This is a composite part that including PgadA, a pH sensitive promoter, a ribozyme sequence, RiboJ, a RBS and a GFP sequence. Promoter gadA is a pH sensitive promoter from E. coli, described by Dundee iGEM 2016(BBa_K1962013). For the part of RiboJ, it 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 and the iGEM team William and Mary 2016 has reported that RiboJ could increase the expression of the downstream gene.
Function of gadA promoter
Our team, NCKU_Tainan 2018 has decided to use gadA promoter as one of the promoter in our pH sensing system but there has a problem, the fluorescent intensity is too low to be observed. To solve this problem, we have added RiboJ at the downstream of gadA promoter to improve the biobrick. In result, the fluorescence intensity of the strain that contains RiboJ is more than the strain that without RiboJ. In conclusion, we have successfully strengthen the signal and increase the specificity of gadA promoter. Thus, we could determine the pH condition of the medium when the color of the medium turns from turbid yellow to green.
Colony PCR to confirm the size
Fluorescence intensity measurement of PgadA
PgadA was previously reported to be induced under neutral and mild acidic environment. We measure the fluorescence intensity for 14 hours. We pre-cultured the strain and incubate the strain with pH modified M9 medium (the pH value is modified with 1M HCl). The induction of PgadA is observed under neutral and mild expression.
Fig 1. The data shows the fluorescence intensity (absorbance: 485 nm, excitation: 535 nm) expressed by PgadA in different pH.
Improvement of Biobrick
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. The 2016 iGEM William and Mary team 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.
Fig 2. 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 3. The fluorescence of PgadA and PgadA-RiboJ in pH 6 and pH 7.
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 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.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI.rc site found at 1020
Kalen P Clifton, Ethan M Jones, Sudip Paudel, John P Marken, Callan E Monette, Andrew D Halleran, Lidia Epp, Margaret S Saha. (2018,May.9). The Genetic Insulator RiboJ Increases Expression of Insulated Genes. bioRxiv