Difference between revisions of "Part:BBa K1890011"
 
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<h2>Indroduction</h2>
 
<h2>Indroduction</h2>
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mVenus is a variant of yellow fluorescent protein (YFP) which contains several mutations that make it more stable and provide an improved speed and efficiency of maturation [1]. We expressed it under the control of the strong constitutive promoter <partinfo>BBa_J23100</partinfo> and RBS <partinfo>BBa_B0030</partinfo>.
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mVenus is a variant of yellow fluorescent protein (YFP) which contains several mutations that make it more stable and provide an improved speed and efficiency of maturation. It has its main emission peak at 530 nm and main excitation peak at 515 nm [1]. We expressed it under the control of the strong constitutive promoter <partinfo>BBa_J23100</partinfo> and RBS <partinfo>BBa_B0030</partinfo>.
  
 
<h2><span class='h3bb'>Sequence and Features</span></h2>
 
<h2><span class='h3bb'>Sequence and Features</span></h2>
 
<partinfo>BBa_K1890011 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1890011 SequenceAndFeatures</partinfo>
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<h2>Characterization</h2>
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This part was expressed in <i>E. coli</i> strain BL21. After transformation, the following experiments were performed to verify the fluorescence:
 +
* Measurement of emission spectrum.
 +
* Viability assay.
 +
 +
<h3>Emission</h3>
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In order to make sure that mVenus was expressed and functioning properly, the emission spectra was recorded in a plate reader (Figure 1). The cells were grown in LB medium and washed in PBS. For measurement an excitation wavelength of 510 nm was used, rather than 515 nm, due to bandwidth limitations of the plate reader used.
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<html>
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    <figure>
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    <center><img src="https://static.igem.org/mediawiki/parts/7/7f/T--TU_Delft--Spectrum_mVenus.png" alt="mVenus" width="60%">
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        <figcaption><b>Figure 1.</b> Emission spectrum of mVenus.</figcaption></center>
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</figure>
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</html>
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Because of the fact that the excitation and emission wavelength are so close together, it was not possible to measure emissions under 530 nm without being within the bandwidth of the excitation source. Apart from that, the spectrum is as expected according to Nagai <i>et al.</i> (2002), from which we can conclude that mVenus is expressed and functioning properly.
 +
 +
<h3>Viability assay</h3>
 +
To investigate whether the constitutive expression of this fluorophore affected the cell growth, we performed a growth study. Cells expressing mVenus were compared with cells expressing GFP under the same promoter and RBS (<partinfo>K1890020</partinfo>).
 +
An overnight culture in eM9 medium was inoculated in fresh eM9 to an OD of 0.1 in a 96 well plate. The emission at 522 nm was measured every 15 minutes. Measurements were done in quadruplicate with pure eM9 as a blank. Figure 2 shows the 24 hour measurement of optical density and fluorescence intensity.
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 +
<html>
 +
    <figure>
 +
    <center><img src="https://static.igem.org/mediawiki/2016/c/c1/T--TU_Delft--growth_mVe_GFP.png" alt="mVenus" width="100%">
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        <figcaption><b>Figure 2.</b> Growth and fluorescence intensity of <i>E. coli</i> expressing GFP (top) or mVenus (bottom).</figcaption></center>
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</figure>
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</html>
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Cells expressing mVenus seem to be having a longer lag phase before exponential growth starts than the ones expressing GFP. Also, they reach a lower final OD than the ones expressing GFP. This phenomenon was also observed while preparing overnight cultures. Cultures expressing mVenus typically took one day longer to reach the same OD as the ones expressing other plasmids. Concluding, constitutive expression of mVenus might be slightly harmful for the cell. This not result in major problems, as the cells could still be cultivated successfully.
  
 
<h2>References</h2>
 
<h2>References</h2>

Latest revision as of 11:41, 19 October 2016


mVenus with strong consitutive promoter and RBS

Indroduction

mVenus is a variant of yellow fluorescent protein (YFP) which contains several mutations that make it more stable and provide an improved speed and efficiency of maturation. It has its main emission peak at 530 nm and main excitation peak at 515 nm [1]. We expressed it under the control of the strong constitutive promoter BBa_J23100 and RBS BBa_B0030.

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]

Characterization

This part was expressed in E. coli strain BL21. After transformation, the following experiments were performed to verify the fluorescence:

  • Measurement of emission spectrum.
  • Viability assay.

Emission

In order to make sure that mVenus was expressed and functioning properly, the emission spectra was recorded in a plate reader (Figure 1). The cells were grown in LB medium and washed in PBS. For measurement an excitation wavelength of 510 nm was used, rather than 515 nm, due to bandwidth limitations of the plate reader used.

mVenus
Figure 1. Emission spectrum of mVenus.
Because of the fact that the excitation and emission wavelength are so close together, it was not possible to measure emissions under 530 nm without being within the bandwidth of the excitation source. Apart from that, the spectrum is as expected according to Nagai et al. (2002), from which we can conclude that mVenus is expressed and functioning properly.

Viability assay

To investigate whether the constitutive expression of this fluorophore affected the cell growth, we performed a growth study. Cells expressing mVenus were compared with cells expressing GFP under the same promoter and RBS (BBa_K1890020). An overnight culture in eM9 medium was inoculated in fresh eM9 to an OD of 0.1 in a 96 well plate. The emission at 522 nm was measured every 15 minutes. Measurements were done in quadruplicate with pure eM9 as a blank. Figure 2 shows the 24 hour measurement of optical density and fluorescence intensity.

mVenus
Figure 2. Growth and fluorescence intensity of E. coli expressing GFP (top) or mVenus (bottom).

Cells expressing mVenus seem to be having a longer lag phase before exponential growth starts than the ones expressing GFP. Also, they reach a lower final OD than the ones expressing GFP. This phenomenon was also observed while preparing overnight cultures. Cultures expressing mVenus typically took one day longer to reach the same OD as the ones expressing other plasmids. Concluding, constitutive expression of mVenus might be slightly harmful for the cell. This not result in major problems, as the cells could still be cultivated successfully.

References

[1] Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).