Regulatory

Part:BBa_K4882000

Designed by: Yuqi Fu   Group: iGEM23_Hangzhou-SDG   (2023-06-09)
Revision as of 13:09, 4 October 2023 by Yuqi Fu (Talk | contribs)


Pmcl1 (short), a truncated version of hemolymph inducible promoter from Metarhizium anisopliae

The Pmcl1 promoter is responsible for regulating the collagen-like protein MCL1 in M. anisopliae. Pmcl1 is a hemolymph inducible promoter that turns on only in insect hemolymph (Wang et al., 2006). iGEM16_NYMU-Taipei registered the full-length Pmcl1 as [1]. Compared to the full-length Pmcl1 (2764bp), a truncated, shorter version of Pmcl1 (1586bp) can lead to a twofold increase in downstream gene expression (Kanjo et al., 2019).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 489
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 45
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

M. anisopliae is an entomopathogenic fungus widely used as a biopesticide. However, it is less efficient than most chemical pesticides. Genetical engineering is the method to improve the efficiency and safety of M. anisopliae. Pmcl1 (short) can be used to control the expression of exogenous toxins and suicide switches.

Characterization

2023 Hangzhou-SDG Team characterized this part with the expression of the downstream genes

KillerRed (BBa_K1184000) and SuperNova (BBa_K4882008) are red fluorescent proteins that produce reactive oxygen species (ROS) in the presence of light. They can be a part of the suicide switch for M. anisopliae when being connected after the hemolymph inducible promoter Pmcl1/Pmcl1(short).

Larvae of Galleria mellonella were killed by engineered M. anisopliae. The corpses were placed under sunshine for days until spores formed outside the insect body. Spores were collected by vertexing and the concentrations of spore suspensions were checked under a microscope.

Figure 1. A. The amount of spore formation by M. anisopliae containing KillerRed on G. mellonella corpses; B. The amount of spore formation by M. anisopliae containing SuperNova on G. mellonella corpses. **: p < 0.01.

Results showed that suicide switches consisting of Pmcl1(short) led to significantly fewer spores than Pmcl1(full-length) did. This was due to the higher expression of the downstream gene.

References

Kanjo, K.; Surin, S. I.; Gupta, T.; Dhanasingh, M.; Singh, B.; Saini, G. K. Truncated, Strong Inducible Promoter PMCL1 from Metarhizium Anisopliae. 3 Biotech 2019, 9 (3). DOI:10.1007/s13205-019-1610-2.

Wang, C., & St. Leger, R. J. (2006). A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses. Proceedings of the National Academy of Sciences of the United States of America, 103, 6647-6652.

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