Difference between revisions of "Part:BBa J10057"

 
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Designed and built by Natalie Kuldell for BioBuilder teacher's kit. Strong constitutive promoter, medium RBS and the lacZa.GFP fusion protein.
 
Designed and built by Natalie Kuldell for BioBuilder teacher's kit. Strong constitutive promoter, medium RBS and the lacZa.GFP fusion protein.
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===Usage and Biology===
 
  
 
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<partinfo>BBa_J10057 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_J10057 SequenceAndFeatures</partinfo>
  
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===Usage and Biology===
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==Lambert_GA 2019 Characterization==
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Lambert_GA 2019 tested several combinations of constitutive promoters and ribosomal binding sites to characterize each by measuring enzyme activity and therefore protein expression. The gene expressed, LacZ, codes for β-galactosidase (β-gal), which typically breaks down lactose. Instead of using lactose, we added the sugar ONPG (Ortho-Nitrophenyl-β-galactoside). β-gal breaks ONPG down into galactose and ONP (Ortho-Nitrophenol), which has a yellow color. If there is more ONP present, there is more enzymatic activity and therefore more expression of LacZ. We used a plate reader to measure absorbance at 420 nm, measuring yellow color, and 600nm, measuring cell density. We inputted those absorbance values into the Miller unit formula to calculate enzymatic activity per cell per milliliter.
  
 
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Revision as of 03:14, 21 October 2019


strong promoter + med RBS + lacZa.GFP fusion

Designed and built by Natalie Kuldell for BioBuilder teacher's kit. Strong constitutive promoter, medium RBS and the lacZa.GFP fusion protein.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 940

Usage and Biology

Lambert_GA 2019 Characterization

Lambert_GA 2019 tested several combinations of constitutive promoters and ribosomal binding sites to characterize each by measuring enzyme activity and therefore protein expression. The gene expressed, LacZ, codes for β-galactosidase (β-gal), which typically breaks down lactose. Instead of using lactose, we added the sugar ONPG (Ortho-Nitrophenyl-β-galactoside). β-gal breaks ONPG down into galactose and ONP (Ortho-Nitrophenol), which has a yellow color. If there is more ONP present, there is more enzymatic activity and therefore more expression of LacZ. We used a plate reader to measure absorbance at 420 nm, measuring yellow color, and 600nm, measuring cell density. We inputted those absorbance values into the Miller unit formula to calculate enzymatic activity per cell per milliliter.