Difference between revisions of "Part:BBa K4340605"
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==E.coli (sfGFP_pET11a) growth curve==
 
==E.coli (sfGFP_pET11a) growth curve==
  
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https://static.igem.wiki/teams/4340/wiki/gfp-abs.png
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      <p>These are the results of our sfGFP_pET11a test. 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 sfGFP_pET11a plasmid.</p>
  
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Figure 1a. Line plot showing GFP Fluorescence emission rate across time (hours), with different culturing mediums. Error bar representing Mean±SE.
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===1. Fluorescence test===
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            [[File:glsa-fl-ph5.png |400px|thumb|center| Figure 1. The Fluorescence Rate in pH 5 initial environment of sfGFP_pET11a and Pasr-glsA_pET11a.]]
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          [[File:glsa-fl-ph7.png |400px|thumb|center| Figure 2. The Fluorescence Rate in pH 7 initial environment of sfGFP_pET11a and Pasr-glsA_pET11a.]]
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            [[File:glsa-fl-ph9.png |400px|thumb|center| Figure 3. The Fluorescence Rate in pH 9 initial environment of sfGFP_pET11a and Pasr-glsA_pET11a.]]
  
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https://static.igem.wiki/teams/4340/wiki/gfp-fluroscence-emission.png
 
  
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Figure 1b. Line plot showing GFP Fluorescence emission rate across time (hours), with different culturing mediums. Error bar representing Mean±SD.
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       <p>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 eighth 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.</p>
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       <p>To test whether E. coli can grow and express GFP in the hydroponic nutrient solutions, we compared the growth of E. coli and GFP expression in the hydroponic solution, LB medium, and water. We calculated the standard error (SE), with n=3:</p>
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https://static.igem.wiki/teams/4340/wiki/se-treatment.png
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where σ is the standard deviation, and n is the number of trials.
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      <p>We found that the E. coli transformed with GFP is more suitable to grow in LB> hydroponic solution> pure water. As expected, the Absorbance OD600 of cell culture in LB medium is the highest. The absorbance in the hydroponic solution is lower than LB, however, it is higher than pure water, suggesting the E.coli can grow with the hydroponic solution.</p>
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      <p>The absorbance in the hydroponic solution reached a peak at the 25th hour, which shows that the E.coli grew and multiplied on the first day. Then, in the 25th to the 45th hour, the value decreases to around 0.1. After that, from the 45th to the 100th hour, the value remains stable. At the 100th to 180th hour, the value increases steadily. These suggest E. coli can grow and survive in hydroponic solution over days.</p>
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      <p>The group's absorbance in pure water peaked at the 25th hour and decreased between the 25th to the 70th hour. This demonstrates that on the first day, the E.coli can still survive in pure water, but in the following hours the E.coli died out.</p>
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      <p>The trends of GFP fluorescence emission rate correspond to the trends of absorbance rate, showing a positive correlation between GFP protein expression and the growth of E. Coli. GFP fluorescence was highest in LB, but it is still present in hydroponic solution.</p>
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      <p>To sum up, the data indicate that E. Coli cells can survive within hydroponic systems. The data indicate that E. coli cells can survive and express protein within hydroponic systems, as demonstrated by the growth and GFP expression. (Figures 1a and 1b) </p>
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      <p>Compared to hydroponic water and LB medium, the rank of growing environments from most to least suitable E. coli transformed with GFP is LB> hydroponic water> pure water. The Absorbance rate of cell culture in the LB medium is the highest, the recorded rate in the hydroponic water is lower than LB group, however, it is higher than in pure water.</p>
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      <p>The Absorbance rate in LB medium is the highest and keeps increasing throughout the 180 hours. This indicates that the LB medium is suitable for E.coli to grow.</p>
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      <p>The Absorbance rate in hydroponic water reached a peak at the 25th hour, which shows that the E.coli survived and multiplied on the first day. Then, in the 25th to the 45th hour, the value decreases to around 0.1. After that, from the 45th to the 100th hour, the value remains stable. At the 100th to 180th hour, the value increases steadily.</p>
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      <p>The group in pure water has reached its peak at the 25th hour, and the rate decreases between the 25th to the 70th hour. In the following 100 hours, the rate slightly fluctuates. This demonstrates that on the first day, the E.coli can still survive in pure water, but in the following hours, the E.coli died.</p>
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<p>To sum up, the data indicates that E. coli cells can survive and express protein within hydroponic systems, as demonstrated by the GFP expression. (Figure 1)</p>  
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 00:41, 12 October 2022


sfGFP

sfGFP(BBa_K2762014) is able to show fluorescence in an acidic environment. We use this plasmid to test the growth curve in our hydroponic system.

E.coli (sfGFP_pET11a) growth curve

These are the results of our sfGFP_pET11a test. 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 sfGFP_pET11a plasmid.

1. Fluorescence test

Figure 1. The Fluorescence Rate in pH 5 initial environment of sfGFP_pET11a and Pasr-glsA_pET11a.
Figure 2. The Fluorescence Rate in pH 7 initial environment of sfGFP_pET11a and Pasr-glsA_pET11a.
Figure 3. The Fluorescence Rate in pH 9 initial environment of sfGFP_pET11a and Pasr-glsA_pET11a.


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 eighth 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.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1283
    Illegal PstI site found at 5809
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1238
    Illegal PstI site found at 5809
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1349
    Illegal BamHI site found at 1205
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1283
    Illegal PstI site found at 5809
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1283
    Illegal PstI site found at 5809
    Illegal NgoMIV site found at 1381
    Illegal NgoMIV site found at 2969
    Illegal NgoMIV site found at 3129
    Illegal NgoMIV site found at 3483
    Illegal AgeI site found at 752
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 5635
    Illegal SapI site found at 4552