Difference between revisions of "Part:BBa K2675056"
 
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In this composite part sfGFP-LVAtag ([[Part:BBa_K2675006|BBa_K2675006]]) was equipped by a custom made RBS ([[Part:BBa_K2675017|BBa_K2675017]]) and placed under the control of the constitutive promoter ([[Part:BBa_J23110|BBa_J23110]]).
 
In this composite part sfGFP-LVAtag ([[Part:BBa_K2675006|BBa_K2675006]]) was equipped by a custom made RBS ([[Part:BBa_K2675017|BBa_K2675017]]) and placed under the control of the constitutive promoter ([[Part:BBa_J23110|BBa_J23110]]).
  
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The pictures of ''E. coli'' cells harbouring this part show a very strong sfGFP-LVAtag expression by this this part [[Part:BBa_K2675056|BBa_K2675056]] and no sfGFP-LVAtag in the absence of the promoter and the RBS ([[Part:BBa_K2675081|BBa_K2675081]]).
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The pictures of ''E. coli'' cells harbouring this part show a very strong sfGFP-LVAtag expression. In contrast, no sfGFP-LVAtag is observed in the absence of the promoter and the RBS ([[Part:BBa_K2675081|BBa_K2675081]]).
  
 
[[File: T--Evry Paris-Saclay--Drops-on-plate BBa K2675081.png|600px]]
 
[[File: T--Evry Paris-Saclay--Drops-on-plate BBa K2675081.png|600px]]
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We used this part as a positive control of sfGFP-LVAtag expression and as a negative control of promoter regulation.
 
We used this part as a positive control of sfGFP-LVAtag expression and as a negative control of promoter regulation.
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===Characterization by Evry Paris-Saclay 2019===
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The LVA degradation tag (AANDENYALVA), that equips the superfolder GFP (sfGFP) in this expression cassette, is a ssrA [https://parts.igem.org/Protein_domains/Degradation/ tag] that accelerates protein degradation in ''Escherichia coli'' at 37°C [1]. It is used in synthetic biology to rapidly degrade reporter genes and thus finely observe the synthetic gene networks dynamics.
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We have characterized this part by measuring the fluorescence of ''E. coli'' cells harboring it. We followed over time 3 different widely used laboratory ''E. coli'' strains: the wild-type MG1655 strain, the cloning strain DH5α and the protein overexpression strain BL21(DE3). The fluorescence values were normalised by OD600nm and the results are presented in figures 2 and 4. The data and error bars are the mean and standard deviation of at least three measurements (three biological replicates).
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The row data, provided in [https://static.igem.org/mediawiki/parts/4/48/T--Evry_Paris-Saclay--Ecoli.xls excel] format, were generated as OD600nm and arbitrary fluorescence units. Using the calibration curves presented in Figure 3 (and as an [https://static.igem.org/mediawiki/parts/e/e9/T--Evry_Paris-Saclay--Data_Analysis_Fluoresceine_Standard_Curve.xls excel] file too), we converted the arbitrary units into Molecules of Equivalent FLuorescein (MEFL) / particle (Figure 4).
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[[File: T--Evry_Paris-Saclay--MG1655-AU.png|800px]]
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[[File: T--Evry_Paris-Saclay--DH5alpha-AU.png|800px]]
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[[File: T--Evry_Paris-Saclay--BL21-AU.png|800px]]
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Figure 2. ''In vivo'' characterisation of Green Fluorescent Protein expression by different ''E. coli'' cells harbouring [[Part:BBa_K1330003|BBa_K1330003]], [[Part:BBa_K2675066|BBa_K2675066]] or this part [[Part:BBa_K2675056|BBa_K2675056]].
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[[File: T--Evry_Paris-Saclay--ParticleStdCurve-Clario.png|800px]]
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[[File: T--Evry_Paris-Saclay--FluoresceinStdCurve-Clario.png|800px]]
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Figure 3. Particle and Fluorescein standard curves.
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[[File: T--Evry_Paris-Saclay--MG1655-MEFL.png|800px]]
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[[File: T--Evry_Paris-Saclay--DH5alpha-MEFL.png|800px]]
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[[File: T--Evry_Paris-Saclay--BL21-MEFL.png|800px]]
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Figure 4. ''In vivo'' characterisation of Green Fluorescent Protein expression by different ''E. coli'' cells harbouring [[Part:BBa_K1330003|BBa_K1330003]], [[Part:BBa_K2675066|BBa_K2675066]] or this part [[Part:BBa_K2675056|BBa_K2675056]].
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As negative control, we used [[Part:BBa_K1330003|BBa_K1330003]], a part containing just the [[Part:BBa_J23110|BBa_J23110]] promoter in pSB1C3 backbone.
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As positive control, we used [[Part:BBa_K2675066|BBa_K2675066]], a composite part containing the sfGFP non equipped with a degradation tag ([[Part:BBa_K2675005|BBa_K2675005]]) placed under the control of the constitutive promoter ([[Part:BBa_J23110|BBa_J23110]]) too.
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The cell measurements results presented in Figures 2 and 4 show the dynamics of fluorescence detection and it's strong dependance on the presence of the LVA degradation tag. In early exponential phase the difference between this part [[Part:BBa_K2675056|BBa_K2675056]] and the positive control ([[Part:BBa_K2675066|BBa_K2675066]]) is very limited. The influence of the LVA degradation tag starts to be visible in middle exponential phase, to reach its maximum in stationary phase. These observations can be clearly made for all 3 ''E. coli'' strains studied.
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No fluorescence was detected for the negative control, as expected and the growth curves are similar.
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===References===
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[1] Purcell O, Grierson CS, Bernardo Md, Savery NJ. Temperature dependence of ssrA-tag mediated protein degradation. J Biol Eng (2012) 6, 10.
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Latest revision as of 20:42, 16 October 2019


sfGFP-LVAtag expression casette under the control of J23110 promoter

This part is an sfGFP-LVAtag (BBa_K2675006) expression cassette under the control of the constitutive promoter BBa_J23110.

Usage and Biology

In this composite part sfGFP-LVAtag (BBa_K2675006) was equipped by a custom made RBS (BBa_K2675017) and placed under the control of the constitutive promoter (BBa_J23110).

The pictures of E. coli cells harbouring this part show a very strong sfGFP-LVAtag expression. In contrast, no sfGFP-LVAtag is observed in the absence of the promoter and the RBS (BBa_K2675081).

T--Evry Paris-Saclay--Drops-on-plate BBa K2675081.png

Figure 1. Pictures of E. coli cells harbouring an empty pSB1C3 backbone, BBa_K2675081 or this part BBa_K2675056.

We used this part as a positive control of sfGFP-LVAtag expression and as a negative control of promoter regulation.

Characterization by Evry Paris-Saclay 2019

The LVA degradation tag (AANDENYALVA), that equips the superfolder GFP (sfGFP) in this expression cassette, is a ssrA tag that accelerates protein degradation in Escherichia coli at 37°C [1]. It is used in synthetic biology to rapidly degrade reporter genes and thus finely observe the synthetic gene networks dynamics.

We have characterized this part by measuring the fluorescence of E. coli cells harboring it. We followed over time 3 different widely used laboratory E. coli strains: the wild-type MG1655 strain, the cloning strain DH5α and the protein overexpression strain BL21(DE3). The fluorescence values were normalised by OD600nm and the results are presented in figures 2 and 4. The data and error bars are the mean and standard deviation of at least three measurements (three biological replicates).

The row data, provided in excel format, were generated as OD600nm and arbitrary fluorescence units. Using the calibration curves presented in Figure 3 (and as an excel file too), we converted the arbitrary units into Molecules of Equivalent FLuorescein (MEFL) / particle (Figure 4).

T--Evry Paris-Saclay--MG1655-AU.png

T--Evry Paris-Saclay--DH5alpha-AU.png

T--Evry Paris-Saclay--BL21-AU.png

Figure 2. In vivo characterisation of Green Fluorescent Protein expression by different E. coli cells harbouring BBa_K1330003, BBa_K2675066 or this part BBa_K2675056.


T--Evry Paris-Saclay--ParticleStdCurve-Clario.png

T--Evry Paris-Saclay--FluoresceinStdCurve-Clario.png

Figure 3. Particle and Fluorescein standard curves.


T--Evry Paris-Saclay--MG1655-MEFL.png

T--Evry Paris-Saclay--DH5alpha-MEFL.png

T--Evry Paris-Saclay--BL21-MEFL.png

Figure 4. In vivo characterisation of Green Fluorescent Protein expression by different E. coli cells harbouring BBa_K1330003, BBa_K2675066 or this part BBa_K2675056.


As negative control, we used BBa_K1330003, a part containing just the BBa_J23110 promoter in pSB1C3 backbone.

As positive control, we used BBa_K2675066, a composite part containing the sfGFP non equipped with a degradation tag (BBa_K2675005) placed under the control of the constitutive promoter (BBa_J23110) too.


The cell measurements results presented in Figures 2 and 4 show the dynamics of fluorescence detection and it's strong dependance on the presence of the LVA degradation tag. In early exponential phase the difference between this part BBa_K2675056 and the positive control (BBa_K2675066) is very limited. The influence of the LVA degradation tag starts to be visible in middle exponential phase, to reach its maximum in stationary phase. These observations can be clearly made for all 3 E. coli strains studied.

No fluorescence was detected for the negative control, as expected and the growth curves are similar.

References

[1] Purcell O, Grierson CS, Bernardo Md, Savery NJ. Temperature dependence of ssrA-tag mediated protein degradation. J Biol Eng (2012) 6, 10.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 90
  • 1000
    COMPATIBLE WITH RFC[1000]