Team:RDFZ-China/Contribution

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Contribution

Overview:

1. The construction of PcaU-pPCA， PcauAM-p3b5c/p3b5b and lacI-TPH1 system.
2. The analysis of fluorence signal data of PcaU-pPCA and PcauAM-p3b5c/p3b5b.
3. The analysis of TPH1 enzymatic activity.

1. Construction of sensing system

1.1 PcaU-pPCA::

PcaU-pPCA is a PCA sensing system design by UMaryteam in 2015(Part:BBa K2825002). Pcau sequence activator will express a protein can be stablize and activate by PCA, and the Pcau protein with PCA can activate the downstream pPCA promoter. pPCA promoter is designed have very low expression when PCA is not present.

Figure 1, 2, 3 show the construction result of PcaU-pPCA

1.2 PcaUAM-p3B5C/p3B5B:

PcaUAM-p3B5C system was uploaded on parts library by 2018 UNAL team( BBa_K3317007). PcaUAM system has functions similar to PcaU system. but this activator can only activate downstream p3b5b or p3b5c promoter.

We conducted a point mutation on p3B5C and derived the p3B5B promoter. Constructed with the PcauAM regulator protein, we conducted experiments to examine the response of this part against PCA concentration. We uploaded this part BBa_K3395015 and the experiment data to the parts library.

The figures 4, 5, 6 show the construction of PcaUAM-p3B5B system

1.3 LacI-hTPH1:

LacI-hTPH1 is a system generating TPH enzymes from the human body. This system can be activated by IPTG. which was designed by previous IGEM team UCL in 2015.

The figure 10,11 and 12 shows the construction results of LacI-TPH1 system that our team use to detect the enzymatic activity of hTPH1(the trptophan hydroxylase in human).

2. The analysis of- fluorence signal data

2.1 Sensing system

We conducted fluorence assay on the sensing systems we constructed against the PCA gradient. Our design can visually and clearly show the sensitivity and effectiveness of the pcau sensor by adding GFP/YFP genes to the downstream and examining the fluorescence data.

The results we gained may be a good reference to other people considering using those parts. We also used the data to create a dose-response curve for the p3B5C sensor, for the effectiveness and sensitivity of the whole system, including pLacIQ, PcaUAM, p3B5C, and EYFP.

2.2 TPH1 enzymatic assay

The data in the right of figure 15 are the tested data of PcaU-pPCA-GFP under emission light 535nm. We make a serial dilution of PCA to do further tasking on PcaU-pPCA control system. We found similar property that when PCA molecular is not added, the fluorescence intensity/bacterial concentration of pcauU-pPCA is about 36000. And the GFP protein expression of the sensing systems has increased since the concentration of PCA increase, and the value of F/A is increased when reflected in the image. The data shows pPCA promoter can be controlled by the pcau activator and the concentration of PCA can be regulated and controlled by the concentration of PCA.

3. The analysis of TPH1 enzymatic activity

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.

By the enzymatic activity assay, we can show 5-HTP concentration varied by time and different concentration of enzymes. We build the model of TPH1 enzyme activity which will be explained in the model page. The data shows our TPH1 enzyme functions very well when trptophan and other catalases are present is the solution. The data and the model we built may be helpful for the community.