Difference between revisions of "Part:BBa K2967028"
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| + | Expressing vector has been constructed using pcold-1 backbone. We demonstrate western blotting to show the expression of myrosinase. Expressing vector harboring myrosinase gene is transformed into BL21 strain using chemical method. After incubating at 37℃ for 12h, CFU is inoculated to LB medium followed by 12h incubation. The culture is then diluted to OD600=0.2 and let grow to around 0.5. 1 mM IPTG is added to the culture to induce protein expression followed by 12h incubation. Cells are washed and collected, ready for western blotting. The molecular weight of protein myrosinase is 57.46 kDa. | ||
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| + | Figure 1. Diagram for myrosinase expressing plasmid in pcold-1. Promoter cspA, a super strong promoter when incubating at low temperature. RBS, ribosome binding site, downstream is gene myrosinase. | ||
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| + | Figure 2. Expressing vector harboring myrosinase gene is transformed into BL21 strain using chemical method. After induction of IPTG, the culture is incubated at 37℃ overnight. The concentration of protein loaded on this two lanes are different. | ||
Revision as of 10:57, 6 October 2019
Expressing vector for YebF-Myrosinase
Expressing vector has been constructed using pcold-1 backbone. We demonstrate western blotting to show the expression of myrosinase. Expressing vector harboring myrosinase gene is transformed into BL21 strain using chemical method. After incubating at 37℃ for 12h, CFU is inoculated to LB medium followed by 12h incubation. The culture is then diluted to OD600=0.2 and let grow to around 0.5. 1 mM IPTG is added to the culture to induce protein expression followed by 12h incubation. Cells are washed and collected, ready for western blotting. The molecular weight of protein myrosinase is 57.46 kDa.
Figure 1. Diagram for myrosinase expressing plasmid in pcold-1. Promoter cspA, a super strong promoter when incubating at low temperature. RBS, ribosome binding site, downstream is gene myrosinase.
Figure 2. Expressing vector harboring myrosinase gene is transformed into BL21 strain using chemical method. After induction of IPTG, the culture is incubated at 37℃ overnight. The concentration of protein loaded on this two lanes are different.