Difference between revisions of "Chassis/Cell-Free Systems"

(→‎Introduction)
(→‎Cell-Free Systems)
Line 1: Line 1:
 
__NOTOC__
 
__NOTOC__
−
=Cell-Free Systems=
 
−
 
 
==Introduction==
 
==Introduction==
−
'''Cell-Free Systems (CFS)''' involve the in-vitro expression of genes into proteins. These systems can serve as a compatible chassis for the various parts and devices from the Registry of Standard Biological Parts.
+
'''Cell-Free Systems (CFS)''' involve the in-vitro expression of genes into proteins. These systems can serve as a compatible chassis for the various parts and devices from the [https://parts.igem.org/wiki/index.php/Main_Page Registry of Standard Biological Parts].
  
−
Coupled transcription-translation systems usually combine a bacteriophage RNA polymerase and promoter with eukaryotic or prokaryotic extracts. In addition, the PURE system has been developed as a reconstituted CFS for synthesizing proteins using recombinant elements.
+
Coupled transcription-translation systems usually combine a bacteriophage RNA polymerase and promoter with eukaryotic or prokaryotic extracts. In addition, the PURE system has been developed as a reconstituted CFS for synthesizing proteins using recombinant elements <cite>1</cite>.
 
<br>
 
<br>
 
<br>
 
<br>
Line 25: Line 23:
 
<br>
 
<br>
 
'''Specifications for CFS characterization'''<br>
 
'''Specifications for CFS characterization'''<br>
−
The following are several parameters that will help us understand the advantages and disadvantages associated with a particular chassis housing the gene expression machinery.
+
Several parameters are identified to help us understand the advantages and disadvantages associated with using a particular chassis.
 
{| border="1"
 
{| border="1"
 
|-
 
|-
 
|style="background:#ffffcc"|'''Properties'''
 
|style="background:#ffffcc"|'''Properties'''
−
||<center>'''Definitions'''</center>
+
|width=75%|<center>'''Definitions'''</center>
−
|-
+
−
|style="background:#ffffcc"|Rise time
+
−
||Measure of time from start of reaction to the point when expression rate first reaches the steady state value.
+
 
|-
 
|-
 
|style="background:#ffffcc"|Peak time
 
|style="background:#ffffcc"|Peak time
 
||Measure of time from start of reaction to the point when expression rate reaches the maximum value.
 
||Measure of time from start of reaction to the point when expression rate reaches the maximum value.
−
|-
 
−
|style="background:#ffffcc"|Settling time
 
−
||Measure of time from start of reaction to the point when expression rate reaches the steady state and does not escape from it for a prolonged period of time.
 
 
|-
 
|-
 
|style="background:#ffffcc"|Stability of synthesized protein
 
|style="background:#ffffcc"|Stability of synthesized protein
Line 50: Line 42:
 
|-
 
|-
 
|}
 
|}
−
 
+
<br>
 
==Cell-Free Systems investigated==
 
==Cell-Free Systems investigated==
−
 
+
Please click on each system for its individual characterization.
−
*[[Chassis/Cell-Free_Systems/Commercial_E.Coli_T7_S30_extract | Home-made E.Coli T7 S30 Extract]]
+
{| border="0" width="100%"
−
*[[Chassis/Cell-Free_Systems/Home made_E.Coli_S30_extract | Home made E.Coli S30 Extract]]
+
|- align=center
−
*[[Chassis/Cell-Free_Systems/Commercial_E.Coli_S30_extract | Commercial E.Coli S30 Extract]]
+
||<font face=georgia color=#330033 size=4>Homemade ''E. coli'' S30</font>
−
* Vesicles ...
+
||<font face=georgia color=#330033 size=4>Commercial ''E. coli'' S30</font>
−
 
+
||<font face=georgia color=#330033 size=4>Commercial ''E. coli'' T7 S30</font>
−
 
+
||<font face=georgia color=#330033 size=4>Vesicle-encapsulated</font>
−
 
+
|-
−
 
+
|}
 +
<br>
 +
'''Different compartmentalization strategies'''
 +
{| border="1"
 +
|-
 +
|style="background:#ccffff"|Batch-mode
 +
||Transcription-translation reaction is carried out in bulk solution.
 +
|-
 +
|style="background:#ccffff"|Continuous-exchange
 +
||Transcription-translation reaction is separated from feeding solution by a dialysis membrane.
 +
|-
 +
|style="background:#ccffff"|Vesicle-encapsulated
 +
||The reaction is separated from feeding solution by a phospholipid bilayer. More reliable exchange of materials is established by inserting a non-specific pore protein into the phospholipid bilayer.<cite>2</cite>
 +
|-
 +
|}
 +
<br>
 +
'''DNA constructs selected for CFS characterization'''
 +
*A simple constitutive gene expression device that reguires an ''E. coli'' RNA polymerase [https://parts.igem.org/wiki/index.php/Part:BBa_I13522 BBa_I13522]
 +
*A simple constitutive gene expression device that requires a T7 bacteriophage RNA polymerase [https://parts.igem.org/wiki/index.php/Part:BBa_E7104 BBa_E7104]
 +
*An inducible gene expression device that is well-characterized in the registry [https://parts.igem.org/wiki/index.php/Part:BBa_T9002 BBa_T9002]
 +
<br>
 
==References==
 
==References==
−
 
+
<biblio>
−
* ...
+
#1 pmid=16076456
−
* ...
+
#2 pmid=16224117
−
* ...
+
#3 pmid=14559971
 +
#4 pmid=15183761
 +
</biblio>

Revision as of 00:01, 21 October 2007

Introduction

Cell-Free Systems (CFS) involve the in-vitro expression of genes into proteins. These systems can serve as a compatible chassis for the various parts and devices from the Registry of Standard Biological Parts.

Coupled transcription-translation systems usually combine a bacteriophage RNA polymerase and promoter with eukaryotic or prokaryotic extracts. In addition, the PURE system has been developed as a reconstituted CFS for synthesizing proteins using recombinant elements 1.

Advantages and disadvantages of CFS

Advantages
Disadvantages
Non-infectious because of non-proliferative nature Short expression lifespan since system cannot replicate
Process is quick and simple requiring only preparation of cell extract and feeding solution and subsequent addition of DNA template Expensive because of the constant need for nutrient and energy supply
Quality control can be achieved easily using modified reaction conditions such as addition of accessory elements or inhibitory factors Less characterization and experience of use in the laboratories compared to E. coli


Specifications for CFS characterization
Several parameters are identified to help us understand the advantages and disadvantages associated with using a particular chassis.

Properties
Definitions
Peak time Measure of time from start of reaction to the point when expression rate reaches the maximum value.
Stability of synthesized protein Measure of the half-life of a given protein (e.g. GFP) in the chassis.
Total expression capacity Measure of the total expression of a chassis for a given DNA construct template. This should take into account the degradation of synthesized protein.
Expression lifespan Measure of time that expression occurs for a given DNA construct template until protein degradation overrides protein synthesis.


Cell-Free Systems investigated

Please click on each system for its individual characterization.

Homemade E. coli S30 Commercial E. coli S30 Commercial E. coli T7 S30 Vesicle-encapsulated


Different compartmentalization strategies

Batch-mode Transcription-translation reaction is carried out in bulk solution.
Continuous-exchange Transcription-translation reaction is separated from feeding solution by a dialysis membrane.
Vesicle-encapsulated The reaction is separated from feeding solution by a phospholipid bilayer. More reliable exchange of materials is established by inserting a non-specific pore protein into the phospholipid bilayer.2


DNA constructs selected for CFS characterization

  • A simple constitutive gene expression device that reguires an E. coli RNA polymerase BBa_I13522
  • A simple constitutive gene expression device that requires a T7 bacteriophage RNA polymerase BBa_E7104
  • An inducible gene expression device that is well-characterized in the registry BBa_T9002


References

<biblio>

  1. 1 pmid=16076456
  2. 2 pmid=16224117
  3. 3 pmid=14559971
  4. 4 pmid=15183761

</biblio>