Difference between revisions of "Part:BBa K3037009"

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.

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

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.

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 151
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 393
Illegal XhoI site found at 477
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 14: / Line 14: @@
 |'''Backbone'''
 |[https://parts.igem.org/Help:2019_DNA_Distribution pSB1C3<br>]
+|-
+|'''Experimental Backbone'''
+|[https://parts.igem.org/Part:BBa_K3037000 pOCC97<br>]
 |-
 |'''Submitted by'''
@@ Line 22: / Line 25: @@
 == Overview ==
-The TU Dresden 2019 team design this biobrick using the [https://parts.igem.org/Part:BBa_K823038 BBa_K823038] [https://2019.igem.org/Team:TU_Dresden/Parts (more information).]
+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 pSB1C3 vector for transformation and expressed in <span style="font-style: italic;">Escherichia coli</span>.
+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.
-https://parts.igem.org/Assembly_standard_25
+=== Biology ===
-The HRP was made from BBa_K1800002 but adapted to the RFC 25 standard
+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 ==
-The Strep-tag [https://parts.igem.org/Part:BBa_K823038 BBa_K823038] was used for purification.
-The protocol used was “Expression and purification of proteins using Strep-Tactin” of IBA Lifescience using buffers without EDTA. From the results of the purifications we conclude that the Strep-tag doesn't works for column purification, and should be used only for Western Blots as it was meant to be.
+=== HRP ===
-<gallery mode="packed", caption="Purification of proteins", widths=400px, heights=200px>
+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.]
-File:T--TU_Dresden--Strep-tag_purification_BBa_3037009.png|<span style="color:#0000ff"> ''Strep-tactin column purification''  </span> (Purification of the composite BioBrick BBa_K3037009)
-</gallery>
+=== Strep-tag===
+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.
+[[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 ==
+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.
 <partinfo>BBa_K3037009 SequenceAndFeatures</partinfo>