Team:Berkeley/Project/Vesicle Buster

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Revision as of 04:23, 26 October 2010

Overview

The Vesicle Buster is designed to degrade the vesicle membrane after lysis by degrading the phospholipids and creating pores in the membrane. It allows the payload that has been released into the vesicle by Self-lysis to move to the cytoplasm of the Choanoflagellate.

Construct

Pcon: constitutive promoter
Perfrinogen O (PFO): a protein from Clostridium perfringens that oligomerizes to form pores in cholesterol-containing membranes
Phospholipase C (PLC): a phospholipase from Clostridium perfringens that degrades eukaryotic phospholipids
Degradation tag (ssDeg): Eukaryotic degradation tag
Pre-pro: a sequence that targets proteins to the periplasm of E. coli

From Mammalian to Lower Metazoan Delivery

We derived the vesicle buster device from a construct built in the Anderson Lab that has been assayed in a mammalian system. Here's an outline of the key features of the device that made it usable in our delivery scheme, and change we made tailor it to our scheme.

1)Constitutively expressed

2)Specific to Eukaryotes

3)Not Specific to any particular Eukaryote

4)Degrades the vesicle, but not the host

Because of the short time window between ingestion and digestion, the vesicle buster had to be constitutively expressed and ready to act upon self lysis. Stable expression of the vesicle buster was accomplished by placing it under the control of Pcon, a constitutive promoter.

Challenge 2: Toxicity to bacteria Since the bacteria stably express the vesicle buster, the device also cannot harm the bacteria and must act only on the choanoflagellate’s membrane. This specificity was satisfied by using PFO and PLC. PFO acts only on a cholesterol-based membrane and does not affect E. coli’s membrane, which is cholesterol-free. PLC also targets phsopholipids found only in eukaryotic membranes. Finally, once the food vesicle is opened and its contents are released into the cytoplasm, PLC and PFO must be prevented from breaking down any other membrane and creating further damage to the choanoflagellate. For this reason, degradation tags were added to these enzymes.

Payload Delivery Device

@@ Line 26: / Line 26: @@
 <font size=5>From Mammalian to Lower Metazoan Delivery</font>
+We derived the vesicle buster device from a construct built in the Anderson Lab that has been assayed in a mammalian system. Here's an outline of the key features of the device that made it usable in our delivery scheme, and change we made tailor it to our scheme.
-<font size=5>Payload Delivery Device</font>
+)Constitutively expressed
+)Specific to Eukaryotes
+)Not Specific to any particular Eukaryote
-Vesicle Buster
+)Degrades the vesicle, but not the host
-We derived the vesicle buster device from a construct built in the Anderson Lab and intended to be used in a mammalian system. There were several design challenges the vesicle buster had to satisfy in order to properly function in our delivery scheme. Because of the short time window between ingestion and digestion, the vesicle buster had to be constitutively expressed and ready to act upon self lysis. Stable expression of the vesicle buster was accomplished by placing it under the control of Pcon, a constitutive promoter. Since the bacteria stably express the vesicle buster, the device also cannot harm the bacteria and must act only on the choanoflagellate’s membrane. This specificity was satisfied by using PFO and PLC. PFO acts only on a cholesterol-based membrane and does not affect E. coli’s cell wall. PLC also targets phsopholipids found only in eukaryotic membranes. Finally, once the food vesicle is opened and its contents are released into the cytoplasm, PLC and PFO must be prevented from breaking down any other membrane and creating further damage to the choanoflagellate. For this reason, degradation tags were added to these enzymes.
+Because of the short time window between ingestion and digestion, the vesicle buster had to be constitutively expressed and ready to act upon self lysis. Stable expression of the vesicle buster was accomplished by placing it under the control of Pcon, a constitutive promoter.
+Challenge 2: Toxicity to bacteria
+Since the bacteria stably express the vesicle buster, the device also cannot harm the bacteria and must act only on the choanoflagellate’s membrane. This specificity was satisfied by using PFO and PLC. PFO acts only on a cholesterol-based membrane and does not affect E. coli’s membrane, which is cholesterol-free. PLC also targets phsopholipids found only in eukaryotic membranes. Finally, once the food vesicle is opened and its contents are released into the cytoplasm, PLC and PFO must be prevented from breaking down any other membrane and creating further damage to the choanoflagellate. For this reason, degradation tags were added to these enzymes.
+<font size=5>Payload Delivery Device</font>