Difference between revisions of "Part:BBa K3767001"
 
(14 intermediate revisions by 2 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K3767001 short</partinfo>
 
<partinfo>BBa_K3767001 short</partinfo>
  
−
Alkaline phosphatases are a group of isoenzymes that catalyze the hydrolysis of organic phosphate esters present in the extracellular space [[Part:BBa_K3767001#References|<sup>[1]</sup>]] . This catalytic activity is commonly used in diagnostic tests as the cleavage of the phosphate ester produces fluorescence visible to the naked eye. [[File:BBa K3767001 Coloured PhoA x40.png|thumb|500px| <b> Figure 1: 3D representation of the alkaline phosphatase. </b> A and B domains shown in red and green respectively. Zinc and Magnesium highlighted in grey and blue respectively]]  
+
<h1> PhoA </h1>
 +
Alkaline phosphatases are a group of isoenzymes that catalyze the hydrolysis of organic phosphate esters present in the extracellular space[[Part:BBa_K3767001#References|<sup>[1]</sup>]] . This catalytic activity is commonly used in diagnostic tests as the cleavage of the phosphate ester produces fluorescence visible to the naked eye. [[File:BBa K3767001 Coloured PhoA x40.png|thumb|200px| <b> Figure 1: 3D representation of the alkaline phosphatase. </b> A and B domains shown in red and green respectively. Zinc and Magnesium highlighted in grey and blue respectively]]  
  
 
This part is being registered as an improvement on a previously registered part [[Part:BBa_K1216001| BBa_K1216001]]. Our contribution altered the sequence to improve enzymatic activity and expression rates in E. Coli.  
 
This part is being registered as an improvement on a previously registered part [[Part:BBa_K1216001| BBa_K1216001]]. Our contribution altered the sequence to improve enzymatic activity and expression rates in E. Coli.  
  
 
<!-- Add more about the biology of this part here-->
 
<!-- Add more about the biology of this part here-->
−
===Usage and Biology===
+
<h3> Usage and Biology </h3>
  
 
Alkaline phosphatase are plasma membrane-bound glycoproteins which are capable of hydrolyzing monophosphate esters resulting in the release of inorganic phosphate[[Part:BBa_K3767001#References|<sup>[3]</sup>]]. Dephosphorylation activity is catalyzed by two Zn2+ and one Mg2+ ions within the active site with both ions being required for enzymatic activity. In addition to this, both metal ions aid in structure conformation stability and subunit-subunit interactions.  
 
Alkaline phosphatase are plasma membrane-bound glycoproteins which are capable of hydrolyzing monophosphate esters resulting in the release of inorganic phosphate[[Part:BBa_K3767001#References|<sup>[3]</sup>]]. Dephosphorylation activity is catalyzed by two Zn2+ and one Mg2+ ions within the active site with both ions being required for enzymatic activity. In addition to this, both metal ions aid in structure conformation stability and subunit-subunit interactions.  
  
 
The varying reaction mechanisms of this enzyme are well-conserved across animal and bacteria variants with either Ser, Thr, or Cys being modified within the active site during transfer (the residue used depends on the activity type)[[Part:BBa_K3767001#References|<sup>[4]</sup>]]. Additionally, the final phosphate receptor is conserved being either H2O, a secondary substrate, or another hydroxyl group within the same molecule. An illustration of the transfer of the phosphate group is depicted below.  
 
The varying reaction mechanisms of this enzyme are well-conserved across animal and bacteria variants with either Ser, Thr, or Cys being modified within the active site during transfer (the residue used depends on the activity type)[[Part:BBa_K3767001#References|<sup>[4]</sup>]]. Additionally, the final phosphate receptor is conserved being either H2O, a secondary substrate, or another hydroxyl group within the same molecule. An illustration of the transfer of the phosphate group is depicted below.  
 +
  
 
[[File:BBa K3767001 3D phoA Reaction Mechanism.png|400px|center|thumb| <b> Figure 2. Reaction mechanism of Alkaline Phosphatase </b> [[Part:BBa_K3767001#References|<sup>[4]</sup>]].]]
 
[[File:BBa K3767001 3D phoA Reaction Mechanism.png|400px|center|thumb| <b> Figure 2. Reaction mechanism of Alkaline Phosphatase </b> [[Part:BBa_K3767001#References|<sup>[4]</sup>]].]]
 +
 +
 +
<h2> Additional characterisation by Sheffield iGEM 2024 </h2>
 +
Our team cloned PhoA into a pet21a(+) derivative plasmid backbone containing a c-terminal 6xHis tag. Protein production strain BL21 Lemo was used and protein was purified via Immobilised Metal Affinity Chromatography (IMAC), and relevant fractions were analysed via SDS-PAGE electrophoresis.
 +
PhoA yield was lower than PafA from flavobacterium johnsoniae (<html><a href="https://parts.igem.org/Part:BBa_K5172000">BBa_K5172000</a></html>) but still significant in elutions 4-6 (see figure 3) . Elutions with proteins were pooled and the buffer was exchanged into a glycerol-based buffer.
 +
 +
<html><img src= "https://static.igem.wiki/teams/5172/registry-folder/phoafigure3.png" ></html>
 +
 +
<i>Figure 3: SDS-PAGE results of PhoA and the PafA post-protein purification. The flow-through (FT), binding buffer (BB), wash buffer (WB), and elutions (E1-10) were all run on the gel to identify which elutions contained protein.</i>
 +
 +
Size exlusion chromatography was done to remove extra banding, signifying other protein impurities. There were less protein impurities post-SEC (see figure 4).
 +
 +
<html><img src= "https://static.igem.wiki/teams/5172/registry-folder/phoafigure4.png" ></html>
 +
 +
<i>Figure 4: SDS-PAGE results of PafA WT and PhoA post-SEC.</i>
 +
 +
Purification steps expose the proteins to harsh conditions, such as Imidazole, that could cause them to unfold, reducing the accuracy of upcoming characterisation assays. Circular dichroism (CD) experiments were carried out to ensure the proteins were folded correctly. Circular dichroism spectroscopy can show the stability of the proteins by determining the temperature at which they aggregate out of solution or unfold. For our uses, we primarily used CD to deduce the thermostability of our proteins.
 +
 +
Figures 5 shows the CD results plotting ellipticity against wavelength for PafA and PhoA respectively. At 222nm, and 208nm , dips are present, indicating that both phosphatases are consistent primarily of alpha helices. As the temperatures increase, plots flatten out as the protein aggregates from the solution or unfolds. Both PafA and PhoA can be seen to fully aggregate out of solution by 70℃, seen in figure 5.
 +
 +
Figure 5A shows the CD results plotting ellipticity against wavelength for PhoA. The results show a repeat at 25℃, “25 E2” was due to us testing elution 2 of PhoA from the Nickel Column Purification Step, however as shown on the graph the plot is relatively flattened which suggested to us that no protein was present in this sample. Therefore we started the CD process again with another elution and the plot looked closer to a typical CD plot. 
 +
 +
 +
<html><img src= "https://static.igem.wiki/teams/5172/registry-folder/phoafigure5.png" ></html>
 +
 +
<i>Figure 5: (A) CD results for PhoA, (B) & (C) a comparison of ellipticity to temperature at each wavelength for alpha helices on PhoA. (D) Percentage of maximum ellipticity (%) against temperature (℃) for normalised PhoA and PafA.</i>
 +
 +
<h3> Determining the activity of PafA compared to PhoA </h3>
 +
 +
Absorbance measurements, as shown in Figures 6 and 7, were converted to the concentration of pNP using their determined linear relationship. Gradients of the linear section of the line were determined using a Matlab script that identified the gradient between every successive 5 points, and outputted the maximum gradient, capturing the maximum velocity of the reaction without being thrown off by artefacts near the beginning of each time series. By automating this process, we saved time and increased accuracy against the traditional technique of plotting all the concentrations of pNP against time graphs and determining gradients by hand- a method that is limited by determining qualitatively where the linear section ends and the graphs begin to level out. The gradients were normalised by enzyme concentration and were plotted against pNPP (substrate concentration) and GraphPad Prism was used to fit the data to a Michaelis-Menten curve (Figure 8).
 +
 +
<html><img src= "https://static.igem.wiki/teams/5172/registry-folder/phoafigure6b.png" ></html>
 +
 +
 +
<i>Figure 6: As the concentration of pNPP substrate increases, PafA WT shows a greater rate of catalysis and is able to react more substrate before plateauing.</i>
 +
 +
<html><img src= "https://static.igem.wiki/teams/5172/registry-folder/phoafigure7.png" ></html>
 +
 +
 +
 +
<i>Figure 7: As the concentration of pNPP substrate increases, PhoA shows a greater rate of catalysis and is able to react more substrate before plateauing</i>
 +
 +
<html><img src= "https://static.igem.wiki/teams/5172/registry-folder/phoafigure8a.png" ></html>
 +
 +
<i>Figure 8: Michaellis-Menten fit on GraphPad Prism for [pNPP] (mM) against velocity of reaction divided by enzyme concentration (v/[E]) (min-1).  A. PafA at an enzyme concentration of 1nM, and B. PhoA at an enzyme concentration of 1nM. </i>
 +
 +
 +
 +
Figure 8 and Prism showed that PafA has a Vmax of 13210 min-1± 720 (95% Confidence interval (CI)), and Km is 0.02236 mM± 0.00524 (95% CI). PhoA has a Vmax of 6774 min-1 ± 405 (95% CI) and Km of 0.01549 mM ± 0.014422(95% CI), showing that PafA was more active than PhoA  (BBa_K3767001), making PafA (BBa_K5172000) the most active phosphatase currently on the registry.
 +
 +
For more information on PafA from F.Johnsoniae:
 +
(<html><a href="https://parts.igem.org/Part:BBa_K5172000">BBa_K5172000</a></html>)
  
 
<!-- -->
 
<!-- -->
Line 20: Line 74:
 
<partinfo>BBa_K3767001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3767001 SequenceAndFeatures</partinfo>
  
−
==Characterization==
 
  
−
Description
 
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 12:23, 2 October 2024


Alkaline Phosphatase optimized for E. Coli w/ 40x catalytic activity

PhoA

Alkaline phosphatases are a group of isoenzymes that catalyze the hydrolysis of organic phosphate esters present in the extracellular space[1] . This catalytic activity is commonly used in diagnostic tests as the cleavage of the phosphate ester produces fluorescence visible to the naked eye.
Figure 1: 3D representation of the alkaline phosphatase. A and B domains shown in red and green respectively. Zinc and Magnesium highlighted in grey and blue respectively

This part is being registered as an improvement on a previously registered part BBa_K1216001. Our contribution altered the sequence to improve enzymatic activity and expression rates in E. Coli.

Usage and Biology

Alkaline phosphatase are plasma membrane-bound glycoproteins which are capable of hydrolyzing monophosphate esters resulting in the release of inorganic phosphate[3]. Dephosphorylation activity is catalyzed by two Zn2+ and one Mg2+ ions within the active site with both ions being required for enzymatic activity. In addition to this, both metal ions aid in structure conformation stability and subunit-subunit interactions.

The varying reaction mechanisms of this enzyme are well-conserved across animal and bacteria variants with either Ser, Thr, or Cys being modified within the active site during transfer (the residue used depends on the activity type)[4]. Additionally, the final phosphate receptor is conserved being either H2O, a secondary substrate, or another hydroxyl group within the same molecule. An illustration of the transfer of the phosphate group is depicted below.


Figure 2. Reaction mechanism of Alkaline Phosphatase [4].


Additional characterisation by Sheffield iGEM 2024

Our team cloned PhoA into a pet21a(+) derivative plasmid backbone containing a c-terminal 6xHis tag. Protein production strain BL21 Lemo was used and protein was purified via Immobilised Metal Affinity Chromatography (IMAC), and relevant fractions were analysed via SDS-PAGE electrophoresis. PhoA yield was lower than PafA from flavobacterium johnsoniae (BBa_K5172000) but still significant in elutions 4-6 (see figure 3) . Elutions with proteins were pooled and the buffer was exchanged into a glycerol-based buffer.

Figure 3: SDS-PAGE results of PhoA and the PafA post-protein purification. The flow-through (FT), binding buffer (BB), wash buffer (WB), and elutions (E1-10) were all run on the gel to identify which elutions contained protein.

Size exlusion chromatography was done to remove extra banding, signifying other protein impurities. There were less protein impurities post-SEC (see figure 4).

Figure 4: SDS-PAGE results of PafA WT and PhoA post-SEC.

Purification steps expose the proteins to harsh conditions, such as Imidazole, that could cause them to unfold, reducing the accuracy of upcoming characterisation assays. Circular dichroism (CD) experiments were carried out to ensure the proteins were folded correctly. Circular dichroism spectroscopy can show the stability of the proteins by determining the temperature at which they aggregate out of solution or unfold. For our uses, we primarily used CD to deduce the thermostability of our proteins.

Figures 5 shows the CD results plotting ellipticity against wavelength for PafA and PhoA respectively. At 222nm, and 208nm , dips are present, indicating that both phosphatases are consistent primarily of alpha helices. As the temperatures increase, plots flatten out as the protein aggregates from the solution or unfolds. Both PafA and PhoA can be seen to fully aggregate out of solution by 70℃, seen in figure 5.

Figure 5A shows the CD results plotting ellipticity against wavelength for PhoA. The results show a repeat at 25℃, “25 E2” was due to us testing elution 2 of PhoA from the Nickel Column Purification Step, however as shown on the graph the plot is relatively flattened which suggested to us that no protein was present in this sample. Therefore we started the CD process again with another elution and the plot looked closer to a typical CD plot.


Figure 5: (A) CD results for PhoA, (B) & (C) a comparison of ellipticity to temperature at each wavelength for alpha helices on PhoA. (D) Percentage of maximum ellipticity (%) against temperature (℃) for normalised PhoA and PafA.

Determining the activity of PafA compared to PhoA

Absorbance measurements, as shown in Figures 6 and 7, were converted to the concentration of pNP using their determined linear relationship. Gradients of the linear section of the line were determined using a Matlab script that identified the gradient between every successive 5 points, and outputted the maximum gradient, capturing the maximum velocity of the reaction without being thrown off by artefacts near the beginning of each time series. By automating this process, we saved time and increased accuracy against the traditional technique of plotting all the concentrations of pNP against time graphs and determining gradients by hand- a method that is limited by determining qualitatively where the linear section ends and the graphs begin to level out. The gradients were normalised by enzyme concentration and were plotted against pNPP (substrate concentration) and GraphPad Prism was used to fit the data to a Michaelis-Menten curve (Figure 8).


Figure 6: As the concentration of pNPP substrate increases, PafA WT shows a greater rate of catalysis and is able to react more substrate before plateauing.


Figure 7: As the concentration of pNPP substrate increases, PhoA shows a greater rate of catalysis and is able to react more substrate before plateauing

Figure 8: Michaellis-Menten fit on GraphPad Prism for [pNPP] (mM) against velocity of reaction divided by enzyme concentration (v/[E]) (min-1). A. PafA at an enzyme concentration of 1nM, and B. PhoA at an enzyme concentration of 1nM.


Figure 8 and Prism showed that PafA has a Vmax of 13210 min-1± 720 (95% Confidence interval (CI)), and Km is 0.02236 mM± 0.00524 (95% CI). PhoA has a Vmax of 6774 min-1 ± 405 (95% CI) and Km of 0.01549 mM ± 0.014422(95% CI), showing that PafA was more active than PhoA (BBa_K3767001), making PafA (BBa_K5172000) the most active phosphatase currently on the registry.

For more information on PafA from F.Johnsoniae: (BBa_K5172000)


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
    COMPATIBLE WITH RFC[1000]



References


1. Lowe, D., Sanvictores, T., and John, S. (2021) Alkaline Phosphatase, StatPearls Publishing, [online] http://www.ncbi.nlm.nih.gov/pubmed/29083622 (Accessed June 8, 2021)
2. Part:BBa K1216001 - parts.igem.org [online] https://parts.igem.org/Part:BBa_K1216001#References (Accessed June 8, 2021)
3. Sharma, U., Pal, D., and Prasad, R. (2014) Alkaline phosphatase: An overview. Indian J. Clin. Biochem. 29, 269–278
4. Millán, J. L. (2006) Alkaline phosphatases. Purinergic Signal. 2, 335–341
5. Du, M. H. L., Lamoure, C., Muller, B. H., Bulgakov, O. V., Lajeunesse, E., Ménez, A., and Boulain, J. C. (2002) Artificial evolution of an enzyme active site: Structural studies of three highly active mutants of Escherichia coli alkaline phosphatase. J. Mol. Biol. 316, 941–953