BASIC PARTS

In our experiment, signal peptide (SS) was used. SS was a short peptide that could secrete proteins outside the cell. We constructed different gene circuits as shown in Figure 1 to verify the function of SS. The results showed that When SS was not present, fluorescence diffused in the cell (positive control). When SS was present (4609 and 5105), the fluorescence was displayed on the cell membrane, indicating that SS could secrete proteins outside the cell.

The anchor protein, PETase and MHETase in this project were characterized by yeGFP and the related results were presented in the composite parts.

Our part BBa_K33829011 is a recombinant yeGFP improved from the part reporter GFP (BBa_K3402000). We optimized the codon and added a stronger promoter (BBa_K3829001) and terminator (BBa_K3829000). In our project, yeGFP was used to screen anchored proteins.
Through restriction enzyme digestion verification and sequencing, the plasmid containing P-GAPDH was successfully constructed (Figure 3).

Fig.5 Verification of recombinant plasmids by restriction enzyme digestion. M: DL 15000 DNA Marker
1：Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-5105-T double enzyme digestion (Xba Ⅰ & EcoR Ⅰ)
The plasmid was linearized and transferred to Candida tropicalis. The transformants were screened out by uracil deficiency. And then cultivated the transformants and observed the fluorescence with confocal laser scanning microscopy (CLSM). As a result, green fluorescence was observed on the cell surface, indicating that yeGFP was expressed. (Figure 4). Therefore, it was demonstrated that P-GAPDH could effectively transcribe the downstream genes in Candida tropicalis.

Fig.6 Representative images of yeGFP (Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-5105-T) expression. The yeast morphology observed under the bright field (Left). The yeast morphology observed under green fluorescence excitation wavelength (Middle). Merged image (Right).

Fig.9 Verification of recombinant plasmids with restriction enzyme digestion. M:DL 15000 DNA Maker
1：Plasmid Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-4609-T double enzyme digestion (EcoR Ⅰ &Xba Ⅰ)
After the plasmid was successfully constructed, it was introduced into the target strain Candida tropicalis. And then the expression of yeGFP and the position of fluorescent was confirmed with CLSM (Figure 8).

Fig.10 Representative images of yeGFP (Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-4609-T) expression. The yeast morphology observed under the bright field (Left). The yeast morphology observed under green fluorescence excitation wavelength (Middle). Merged image (Right).

Anchor proteins 4609 and 5105 were screened by model prediction and yeGFP characterization. We then used them for the following experiment. On the basis of BBa_K3829010 and BBa_K3829011, we replaced PETase with yeGFP to obtain the composite parts BBa_K3829012 and BBa_K3829013.

Fig.11 The structure of the gene circuit.
The overall enzyme activity of PETase was measured. Firstly, a crude test was carried out to show the enzyme activity at different temperatures. Then, p-nitrophenol absorption was measured because PETase could also catalyze substrates into p-nitrophenol. The results showed that PET-4609 and 5105 performed better than the wild type ATCC20336 and cytPET (Figure 12).

Fig.12 Determination of enzyme activity of PETase.
It was acknowledged that PETase was able to degrade PET into TPA and MHET. According to the HPLC detection results, the product content of the degraded PET powder of each strain were plotted (Figure 13). It was demonstrated that target products were not detected in the group of ATCC 20336. Compared with the control strain, degradation products were obviously presented in the strain PET-4609 and PET-5105, indicating PETase is displayed on the surface of Candida tropicalis with high enzyme activity. However, several products were detected in the strain cytPET which should not be detected theoretically. It was speculated that a part of PETase is released due to the lysis of cells.

Fig.13 Hydrolysate content of PET powder. Content of PET powder: 10 mg, thallus: OD=5, reaction system: 1 mL, reaction time: 18 h.
In conclusion, these results demonstrated that the surface display system was indeed able to degrade PET, which was consistent with the previous results.

Name	Part Number	Part Description
P_-GAPDH	BBa_K3829001	Promoter
T_-GAPDH	BBa_K3829000	Terminator
V5-Tag	BBa_K3829004	V5-Tag
PETase	BBa_K3829008	Protein domain of PETase
MHETase	BBa_K3829009	Protein domain of MHETase
yeGFP	BBa_K3829002	Reporter
SS	BBa_K3829005	Signal peptide
Anchor protein 4609	BBa_K3829006	Anchor protein 4609
Anchor protein 5105	BBa_K3829007	Anchor protein 5105
P-ss-yeGFP-V5tag-Anchor protein 4609-T	BBa_K3829010	Surface display system of yeGFP with anchor protein 4609
P-ss-yeGFP-V5tag-Anchor protein 5105-T	BBa_K3829011	Surface display system of yeGFP with anchor protein 5105
P-ss-PETase-V5tag-Anchor protein 4609-T	BBa_K3829012	Surface display system of PETase with anchor protein 4609
P-ss-PETase-V5tag-Anchor protein 5105-T	BBa_K3829013	Surface display system of PETase with anchor protein 5105
P-ss-MHETase-V5tag-Anchor protein 5105-T	BBa_K3829014	Surface display system of MHETase with anchor protein 5105

Team:IvyMaker-China/Parts