Team:Berkeley/Project/Vesicle Buster

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Latest revision as of 23:46, 27 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.

NOTE: Since this construct contains parts that are Risk Group 2, we were not allowed to submit it to the Registry.

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 the changes we made tailor it to our scheme.

Constitutively expressed: We added a constitutive promoter (Pcon) to the device because there is only a short time window between ingestion and digestion, meaning the vesicle buster must be constitutively expressed so it's ready to act right after self lysis occurs.
Specific to Eukaryotes: 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. Bacteria cell membranes are different from eukaryotic membranes in several ways: bacteria membranes don't contain cholesterol, while eukaryotic membranes do, and bacteria membranes are composed of different phospholipids. The design of the Vesicle Buster exploits these differences by including proteins that act specifically on eukaryotic membranes, leaving the bacteria's membranes unharmed. 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.
Not Specific to any particular Eukaryote: Since PFO and PLC act on key components of eukaryotic membranes, this device has the potential to be effective in a wide variety of hosts, ranging from mammalian cells to Choanoflagellates. We hope to use the device to try Payload delivery in other Lower Metazoans in addition to Choanoflagellates.
Degrades the vesicle, but not the host: Although the PFO and PLC proteins are designed to degrade the vesicle membrane, the vesicle-buster device should not degrade the membrane of the choanoflagellates as well. To prevent this, there are degradation tags on PLC and PFO. PLC and PFO are degraded once they escape the vesicle and are released into the cytoplasm, preventing them from being toxic to the Choanoflagellates.

@@ Line 10: / Line 10: @@
 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.
+NOTE: Since this construct contains parts that are Risk Group 2, we were not allowed to submit it to the Registry.
 <font size=5>'''Construct'''</font>
@@ Line 29: / Line 31: @@
 #'''Constitutively expressed:''' We added a constitutive promoter (Pcon) to the device because there is only a short time window between ingestion and digestion, meaning the vesicle buster must be constitutively expressed so it's ready to act right after self lysis occurs.
+#'''Specific to Eukaryotes:''' 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. Bacteria cell membranes are different from eukaryotic membranes in several ways: bacteria membranes don't contain cholesterol, while eukaryotic membranes do, and bacteria membranes are composed of different phospholipids. The design of the Vesicle Buster exploits these differences by including proteins that act specifically on eukaryotic membranes, leaving the bacteria's membranes unharmed. 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.
-#'''Specific to Eukaryotes:''' Bacteria cell membranes are different from eukaryotic membranes in several ways. For example, bacteria membranes don't contain cholesterol, while eukaryotic membranes do. Their membranes are also composed of different phospholipids. The design of the Vesicle Buster exploits these differences by including proteins that act specifically on eukaryotic membranes (see details of PFO and PLC mechanism above), leaving the bacteria's membranes unharmed.
 #'''Not Specific to any particular Eukaryote:''' Since PFO and PLC act on key components of eukaryotic membranes, this device has the potential to be effective in a wide variety of hosts, ranging from mammalian cells to Choanoflagellates. We hope to use the device to try Payload delivery in other Lower Metazoans in addition to Choanoflagellates.
+#'''Degrades the vesicle, but not the host:''' Although the PFO and PLC proteins are designed to degrade the vesicle membrane, the vesicle-buster device should not degrade the membrane of the choanoflagellates as well. To prevent this, there are degradation tags on PLC and PFO. PLC and PFO are degraded once they escape the vesicle and are released into the cytoplasm, preventing them from being toxic to the Choanoflagellates.
-#'''Degrades the vesicle, but not the host:''' Although the PFO and PLC proteins are designed to degrade the vesicle membrane, ideally they wouldn't degrade the membrane of the choanoflagellates as well. To prevent this, there are degradation tags on PLC and PFO, so that the Choanoflagellates will degrade them once they've busted the vesicle and have been released into the cytoplasm along with the payload, preventing them from being toxic to the Choanoflagellates.
-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>