Difference between revisions of "Part:BBa J364007"

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Test Device 4 expresses GFP under the control of a constitutive promoter from the Anderson collection.
 
Test Device 4 expresses GFP under the control of a constitutive promoter from the Anderson collection.
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===Interlab 2022 Experiment 3 Device===
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* Test Device 4 for Experiment 3 of the 2022 iGEM Interlab
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{{Template:Collection/Interlab_2022/Experiment_3/Partof}}
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<!-- Add more about the biology of this part here -->
 
<!-- Add more about the biology of this part here -->
 
===Usage and Biology===
 
===Usage and Biology===
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E.coli GFP expression in LB and TB media：
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<html>
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https://parts.igem.org/File:Figures_of_Device_4_and_Negative_control.xls
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<h3> Georgia State University 2019 Characterization</h3>
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<p>A variety of culture media are available to support the bacteria commonly used in synthetic biology. Lysogeny broth (LB) is the most commonly used media in biology research. It is easy to make and compatible with most microorganisms. Terrific Broth (TB), a high nutrition solution, which has extra glycerol compared to LB media can also support E. coli and other cultures. We proposed to test the growth and expression of a foreign transgene in E. coli in two different media: LB and TB. In order to accurately measure the growth conditions, we tested both Abs600 (an indirect measure of cell density) and fluorescence data (expression of the GFP transgene) over a 6 hour time course. We chose to focus on the first 6 hours of the linear growth phase using some of the interlab test devices from the iGEM registry because we had seen some anomalies in the growth and expression curves over time. We hoped to identify the better medium to use to optimize expression of the transgene.</p>
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<img src="https://2019.igem.org/wiki/images/0/07/T--Georgia_State--contrib1.png"></img>
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</div>
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<div class=col-6>
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  <img src="https://2019.igem.org/wiki/images/4/4e/T--Georgia_State--contrib2.png"></img>
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</div>
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</div>
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<div class="row">
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<div class=col-6>
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  <img src="https://2019.igem.org/wiki/images/e/eb/T--Georgia_State--contrib3.png"></img>
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</div>
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<div class=col-6>
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  <img src="https://2019.igem.org/wiki/images/9/95/T--Georgia_State--contrib4.png"></img>
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</div>
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</div>
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<div class="row">
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<div class=col-12>
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  <img src="https://2019.igem.org/wiki/images/b/b2/T--Georgia_State--contrib5.png"></img>
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</div>
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</div>
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<p>As seen in Figure 3, the production of GFP in LB medium increased steadily over the first two hours of incubation and was maintained at a high level for the remaining time points. This reflected increases in both the cell density and the fluorescence. The increase in the ratio indicates that not only were more cells growing and producing fluorescent protein but that the amount of fluorescence per cell was increased and remained high. By comparison , the cultures maintained in TB did not show the same sustained increase in fluorescence per cell over the time course. In fact, as seen in Figure 4, the fluorescence ratio decreased after the 2 hour time point. This was reflected in a flattening of the raw fluorescence measured while the Abs600 continued to increase.
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Also, comparing the curve of different starting cell concentrations in Figure 4, the peak value of Fluo/Abs value in OD 0.10 was significantly lower than the value in OD 0.02, which indicates the higher bacteria concentration was a negative factor for the protein-producing efficiency in TB.
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Based on our results, the protein-producing efficiency and protein production amount of bacteria in LB media was distinctly greater than those grown in TB media. For those researchers aiming for more rapid growth of cells, TB may be the better choice, but our results indicate that this rapid increase in cell density may compromise efficiency of protein production, at least for GFP. If more functional protein is the goal, LB may be the better choice of growth medium.</p>
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<a href="https://parts.igem.org/File:Figures_of_Device_4_and_Negative_control.xls">E.coli GFP expression in LB and TB media:</a>
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</html>
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 00:15, 6 July 2022


Test Device 4 for 2018 InterLab Study

Test Device 4 expresses GFP under the control of a constitutive promoter from the Anderson collection.


Interlab 2022 Experiment 3 Device

  • Test Device 4 for Experiment 3 of the 2022 iGEM Interlab

The following device is used in the iGEM 2022 InterLaboratory Study for Experiment 3.



Usage and Biology

Georgia State University 2019 Characterization

A variety of culture media are available to support the bacteria commonly used in synthetic biology. Lysogeny broth (LB) is the most commonly used media in biology research. It is easy to make and compatible with most microorganisms. Terrific Broth (TB), a high nutrition solution, which has extra glycerol compared to LB media can also support E. coli and other cultures. We proposed to test the growth and expression of a foreign transgene in E. coli in two different media: LB and TB. In order to accurately measure the growth conditions, we tested both Abs600 (an indirect measure of cell density) and fluorescence data (expression of the GFP transgene) over a 6 hour time course. We chose to focus on the first 6 hours of the linear growth phase using some of the interlab test devices from the iGEM registry because we had seen some anomalies in the growth and expression curves over time. We hoped to identify the better medium to use to optimize expression of the transgene.

As seen in Figure 3, the production of GFP in LB medium increased steadily over the first two hours of incubation and was maintained at a high level for the remaining time points. This reflected increases in both the cell density and the fluorescence. The increase in the ratio indicates that not only were more cells growing and producing fluorescent protein but that the amount of fluorescence per cell was increased and remained high. By comparison , the cultures maintained in TB did not show the same sustained increase in fluorescence per cell over the time course. In fact, as seen in Figure 4, the fluorescence ratio decreased after the 2 hour time point. This was reflected in a flattening of the raw fluorescence measured while the Abs600 continued to increase. Also, comparing the curve of different starting cell concentrations in Figure 4, the peak value of Fluo/Abs value in OD 0.10 was significantly lower than the value in OD 0.02, which indicates the higher bacteria concentration was a negative factor for the protein-producing efficiency in TB. Based on our results, the protein-producing efficiency and protein production amount of bacteria in LB media was distinctly greater than those grown in TB media. For those researchers aiming for more rapid growth of cells, TB may be the better choice, but our results indicate that this rapid increase in cell density may compromise efficiency of protein production, at least for GFP. If more functional protein is the goal, LB may be the better choice of growth medium.

E.coli GFP expression in LB and TB media:

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 705