Composite

Part:BBa_K4350142

Designed by: Trevor Fox   Group: iGEM22_UBC-Okanagan   (2022-09-30)

Usage of this Transcriptional Unit by the 2022 UBC-Okanagan iGEM Team

A transcriptional unit for the constitutive overexpression of fungal hispidin-3-hydroxylase from Neonothopanus nambi. This level 1 MoClo part is compatible for assembly in a multigene construct through Type IIS golden gate cloning. Utilizing the Plant Golden Gate MoClo Kit syntax, this part forms the third transcriptional unit in a level 2 multigene construct (Vasudevan et al., 2019).

The PlacIQ promoter is a strong constitutive promoter that is suitable for heterologous expression in E. coli and Synechocystis sp. PCC 6803 (Vasudevan et al., 2019). It is a modified form of the PlacI promoter, which is responsible for expression of the lacI gene in E. coli (Vasudevan et al., 2019). This sequence also includes a 5’ UTR, making it suitable for assembly directly upstream of the coding sequence (Vasudevan et al., 2019).

nnH3H is an NAD(P)H-dependent monooxygenase that catalyzes the formation of 3-hydroxyhispidin from hispidin (Kotlobay et al., 2018). It is the second enzyme in the fungal bioluminescence pathway, responsible for the formation of fungal luciferin, the substrate of the light-emitting reaction in bioluminescent fungi (Kotlobay et al., 2018). This enzyme connects the fungal bioluminescence pathway with a common fungal metabolite, hispidin (Kotlobay et al., 2018).

TpheA-1 is the terminator for the pheA gene from E. coli, although it has demonstrated functionality in Synechocystis sp. PCC 6803 as well (Vasudevan et al., 2019).

This construct was assembled by UBCO iGEM in 2022. The H3H coding sequence was codon optimized for Synechocystis sp. PCC 6803 and acquired through IDT DNA synthesis as linear fragments with Type IIS restriction sites for assembly into a level 0 part. The PlacIQ promoter and TpheA-1 terminator were acquired from the CyanoGate Kit as level 0 parts compatible with MoClo assembly.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1391
    Illegal BsaI site found at 1569


References

  • Kotlobay, A. A., Sarkisyan, K. S., Mokrushina, Y. A., Marcet-Houben, M., Serebrovskaya, E. O., Markina, N. M., Gonzalez Somermeyer, L., Gorokhovatsky, A. Y., Vvedensky, A., Purtov, K. V., Petushkov, V. N., Rodionova, N. S., Chepurnyh, T. V., Fakhranurova, L. I., Guglya, E. B., Ziganshin, R., Tsarkova, A. S., Kaskova, Z. M., Shender, V., … Yampolsky, I. V. (2018). Genetically encodable bioluminescent system from fungi. Proceedings of the National Academy of Sciences, 115(50), 12728–12732. https://doi.org/10.1073/pnas.1803615115
  • Vasudevan, R., Gale, G. A. R., Schiavon, A. A., Puzorjov, A., Malin, J., Gillespie, M. D., Vavitsas, K., Zulkower, V., Wang, B., Howe, C. J., Lea-Smith, D. J., & McCormick, A. J. (2019). CyanoGate: A modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax. Plant Physiology, 180(1), 39–55. https://doi.org/10.1104/pp.18.01401
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