Difference between revisions of "Part:BBa K2967028"

 
 
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__NOTOC__
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<partinfo>BBa_K2967028 short</partinfo>
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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.
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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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'''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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https://static.igem.org/mediawiki/parts/thumb/f/fb/T--NEU_China--result--myro-wb.png/600px-T--NEU_China--result--myro-wb.png
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'''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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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2967028 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K2967028 parameters</partinfo>
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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.

800px-T--NEU_China--part--Myro_vector.png

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.

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℃ 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