Part:BBa_B0035

RBS (B0030 derivative)

-- No description --

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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.

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 the combination of BBa_B0035 (reference RBS) with BBa_J23115 (reference promoter) 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 have a negligible effect on expression.

After we calculated Miller units, the data showed the reference’s level of expression was between the weak and medium promoter’s. As a result, the reference was graphed between the weak and medium promoter.