Engineering success #

Part 1 Design of scaffold for surface display #

1.1 Background #

Last year, we used the surface display system to display PETase and MHETase separately. To increase the effectiveness and convenience of the degradation of PET plastic, we plan to display MHETase and PETase at the same time.

Goal: Develop a self-assembled multi-enzyme display system of Candida tropicalis.

1.2 Research #

To attain co-display, we combined our display system with two selective protein binding systems, SpyTag-SpyCatcher and SnoopTag-SnoopCatcher.

Tag-Catcher system #

Tag-Catcher system include two pairs of tags and catchers: SpyTag/SpyCatcher and SnoopTag/SnoopCatcher. They are power bio-conjugation tools developed by Oxford researchers. Both tags and catchers can be connected to a peptide through covalent bonds. Additionally, when united, they spontaneously form irreversible isopeptide connections with one another.

Therefore, it is worthy to exploit this system in vivo because we need to target both of our enzymes on the surface of yeast at the same time.

Fluorescence intensity #

In our experiments, we intended to use fluorescent proteins, GFP and RFP, to help us construct the system. For fluorescent proteins, fluorescence intensity is a crucial index to measure the activity of the target in a fluorescence detection method. Its unit is arbitrary, and it is proportional to the concentration of the target. By detecting the color and intensity of fluorescence, we can verify the amount of enzymes expressed on the cell surface and the type of enzymes, therefore achieving our goal of building a double-enzyme display system.

1.3 Imagine and Design #

On the surface of Candida tropicalis, we can use SpyTag & SpyCatcher and SnoopTag & SnoopCatcher pairs to carry PETase and MHETase, respectively.

Step1: First, we can use different colors of fluorescent proteins, RFP and GFP, for example, to connect with Tag and Catcher. Then, by detecting surface fluorescence we can verify the plausibility of simultaneous display of PETase and MHETase.

Step2: After that, display GFP and RFP with MHETase and PETase.

1.4 Build #

In our experiment, GFP and RFP were used to indicate the successful construction of Spycatcher/Spytag and Snoopcatcher/Snooptag systems. We initially tried two catcher types with a ratio of 1:3.

1.5 Test #

GFP+ and RFP+ suggested the successful construction of Spycatcher/Spytag system and Snoopcatcher/Snooptag system.

1.6 Learn #

When replacing RFP and GFP with MHETase and PETase, we did not observe immunofluorescence with secondary antibodies that should theoretically bind specifically to the V5 tag. To analyze whether PETase-spytag and MHETase-snooptag fused protein folded correctly, we constructed a model of the fusion protein, we used prediction software such as trRosetta and ITASSER to construct the structure. The evaluation results of the two models shows the structure is convincing. So, no enzyme activity could be a steric hindrance between the fusion protein and the scaffold (See Modeling for details, https://2022.igem.wiki/ivymaker-china/model.html).

Similarly, we used I-TASSER to model our “CBM-SC-SC-SNC-SC-V5-7813” scaffold (See Modeling for details). When the display system is constructed, immunofluorescence cannot be detected, presumably as the V5 tag has been obstructed. To verify the theory, we predicted the model of the overall protein using the I-TASSER server and discovered that the V5 tag is truly embedded by other proteins.

It can be seen from the figure that the red component (V5 tag) is blocked by other components, meaning the V5 tag cannot function ideally as designed. We presumed the V5 tag would be available if it was located at the sequence's beginning, as the catchers may have a larger size that blocks the V5 tag if it is located at the end of the sequence.

1.7 Improve #

To make V5 tag and in turn immunofluorescence visible, we changed V5 tag’s position in our sequence, to the front of the plasmid. This edition means V5 tag transcription takes place before catchers’ transcription, lowering the possibility that large seized catcher protein obstructing V5-tag. After altering the V5 tag’s location, we predicted the model again using I-TASSER to ensure its feasibility.

All in all, we changed the position of V5 tag and successfully improve the condition.

Part 2 (Improvement) Optimization of scaffold for surface display #

2.1 Research and Design #

To improve PET plastic degradation efficiency, we imitated cellulosome and controlled the ratio and display sequence of PETase and MHETase enzymes.

Step 1: Optimize the scaffold by exchanging tag and catcher to reduce the molecular mass of the proteins. Notes: Tag is much smaller than catcher.

Step 2: Optimize PETase and MHETase surface display Ratios

Step 3: Fast-PETase with higher enzyme activity were used rather than wild-type PETase

2.2 Build #

We exchanged the position of the tags and catchers. In addition, we also changed V5 tag's position prevent it being covered by the protein. We built the scaffolds with Spy tag and Snoop tag that bind to Spy catcher and Snoop catcher to display the two enzymes. The construction changed from "SP-CBM-SC-SC-SNC-SC-V5-7813" to "SP-CBM-V5-ST-ST-SNT-ST-7813". Our predicted model revealed it was feasible and actual wet experiment proved its viability.

2.3 Test #

By observing with fluorescence microscope, we successfully detect the immunofluorescence (FITC-Fluorescein isothiocyanate isomer) outside the yeast showing the functionality of our optimized system.

By swapping the positions of tags and catchers, we can successfully detect the activity of PETase. And according to the latest report, we have synthesized Fast-PETase. The results showed that the activity of Fast-PETase is indeed higher than that of wild-type PETase.

We also measured the effectiveness of FAST-PETase more directly by testing its effect with degrading PET powder. Specifically, we took the following steps. First, we collected an appropriate amount of cultivated strains and washed it three times with 50 mM glycine-NaOH (pH 9.0-10) buffer. Second, the bacteria were incubated with 1 mL buffer containing 50 mM glycine-NaOH (pH 9.0) and 10 mg PET powder at 30℃ with a shaking speed of 900 r /min. Third, the reaction was terminated by diluting the aqueous solution with 18 mM phosphate buffer (pH 2.5) containing 10% (v/v) DMSO followed by heat treatment (85°C, 10 min). Fourth, the supernatant obtained by centrifugation (15,000 × g, 10 min) was analyzed by HPLC. The result shown in the figure below reflect a significantly larger concentration of degraded PET, MHET with FAST-PETase than wild PETase, consistent under different OD codition,

Besides, w e optimized the ratios of spytag and snooptag. We started with a ratio of snooptag: spytag=1:3. In order to obtain better catalytic effect, we optimized its proportion and successfully constructed scaffolds with different proportions.

We tested the effect of different ratios by HPLC, and found that the ratio of 2:1 performed the best among all the groups.

The results of degraded PET film further confirmed the results.

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