Difference between revisions of "Part:BBa K3388014"

 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3388014 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3388014 SequenceAndFeatures</partinfo>
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<p>The Characteristic of UGT89C1 Protein：
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Molecular weight（kD）：48.12
 +
Isoelectric point：5.77
 +
Large Average Hydrophilicity：-0.034
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Instability index：38.49
 +
Half life period：>10h
 +
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1.plasmid construction
 +
Due to the influence of the COVID-19 epidemic, we have little time to do the cloning.
 +
Therefore, we sent our plasmid file to Genescript, which helped us optimize the gene sequence and cloned it into the target vector.
 +
We asked Genescript to clone UGT89C1 gene into pCDFDuet, and send us freeze-dried plasmid and glycerol bacteria.
 +
 +
2. Gel imaging
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According to the instructions, we performed gel imaging on Escherichia coli DE3 transformed with pGEX-UGT78D2 plasmid and pCDF-UGT89C1 plasmid. As shown in the figure, the band of the sample was compared with marker, which proved that we successfully introduced the target gene into bacteria.
 +
<img src="https://parts.igem.org/File:T--NWU-CHINA-B--ningjiao.jpg" />
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3.Glycosylation module experiment
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3.1Strains and growth
 +
Using E. coli DE3 as the expression host, 2ml seed solution was prepared. Take the 100-fold diluted seed solution, transfer it to liquid LB medium, and grow at 37°C so that its OD value reaches 0.8.
 +
3.2Add 10ul IPTG with concentrations of 0.2mM, 0.4Mm, 0.6mM, 0.8mM and 1.0mM to 10ml liquid LB medium in a 150ml conical flask. Growing at 18℃ for 20 hours, measuring and recording the OD of bacterial liquid at different IPTG concentrations every 1 hour from the first hour, for a total of 20 hours. After 20 hours, the proteins with different IPTG concentrations were purified (GST tag protein affinity chromatography kit, a small amount of extraction), and the concentrations were measured by biodrop and recorded.
 +
3.3 Induction of protein expression
 +
Induce a large amount of protein expression with the optimal inducer concentration. After 20 hours, purify the protein at each IPTG concentration (GST-tagged protein affinity chromatography kit and 6xHis-tagged protein kit, mass extraction), measure the concentration with nanodrop, and record.</p>
  
  

Revision as of 02:52, 28 October 2020


pCDF-UGT89C1

It is used to synthesize kaempferol -3-O- glucose -7-O- rhamnose with astragaloside and UDP- rhamnose as substrates.Its resistance gene is streptomycin.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 880
    Illegal XhoI site found at 1045
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 762
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 985

The Characteristic of UGT89C1 Protein： Molecular weight（kD）：48.12 Isoelectric point：5.77 Large Average Hydrophilicity：-0.034 Instability index：38.49 Half life period：>10h 1.plasmid construction Due to the influence of the COVID-19 epidemic, we have little time to do the cloning. Therefore, we sent our plasmid file to Genescript, which helped us optimize the gene sequence and cloned it into the target vector. We asked Genescript to clone UGT89C1 gene into pCDFDuet, and send us freeze-dried plasmid and glycerol bacteria. 2. Gel imaging According to the instructions, we performed gel imaging on Escherichia coli DE3 transformed with pGEX-UGT78D2 plasmid and pCDF-UGT89C1 plasmid. As shown in the figure, the band of the sample was compared with marker, which proved that we successfully introduced the target gene into bacteria. <img src="https://parts.igem.org/File:T--NWU-CHINA-B--ningjiao.jpg" /> 3.Glycosylation module experiment 3.1Strains and growth Using E. coli DE3 as the expression host, 2ml seed solution was prepared. Take the 100-fold diluted seed solution, transfer it to liquid LB medium, and grow at 37°C so that its OD value reaches 0.8. 3.2Add 10ul IPTG with concentrations of 0.2mM, 0.4Mm, 0.6mM, 0.8mM and 1.0mM to 10ml liquid LB medium in a 150ml conical flask. Growing at 18℃ for 20 hours, measuring and recording the OD of bacterial liquid at different IPTG concentrations every 1 hour from the first hour, for a total of 20 hours. After 20 hours, the proteins with different IPTG concentrations were purified (GST tag protein affinity chromatography kit, a small amount of extraction), and the concentrations were measured by biodrop and recorded. 3.3 Induction of protein expression Induce a large amount of protein expression with the optimal inducer concentration. After 20 hours, purify the protein at each IPTG concentration (GST-tagged protein affinity chromatography kit and 6xHis-tagged protein kit, mass extraction), measure the concentration with nanodrop, and record.