Difference between revisions of "Part:BBa K3829012"
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<p>Anchor proteins 4609 was screened by model prediction and yeGFP characterization. We then used it for the following experiment. On the basis of <a href="https://parts.igem.org/Part:BBa_K3829010">BBa_K3829010</a>, we replaced PETase with yeGFP to obtain the composite parts BBa_K3829012.</p> | <p>Anchor proteins 4609 was screened by model prediction and yeGFP characterization. We then used it for the following experiment. On the basis of <a href="https://parts.igem.org/Part:BBa_K3829010">BBa_K3829010</a>, we replaced PETase with yeGFP to obtain the composite parts BBa_K3829012.</p> | ||
| + | <img src="https://2021.igem.org/wiki/images/5/5c/T--IvyMaker-China--Lab-51.png" style = "width:70%;"> | ||
| + | <br/><b>Fig.1</b> The structure of the gene circuit. | ||
| + | <br/> | ||
| + | <p>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 2).</p> | ||
| + | <img src="https://2021.igem.org/wiki/images/d/d2/T--IvyMaker-China--Lab-22.jpg" style = "width:50%;"> | ||
| + | <img src="https://2021.igem.org/wiki/images/d/d2/T--IvyMaker-China--Lab-22.jpg" style = "width:50%;"> | ||
| + | <br/><b>Fig.2</b> Determination of enzyme activity of PETase. | ||
| + | <br/> | ||
| + | <p>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.</p> | ||
| − | + | <img src="https://2021.igem.org/wiki/images/2/20/T--IvyMaker-China--Lab-39-new.png" style = "width:70%;"> | |
| − | + | <br/><b>Fig.3</b> Hydrolysate content of PET powder. Content of PET powder: 10 mg, thallus: OD=5, reaction system: 1 mL, reaction time: 18 h. | |
| + | <br/> | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
| − | <partinfo>BBa_K3829012 | + | <partinfo>BBa_K3829012 Sequence And Features</partinfo> |
Revision as of 06:06, 21 October 2021
P-ss-PETase-V5tag-Anchor protein 4609-T
The composite Part is used to express PETase. SS secretes PETase out of the cell, while anchor protein anchor 4609 anchor it on the cell surface. This part is based on BBa_K3829010, replacing GFP with PETase.
Anchor proteins 4609 was screened by model prediction and yeGFP characterization. We then used it for the following experiment. On the basis of BBa_K3829010, we replaced PETase with yeGFP to obtain the composite parts BBa_K3829012.
Fig.1 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 2).
Fig.2 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.3 Hydrolysate content of PET powder. Content of PET powder: 10 mg, thallus: OD=5, reaction system: 1 mL, reaction time: 18 h.
Sequence and Features