Part:BBa_J10050

weak promoter + weak RBS + lacZa.GFP fusion

Designed and built by Natalie Kuldell for BioBuilder teacher's kit. Weak constitutive promoter, weak 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 941
  

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.

Strain Identification Number	Promoter Part Number	RBS Part Number	Relative Strength of Promoter/RBS
R (positive control)	BBa_J23115	BBa_B0035	Reference/Reference
1	BBa_J23113	BBa_B0031	Weak/Weak
2	BBa_J23113	BBa_B0032	Weak/Medium
3	BBa_J23113	BBa_B0034	Weak/Strong
4	BBa_J23106	BBa_B0031	Medium/Weak
5	BBa_J23106	BBa_B0032	Medium/Medium
6	BBa_J23106	BBa_B0034	Medium/Strong
7	BBa_J23119	BBa_B0031	Strong/Weak
8	BBa_J23119	BBa_B0032	Strong/Medium
9	BBa_J23119	BBa_B0034	Strong/Strong

Figure 1: The highlighted points in yellow represent BBa_J10050 (weak promoter/ weak RBS) compared to the remaining nine combinations highlighted in blue. Combinations showing promoters with the same strength, but different RBS, share similar expression. An increase in promoter strength results in an increase in expression; on the other hand, changes in RBS strength has a negligible effect on expression.