Difference between revisions of "Part:BBa K4340609"

Revision as of 00:56, 12 October 2022 (view source) Gina Liu (Talk | contribs) ← Older edit		Latest revision as of 14:50, 12 October 2022 (view source) Gina Liu (Talk | contribs) (→‎Experiment 2: Western blot)
(19 intermediate revisions by the same user not shown)
Line 7:		Line 7:
	==genetic pH shooting system (BBa_K4340609) and pET11a empty vector pH maintenance functional test==		==genetic pH shooting system (BBa_K4340609) and pET11a empty vector pH maintenance functional test==
−	===Experiment 1: pH change and OD value===	+	===Experiment 1: pH change===
−	<table>	+
−	<tr>	+
−	<td>	+
−	[[File:PHS-OD-5.png|400px|thumb|center|Figure 1. The OD change of genetic pH shooting system_pET11a in the pH 5 initial environment]]	+
−	</td>	+	[[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]]
−	<td>	+
−	[[File:glsa-od-ph5.png  |400px|thumb|center| Figure 4. The OD changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 5 environment.]]
−	</td>
−	</tr>
−	<tr>
−	[[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:PHS-pH-6.png|400px|thumb|center|Figure 2. The pH change of genetic pH shooting system_pET11a in the pH 6 initial environment]]
−		+	[[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]]
−	</tr>	+
−	<tr>	+
−	<td>	+
−		+
−	[[File:PHS-OD-7.png|400px|thumb|center|Figure 3. The OD change of genetic pH shooting system_pET11a in the pH 7 initial environment]]	+
−	</td>	+
−	<td>	+
−	[[File:glsa-od-ph7.png |400px|thumb|center| Figure 5. The OD changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 7 environment.]]	+
−	</td>	+
−	</tr>	+
−	<tr>	+
−	[[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]]	+
−	</tr>	+
−	<tr>	+
−	<td>	+
−	[[File:PHS-OD-9.png|400px|thumb|center|Figure 10. The OD change of genetic pH shooting system_pET11a in the pH 9 initial environment]]	+
−	</td>	+
−	<td>	+
−	[[File:glsa-od-ph9.png |400px|thumb|center| Figure 6. The OD changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 9 environment.]]	+
−	</td>
−	</tr>
−	<table>
−	[[File:PHS 1.png|200px|thumb|center|Photo 1: The genetic pH shooting transformed E.coli plate.]]
−	[[File:PHS 2.png|200px|thumb|center|Photo 2: 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. (Figure 1&2)</p>	+	[[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]]
−	<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 3)</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 4&5) </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 7, 8&9)</p>	+
−	===Experiment 2: Western blot===	+	[[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 westernblot.png|600px|thumb|center|Figure 11. 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>
−	<!-- Add more about the biology of this part here
−	===1. pH change test===	+	[[File:PHS 2.png|400px|thumb|center|Photo 1: The pH adjusted LB broth for pH changes test.]]
−	[[File:Glsa-ph-ph5.png|400px|thumb|center| Figure 1. The pH change in 24 hours of glsA compared to sfGFP (BBa_K4340605)  as the control group starting from initial pH 5.]]	+
−	[[File:Glsa-ph-ph7.png |400px|thumb|center| Figure 2. The pH change in 24 hours of glsA compared to sfGFP (BBa_K4340605)  as the control group starting from initial pH 7.]]	+
−	[[File:glsa-ph-ph9.png |400px|thumb|center| Figure 3. The pH change in 24 hours of glsA compared to sfGFP (BBa_K4340605)  as the control group starting from initial pH 9.]]	+
−	<p>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.</p>
−	[[File:GlsA plate.png|200px|thumb|center|Photo 1: The glsA transformed E.coli plate.]]	+	<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>
−	[[File:GlsA plate2.png|400px|thumb|center|Photo 2: The glsA transformed E.coli plate.]]	+
		+	<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>
−	===2. OD change test===	+	<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>
−	<p>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.</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>
		+
		+	<!-- Add more about the biology of this part here

---

Latest revision as of 14:50, 12 October 2022

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. We compared our pH changes, and OD changes to validate the improvement of glsA part.

genetic pH shooting system (BBa_K4340609) and pET11a empty vector pH maintenance functional test

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