Difference between revisions of "Part:BBa K2963033:Design"

 
 
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===Design Notes===
 
===Design Notes===
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We use the "Ptac-lacO-lacO-rbs-racE-T7t" part in order to reduce the leaked expression of racE.
 
  
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The strength of <i>racE</i> gene expression affects the proportion of D/L glutamate monomer in γ-PGA. In our project, we wanted to regulate the expression of the <i>racE</i> gene to produce γ-PGA with different D/L-glutamate monomer ratios.
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Tac Promoter contains a kind of operator gene called <i>lacO</i>. We changed the number of operator gene in tac promoter. We used this part to produce γ-PGA with different D/L glutamic acid monomer ratios. Analysis of the results of the preliminary fermentation experiments show that we can roughly produce γ-PGA containing different ratios of D/L glutamic acid monomers. More detailed experimental steps and experimental results please visit our experiment page.
  
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Determination of glutamate racemase activity:
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The cells ferment for 24 h, and the fermentation broth is centrifuged at 4°C. Collect the cells at 10000 rpm. Wash and centrifuge the cells using 0.85% physiological saline. Repeat the wash 3 times.
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The cells are suspended in p H 8.0, 0.1 M Tris-HCl buffer. Sonicate for 10 min in an ice bath. Centrifuge at 12000 rpm for 30 min, and the supernatant (crude enzyme solution) is taken for immediate determination of the relevant enzyme activity.
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Using 100 μL of crude enzyme solution reacts with 100 μL of substrate (0.5 g/L L-glutamic acid) at 30 ° C for 30 min and the reaction is terminated by boiling water bath. The content of D-glutamic acid in the reaction solution is determined by a high performance liquid phase method.
  
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===Source===
 
  
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racE from Bacillus Genome.
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===Source===
  
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===References===
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<i>racE</i> from <i>Bacillus</i> Genome.

Latest revision as of 11:34, 20 October 2019


Ptac(two lacOs)-racE- Producing γ-PGA with different D/L monomer ratio


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 971
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The strength of racE gene expression affects the proportion of D/L glutamate monomer in γ-PGA. In our project, we wanted to regulate the expression of the racE gene to produce γ-PGA with different D/L-glutamate monomer ratios. Tac Promoter contains a kind of operator gene called lacO. We changed the number of operator gene in tac promoter. We used this part to produce γ-PGA with different D/L glutamic acid monomer ratios. Analysis of the results of the preliminary fermentation experiments show that we can roughly produce γ-PGA containing different ratios of D/L glutamic acid monomers. More detailed experimental steps and experimental results please visit our experiment page.

Determination of glutamate racemase activity: The cells ferment for 24 h, and the fermentation broth is centrifuged at 4°C. Collect the cells at 10000 rpm. Wash and centrifuge the cells using 0.85% physiological saline. Repeat the wash 3 times. The cells are suspended in p H 8.0, 0.1 M Tris-HCl buffer. Sonicate for 10 min in an ice bath. Centrifuge at 12000 rpm for 30 min, and the supernatant (crude enzyme solution) is taken for immediate determination of the relevant enzyme activity. Using 100 μL of crude enzyme solution reacts with 100 μL of substrate (0.5 g/L L-glutamic acid) at 30 ° C for 30 min and the reaction is terminated by boiling water bath. The content of D-glutamic acid in the reaction solution is determined by a high performance liquid phase method.


Source

racE from Bacillus Genome.