Part:BBa_K2762015

P_gadA-B0034-GFP

Background

The pH sensing system, our side project, is a system that allows us to monitor the pH in the surrounding medium in our device at any time by observing the color change of the medium.

We selected two pH sensitive promoter from E. coli: P_asr and P_gadA. P_gadA will be induced under neutral condition while P_asr will be induced under acidic condition. We cloned a GFP and sfGFP gene downstream of these promoters respectively, whose product will express green fluorescence once the promoter has been activated. For the design of P_gadA sensing system, we took the previous constructed P_gadA biobrick BBa_K1962013 from 2016 iGEM Dundee team as our reference. We also improve the P_gadA biobrick to enhance the expression of GFP.

In conclusion, when the color of the medium turns from turbid yellow to green, it indicates the pH of the medium has altered so we can determine the pH condition of the medium.

Characterization

Fluorescence intensity measurement of P_gadA

P_gadA 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 P_gadA is observed under neutral and mild expression.

Fig 1. The data shows the fluorescence intensity (absorbance: 485 nm, excitation: 535 nm) expressed by P_gadA in M9 medium with 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, P_gadA (BBa_K1962013). We found out that although P_gadA can be induced under neutral and weak acidic condition. But the fluorescence 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 P_gadA.

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 well 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 P_gadA 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 fluorescence 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 fluorescence intensity (absorbance: 480 nm, excitation: 510 nm) in a short period of time. The pH value of M9 medium is adjusted with 1M HCl.

Fig 2. The fluorescence intensity expressed by P_gadA (BBa_K1962013) and P_gadA with RiboJ (BBa_K2762016) of 14 hours in M9 medium with four different pH value.

Fig 3. The fluorescence of P_gadA and P_gadA-RiboJ in pH 6 and pH 7.

We observe that with the RiboJ sequence, the fluorescence intensity has risen dramatically at the 14^th hour. We also compare the fluorescence at 3^rd, 6^th, and 9^th 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 fluorescence protein is increased. We can also conclude that RiboJ could also strengthen the signal and increase the specificity of the downstream gene of it. We improve the P_gadA part so the difference of the surrounding pH condition can be observed easier.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 937