Difference between revisions of "Part:BBa K1065310"
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<partinfo>BBa_K1065310 short</partinfo> | <partinfo>BBa_K1065310 short</partinfo> | ||
| − | 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. Dark causes the turning off, thus inhibits the production of the reporter.<BR><BR>Everything is under the control of the constitutive promoter J23100 (Anderson family). | + | 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. Dark causes the turning off, thus inhibits the production of the reporter.<BR><BR>Everything is 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 transcription 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.<BR><BR><BR>SAFETY NOTES: this part does not have safety concerns. | This part was cloned and successfully characterized by UNITN-Trento 2013 iGEM team in order to test protein transcription 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.<BR><BR><BR>SAFETY NOTES: this part does not have safety concerns. | ||
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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> | ||
| − | We tested different light sources in order to define | + | 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: <BR>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 chromoprotein, 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 chromoprotein, 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. | |
</p></center> | </p></center> | ||
Revision as of 16:00, 24 September 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 and an inverter cassette (cI and pLambda) which are needed to produce the reporter (amilCP) when blue light (470 nm) is present. Dark causes the turning off, thus inhibits the production of the reporter.
Everything is 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 transcription 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.
SAFETY NOTES: this part does not have safety concerns.
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).
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.
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 NEB10b.
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 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);
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 chromoprotein, 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 experiment setup involving different types of light
Induction test results:
Figure 3 and 4. Massive induction under illumination and prevented transcription at 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 massively 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 took some measurement at the 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).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Unknown
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1354
Illegal BsaI.rc site found at 218