Composite

Part:BBa_K3037009

Designed by: Arnau Pérez Roig   Group: iGEM19_TU_Dresden   (2019-10-15)
Revision as of 20:50, 21 October 2019 by ArnauP (Talk | contribs) (→‎Sequence)

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 design this biobrick using the BBa_K823038 (more information).

HRP+Strep-tag was inserted into the pSB1C3 vector for transformation and expressed in Escherichia coli.

The HRP was made from BBa_K1800002 but adapted to the Freiburg RFC25 standard

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 read out but however upon addition of suitable substrate, HRP oxidizes this substrate and yields a colour change and this can be spectrophotometrically analysed. One such common example for chromogenic substrate is TMB (3,3',5,5'-Tetramethylbenzidine) that HRP oxidizes. Added advantage of using HRP includes its stability and small size, hence reducing the interference during conjugation reactions such as for secondary antibody detection and moreover it is economical in comparison with other alternative enzymes like the alkaline phosphatase.

Characterization

The Strep-tag 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.

T--TU Dresden--Strep-tag purification BBa 3037009.png

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]


[edit]
Categories
Parameters
None