Part:BBa_K1364013

Pveg - RBS - Antifungal GAFP-1 - RBS - Antifungal D4E1 - Double terminator

This part is designed for the co-expression of two different peptides with anti-fungal activites : D4E1 and GAFP-1. The Gastrodia anti-fungal protein (GAFP-1) is a mannose-binding lectin which is able to inhibit the growth of multiples species of plant pathogenic fungi. D4E1 is a linear synthetic peptide of 17 amino acids which has shown to have antifungal activities by complexing with a sterol present in conidia's wall of a variety of fungi.

Design
This part is composed of the translational unit of GAFP-1 (K1364002) and the translational unit of D4E1 (K1364003). The same RBS are used before both coding sequences (K78002 Strong RBS). The operon is under the control of a constitutive promotor of Bacillus subtilis : Pveg (K7823003) and includes also a double terminator (B0015).

For more information on each antifugal peptides, see the dedicated pages on the Registry :
K1364002 for GAFP-1 https://parts.igem.org/Part:BBa_K1364002
K1364003 for D4E1 https://parts.igem.org/Part:BBa_K1364003

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 304
  Illegal NgoMIV site found at 499
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional characterization of the BioBrick To test this part, conidia of different fungus are harvested with a drop of tween, counted and diluted. The culture medium is a "Sap-like" medium consiting of LB + agar + different sugars (see our wiki to have more information). The incubation is carried out at Room Temperature during several days. Transformed strains expressing BBa_K1364013 and WT bacteria are tested. As shown on the following figure the strain expressing D4E1+GAFP-1 has shown an antifungal activity on Trichoderma reseei.

The antifungal activity of this part has also been tested on model plants (Nicotiana benthamiana) with a pyhtopathogenic fungus (Sclerotinia sclerotiorum). The lesion size and the percentage of sites showing necrosis has been studied 40h after the infection.