![Image](https://static.igem.org/mediawiki/2017/5/55/EL5.png)
Figure1: The of the wild type E.coli has no connection with the lysine concentration.
![Image](https://static.igem.org/mediawiki/2017/b/be/EL6.png)
Figure2: The GFP fluorescence of the wild type E.coli has no connection with the lysine concentration.
(10 intermediate revisions by the same user not shown) | |||
Line 75: | Line 75: | ||
<div class="row animate-box"> | <div class="row animate-box"> | ||
<div class="col-md-8 col-md-offset-2 text-center fh5co-heading"> | <div class="col-md-8 col-md-offset-2 text-center fh5co-heading"> | ||
− | <h2>Necessity of E.coli ΔLysA</h2> | + | <h2>1.Necessity of E.coli ΔLysA</h2> |
− | <p> After we have verified that lysine can be the signal upstream the sensor, what we need to do next is to conform the connection between the lysine concentration and the growth of E.coli ΔLysA. Before that, we have to make sure that the growth of wild type E.coli has no connection with lysine concentration. Part BBa_K876070 was transformed into wild type E.coli to test its OD600 and GFP fluorescence every hour with microplate reader. Lysine was added in increments of 0.5ug/mL to establish a range of 0-3ug/mL. It shows that the growth of wild type E.coli has no connection with lysine concentration. From this, we can say that it’s necessary for E.coli ΔLysA to be the signal receiver of lysine. | + | <p> After we have verified that lysine can be the signal upstream the sensor, what we need to do next is to conform the connection between the lysine concentration and the growth of E.coli ΔLysA. Before that, we have to make sure that the growth of wild type E.coli has no connection with lysine concentration. Part <a href="http://parts.igem.org/Part:BBa_K876070">BBa_K876070</a> was transformed into wild type E.coli to test its OD600 and GFP fluorescence every hour with microplate reader. Lysine was added in increments of 0.5ug/mL to establish a range of 0-3ug/mL. It shows that the growth of wild type E.coli has no connection with lysine concentration. From this, we can say that it’s necessary for E.coli ΔLysA to be the signal receiver of lysine. |
</p> | </p> | ||
+ | </div> | ||
<div class="container-fluid js-tm-page-content" data-page-no="4" data-page-type="gallery"> | <div class="container-fluid js-tm-page-content" data-page-no="4" data-page-type="gallery"> | ||
<div class="tm-img-gallery-container tm-img-gallery-container-3"> | <div class="tm-img-gallery-container tm-img-gallery-container-3"> | ||
Line 84: | Line 85: | ||
<div class="grid-item"> | <div class="grid-item"> | ||
<figure class=""> | <figure class=""> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/5/55/EL5.png" alt="Image" class="img-fluid tm-img"><br><font size=2> |
+ | Figure1: The of the wild type E.coli has no connection with the lysine concentration.</font><br> | ||
</div> | </div> | ||
<div class="grid-item"> | <div class="grid-item"> | ||
<figure class=""> | <figure class=""> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/b/be/EL6.png" alt="Image" class="img-fluid tm-img"><br><font size=2> |
+ | Figure2: The GFP fluorescence of the wild type E.coli has no connection with the lysine concentration.</font><br> | ||
</div> | </div> | ||
Line 102: | Line 105: | ||
<p> To explore the connection between the lysine concentration and the growth of E.coli ΔLysA, we first cultured E.coli ΔLysA in LB medium till its OD600 has reached 0.4. Then aliquots of 100ul were transferred into M9 medium to deplete lysine. After 10h, aliquots of 100ul were transferred into M9 medium with lysine. Lysine was added in increments of 0.5ug/mL to establish a range of 0-3ug/mL. OD600 was measured with microplate reader every hour. The result shows that the OD600 of E.coli ΔLysA has a linear relation with lysine concentration. Thus, we have proved our lysine sensor to be feasible. | <p> To explore the connection between the lysine concentration and the growth of E.coli ΔLysA, we first cultured E.coli ΔLysA in LB medium till its OD600 has reached 0.4. Then aliquots of 100ul were transferred into M9 medium to deplete lysine. After 10h, aliquots of 100ul were transferred into M9 medium with lysine. Lysine was added in increments of 0.5ug/mL to establish a range of 0-3ug/mL. OD600 was measured with microplate reader every hour. The result shows that the OD600 of E.coli ΔLysA has a linear relation with lysine concentration. Thus, we have proved our lysine sensor to be feasible. | ||
</p> | </p> | ||
+ | </div> | ||
<div class="container-fluid js-tm-page-content" data-page-no="4" data-page-type="gallery"> | <div class="container-fluid js-tm-page-content" data-page-no="4" data-page-type="gallery"> | ||
<div class="tm-img-gallery-container tm-img-gallery-container-3"> | <div class="tm-img-gallery-container tm-img-gallery-container-3"> | ||
Line 108: | Line 112: | ||
<div class="grid-item"> | <div class="grid-item"> | ||
<figure class=""> | <figure class=""> | ||
− | <img src="https://static.igem.org/mediawiki/2017/a/aa/EL3.png" alt="Image" class="img-fluid tm-img"> | + | <img src="https://static.igem.org/mediawiki/2017/a/aa/EL3.png" alt="Image" class="img-fluid tm-img"><br><font size=2> |
+ | Figure3:The OD600 of the E.coli ΔLysA is induced by the lysine concentration.</font></br> | ||
</div> | </div> | ||
<div class="grid-item"> | <div class="grid-item"> | ||
<figure class=""> | <figure class=""> | ||
− | <img src="https://static.igem.org/mediawiki/2017/f/f3/EL4.png" alt="Image" class="img-fluid tm-img"> | + | <img src="https://static.igem.org/mediawiki/2017/f/f3/EL4.png" alt="Image" class="img-fluid tm-img"><br><font size=2> |
+ | Figure4:The GFP fluorescence of the E.coli ΔLysA is induced by the lysine concentration.</font></br> | ||
</div> | </div> | ||
Line 123: | Line 129: | ||
<div class="row animate-box"> | <div class="row animate-box"> | ||
<div class="col-md-8 col-md-offset-2 text-center fh5co-heading"> | <div class="col-md-8 col-md-offset-2 text-center fh5co-heading"> | ||
− | <h2>Confirmation in Models</h2> | + | <h2>2.Confirmation in Models</h2> |
<p> After we have confirmed that the E.coli ΔLysA can response and transform lysine signal to the fluorescent signal, what we need to do is to amplify the fluorescent signal and improve the sensitivity. To solve this problem and make sure that our own-designed circuit works well, we need to test the effect of the strong promoter, cyclic amplifier and dual fluorescence system respectively. The verified experiments are as follows. | <p> After we have confirmed that the E.coli ΔLysA can response and transform lysine signal to the fluorescent signal, what we need to do is to amplify the fluorescent signal and improve the sensitivity. To solve this problem and make sure that our own-designed circuit works well, we need to test the effect of the strong promoter, cyclic amplifier and dual fluorescence system respectively. The verified experiments are as follows. | ||
</p> | </p> | ||
Line 131: | Line 137: | ||
<div class="row animate-box"> | <div class="row animate-box"> | ||
<div class="col-md-8 col-md-offset-2 text-center fh5co-heading"> | <div class="col-md-8 col-md-offset-2 text-center fh5co-heading"> | ||
− | <h2> | + | <h2>2.1 Strong Promoter</h2> |
− | <p> To confirm that the strong constitutive promoter can induce plux promoter, we designed the biobrick BBa_K2305004. The purpose of this biobrick is to produce as much green fluorescence as possible by inducing the induced promoter plux, to ensure a stronger expression in E.coli. | + | <p> To confirm that the strong constitutive promoter can induce plux promoter, we designed the biobrick <a href="http://parts.igem.org/Part:BBa_K2305004">BBa_K2305004</a>. The purpose of this biobrick is to produce as much green fluorescence as possible by inducing the induced promoter plux, to ensure a stronger expression in E.coli. |
</p> | </p> | ||
<center> | <center> | ||
<img src="https://static.igem.org/mediawiki/2017/9/9e/SP.png" alt="Free HTML5 Bootstrap Template" class="img-responsive img-rounded"> | <img src="https://static.igem.org/mediawiki/2017/9/9e/SP.png" alt="Free HTML5 Bootstrap Template" class="img-responsive img-rounded"> | ||
</center> | </center> | ||
− | <p> We added a strong promoter on the basis of the previous part BBa_J37032. Parts from BBa_J23100 to BBa_J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. With the addition of the strong constitutive promoter upstream the plux, we hope the plux promoter can be induced and therefore express more GFP. As the figure shows, in the first eight hours the OD600 value grew well, and the new part reached a higher GFP expression in E.coli than the previous part. Importantly, the new part has amplified the expression of GFP for more than 10 times. So we can say that with the existence of the strong constitutive promoter, the expression of GFP has been amplified. So it’s effective for us to put plux promoter upstream the route, in other to amplify the detecting signal. | + | <p> We added a strong promoter on the basis of the previous part <a href="http://parts.igem.org/Part:BBa_J37032">BBa_J37032</a>. Parts from <a href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a> to <a href="http://parts.igem.org/Part:BBa_J23119">BBa_J23119</a> are a family of constitutive promoter parts isolated from a small combinatorial library. With the addition of the strong constitutive promoter upstream the plux, we hope the plux promoter can be induced and therefore express more GFP. As the figure shows, in the first eight hours the OD600 value grew well, and the new part reached a higher GFP expression in E.coli than the previous part. Importantly, the new part has amplified the expression of GFP for more than 10 times. So we can say that with the existence of the strong constitutive promoter, the expression of GFP has been amplified. So it’s effective for us to put plux promoter upstream the route, in other to amplify the detecting signal. |
</p> | </p> | ||
− | + | </div> | |
<div class="container-fluid js-tm-page-content" data-page-no="4" data-page-type="gallery"> | <div class="container-fluid js-tm-page-content" data-page-no="4" data-page-type="gallery"> | ||
<div class="tm-img-gallery-container tm-img-gallery-container-3"> | <div class="tm-img-gallery-container tm-img-gallery-container-3"> | ||
Line 146: | Line 152: | ||
<div class="grid-item"> | <div class="grid-item"> | ||
<figure class=""> | <figure class=""> | ||
− | <img src="https://static.igem.org/mediawiki/2017/6/65/SP5.png" alt="Image" class="img-fluid tm-img"> | + | <img src="https://static.igem.org/mediawiki/2017/6/65/SP5.png" alt="Image" class="img-fluid tm-img"><br><font size=2> |
+ | Figure5: The OD600 of BBa_J37032 and BBa_K2305004.</font></br> | ||
</div> | </div> | ||
<div class="grid-item"> | <div class="grid-item"> | ||
<figure class=""> | <figure class=""> | ||
− | <img src="https://static.igem.org/mediawiki/2017/d/dd/SP6.png" alt="Image" class="img-fluid tm-img"> | + | <img src="https://static.igem.org/mediawiki/2017/d/dd/SP6.png" alt="Image" class="img-fluid tm-img"><br><font size=2> |
+ | Figure6: The part with strong promoter has about 10 times larger GFP fluorescence than the part without the strong promoter.</font></br> | ||
</div> | </div> | ||
Line 161: | Line 169: | ||
<div class="row animate-box"> | <div class="row animate-box"> | ||
<div class="col-md-8 col-md-offset-2 text-center fh5co-heading"> | <div class="col-md-8 col-md-offset-2 text-center fh5co-heading"> | ||
− | <h2>2.Cyclic Amplifier</h2> | + | <h2>2.2 Cyclic Amplifier</h2> |
<center> | <center> | ||
<img src="https://static.igem.org/mediawiki/2017/1/1c/%E5%BE%AA%E7%8E%AF%E6%94%BE%E5%A4%A7%E8%B7%AF%E7%BA%BF%E5%9B%BE.png" alt="Free HTML5 Bootstrap Template" class="img-responsive img-rounded"> | <img src="https://static.igem.org/mediawiki/2017/1/1c/%E5%BE%AA%E7%8E%AF%E6%94%BE%E5%A4%A7%E8%B7%AF%E7%BA%BF%E5%9B%BE.png" alt="Free HTML5 Bootstrap Template" class="img-responsive img-rounded"> | ||
</center> | </center> | ||
− | <p> The luxI downstream the induced promoter plux will be encoded to produce LuxI, which can combine with LuxR to promote plux. It can be used to form a positive feedback, so that the input signal can be further amplified.<br> In order to verify the effect of the cyclic amplifier on the fluorescent signal, we designed two gene circuits. Without luxI, BBa_K2305005 cannot produce LuxI, so it can not constitute a cyclic amplifier. But BBa_K2305016 can do it, then we compare these two gene circuits to verify the cyclic amplifier.</p> | + | <p> The luxI downstream the induced promoter plux will be encoded to produce LuxI, which can combine with LuxR to promote plux. It can be used to form a positive feedback, so that the input signal can be further amplified.<br> In order to verify the effect of the cyclic amplifier on the fluorescent signal, we designed two gene circuits. Without luxI, <a href="http://parts.igem.org/Part:BBa_K2305005">BBa_K2305005</a> cannot produce LuxI, so it can not constitute a cyclic amplifier. But <a href="http://parts.igem.org/Part:BBa_K2305016">BBa_K2305016</a> can do it, then we compare these two gene circuits to verify the cyclic amplifier.</p> |
<center> | <center> | ||
<img src="https://static.igem.org/mediawiki/2017/8/8b/CA4.png" alt="Free HTML5 Bootstrap Template" class="img-responsive img-rounded"> | <img src="https://static.igem.org/mediawiki/2017/8/8b/CA4.png" alt="Free HTML5 Bootstrap Template" class="img-responsive img-rounded"> | ||
Line 172: | Line 180: | ||
<img src="https://static.igem.org/mediawiki/2017/8/88/CA5.png" alt="Free HTML5 Bootstrap Template" class="img-responsive img-rounded"> | <img src="https://static.igem.org/mediawiki/2017/8/88/CA5.png" alt="Free HTML5 Bootstrap Template" class="img-responsive img-rounded"> | ||
</center> | </center> | ||
− | <p> The results of the validation show that the GFP fluorescence of BBa_K2305005 did not change over time. The fluorescence intensity of BBa_K2305016 remarkably increased over time, and the growth rate has increased after seventh hours later. Therefore, it can be shown that the expression of GFP protein, as significantly increased after the addition of the cyclic amplifier, which will have a greater effect on our detection. | + | <p> The results of the validation show that the GFP fluorescence of <a href="http://parts.igem.org/Part:BBa_K2305005">BBa_K2305005</a> did not change over time. The fluorescence intensity of <a href="http://parts.igem.org/Part:BBa_K2305016">BBa_K2305016</a> remarkably increased over time, and the growth rate has increased after seventh hours later. Therefore, it can be shown that the expression of GFP protein, as significantly increased after the addition of the cyclic amplifier, which will have a greater effect on our detection. |
</p> | </p> | ||
+ | </div> | ||
<div class="container-fluid js-tm-page-content" data-page-no="4" data-page-type="gallery"> | <div class="container-fluid js-tm-page-content" data-page-no="4" data-page-type="gallery"> | ||
<div class="tm-img-gallery-container tm-img-gallery-container-3"> | <div class="tm-img-gallery-container tm-img-gallery-container-3"> | ||
Line 180: | Line 189: | ||
<div class="grid-item"> | <div class="grid-item"> | ||
<figure class=""> | <figure class=""> | ||
− | <img src="https://static.igem.org/mediawiki/2017/3/3c/CA2.png" alt="Image" class="img-fluid tm-img"> | + | <img src="https://static.igem.org/mediawiki/2017/3/3c/CA2.png" alt="Image" class="img-fluid tm-img"><br><font size=2> |
+ | Figure7: The OD600 of BBa_K2305005 and BBa_K2305016.</font></br> | ||
</div> | </div> | ||
<div class="grid-item"> | <div class="grid-item"> | ||
<figure class=""> | <figure class=""> | ||
− | <img src="https://static.igem.org/mediawiki/2017/5/5a/CA3.png" alt="Image" class="img-fluid tm-img"> | + | <img src="https://static.igem.org/mediawiki/2017/5/5a/CA3.png" alt="Image" class="img-fluid tm-img"><br><font size=2> |
+ | Figure8: The part with the cyclic amplifier has about 10 times larger GFP fluorescence than the part without the cyclic amplifier.</font></br> | ||
</div> | </div> | ||
Line 197: | Line 208: | ||
<div class="row animate-box"> | <div class="row animate-box"> | ||
<div class="col-md-8 col-md-offset-2 text-center fh5co-heading"> | <div class="col-md-8 col-md-offset-2 text-center fh5co-heading"> | ||
− | <h2> | + | <h2>2.3 Dual Fluorescence System</h2> |
− | <p> We inserted the lacl gene into the downstream of the plux gene. LacI can inhibit the expression of the plac promoter to inhibit the expression of the RFP gene, a reporter downstream the plac promoter. Because of the existence of the strong promoter and the cyclic amplifier, the expression and the fluorescence intensity of the GFP will increase along with the growth of the E.coli ΔLysA, which is promoted by lysine. On the contrary, the expression and the fluorescence intensity of the RFP are inhibited. Then we used the GFP /RFP fluorescence intensity ratio to represent the output signal to improve the sensitivity.</p> | + | <p> We inserted the lacl gene into the downstream of the plux gene. LacI can inhibit the expression of the plac promoter to inhibit the expression of the RFP gene, a reporter downstream the plac promoter. Because of the existence of the strong promoter and the cyclic amplifier, the expression and the fluorescence intensity of the GFP will increase along with the growth of the E.coli ΔLysA, which is promoted by lysine. On the contrary, the expression and the fluorescence intensity of the RFP are inhibited. Then we used the GFP/RFP fluorescence intensity ratio to represent the output signal to improve the sensitivity.</p> |
<center> | <center> | ||
<img src="https://static.igem.org/mediawiki/2017/1/1f/DF1.png" alt="Free HTML5 Bootstrap Template" class="img-responsive img-rounded"> | <img src="https://static.igem.org/mediawiki/2017/1/1f/DF1.png" alt="Free HTML5 Bootstrap Template" class="img-responsive img-rounded"> | ||
Line 205: | Line 216: | ||
<img src="https://static.igem.org/mediawiki/2017/4/45/DF2.png" alt="Free HTML5 Bootstrap Template" class="img-responsive img-rounded"> | <img src="https://static.igem.org/mediawiki/2017/4/45/DF2.png" alt="Free HTML5 Bootstrap Template" class="img-responsive img-rounded"> | ||
</center> | </center> | ||
− | <p> To test the effect of the dual reporter fluorescence, the genetic circuits we designed are as shown. The pbad is a promoter induced by L-arabinose. We simulate the response of E.coli ΔLysA to the lysine concentration with the response of pbad promoter to the L-arabinose concentration. With the increase of the lysine concentration, the transcriptional efficiency of the pbad increases, therefore the expression of the lacl and GFP will also increase. On the contrary, the expression of RFP is low, because LacI can inhibit the plac promoter. Then we use the GFP /RFP fluorescence intensity ratio to represent the concentration of the L-arabinose. And we have worked out the relationship of the GFP expression of BBa_K584000 and the L-arabinose concentration, as well as the connection between the fluorescence ratio of the BBa_K2305002 and the L-arabinose concentration, in order to verify that the dual reporter fluorescence can improve the sensitivity of our detection. | + | <p> To test the effect of the dual reporter fluorescence, the genetic circuits we designed are as shown. The pbad is a promoter induced by L-arabinose. We simulate the response of E.coli ΔLysA to the lysine concentration with the response of pbad promoter to the L-arabinose concentration. With the increase of the lysine concentration, the transcriptional efficiency of the pbad increases, therefore the expression of the lacl and GFP will also increase. On the contrary, the expression of RFP is low, because LacI can inhibit the plac promoter. Then we use the GFP/RFP fluorescence intensity ratio to represent the concentration of the L-arabinose. And we have worked out the relationship of the GFP expression of <a href="http://parts.igem.org/Part:BBa_K584000">BBa_K584000</a> and the L-arabinose concentration, as well as the connection between the fluorescence ratio of the <a href="http://parts.igem.org/Part:BBa_K2305002">BBa_K2305002</a> and the L-arabinose concentration, in order to verify that the dual reporter fluorescence can improve the sensitivity of our detection. |
</p> | </p> | ||
</div> | </div> | ||
Line 212: | Line 223: | ||
<div class="row animate-box"> | <div class="row animate-box"> | ||
<div class="col-md-8 col-md-offset-2 text-center fh5co-heading"> | <div class="col-md-8 col-md-offset-2 text-center fh5co-heading"> | ||
− | <h2>The Test of Whole Route</h2> | + | <h2>3.The Test of Whole Route</h2> |
− | <p> After we had proved that the strong promoter, cyclic amplifier and the dual fluorescence system can enhance the signal and improve the sensitivity, we transformed the whole genetic circuit into the E.coli ΔLysA. It was cultured in LB medium till its OD600 had reached 0.4. Then aliquots of 100ul were transferred into M9 medium to deplete lysine. After 10h, aliquots of 100ul were transferred into M9 medium with lysine. Lysine was added in increments of 0.5ug/mL to establish a range of 0-3ug/mL. We detected the OD600 and the GFP and RFP fluorescence every hour with microplate reader. The control group was the E.coli ΔLysA with the part | + | <p> After we had proved that the strong promoter, cyclic amplifier and the dual fluorescence system can enhance the signal and improve the sensitivity, we transformed the whole genetic circuit <a href="http://parts.igem.org/Part:BBa_K2305000">BBa_K2305000</a> into the E.coli ΔLysA. It was cultured in LB medium till its OD600 had reached 0.4. Then aliquots of 100ul were transferred into M9 medium to deplete lysine. After 10h, aliquots of 100ul were transferred into M9 medium with lysine. Lysine was added in increments of 0.5ug/mL to establish a range of 0-3ug/mL. We detected the OD600 and the GFP and RFP fluorescence every hour with microplate reader. The control group was the E.coli ΔLysA with the part <a href="http://parts.igem.org/Part:BBa_K876070">BBa_K876070</a>. |
</p> | </p> | ||
</div> | </div> |
After we have verified that lysine can be the signal upstream the sensor, what we need to do next is to conform the connection between the lysine concentration and the growth of E.coli ΔLysA. Before that, we have to make sure that the growth of wild type E.coli has no connection with lysine concentration. Part BBa_K876070 was transformed into wild type E.coli to test its OD600 and GFP fluorescence every hour with microplate reader. Lysine was added in increments of 0.5ug/mL to establish a range of 0-3ug/mL. It shows that the growth of wild type E.coli has no connection with lysine concentration. From this, we can say that it’s necessary for E.coli ΔLysA to be the signal receiver of lysine.
To explore the connection between the lysine concentration and the growth of E.coli ΔLysA, we first cultured E.coli ΔLysA in LB medium till its OD600 has reached 0.4. Then aliquots of 100ul were transferred into M9 medium to deplete lysine. After 10h, aliquots of 100ul were transferred into M9 medium with lysine. Lysine was added in increments of 0.5ug/mL to establish a range of 0-3ug/mL. OD600 was measured with microplate reader every hour. The result shows that the OD600 of E.coli ΔLysA has a linear relation with lysine concentration. Thus, we have proved our lysine sensor to be feasible.
After we have confirmed that the E.coli ΔLysA can response and transform lysine signal to the fluorescent signal, what we need to do is to amplify the fluorescent signal and improve the sensitivity. To solve this problem and make sure that our own-designed circuit works well, we need to test the effect of the strong promoter, cyclic amplifier and dual fluorescence system respectively. The verified experiments are as follows.
To confirm that the strong constitutive promoter can induce plux promoter, we designed the biobrick BBa_K2305004. The purpose of this biobrick is to produce as much green fluorescence as possible by inducing the induced promoter plux, to ensure a stronger expression in E.coli.
We added a strong promoter on the basis of the previous part BBa_J37032. Parts from BBa_J23100 to BBa_J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. With the addition of the strong constitutive promoter upstream the plux, we hope the plux promoter can be induced and therefore express more GFP. As the figure shows, in the first eight hours the OD600 value grew well, and the new part reached a higher GFP expression in E.coli than the previous part. Importantly, the new part has amplified the expression of GFP for more than 10 times. So we can say that with the existence of the strong constitutive promoter, the expression of GFP has been amplified. So it’s effective for us to put plux promoter upstream the route, in other to amplify the detecting signal.
The luxI downstream the induced promoter plux will be encoded to produce LuxI, which can combine with LuxR to promote plux. It can be used to form a positive feedback, so that the input signal can be further amplified.
In order to verify the effect of the cyclic amplifier on the fluorescent signal, we designed two gene circuits. Without luxI, BBa_K2305005 cannot produce LuxI, so it can not constitute a cyclic amplifier. But BBa_K2305016 can do it, then we compare these two gene circuits to verify the cyclic amplifier.
The results of the validation show that the GFP fluorescence of BBa_K2305005 did not change over time. The fluorescence intensity of BBa_K2305016 remarkably increased over time, and the growth rate has increased after seventh hours later. Therefore, it can be shown that the expression of GFP protein, as significantly increased after the addition of the cyclic amplifier, which will have a greater effect on our detection.
We inserted the lacl gene into the downstream of the plux gene. LacI can inhibit the expression of the plac promoter to inhibit the expression of the RFP gene, a reporter downstream the plac promoter. Because of the existence of the strong promoter and the cyclic amplifier, the expression and the fluorescence intensity of the GFP will increase along with the growth of the E.coli ΔLysA, which is promoted by lysine. On the contrary, the expression and the fluorescence intensity of the RFP are inhibited. Then we used the GFP/RFP fluorescence intensity ratio to represent the output signal to improve the sensitivity.
To test the effect of the dual reporter fluorescence, the genetic circuits we designed are as shown. The pbad is a promoter induced by L-arabinose. We simulate the response of E.coli ΔLysA to the lysine concentration with the response of pbad promoter to the L-arabinose concentration. With the increase of the lysine concentration, the transcriptional efficiency of the pbad increases, therefore the expression of the lacl and GFP will also increase. On the contrary, the expression of RFP is low, because LacI can inhibit the plac promoter. Then we use the GFP/RFP fluorescence intensity ratio to represent the concentration of the L-arabinose. And we have worked out the relationship of the GFP expression of BBa_K584000 and the L-arabinose concentration, as well as the connection between the fluorescence ratio of the BBa_K2305002 and the L-arabinose concentration, in order to verify that the dual reporter fluorescence can improve the sensitivity of our detection.
After we had proved that the strong promoter, cyclic amplifier and the dual fluorescence system can enhance the signal and improve the sensitivity, we transformed the whole genetic circuit BBa_K2305000 into the E.coli ΔLysA. It was cultured in LB medium till its OD600 had reached 0.4. Then aliquots of 100ul were transferred into M9 medium to deplete lysine. After 10h, aliquots of 100ul were transferred into M9 medium with lysine. Lysine was added in increments of 0.5ug/mL to establish a range of 0-3ug/mL. We detected the OD600 and the GFP and RFP fluorescence every hour with microplate reader. The control group was the E.coli ΔLysA with the part BBa_K876070.
Facilis ipsum reprehenderit nemo molestias. Aut cum mollitia reprehenderit. Eos cumque dicta adipisci architecto culpa amet.