Difference between revisions of "Part:BBa K4340603"

Revision as of 13:25, 12 October 2022 (view source) Gina Liu (Talk | contribs) (→‎3. Fluorescence test) ← Older edit		Revision as of 14:31, 12 October 2022 (view source) Gina Liu (Talk | contribs) (→‎Pasr-glsA (BBa_K4340603)pH maintenance functional test) Newer edit →
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	[[FIle:glsa-pcr.png |200px|thumb|center| Figure 10. The real-time PCR test result of Pasr-glsA.]]		[[FIle:glsa-pcr.png |200px|thumb|center| Figure 10. The real-time PCR test result of Pasr-glsA.]]
	<p>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.</p>		<p>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.</p>
		+
		+	===Experiment 1: pH change===
		+
		+	[[File:Compare ph glsa phs ph5.jpeg|400px|thumb|center|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]]
		+
		+
		+	[[File:PHS-pH-6.png|400px|thumb|center|Figure 2. The pH change of genetic pH shooting system_pET11a in the pH 6 initial environment]]
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		+	[[File:Compare ph glsa phs ph7.jpeg|400px|thumb|center|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]]
		+
		+
		+	[[File:PHS-pH-8.png|400px|thumb|center|Figure 4. The pH change of genetic pH shooting system_pET11a in the pH 8 initial environment]]
		+
		+	[[File:Compare ph glsa phs ph9.jpeg|400px|thumb|center|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]]
		+
		+
		+
		+	[[File:PHS 2.png|400px|thumb|center|Photo 1: The pH adjusted LB broth for pH changes test.]]
		+
		+
		+	<p>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)</p>
		+
		+	<p>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)</p>
		+
		+	<p>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) </p>
		+
		+	<p>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)</p>
		+
		+	<p>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.</p>
		+
		+	===Experiment 2: OD changes===
		+
		+	[[File:compare_od_glsa_phs_ph5.png|400px|thumb|center|Figure 1. The OD change of genetic pH shooting system_pET11a and Pasr-glsA_pET11a with pET11a (control) in the pH 5 initial environment]]
		+
		+	[[File:PHS-OD-6.png|400px|thumb|center|Figure 2. The OD change of genetic pH shooting system_pET11a in the pH 6 initial environment]]
		+
		+	[[File:compare_od_glsa_phs_ph7.png|400px|thumb|center|Figure 3. The OD change of genetic pH shooting system_pET11a and Pasr-glsA with pET11a (control) in the pH 7 initial environment]]
		+
		+	[[File:PHS-OD-8.png|400px|thumb|center|Figure 4. The OD change of genetic pH shooting system_pET11a in the pH 8 initial environment]]
		+
		+	[[File:compare_od_glsa_phs_ph9.png|400px|thumb|center|Figure 5. The OD change of genetic pH shooting system_pET11a and Pasr-glsA with pET11a (control) in the pH 9 initial environment]]
		+
		+	<p>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.</p>
		+
		+	===Experiment 2: 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)]]
		+
		+	<p>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.</p>
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Revision as of 14:31, 12 October 2022

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.

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.

Experiment 1: pH change

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

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

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