Part:BBa_K4340609

genetic pH shooting system

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.

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 change of genetic pH shooting system_pET11a in the pH 6 initial environment.

Figure 3. The pH change of genetic pH shooting system_pET11a 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 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. (Figure 1)

In the initial pH 6 environment, the convergence of the genetic pH shooting system to neutral pH performed well in 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)

       
       
       Figure 5. The OD change of genetic pH shooting system_pET11a in the pH 5 initial environment.
       Figure 6. The OD change of genetic pH shooting system_pET11a in the pH 6 initial environment.

Figure 7. The OD change of genetic pH shooting system_pET11a in the pH 7 initial environment.

<figure> <figcaption>Figure 8. The OD change of genetic pH shooting system_pET11a in the pH 8 initial environment.</figcaption> </figure>

<figure> " <figcaption>Figure 9. The OD change of genetic pH shooting system_pET11a in the pH 9 initial environment.</figcaption> </figure>

For the OD change of the genetic pH shooting system (Figure 7), The highest OD value is in the pH 7 environment, followed by pH 8, pH 6, pH 5, and pH 9. The OD value of pH 9 is clearly lower than the other pH groups (pH 5, 6, 7, 8, and 9 of genetic pH shooting system), which shows that the transforemd E.coli might not grow as well as E.coli with empty pET11a vector since it has to produce alkaline.

Experiment 2: Western blot

<figure>

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<center><figcaption>Figure 11. Our western blot result shows the protein expression in different pH environments. (glsA for Pasr-glsA, pHS for genetic pH shooting system)</figcaption> </figure>

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.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 922
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1191