Difference between revisions of "Part:BBa K3037009"
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== Overview == | == Overview == | ||
| − | The TU Dresden 2019 team | + | The [https://2019.igem.org/Team:TU_Dresden TU Dresden 2019 team] designed this biobrick using the [https://parts.igem.org/Part:BBa_K823038 BBa_K823038] for the Strep-tag and [https://parts.igem.org/Part:BBa_K3037007 BBa_K3037007] for HRP [https://2019.igem.org/Team:TU_Dresden/Parts (more information).] |
| − | HRP+Strep-tag was inserted into the | + | HRP+Strep-tag was inserted into the pOCC97 [https://parts.igem.org/Part:BBa_K3037000 BBa_K3037000] vector for transformation and expressed in <span style="font-style: italic;">Escherichia coli</span> pRARE T7. |
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=== Biology === | === Biology === | ||
| − | The metalloenzyme horse radish peroxide is widely used in many biochemical and immunological applications. This | + | The metalloenzyme horse radish peroxide is widely used in many biochemical and immunological applications. This enzyme on its own does not give any visual readout but however upon addition of a suitable substrate, HRP oxidizes it and yields a colour change that can be spectrophotometrically analysed. One such common example for chromogenic substrate is TMB (3,3',5,5'-Tetramethylbenzidine) that HRP oxidizes. Additionally, an advantage of using HRP includes its stability and small size, hence reducing the interference during conjugation reactions such as for secondary antibody detection. Furthermore, it is economical in comparison with other alternative enzymes like Alkaline Phosphatase. |
== Characterization == | == Characterization == | ||
| − | |||
| − | The protocol used was “Expression and purification of proteins using Strep-Tactin” of IBA Lifescience | + | === HRP === |
| + | |||
| + | Since the HRP-Strep part is a composite part, the correct functioning of the HRP activity was already characterized individually. Please check for that the characterization of this BioBrick [https://parts.igem.org/Part:BBa_K3037007 BBa_K3037007.] | ||
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| + | === Strep-tag=== | ||
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| + | The Strep-tag [https://parts.igem.org/Part:BBa_K823038 BBa_K823038] was supposed to be used for purification. | ||
| + | |||
| + | The protocol used was “Expression and purification of proteins using Strep-Tactin” of IBA Lifescience [1] preparing the same buffers described in this protocols without EDTA to not harm the activity of HRP. From the results of the purifications we concluded that the Strep-tag is not working properly for column purification and should therefore only be used for Western Blots. See more information regarding this in the original registry of this BioBrick ([https://parts.igem.org/Part:BBa_K823038 BBa_K823038)], and see Figure 1 for this concrete construct. | ||
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| + | [[File:T--TU_Dresden--Strep-tag_purification_BBa_3037009.png|center|400px|thumb|none| Figure 1: SDS-page showing the unsuccessful purification of the HRP-strep construct. The elution fractions do not contain our construct of interest.]] | ||
| + | |||
| + | == References== | ||
| − | + | https://www.iba-lifesciences.com/isotope/2/2-3206-100-Manual-Twin--Strep-tag.pdf | |
== Sequence == | == Sequence == | ||
Latest revision as of 02:03, 22 October 2019
HRP+Strep-tag
| HRP+Strep-tag | |
|---|---|
| Function | Expression |
| Use in | Escherichia coli |
| RFC standard | Freiburg RFC25 standard |
| Backbone | pSB1C3 |
| Experimental Backbone | pOCC97 |
| Submitted by | Team: TU_Dresden 2019 |
Overview
The TU Dresden 2019 team designed this biobrick using the BBa_K823038 for the Strep-tag and BBa_K3037007 for HRP (more information).
HRP+Strep-tag was inserted into the pOCC97 BBa_K3037000 vector for transformation and expressed in Escherichia coli pRARE T7.
Biology
The metalloenzyme horse radish peroxide is widely used in many biochemical and immunological applications. This enzyme on its own does not give any visual readout but however upon addition of a suitable substrate, HRP oxidizes it and yields a colour change that can be spectrophotometrically analysed. One such common example for chromogenic substrate is TMB (3,3',5,5'-Tetramethylbenzidine) that HRP oxidizes. Additionally, an advantage of using HRP includes its stability and small size, hence reducing the interference during conjugation reactions such as for secondary antibody detection. Furthermore, it is economical in comparison with other alternative enzymes like Alkaline Phosphatase.
Characterization
HRP
Since the HRP-Strep part is a composite part, the correct functioning of the HRP activity was already characterized individually. Please check for that the characterization of this BioBrick BBa_K3037007.
Strep-tag
The Strep-tag BBa_K823038 was supposed to be used for purification.
The protocol used was “Expression and purification of proteins using Strep-Tactin” of IBA Lifescience [1] preparing the same buffers described in this protocols without EDTA to not harm the activity of HRP. From the results of the purifications we concluded that the Strep-tag is not working properly for column purification and should therefore only be used for Western Blots. See more information regarding this in the original registry of this BioBrick (BBa_K823038), and see Figure 1 for this concrete construct.
References
https://www.iba-lifesciences.com/isotope/2/2-3206-100-Manual-Twin--Strep-tag.pdf
Sequence
NOTE: Please be aware, that by combining all the basic parts used for this composite part, the registry automatically inserted a RFC23 scar. However, the design and our cloning strategy is based on RFC25.
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 151
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 393
Illegal XhoI site found at 477 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]