Team:NCKU Tainan/Improve
Improve
Part Improvement
After analyzing the experiment result for our pH-sensing system, we have concluded that we should try to improve one of our pH-sensitive promoters, PgadA. We found out that although PgadA can be induced under neutral and weak acidic condition. But the fluorescent intensity is too slow to be observed. We planned to increase the fluorescent intensity and shorten the time interval of induction. We thus add a RiboJ sequence at the downstream of the PgadA.
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. The mechanism of RiboJ is that the upstream sequence as will as RiboJ sequence will cleave after the transcription. The cleavage of promoter and RiboJ sequence aids the design of predictable genetic constructs. In 2016, the iGEM team William and Mary has reported that RiboJ could increase the expression of the downstream gene. Hence, we decided to enhance the expression of PgadA by adding a gene, RiboJ at the downstream of promoter gadA.
To determine the function and compare the improvement of the parts, we have measured the fluorescent intensity (fluorescence (a.u.) / O.D.600) of the construct with and without RiboJ in different pH environment. We incubated the bacteria in pH adjusted M9 medium and measure the fluorescent intensity (absorbance: 480nm, excitation: 510 nm) in a short period of time. The pH value of M9 medium is adjusted with HCl.
Experiment Result
Fig.1 The fluorescent intensity expressed by PgadA and Pgada with RiboJ of 14 hours in four different pH value of M9 medium.
Fig. 2 The fluorescence of 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.