Results - Blue Light

We decided to develop a photo-inducible genetic circuit that triggers the production of Ethylene in presence of blue light (470 nm), and stops at dark.

We thought to use blue light as our inducer because it would fit perfectly to our very own B. fruity vending machine, being the easiest way to control a genetic device in a totally automated scaffold. All parts have been transformed and characterized in E. coli (strain NEB10b).

The device

We aimed at first to get ethylene production at blue light (470 nm) and the off state at dark, so we went for the design of a blue light dependent device that includes an inverter cassette.

BBa_K1065310

We engineered in E. coli a blue-light sensor composed by:

• Anderson promoter BBa_J23100;
• the blue light receptor YF1, which consist of YtvA from B. subtilis fused to a kinase domain (fixL) from B. japonicum (Möglich A, Ayers RA, Moffat K. 2008);
• its response regulator, FixJ;
• a downstream promoter PfixK2, which is turned off by phosphorylated FixJ;
• an inverter cassette composed of cI and Plambda;
• a reporter (chromoprotein amilCP), which was later substituted by EFE (our ethylene forming enzyme).

To assemble this device we used the following parts from the registry:

• Bba_J23100 from Berkeley 2006 iGEM team;
• Bba_K592016 from Uppsala 2011 iGEM team;
• BBa_K592020 from Uppsala 2011 iGEM team.

We characterized this circuit along with the version without the inverter cassette (activated at dark and inhibited by blue light). Thus we also created the part:

BBa-Bba_K1065302

Different sources of blue light induces AmilCP production in the "inverted circuit"

We first assembled the “inverted circuit” with a blue chromo-protein (AmilCP) downstream instead of EFE to obtain easy-to-watch and clear characterization results.
At first we compared the induction power of several light sources:

• 1 LED blue light;
• 1 blue light bulb;
• 1 white light bulb.

Then we decided to use only blue LED and normal light as inducers in further tests.