Difference between revisions of "Part:BBa K3767002"
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<partinfo>BBa_K3767002 short</partinfo> | <partinfo>BBa_K3767002 short</partinfo> | ||
| − | + | This part consists of a developed ScFv sequence (BBa_K3767000) based on previous research conducted by Ghosh and Huber [[Part:BBa_K3767002#References|<sup>[1]</sup>]] using SAbPred’s ABodyBuilder, Clusterpro supercomputer and PyMol to determine if the ScFv would bind to the target OspA surface protein. The part also consists of an improved Alkaline Phosphatase(BBa_K3767001) linked to the ScFv sequence via (GGGS)<sub>3</sub> linkers at the C-terminal. | |
| − | This part consists of a developed ScFv sequence (BBa_K3767000) based on previous research conducted by Ghosh and Huber [[Part:BBa_K3767002#References|<sup>[1]</sup>]] using SAbPred’s ABodyBuilder, Clusterpro supercomputer and PyMol to determine if the ScFv would | + | [[File:BBa K3767000 Anti-OspA 3-24 ScFv.png|thumb|340px| Figure 2: Anti-OspA 3-24 ScFv. Heavy chain regions in blue, light chain regions in purple and ScFv sequence in green.]] [[File:BBa K3767001 phoA colored (flipped).png|300px|center|thumb| 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]] |
===Usage and Biology/Background=== | ===Usage and Biology/Background=== | ||
| − | Alkaline Phosphatases (phoA) are ubiquitous membrane bound glycoprotiens that catalyze the hydrolysis of phosphate monoesters at basic pH levels with release of inorganic phosphate[[Part:BBa_K3767002#References|<sup>[2]</sup>]]. They are used as reporter enzymes in different assays such as Western Blotting and in situ hybridization[[Part:BBa_K3767002#References|<sup>[3]</sup>]]. The phoA was linked to the ScFv sequence as a chromogenic reagent, meaning the when the phoA is mixed | + | Alkaline Phosphatases (phoA) are ubiquitous membrane bound glycoprotiens that catalyze the hydrolysis of phosphate monoesters at basic pH levels with release of inorganic phosphate[[Part:BBa_K3767002#References|<sup>[2]</sup>]]. They are used as reporter enzymes in different assays such as Western Blotting and in situ hybridization[[Part:BBa_K3767002#References|<sup>[3]</sup>]]. The phoA was linked to the ScFv sequence as a chromogenic reagent, meaning the when the phoA is mixed with 5-Bromo-4-Chloro-3-indolyl phosphate (BCIP) expresses a blue color in the 615 nm range which is visible to the naked eye. Alkaline phosphatase is linked to the C-terminus of the ScFv’s light chain through (GGGS) [glycine] linkers. Thus, the ScFv is responsible for binding to OspA and will only express color once the ScFv binds to the target OspA protein. [[File:PhoA substrates.png|500px|center|thumb| Figure 3. General mechanism of phosphate monoester hydrolysis via the catalysis from phoA[[Part:BBa_K3767002#References|<sup>[3]</sup>]].]] |
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| + | <partinfo>BBa_K3767002 SequenceAndFeatures</partinfo> | ||
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| + | <!-- Uncomment this to enable Functional Parameter display | ||
| + | ===Functional Parameters=== | ||
| + | <partinfo>BBa_K3767002 parameters</partinfo> | ||
| + | <!-- --> | ||
===References=== | ===References=== | ||
Latest revision as of 21:19, 18 October 2021
3-24 ScFv Sequence linked to an Alkaline Phosphatase (phoA) to aid in OpsA binding detection
This part consists of a developed ScFv sequence (BBa_K3767000) based on previous research conducted by Ghosh and Huber [1] using SAbPred’s ABodyBuilder, Clusterpro supercomputer and PyMol to determine if the ScFv would bind to the target OspA surface protein. The part also consists of an improved Alkaline Phosphatase(BBa_K3767001) linked to the ScFv sequence via (GGGS)3 linkers at the C-terminal.
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Usage and Biology/Background
Alkaline Phosphatases (phoA) are ubiquitous membrane bound glycoprotiens that catalyze the hydrolysis of phosphate monoesters at basic pH levels with release of inorganic phosphate[2]. They are used as reporter enzymes in different assays such as Western Blotting and in situ hybridization[3]. The phoA was linked to the ScFv sequence as a chromogenic reagent, meaning the when the phoA is mixed with 5-Bromo-4-Chloro-3-indolyl phosphate (BCIP) expresses a blue color in the 615 nm range which is visible to the naked eye. Alkaline phosphatase is linked to the C-terminus of the ScFv’s light chain through (GGGS) [glycine] linkers. Thus, the ScFv is responsible for binding to OspA and will only express color once the ScFv binds to the target OspA protein.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
1. Ghosh, S., and Huber, B. T. (2007) Clonal diversification in OspA-specific antibodies from peripheral circulation of a chronic Lyme arthritis patient. J. Immunol. Methods. 321, 121–134
2. Sharma,U., Pal,D., and Prasad,R. (2013) Alkaline Phospatase: An Overview, Indian J Clin Biochem, [Online] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4062654/, (accessed June 12 2021)
3. Part:BBa K1216001 - parts.igem.org [online] https://parts.igem.org/Part:BBa_K1216001#References (Accessed June 12, 2021)
4. 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