Our project aims to develop a more efficient and safer fungal biopesticide. We successfully introduced the insect-specific toxin, LqhIT2, to Metarhizium anisopliae ACCC30104, and it significantly improved the virulence of the fungus towards pests. To achieve this, we introduced BBa_K4882000, BBa_K4882001, BBa_K4882002, BBa_K4882006, and BBa_K4882007. To ensure biosafety, we tested different combinations of Pmcl1/Pmcl1(short) and SuperNova/KillerRed as suicide switches. The results showed that Pmcl1(short)-SuperNova was the most effective suicide switch combination. To achieve this, we introduced BBa_K4882000, BBa_K4882003, BBa_K4882004, BBa_K4882005, BBa_K4882008, BBa_K4882009, and BBa_K4882010.
| Name | Type | Description | Length |
| BBa_K4882000 | Basic | Pmcl1 (short) | 1586 bp |
| BBa_K4882001 | Basic | LqhIT2 | 189 bp |
| BBa_K4882002 | Basic | Mcl1 signal peptide | 57 bp |
| BBa_K4882003 | Composite | SuperNova-SV40 NLS | 738 bp |
| BBa_K4882004 | Composite | Pmcl1-SuperNova-SV40 NLS-TtrpC | 3744 bp |
| BBa_K4882005 | Composite | Pmcl1 (short)-SuperNova-SV40 NLS-TtrpC | 2558 bp |
| BBa_K4882006 | Composite | Pmcl1 (short)-MCL1ss-LqhIT2-TtrpC | 2063 bp |
| BBa_K4882007 | Composite | Mcl1 signal peptide-LqhIT2 | 246 bp |
| BBa_K4882008 | Basic | SuperNova | 717 bp |
| BBa_K4882009 | Composite | Pmcl1 (short)-KillerRed-SV40 NLS-TtrpC | 2564 bp |
| BBa_K4882010 | Composite | Pmcl1-KillerRed-SV40 NLS-TtrpC | 3750 bp |
New Improved Part
Pmcl1(short) (BBa_K4882000) is an improved version of Pmcl1 (BBa_K2040100). We proved that a short version of this part (1586bp) can lead to a higher expression of the downstream genes.
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. iGEM16_NYMU-Taipei registered the full-length Pmcl1 as BBa_K2040100. We tested Pmcl1 and Pmcl1 (short) by connecting them with KillerRed (BBa_K1184000) and SuperNova (BBa_K4882008) to form suicide switches for M. anisopliae. We used engineered M. anisopliae to infect larvae of Galleria mellonella, and checked the amount of spore formation in each group. 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.
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.