Team:Freiburg/SampleData

From 2011.igem.org

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== '''How Our System Works''' ==
== '''How Our System Works''' ==
-
[[File:Freiburg2011_sampledatapicture100000000000000.jpg]]
+
[[File:Freiburg2011_sample_data_picture2011.jpg]]
<br>
<br>
 +
'''How our system works:'''
 +
<br>
-
<ins>'''''Example for red light induced expression'''''</ins>
 
-
<br>
 
-
;'''1.A)''' Exposure of bacterial culture to red light (705 nm)
+
;'''1.A)''' Exposure of bacterial culture to red light (650 nm)
:Red light inducible promoter leads to expression of His-tagged protein of interest.
:Red light inducible promoter leads to expression of His-tagged protein of interest.
<br>
<br>
 +
;'''1.B)''' Exposure of bacterial culture to blue light (470nm)
 +
:Blue light leads to a conformational change of the blue light receptor (LovTAP) and the expression of a His-tagged protein.
 +
 +
<br/>
;'''2.)''' Exposure of bacterial culture to green light (532 nm)
;'''2.)''' Exposure of bacterial culture to green light (532 nm)
-
:Green light inducible promoter leads to expression of the "Precipitator"
+
:Green light inducible promoter leads to expression of the "Precipitator".
<br>
<br>
;'''3.)''' Precipitator-Protein of interest complex
;'''3.)''' Precipitator-Protein of interest complex
-
:His-tag of the protein of interest is bound by the Nickel "Precipitator" complex  
+
:His-tag of the protein of interest is bound by the Nickel "Precipitator" complex.
<br>
<br>
;'''4.)''' Cell lysis
;'''4.)''' Cell lysis
-
:Cells are autolysed by simply being heated for about 1 hour to 42°C
+
:Cells are autolysed by simply being heated for about 1 hour to 42°C.
<br>
<br>
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1.  
1.  
-
[http://partsregistry.org/Part:BBa_K608408 BBa_K608408] GST<br/>
+
[http://partsregistry.org/Part:BBa_K608408 BBa_K608408] '''GST'''<br/>
The GST-tag was PCR amplified from a pGEX vector with overhang primers including the iGEM restriction sites and then pasted into the iGEM vector. To verify the functionality of the construct we cloned it before a GFP sequence and expressed it with an IPTG inducible vector. Results see partsregistry page. The submitted sequence was partially confirmed by sequencing.
The GST-tag was PCR amplified from a pGEX vector with overhang primers including the iGEM restriction sites and then pasted into the iGEM vector. To verify the functionality of the construct we cloned it before a GFP sequence and expressed it with an IPTG inducible vector. Results see partsregistry page. The submitted sequence was partially confirmed by sequencing.
 +
2. [http://partsregistry.org/Part:BBa_K608406 BBa_K608406]
2. [http://partsregistry.org/Part:BBa_K608406 BBa_K608406]
-
'''Precipitator
+
'''Precipitator'''
-
'''
+
The Precipitator is a new artificially designed LRR protein. It is meant as protein that binds Nickel ions with Histidines grouped on its surface. The bound Nickel can then precipitate His-tagged proteins. In our Lab in a Cell it should function as an adaptor between the plastic surface of pipettes and the His-tagged protein. Please look at our detailed description of the design layout in our modeling section. The sequence was synthesized and cloned into the iGEM vector. The submitted sequence was fully confirmed by sequencing.<br/>
The Precipitator is a new artificially designed LRR protein. It is meant as protein that binds Nickel ions with Histidines grouped on its surface. The bound Nickel can then precipitate His-tagged proteins. In our Lab in a Cell it should function as an adaptor between the plastic surface of pipettes and the His-tagged protein. Please look at our detailed description of the design layout in our modeling section. The sequence was synthesized and cloned into the iGEM vector. The submitted sequence was fully confirmed by sequencing.<br/>
-
3. [http://partsregistry.org/Part:BBa_K608404 BBa_K608404] IPTG-inducible Promoter with plastic binding domain-tagged GFP  
+
3. [http://partsregistry.org/Part:BBa_K608404 BBa_K608404] '''IPTG-inducible Promoter with plastic binding domain-tagged GFP'''
 +
 
 +
The plastic binding domain (pbd) enables the isolation of the Precipitator-His-tagged-protein-complex. The pbd is a small amino acid sequence that can bind plastic, probably due to nonspecific hydrophobic interactions. As hydrophobic proteins can cause problems in E.coli it should be expressed by an inducible promoter. To test the binding interactions of the plastic binding domain it was fused to GFP via a short linker. In our "lab in a cell" model the pbd is part of the Precipitator.
'''Also new parts'''  
'''Also new parts'''  
-
[http://partsregistry.org/Part:BBa_K608101 BBa_K608101]
+
for the '''green light receptor system'''
-
CcaR, green light response regulator
+
[http://partsregistry.org/Part:BBa_K608102 BBa_K608102]
[http://partsregistry.org/Part:BBa_K608102 BBa_K608102]
-
CcaS, green light receptor
+
'''CcaS''', green light receptor
 +
 
 +
CcaS is a cyanobacteriochrome which functions as the receptor for green light.
 +
After light of the wavelength of 532 nm is exposed to the CcaS receptor, it changes its conformation undergoing autophosphorylation. The phosphate group is transfered to the response regulator CcaR. Once phosphorylated, CcaR can bind to the specific promoter region of cpcG and activate gene expression.
 +
 
 +
[http://partsregistry.org/Part:BBa_K608101 BBa_K608101]
 +
'''CcaR''', green light response regulator
 +
 
 +
CcaR is the response regulator of the green light receptor CcaS.
 +
It belongs to the family of OmpR regulator class. CcaR consists of an N-terminal receiver domain that can be
 +
phosphorylated by CcaS and a C-terminal DNA-binding domain that binds directly to the promoter region of cpcG2.
== '''Pre-existing Parts''' ==
== '''Pre-existing Parts''' ==
-
==<span style="color:orange;">Lysis cassette</span>==
+
<span style="color:orange;">Lysis cassette</span>
'''The Concept'''<br>
'''The Concept'''<br>
Line 79: Line 94:
<br>
<br>
<br>
<br>
-
<br/>
+
 
 +
----
 +
 
[http://partsregistry.org/Part:BBa_K608151 BBa_K608151]
[http://partsregistry.org/Part:BBa_K608151 BBa_K608151]
'''translational unit of pcyA'''
'''translational unit of pcyA'''
Line 93: Line 110:
== '''We've Also Characterized the Following Parts''' ==
== '''We've Also Characterized the Following Parts''' ==
-
 
+
Here we cloned different promoters with different RBS for individual gene expression.
-
[http://partsregistry.org/Part:BBa_K608351 BBa_K608351]
+
-
(correct) temperature sensitive promoter
+
-
 
+
-
[http://partsregistry.org/Part:BBa_K608352 BBa_K608352]
+
-
Bacteriophage Lysis Cassette with RBS
+
[http://partsregistry.org/Part:BBa_K608002 BBa_K608002]
[http://partsregistry.org/Part:BBa_K608002 BBa_K608002]
-
strong Promotor and strong RBS
+
strong Promoter and strong RBS
[http://partsregistry.org/Part:BBa_K608003 BBa_K608003]
[http://partsregistry.org/Part:BBa_K608003 BBa_K608003]
-
strong Promotor , medium RBS
+
strong Promoter , medium RBS
[http://partsregistry.org/Part:BBa_K608004 BBa_K608004]
[http://partsregistry.org/Part:BBa_K608004 BBa_K608004]
-
strong Promotor , weak RBS
+
strong Promoter , weak RBS
[http://partsregistry.org/Part:BBa_K608005 BBa_K608005]
[http://partsregistry.org/Part:BBa_K608005 BBa_K608005]
-
medium Promotor , strong RBS  
+
medium Promoter , strong RBS  
[http://partsregistry.org/Part:BBa_K608006 BBa_K608006]
[http://partsregistry.org/Part:BBa_K608006 BBa_K608006]
-
medium Promotor , medium RBS
+
medium Promoter , medium RBS
[http://partsregistry.org/Part:BBa_K608007 BBa_K608007]
[http://partsregistry.org/Part:BBa_K608007 BBa_K608007]
-
medium Promotor , weak RBS
+
medium Promoter , weak RBS
-
==<span style="color:grey;">Precipitator</span>==
+
----
-
==<span style="color:orange;">Lysis cassette</span>==
 
-
'''The Concept'''<br>
 
-
<br>
 
-
The idea behind our lysis cassette was to be able to achieve cell lysis by simply heating the bacteria to 42°C for a short period of time. This was to be accomplished by combining the biobricks [http://partsregistry.org/Part:BBa_K098995 BBa_K098995] (temperature sensitive promoter) and [http://partsregistry.org/Part:BBa_K124017 BBa_K124017] (Bacteriophage λ lysis genes). Sequencing of these parts (carried out by GATC Biotech GmbH) showed some fundamental inconsistencies leading to a lot of time spent on fixing them.
 
-
<br>
+
To quantify the strength of our PR-constructs we cloned GFP and RFP behind it<br/>
 +
and quantified the protein output.<br/>
 +
 
 +
 
 +
[http://partsregistry.org/Part:BBa_K608008 BBa_K608008]
 +
constitutive strong promoter with medium RBS and GFP
 +
 
 +
[http://partsregistry.org/Part:BBa_K608009 BBa_K608009]
 +
Strong promoter with weak RBS and GFP
 +
 
 +
[http://partsregistry.org/Part:BBa_K608010 BBa_K608010]
 +
Medium promoter with strong RBS and GFP
 +
 
 +
[http://partsregistry.org/Part:BBa_K608011 BBa_K608011]
 +
Medium promoter with medium RBS and GFP
 +
 
 +
[http://partsregistry.org/Part:BBa_K608012 BBa_K608012]
 +
Medium promoter with weak RBS and GFP
 +
 
 +
----
 +
 
 +
[http://partsregistry.org/Part:BBa_K608013 BBa_K608013]
 +
strong Promoter with strong RBS and RFP
 +
 
 +
[http://partsregistry.org/Part:BBa_K608014 BBa_K608014 ]
 +
PR with RFP
 +
 
 +
[http://partsregistry.org/Part:BBa_K608015 BBa_K608015]
 +
PR with RFP
 +
 
 +
[http://partsregistry.org/Part:BBa_K608016 BBa_K608016]
 +
PR with RFP
 +
 
 +
[http://partsregistry.org/Part:BBa_K608017 BBa_K608017]
 +
PR with RFP
 +
 
 +
[http://partsregistry.org/Part:BBa_K608018 BBa_K608018]
 +
PR with RFP
 +
 
-
The sequences were corrected and the new biobricks [http://partsregistry.org/Part:BBa_K608351 BBa_K608351] and [http://partsregistry.org/Part:BBa_K608352 BBa_K608352] were sent to the registry, although the novel temperature sensitive lysis cassette composite's sequence was verified some 18 hours before the Wiki-freeze, so that it could not be sent to the registry on time. Nevertheless, some preliminary OD-measurement results did hint at the expected functionality of the newly developed composite part.
 
-
<br>
 
-
For more Information, please check our description page https://2011.igem.org/Team:Freiburg/Description#Part_Design_2
 
-
<br>
 
-
<br>
 
{{:Team:Freiburg/Templates/footer}}
{{:Team:Freiburg/Templates/footer}}

Latest revision as of 03:52, 22 September 2011


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!


Contents

Sample Data

How Our System Works

Freiburg2011 sample data picture2011.jpg


How our system works:


1.A) Exposure of bacterial culture to red light (650 nm)
Red light inducible promoter leads to expression of His-tagged protein of interest.


1.B) Exposure of bacterial culture to blue light (470nm)
Blue light leads to a conformational change of the blue light receptor (LovTAP) and the expression of a His-tagged protein.


2.) Exposure of bacterial culture to green light (532 nm)
Green light inducible promoter leads to expression of the "Precipitator".


3.) Precipitator-Protein of interest complex
His-tag of the protein of interest is bound by the Nickel "Precipitator" complex.


4.) Cell lysis
Cells are autolysed by simply being heated for about 1 hour to 42°C.


And thus the Precipitator-Protein of interest complex is in solution. It can now bind to the surface of serological pipettes (made of polystyrene) with the help of a hydrophobic plastic binding domain, and stabilize the protein of interest long enough to endure two wash steps to elute it.


Our Favorite New Parts

1. [http://partsregistry.org/Part:BBa_K608408 BBa_K608408] GST

The GST-tag was PCR amplified from a pGEX vector with overhang primers including the iGEM restriction sites and then pasted into the iGEM vector. To verify the functionality of the construct we cloned it before a GFP sequence and expressed it with an IPTG inducible vector. Results see partsregistry page. The submitted sequence was partially confirmed by sequencing.


2. [http://partsregistry.org/Part:BBa_K608406 BBa_K608406] Precipitator

The Precipitator is a new artificially designed LRR protein. It is meant as protein that binds Nickel ions with Histidines grouped on its surface. The bound Nickel can then precipitate His-tagged proteins. In our Lab in a Cell it should function as an adaptor between the plastic surface of pipettes and the His-tagged protein. Please look at our detailed description of the design layout in our modeling section. The sequence was synthesized and cloned into the iGEM vector. The submitted sequence was fully confirmed by sequencing.

3. [http://partsregistry.org/Part:BBa_K608404 BBa_K608404] IPTG-inducible Promoter with plastic binding domain-tagged GFP

The plastic binding domain (pbd) enables the isolation of the Precipitator-His-tagged-protein-complex. The pbd is a small amino acid sequence that can bind plastic, probably due to nonspecific hydrophobic interactions. As hydrophobic proteins can cause problems in E.coli it should be expressed by an inducible promoter. To test the binding interactions of the plastic binding domain it was fused to GFP via a short linker. In our "lab in a cell" model the pbd is part of the Precipitator.

Also new parts

for the green light receptor system

[http://partsregistry.org/Part:BBa_K608102 BBa_K608102] CcaS, green light receptor

CcaS is a cyanobacteriochrome which functions as the receptor for green light. After light of the wavelength of 532 nm is exposed to the CcaS receptor, it changes its conformation undergoing autophosphorylation. The phosphate group is transfered to the response regulator CcaR. Once phosphorylated, CcaR can bind to the specific promoter region of cpcG and activate gene expression.

[http://partsregistry.org/Part:BBa_K608101 BBa_K608101] CcaR, green light response regulator

CcaR is the response regulator of the green light receptor CcaS. It belongs to the family of OmpR regulator class. CcaR consists of an N-terminal receiver domain that can be phosphorylated by CcaS and a C-terminal DNA-binding domain that binds directly to the promoter region of cpcG2.

Pre-existing Parts

Lysis cassette

The Concept

The idea behind our lysis cassette was to be able to achieve cell lysis by simply heating the bacteria to 42°C for a short period of time. This was to be accomplished by combining the biobricks [http://partsregistry.org/Part:BBa_K098995 BBa_K098995] (temperature sensitive promoter) and [http://partsregistry.org/Part:BBa_K124017 BBa_K124017] (Bacteriophage λ lysis genes). Sequencing of these parts (carried out by GATC Biotech GmbH) showed some fundamental inconsistencies leading to a lot of time spent on fixing them.


The sequences were corrected and the new biobricks [http://partsregistry.org/Part:BBa_K608351 BBa_K608351] and [http://partsregistry.org/Part:BBa_K608352 BBa_K608352] were sent to the registry, although the novel temperature sensitive lysis cassette composite's sequence was verified some 18 hours before the Wiki-freeze, so that it could not be sent to the registry on time. Nevertheless, some preliminary OD-measurement results did hint at the expected functionality of the newly developed composite part.


For more Information, please check our description page https://2011.igem.org/Team:Freiburg/Description#Part_Design_2

[http://partsregistry.org/Part:BBa_K608151 BBa_K608151] translational unit of pcyA


We designed this composite in order to gain the enzyme pcyA (phycocyanobilin-ferredoxin oxidoreductase),
this design is necessary if the part is going be integrated into an assembly of various genes.
The enzyme plays an important role in the production of the PCB chromophore (phycocyanobilin)
which is essential for the green and red light receptor system.
Sequencing confirmed that the part is correct in the pSB1C3 vector.

We've Also Characterized the Following Parts

Here we cloned different promoters with different RBS for individual gene expression.


[http://partsregistry.org/Part:BBa_K608002 BBa_K608002] strong Promoter and strong RBS

[http://partsregistry.org/Part:BBa_K608003 BBa_K608003] strong Promoter , medium RBS

[http://partsregistry.org/Part:BBa_K608004 BBa_K608004] strong Promoter , weak RBS

[http://partsregistry.org/Part:BBa_K608005 BBa_K608005] medium Promoter , strong RBS

[http://partsregistry.org/Part:BBa_K608006 BBa_K608006] medium Promoter , medium RBS

[http://partsregistry.org/Part:BBa_K608007 BBa_K608007] medium Promoter , weak RBS


To quantify the strength of our PR-constructs we cloned GFP and RFP behind it
and quantified the protein output.


[http://partsregistry.org/Part:BBa_K608008 BBa_K608008] constitutive strong promoter with medium RBS and GFP

[http://partsregistry.org/Part:BBa_K608009 BBa_K608009] Strong promoter with weak RBS and GFP

[http://partsregistry.org/Part:BBa_K608010 BBa_K608010] Medium promoter with strong RBS and GFP

[http://partsregistry.org/Part:BBa_K608011 BBa_K608011] Medium promoter with medium RBS and GFP

[http://partsregistry.org/Part:BBa_K608012 BBa_K608012] Medium promoter with weak RBS and GFP

[http://partsregistry.org/Part:BBa_K608013 BBa_K608013] strong Promoter with strong RBS and RFP

[http://partsregistry.org/Part:BBa_K608014 BBa_K608014 ] PR with RFP

[http://partsregistry.org/Part:BBa_K608015 BBa_K608015] PR with RFP

[http://partsregistry.org/Part:BBa_K608016 BBa_K608016] PR with RFP

[http://partsregistry.org/Part:BBa_K608017 BBa_K608017] PR with RFP

[http://partsregistry.org/Part:BBa_K608018 BBa_K608018] PR with RFP




                       

Retrieved from "http://2011.igem.org/Team:Freiburg/SampleData"