Difference between revisions of "Part:BBa K1065310"

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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>
 
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>
 
we characterized ths part in E. coli using cells NEB10b<BR><BR>
 
we characterized ths part in E. coli using cells NEB10b<BR><BR>
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<html><center><img style="width:900px;"src="https://static.igem.org/mediawiki/2013/thumb/5/54/Tn-2013.png/800px-Tn-2013.png"></center><BR><BR></html>
  
 
===Testing different light sources===
 
===Testing different light sources===

Revision as of 14:17, 23 September 2013

Blue light circuit with inverter for the production of amilCP

This part is a blue light sensing device: it consists of a Blue light sensor with its response regulator (Bba_K592016), the RR dependent promoter (Bba_K592006) 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 a constitutive promoter (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 is 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 instead 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 ths 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 in which conditions our device is actually switched on and which condition effectively turns the transcription off. 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 condition:
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). We could notice also that 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).

sometimes we observed amilCP production even in the dark control, so we could assume that the circuit doesn’t act like a perfectly controlled switch probably because Plambda is actually a strong promoter but CI transcription is at the end of a pretty long cascade that is likely to produce low CI, so there isn't enough inverter to block Plambda.

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 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


Functional Parameters