Difference between revisions of "Team:Technion Israel/Project/Results"
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<h2>Introduction</h2> | <h2>Introduction</h2> | ||
| + | <div id="expression" name="expression_results"> | ||
<h2>Expression</h2> | <h2>Expression</h2> | ||
<p>After successful overexpression of the 3ɑ-HSD enzyme under pT7 promoter (<a href="http://parts.igem.org/Part:BBa_K1674002" target="_blank">BBa_K1674002</a>), we conducted a series of experiments based on the 3ɑ-HSD activity measurement protocol, where we measured NADPH fluorescence over time added to E.coli lysates. Every experiment helped us understand a different aspect of the dihydrotestosterone (DHT) reduction reaction using our clones, and characterize the plasmid.</p> | <p>After successful overexpression of the 3ɑ-HSD enzyme under pT7 promoter (<a href="http://parts.igem.org/Part:BBa_K1674002" target="_blank">BBa_K1674002</a>), we conducted a series of experiments based on the 3ɑ-HSD activity measurement protocol, where we measured NADPH fluorescence over time added to E.coli lysates. Every experiment helped us understand a different aspect of the dihydrotestosterone (DHT) reduction reaction using our clones, and characterize the plasmid.</p> | ||
<h3>Enzymatic activity as a function of DHT concentration</h3> | <h3>Enzymatic activity as a function of DHT concentration</h3> | ||
| + | <p><i>The protocol for this assay can be seen <a href="https://static.igem.org/mediawiki/2015/7/7e/Technion_Israel_2015_protocols_3aHSDactivity.pdf" target="_blank">here</a>.</i></p> | ||
<p>To get a basic idea of the kinetics of 3ɑ-HSD enzymatic reaction, we first wanted to examine the effect of increase in initial substrate concentration (DHT). Therefore, we sonicated <i>E. coli</i> BL21 cells containing <a href="http://parts.igem.org/Part:BBa_K1674002" target="_blank">BBa_K1674002</a> after two hours of induction with IPTG, added 150 µM NADPH to the lysates in a 96-well plate, and inserted into a plate reader pre-heated to 37℃ for 30 minutes to allow for result stabilization. 37℃ mimics human body temperature, which is the environment in which the enzyme will be working in our final product, and is in the optimal temperature range for the enzyme activity.(SOURCE)</p> | <p>To get a basic idea of the kinetics of 3ɑ-HSD enzymatic reaction, we first wanted to examine the effect of increase in initial substrate concentration (DHT). Therefore, we sonicated <i>E. coli</i> BL21 cells containing <a href="http://parts.igem.org/Part:BBa_K1674002" target="_blank">BBa_K1674002</a> after two hours of induction with IPTG, added 150 µM NADPH to the lysates in a 96-well plate, and inserted into a plate reader pre-heated to 37℃ for 30 minutes to allow for result stabilization. 37℃ mimics human body temperature, which is the environment in which the enzyme will be working in our final product, and is in the optimal temperature range for the enzyme activity.(SOURCE)</p> | ||
<p>In previous experiments we noticed fluctuations in fluorescence during the first 15-30 minutes after the addition of NADPH, even in negative controls, which we assume occurs due to a reaction of NADPH with the phosphate buffer, resulting in a new equilibrium state between NADPH and NADP<sup>+</sup>.</p> | <p>In previous experiments we noticed fluctuations in fluorescence during the first 15-30 minutes after the addition of NADPH, even in negative controls, which we assume occurs due to a reaction of NADPH with the phosphate buffer, resulting in a new equilibrium state between NADPH and NADP<sup>+</sup>.</p> | ||
| Line 97: | Line 98: | ||
<p>After 30 minutes we added DHT to each well in various concentrations and measured NADPH fluorescence during 5.5 hours. In a logarithmic time scale we can see linear behavior of NADPH degradation, where the slope represents the degradation rate (Figure 1). When comparing the different <i>E. coli</i> BL21 strains, with and without the 3ɑ-HSD gene, we can see clearly that the graph slope is steeper in presence of 3ɑ-HSD, implying faster NADPH degradation rate due to the specific enzymatic activity. </p> | <p>After 30 minutes we added DHT to each well in various concentrations and measured NADPH fluorescence during 5.5 hours. In a logarithmic time scale we can see linear behavior of NADPH degradation, where the slope represents the degradation rate (Figure 1). When comparing the different <i>E. coli</i> BL21 strains, with and without the 3ɑ-HSD gene, we can see clearly that the graph slope is steeper in presence of 3ɑ-HSD, implying faster NADPH degradation rate due to the specific enzymatic activity. </p> | ||
| − | <figure><img class="img_center" src=""/><figcaption>Figure 1 - NADPH degradation rate over time in logarithmic scale with initial concentration of 40 µM DHT | + | <figure><img class="img_center" src="https://static.igem.org/mediawiki/2015/6/61/Technion_Israel_2015_activity_results_figure1.png"/><figcaption>Figure 1 - NADPH degradation rate over time in logarithmic scale with initial concentration of 40 µM DHT</figcaption></figure> |
| + | <p>If we take the slope from each DHT concentration graph, we can describe the reaction rate as a function of the substrate concentration (Figure 2). It seems that the reaction rate in the absence of 3ɑ-HSD enzyme stays relatively constant with increasing DHT concentrations, whereas it rises logarithmically in the presence of the enzyme. From the results presented in Figure 2, we can assume that the enzymatic reaction rate reaches saturation at a DHT concentration between 40-60 µM. | ||
| + | This result is compatible with the Michaelis-Menten enzyme kinetics model, which we described in detail in our modeling section.</p> | ||
| − | <h2>Secretion</h2> | + | <figure><img class="img_center" src="https://static.igem.org/mediawiki/2015/9/97/Technion_Israel_2015_activity_results_figure2.png"/><figcaption> Figure 2 - Reaction rate vs. DHT concentration</figcaption></figure> |
| − | <h2>Cofactor</h2> | + | |
| − | <h2>Integrative experiments | + | <h3>Enzymatic activity as a function of NADPH concentration</h3> |
| + | <p><i>The protocol for this assay can be seen <a href="https://static.igem.org/mediawiki/2015/7/7e/Technion_Israel_2015_protocols_3aHSDactivity.pdf" target="_blank">here</a>.</i></p> | ||
| + | <p>In the next experiment, we wanted to check the reaction rate dependence on the cofactor. Therefore, we performed the same procedure, this time with a constant DHT concentration of 50 µM, and varying NADPH concentrations. We observed a decrease in the reaction rate with an increase in NADPH concentration in presence of 3ɑ-HSD enzyme (Figure 3), contrary to substrate dependency. We were surprised by the difference between the effects of concentrations of substrate and cofactor, so we went back to our model to get some answers. </p> | ||
| + | <p>We hypothesize that during the 30 minutes of stabilization, some of the NADPH molecules were converted to NADP+ by other enzymes from the lysate which then could bind to 3ɑ-HSD and inhibits its activity. It is possible that in high concentration of NADPH, after 30 minutes, a large fraction of the 3ɑ-HSD molecules are bound to NADP<sup>+</sup> and hence the reaction in the direction of DHT reduction is inhibited. </p> | ||
| + | <figure><img class="img_center" src="https://static.igem.org/mediawiki/2015/f/fc/Technion_Israel_2015_activity_results_figure3.png"/><figcaption> Figure 3 - Reaction rate vs. NADPH concentration</figcaption></figure> | ||
| + | |||
| + | </div> | ||
| + | <div id="secretion" name="secretion_results"><h2>Secretion</h2> | ||
| + | </div> | ||
| + | <div id="cofactor" name="cofactor_results"><h2>Cofactor</h2> | ||
| + | <div id="integrative" name="integrative_results"><h2>Integrative experiments</h2> | ||
| + | |||
| + | <div id="shelf-life" name="shelf-life_results"><h2>Shelf-life</h2> | ||
Revision as of 05:38, 18 September 2015
Results
Introduction
Expression
After successful overexpression of the 3ɑ-HSD enzyme under pT7 promoter (BBa_K1674002), we conducted a series of experiments based on the 3ɑ-HSD activity measurement protocol, where we measured NADPH fluorescence over time added to E.coli lysates. Every experiment helped us understand a different aspect of the dihydrotestosterone (DHT) reduction reaction using our clones, and characterize the plasmid.
Enzymatic activity as a function of DHT concentration
The protocol for this assay can be seen here.
To get a basic idea of the kinetics of 3ɑ-HSD enzymatic reaction, we first wanted to examine the effect of increase in initial substrate concentration (DHT). Therefore, we sonicated E. coli BL21 cells containing BBa_K1674002 after two hours of induction with IPTG, added 150 µM NADPH to the lysates in a 96-well plate, and inserted into a plate reader pre-heated to 37℃ for 30 minutes to allow for result stabilization. 37℃ mimics human body temperature, which is the environment in which the enzyme will be working in our final product, and is in the optimal temperature range for the enzyme activity.(SOURCE)
In previous experiments we noticed fluctuations in fluorescence during the first 15-30 minutes after the addition of NADPH, even in negative controls, which we assume occurs due to a reaction of NADPH with the phosphate buffer, resulting in a new equilibrium state between NADPH and NADP+.
After 30 minutes we added DHT to each well in various concentrations and measured NADPH fluorescence during 5.5 hours. In a logarithmic time scale we can see linear behavior of NADPH degradation, where the slope represents the degradation rate (Figure 1). When comparing the different E. coli BL21 strains, with and without the 3ɑ-HSD gene, we can see clearly that the graph slope is steeper in presence of 3ɑ-HSD, implying faster NADPH degradation rate due to the specific enzymatic activity.
If we take the slope from each DHT concentration graph, we can describe the reaction rate as a function of the substrate concentration (Figure 2). It seems that the reaction rate in the absence of 3ɑ-HSD enzyme stays relatively constant with increasing DHT concentrations, whereas it rises logarithmically in the presence of the enzyme. From the results presented in Figure 2, we can assume that the enzymatic reaction rate reaches saturation at a DHT concentration between 40-60 µM. This result is compatible with the Michaelis-Menten enzyme kinetics model, which we described in detail in our modeling section.
Enzymatic activity as a function of NADPH concentration
The protocol for this assay can be seen here.
In the next experiment, we wanted to check the reaction rate dependence on the cofactor. Therefore, we performed the same procedure, this time with a constant DHT concentration of 50 µM, and varying NADPH concentrations. We observed a decrease in the reaction rate with an increase in NADPH concentration in presence of 3ɑ-HSD enzyme (Figure 3), contrary to substrate dependency. We were surprised by the difference between the effects of concentrations of substrate and cofactor, so we went back to our model to get some answers.
We hypothesize that during the 30 minutes of stabilization, some of the NADPH molecules were converted to NADP+ by other enzymes from the lysate which then could bind to 3ɑ-HSD and inhibits its activity. It is possible that in high concentration of NADPH, after 30 minutes, a large fraction of the 3ɑ-HSD molecules are bound to NADP+ and hence the reaction in the direction of DHT reduction is inhibited.
