Team:RDFZ-China/Composite Parts

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Composite Parts

1. pTac-TPH1 (BBa_K3395004):

We decided to use TPH1 -We are looking for additional tryptophan hydroxylase to catalyze 5-HTP synthesis. According to the literature,we selected human-source TPH1 with high enzymatic activity and relatively complete sequences - TPH1（BBa_K3395000) and successfully constructed pTac-TPHI（BBa_K3395004).

2. Design

pTac-TPH1 is constructed on pET28-b plasmid. We transformed pET28b_TPH1 vector into E .coli BL21 for expression.

3. TPH1 activity assay

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.

1. pcauam_p3B5B(BBa_K3395015):

We decided to use p3B5B as an additional promoter that responds to PcauAM. With p3B5B BBa_K3395009, it's regulatory protein PcauAM BBa_K3395006 and a EYFP reporter BBa_K3395007, successfully constructed pcauam_p3B5B BBa_K3395015

2. Design:

We constructed pcauam_p3B5B into the low copy p15A plasmid. We transformed pET28b_pcauam_p3B5B vector into E .coli BL21 for testing.

3. Fluorescence assay:

PcauAM-YFP: excitation:485nm, emission:525nm:

The data in figure 15 are the tested data of PcaUAM-YFP under emission light 535nm.

When PCA molecular is not added, the fluorescence intensity divided by bacterial concentration of pcauAM-p3b5b and pcauAM-p3b5c are both about 3000. And when PCA is added, the YFP protein expression of the two sensing systems has increased to different degrees, and the value of F/A is increased when reflected in the image. The data shows both p3b5b and p3b5c promoter can be controlled by the pcauAM activator. And under the horizontal comparison between p3b5b and p3b5c promoter, p3b5b promoter have much higher sensitivity than p3b5c promoter.