Team:NYMU-Taipei/Project/Inhibition/Prohibition

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Revision as of 13:05, 27 September 2013

National Yang Ming University

1 Polar Filament Inhibition

Polar Filament Inhibition

Background

　　After N. ceranae invades epithelial cells of a bee's midgut, it will soon reproduce in two days and new generation appears. Since its lifecycle is so short that there is no time for biological methods to react and inhibit the development of the pathogen, we choose to inhibit it before entering bees' epithelial cells, which will be before N. ceranae’s germination. When N. ceranae's spore moves to bees' midgut, it will start to germinate and develop a structure called polar filament. This structure will then bind to epithelial cells of bees' midgut and poke a hole at the membrane of an epithelial cell, causing the bee eventually dead. In order to inhibit polar filament's development, we find out that there is a protein called PTP1 is highly relevant to the binding of the polar filament and epithelial cells. So we decide to suppress the growth of the polar filament by obstruct the production of PTP1. In the research that is done by other scientist, they have found out that in the process of PTP1 formation a mannose will bind to the protein. So we use mannosidase to prevent N. ceranae from producing proper PTP1 further to stop its polar filament to bind to the epithelial cell and of course, stop the infection of N. ceranae.

Mannosidase

2-O-α-mannosyl-D-glycerate degradation pathway in MG1655

　　MngB, a passage of gene in Escherichia coli K-12 substr MG1655, can produce alpha-mannosidase. It is an enzyme that turns 2-O-(6-phospho-α-mannosyl)-D-glycerate into mannose-6-phosphate and glycerate, which is the reverse reaction of the post-translation (glycosylation) on polar filaments of Fungi. This degradation pathway is often seen in many organisms, and the reason why we choose from MG1655 is that this species of E-coli are able to live in bees' midgut. Besides MG1655 is also the bacteria that we are going to transform our whole circuit into. This decision make our experiment more easier and more certain that this enzyme can well perform in our Bee. coli. . We tent to overexpress this gene in order to inhibit the production of the PTP1 in the polar filament of N. ceranae, the chosen target fungi that we are eager to eliminate.

Circuit Design

　　Since we cannot be sure when the spore of N. ceranae will start growing polar filament, also not having an accurate sensor to know when N. ceranae invade, we decided to add a constitutive promoter in front of mngB to let it express continuously.
The circuit design is as below:

circuit design

Experiment

Reference

link:[http://ecocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=EG13236-MONOMER alpha mannosidase]

Retrieved from "http://2013.igem.org/Team:NYMU-Taipei/Project/Inhibition/Prohibition"

@@ Line 2: / Line 2: @@
 =Polar Filament Inhibition=
-==background==
+==Background==
-　　After ''N. ceranae'' invades the epithelial cell of bee's midgut it will soon reproduce in two days and new generation appears. Since it's lifecycle is really short that there are no time for biological methods to react and inhibit the pathogen's development, we choose to inhibit it before it enter's the bee's epithelial cell, which will be before ''N. ceranae''’s germination. When ''N. ceranae'''s spore moves to bee's midgut it will start to germinate and develop a structure called polar filament. This structure will then bind to the epithelial cell of the bee's midgut and poke a hole at the membrane of epithelial cell, causing the bee eventually dead.In order to inhibit polar filament's development, we have found out that there is a protein called PTP1 which is highly relevant to the binding of the polar filament and the epithelial cell. So we decided to suppress the growth of the polar filament by obstruct the production of PTP1. In the research that is done by other scientist, they have found out that in the process of PTP1 formation a mannose  will bind to the protein. So we use mannosidase to prevent ''N. ceranae'' from producing proper PTP1 further to stop its polar filament to bind to the epithelial cell and of course, stop the infection of ''N. ceranae''.
+　　After ''N. ceranae'' invades epithelial cells of a bee's midgut, it will soon reproduce in two days and new generation appears.  Since its lifecycle is so short that there is no time for biological methods to react and inhibit the development of the pathogen, we choose to inhibit it before entering bees' epithelial cells, which will be before ''N. ceranae''’s germination.  When ''N. ceranae'''s spore moves to bees' midgut, it will start to germinate and develop a structure called polar filament.  This structure will then bind to epithelial cells of bees' midgut and poke a hole at the membrane of an epithelial cell, causing the bee eventually dead.  In order to inhibit polar filament's development, we find out that there is a protein called PTP1 is highly relevant to the binding of the polar filament and epithelial cells.  So we decide to suppress the growth of the polar filament by obstruct the production of PTP1. In the research that is done by other scientist, they have found out that in the process of PTP1 formation a mannose  will bind to the protein. So we use mannosidase to prevent ''N. ceranae'' from producing proper PTP1 further to stop its polar filament to bind to the epithelial cell and of course, stop the infection of ''N. ceranae''.
-==mannosidase==
+==Mannosidase==
 [[File:NYMU_Taipei_MngB pathway.PNG|right|thumb|325px|2-O-α-mannosyl-D-glycerate degradation pathway in MG1655]]
-　　Mannosidase is an enzyme that can be found in many different organisms. It is an enzyme that turns 2-O-(6-phospho-α-mannosyl)-D-glycerate into mannose-6-phosphate and glycerate, which is the reverse reaction of the reaction that mentioned before in the PTP1's formation. This reaction is part of the 2-O-α-mannosyl-D-glycerate degradation pathway, which is often seen in many organisms.We choose to clone the mannosidase gene that is from Escherichia coli K-12 substr. MG1655 which is also the bacteria that we are going to transform our parts into and form our devise. This can let our experiment become more easier and be more sure that the enzyme can have the right function in our Bee. coli. This gene is called mngB. We overexpress this gene in order to inhibit the production of the PTP1 in the polar filament of ''N. ceranae''.<br>
+　　MngB, a passage of gene in ''Escherichia coli'' K-12 substr MG1655, can produce alpha-mannosidase.  It is an enzyme that turns 2-O-(6-phospho-α-mannosyl)-D-glycerate into mannose-6-phosphate and glycerate, which is the reverse reaction of the post-translation (glycosylation) on polar filaments of Fungi.  This degradation pathway is often seen in many organisms, and the reason why we choose from MG1655 is that this species of ''E-coli'' are able to live in bees' midgut. Besides MG1655 is also the bacteria that we are going to transform our whole circuit into.  This decision make our experiment more easier and more certain that this enzyme can well perform in our ''Bee. coli''.  . We tent to overexpress this gene in order to inhibit the production of the PTP1 in the polar filament of ''N. ceranae'', the chosen target fungi that we are eager to eliminate.<br>
@@ Line 14: / Line 15: @@
-==circuit design==
+==Circuit Design==
 　　Since we cannot be sure when the spore of ''N. ceranae'' will start growing polar filament, also not having an accurate sensor to know when ''N. ceranae'' invade, we decided to add a constitutive promoter in front of mngB to let it express continuously. <br>The circuit design is as below:<br>
 [[File:NYMU_Taipei_mannosidase circuit.PNG|left|thumb|350px|circuit design]]
-==experiment==
+==Experiment==
-==reference==
+==Reference==
 link:[http://ecocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=EG13236-MONOMER alpha mannosidase]