Team:UNITN-Trento/Project/Blue light

Results - Blue Light

We decided to develop a photoinducible genetic circuit that triggers the production of ethylene in the presence of blue light (470 nm), and blocks the production of ethylene in the dark.

We decided to use blue light as our inducer because a photoinducible system is highly compatible with our very own B. fruity vending machine. All parts have been transformed and characterized in E. coli NEB10β.

The device

We wanted to produce ethylene with blue light (470 nm) and have an off state in the dark, so we designed a blue light dependent device that includes an inverter cassette.

BBa_K1065310

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

• Anderson promoter BBa_J23100;
• the blue light receptor YF1, which consists of YtvA from B. subtilis fused to a kinase domain (FixL) from B. japonicum; (Möglich A., J Mol Biol. 2009, 385(5): 1433–1444)(Ohlendorf R., J Mol Biol. 2012, 414: 534-542)
• 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), subsequently EFE was incorporated.

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 in the dark and inhibited by blue light).

BBa_K1065302
Different sources of blue light induces amilCP production in the "inverted circuit"

We first assembled the “inverted circuit” with a blue chromoprotein (amilCP) downstream instead of EFE to obtain easy-to-observe and clear characterization results.
At first we compared the induction power of several light sources (Figure 1):

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

Fig. 1: Different light sources induction power. We had massive production of amilCP with LED light (4) and white light (3). Instead exposure to the blue light bulb (2) induced a little less but still much more than the dark control (1): probably white light worked as well because white light contains the right wavelength (470 nm) while the blue bulb wavelength range is unknown.

Therefore we decided to use only blue LED and normal light as inducers in further tests (Figure 2).

Fig. 2: Light successfully induced AmilCP production. We started the characterization test by splitting the cultures at an O.D. of 0.7 into 3 put at 3 different conditions: blue LED induced culture (1), normal light induced culture (2), dark control (3). We induced O/N at 37°C. After overnight incubation the differences in the cultures were very clear.

Moreover, amilCP has an absorbance peak at 588 nm so we measured the absorbance peak at the UV-VIS spectrometer (PerkinElmer lambda 25) in order to have more quantitative data (Figure 3). We sonicated the samples for 10 seconds and resuspended the pellets in 2 ml of PBS.

Fig. 3: by measuring the absorbance of the three samples (588 nm), we observed a substantial difference between the dark control and the other two samples. This is a quantitative demonstration that the production of amilCP in the dark was strongly repressed. The graph also shows that white light induction is a little less efficient.

We carried out several tests in order to demonstrate the reproducibility of the behavior. Sometimes we observed amilCP production even in the dark control. Therefore it seems that the circuit doesn’t act like a perfectly controlled switch and/or there are other unidentified variables that impact the system. We can speculate on some potential causes:

• the inverter cassette may not function as expected;
• not enough cI is produced due to its position in the circuitry.

To further probe our system we tested also the circuit without the inverter. This device should produce the GOI in the dark, as opposed to the previous system which was in the light. We extracted the part BBa_K952003 which contains the circuit with the reporter amilGFP (yellow fluorescent protein) as GOI.

BBa_K952003

The part extracted from the registry was missing a RBS sequence, resulting in a nonfunctional part. We improved this part by inserting the missing RBS via mutagenic PCR.

BBa_K1065305

In order to have it tested and characterized, we also added the pLac promoter ahead as shown here (BBa_K1065302).

Better defined switch observed in the circuit without inverter

In order to demonstrate the actual enhancement of the device we induced both the improved circuit and the original part (to test it we created the part BBa_K1065300). So we compared samples depending on two factors: induction/non induction and RBS/no-RBS. We also took some quantitative measurements with a Cary Eclipse Varian fluorimeter considering that amilGFP is a fluorescent protein that is excitated at 503 nm and emits at 512 nm. For measurements we resuspended sonicated sample pellets in 2 ml of PBS.

Fig. 4: Yellow shade appears only in the induced sample with RBS: data confirmed by fluorescence measurements. After the culture with BBa_K1065302 reached OD= 0.7 we split it into 2 samples of 10ml at 37°C: induced sample in the dark (1, right panel) and blue light exposed control(2, right panel). We also made 2 sample (3 and 4, right panel) at the same conditions from a culture transformed with BBa_K1065300, in order to compare the original part to the improved one. amilGFP production is produced only in the induced sample with BBa_K1065302. In the left panel, fluorescence spectra show that BBa_K1065302 induced sample (purple) produces amilGFP and the control sample (green) has a basal expression. As expected the part without the RBS doesn't produce amilGFP neither in the dark (blue) nor under illumination (red). Our part with RBS is undeniably improved and works as expected. Summary

We achieved a successful characterization of both circuits with and without the inverter. There was a substantial difference between the controls and the induced samples. There were some issues in implementing BBa_K1065310. Nevertheless, the construct that did not contain the inverter (BBa_K1065302) provided the desired functionality.

There are a few things that we can try to improve the device such as adding a terminator after pFixK2, substituting J23100 with pLac promoter or improving the transcription of cI. We are working on this now.

We used this circuit to build a device that produces ethylene in response to blue light. For more info check our ethylene page