Difference between revisions of "Part:BBa K3767001"

Revision as of 16:13, 16 June 2021

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

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.

3D representation of the alkaline phosphatase

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 1. Reaction mechanism of Alkaline Phosphatase.

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
COMPATIBLE WITH RFC[1000]

Characterization

Description

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. 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

@@ Line 5: / Line 5: @@
 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| 3D representation of the alkaline phosphatase]]
+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-->
 ===Usage and Biology===
-Alkaline phosphatases are used as reporter enzymes in different assays such as Western Blotting and in situ hybridization[[Part:BBa_K1216001#References|<sup>[1]</sup>]]. Testing human blood for Alkaline Phosphatase levels is a routine test that can reveal different conditions[[Part:BBa_K1216001#References|<sup>[2]</sup>]].
+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. [[File:BBa K3767001 3D phoA Reaction Mechanism.png|frame|center|50px|Figure 1. Reaction mechanism of Alkaline Phosphatase.]]
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