Part:BBa_K1065305

Blue light sensor without inverter for the production of amilGFP

This part is an 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.
This part was cloned and successfully improved by UNITN-Trento 2013 iGEM team. We characterized the circuit with the control of pLac promoter (to do that we created the part BBa_K1065302) in order to test protein production 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.
For characterization tests we used our part BBa_K1065302 that includes pLac upstream of the improved part whith RBS.

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 in the dark and not under illumination. BBa_K1065300 (constituted by the original part BBa_K952003 and 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 split 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 BBa_K1065302 (red) produced the greatest amount of chromoprotein; Cultures of BBa_K1065302 exposed to light showed a basal expression of amilGFP (green). As expected cultures of the part without RBS, BBa_K1065300 showed no activity at all both in the dark (orange) and with light (blue).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 562
  Illegal NgoMIV site found at 634
  Illegal NgoMIV site found at 724
  Illegal NgoMIV site found at 742
  Illegal NgoMIV site found at 1254
  Illegal NgoMIV site found at 1547
  Illegal NgoMIV site found at 1641
  Illegal AgeI site found at 276
  Illegal AgeI site found at 1422
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1311
  Illegal BsaI.rc site found at 175