Generator

Part:BBa_K1364013

Designed by: Aurélie Kanitzer   Group: iGEM14_Toulouse   (2014-10-01)

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 http://parts.igem.org/Part:BBa_K1364002
K1364003 for D4E1 http://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.

Resultfong.jpg Results_fong_2.jpg

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.

Results_d4%2B_gafp1.jpg

[edit]
Categories
//chassis/prokaryote/bsubtilis
Parameters
n/aPveg - RBS - Antifungal GAFP-1 - RBS - Antifungal D4E1 - Double terminator