Composite

Part:BBa_K3196011

Designed by: Hao Qiu   Group: iGEM19_HUST-China   (2019-10-16)


AOX1-Kozak-FLO10-SLAC-His tag-AOX1 Terminator

Characterization

This is a composite part that used to degraded lignin. SLAC is a multicopper oxidase isolated from S. coelicolor , capable of catalyzing one-electron oxidation of a wide range of substrates to generate radicals while concomitantly reducing molecular oxygen to water[1].

FLO10 is signal peptide suitable for expression in pichia pastoris Lectin-like protein with similarity to Flo1 p，thought to be expressed andinvolved in flocculation.[2]

P. pastoris is usually the preferred host for the production of industrial enzymes.

Figure1.This is the pathway of this composite part.

DNA Gel Electrophoretic

To confirm the function of this part, first we confirm that the gene is transferred to P. pastoris GS115 successfully.

1.DNA extraction of the E.coli plasmid and verification of the right fragment.

2.Prepare the competent cells of P. pastoris GS115.

3.Electro transformation.

4.Yeast genome extraction and PCR verification.

As the picture shows, we have constructed the engineering bacteria successfully.

Figure1:This is the DNA Gel Electrophoretic after the PCR of engineering P. pastoris GS115 genomo.


SDS-PAGE

Second, we cultured the engineering P. pastoris GS115(FLO10-SLAC)in the buffered glycerol-complex medium (BMGY) and induced it in buffered minimal methanol medium (BMM).

Figure3:The SDS-page result shows that engineering P. pastoris GS115(FLO10-SLAC) have successfully secrete SLAC protein into superfluous liquid.


Enzyme Activity

Laccase activity was determined at room temperature (22–25 °C) using ABTS. Oxidation of ABTS (1 mM) was measured at 420 nm (ε = 36,000 M−1 cm−1) in 20 mM acetate buffer (pH 4.0).

By using this formula: 〖activity=(A2−A1)〗∕t∗11244

We obtain the follow figure that represent the enzyme activity changes with time.

Figure4:This is the engineering P. pastoris GS115(FLO10-SLAC) enzyme activity curve.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 937
    Illegal BamHI site found at 948
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1387


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