Overview
One of our goals is to create a better L-arabinose induced constitutive promoter. Here, we used a constitutive promoter (BioBrick_BBa_J23106) to co-regulate with pBad promoter (BBa_K206000). The pBad promoter is a positively regulated promoter, which will be inhibited by araC without the presence of L-arabinose. The L-arabinose can bind with araC and turn araC from a promoter inhibitor to a promoter inducer.
In our kill switch design, we hope to generate a stronger pBad promoter with greater expression rate and wider inducing range. With the regulation of both constitutive promoter and L-arabinose induced pBad promoter, we hope it can help the pBad to increase its expression ability.
Fig 1. A schematic of our biobrick construction.
In other words, we are trying to improve BioBrick_BBa_K206000. We want to ligate pBad promoter with a constitutive promoter to form a tandem promoter. We hypothesized that by adding a constitutive promoter beside pBad, the expression of the tandem promoter will be better than the normal pBad promoter (BioBrick_BBa_K206000). We compared the intensity of RFP gene constructed behind the pBad promoter and pBad-J23106 tandem promoter to see if the tandem promoter has better expression rate.
Experiment Results
RFP Intensity Experiment
We made a series of liquid cultures with different concentrations of L-arabinose. Then, we cultured
E. coli DH5-alpha with pBad and pBad-J23106 separately. After making the bacteria shake at 24°C for 24 hrs, we tested the RFP intensity and OD600 of these liquid cultures.
Fig 2. The comparison between pBad and pBad-J23106 after 24hrs induction.
According to the figure, we can see that pBad-J23106 significantly outshines the original BioBrick_BBa_K206000. When the L-arabinose is lower than 0.025 M, both the pBad and pBad-J23106 promoters were induced. The RFP intensity of the improved part pBad-J23106 reached about 17000 at the L-arabinose concentration of 0.025, which is eight times higher than the original part pBad. The expression rate of pBad-J23106 still exceeds the origin part pBad at the concentration of L-arabinose higher than 0.025. We implemented this design in our kil-switch. Please visit our project
Results page for more information.
References
[1] Öztürk, S., Ergün, B. G., & Çalık, P. (2017). Double promoter expression systems for recombinant protein production by industrial microorganisms. Applied microbiology and biotechnology, 101(20), 7459–7475.
