Team:NCKU Tainan/Improve
Improve
Part Improvement
During the designation of the experiment for our pH alert system, we have concluded that we should try to improve the expression of our pH-sensitive promoter, promoter gadA. The motivation for us to do this is because our team think that if we can build a system which could observe the situation in our device by looking at the color change of the medium, it should be an economic friendly as there is no more extra charges to send your sample to someway for checking and also wasting a lot of time for waiting the result. But according to the expression of promoter gadA from the previous team did, it is not enough to be observed by using our eyes.
Based on our research, RiboJ first described as it is an insulator in genetic construct but there has no any data shown this insulation effected the downstream genetic parts. In 2018, there was a team has proved that RiboJ could increase the expression of downstream gene and they are from collage of William and Mary. Hence, our team, NCKU_Tainan has desided to enhance the expression of promoter gadA by adding a gene, RiboJ at the downstream of promoter gadA.
For the function determination, we have measured the fluorescent density of the plasmid with and without RiboJ in different pH environment. We incubated the bacteria in pH adjacent M9 medium and measure the fluorescent intensity (absorbance: 480nm, excitation: 510 nm) in a short period of time.
Experiment Result
Figure 1: The fluorescent intensity expressed by PgadA of 24 hours in four different pH value of M9 medium.
Figure 2: The fluorescent intensity expressed by PgadA-RiboJ of 24 hours in four different pH value of M9 medium.
Figure 3: The fluorescent intensity between PgadA and PgadA-RiboJ in pH 6 and pH 7.
Conclusion
Based on our functional determination test results, the improvement that we did has succeeded. As you could see, E. coli that carrying promoter gadA with RiboJ (Figure 2) has the higher fluorescent level than the E. coli that carrying promoter gadA but without RiboJ (Figure 1). From our experiment result, we also could conclude that RiboJ not only can enhance expression but it could also strengthen the signal and increase the specificity of the downstream gene of it. So, our team has successfully improve the part of promoter gadA with the addition of RiboJ gene.