Team:RDFZ-China/Engineering

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Engineering success

Overview

1.PcaUAM-p3B5b Sensor (BBa_K3395015)

2.TPH1(BBa_K3395000) and musTPH1(BBa_K3395001)

Comparing the data we got through several tests about the sensitivity of different kinds of sensing systems. All three systems show different positive sensitivity when PCA is added. However, after integrating many considerations, we constructed the PcaUAM sensor which is made of upstream PcauAM expressor and downstream p3b5c promoter, from p3b5c promoter we conducted a point mutation and derived the p3b5b promoter, and we found out that it is the combination that functions the best.

1.PcaUAM-p3B5b Sensor (BBa_K3395015)

The reason why we don't choose the original promoter with PcaU expressor is that the data proved the original promoter in downstream still presents a high binding rate with RBS in the circumstance without PCA, which will cause the downstream expressor express without control.

From the figure, we can see the 2 groups of repeat experiments both present that fluorescence intensity in our Pcau-original-puc57 plasmid is much higher than our empty plasmid, which denotes the leaking of downstream sequence. This will make our modeling and future work very difficult. Because we can not know the amount of 5-HTP if we build this system and TPH1 effector together.

Because the difference between the other 2 sensing systems only includes the difference in the downstream promoter. Sensitivity is our primary focus. Our design can visually and clearly show the sensitivity and effectiveness of the pcau sensor by replacing TPH, which is hard to detect, with to GFP/YFP gene which is a reporter gene that is easier to be visualized.

The data here clearly represents that at the emission wavelength of 535 nm, the sensitivity of p3b5b downstream promoter is higher than the p3b5c promoter. We can also use the data to create a dose-response curve for the p3B5b sensor, so that we are able to detect the effectiveness and sensitivity of the whole system, including pLacIQ, PcaUAM, p3B5B, and EYFP.

The reason why BN056 empty plasmid has high fluorescence intensity maybe because that BN056 plasmid has a GFP sequence. We have cut and thrown away during the experiment of building PcauAM-p3b5b/p3b5c-BN056 plasmid.

Above all, we conducted a point mutation on the existing p3b5c promoter and got the p3b5b promoter. With it, PcauAM-p3b5b-BN056 is the best combination we get through the whole experiment of the sensing system.

Here shows the figure of our design on the PcauAM-p3b5b system:

2.TPH1 (BBa_K3395000) & musTPH1 (BBa_K3395001)

For TPH1 engineering, we designed and characterized pTac-TPHI (BBa_K3395004) and pTac-musTPHI (BBa_K3395005). Because we only need the two pathways to characterize and to determine whether TPH1 sequence is functional, we only designed a pair of restriction enzyme cutting sites for pTac-TPHI. mus-TPH1 is the tryptophan hydroxylase in mice. It is known to catalyze the same reaction human TPH1 did. Although the full experimentation data haven’t been completed on this biobrick, musTPH1 is proven to have high expression in the bacteria strain E.coli MC1061 with proper activity to produce concentration greater than 1mM under certain conditions by the article Park D H, Stone D M, Kim K S, et al. Characterization of recombinant mouse tryptophan hydroxylase expressed in Escherichia coli[J]. Molecular and Cellular Neuroscience, 1994, 5(1): 87-93.

TPH1 sequence can express tryptophan hydroxylase, and this enzyme can catalyze a common amino acid, tryptophan, into 5-HTP, which will be transported to our brain and alleviate depression. First, we successfully constructed TPH1 and LacI sequence on pet28b plasmid, and use IPTG to activate the downstream expression. Then we conducted enzymatic activity assay on it according to a protocol “A continuous fluorescence assay for Tryptophan Hydroxylase” Moran and Fitzpatrick, AnalyticalBiochemistry 266, 148–152 (1999). We derived 5-HTP concentration from the fluorescence result. From the data, TPH1 enzymes express and work very well in the substrate solution. But the lacI control system didn't work, because the curve of enzymatic reaction with IPTG negative and positive can highly overlap each other. But the tendency of our result can be assumed to attach Mistral kinetics theory, which means the relationship between the Max velocity and the concentration of an enzyme can be calculated.

The column 1-6 is the serial dilution of enzyme concentration, where enzyme concentration increases from column 1 to 6

We planned to use musTPH1 as another effector. However, since the process of building musTPH1 sequence on our backbone hasn't been successful, we will construct musTPH1 in the future, and test its function.

Reference

1.Park D H, Stone D M, Kim K S, et al. Characterization of recombinant mouse tryptophan hydroxylase expressed in Escherichia coli[J]. Molecular and Cellular Neuroscience, 1994, 5(1): 87-93.