Team:UC Chile/In Vitro Channeling

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                <h1>Whaterversisome</h1>
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    <h1>In Vitro Channeling</h1>
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In vitro channeling are going to generate different compounds using a chain of recombinant Carboxysomes, in our case Whateversisomes. Employing one enzyme corresponding to one Carboxysome, we are looking forward to create synthetic organelles that maximize the encounter probability between the substrate and the enzyme. As a proof of concept experiment, we want to use the enzymes B-D-galactosidase and D-galactose dehydrogenase, which have the characteristic that generate products easily measured by spectrophotometer and the reactant of one is the product of the other.
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We are aiming to improve the capacity of producing different compounds through the target of any desirable enzyme inside the Carboxysome. Thus, we can have a desirable product.<br><br>
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In vitro channeling is the way of getting these products as a combinatorial in vitro system, in  which each Carboxysome has an enzyme associated to create an optimized net pathways for specific production of compounds. As above, we could actually model an entire bacteria metabolism (<a href="https://2013.igem.org/Team:UC_Chile/Mathematical_Model">link</a>) .
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    <img src="https://static.igem.org/mediawiki/2013/e/e6/Team_UC_Chile_In_Vitro_1.jpg">
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    <div class="legend"><i>In-Vitro</i> step by step</div>
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<h3>Proof of concept: Experimental design</h3>
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To prove both the in vitro reactions which means the in vitro channeling, we want to target into Carboxysome two different enzymes: the B-D-galactosidase and the B-D-galactose dehydrogenase. These enzymes catalyze the following reactions, respectively <a href="#Referencias">(1,2)</a>:<br><br>
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<div class="ecuation">lactose + H<span class="subind">2</span>O <==> β-D-galactose + β-D-glucose</div><br>
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<div class="ecuation">β-D-galactose + NAD<span class="supind">+</span> <==> D-galactono-1,4-lactone + NADH + H<span class="supind">+</span></div><br>
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In order to achieve this, we needed to create a construct with the large subunit of RuBisCO, our targeting signal <a href="https://2013.igem.org/Team:UC_Chile/Targeting">(link)</a>, which is joined with the B-D-galactosidase and another with the B-D-galactose dehydrogenase instead. The process of these reactions could be measured by spectrophotometer.
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<h3>b-D-Galactosidase</h3>
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<div class="ecuation">lactose + H<span class="subind">2</span>O <==> β-D-galactose + β-D-glucose</div><br>
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b-D-Galactosidase is a monomer enzyme that catalyzes the reaction described above <a href="#Referencias">(1)</a>. Instead of lactose, X-Gal could be used in order to get a better way of measuring the reaction as it is occurring.<br><br>
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X-Gal, a synthetic compound, could be hydrolyzed by b-D-galactosidase and subsequently transformed into galactose and 5-bromo-4-chloro-3-hydroxyindole. The last compound forms an insoluble blue compound, 5,5’-dibromo-4,4’-dichloro indigo, via a dimerization and oxidation sequence <a href="#Referencias">(3)</a>. By the appearance of this blue color in the petri dish, it is possible to determine if the enzymatic reaction is working. Moreover, this compound has its absorbance peak at 650 nm <a href="#Referencias">(4)</a>, therefore by using a calibration curve, the absorbance could be used to calculate the concentration of the newly generated product. This also allows us to calculate the kinetic of the reaction. More information about this procedure is available at the protocols .<br>
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<h3>B-D-galactose dehydrogenase</h3>
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<div class="ecuation">β-D-galactose + NAD<span class="supind">+</span> <==> D-galactono-1,4-lactone + NADH + H<span class="supind">+</span></div><br>
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B-D-galactose dehydrogenase is a monomer enzyme that catalyzes the reaction described above <a href="#Referencias">(2)</a>. This reaction could be followed by spectrophotometer at 340 nm in which NADH has its absorbance peak <a href="#Referencias">(5)</a>. The rest is done equally as before.<br>
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<h3>In vitro channeling</h3>
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The last step would be proving if both Carboxysomes are able to interact with each other. For this test both Whateversisomes has to be in the same petri dish. We are looking to use lactose as the first substrate and measured the NADH appearance as the final product.<br><br>
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The kinetic of the reaction can be measured as described above with a spectrophotometer. This approach will let us evaluate if the communication was achieved between both structures.<br>
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    <b>References:</b>
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    <ul>
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        <li><span>1. Metacyc, Enciclopedia of Metabolic Pathways [internet principal page]. United States. Metacyc Enzyme: b-galactosidase. [consult day: august 15, 2013]. Available in:</span><br><a href="http://metacyc.org/META/new-image?type=ENZYME&object=BETAGALACTOSID-CPLX">http://metacyc.org/META/new-image?type=ENZYME&object=BETAGALACTOSID-CPLX</a></li>
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        <li><span>2. Metacyc, Enciclopedia of Metabolic Pathways [internet principal page]. United States. Metacyc Enzyme: Galactose dehydrogenase. [consult day: august 15, 2013]. Available in:</span><br><a href="http://metacyc.org/META/new-image?type=ENZYME&object=MONOMER-12877">http://metacyc.org/META/new-image?type=ENZYME&object=MONOMER-12877</a></li>
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        <li><span>3. Carbosynth [internet principal page]. United Kingdoms. 5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside (X-Gal). [consult day: august 15, 2013]. Available in:</span><br><a href="http://www.carbosynth.com/carbosynth/website.nsf/(w-productdisplay)/E82ABA08F88C953A80256A6B00359988">http://www.carbosynth.com/carbosynth/website.nsf/(w-productdisplay)/E82ABA08F88C953A80256A6B00359988</a></li>
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        <li><span>4. Cox, S. Handbook of Pharmaceutical Biotechnology. John Wiley & Sons, Inc. United States, 1° Edition, 2007.</span></li>
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        <li><span>5. BMG labtech [internet principal page]. Germany. Detection of NADH and NADPH with the Omega’s High Speed, Full UV/Vis Absorbance Spectrometer. [consult day: august 15, 2013]. Available in:</span><br><a href="http://www.bmglabtech.com/application-notes/absorbance/spectrometer-nadh-nadph-170.cfm">http://www.bmglabtech.com/application-notes/absorbance/spectrometer-nadh-nadph-170.cfm</a></li>
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Latest revision as of 03:39, 28 September 2013

Wiki-IGEM

In Vitro Channeling

In vitro channeling are going to generate different compounds using a chain of recombinant Carboxysomes, in our case Whateversisomes. Employing one enzyme corresponding to one Carboxysome, we are looking forward to create synthetic organelles that maximize the encounter probability between the substrate and the enzyme. As a proof of concept experiment, we want to use the enzymes B-D-galactosidase and D-galactose dehydrogenase, which have the characteristic that generate products easily measured by spectrophotometer and the reactant of one is the product of the other.
We are aiming to improve the capacity of producing different compounds through the target of any desirable enzyme inside the Carboxysome. Thus, we can have a desirable product.

In vitro channeling is the way of getting these products as a combinatorial in vitro system, in which each Carboxysome has an enzyme associated to create an optimized net pathways for specific production of compounds. As above, we could actually model an entire bacteria metabolism (link) .
In-Vitro step by step

Proof of concept: Experimental design

To prove both the in vitro reactions which means the in vitro channeling, we want to target into Carboxysome two different enzymes: the B-D-galactosidase and the B-D-galactose dehydrogenase. These enzymes catalyze the following reactions, respectively (1,2):

lactose + H2O <==> β-D-galactose + β-D-glucose

β-D-galactose + NAD+ <==> D-galactono-1,4-lactone + NADH + H+

In order to achieve this, we needed to create a construct with the large subunit of RuBisCO, our targeting signal (link), which is joined with the B-D-galactosidase and another with the B-D-galactose dehydrogenase instead. The process of these reactions could be measured by spectrophotometer.

b-D-Galactosidase

lactose + H2O <==> β-D-galactose + β-D-glucose

b-D-Galactosidase is a monomer enzyme that catalyzes the reaction described above (1). Instead of lactose, X-Gal could be used in order to get a better way of measuring the reaction as it is occurring.

X-Gal, a synthetic compound, could be hydrolyzed by b-D-galactosidase and subsequently transformed into galactose and 5-bromo-4-chloro-3-hydroxyindole. The last compound forms an insoluble blue compound, 5,5’-dibromo-4,4’-dichloro indigo, via a dimerization and oxidation sequence (3). By the appearance of this blue color in the petri dish, it is possible to determine if the enzymatic reaction is working. Moreover, this compound has its absorbance peak at 650 nm (4), therefore by using a calibration curve, the absorbance could be used to calculate the concentration of the newly generated product. This also allows us to calculate the kinetic of the reaction. More information about this procedure is available at the protocols .

B-D-galactose dehydrogenase

β-D-galactose + NAD+ <==> D-galactono-1,4-lactone + NADH + H+

B-D-galactose dehydrogenase is a monomer enzyme that catalyzes the reaction described above (2). This reaction could be followed by spectrophotometer at 340 nm in which NADH has its absorbance peak (5). The rest is done equally as before.

In vitro channeling

The last step would be proving if both Carboxysomes are able to interact with each other. For this test both Whateversisomes has to be in the same petri dish. We are looking to use lactose as the first substrate and measured the NADH appearance as the final product.

The kinetic of the reaction can be measured as described above with a spectrophotometer. This approach will let us evaluate if the communication was achieved between both structures.
References:

Retrieved from "http://2013.igem.org/Team:UC_Chile/In_Vitro_Channeling"