Coding

Part:BBa_K2485001

Designed by: Michael Herschl   Group: iGEM17_Stanford-Brown   (2017-07-29)
Revision as of 20:30, 21 October 2019 by Hjy199999666 (Talk | contribs) (→‎Contribution: WHU-China 2019)

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BirA with 6x His Tag

BirA is a biotinylation enzyme in E. Coli. It can both facilitate the addition of biotin to other proteins in the presence of ATP and act as a repressor of the biotin operon.

Contribution: WHU-China 2019

Group: WHU-China 2019
Authors: Jiongyi He
Summary: We confirmed the sequence and function of BirA with 6x His Tag (BBa_K2485001) in vivo.
Documentation:


It is universally acknowledged that Avi-tag can be specifically recognized by BirA enzyme in vitro/vivo and attach a biotin to the protein. So we constructed three plasmids and expressed them separately: pet28a-BirA (pB), pet28a-sfGFP+Avitag (pGa) and pet28a-sfGFP+Avitag-BirA (pBGa). The sequence of BirA is from BBa_K2485001. After purification by Ni resin and chromatography, we got purified products from pB, pGa and pBGa. sfGFP+Avi-tag is the part BBa_K3098015.

According to the result of SDS-PAGE below, we can know that the BirA enzyme has 6x His-tag since it can be purified by Ni-resin. Besides, we got sfGFP from pGa and pBGa.

(M: Marker; BirA: the lysate supernatant of pB purified by Ni-resin; GFP: the lysate supernatant of pGa purified by Ni-resin; LS: the lysate supernatant of pBGa; Ni: the lysate supernatant of pBGa purified by Ni-resin; Chro: the eluent of Ni-resin of pBGa purified by chromatography; M: Marker)



Then we measured protein concentration by Bradford method and tried to measure the biotin levels of products of pGa and pBGa by HABA/Avidin reagent under OD500.

HABA/Avidin is suitable for the spectrophotometric determination of in a diverse range of sample types. This method is based on the binding of the dye HABA to avidin and the ability of biotin to displace the dye in stoichiometric proportions. This displacement of dye is accompanied by a change in absorbance at A500 which has a known extinction coefficient. Here is the method:

Table 1 Method of HABA/Avidin Measurement


The results of measurement of HABA/Avidin reagent:

Bira data.png


According to the results, we know that the sfGFP produced by pBGa are biotinylated well. The results are convincing. Therefore, the function of BBa_2485001—BirA with 6x His Tag is confirmed.

Table 2 Data from Bradford And HABA/Avidin Measurements


Reference:
[1] Ashraf S S, Benson R E, Payne E S, et al. A novel multi-affinity tag system to produce high levels of soluble and biotinylated proteins in Escherichia coli [J]. Protein Expression and Purification, 2004, 33(2):0-245.
[2] Chapman-Smith A, Cronan J E. In vivo enzymatic protein biotinylation [J]. Biomolecular Engineering, 2000, 16(1-4):119-125.

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]


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