Part:BBa_K4340603

Pasr-glsA

Pasr-glsA (BBa_K4340603)pH maintenance functional test

This is a plasmid that can neutralize an acidic environment with ammonia.

These are the results of our Pasr-glsA in pET11a plasmid test (comparing with sfGFP_pET11a). We measured the pH, OD 600, and fluorescence of the cell culture of the transformed E.coli. We use these three indicators to validate the pH neutralizing function and the survival and growth curve of the E.coli transformed with Pasr-glsA_pET11a plasmid.

Experiment 1: pH change

Figure 1. The pH change of genetic pH shooting system_pET11a and Pasr-glsA-pET11a against empty pET11a vector 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 against empty pET11a vector 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 against empty pET11a vector control in the pH 9 initial 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. 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.

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

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

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.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 983
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 983
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 983
  Illegal AgeI site found at 1558
  
• 1000
  COMPATIBLE WITH RFC[1000]