Difference between revisions of "Part:BBa K1065310"

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<partinfo>BBa_K1065310 short</partinfo>
 
<partinfo>BBa_K1065310 short</partinfo>
  
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This part is a blue light sensing device: it consists of the Blue light sensor YF1 with its response regulator FixJ, the RR dependent promoter pFixK2 and an inverter cassette (cI and pLambda) which are needed to produce the reporter (amilCP) when blue light (470 nm) is present. YF1 and FixJ production are under the control of the constitutive promoter J23100 (Anderson family). <BR>
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This part is a blue light sensing device: it consists of the Blue light sensor YF1 with its response regulator FixJ, the RR dependent promoter pFixK2 <html><a href="#ref1" id="ret_ref1">[1]</a></html>, <html><a href="#ref2">[2]</a></html> and an inverter cassette (cI and pLambda) <html><a href="#ref3">[3]</a></html> which are needed to produce the reporter (amilCP) <html><a href="#ref4">[4]</a></html> when blue light (470 nm) is present . YF1 and FixJ production are under the control of the constitutive promoter J23100 (Anderson family). <BR>
  
 
This part was cloned and successfully characterized by UNITN-Trento 2013 iGEM team in order to test protein production and then replace the blue chromoprotein with an ethylene forming enzyme (EFE). The final goal was to design an ethylene producing device that is induced by blue light to control and speed up fruit ripening.<BR><BR> Parts from 2011 Uppsala-Sweden team and 2006 Berkeley team were used: <partinfo>BBa_J23100</partinfo>, <partinfo>BBa_K592016</partinfo>, <partinfo>BBa_K592020</partinfo>.
 
This part was cloned and successfully characterized by UNITN-Trento 2013 iGEM team in order to test protein production and then replace the blue chromoprotein with an ethylene forming enzyme (EFE). The final goal was to design an ethylene producing device that is induced by blue light to control and speed up fruit ripening.<BR><BR> Parts from 2011 Uppsala-Sweden team and 2006 Berkeley team were used: <partinfo>BBa_J23100</partinfo>, <partinfo>BBa_K592016</partinfo>, <partinfo>BBa_K592020</partinfo>.
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<center>https://static.igem.org/mediawiki/2013/d/d7/Tn-2013_For-partsss.png</center>
 
<center>https://static.igem.org/mediawiki/2013/d/d7/Tn-2013_For-partsss.png</center>
 
===Usage and Biology===
 
===Usage and Biology===
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YF1, the blue light sensor, is a fusion protein of the LOV blue light sensor domain of <i>Bacillus subtilis</i> (YtvA) and FixL histidine kinase domain (from <i>Bradyrhizobium japonicum</i>).<BR>In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing the expression of the inverter cI. cI inhibits pLambda activity thus amilCP transcription.<BR>Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2: the outcome is AmilCP production.<BR>
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YF1, the blue light sensor, is a fusion protein of the LOV blue light sensor domain of <i>Bacillus subtilis</i> (YtvA) and FixL histidine kinase domain (from <i>Bradyrhizobium japonicum</i>) <html><a href="#ref1">[1]</a></html> <html><a href="#ref2">[2]</a></html>.<BR>In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing the expression of the inverter cI. cI inhibits pLambda activity thus amilCP transcription <html><a href="#ref4">[4]</a></html>.<BR>Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2: the outcome is AmilCP production.<BR>
 
<BR>
 
<BR>
 
We characterized this part in <i>E. coli</i> using cells NEB10beta.<BR><BR>
 
We characterized this part in <i>E. coli</i> using cells NEB10beta.<BR><BR>
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<center><p style="width:600px; margin-bottom:60px; text-align:justify">
 
<center><p style="width:600px; margin-bottom:60px; text-align:justify">
 
<b>Figure 1. Different light sources induction power</b>  
 
<b>Figure 1. Different light sources induction power</b>  
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We tested different light sources in order to define the best conditions to switch on and off the circuit.  We grew a culture of transformed cells until it reached an OD = 0.7 (after, then we split the culture in 4 samples (5ml) and exposed them to different conditions: dark control (the glass tube was wrapped with aluminum foil) (1); blue light bulb (2); white light (3); blue LED light (4);<BR>
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We tested different light sources in order to define the best conditions to switch on and off the circuit.  We grew a culture of NEB10beta cells transformed with part BBa_K1065310 until it reached an OD = 0.7 (after, then we split the culture in 4 samples (5ml) and exposed them to different conditions: dark control (the glass tube was wrapped with aluminum foil) (1); blue light bulb (2); white light (3); blue LED light (4);<BR>
 
After an induction period of about 8 hours, in which cultures grew at 37 degrees with stirring, we centrifuged cultures and observed that blue LED, blue bulb and white light all induced successfully the transcription of the reporter, instead the dark control stayed uncoloured. White light worked as an activator, probably because it includes the right wavelength (470 nm).
 
After an induction period of about 8 hours, in which cultures grew at 37 degrees with stirring, we centrifuged cultures and observed that blue LED, blue bulb and white light all induced successfully the transcription of the reporter, instead the dark control stayed uncoloured. White light worked as an activator, probably because it includes the right wavelength (470 nm).
 
Blue bulb illumination provoked a little less efficient induction.
 
Blue bulb illumination provoked a little less efficient induction.
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<b>Figure 2. Our experimental setup involving different types of light</b>
 
<b>Figure 2. Our experimental setup involving different types of light</b>
 
</p></center></html><BR><BR>
 
</p></center></html><BR><BR>
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===Induction test results:===
 
===Induction test results:===
  
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<center><p style="width:600px; margin-bottom:60px; text-align:justify">
 
<center><p style="width:600px; margin-bottom:60px; text-align:justify">
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<b>Figure 5. Absorbance spectra of induced samples and control:</b> Since amilCP is a chromoprotein that absorbes in the UV/VIS range (peak at 588), we pelletted the samples, sonicated and resuspended in 2ml of PBS, then we took some measurements at the UV-VIS spectrometer (PerkinElmer lambda 25) to have some quantitative data of the induction experiment. Data shown are relative to three samples induced for 9 hours. We considered a range between 400 nm and 700 nm. The graph shows that dark (green) significantly inhibited the device. It is also confirmed that white light (red) somehow induced a little less than the blue LE (blue). </p></center>
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<b>Figure 5. Absorbance spectra of induced samples and control:</b> Since amilCP is a chromoprotein that absorbes in the UV/VIS range (peak at 588), we pelletted the samples, sonicated and resuspended in 2ml of PBS, then we took some measurements at the UV-VIS spectrometer (PerkinElmer lambda 25) to have some quantitative data of the induction experiment. Data shown are relative to three samples induced for 9 hours. We considered a range between 400 nm and 700 nm. The graph shows that dark (green trace) significantly inhibited the device. It is also confirmed that white light (red trace) somehow induced a little less than the blue LE (blue trace). </p></center>
 
</html>
 
</html>
 
<html>NOTE: To our surprise, a few times we observed amilCP production even in the dark control, suggesting that the circuit doesn’t act like a perfectly controlled switch. The reason for this could be found in the fact that Plambda is a strong promoter and probably the circuit is not always producing enough CI.  CI transcription is infact at the end of a long cascade that is likely to produce low CI, so there isn't enough inverter to block Plambda.</html>
 
<html>NOTE: To our surprise, a few times we observed amilCP production even in the dark control, suggesting that the circuit doesn’t act like a perfectly controlled switch. The reason for this could be found in the fact that Plambda is a strong promoter and probably the circuit is not always producing enough CI.  CI transcription is infact at the end of a long cascade that is likely to produce low CI, so there isn't enough inverter to block Plambda.</html>
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<partinfo>BBa_K1065310 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1065310 SequenceAndFeatures</partinfo>
  
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===Functional Parameters===
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===References===
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<partinfo>BBa_K1065310 parameters</partinfo>
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<html>
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<ol>
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<li> <a id="ref1"></a> Moglich A, Ayers RA and Moffat K. (2009) Design and Signaling Mechanism of Light-Regulated Histidine Kinases. J. Mol. Bio. 385, 5, 1433-1444.</li>
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<li><a id="ref2"></a> Ohlendorf, R., Vidavski, R.R., Eldar, A., Moffat, K. & Möglich, A.(2012). From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. J. Mol. Biol., 416: 534: 542</li>
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<li><a id="ref3"></a> Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001</li>
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<li><a id="ref4"></a> Alieva, N. O., et al. 2008. Diversity and evolution of coral fluorescent proteins. PLoS One 3:e2680.</li>
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</ol>
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</html>
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Latest revision as of 22:29, 4 October 2013

Blue light circuit with inverter for the production of amilCP

This part is a blue light sensing device: it consists of the Blue light sensor YF1 with its response regulator FixJ, the RR dependent promoter pFixK2 [1], [2] and an inverter cassette (cI and pLambda) [3] which are needed to produce the reporter (amilCP) [4] when blue light (470 nm) is present . YF1 and FixJ production are under the control of the constitutive promoter J23100 (Anderson family).

This part was cloned and successfully characterized by UNITN-Trento 2013 iGEM team in order to test protein production and then replace the blue chromoprotein with an ethylene forming enzyme (EFE). The final goal was to design an ethylene producing device that is induced by blue light to control and speed up fruit ripening.

Parts from 2011 Uppsala-Sweden team and 2006 Berkeley team were used: BBa_J23100, BBa_K592016, BBa_K592020.

SAFETY NOTES: this part does not have safety concerns.

Tn-2013_For-partsss.png

Usage and Biology

YF1, the blue light sensor, is a fusion protein of the LOV blue light sensor domain of Bacillus subtilis (YtvA) and FixL histidine kinase domain (from Bradyrhizobium japonicum) [1] [2].
In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing the expression of the inverter cI. cI inhibits pLambda activity thus amilCP transcription [4].
Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2: the outcome is AmilCP production.

We characterized this part in E. coli using cells NEB10beta.



Testing different light sources

Figure 1. Different light sources induction power We tested different light sources in order to define the best conditions to switch on and off the circuit. We grew a culture of NEB10beta cells transformed with part BBa_K1065310 until it reached an OD = 0.7 (after, then we split the culture in 4 samples (5ml) and exposed them to different conditions: dark control (the glass tube was wrapped with aluminum foil) (1); blue light bulb (2); white light (3); blue LED light (4);
After an induction period of about 8 hours, in which cultures grew at 37 degrees with stirring, we centrifuged cultures and observed that blue LED, blue bulb and white light all induced successfully the transcription of the reporter, instead the dark control stayed uncoloured. White light worked as an activator, probably because it includes the right wavelength (470 nm). Blue bulb illumination provoked a little less efficient induction.

Figure 2. Our experimental setup involving different types of light



Induction test results:





Figure 3 and 4. Enhanced induction upon illumination and prevented transcription in the dark: liquid cultures and pellets. The images show the result of the test after an exposition time of 10 hours. As we can notice from the pellets, blue chromoprotein production occurred only in the blue LED exposed sample (1) and in the one exposed to regular light (2); dark definitely prevented amilCP to be produced (3). However from the liquid cultures we can infer that the blue LED worked more efficiently than the white light.

experiment results confirmed by absorbance measurements

Figure 5. Absorbance spectra of induced samples and control: Since amilCP is a chromoprotein that absorbes in the UV/VIS range (peak at 588), we pelletted the samples, sonicated and resuspended in 2ml of PBS, then we took some measurements at the UV-VIS spectrometer (PerkinElmer lambda 25) to have some quantitative data of the induction experiment. Data shown are relative to three samples induced for 9 hours. We considered a range between 400 nm and 700 nm. The graph shows that dark (green trace) significantly inhibited the device. It is also confirmed that white light (red trace) somehow induced a little less than the blue LE (blue trace).

NOTE: To our surprise, a few times we observed amilCP production even in the dark control, suggesting that the circuit doesn’t act like a perfectly controlled switch. The reason for this could be found in the fact that Plambda is a strong promoter and probably the circuit is not always producing enough CI. CI transcription is infact at the end of a long cascade that is likely to produce low CI, so there isn't enough inverter to block Plambda.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 605
    Illegal NgoMIV site found at 677
    Illegal NgoMIV site found at 767
    Illegal NgoMIV site found at 785
    Illegal NgoMIV site found at 1297
    Illegal NgoMIV site found at 1590
    Illegal NgoMIV site found at 1684
    Illegal AgeI site found at 319
    Illegal AgeI site found at 1465
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1354
    Illegal BsaI.rc site found at 218

References

  1. Moglich A, Ayers RA and Moffat K. (2009) Design and Signaling Mechanism of Light-Regulated Histidine Kinases. J. Mol. Bio. 385, 5, 1433-1444.
  2. Ohlendorf, R., Vidavski, R.R., Eldar, A., Moffat, K. & Möglich, A.(2012). From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. J. Mol. Biol., 416: 534: 542
  3. Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001
  4. Alieva, N. O., et al. 2008. Diversity and evolution of coral fluorescent proteins. PLoS One 3:e2680.