Composite

Part:BBa_K3332055

Designed by: Yangqi Deng   Group: iGEM20_XMU-China   (2020-10-17)
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J23100-RBS-GOX-BrkA-terminator

We anchored GOX protein onto membranes through BrkA to catalyze the reaction of degradating glyphosate to form glyoxalic acid and AMPA. We use K880005 to construct the expression system and anchor GOX on the surface of E.coli.


Biology

        BrkA is an anchor protein from Bordetella pertussis, which has β-barrel structure. It can anchor its passenger protein to the cell membrane and has been widely used in cell-surface display. GOX, also known as EcAKR4-1, is found in Echinochloa colona. It can decompose glyphosate into AMPA and glyoxylic acid. GOX is fused at C terminal with BrkA to get GOX-BrkA and we hope GOX can be displayed on the surface of E. coli successfully and still active.[1] [2]

Fig 1. Mechanism of GOX-BrkA on the surface of E. coli


Usage

        Here, we used BBa_K880005 to construct the expression system and obtained the composite part BBa_K3332055, which may achieve surface display of GOX on our engineering bacteria.

Fig 2. Gene circuit of GOX-BrkA

Characterization

1. Identification

        After receiving the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure3.

Fig 3. DNA gel electrophoresis of restriction digest products of iNap-BrkA-pSB1C3 (Xba I & Pst I sites)

2. Ability of degrading NADPH

        After transforming glyphosate into glyoxylic acid with the function of GOX, we use GRHPR, a glyoxylate reductase from human liver, to reduce glyoxylic acid. GRHPR can convert glyoxylic acid when NADPH is consumed as cofactor. NADPH is a suitable target compound that can be detected by the signal of 340. And when NADPH is consumed, OD340 declines.

        We mixed glyphosate solution, NADPH solution, purified GRHPR protein dissolved in Tris-HCl(pH=7.5) and bacteria solution carrying GOX-BrkA. Then, we immediately detected OD340 by using TECAN® Infinite M200 Pro to see the effect of GOX fused with anchor protein.

        When using GOX-BrkA bacteria solution, we successfully found OD340 decrease as time went on. In blank control samples (replace GOX-BrkA bacteria with J23100-RBS(BBa_K880005) bacteria) and negative control samples (replace GOX-BrkA bacteria with GOX-Histag bacteria) , we can see the decrease is less than the GOX-BrkA samples. The results prove that GOX-BrkA can be displayed on the surface and convert glyphosate as normal, which is shown in figure 5.

Fig 4. OD340-Time curve of three fusion protein of GOX and anchor protein


References

  1. ↑ Pan L, Yu Q, Han H, et al. Aldo-keto Reductase Metabolizes Glyphosate and Confers Glyphosate Resistance in Echinochloa colona[J]. Plant Physiol, 2019, 181(4): 1519-1534.
  2. ↑ http://2016.igem.org/Team:TJUSLS_China


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 355
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 574
    Illegal NgoMIV site found at 1304
    Illegal NgoMIV site found at 1733
    Illegal NgoMIV site found at 2369
    Illegal AgeI site found at 644
    Illegal AgeI site found at 721
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2337


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