Difference between revisions of "Part:BBa K1189037"

Revision as of 03:26, 29 October 2013

His tag + Human ferritin di-subunit with E coil under LacI promoter

This part was created by fusing the heavy chain and light chains (BBa_K1189024 BBa_K1189025) of human ferritin together (sequences from: P02794 and P02792 [UniParc]). It is expressed under the lacI promoter (BBa_J04500) and has a his-tag for protein purification. An E-coil (BBa_K1189011) is included in order to allow binding of parts containing the respective K-coil (BBa_K1189010). Characterization of this part was done primarily with commercially purchased ferritin, which is structurally very similar to this recombinant ferritin (Figure 1).

This construct can be used as a reporter through a modification of the iron core to form Prussian Blue (Figure 2). The resulting molecule can then catalyze the formation of radicals from hydrogen peroxide, which can then cause a colour change in substrates such as TMB or ABTS (Figure 3) (Zhang et al., 2013).

This protein is a highly robust protein, remaining stable under extreme pH, temperature, and denaturing conditions. It is also highly accepting of fusion proteins, as it continues to form the nanoparticle despite fusions to both N-terminus and C-terminus. In addition, proteins fused to this protein have been found to be stabilized due to the fusion.

Ferritin — **Figure 1.** Ribbon visualization of a fully assembled ferritin protein.

Substrate Colours — **Figure 2.** Image of the colours of ABTS and TMB (10 mg/mL for both) after reacting with Prussian blue ferritin.

Prussian Blue Synthesis — **Figure 3.** Comparison image of commercial ferritin to Prussian blue ferritin after the synthesis reaction. The synthesis reaction took place over a 12 hour time period.

Applications of BBa_K1189037

The main purpose of this page is to display the characterization data of Prussian blue chemically modified commercial horse spleen ferritin as a catalyst. This ferritin is extremely similar to our constructed ferritin and we do not anticipate any differences in their properties. Next we show how our own constructed ferritin displays the same catalytic activity. At the end we show how the coil found in this part can bind to coils found on our other parts.

Kinetic Analysis of Prussian Blue Ferritin

We performed a kinetic analysis of our Prussian blue ferritin. We included a comparison of Prussian blue horse spleen ferritin to regular horse spleen ferritin for both TMB and ABTS (Figures 1 and 2). For both of the substrates we can see that normal ferritin has a very low catalytic activity compared to our modified ferritin. Using this data were able to determine the Michaelis-Menten catalytic constants for Prussian blue ferritin with different substrates.

Prussian Blue Ferritin and TMB — **Figure 1.** Measurements of the absorbance of the 650nm light by the substrate TMB over a period of 600 seconds. 6 µL of 10 mg/mL substrate was used in a 242 µL reaction volume.Commercial Prussian blue ferritin ( 10 µL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 µL of 0.047 mg/mL sample). Negative controls are TMB and hydrogen peroxide, and TMB only. Standard error of the mean bars are based on a sample size where n=8. Substrate and hydrogen peroxide sample data is not clearly visible as it is in line with the substrate only data.

Prussian Blue Ferritin and ABTS — **Figure 2.** Measurements of the absorbance of the 415nm light by the substrate ABTS over a period of 600 seconds. 8 µL of 10 mg/mL substrate was used in a 242 µL reaction volume. Commercial Prussian blue ferritin ( 10 µL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 µL of 0.047 mg/mL sample). Negative controls are ABTS and hydrogen peroxide, and ABTS only. Standard error of the mean bars are based on a sample size where n=8.

In order to complete our kinetic analysis we had to determine the catalytic properties of our Prussian blue ferritin according to the Michaelis-Menten kinetic model. For these tests we varied the colourimetric substrate concentrations (ABTS and TMB) (Figures 3 and 4). We also varied the hydrogen peroxide concentration in association with TMB as this the first chemical compound that will react in the system (Figure 5).

Michaelis-Menten Plot for Prussian Blue Ferritin with ABTS — **Figure 3.** Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of ABTS. Absorbance readings were taken at 415 nm. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data (eg. Figure 6).

Michaelis-Menten Plot for Prussian Blue Ferritin with TMB — **Figure 4.** Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of TMB. Absorbance readings were taken at 650 nm. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data (eg. Figure 5).

Michaelis-Menten Plot for Prussian Blue Ferritin Based on Hydrogen Peroxide (with TMB) — **Figure 5.** Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of hydrogen peroxide. Absorbance readings were taken at 650 nm which measure the breakdown of TMB. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data.

Table 1. Catalytic constants for our Prussian blue ferritin

Catalyst	Enzyme Concentration (M)	Substrate	K_m (mM)	V_max (Ms^-1)	K_cat (s^-1)	K_cat/K_m (M^-1s^-1)
Prussian Blue Ferritin	1.31 x 10^-9	ABTS	0.448	1.25 x 10^-8	9.51	2.12 x 10⁴
Prussian Blue Ferritin	1.31 x 10^-9	TMB	0.0432	1.12 x 10^-7	85.3	1.97 x 10⁶
Prussian Blue Ferritin	1.31 x 10^-9	H₂O₂ (TMB)	0.0176	1.31 x 10^-8	11.1	6.28 x 10⁵

pH Optimization of Prussian blue Ferritin

We also performed a pH optimization of our Prussian blue ferritin using the substrates TMB and ABTS (Figure 6 and 7). The results show TMB to be a much more robust substrate, showing high catalytic activity across a more broad range of pH compared to ABTS.

ABTS pH Optimization — **Figure 6.** pH optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

TMB pH Optimization — **Figure 7.** pH optimization of commercial Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

Temperature Optimization of Prussian Blue Ferritin

Another aspect of our analysis was determining the optimal temperature for catalytic activity of Prussian blue ferritin (Figure 8 and 9). This showed us that the Prussian blue reporter has a much higher catalytic activity at higher temperatures.

ABTS Temperature Optimization — **Figure 8.** Temperature optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

TMB Temperature Optimization — **Figure 9.** Temperature optimization of commerical Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

Prussian Blue Ferritin on Nitrocellulose

The next aspect of our analysis was to see how Prussian blue ferritin would act in a catalytic sense on nitrocellulose (Figures 10 and 11). From these results we can that TMB is a better substrate on for use on nitrocellulose(Figure 11). With this substrate we saw a result from only 5 ng of Prussian blue ferritin present on the nitrocellulose.

Prussian Blue Ferritin and ABTS on Nitrocellulose — **Figure 10.** Blots of Prussian blue ferritin on nitrocellulose (5 µL samples) that are reacted with ABTS (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.

Prussian Blue Ferritin and TMB on Nitrocellulose — **Figure 11.** Blots of Prussian blue ferritin on nitrocellulose (5 µL samples) that are reacted with TMB (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.

Making Prussian Blue out of This Part

We applied a scaled down Prussian blue synthesis experiment to our own ferritinOur own Prussian blue ferritin was then exposed to the TMB substrate (Figure 12). From the results we can see that the ferritin with the E-coil attached had excellent catalytic activity.

Creating Prussian Blue Ferritin out of our Own Ferritin — **Figure 12.** Measurements of the coloured substrate TMB (10 mg/mL) at 650 nm over a 600 second time period for our own Prussian blue ferritin and unmodified ferritin. Sample volume was 242 µL. Controls for this experiment include bovine serum albumin (1 mg/mL)and the substrate solution by itself. Due to limitations on the protein available only one replicate was performed. Zero time points do not have low absorbance as colour change was rapid and began before measurements started.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1307

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K1189037 short</partinfo>
-DUNNO
+	<p>This part was created by fusing the heavy chain and light chains (<partinfo>BBa_K1189024</partinfo> <partinfo>BBa_K1189025</partinfo>) of human ferritin together (sequences from: P02794 and P02792 [UniParc]). It is expressed under the lacI promoter (<partinfo>BBa_J04500</partinfo>) and has a his-tag for protein purification. An E-coil (<partinfo>BBa_K1189011</partinfo>) is included in order to allow binding of parts containing the respective K-coil (<partinfo>BBa_K1189010</partinfo>). Characterization of this part was done primarily with <html><a href="http://www.sigmaaldrich.com/catalog/product/sigma/f4503?lang=en&region=CA">commercially purchased ferritin</a></html>, which is structurally very similar to this recombinant ferritin (Figure 1).  </p>
+	<p>This construct can be used as a reporter through a modification of the iron core to form Prussian Blue (Figure 2). The resulting molecule can then catalyze the formation of radicals from hydrogen peroxide, which can then cause a colour change in substrates such as TMB or ABTS (Figure 3) (Zhang <i> et al.,</i> 2013).</p>
+<p> This protein is a highly robust protein, remaining stable under extreme pH, temperature, and denaturing conditions. It is also highly accepting of fusion proteins, as it continues to form the nanoparticle despite fusions to both N-terminus and C-terminus. In addition, proteins fused to this protein have been found to be stabilized due to the fusion.
+</p>
+<html>
+<br>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/1/18/UCalgary2013TRFerritinrender2png.png" alt="Ferritin" width="300" height="300">
+<figcaption>
+	<p><b>Figure 1.</b> Ribbon visualization of a fully assembled ferritin protein.</p>
+</figcaption>
+</figure>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/a/a9/UCalgary2013TRSubstratecolour.png" alt="Substrate Colours" width="250" height="300">
+<figcaption>
+	<p><b>Figure 2.</b> Image of the colours of ABTS and TMB (10 mg/mL for both) after reacting with Prussian blue ferritin.</p>
+</figcaption>
+</figure>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/c/c7/UCalgary2013TRPrussianblueferritinsynthesis.png" alt="Prussian Blue Synthesis" width="400" height="200">
+<figcaption>
+	<p><b>Figure 3.</b> Comparison image of commercial ferritin to Prussian blue ferritin after the synthesis reaction. The synthesis reaction took place over a 12 hour time period. </p>
+</figcaption>
+</figure>
+</html>
+===Applications of BBa_K1189037===
+<p>The main purpose of this page is to display the characterization data of Prussian blue chemically modified commercial horse spleen ferritin as a catalyst. This ferritin is extremely similar to our constructed ferritin and we do not anticipate any differences in their properties. Next we show how our own constructed ferritin displays the same catalytic activity. At the end we show how the coil found in this part can bind to coils found on our other parts. </p>
+<b>Kinetic Analysis of Prussian Blue Ferritin</b>
+<html>
+<p>We performed a kinetic analysis of our Prussian blue ferritin. We included a comparison of Prussian blue horse spleen ferritin to regular horse spleen ferritin for both TMB and ABTS (Figures 1 and 2). For both of the substrates we can see that normal ferritin has a very low catalytic activity compared to our modified ferritin. Using this data were able to determine the Michaelis-Menten catalytic constants for Prussian blue ferritin with different substrates.</p>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/3/36/UCalgary2013TRTmb6ulgraph.png" alt="Prussian Blue Ferritin and TMB" width="800" height="439">
+<figcaption>
+	<p><b>Figure 1.</b> Measurements of the absorbance of the 650nm light by the substrate TMB over a period of 600 seconds. 6 µL of 10 mg/mL substrate was used in a 242 µL reaction volume.Commercial Prussian blue ferritin ( 10 µL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 µL of 0.047 mg/mL sample). Negative controls are TMB and hydrogen peroxide, and TMB only. Standard error of the mean bars are based on a sample size where n=8. Substrate and hydrogen peroxide sample data is not clearly visible as it is in line with the substrate only data. </p>
+</figcaption>
+</figure>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/1/15/UCalgary2013TRABTS8ulgraph.png" alt="Prussian Blue Ferritin and ABTS" width="800" height="433">
+<figcaption>
+	<p><b>Figure 2.</b> Measurements of the absorbance of the 415nm light by the substrate ABTS over a period of 600 seconds. 8 µL of 10 mg/mL substrate was used in a 242 µL reaction volume. Commercial Prussian blue ferritin ( 10 µL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 µL of 0.047 mg/mL sample). Negative controls are ABTS and hydrogen peroxide, and ABTS only. Standard error of the mean bars are based on a sample size where n=8.</p>
+</figcaption>
+</figure>
+<p>In order to complete our kinetic analysis we had to determine the catalytic properties of our Prussian blue ferritin according to the Michaelis-Menten kinetic model. For these tests we varied the colourimetric substrate concentrations (ABTS and TMB) (Figures 3 and 4). We also varied the hydrogen peroxide concentration in association with TMB as this the first chemical compound that will react in the system (Figure 5).</p>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/2/20/UCalgary2013TRPBFABTSmichaelismentengraph.png" alt="Michaelis-Menten Plot for Prussian Blue Ferritin with ABTS" width="800" height="436">
+<figcaption>
+	<p><b>Figure 3.</b> Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of ABTS. Absorbance readings were taken at 415 nm. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data (eg. Figure 6).</p>
+</figcaption>
+</figure>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/c/c7/UCalgary2013TRPBFTMBmichaelismentengraph.png" alt="Michaelis-Menten Plot for Prussian Blue Ferritin with TMB" width="800" height="435">
+<figcaption>
+	<p><b>Figure 4.</b> Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of TMB. Absorbance readings were taken at 650 nm. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data (eg. Figure 5).</p>
+</figcaption>
+</figure>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/e/e5/UCalgary2013TRPBFTMBGHydrogenperoxidemichaelismentengraph.png" alt="Michaelis-Menten Plot for Prussian Blue Ferritin Based on Hydrogen Peroxide (with TMB)" width="800" height="434">
+<figcaption>
+	<p><b>Figure 5.</b> Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of hydrogen peroxide. Absorbance readings were taken at 650 nm which measure the breakdown of TMB. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data.</p>
+</figcaption>
+</figure>
+<center><b>Table 1.</b> Catalytic constants for our Prussian blue ferritin</center>
+<center><table width="800" border="1">
+  <tr>
+    <td>Catalyst</td>
+    <td>Enzyme Concentration (M)</td>
+    <td>Substrate</td>
+    <td>K<sub>m</sub> (mM)</td>
+    <td>V<sub>max</sub> (Ms<sup>-1</sup>)</td>
+    <td>K<sub>cat</sub> (s<sup>-1</sup>)</td>
+    <td>K<sub>cat</sub>/K<sub>m</sub> (M<sup>-1</sup>s<sup>-1</sup>)</td>
+  </tr>
+  <tr>
+    <td>Prussian Blue Ferritin</td>
+    <td>1.31 x 10<sup>-9</sup></td>
+    <td>ABTS</td>
+    <td>0.448</td>
+    <td>1.25 x 10<sup>-8</sup></td>
+    <td>9.51</td>
+    <td>2.12 x 10<sup>4</sup></td>
+  </tr>
+  <tr>
+    <td>Prussian Blue Ferritin</td>
+    <td>1.31 x 10<sup>-9</sup></td>
+    <td>TMB</td>
+    <td>0.0432</td>
+    <td>1.12 x 10<sup>-7</sup></td>
+    <td>85.3</td>
+    <td>1.97 x 10<sup>6</sup></td>
+  </tr>
+  <tr>
+    <td>Prussian Blue Ferritin</td>
+    <td>1.31 x 10<sup>-9</sup></td>
+    <td>H<sub>2</sub>O<sub>2 </sub> (TMB)</td>
+    <td>0.0176</td>
+    <td>1.31 x 10<sup>-8</sup></td>
+    <td>11.1</td>
+    <td>6.28 x 10<sup>5</sup></td>
+  </tr>
+</table></center>
+<b>pH Optimization of Prussian blue Ferritin</b>
+<p>We also performed a pH optimization of our Prussian blue ferritin using the substrates TMB and ABTS (Figure 6 and 7). The results show TMB to be a much more robust substrate, showing high catalytic activity across a more broad range of pH compared to ABTS.</p>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/c/ca/UCalgary2013TRABTSphoptimization.png" alt="ABTS pH Optimization" width="800" height="431">
+<figcaption>
+	<p><b>Figure 6.</b> pH optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility. </p>
+</figcaption>
+</figure>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/1/1c/UCalgary2013TRTMBphoptimization.png" alt="TMB pH Optimization" width="800" height="444">
+<figcaption>
+	<p><b>Figure 7.</b> pH optimization of commercial Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility. </p>
+</figcaption>
+</figure>
+<b>Temperature Optimization of Prussian Blue Ferritin</b>
+<p>Another aspect of our analysis was determining the optimal temperature for catalytic activity of Prussian blue ferritin (Figure 8 and 9). This showed us that the Prussian blue reporter has a much higher catalytic activity at higher temperatures.</p>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/5/57/UCalgary2013TRABTStemperatureoptimization.png" alt="ABTS Temperature Optimization" width="800" height="437">
+<figcaption>
+	<p><b>Figure 8.</b> Temperature optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility. </p>
+</figcaption>
+</figure>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/6/63/UCalgary2013TRTMBtemperatureoptimization.png" alt="TMB Temperature Optimization" width="800" height="440">
+<figcaption>
+	<p><b>Figure 9.</b> Temperature optimization of commerical Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility. </p>
+</figcaption>
+</figure>
+<b>Prussian Blue Ferritin on Nitrocellulose</b>
+<p>The next aspect of our analysis was to see how Prussian blue ferritin would act in a catalytic sense on nitrocellulose (Figures 10 and 11). From these results we can that TMB is a better substrate on for use on nitrocellulose(Figure 11). With this substrate we saw a result from only 5 ng of Prussian blue ferritin present on the nitrocellulose.</p>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/5/53/UCalgary2013TRABTSnitrocellulose.png" alt="Prussian Blue Ferritin and ABTS on Nitrocellulose" width="701" height="600">
+<figcaption>
+	<p><b>Figure 10.</b> Blots of Prussian blue ferritin on nitrocellulose (5 µL samples) that are reacted with ABTS (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.</p>
+</figcaption>
+</figure>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/c/cd/UCalgary2013TRTMBnitrocellulose.png" alt="Prussian Blue Ferritin and TMB on Nitrocellulose" width="693" height="600">
+<figcaption>
+	<p><b>Figure 11.</b> Blots of Prussian blue ferritin on nitrocellulose (5 µL samples) that are reacted with TMB (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.</p>
+</figcaption>
+</figure>
+<b>Making Prussian Blue out of This Part</b>
+<p>We applied a scaled down Prussian blue synthesis experiment to our own ferritinOur own Prussian blue ferritin was then exposed to the TMB substrate (Figure 12). From the results we can see that the ferritin with the E-coil attached had excellent catalytic activity.</p>
+<figure>
+<img src="https://static.igem.org/mediawiki/2013/8/89/UCalgary2013TRRecombinantPrussianBlueFerritin.png" alt="Creating Prussian Blue Ferritin out of our Own Ferritin" width="800" height="511">
+<figcaption>
+	<p><b>Figure 12.</b> Measurements of the coloured substrate TMB (10 mg/mL) at 650 nm over a 600 second time period for our own Prussian blue ferritin and unmodified ferritin. Sample volume was 242 µL. Controls for this experiment include bovine serum albumin (1 mg/mL)and the substrate solution by itself.  Due to limitations on the protein available only one replicate was performed. Zero time points do not have low absorbance as colour change was rapid and began before measurements started.</p>
+</figcaption>
+</figure>
+<br>
+</html>
-<!-- Add more about the biology of this part here
-===Usage and Biology===
 <!-- -->