Difference between revisions of "Part:BBa K1065310"

Revision as of 09:11, 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

Characterization

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.

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

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 <partinfo>BBa_K1065310 parameters</partinfo>
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