Difference between revisions of "Part:BBa K4375021"

 
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<partinfo>BBa_K4375021 short</partinfo>
 
<partinfo>BBa_K4375021 short</partinfo>
  
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This device is meant for the light-inducible production of mCherry (BBa_I0500), a pore-forming toxin. To achieve this, we used a modified arabinose operator system, where the transcription factor, AraC, is engineered to homodimerise by blue-light instead of arabinose binding. Its called BLADE and described in BBa_K4375003, and it is produced under the strong constitutive promoter J23101*.
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This device is meant for the light-inducible production of mCherry (BBa_I0500), a red fluorescent protein. To achieve this, we used a modified arabinose operator system, where the transcription factor, AraC, is engineered to homodimerize upon blue-light illumination instead of arabinose binding. This system is called BLADE (BBa_K4375003), and it is produced under the strong constitutive promoter J23101*.
  
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==Characteristics==
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[[File: BBa_K4375021_BLADEmCherry_setup.png|500px|]]
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Fig. 1) Blue-light induced mCherry production characterization; A) Blue-light induced mCherry production with our first illumination setup; B) Blue-light intensity dependent mCherry production with our first illumination setup
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Team ELTE has developed two types of illumination devices, both capable of emitting blue light around 450 nm. (To know about them, please refer to our Hardware page) The first is a quite simple device, with whom we could monitor the mCherry production over time at a given light intensity. Fig. 1/A shows the results of this experiment. After 7,5 hours of irradiation (at 37 °C), we observed an 8.25 fold increase in fluorescence, which was significantly less than it was described in the literature. (15x) However, this could be easily explained by the difference between light sources since Romano et. al used an illumination setup which produces an irradiance of 5 W*m^−2. [1] We have not obtained such information about our device yet, therefore, these results cannot be compared exactly.
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Our other illumination device is capable of irradiating a 96-well plate and is highly programmable. With it, we could investigate the light intensity-dependent nature of our protein production. (Fig. 1/B) We could detect differences between illuminated and dark samples during this experiment, but significantly less than we previously did. This also could be reasoned by the different light sources or the different temperatures which we used for the protein expression. (37 vs. 25 °C) Moreover, we could not put our second device into an incubator, the culture was not as oxygenated as in the first case.
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Later we hypothesised, that the difference could be explained by the different bacterial culturing protocols, which could affect the number of plasmid copies. The previous measurement was conducted with a freshly transformed culture, but in the other case, we used frozen glycerol bacteria stock.
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We observed a slight difference between protein productions at different light intensities, although the correlation was not directly proportional. It seemed, over 57% of power there is no substantial difference between samples.
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==Usage and Biology==
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This device is a blue light-activatable system, which is based on the BLADE protein. Upon illumination, the BLADE binds to the AraBAD promoter and mCherry is expressed. This allows the monitoring of the protein levels upon blue light illumination.
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[[File: BBa_K4375021_BLADE_mCherry.png|500px|]]
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Figure 2 The mCherry protein sequence after BLADE promoter scheme.
  
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===Usage and Biology===
 
  
 
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Revision as of 10:21, 10 October 2022


Blue light inducible production of mCherry (BLADE)

This device is meant for the light-inducible production of mCherry (BBa_I0500), a red fluorescent protein. To achieve this, we used a modified arabinose operator system, where the transcription factor, AraC, is engineered to homodimerize upon blue-light illumination instead of arabinose binding. This system is called BLADE (BBa_K4375003), and it is produced under the strong constitutive promoter J23101*.

Characteristics

BBa K4375021 BLADEmCherry setup.png

Fig. 1) Blue-light induced mCherry production characterization; A) Blue-light induced mCherry production with our first illumination setup; B) Blue-light intensity dependent mCherry production with our first illumination setup

Team ELTE has developed two types of illumination devices, both capable of emitting blue light around 450 nm. (To know about them, please refer to our Hardware page) The first is a quite simple device, with whom we could monitor the mCherry production over time at a given light intensity. Fig. 1/A shows the results of this experiment. After 7,5 hours of irradiation (at 37 °C), we observed an 8.25 fold increase in fluorescence, which was significantly less than it was described in the literature. (15x) However, this could be easily explained by the difference between light sources since Romano et. al used an illumination setup which produces an irradiance of 5 W*m^−2. [1] We have not obtained such information about our device yet, therefore, these results cannot be compared exactly.

Our other illumination device is capable of irradiating a 96-well plate and is highly programmable. With it, we could investigate the light intensity-dependent nature of our protein production. (Fig. 1/B) We could detect differences between illuminated and dark samples during this experiment, but significantly less than we previously did. This also could be reasoned by the different light sources or the different temperatures which we used for the protein expression. (37 vs. 25 °C) Moreover, we could not put our second device into an incubator, the culture was not as oxygenated as in the first case.

Later we hypothesised, that the difference could be explained by the different bacterial culturing protocols, which could affect the number of plasmid copies. The previous measurement was conducted with a freshly transformed culture, but in the other case, we used frozen glycerol bacteria stock.

We observed a slight difference between protein productions at different light intensities, although the correlation was not directly proportional. It seemed, over 57% of power there is no substantial difference between samples.


Usage and Biology

This device is a blue light-activatable system, which is based on the BLADE protein. Upon illumination, the BLADE binds to the AraBAD promoter and mCherry is expressed. This allows the monitoring of the protein levels upon blue light illumination.


BBa K4375021 BLADE mCherry.png


Figure 2 The mCherry protein sequence after BLADE promoter scheme.


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 29
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1174
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1009
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 991