Part:BBa_K4882000
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). This is the improved part of the full-length Pmcl1 which was registered by iGEM16_NYMU-Taipei as BBa_K2040100. Compared to the full-length Pmcl1 (2764bp), a truncated, shorter version of Pmcl1 (1586bp) can lead to an increase in downstream gene expression.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 489
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 45
- 1000COMPATIBLE 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. Fewer spores meant more cells committed suicide.
Results showed that suicide switches consisting of Pmcl1(short) led to significantly fewer spores than Pmcl1(full-length) (p < 0.01). This was due to the higher expression of the downstream genes. This proved that Pmcl1 (short) is an improved version of Pmcl1 (BBa_K2040100).
References
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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