Method for inducing protein interaction and gene expression by pesticide
A technology for inducing protein and gene expression, applied in the direction of microorganism-based methods, chemical instruments and methods, biochemical equipment and methods, etc., to achieve the effect of increasing usage
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Embodiment 1
[0047] A method for pesticide-induced protein interaction and gene expression, in which mandipropamid induces complementation of three fragments of green fluorescent protein, the steps are:
[0048] Step 1: Construct plasmid 2 based on plasmid 1, ABI-CP N-terminal fusion GFP10, PYR1 MANDI Carboxy-terminally fused to GFP11.
[0049] Step 2: Plasmid 3 was constructed to induce expression of GFP1-9 fragments in Escherichia coli controlled by T7 promoter_lacO to match the IPTG induction system of Escherichia coli BL21 (DE3) strain.
[0050] Step 3: Transform plasmids 2 and 3 into Escherichia coli BL21(DE3) strain.
[0051] Step 4, detection of the expression products of plasmids 2 and 3 induced by mandipropamid in Escherichia coli: take the positive clones of Escherichia coli BL21 (DE3) strain background and shake the bacteria, wait for OD 600 When it reaches 0.40-0.60, add IPTG (isopropylthiogalactopyranoside) with a final concentration of 1mM to the bacterial solution to induc...
Embodiment 2
[0054] Mandipropamid induces the expression of the gene encoding green fluorescent protein:
[0055] Step 1: Construction of Plasmid 4 to Combine Split T7 RNA Polymerase with PYR1 in E. coli MANDI Fusion expression with ABI-CP respectively;
[0056] Step 2: construct plasmid 5, use the T7 promoter to drive the expression of the GFP-encoded gene, and detect the activity of mandipropamid-induced T7 RNA polymerase in Escherichia coli;
[0057] Step 3: transform plasmids 4 and 5 into E. coli Top10 strain. Note that strains with endogenous T7 RNA polymerase cannot be used, such as BL21(DE3) strains;
[0058] Step 4: Detect that the expression products of plasmids 4 and 5 are induced by mandipropamid in E. coli: Take the positive clones of the background of E. coli Top10 strains and shake them until OD 600 When it reaches 0.40-0.60, add mandipropamid with a final concentration gradient of 0.1μM, 1μM, 10μM, 100μM, 200μM, 400μM to the bacterial solution, and use the following param...
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