Difference between revisions of "Part:BBa K4340609"
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<partinfo>BBa_K4340609 short</partinfo>
 
<partinfo>BBa_K4340609 short</partinfo>
  
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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.
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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.
  
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==genetic pH shooting system (BBa_K4340609) and pET11a empty vector pH maintenance functional test==
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==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 and OD value===
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<table>
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<tr>
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<td>
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        [[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]]
  
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        [[File:PHS-pH-5.png|400px|thumb|center|Figure 1. The pH change of genetic pH shooting system_pET11a in the pH 5 initial environment]]
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</td>
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        [[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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<td>
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        [[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.]]
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</td>
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</tr>
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<tr>
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          [[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]]         
  
 
        
 
        
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        [[File:PHS-pH-7.png|400px|thumb|center|Figure 3. The pH change of genetic pH shooting system_pET11a in the pH 7 initial environment]]
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</tr>
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<tr>
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<td>
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        [[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]]
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        [[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]]
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</td>
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<td>
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        [[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.]]
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</td>
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</tr>
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<tr>
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        [[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]]
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</tr>
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<tr>
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<td>
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        [[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]]
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</td>
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<td>
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        [[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.]]
  
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        [[File:PHS-pH-9.png|400px|thumb|center|Figure 5. The pH change of genetic pH shooting system_pET11a in the pH 9 initial environment]]
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</td>
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</tr>
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<table>
 
[[File:PHS 1.png|200px|thumb|center|Photo 1: The genetic pH shooting transformed E.coli plate.]]
 
[[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.]]
 
[[File:PHS 2.png|200px|thumb|center|Photo 2: The pH adjusted LB broth for pH changes test.]]
  
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       <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)</p>
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       <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>
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       <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>
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       <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>
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       <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>
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       <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>
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       <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>
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       <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>
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        [[File:PHS-OD-5.png|400px|thumb|center|Figure 6. The OD change of genetic pH shooting system_pET11a in the pH 5 initial environment]]
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        [[File:PHS-OD-6.png|400px|thumb|center|Figure 7. The OD change of genetic pH shooting system_pET11a in the pH 6 initial environment]]       
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===Experiment 2: Western blot===
  
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          [[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)]]
  
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         [[File:PHS-OD-7.png|400px|thumb|center|Figure 8. The OD change of genetic pH shooting system_pET11a in the pH 7 initial environment]]
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         <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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        [[File:PHS-OD-8.png|400px|thumb|center|Figure 9. The OD change of genetic pH shooting system_pET11a in the pH 8 initial environment]]
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===1. pH change test===
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          [[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.]]
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          [[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.]]
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            [[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.]]
  
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      <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>
  
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        [[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]]
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[[File:GlsA plate.png|200px|thumb|center|Photo 1: The glsA transformed E.coli plate.]]
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[[File:GlsA plate2.png|400px|thumb|center|Photo 2: The glsA transformed E.coli plate.]]
  
  
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       <p>For the OD change of the genetic pH shooting system (Figure 7), The highest OD value is in the pH 7 environment, followed by pH 8, pH 6, pH 5, and pH 9. The OD value of pH 9 is lower than the other pH groups (pH 5, 6, 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 empty pET11a vector since it has to produce alkaline.</p>
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===2. OD change test===
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       <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>
  
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===Experiment 2: Western blot===
 
  
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          [[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)]]
 
  
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        <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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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  

Revision as of 00:56, 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 and OD value

Figure 2. The OD change of genetic pH shooting system_pET11a in the pH 6 initial environment


Figure 4. The OD change of genetic pH shooting system_pET11a in the pH 8 initial environment
Figure 1. The OD change of genetic pH shooting system_pET11a in the pH 5 initial environment
Figure 4. The OD changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 5 environment.
Figure 3. The OD change of genetic pH shooting system_pET11a in the pH 7 initial environment
Figure 5. The OD changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 7 environment.
Figure 10. The OD change of genetic pH shooting system_pET11a in the pH 9 initial environment
Figure 6. The OD changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 9 environment.
Photo 1: The genetic pH shooting transformed E.coli plate.
Photo 2: 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. (Figure 1&2)

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)

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)

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)

Experiment 2: Western blot

Figure 11. 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
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 922
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1191