Difference between revisions of "Part:BBa K3332055"

Revision as of 19:42, 26 October 2020

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