Difference between revisions of "Part:BBa K1769006"

Revision as of 08:21, 18 September 2015

Dimeric FYVE (without stop codon)

Dimeric Hrs FYVE is a small binding molecule that can bind to phosphatidylinositol 3-phosphate (PtdIns(3)P), a receptor that exists on endosome. Since it is more stable then monomeric FYVE, it is widely used to recognize and locate PtdIns(3)P.

Usage and Biology

Many pathogen, such as Plasmodium falciparum that causes malaria and Phytophthora spp. that cause a wide range of plant disaeses,inhibits the immune system of host cell by secreting effector proteins. These effector protein translocate into host cell by binding to a outer membrane receptor, PI3P. This biobrick can not only serve as a useful tool to study the signal transduction pathway of PI3P but can also be a weapon to stop these pathogen's infection.

Introduction

To stop the effector protein secreted by P.infestans from binding to PI3P receptor and translocate into the cell, we designed a competitive inhibitor called FYVE protein domain. This protein domain was obtained from Hrs gene from mice. However, a monomeric FYVE protein domain is not stable in plant cell and the affinity for PI3P is not high enough to compete with effector protien, Avr3, as you can see in figure1. Therefore we designed a linker region to connect the two protein domain together.(Fig2)

Fig1. Competition model of monomeric FYVE and Avr3,a pathegon effector protein

Fig2. Competition model of dimeric FYVE and Avr3,a pathegon effector protein

Results

We test this part in BY-2 tobacoo cell line. First we construct this part in vector PSAT6. It has a promoter CAMV35S that act as a consitiutive promoter in plat cells. To see if the dimeric FYVE protein domain really binds to PI3P receptor on the endosome of plant cell, we added a Phosphoinositide-3-kinase(PI3K) inhibitor, wortmannin, that can inhibit PI3K from turning PI3P precursor into PI3P.

The picture on the left shows that after we introduce dimeric FYVE into BY-2 tobacoo cell line, the FYVE concentrated in endosome and cell membrane. However, one hour after we added 10^-6M wortmannin, dimeric FYVE redistributed in cytoplasm.

Sequence and Features