• genetic pH shooting system (BBa_K4340609)
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genetic pH shooting system (BBa_K4340609) pSB1C3 redesigns References


genetic pH shooting system validation

To improve Pasr-glsA, we designed a pH shooting system which contains two circuits: ASC (acid shooting circuit) and BSC (base shooting circuit).

Experiment 1: pH and OD change of genetic pH shooting system and Pasr-glsA compared with control

In our experiment, we accomplished a pH change test and monitored its OD value to demonstrate the survival of the bacteria, and ensure that the pH change is conducted by our transformed E.coli. On the other hand, we have conducted a western blot experiment to validate the expression of glsA in pH 5, 6, and 7; the expression of ldhA in pH 5, 7, and 9 by testing the existence of the related protein.

1. pH change test
Figure 1. The pH change of genetic pH shooting system_pET11a and Pasr-glsA_pET11a with pET11a (control) in the pH 5 initial environment.
Figure 2. The pH change of genetic pH shooting system_pET11a in the pH 6 initial environment.
Figure 3. The pH change of genetic pH shooting system_pET11a and Pasr-glsA_pET11a with pET11a (control) in the pH 7 initial environment.
Figure 4. The pH change of genetic pH shooting system_pET11a in the pH 8 initial environment.
Figure 5. The pH change of genetic pH shooting system_pET11a and Pasr-glsA_pET11a with pET11a (control) in the pH 9 initial environment.

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. However, compared with Pasr-glsA, this system has less efficiency in acidic environment adjusting.(Figure 1)

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 2)

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 3)

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 4&5)

To sum up, the genetic pH shooting system worked and optimize the Pasr-glsA construct, with an alkaline adjusting system and a stable pH neutralizing ability.


2. OD change test

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. Figure 1. The OD change of genetic pH shooting system_pET11a and Pasr-glsA_pET11a 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_pET11a with pET11a (control) in the pH 9 initial environment

Overall, the OD of the genetic pH shooting system is higher than the Pasr-glsA. This demonstrated that the genetic pH shooting system worked to survive better in an acidic and alkaline environment. Particularly, the OD curve of the pH shooting system is significantly higher than the Pasr-glsA in a pH 9 environment, indicating that the base circuit facilitated the E.coli growth in an alkaline environment.

For the OD change of the genetic pH shooting system, The highest OD value is in the pH 7 environment, followed by pH 8, and pH 9. The OD value of pH 9 is lower than the other pH groups (pH 7, 8, and 9 of the genetic pH shooting system), which shows that the transformed E.coli might not grow as well as E.coli with an empty pET11a vector since it has to produce alkaline.


Experiment 2: Western Blot and Real-time PCR

We also did real-time PCR to validate if our ideal genes have been expressed through the CT value of the mRNA.

Prepare protein for western blot

We first collected 50mL of the sample (LB cell culture), and centrifuged them to isolate the LB broth and the cells. Then, we used sonication to break the cells so the protein will dissolve in the solution. Therefore, we could separate the protein from other substances in the cells. Through this, we extracted the protein before starting to do a western blot. Next, we added the loading buffer into the protein solution for preparing the following experiment and stored it at -80 celsius.

The next day, we ran the gel at 80V for 2 hours and transferred it to a membrane. We used dry milk powder for blocking and soaked the membrane in an antibody solution. After washing it multiple times with a buffer solution, the membrane was ready for chemiluminescence photography.

Figure 1. 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 blurry band of the 10ul samples. But overall, it is clear that the glsA in the pH5 environment expresses the best.

sfGFP_pSB1C3, asr-glsA_pSB1C3, and genetic pH shooting system_pSB1C3 plasmids validation (Redesigned)

We designed these three constructs with pET11a vectors. However, we found that IPTG induction, which is required in a T7 system for higher efficiency and yield, is not ideal for our hydroponic system. It would be difficult for our dried E.coli powder to go through such an induction procedure and therefore increase the difficulties to promote our product to the public. Hence, after we discussed with our technical assistant [Refer to Integrated Human Practice 1], we redesigned our plasmids with the pSB1C3 system, an optimal choice for our genetic pH shooting system which is composed of two circuits.

Eventually, we received our plasmids design in September and performed a basic functional test to test the function of our genetic pH shooting system_pSB1C3 with sfGFP_pSB1C3 as the control group in LB medium of pH 5, 7, and 9; we also tested Pasr-glsA_pSB1C3 in LB medium of pH 5 and 7. Besides, we also used three pH cloud meters, which can store data automatically on a cloud drive, in the hydroponic system to record the pH changes from pH 5.

Experiment 1: pH changes in LB medium, pH 5, 7, and 9

Photo 1. The cell culture in pH 5, genetic pH shooting system, Pasr-glsA, sfGFP in pSB1C3 vector (from left to right) under UV light.
Figure 1. The pH changes by the newly designed pH system compared with the initial pH 5 environment between glsA, pH shooting system, and control (pET11a empty vector).
Figure 2. The pH changes by the original pH system compared with the initial pH 5 environment between glsA, pH shooting system, and control.
Figure 3. The pH changes by the newly designed pH system compared with the initial pH 7 environment between glsA, pH shooting system, and control.
Figure 4. The pH changes by the original pH system compared with the initial pH 7 environment between glsA, pH shooting system, and control (pET11a empty vector)
Figure 5. The pH changes by the newly designed pH system compared with the initial pH 9 environment between the pH shooting system and control (pET11a empty vector)
Figure 6. The pH changes by the original pH system compared with the initial pH 9 environment between genetic pH shooting system-pET11a and control (pET11a empty vector)

In relative initial pH levels, the new glsA and pH shooting constructs regulated pH as efficiently as the original constructs within the first ten hours. When the initial pH is 5, by the tenth hour, both Pasr-glsA-pSB1C3 and genetic pH shooting system-pSB1C3 were working well due to the acid promoter (asr) of their respective glsA genes. When the initial pH is 7, the pH was roughly neutral and unchanged at the tenth hour. When the initial pH is 9, the ldhA gene of the genetic pH shooting system-pSB1C3 was expressed through its base promoter, P-atp2. It worked well in the first ten hours, lowering the pH by 0.8. In all three cases, the lines converged by the 24th hour. This can be explained by expected non-apoptotic cell death, in which the cells may discharge substances that can alter pH. In general, the results show that the newly designed constructs can function well without IPTG induction, so it is more convenient for the implementation of our transformed E.coli in plant growth.

Experiment 2: pH changes in a hydroponic system, pH 5, 7, and 9


Photo 2. The pH monitoring system set up

We have set up a system with three pH meters and a Wi-Fi module to test the pH of the hydroponic system for the whole day. The pH meter will mark the data and the Wi-Fi module will send it to the internet, so we can monitor the value whenever we want.

photo 3. the online pH value collecting system

After 120 hours, we finally got our data.

Figure 1. Comparison of Pasr-glsA-pSB1C3, genetic pH shooting system-pSB1C3, and control under an acidic environment of a hydroponic system

It can be seen from figure 1 that when glsA and genetic pH shooting system transformed cells were present, the pH was stable at around the 6.5 range. Although the line might not be so smooth due to the instability of the current in the pH meter, the overall trend shows an expected, stable result. When it comes to the control, the pH value keeps rising continuously, which is deleterious to the germination and growth of plants.

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