Part:BBa_K3805329:Experience

1. Construction of pathways

We obtain the gene fragments of araC and pBAD promoter by PCR from the plasmid given to us by Sichuan University, and insert the required enzyme digestion sites at both ends. After the completion of PCR, electrophoresis is performed to determine the size of DNA, and then DNA fragments are recovered. Finally, we use the restriction endonucleases for digestion, and then ligate the araC and pBAD promoter fragments into PUC19 plasmids using T4 ligase. Subsequently, in order to verify the inhibitory effect of araC on pBAD promoter, we insert GFP (green fluorescent protein) downstream of pBAD promoter using the same method mentioned above. The intensity of GFP fluoresence represents the transcriptional level of pBAD promoter.

Figure 1 proinsulin normal 1+3-pathways

Figure 2 proinsulin mutant 1 1+3-pathways

Figure 3 proinsulin mutant 2 1+3-pathways

2.Verification of imported gene pathways

We transform E. coli BL21(DE3) competent cells with the prepared plasmid for overnight culture. Then, we confirm that the constructed plasmid has been successfully transformed E. coli Trans 5α by colony PCR as well as PCR using the extracted plasmid as the template. Finally, we preserve the engineered bacteria in glycerol and store them at -80℃ for standby.

3. Verification of the inhibitory effect of AraC on pBAD promoter

(1) Inhibitory effect of araC on pBAD promoter

We select E. coli BL21(DE3) strain control, proinsulin normal E. coli BL21(DE3) strain, proinsulin mutant 1 E. coli BL21(DE3) strain and proinsulin mutant 2 E. coli BL21(DE3) strain as the four groups of experiment strains. We add 0.6μL 500mM/mL IPTG for each 200μL bacterium to induce the expression of proinsulin and araC fusion protein downstream of the lac operon, then incubate the bacterium at 37℃,200rpm, and the OD600 value and fluorescence intensity of the broth are measured every 30 min.

(2) Fusion expression of araC and proinsulin

We add 60μL 500mM/L IPTG for each 60 mL proinsulin normal strain, proinsulin mutant 1 E. coli BL21(DE3) strain and proinsulin mutant 2 E. coli BL21(DE3) strain to induce the expression of proinsulin and araC fusion protein downstream of the lac operon for 4h. The three fusion expression proteins are extracted using his-tag protein extraction kit and verified by SDS-PAGE gel.

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