Team:ETH Zurich/Design

From 2012.igem.org

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=== Expression of the proteins PabA & PabB/C ===
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== Design ==
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TOP10 colonies were transformed with <partinfo>BBa_K137055</partinfo> , <partinfo>BBa_S04039</partinfo> and <partinfo>BBa_K909014</partinfo>. The transformed cells as well as the TOP10 WT were cultivated ON in falcon tubes containing 4 mL of LB. After normalization to an OD of 0.15, the cells were lysed and mixed with 6X Laemmli buffer.
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=== UVR8 ===
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Three different fusion strategies were carried out to verify the best UVR8 dTetR fusion.
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PabA has a size of 21,656 kDa (193 aa), PabB/C of 71,743 kDA (628 aa).
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SDS PAGE
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== Design of the single parts==
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=== YcgZ promoter with multiple BluR (YcgE) operator sites ===
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One of our approaches was to make use of the Blue light sensitive protein BluF (YcgF) which is activation upon blue light radiation, then dimerizes and inactivates BluR (YcgE), a transcriptional repressor which binds to the YcgZ promoter. We cloned the ''lacZ'' downstream to the YcgZ promoter and tested the beta-galactosidase activity making use of the Miller Assay. As we could not see any difference between the different K.O. strains we suggested that the endogenous amounts of activator and repressor was not enough to lead to a clear difference.
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Leakiness. Tschowri DNA footprint assay.. Identified operator sites. picture.... link to partsregistry. bliblanrfhiuqifufihuwe
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=== UVR8 - dTetR fusion: a UV sensing protein ===
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Three different fusion strategies were carried out to verify the best UVR8 dTetR fusion [[http://partsregistry.org/Part:BBa_K909008 link to the partsregistry]]. Tet DBD stands for TetR DNA binding domain.  
1. Full length UVR8 without a linker
1. Full length UVR8 without a linker
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[[File:Uvr83rdeth
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.jpg|frameless|400px|center]]
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[[File:Ethuvr81.jpg|frameless|400px|center]]
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2. Full length UVR8 + [GGS]x2 linker (+6 aa)
2. Full length UVR8 + [GGS]x2 linker (+6 aa)
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[[File:Uvr82ndeth.jpg|frameless|400px|center]]
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[[File:Uvr83rdeth.jpg|frameless|400px|center]]
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3. Truncated UVR8 (-13 aa which are not seen in the 3d structure)
3. Truncated UVR8 (-13 aa which are not seen in the 3d structure)
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[[File:Ethuvr81.jpg|frameless|400px|center]]
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[[File:Uvr82ndeth.jpg|frameless|400px|center]]
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[[File:Uvr8uv.jpg|frameless|400px|center]]
[[File:Uvr8uv.jpg|frameless|400px|center]]
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You can find the split [http://partsregistry.org/Part:BBa_K909007 TetR DNA binding domain containing a BamHI restriction site] which allows it to be fused to any protein of interest containing the corresponding restriction site. This allows you to test if a protein of interest is able to dimerize.
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=== p-ABA generator ===
=== p-ABA generator ===
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We constructed one operon consisting of the pabA and pabB/C gene from Lactococcus Lactis to overexpress p-ABA.
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We constructed one [http://partsregistry.org/Part:BBa_K909015 pab operon] consisting of the pabA and pabB/C gene from ''Lactococcus lactis'' to overexpress p-ABA.
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Therefore we used the biobrick parts K137055 and S04039 as a template.
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Therefore we used the biobrick parts [http://partsregistry.org/Part:BBa_K137055 BBa_K137055] and [http://partsregistry.org/Part:BBa_S04039 BBa_S04039] as a template to construct the wished composites.
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In a second step the pab operon was cloned downstream to the Tet promoter so that derepression of UVR8 leads to p-ABA expression as a response to UV radiation.
+
 
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[http://partsregistry.org/Part:BBa_K909016 Biobrick K909016] will be used to show that p-ABA is produced, while the operon can be cloned downstream to any promoter.
 +
 
 +
 
 +
UVR8 - system:
 +
In a second step the pab operon will be cloned downstream of the pTet promoter. Derepression of UVR8 by UV irradiation leads then to p-ABA expression.
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 +
Decoder:
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In a second step the pab operon will be cloned downstream of the [http://partsregistry.org/Part:BBa_K909011  PL promoter with operator sites TetO<sub>1</sub> and LacO<sub>1</sub>]. In case blue and red light is present, p-ABA will be produced as a blue and red light response.
=== Dual input promoters ===
=== Dual input promoters ===
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The decoder  
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In our case we want the [[Team:ETH_Zurich/Modeling/LovTAP-Cph1| decoder]]  to have AND logics. Therefore we designed 3 different dual input promoters, all of them derived from the λ-phage promoters P<sub>R</sub> and P<sub>L</sub>:
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1. [http://partsregistry.org/Part:BBa_K909012  Modified P<sub>R</sub> promoter with operator sites O<sub>R1</sub>,O<sub>R2</sub> and LacO<sub>1</sub>]
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2. [http://partsregistry.org/Part:BBa_K909013 Modified P<sub>R</sub> promoter with operator sites O<sub>R1</sub>,O<sub>R2</sub> and TetO<sub>1</sub>]
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3. [http://partsregistry.org/Part:BBa_K909011 Modified P<sub>L</sub> promoter with operator sites TetO<sub>1</sub> and LacO<sub>1</sub>]
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Truth table
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[[File:Promoterr.jpg|500px|center]]
[[File:Promoterr.jpg|500px|center]]

Latest revision as of 00:19, 27 September 2012

Eth ecolipseeth logo.png
Eth igem logo.png

Contents

Expression of the proteins PabA & PabB/C

TOP10 colonies were transformed with <partinfo>BBa_K137055</partinfo> , <partinfo>BBa_S04039</partinfo> and <partinfo>BBa_K909014</partinfo>. The transformed cells as well as the TOP10 WT were cultivated ON in falcon tubes containing 4 mL of LB. After normalization to an OD of 0.15, the cells were lysed and mixed with 6X Laemmli buffer.

PabA has a size of 21,656 kDa (193 aa), PabB/C of 71,743 kDA (628 aa).


SDS PAGE


Design of the single parts

YcgZ promoter with multiple BluR (YcgE) operator sites

One of our approaches was to make use of the Blue light sensitive protein BluF (YcgF) which is activation upon blue light radiation, then dimerizes and inactivates BluR (YcgE), a transcriptional repressor which binds to the YcgZ promoter. We cloned the lacZ downstream to the YcgZ promoter and tested the beta-galactosidase activity making use of the Miller Assay. As we could not see any difference between the different K.O. strains we suggested that the endogenous amounts of activator and repressor was not enough to lead to a clear difference.

Leakiness. Tschowri DNA footprint assay.. Identified operator sites. picture.... link to partsregistry. bliblanrfhiuqifufihuwe


UVR8 - dTetR fusion: a UV sensing protein

Three different fusion strategies were carried out to verify the best UVR8 dTetR fusion http://partsregistry.org/Part:BBa_K909008 link to the partsregistry. Tet DBD stands for TetR DNA binding domain.

1. Full length UVR8 without a linker

Ethuvr81.jpg


2. Full length UVR8 + [GGS]x2 linker (+6 aa)

Uvr83rdeth.jpg


3. Truncated UVR8 (-13 aa which are not seen in the 3d structure)

Uvr82ndeth.jpg
Uvr8uv.jpg

You can find the split [http://partsregistry.org/Part:BBa_K909007 TetR DNA binding domain containing a BamHI restriction site] which allows it to be fused to any protein of interest containing the corresponding restriction site. This allows you to test if a protein of interest is able to dimerize.



p-ABA generator

We constructed one [http://partsregistry.org/Part:BBa_K909015 pab operon] consisting of the pabA and pabB/C gene from Lactococcus lactis to overexpress p-ABA. Therefore we used the biobrick parts [http://partsregistry.org/Part:BBa_K137055 BBa_K137055] and [http://partsregistry.org/Part:BBa_S04039 BBa_S04039] as a template to construct the wished composites.

[http://partsregistry.org/Part:BBa_K909016 Biobrick K909016] will be used to show that p-ABA is produced, while the operon can be cloned downstream to any promoter.


UVR8 - system: In a second step the pab operon will be cloned downstream of the pTet promoter. Derepression of UVR8 by UV irradiation leads then to p-ABA expression.

Decoder:

In a second step the pab operon will be cloned downstream of the [http://partsregistry.org/Part:BBa_K909011 PL promoter with operator sites TetO1 and LacO1]. In case blue and red light is present, p-ABA will be produced as a blue and red light response.


Dual input promoters

In our case we want the decoder to have AND logics. Therefore we designed 3 different dual input promoters, all of them derived from the λ-phage promoters PR and PL:

1. [http://partsregistry.org/Part:BBa_K909012 Modified PR promoter with operator sites OR1,OR2 and LacO1]

2. [http://partsregistry.org/Part:BBa_K909013 Modified PR promoter with operator sites OR1,OR2 and TetO1]

3. [http://partsregistry.org/Part:BBa_K909011 Modified PL promoter with operator sites TetO1 and LacO1]


Truth table


Promoterr.jpg

UVR8

UVR8


References

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  • Christie, J. M., Salomon, M., Nozue, K., Wada, M., & Briggs, W. R. (1999): LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proceedings of the National Academy of Sciences of the United States of America, 96(15), 8779–83.
  • Christie, J. M., Arvai, A. S., Baxter, K. J., Heilmann, M., Pratt, A. J., O’Hara, A., Kelly, S. M., et al. (2012). Plant UVR8 photoreceptor senses UV-B by tryptophan-mediated disruption of cross-dimer salt bridges. Science (New York, N.Y.), 335(6075), 1492–6.
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