Difference between revisions of "Part:BBa K4340609"
(→‎Experiment 2: Western blot)
 
(8 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-pH-5.png|400px|thumb|center|Figure 1. The pH change of genetic pH shooting system_pET11a in the pH 5 initial environment]]
+
−
</td>
+
−
<td>
+
−
        [[File:Glsa-ph-ph5.png|400px|thumb|center|Figure 2. The pH changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 5 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]]
−
</tr>
+
−
<tr>
+
−
<td>
+
  
−
          [[File:PHS-pH-6.png|400px|thumb|center|Figure 3. The pH change of genetic pH shooting system_pET11a in the pH 6 initial environment]]       
 
  
−
</td>   
+
          [[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]]      
−
</tr>
+
−
<tr>
+
−
<td>
+
−
       
+
−
        [[File:PHS-pH-7.png|400px|thumb|center|Figure 4. The pH change of genetic pH shooting system_pET11a in the pH 7 initial environment]]
+
−
</td>
+
−
<td>
+
−
        [[File:Glsa-ph-ph7.png |400px|thumb|center| Figure 5. The pH changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 7 environment.]]
+
−
</td>
+
−
</tr>
+
−
<tr>
+
−
<td>
+
−
        [[File:PHS-pH-8.png|400px|thumb|center|Figure 6. The pH change of genetic pH shooting system_pET11a in the pH 8 initial environment]]
+
−
</td>
+
−
</tr>
+
−
<tr>
+
−
<td>
+
−
        [[File:PHS-pH-9.png|400px|thumb|center|Figure 7. The pH change of genetic pH shooting system_pET11a in the pH 9 initial environment]]
+
−
</td>
+
−
<td>
+
−
        [[File:glsa-ph-ph9.png |400px|thumb|center| Figure 8. The pH changes of Pasr-glsA-pET11a and sfGFP-pET11a transformed E.coli in a pH 9 environment.]]
+
  
−
</td>
+
        [[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>
+
−
<table>
+
−
<table>
+
−
<tr>
+
−
<td>
+
−
[[File:PHS 1.png|400px|thumb|center|Photo 1: The genetic pH shooting plasmid product.]]
+
−
</td>
+
−
<td>
+
−
[[File:PHS 2.png|400px|thumb|center|Photo 2: The pH adjusted LB broth for pH changes test.]]
+
−
</td>
+
−
</tr>
+
−
</table>
+
  
−
       <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>
+
 
−
       <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>
+
        [[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 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>
+
        [[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===
 
===Experiment 2: OD changes===
  
−
<table>
 
−
<tr>
 
−
<td>
 
 
         [[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: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]]
−
</td>
+
 
−
<td>
+
−
</tr>
+
−
<tr>
+
−
<td>
+
 
           [[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-OD-6.png|400px|thumb|center|Figure 2. The OD change of genetic pH shooting system_pET11a in the pH 6 initial environment]]         
−
</td> 
 
−
</tr>
 
−
<tr>
 
−
<td>
 
 
          
 
          
 
         [[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: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]]
−
</td>
+
 
−
</tr>
+
−
<tr>
+
−
<td>
+
 
         [[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:PHS-OD-8.png|400px|thumb|center|Figure 4. The OD change of genetic pH shooting system_pET11a in the pH 8 initial environment]]
−
</td>
+
 
−
</tr>
+
−
<tr>
+
−
<td>
+
 
         [[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]]
 
         [[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]]
  
−
</td>
+
<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.
−
</tr>
+
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>
−
<table>
+
  
 
===Experiment 2: Western blot===
 
===Experiment 2: Western blot===
Line 103: Line 56:
  
 
         <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>
 
         <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
 
<!-- 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

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


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


Figure 4. The pH change of genetic pH shooting system_pET11a in the pH 8 initial environment
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


Photo 1: 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. 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

Figure 1. The OD change of genetic pH shooting system_pET11a and Pasr-glsA_pET11a with pET11a (control) in the pH 5 initial environment
Figure 2. The OD change of genetic pH shooting system_pET11a in the pH 6 initial environment
Figure 3. The OD change of genetic pH shooting system_pET11a and Pasr-glsA with pET11a (control) in the pH 7 initial environment
Figure 4. The OD change of genetic pH shooting system_pET11a in the pH 8 initial environment
Figure 5. The OD change of genetic pH shooting system_pET11a and Pasr-glsA with pET11a (control) in the pH 9 initial environment

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

Figure 6. 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