Difference between revisions of "Part:BBa K4340613"
 
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This is a gene that encodes protein ldhA and produces lactic acid. Gene accession code: 946315
 
This is a gene that encodes protein ldhA and produces lactic acid. Gene accession code: 946315
  
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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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ldhA is the gene code of the alkaline shooting circuit (with P-atp2 promoter) in the genetic pH shooting system.
  
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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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        [[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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        [[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:PHS-pH-7.png|400px|thumb|center|Figure 1. The pH change of genetic pH shooting system_pET11a in the pH 7 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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         [[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-pH-8.png|400px|thumb|center|Figure 2. The pH change of genetic pH shooting system_pET11a in the pH 8 initial 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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         [[File:PHS-pH-9.png|400px|thumb|center|Figure 3. The pH change of genetic pH shooting system_pET11a in the pH 9 initial environment]]
 
          
 
          
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[[File:PHS 2.png|400px|thumb|center|Photo 1: The pH adjusted LB broth for pH changes test.]]
  
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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 1) </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 2 & 3)</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)</p>
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         [[File:PHS-OD-7.png|400px|thumb|center|Figure 4. The OD change of genetic pH shooting system_pET11a in the pH 7 initial environment]]
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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 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 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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        [[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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         [[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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         [[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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         [[File:PHS-OD-8.png|400px|thumb|center|Figure 5. The OD change of genetic pH shooting system_pET11a in the pH 8 initial environment]]
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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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      <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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===Experiment 2: Western blot===
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        [[File:PHS-OD-9.png|400px|thumb|center|Figure 6. The OD change of genetic pH shooting system_pET11a in the pH 9 initial environment]]
  
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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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      <p>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>
  
 
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Latest revision as of 10:17, 11 October 2022


ldhA

This is a gene that encodes protein ldhA and produces lactic acid. Gene accession code: 946315

ldhA is the gene code of the alkaline shooting circuit (with P-atp2 promoter) in the genetic pH shooting system.

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

Experiment 1: pH change and OD value

Figure 1. The pH change of genetic pH shooting system_pET11a in the pH 7 initial environment


Figure 2. The pH change of genetic pH shooting system_pET11a in the pH 8 initial environment


Figure 3. The pH change of genetic pH shooting system_pET11a in the pH 9 initial environment
Photo 1: The pH adjusted LB broth for pH changes test.

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 1)

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 2 & 3)

Figure 4. The OD change of genetic pH shooting system_pET11a in the pH 7 initial environment


Figure 5. The OD change of genetic pH shooting system_pET11a in the pH 8 initial environment


Figure 6. The OD change of genetic pH shooting system_pET11a in the pH 9 initial 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.

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 559
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 828