Difference between revisions of "Team:Bielefeld-CeBiTec/Project/toolbox/labeling"

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Protein localization <i>in vivo</i> can be performed by labeling the target protein with a
 
Protein localization <i>in vivo</i> can be performed by labeling the target protein with a
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fluorescent protein like <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photolysis#green fluorescent protein GFP">green fluorescent protein (GFP)</a> or <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photolysis#GFP"> red fluorescent protein (RFP) </a>.
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fluorescent protein like <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photolysis#green fluorescent protein GFP">green fluorescent protein (GFP)</a> or <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photolysis#GFP"> red fluorescent protein (RFP)</a>.
 
The labeling is done by a translational fusion of the CDS from the fluorescent protein C-
 
The labeling is done by a translational fusion of the CDS from the fluorescent protein C-
 
or N&#x2011;terminal with a <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photolysis#Fusionprotein">short linker</a> to the CDS from the target protein. But the labeling is
 
or N&#x2011;terminal with a <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photolysis#Fusionprotein">short linker</a> to the CDS from the target protein. But the labeling is
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production of 2&#x2011;phosphoglycolate when O<sub>2</sub> is present, RuBisCo is a very inefficient catalyst.  
 
production of 2&#x2011;phosphoglycolate when O<sub>2</sub> is present, RuBisCo is a very inefficient catalyst.  
 
CO<sub>2</sub> and O<sub>2</sub> are competitive substrates in the two reactions and only the production of  
 
CO<sub>2</sub> and O<sub>2</sub> are competitive substrates in the two reactions and only the production of  
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3&#x2011;phosphoglycerate leads to CO<sub>2</sub> fixation. [Andersson 2008, Jordan 1981].
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3&#x2011;phosphoglycerate leads to CO<sub>2</sub> fixation. [Andersson 2008, Jordan 1981]. To create a compartment with a higher CO<sub>2</sup> enviroment the iGEM team Bielefeld CeBiTec created an artifical compartment the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Project/CO2-fixation/Carboxysome"> carboxysome</a>. We want to see where the RuBisCo is located in the cell, inside the carboxysome or in the whole cytoplasm.
 
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Revision as of 12:13, 28 August 2017

Labeling

short summary

As part of the toolbox, the labeling of a protein in vivo is a useful tool that allows the investigation of a protein in its native environment. As a label for our target protein we use the fluorescent amino acid L‑(7‑hydroxycoumarin‑4‑yl) ethylglycine (CouAA) that is incorporated by an orthogonal t-RNA/aminoacyl‑synthethase pair at a defined position.
To demonstrate this tool we want to find out if the ribulose 1,5‑bisphosphat carboxylase oxygenase (RuBisCo) is located in the carboxysome, an artificial compartment surrounded by proteins and used by the iGEM Team CeBiTec 2014 to increase the activity of the RuBisCo. The carboxysome has already been labeled with green fluorescent protein (GFP) and we want to co-localizate the RuBisCo labeled with an genetically encoded fluorescent amino acid L‑(7‑hydroxycoumarin‑4‑yl) ethylglycine and in comparison labeled with red fluorescent protein (RFP).

Labeling of a protein in vivo

Protein localization in vivo can be performed by labeling the target protein with a fluorescent protein like green fluorescent protein (GFP) or red fluorescent protein (RFP). The labeling is done by a translational fusion of the CDS from the fluorescent protein C- or N‑terminal with a short linker to the CDS from the target protein. But the labeling is limited to the C- or N‑terminus and due to its size GFP (29 kDa [Charbon 2011]) could be bigger than the target protein and be a hindrance. Both could cause a significant change of the structure of the target protein or a loss of function,especially if the protein is part of an assembly in a larger complex or oligomer [Charbon 2011, Wang 2006].
The usage of a genetically encoded fluorescent amino acid would circumvent these problems and deliver a tool to study protein localization and function in vivo and in vitro. An orthogonal t‑RNA/aminoacyl‑tRNA synthetase pair allows the incorporation of amino acids in response to the amber stop codon (TAG) selectively at a defined position in the protein [Charbon 2011].

L‑(7‑hydroxycoumarin‑4‑yl) ethylglycine (CouAA)

The fluorescent amino acid L‑(7‑hydroxycoumarin‑4‑yl) (CouAA) ethylglycine is relatively small, has a high fluorescence quantum yield and relatively large Stoke's shift. It is also solvent polar and pH-sensitive so it can indicate pH-changes in the cell [Wang 2006]. The translational incorporation of CouAA with an aaRS was shown by Schultz 2006 and Charbon 2011,2012 into different proteins.
  • Name: L‑(7‑hydroxycoumarin‑4‑yl) ethylglycine
  • Short: CouAA
  • CAS: 905442‑42‑4
  • MW: 263.25
  • Storage: -20 °C
  • Source: Bachem
  • Prize: 1g - £590.00
  • Function: Fluorescent amino acid

Figure 1: Structure of CouAA
Structure of the fluorescent amino acid L‑(7‑hydroxycoumarin‑4‑yl) ethylglycine (CouAA).


The amino acid is suitable for in vivo and in vitro localization, even for localization in SDS‑PAGES. The extinction and emission spectrum of CouAA is shown in Figure 2:

Figure 2: Fluorescence spectrum of CouAA
Adsorption and fluorescence spectrum of L‑(7‑hydroxycoumarin‑4‑yl) ethylglycine. [Wang 2006].

The relative fluorescence signal of CouAA decreases over time when irradiated with the excitation wavelength. This effect is called photobleaching and occurs in more or less in all fluorophores. This photobleaching effect is shown in Figure 3 for the labeling of the bacterial tubulin FtsZ with CouAA [Charbon 2011].

Figure 3: Photobleaching of CouAA
The in vivo dynamic properties of FtsZ10CouAA. The graph represents the data corrected for photobleaching due to image acquisition for unbleached (green) and bleached (blue) regions; the red line represents the theoretical recovery curve fit. FtsZ10CouAA (The labeled protein) half-time recovery is 12(+-5) s (mean ±standard deviation); 11.6 s in the example shown. [Charbon 2011].

Colocalisation of the ribulose 1,5‑bisphosphate carboxylase oxygenase and the carboxysome

Ribulose 1,5‑bisphosphate carboxylase oxygenase (RuBisCo)

The protein we want to label with CouAA is the ribulose 1,5‑bisphosphate carboxylase oxygenase (RuBisCo). RuBisCo catalyzes the incorporation of inorganic CO2 to ribulose 1,5‑bisphosphate to form two 3‑phosphoglycerate molecules. The catalyzed reaction is shown in Figure 4. [Andersson 2008].

Figure 4: RuBisCo reaction
Reaction catalyzed by Ribulose 1,5-bisphosphat Carboxylase Oxygenase (RuBisCo). Ribulose 1,5‑bisphosphate is converted in two molecules 3‑phophoglycerate.

Due to its numerous side reactions, for example the oxygenase activity resulting in the production of 2‑phosphoglycolate when O2 is present, RuBisCo is a very inefficient catalyst. CO2 and O2 are competitive substrates in the two reactions and only the production of 3‑phosphoglycerate leads to CO2 fixation. [Andersson 2008, Jordan 1981]. To create a compartment with a higher CO2 enviroment the iGEM team Bielefeld CeBiTec created an artifical compartment the carboxysome. We want to see where the RuBisCo is located in the cell, inside the carboxysome or in the whole cytoplasm.