Part:BBa_K4882006

Pmcl1 (short)-MCL1ss-LqhIT2-TtrpC

This is a complete expression cassette consisting of a hemolymph inducible promoter Pmc1(short), a Mcl1 secretory signal peptide fused with an insect-specific toxin LqhIT2, and a trpC terminator.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 489
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1731
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 45
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Metarhizium anisopliae is an entomopathogenic fungus widely used as a biopesticide. However, it is less efficient than most chemical pesticides. To improve the efficiency of M. anisopliae as a fungal biopesticide, we choose to introduce the LqhIT2 toxin into the fungus. To promote the secretion of the toxin, a Mcl1ss is linked at the 5’ end of the LqhIT2 gene. We put Mcl1ss-LqhIT2 (BBa_K4882007) downstream of Pmcl1 (short, BBa_K4882000). This design limited the expression of LqhIT2 inside the insect body and improved the biosafety of our product. A trpC terminator was also included to form the complete expression cassette.

Figure 1. Gel electrophoresis of colony PCR products for verification of correct transformation of plasmid pBARGEP1-Pmcl1(short)-Mcl1ss-LqhIT2-BenA into E .coli DH5α.

Characterization

2023 Hangzhou-SDG Team characterized the virulence of this part against pests

We tested the virulence of the engineered M. anisopliae on the larvae of the greater wax moth (Galleria mellonella). Different concentrations of spore suspensions (1 × 10^7, 1 × 10^6, 1 × 10^5 spores/mL) from the original strain and the strain expressing LqhIT2 were inoculated to different groups.

Figure 2: The larvae of G. mellonella infected by LqhIT2 M. anisopliae. A. Alive on day 1; B. Dead on day 4; C. Hyphae appeared on the surface on day 7; D. Hyphae covered the body on day 10.

Figure 3: The survival rate curve of WT and LqhIT2 groups inoculated with 1 × 10^7 spores/mL.

Table 1. The median lethal cell density (LC50, spores/mL) on day 5 and the median time to death (LT50, day) under 1 × 10^7 spores/mL of each strain, calculated using SPSS 26.0.0.2..

The median lethal cell density (LC50) and the median time to death (LT50) were used to evaluate the virulence of each strain. The LC50 of the LqhIT2 strain is 25.26-fold lower than that of the WT strain, showing a remarkable increase in virulence. The LT50 of the LqhIT2 strain was also significantly lower than that of the WT strain (p < 0.05). To conclude, the toxin, LqhIT2, significantly increased the virulence of M. anisopliae, and made it more efficient as a fungal biopesticide.