To improve glsA (BBa_K4340611) and ldhA (BBa_K4340613) and to design a plasmid that is suitable for the hydroponics system, we designed our genetic pH shooting system. We compared our pH changes, and OD changes to validate the improvement of glsA part.
genetic pH shooting system (BBa_K4340609) and pET11a empty vector pH maintenance functional test
Experiment 1: pH change and OD value
Figure 1. The pH change of genetic pH shooting system_pET11a in the pH 5 initial environment
Figure 2. The pH changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 5 environment.
Figure 3. The pH change of genetic pH shooting system_pET11a in the pH 6 initial environment
Figure 4. The pH change of genetic pH shooting system_pET11a in the pH 7 initial environment
Figure 5. The pH changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 7 environment.
Figure 6. The pH change of genetic pH shooting system_pET11a in the pH 8 initial environment
Figure 7. The pH change of genetic pH shooting system_pET11a in the pH 9 initial environment
Figure 8. The pH changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 9 environment.
Photo 1: The pH adjusted LB broth for pH changes test.
The pH change of the genetic pH shooting system is larger than the control group (pET11a) in the initial pH 5 environment in the first 5 hours, indicating that the genetic pH shooting system worked to converge the pH to neutral pH level. (Figure 1&2)
In the initial pH 6 environment, the convergence of the genetic pH shooting system to neutral pH performed well in the 7th to 9th hours. In the following 15 hours, both the pH levels of the control and genetic pH shooting system group raised to pH 8 due to the possibility of the ammonia generated by the died E.coli. (Figure 3)
In the initial pH 7 environment, the pH curve of both groups are relatively similar, showing that the system does not function in a pH 7 environment, which conforms to the promoter design (Pasr for acidic environment and P-atp2 for alkaline environment) (Figure 4&5)
In both initial pH 8 and pH 9, the pH level of the genetic pH shooting system drops more than the control group (pET11a). This demonstrated that the base shooting circuit functioned to neutralize the alkaline environment. (Figure 7, 8&9)
Experiment 2: OD changes
Figure 1. The OD change of genetic pH shooting system_pET11a and Pasr-glsA_pET11a with pET11a (control) in the pH 5 initial environment
Figure 2. The OD change of genetic pH shooting system_pET11a in the pH 6 initial environment
Figure 3. The OD change of genetic pH shooting system_pET11a and Pasr-glsA with pET11a (control) in the pH 7 initial environment
Figure 4. The OD change of genetic pH shooting system_pET11a in the pH 8 initial environment
Figure 5. The OD change of genetic pH shooting system_pET11a and Pasr-glsA with pET11a (control) in the pH 9 initial environment
Experiment 2: Western blot
Figure 6. Our western blot result shows the protein expression in different pH environments. (glsA for Pasr-glsA, pHS for genetic pH shooting system)
The western blot was able to validate the quality of protein expression of glsA and the pH shooting system. In the experiment, there is a clear band of both the pH shooting system and glsA in 20ul samples at 30 kDa. There is a relatively more blended band of the 10ul samples. As predicted, it is clear that the glsA in the pH5 environment expresses the best.
