pBAD promoter with mCherry for inducible expression of reporter
This construct is composed of arabinose-inducible promoter pBAD and fluorescent protein mCherry. In our project, we used it to characterize the inducibility of arabinose on pBAD activity.
2021 Team Leiden
This construct is used to calibrate the inducibility of pBAD by L-arabinose. The mCherry fluorescent protein was used as reporter and the construct of p2547::mCherry (BBa_K3962339) was used as an internal reference (Fig. 1).
Figure 1. The fluorescence intensity of strains carrying our construct under different arabinose concentration. The measurement was carried out for 20 h. The x-axis shows the time of each measurement. The y-axis shows the fluorescence of mCherry in the arbitrary unit (AU). The blue data points represent the fluorescent intensity of p2547::mCherry strain, red data points represent the pBAD::mCherry strain. The arabinose concentration is shown at the top of each graph.
The results of the plate reader assay show that the inducible promoter pBAD gave varying levels of expression depending on L-arabinose concentration and constitutive promoter p2547 gave constant expression. At higher concentrations, the expression of pBAD is higher than the activity of p2547, and it is lower at concentrations approaching zero. We used this data to fit a linear regression model to determine at which concentration the level of pBAD equals p2547 expression (Fig. 2).
Figure 2. Linear regression of arabinose concentration relative fluorescence of pBAD compared with p2547. The orange dash lines showed how p2547 related to pBAD transcriptional activity at the arabinose concentration of 0.14% (log5-1.22).
The linear regression model showed that at a L-arabinose concentration of 0.14% (w/v), the expression of genes under pBAD is the same as under p2547.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 1205
- 21Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 979
- 1000Illegal SapI site found at 961