Difference between revisions of "Team:BIT/BA2"
| Line 103: | Line 103: | ||
<h2>Strong Promoter</h2> | <h2>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 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> | ||
| + | </div> | ||
| + | <div id="fh5co-blog"> | ||
| + | <div class="container"> | ||
| + | <div class="row animate-box"> | ||
| + | <div class="col-md-8 col-md-offset-2 text-center fh5co-heading"> | ||
| + | <h2>Cyclic Amplifier</h2> | ||
| + | <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.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. | ||
</p> | </p> | ||
</div> | </div> | ||
Revision as of 18:53, 29 October 2017
Necessity of E.coli ΔLysA
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.
Confirmation in Models
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.
Strong Promoter
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.
Cyclic Amplifier
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.
Hire Us!
Facilis ipsum reprehenderit nemo molestias. Aut cum mollitia reprehenderit. Eos cumque dicta adipisci architecto culpa amet.
