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 the previously constructed pH-sensitive promoters by 2016 iGEM Dundee team, PgadA (BBa_K1962013). We found out that although PgadA can be induced under neutral and weak acidic condition. But the fluorescent intensity is too low to be observed. We planned to increase the fluorescent intensity and shorten the time interval of induction. Thus, we added a RiboJ sequence at the downstream of the PgadA.
Based on our research, RiboJ was first described as an insulator in genetic construct but there has no any data shown this insulation affected 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 (BBa_K1962013)and PgadA with RiboJ (BBa_K2762016) 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
We observe that with the RiboJ sequence, the fluorescence intensity has dramatically risen at 14th hour. We also compare the fluorescent at 3rd, 6th, and 9th hour. The fluorescence of the strain that contains RiboJ is more than that does not contain RiboJ. We can conclude that by adding the RiboJ sequence to the construct, the expression of fluorescent protein is increase. We can also could conclude that riboJ could also strengthen the signal and increase the specificity of the downstream gene of it. We improve the PgadA part so the difference of the surrounding pH condition can be observed more easily.