Part:BBa_K1065302
Blue light sensing device without inverter for the production of amilGFP
This part includes the promoter pLac and BBa_K1065305, the improved version of the Blue light sensor device BBa_K952003 (see details in Design notes). In the presence of blue light (470 nm) the production of the reporter amilGFP is inhibited. In the absence of blue light the device is activated, thus producing amilGFP. Everything is under the control of a constitutive promoter pLac.
This part was cloned and successfully characterized by UNITN-Trento 2013 iGEM team in order to test protein transcription and then add an ethylene forming enzyme (EFE) after amilGFP.
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 amilGFP transcription.
Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2: amilGFP is no longer produced.
We characterized this part in E. coli using cells NEB10b.
induction test: successful improvement of the part and defined light dependent ON/OFF switch
To understand if inserting an RBS after pFixK2 would actually improve the part (BBa_K952003) we characterized both our improved part and the original one, testing them under the same conditions. The improved part actually behaved as expected, producing the yellow fluorescent protein at dark and not under illumination. BBa_K1065300 (consisting of the original part BBa_K952003 under pLac) instead doesn't work in both cases: that means that the original part doesn't work and we effectively improved it.
Figure 1. improved part and not improved part after induction: we grew one culture of E. coli (strain NEB10b) transformed with Bba_K1065302 and one with BBa_K1065300, until they reached an OD = 0.7. Then we splitted each culture in two 10ml samples (dark and light). Cells were left in glass tubes at 37 degrees under shaking O/N. After this time lapse we centrifuged the cultures and obtained pellets to compare. We were able to observe a different color in the Bba_K1065302-transformed sample that stayed in the dark (1) when compared to the blue light exposed control (2) and the samples without RBS (3 and 4).
fluorescence measurements confirms previuos testing
After the induction time, 10 mL of cells culture were pelleted, resuspended in 2 mL of PBS, sonicated and the supernatant was used to measure fluorescence spectra. Excitation and Emission wavelengths were 503 nm and 512 nm, respectively. All measurements were taken with a Cary Eclipse Varian fluorimeter.
Figure 2.fluorimetric spectra: Dark induced cultures of
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 770
Illegal NgoMIV site found at 842
Illegal NgoMIV site found at 932
Illegal NgoMIV site found at 950
Illegal NgoMIV site found at 1462
Illegal NgoMIV site found at 1755
Illegal NgoMIV site found at 1849
Illegal AgeI site found at 484
Illegal AgeI site found at 1630 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1519
Illegal BsaI.rc site found at 383
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