Difference between revisions of "Part:BBa K2205002"
 
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Improvement by iGEM ZJUT-China 2021
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Aim
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The deGFP reporter gene is an important component of synthetic biology, and the expression level of this gene can indicate many interesting characteristics of an experiment, such as sensitivity, leakage, etc. DeGFP synthesis rate differs for the same element in different chassis. <br>
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<strong>ZJUT-China 2021</strong> found this part in the Parts, but the synthesis rate of deGFP in the Cell-Free system was lower than that in a cell-based system. <br>
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As a result, according to promoters and RBS suitable for Cell-Free systems reported in literature, through experiments and modeling, ZJUT-China 2021 found <strong>the combination of promoter and RBS</strong> that best expresses deGFP, and we registered it as  <a href="https://parts.igem.org/Part:BBa_K3885311">BBa_K3885311</a> .  <br>
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Improve
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We improved the component in two methods, one was to<strong> replace the promoter</strong>, the other was to<strong> replace RBS</strong>.<br>
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<p style="font-size: 14px;">
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<strong>
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1.Change Promoter</strong>
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<img src="https://static.igem.org/mediawiki/parts/0/08/1-p70.png" width=95% height=70% style="display: block;margin: 10px auto;"/>
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<p style="text-align: center:"> Figure 1. </p>
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<img src="https://static.igem.org/mediawiki/parts/9/92/1p1.png" width=95% height=70% style="display: block;margin: 10px auto;"/>
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<p style="text-align: center:"> Figure 2. Rates of deGFP synthesis in the Cell-Free system, compared with model predictions, for reactions containing one of 3 plasmids, including the combinations of promoters P70a, P70b and P70c with UTRs UTR1, UTR2 and UTR3, at various different plasmid concentrations. </p>
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<br>
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We changed the promoter and measured the synthesis rate of deGFP with the different concentration of the plasmid(Figure 1).<br>
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The figure indicates that <strong>the intensity of promoter is P70a>P70b>P70c</strong>. The relative strength of UTR spans nearly two orders of magnitude.<br>
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<p style="font-size: 14px;">
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<strong>
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2.Change RBS</strong>
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<div style="border: 1px solid #000;width: 50%; background-color: #f9f9f9;">
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<img src="https://static.igem.org/mediawiki/parts/7/75/1u.png" width=95% height=70% style="display: block;margin: 10px auto;"/>
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<p style="text-align: center:"> Figure 3. </p>
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<img src="https://static.igem.org/mediawiki/parts/a/af/1u1.png" width=95% height=70% style="display: block;margin: 10px auto;"/>
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<p style="text-align: center:"> Figure 4. Rates of deGFP synthesis in the Cell-Free system, compared with model predictions, for reactions containing different UTRs with the same promoter, at various different plasmid concentrations. </p>
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<br>
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We changed the RBS and measured the synthesis rate of deGFP with different concentration of the plasmid(Figure 3).<br>
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The figure indicates that<strong> the intensity of UTR is UTR1>UTR2>UTR3</strong>. The relative strength of UTR spans nearly two orders of magnitude.<br>
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<p style="font-size: 16px;">
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<strong>
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Result</strong>
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<div class="flex" style="margin: 0 auto; width: 100%;">
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<div style="border: 1px solid #000;width: 50%; background-color: #f9f9f9;">
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<img src="https://static.igem.org/mediawiki/parts/7/7b/1212.png" width=95% height=70% style="display: block;margin: 10px auto;"/>
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<p style="text-align: center:"> Figure 5. Kinetics of different combinations of P70 and UTR plasmids (5nM). </p>
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<br>
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Finally, the kinetic curves of<strong> 9 different combinations of P70 and UTR </strong>were analyzed for 3 hours at a plasmid concentration of 5nM (Figure 5. A). <br>
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The figure above indicates that<strong> P70a-UTR1-deGFP </strong>is the best combination for deGFP expression in Cell-Free systems.<br>
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Among the 9 combinations, we tested <strong> the reaction endpoint of the plasmid J23100-deGFP and P70a-UTR1-deGFP</strong> , and it was found that the improved part had a fluorescence intensity that was four times higher than the original part (Figure 5. B).
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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 14:29, 21 October 2021


J23100-deGFP

deGFP is a variant of Green Fluorescent Protein (GFP). It was initially designed by Shin and Noireaux (2010) for expression in cell-free protein synthesis systems and is more efficiently translated than other variants.


CFPS Expression of deGFP

T--Newcastle--BB CFPS deGFP sfGFP.png


CFPS Expression of J23100-deGFP and J23100-sfGFP Constructs: Time course for increase in fluorescence intensity of CFPS systems expressing GFP constructs over time. Each data point is an average of triplicate results, and error bars show +/- standard error. CFPS system with no DNA (Red) was used as the negative control.


In vivo Qualitative Comparison

T--Newcastle--BB deGFP.png

FACS Data - Collected by Exeter iGEM 2017 for Newcastle iGEM 2017

<img src="T--Newcastle--BB_deGFP_FACS.png" width="600px"/> Figure 5: Single cell data for E. coli cells expressing deGFP (collected by Exeter iGEM). Columns 1-6: cells expressing deGFP. Columns 7 and 8: wild type Top10 cells. Red parts of the bar are non-fluorescent cells, green parts are fluorescent cells.





Improvement by iGEM ZJUT-China 2021

Aim

The deGFP reporter gene is an important component of synthetic biology, and the expression level of this gene can indicate many interesting characteristics of an experiment, such as sensitivity, leakage, etc. DeGFP synthesis rate differs for the same element in different chassis.
ZJUT-China 2021 found this part in the Parts, but the synthesis rate of deGFP in the Cell-Free system was lower than that in a cell-based system.
As a result, according to promoters and RBS suitable for Cell-Free systems reported in literature, through experiments and modeling, ZJUT-China 2021 found the combination of promoter and RBS that best expresses deGFP, and we registered it as BBa_K3885311 .

Improve

We improved the component in two methods, one was to replace the promoter, the other was to replace RBS.

1.Change Promoter

Figure 1.

Figure 2. Rates of deGFP synthesis in the Cell-Free system, compared with model predictions, for reactions containing one of 3 plasmids, including the combinations of promoters P70a, P70b and P70c with UTRs UTR1, UTR2 and UTR3, at various different plasmid concentrations.


We changed the promoter and measured the synthesis rate of deGFP with the different concentration of the plasmid(Figure 1).
The figure indicates that the intensity of promoter is P70a>P70b>P70c. The relative strength of UTR spans nearly two orders of magnitude.

2.Change RBS

Figure 3.

Figure 4. Rates of deGFP synthesis in the Cell-Free system, compared with model predictions, for reactions containing different UTRs with the same promoter, at various different plasmid concentrations.


We changed the RBS and measured the synthesis rate of deGFP with different concentration of the plasmid(Figure 3).
The figure indicates that the intensity of UTR is UTR1>UTR2>UTR3. The relative strength of UTR spans nearly two orders of magnitude.

Result

Figure 5. Kinetics of different combinations of P70 and UTR plasmids (5nM).


Finally, the kinetic curves of 9 different combinations of P70 and UTR were analyzed for 3 hours at a plasmid concentration of 5nM (Figure 5. A).
The figure above indicates that P70a-UTR1-deGFP is the best combination for deGFP expression in Cell-Free systems.
Among the 9 combinations, we tested the reaction endpoint of the plasmid J23100-deGFP and P70a-UTR1-deGFP , and it was found that the improved part had a fluorescence intensity that was four times higher than the original part (Figure 5. B).

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
    COMPATIBLE WITH RFC[1000]