Part:BBa_K1364009

Pveg - RBS - Antifungal D4E1 - Double Terminator

This expression cassette is designed for the expression of an antifugal peptide, D4E1, and for its secretion in Bacillus subtilis. D4E1 has shown an antifungal activity by complexing with a sterol present in conidinia's wall of a variety of fungi.

Sequence and Features

The part is composed of a constituve promoter (K143012 Pveg), a RBS (K780002 strong RBS), the open reading frame of D4E1 and a double terminator (B0015). The coding sequence of D4E1 is flanked on the N-terminal end with a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process. D4E1 is 17 amino acids long : FKLRAKIKVRLRAKIKL

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
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functionnal 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. Commercial peptides, transformed strains expressing BBa_K1364009 and WT bacteria are tested. As shown on the following figure the strain expressing D4E1 has shown an antifungal activity on Trichoderma reseei. A lower effect of D4E1 was also proved on Aspergillus brasiliansis (see [http://2014.igem.org/Team:Toulouse/Result/experimental-results here])

The antifungal activity of D4E1 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.

More information on our wiki : http://2014.igem.org/Team:Toulouse

References:
Kanniah Rajasekaran, Kurt D. Stromberg, Jeffrey W. Cary, and Thomas E. Cleveland. Broad- Spectrum Antimicrobial Activity in vitro of the Synthetic Peptide D4E1. J. Agric. Food Chem. 2001, Vol. 49, 2799-2803.

A.J De Lucca, J.M Bland, C. Grimm, T.J Jacks. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1. Canadian Journal of Microbiology. 1998, Vol. 44:514-520.