Difference between revisions of "Part:BBa K4340610"
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<p>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.</p > | <p>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.</p > | ||
− | === | + | ===4. Western blot=== |
[[File:PHS westernblot.png|600px|thumb|center|Figure 6. Our western blot result shows the protein expression in different pH environments. (glsA for Pasr-glsA, pHS for genetic pH shooting system)]] | [[File:PHS westernblot.png|600px|thumb|center|Figure 6. Our western blot result shows the protein expression in different pH environments. (glsA for Pasr-glsA, pHS for genetic pH shooting system)]] |
Revision as of 14:52, 12 October 2022
Contents
Pasr
This is a promoter that is activated in an acidic environment.
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
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
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
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
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)
1. pH change test
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
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. Fluorescence test
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. Western blot
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
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 983
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23INCOMPATIBLE WITH RFC[23]Unknown
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 983
Illegal AgeI site found at 1558 - 1000COMPATIBLE WITH RFC[1000]