Difference between revisions of "Part:BBa K2967028"

Latest revision as of 09:20, 16 October 2019

Expressing vector for YebF-Myrosinase

Expressing vector has been constructed using pcold-1 backbone. We demonstrated western blotting to show the expression of myrosinase. Expressing vector harboring myrosinase gene was transformed into BL21 strain using chemical method. After incubating at 37℃ for 12h, CFU was inoculated to LB medium followed by 12h incubation. The culture was 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 were 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.Protein expression of myrosinase gene which transformed in BL21 strain. After induction of IPTG, the culture is incubated at 37℃ overnight. The concentration of protein loaded on this two lanes were different.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 689
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 918
Illegal BsaI site found at 1947

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 <partinfo>BBa_K2967028 short</partinfo>
-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&#8451; 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.
+Expressing vector has been constructed using pcold-1 backbone. We demonstrated western blotting to show the expression of myrosinase. Expressing vector harboring myrosinase gene was transformed into BL21 strain using chemical method. After incubating at 37&#8451; for 12h, CFU was inoculated to LB medium followed by 12h incubation. The culture was then diluted to OD<sub>600</sub> =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 were washed and collected, ready for western blotting. The molecular weight of protein myrosinase is 57.46 kDa.
 https://static.igem.org/mediawiki/parts/thumb/0/05/T--NEU_China--part--Myro_vector.png/800px-T--NEU_China--part--Myro_vector.png
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 https://static.igem.org/mediawiki/parts/thumb/f/fb/T--NEU_China--result--myro-wb.png/600px-T--NEU_China--result--myro-wb.png
-'''Figure 2.Protein expression of myrosinase gene which transformed in BL21 strain.''' After induction of IPTG, the culture is incubated at 37&#8451; overnight. The concentration of protein loaded on this two lanes are different.
+'''Figure 2.Protein expression of myrosinase gene which transformed in BL21 strain.''' After induction of IPTG, the culture is incubated at 37&#8451; overnight. The concentration of protein loaded on this two lanes were different.
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