Coding

Part:BBa_K4340611

Designed by: Chein Yueh Liu   Group: iGEM22_PuiChing_Macau   (2022-10-03)


glsA

glsA pH maintenance functional test

This is a coding part that can neutralize an acidic environment with ammonia. gene code: 946187

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.

1. pH change test

Figure 1. The pH change in 24 hours of glsA compared to sfGFP (BBa_K4340605) as the control group starting from initial pH 5.
Figure 2. The pH change in 24 hours of glsA compared to sfGFP (BBa_K4340605) as the control group starting from initial pH 7.
Figure 3. The pH change in 24 hours of glsA compared to sfGFP (BBa_K4340605) as the control group starting from initial pH 9.

As the Pasr-glsA_pET11a plasmid is an acid shooting circuit that functions at a low pH environment, the pH change of Figure 2 is very significant in that the pH converges to pH 7 after 24 hours. For figure 3, which is in a pH 7 environment, the pH of Pasr-glsA_pET11a culture drops to pH 6.5 in the first three hours and increases firmly from the third to the ninth hour. Then, starting from the ninth to the 24th hour, the pH increases to around pH 7.4. Overall, the Pasr-glsA_pET11a plasmid does not make a shift change in the pH 7 environment, which is the same result as predicted. In Figure 4, which is in a pH 9 environment where Pasr-glsA_pET11a should not function, the pH first drops to around pH 7.4 in the first nine hours and climbs up to pH 7.8 slowly from the ninth hour to the 24th hour. At the same time, the control group sfGFP (BBa_K4340605)follows the same pattern, which indicates that the Pasr-glsA_pET11a does not function in a high pH environment.

Photo 1: The glsA transformed E.coli plate.
Photo 2: The glsA transformed E.coli plate.

2. OD change test

Figure 4. The OD changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 5 environment.
Figure 5. The OD changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 7 environment.
Figure 6. The OD changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 9 environment.

We also tested the OD value of the E.coli transformed with Pasr-glsA_pET11a and the sfGFP_pET11a (as a control group). In the glsA group, E.coli grows best at pH 7, following pH 5, and finally at pH 9. Since Pasr-glsA constructs can only work to neutralize a low pH environment, the result is as predicted. However, in the sfGFP control group, the highest OD600 rate is in the pH7 environment.

3. Fluorescence test

Figure 7. The Fluorescence Rate in pH 5 initial environment of sfGFP_pET11a and Pasr-glsA_pET11a.
Figure 8. The Fluorescence Rate in pH 7 initial environment of sfGFP_pET11a and Pasr-glsA_pET11a.
Figure 9. The Fluorescence Rate in pH 9 initial environment of sfGFP_pET11a and Pasr-glsA_pET11a.

The fluorescence of sfGFP in pH 5 is significantly higher than the fluorescence of Pasr-glsA. It is possibly because the protein size of the sfGFP is smaller than Pasr-glsA, which at the same time produces ammonia to neutralize the environment. In a pH 7 environment, the fluorescence of both sfGFP and Pasr-glsA is relatively similar and reached the same point at the ninth hour. In a pH 9 environment, both fluorescence of sfGFP and Pasr-glsA are low compared to data in pH 5 and 7. This proved that sfGFP and Pasr-glsA, which have the same acid promoter (asr) show low fluorescence in a high-pH environment.

4. real-time PCR result

Figure 10. The real-time PCR test result of Pasr-glsA.

In our real-time quantitative PCR test, we can see that the fold change of glsA in pH 6.0 is the highest, followed by pH 6.0 and pH 7.0. The glsA mRNA expressed in acid and weak acid environment, which demonstrates that glsA expressed appropriately as predicted.

Improvements (genetic pH shooting system) PuiChing_Macau 2022

1. pH change test

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

As the Pasr-glsA_pET11a plasmid is an acid shooting circuit that functions at a low pH environment, the pH change of Figure 2 is very significant in that the pH converges to pH 7 after 24 hours. For figure 3, which is in a pH 7 environment, the pH of Pasr-glsA_pET11a culture drops to pH 6.5 in the first three hours and increases firmly from the third to the ninth hour. Then, starting from the ninth to the 24th hour, the pH increases to around pH 7.4. Overall, the Pasr-glsA_pET11a plasmid does not make a shift change in the pH 7 environment, which is the same result as predicted. In Figure 4, which is in a pH 9 environment where Pasr-glsA_pET11a should not function, the pH first drops to around pH 7.4 in the first nine hours and climbs up to pH 7.8 slowly from the ninth hour to the 24th hour. At the same time, the control group sfGFP (BBa_K4340605)follows the same pattern, which indicates that the Pasr-glsA_pET11a does not function in a high pH environment.


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. 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


We also tested the OD value of the E.coli transformed with Pasr-glsA_pET11a and the sfGFP_pET11a (as a control group). In the glsA group, E.coli grows best at pH 7, following pH 5, and finally at pH 9. Since Pasr-glsA constructs can only work to neutralize a low pH environment, the result is as predicted. However, in the sfGFP control group, the highestOD600 rate is in the pH7 environment.

3. 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 No part name specified with partinfo tag.


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