Team:XJTU-China/Demonstrate
Demonstration
Our team is committed to improving the activity of DTE. We designed a DEcose (Directed Evolved D-psicose) platform to literally build a self-evolving factory for the DTE enzyme, where DE (Directed Evolution) provides us an automatic, high-throughput, repeatable production mode. We also designed a series of devices to demonstrate the platform: the sensing, selecting, coupling and evolving circuit. Here are some of results and explanation of our experiments to demonstrate our circuit really work.
Sensing circuit
Sensing Circuit converts the productivity (cellular concentration) of D-psicose to the expression level of downstream genes. In order to test efficiency of pPsi and PsiR, we did the following two experiments.
Orthogonality of pPsi
When selecting different enzyme, there will be psicose, gluctose, IPTG,and fructose in the medium. Other sugar like fructose may also bind to PsiR and induce gene expression. To illustrate the specify of promoter, we designed this experiment. We added same amout of psicose, fructose,gluctsoe,and IPTG into same amount of same bacteria. Fluorescence intensity and optical density (OD600)was measured every 30min and the curve of fluorescence in single cell strain change with time was plotted. The results were shown below.
Figure 1 Effect of different sugars on pPsi
Figure1 shows that at the same time, the florescence intensity varies with different sugar and psicose is always higher than other sugar. So we could draw this conclusion that pPsi is specific to psicose and not sensitive to other sugar.
Efficiency of pPsi
To qualitatively describe the relationship between fluorescence intensity and psicose concentration, we designed the following experiment to test the efficiency of pPsi. We added different amount of psicose into medium and measured Fluorescence intensity and optical density (OD600) every 15min. The curve of fluorescence in single cell strain change with time was plotted. The results were shown as blow.
Figure 2 concentration of psicose' s effect on pPsi
Figure 2 shows that the fluorescence intensity induced by 50mM of psicose was the highest and the higher the psicose concentration, the fluorescence intensity at the same time will be more intense. We could draw the conclusion that the efficiency of pPsi and concentration of psicose in the medium are positively related.
Selecting Circuit
We have explored the potential to use a series of antibiotic resistance genes as quantitative reporter, which their expression levels are converted into different growth rates of chassis organisms. In order to make the characterization data reusable, we have substituted pPsi promoter to pLac. pLac is a more characterized promoter than D-psicose induced one.
We designed a series of experiments to characterize the function of selecting circuit and to decide which antibiotic is suitable for our system. Take selecting circuit-KanR for example, we put the same quantity of bacteria with selecting circuit into a 96-well plate, added different concentration of Kan and IPTG into the medium and measured the OD600 during 5h. The results were shown as below.
Figure 3-7 Interaction of AB and AB resistance proteins
When the concentration of IPTG locates in a certain range (below 1.5mM), the final OD600 of bacteria will decrease as the concentration of Kan increase. It implies that the bacteria have a high possibility of survival if the expression level of KanR is high, because a higher IPTG concentration can induce more expression of KanR. Moreover, the bacteria which expressed more KanR survived more.
As for selecting circuit-AmpR, only a little expression of AmpR can give bacteria extraordinary resistance. Although we didn’t add any inducer to the medium, the bacteria grown merrily in the ocean of Amp. Selecting circuit-SmR is the same. There is no doubt that Amp and Smr are not competent to screening. We chose KanR to construct our device D.
Coupling circuit
To test whether through hairpin structure, the downstream translational level is quantitatively associated with the upstream. We designed this experiment. In different time periods, we measured RFP and EGFP fluorescence intensity and fitted a curve. The results were shown as below.
Figure 3 relationship between RFP and EGFP fluorescence intensity
Figure 3 shows that when GFP fluorescence intensity is within the scope of 80-230, RFP and EGFP fluorescence intensity is near linear relationship. However, further study should be done to confim this later.
Evolving Circuit
Evolving Circuit summarizes all regulatory mechanisms used above and acts to quantitatively convert different productivities of DTE to a spectrum of growth rates. Utilizing the characterization from Selecting and Coupling circuits, we achieved the suitable antibiotic concentration to exert growth pressure in evolution.
Previous study has shown different activity of DTEs (DTE and DPE, figure4 ). To demostrate our system really work, we constructed evolving circuit plasmid with DTE and DPE separately and transformed them into E.coli.
Figure 4 different activities of DTEs
Firstly, according to sensing circuit, the efficiency of pPsi and concentration of psicose in the medium are positively related. Secondly, if activity of DTEs is higher, there will be more psicose in the medium which results in more gene expression. Thirdly, according to coupling circuit, the fluorescence intensity was qualitatively related to the expression of resistance genes. To characterize the expression of antibiotic resistance gene, we measured florescence intensity at different time and ABS at 600. The curve of fluorescence in single cell strain change with time was plotted. The results were shown as blow.
Figure 5 Changes of fluorescence intensity per cell of E.coli harboring DTEs with different activities
Figure 5 shows that compared with E. coli harboring DPE, florescence intensity per cell of E.coli harboring DTE is always higher no matter under which concentration of antibiotic. In other words, antibiotic gene expressed in E. coli harboring DTE is always higher than DPE, which implied that the activity of DTE is higher than DPE. This also means that our system can distinguish DTEs with different activities. This also demonstrate our system really work.
According to selecting circuit, kanamycin is better than other antibiotics. We set KanR three concentration gradients and measured changes of cell growth of E. coli under these concentration. The results are shown below (figure 6 and 7).
Figure 6 changes of cell growth of E. coli (DTE)
Figure 7 changes of cell growth of E. coli (DPE)
Both figure 6 and 7 show that at the same time, ABS at 600 gradually decreased with the increase of antibiotic concentration. In other words, increasing concentration of antibiotics means increasing the pressure for evolution and the inhibition of bacterial growth is also more obvious.
They also mean that our system can work.