Difference between revisions of "Part:BBa J10051"
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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. | 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. | ||
| + | [[File:T--Lambert GA--tuningalldata.png|800px]] | ||
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Revision as of 16:26, 21 October 2019
weak promoter + strong RBS + lacZa.GFP fusion
Designed and built by Natalie Kuldell for BioBuilder teacher's kit. Weak constitutive promoter, med RBS and the lacZa.GFP fusion protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 939
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.
