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 and optimize the system.

Basic parts #

1. [Contribution] Change the position of V5 Tag (BBa_K3829004) #

What we have learned and want to share with iGEMers: From this session, we understand that in protein expression/surface display systems, protein folding problems need to be considered in particular. And sometimes protein folding problems can be solved by changing the position of tags or proteins.

V5 tag is a basic part used last year (BBa_K3829004), this year we introduced Tag-catcher system. 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.

To make V5 tag and in turn immunofluorescence visible, we changed V5 tag’s position 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.

Finally, the results of changing the position of V5 tag were proved to be effective.

2. [Improvement] Optimization of PETase (BBa_K3829008) #

PETase is one of the parts we used last year (BBa_K3829008), it is a key enzyme for degrading plastics. This year we have improved the enzyme, which is also a contribution to BBa_K3829008. According to the latest report, we have synthesized Fast-PETase. Fast-PETase have been reported to have higher enzyme activity. The results showed that the enzyme activity of Fast-PETase was 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 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 reflected a significantly larger concentration of degraded PET and MHET with FAST-PETase than wild PETase, consistent under different OD conditions.

3. CBM(BBa_K4122006) #

CBM has two functions:

1. hydrophobic properties for better secretion.
2. CBM can improve the catalytic efficiency of carbohydrate active enzymes.

When we introduced CBM in the original system, the first thing we needed to determine was whether CBM had an impact on the surface display system. The experimental results showed that the presence of CBM did not affect the expression of GFP and the localization of anchoring proteins, as well as the determination of immunofluorescence.

Composite parts #

1. [Improvement of Last year’s system] Screen of surface display systems via GFP and RFP (BBa_K4122017) #

Introduction of Tag-Catcher system to co-display PETase and MHETase To attain co-display, we combined our display system with two selective protein binding systems, SpyTag-SpyCatcher and SnoopTag-SnoopCatcher.

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.

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

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.

Although the design in Figure 9 was a failure, it was a crucial part of our investigative process and showed that experiments often did not yield ideal results.

2. [Improvement of co-display system] Exchanging tag and catcher (BBa_K4122025) #

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.

P-SP-PETase-His-Spycatcher-T-ADH2 (BBa_K4122022)

• P-SP-MHETase-cMyc-Snoopcatcher-T-PGK1 (BBa_K4122023)
• SP-CBM-V5-ST-ST-SNT-ST-7813 (BBa_K4122024)

We exchanged the position of the tags and catchers. Combined with the previous experiments 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 proveed its viability.

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

3. [Improvement of co-display system] Change ratios (BBa_K4122025, BBa_K4122027, BBa_K4122028) #

Step 2: Optimize PETase and MHETase surface display Ratios.

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.

Parts Collection #