Team:UNITN-Trento/Project/Blue light

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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 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

The test involved the induction of both, the improved circuit and the original part, in order to demonstrate the actual enhancement of the device. 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: Slight yellow shade appears only in the induced sample with RBS: After the culture with BBa_K1065302 reached OD= 0.7 we split it into 2 samples of 5ml at 37°: blue light exposed control (2) and induced sample at dark (1).We also made 2 sample (3 and 4) at the same conditions from a culture transformed with the original part missing the RBS, in order to compare the original part to the improved one. From both the image and the plot we can confirm that 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 built a device that produces ethylene in response to blue light.

Continue the tour!

[http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Blue_light&action=edit Edit this page] | Main Page

@@ Line 10: / Line 10: @@
 		<span class="tn-title">Results - Blue Light</span>
 		<p>
-			We decided to develop a photo-inducible genetic circuit that triggers the production of Ethylene in the presence of blue light (470 nm), and blocks it in the dark.
+			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.
 		</p>
 		<img class="no-border" src="https://static.igem.org/mediawiki/2013/a/ab/Tn-2013_Photoreceptor_path.png" />
 		<p>
-			We thought to use blue light as our inducer because it would fit perfectly to our very own <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending_Machine">B. fruity vending machine</a>, 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).
+			We decided to use blue light as our inducer because photoinducible system is highly compatible with our very own <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending_Machine">B. fruity vending machine</a>. All parts have been transformed and characterized in <i>E. coli</i> NEB10&beta;.
 		</p>
@@ Line 25: / Line 25: @@
 		<span class="tn-subtitle">The device</span>
 		<p>
-			We wanted to produce ethylene with blue light (470 nm) and have an off state in the dark, so we designed of a blue light dependent device that includes an inverter cassette.
+			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.
 		</p>
@@ Line 31: / Line 31: @@
 		<img src="https://static.igem.org/mediawiki/2013/3/3c/Tn-2013_bluelight_Constructs.jpg" /><br>
 		<p>
-			We engineered in E. coli a blue-light sensor composed by:
+			We engineered in <i>E. coli</i> a blue-light sensor composed of:
 		</p>
 		<ul>
@@ Line 38: / Line 38: @@
 			</li>
 			<li>
-				the blue light receptor YF1, which consist of YtvA from B. subtilis fused to a kinase domain (fixL) from B. japonicum <span class="ref">("Möglich A., J Mol Biol. 2009, 385(5): 1433–1444)</span><span class="ref">(Ohlendorf R., J Mol Biol. 2012, 414: 534-542)</span>;
+				the blue light receptor YF1, which consists of YtvA from <i>B. subtilis</i> fused to a kinase domain (FixL) from <i>B. japonicum</i>; <span class="ref">(Möglich A., J Mol Biol. 2009, 385(5): 1433–1444)</span><span class="ref">(Ohlendorf R., J Mol Biol. 2012, 414: 534-542)</span>
 			</li>
 			<li>
@@ Line 44: / Line 44: @@
 			</li>
 			<li>
-				a downstream promoter PfixK2, which is turned off by phosphorylated FixJ;
+				a downstream promoter pFixK2, which is turned off by phosphorylated FixJ;
 			</li>
 			<li>
-				an inverter cassette composed of cI and Plambda;
+				an inverter cassette composed of cI and pLambda;
 			</li>
 			<li>
-				a reporter (chromoprotein amilCP), which was later substituted by EFE (our ethylene forming enzyme).
+				a reporter (chromoprotein amilCP), subsequently EFE was incorporated.
 			</li>
 		</ul>
@@ Line 70: / Line 70: @@
 		<p>
-			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:
+			We characterized this circuit along with the version without the inverter cassette (activated in the dark and inhibited by blue light).
 		</p>
@@ Line 76: / Line 76: @@
 		<img src="https://static.igem.org/mediawiki/2013/6/68/Tn-2013_Blue_noinverter.jpg" /><br>
-		<span class="tn-quote">If you are interested in all the molecular details of these circuits, please check our <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Datapage">datapage</a></span>
+		<span class="tn-subtitle">Different sources of blue light induces amilCP production in the "inverted circuit"</span>
-		<span class="tn-subtitle">Different sources of blue light induces AmilCP production in the "inverted circuit"</span>
 		<p>
-			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.<br>
+			We first assembled the “inverted circuit” with a blue chromoprotein (amilCP) downstream instead of EFE to obtain easy-to-observe and clear characterization results.<br>
-			At first we compared the induction power of several light sources:
+			At first we compared the induction power of several light sources (<b>Figure 1</b>):
 		</p>
 		<ul>
@@ Line 99: / Line 97: @@
 			<img class="photo" style=" height: 280px;" src="https://static.igem.org/mediawiki/2013/e/e8/Tn-2013_different_lights_pic.jpg" />
 		</div>
-		<span class="caption"><b>Fig. 1: Different light sources induction power.</b> We had massive production of amilCP at LED light (4) and white light (3), instead exposure to the blue light bulb (2) induced a little less but still way more than the dark control (1): probably white light worked as well because it included the right wavelength (470), instead the blue bulb wavelength range is unknown, perhaps the excitation spectrum of this bulb was a little off from 470 nm.</span>
+		<span class="tn-caption"><b>Fig. 1: Different light sources induction power.</b> 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.</span>
 		<p>
-			Then we decided to use only blue LED and normal light as inducers in further tests.
+			Therefore we decided to use only blue LED and normal light as inducers in further tests (<b>Figure 2</b>).
 		</p>
 		<div class="tn-doublephoto-wrap">
@@ Line 108: / Line 106: @@
 			<img class="photo" src="https://static.igem.org/mediawiki/2013/b/ba/Tn-2013_bluelight_noinverter_pic2.jpg" />
 		</div>
-		<span class="caption"><b>Fig. 2: Light successfully induced AmilCP production.</b> We started the characterization test splitting the cultures (OD = 0.7) into 3 samples of 5 ml under 3 different conditions: Blue LED induced culture (1), Normal light induced culture (2), Dark control (3). We induced O/N at 37° and the next day <b>we could admire a very conspicuous difference in color of liquid cultures and pellets</b>.</span>
+		<span class="tn-caption"><b>Fig. 2: Light successfully induced AmilCP production.</b> 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&deg;C. <b>After overnight incubation the differences in the cultures were very clear</b>.
 		<p>
-			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 concrete data. We sonicated samples for 10 seconds and resuspended the pellets in 2 ml of PBS.
+			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 (<b>Figure 3</b>). We sonicated the samples for 10 seconds and resuspended the pellets in 2 ml of PBS.
 		</p>
 		<img src="https://static.igem.org/mediawiki/2013/e/e8/Blue_fluorimeter-plot.jpg" />
-		<span class="caption"><b>Fig. 3:</b> 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 proof that the production of AMILCP at dark was strongly repressed. The graph also proves that white light induction is a little less efficient.</span>
+		<span class="tn-caption"><b>Fig. 3:</b> 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.</span>
 		<p>
-			We carried out several tests in order to demonstrate the reproducibility of the behavior, although sometimes we observed amilCP production even in the dark control, so we could assume that the circuit doesn’t act like a perfectly controlled switch. We can make a speculation on the cause: probably the inverter cassette presence is responsible of this flawed behavior; Plambda is actually a strong promoter but CI transcription is at the end of a pretty long cascade that is likely to produce low CI; this means that there isn’t enough inhibitor to block Plambda activity. In order to confirm our theory <b>we tested also the circuit without the inverter</b>. This device, on the contrary, is designed to get switched on at dark and to be inhibited by blue light though. We extracted the part <a href="http://parts.igem.org/Part:BBa_K952003">BBa_k952003</a>, the circuit with the reporter AMILGFP (yellow fluorescent protein).
+			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:
+			<ul>
+				<li>the inverter cassette may not function as expected;</li>
+				<li>not enough cI is produced due to its position in the circuitry.</li>
+			</ul>
+			To further probe our system <b>we tested also the circuit without the inverter</b>. This device should produce the GOI in the dark, as opposed to the previous system which was in the light. We extracted the part <a href="http://parts.igem.org/Part:BBa_K952003">BBa_k952003</a> which contains the circuit with the reporter amilGFP (yellow fluorescent protein) as GOI.
 		</p>
@@ Line 124: / Line 127: @@
 		<img src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_Norbs_yellowpart.jpeg" />
 		<p>
-			The part extracted from the registry missed a RBS sequence, resulting in a nonfunctional part. We decided to <b>improve this part</b> by inserting the missing RBS with a mutagenesis PCR. The mutagenesis was <b>successfull</b>.
+			The part extracted from the registry was missing a RBS sequence, resulting in a nonfunctional part. We <b>improved this part</b> by inserting the missing RBS via mutagenic PCR.
 		</p>
@@ Line 130: / Line 133: @@
 		<img src="https://static.igem.org/mediawiki/2013/2/20/Tn-2013_Rbs_yellowpart.jpeg" />
 		<p>
-			In order to have it tested and characterized, we also added the pLac promoter ahead as already shown (<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>).
+			In order to have it tested and characterized, we also added the pLac promoter ahead as shown here (<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>).
 		</p>
+		<img src="https://static.igem.org/mediawiki/2013/6/68/Tn-2013_Blue_noinverter.jpg" />
-		<span class="tn-subtitle">Better defined switch observed in the circuit wothout inverter</span>
+		<span class="tn-subtitle">Better defined switch observed in the circuit without inverter</span>
 		<p>
-			The test involved the induction of both, the improved circuit and the original part, in order to demonstrate the actual enhancement of the device. So we compared samples depending on two factors: induction/non induction &amp; RBS/no RBS. We also took some quantitative measurements with a Cary Eclipse Varian fluorimeter considering that amilGFP is a fluorescent protein that emits at 512 nm and absorbs at 503 nm. For measurements we resuspended sonicated samples' pellets in 2 ml of PBS.
+			The test involved the induction of both, the improved circuit and the original part, in order to demonstrate the actual enhancement of the device. 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.
 		</p>
@@ Line 142: / Line 146: @@
 			<img class="photo" src="https://static.igem.org/mediawiki/2013/b/b1/Tn-2013_Part_improvement.jpg" />
 		</div>
-		<span class="caption"><b>Fig. 4: Slight yellow shade appears only in the induced sample with RBS</b>: After the culture with <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> reached OD= 0.7 we split it into 2 samples of 5ml at 37°: blue light exposed control (2) and induced sample at dark (1).We also made 2 sample (3 and 4) at the same conditions from a culture transformed with the original part missing the RBS, in order to compare the original part to the improved one. From both the image and the plot we can confirm that <b>our part with RBS is undeniably improved and works as expected</b>.</span>
+		<span class="tn-caption"><b>Fig. 4: Slight yellow shade appears only in the induced sample with RBS</b>: After the culture with <a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> reached OD= 0.7 we split it into 2 samples of 5ml at 37°: blue light exposed control (2) and induced sample at dark (1).We also made 2 sample (3 and 4) at the same conditions from a culture transformed with the original part missing the RBS, in order to compare the original part to the improved one. From both the image and the plot we can confirm that <b>our part with RBS is undeniably improved and works as expected</b>.</span>
 		<span class="tn-title">Summary</span>
 		<p>
-			<b>We achieved a successful characterization of both circuit with or without the inverter</b>, noticing a substantial difference between controls and induced samples; Nevertheless we couldn’t appreciate this for every test performed on <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_k1065310</a>. Comparing the behavior of the 2 devices, we can notice that, under the conditions that we used, the one without the inverter definitely shows a sharper switch.<br>
+			<b>We achieved a successful characterization of both circuits with and without the inverter</b>. There was a substantial difference between the controls and the induced samples. There were some issues in implementing <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a>. Nevertheless, the construct that did not contain the inverter (<a href="http://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a>) provided the desired functionality.
-			Supposed causes:
-		</p>
-		<ul>
-			<li>as already explained probably the genetic cascade in <a href="http://parts.igem.org/Part:BBa_K1065310">BBa_K1065310</a> that brings the activation of cI isn’t strong enough, resulting in a limited production of inverter thus in the subsequent production of AmilCP;
-			</li>
-			<li>furthermore, we believe that the absence of a terminator after pFixK2 could cause the transcription of several different-sized segments, sometimes including the inverter cassette.
-			</li>
-			<li>a remaining element probably involved in the different behaviors is the different upstream promoters used.
-			</li>
-		</ul>
 		<p>
-			We believe that more improvements need to be provided to the system in order to get it perfected and satisfying:
+		<p>
+			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.
 		</p>
-		<ul>
-			<li>add a terminator after pFixK2;
-			</li>
-			<li>substitute J23100 with pLac promoter;
-			</li>
-			<li>improve the transcription of CI;
-			</li>
-		</ul>
-		<p>
+		<span class="tn-effect">
-			To this day we are in the process of improving it and hopefully have some great enhancements for the championships!!!
+			We built a device that produces ethylene in response to blue light.
-		</p>
+		</span>
-		<p>
-			However we were <b>able to produce ethylene</b> by placing EFE (<a href="http://parts.igem.org/Part:BBa_K1065000">BBa_K1065000</a>) downstream the circuits. Please go ahead to our <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Ethylene">ethylene measurements section</a> for these data.
-		</p>
 	</div>