toolbox for genetic expression in Bacillus subtilis.[2]

For RBS refer toBBa_B0033

PNC-27 is a synthetic protein firstly prepared via solid-phase synthesis. The sequece contains HDM2 binding domain of p53 (residues 12–26)[3], native to human, and CPP leader sequence derived from a leader sequence of the antennapedia protein, which is native to Drosophila pseudoobscura pseudoobscura, or a fruit fly[4]. The NSP4 is the one of six novel signaling peptides designed by rearrangement of the different domains of the dsbAss and pelBss, which are naturally occurring in the E. Coli wild type strains.[5]

For terminator refer toBBa_B1006

References

[1] Aguilera L, Campos E, Giménez R, Badía J, Aguilar J, Baldoma L. (2008). Dual Role of LldR in Regulation of the lldPRD Operon, Involved in l-Lactate Metabolism in Escherichia coli. J Bacteriol190:.https://doi.org/10.1128/jb.02013-07

[2] Guiziou, S., Sauveplane, V., Chang, H. J., Clerté, C., Declerck, N., Jules, M., & Bonnet, J. (2016). A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic acids research, 44(15), 7495–7508. https://doi.org/10.1093/nar/gkw624

[3] Kanovsky, M., Raffo, A., Drew, L., Rosal, R., Do, T., Friedman, F. K., Rubinstein, P., Visser, J., Robinson, R., Brandt-Rauf, P. W., Michl, J., Fine, R. L., & Pincus, M. R. (2001b). Peptides from the amino terminal mdm-2-binding domain of p53, designed from conformational analysis, are selectively cytotoxic to transformed cells. Proceedings of the National Academy of Sciences, 98(22), 12438–12443. https://doi.org/10.1073/pnas.211280698


[4] Davitt, K., Babcock, B. D., Fenelus, M., Poon, C. K., Sarkar, A., Trivigno, V., Zolkind, P. A., Matthew, S. M., Grin'kina, N., Orynbayeva, Z., Shaikh, M. F., Adler, V., Michl, J., Sarafraz-Yazdi, E., Pincus, M. R., & Bowne, W. B. (2014). The anti-cancer peptide, PNC-27, induces tumor cell necrosis of a poorly differentiated non-solid tissue human leukemia cell line that depends on expression of HDM-2 in the plasma membrane of these cells. Annals of clinical and laboratory science, 44(3), 241–248.


[5] Han, S. J., Machhi, S., Berge, M., Xi, G., Linke, T., & Schoner, R. (2017). Novel signal peptides improve the secretion of recombinant Staphylococcus aureus alpha toxinH35L in Escherichia coli. AMB Express, 7(1). https://doi.org/10.1186/s13568-017-0394-1