Figure1: The of the wild type E.coli has no connection with the lysine concentration.
Figure2: The GFP fluorescence of the wild type E.coli has no connection with the lysine concentration.
Team:BIT/Demonstrate/Bio-amplifier
1.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.
Figure1: The of the wild type E.coli has no connection with the lysine concentration.
Figure2: The GFP fluorescence of the wild type E.coli has no connection with the lysine concentration.
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.
Figure3:The OD600 of the E.coli ΔLysA is induced by the lysine concentration.
Figure4:The GFP fluorescence of the E.coli ΔLysA is induced by the lysine concentration.
2.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.
2.1 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.
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.
Figure5: The OD600 of BBa_J37032 and BBa_K2305004.
Figure6: The part with strong promoter has about 10 times larger GFP fluorescence than the part without the strong promoter.
2.2 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.
Figure7: The OD600 of BBa_K2305005 and BBa_K2305016.
Figure8: The part with the cyclic amplifier has about 10 times larger GFP fluorescence than the part without the cyclic amplifier.
2.3 Dual Fluorescence System
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.
Figure9: The OD600 of BBa_K584000.
Figure10: The OD600 of BBa_K2305002.
We constructed the relation between the fluorescence and L-arabinose concentration with the fixed time, and the relation between the fluorescence and time with the fixed L-arabinose concentration.We can see from the figure that the effect of the dual fluorescence is not very obvious. Therefore we will choose another way to deal with this system, which will be shown in our discussion part.
Figure11: The linear relation between the GFP fluorescence and the L-arabinose concentration with fixed time in 6h.
Figure12: The linear relation between the GFP and RFP fluorescence ratio and the L-arabinose concentration with fixed time in 6h.
Figure13: The linear relation between the GFP fluorescence and time with fixed L-arabinose concentration in 6h.
Figure14: The linear relation between the GFP and RFP fluorescence ratio and time with fixed L-arabinose concentration in 6h.
3.The Test of Whole Route
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 circuitBBa_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.
The result is as follows. We built a relationship between the OD600 and the lysine concentration, the GFP and RFP fluorescence ratio respectively. Then we fitted the curve and found that in the third hour, we can get the best linear relation between the GFP and RFP fluorescence ratio and the lysine concentration. The R2 we got was 0.9984. Therefore we chose the third hour to be the time for detecting. The figure is as shown. Then we calculated the LOD(limit of detection). We plug three times of the error into the equation in the third hour, and got the LOD to be 0.000178 ug/ml lysine.
Figure15: The OD600 of of BBa_K2305000 in E.coli ΔLysA induced by the lysine concentration.
Figure16: The GFP fluorescence of BBa_K2305000 in E.coli ΔLysA induced by the lysine concentration.
Figure17:The GFP fluorescence of BBa_K2305000 in E.coli ΔLysA in the third hour.
4. Discussion
Since the GFP and RFP fluorescence ratio can hardly improve the sensitivity of the detection, we decided to use another way to make the dual fluorescence system effective. For BBa_K584000, we devised the growth rate of the GFP fluorescence by the amount of the bacteria, to get a curve about GFP-producing speed of every bacteria. The variate "a" is the amount of the bacteria in 100ul when the OD600 is about 0.1. We assumed the "a" to be 2000. For BBa_K2305002, we calculated the derivative of the GFP and RFP fluorescence respectively, and made a curve of the growth rate of its ratio. The L-arabinose was added 30ul in 7ml LB medium. The part with the dual fluorescence system can react the L-arabinose signal more quickly. Through comparison, we can say that the dual fluorescence system has a higher sensitivity and amplification factor than the part without the dual fluorescence system.
Figure7: The GFP-producing speed of every bacteria with BBa_K584000.
Figure8: For BBa_K2305002, we calculated the derivative of the GFP and RFP fluorescence respectively, and made a curve of the growth rate of its ratio.
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