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Contribution

Contribution of parts

1. Change the position of V5 Tag (BBa_K3829004)

V5 tag is a basic part used last year (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.

Model of scaffold CBM-SC-SC-SNC-SC-V5-7813 predicted by I-TASSER server
Fig.1 Model of scaffold CBM-SC-SC-SNC-SC-V5-7813 predicted by I-TASSER server

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. Through modeling, we figured out that the problem was caused by V5 tag being embedded (Figure 1).

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 beginning of the sequence, as the catchers may have a larger protein 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.

Model of scaffold CBM-V5-SC-SC-SNC-SC-7813 predicted by I-TASSER server
Fig.2 Model of scaffold CBM-V5-SC-SC-SNC-SC-7813 predicted by I-TASSER server
Successful construction of changing the position of V5-tag
Fig.3 Successful construction of changing the position of V5-tag

2. 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 modified 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 confirmed the activity of Fast-PETase was indeed higher than that of wild-type PETase.

The chemical structure of FAST-PETase
Fig.4 The chemical structure of FAST-PETaseReference: Lu, Hongyuan, et al. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature 604.7907 (2022): 662-667. https://www.nature.com/articles/s41586-022-04599-z?s=09
Comparison of enzyme activities of fast and wild PETase
Fig.5 Comparison of enzyme activities of fast and wild 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.

Comparison of FAST-PETase and wild PETase with HPLC analysis of degraded PET
Fig.6 Comparison of FAST-PETase and wild PETase with HPLC analysis of degraded PET

3. Screen of surface display system via GFP(BBa_K3829002)

Last year, we made improvements on existed part reporter GFP（BBa_K3402050) so that it will be more suitable to express in our host Candida tropicalis, which becomes our last year’s part yeGFP:BBa_K3829002. This year we have added more relevant data. We used GFP as a marker of successful construction of spycatcher and spytag syetem, which greatly facilitated our experiments.

The immunofluorescence of GFP
Fig.7 The immunofluorescence of GFP.Left, bright field; Right, GFP fluorescence

Contribution of Reference Papers (MLA)

A. Host selection – Canada Tropicalis

  1. Li, Yujie et al. “Development of a gRNA Expression and Processing Platform for Efficient CRISPR-Cas9-Based Gene Editing and Gene Silencing in calis.” Microbiology spectrum vol. 10,3 (2022): e0005922. doi:10.1128/spectrum.00059-22
  2. Torkko, Juha M et al. “calis expresses two mitochondrial 2-enoyl thioester reductases that are able to form both homodimers and heterodimers.” The Journal of biological chemistry vol. 278,42 (2003): 41213-20. doi:10.1074/jbc.M307664200
  3. Eirich, L Dudley et al. “Cloning and characterization of three fatty alcohol oxidase genes from calis strain ATCC 20336.” Applied and environmental microbiology vol. 70,8 (2004): 4872-9. doi:10.1128/AEM.70.8.4872-4879.2004
  4. Kanai, T et al. “An n-alkane-responsive promoter element found in the gene encoding the peroxisomal protein of calis does not contain a C(6) zinc cluster DNA-binding motif.” Journal of bacteriology vol. 182,9 (2000): 2492-7. doi:10.1128/JB.182.9.2492-2497.2000
  5. Kato, M et al. “Phylogenetic relationship and mode of evolution of yeast DNA topoisomerase II gene in the pathogenic Candida species.” Gene vol. 272,1-2 (2001): 275-81. doi:10.1016/s0378-1119(01)00526-1

B. Spy and Snoop tag and catcher system

  1. van den Berg van Saparoea, H Bart et al. “Combining Protein Ligation Systems to Expand the Functionality of Semi-Synthetic Outer Membrane Vesicle Nanoparticles.” Frontiers in microbiology vol. 11 890. 12 May. 2020, doi:10.3389/fmicb.2020.00890
  2. Lang, Martina et al. “Tagging and catching: rapid isolation and efficient labeling of organelles using the covalent Spy-System in planta.” Plant methods vol. 16 122. 1 Sep. 2020, doi:10.1186/s13007-020-00663-9

C. The PCR method

  1. Waters, Daniel L E, and Frances M Shapter. “The polymerase chain reaction (PCR): general methods.” Methods in molecular biology (Clifton, N.J.) vol. 1099 (2014): 65-75. doi:10.1007/978-1-62703-715-0_7
  2. Green, Michael R, and Joseph Sambrook. “The Basic Polymerase Chain Reaction (PCR).” Cold Spring Harbor protocols vol. 2018,5 10.1101/pdb.prot095117. 1 May. 2018, doi:10.1101/pdb.prot095117

D. E.coli Plasmid display system

  1. Muhamadali, Howbeer et al. “Metabolomic analysis of riboswitch containing E.coli recombinant expression system.” Molecular bioSystems vol. 12,2 (2016): 350-61. doi:10.1039/c5mb00624d
  2. Kim, Chakhee et al. “Inducible plasmid display system for high-throughput selection of proteins with improved solubility.” Journal of biotechnology vol. 329 (2021): 143-150. doi:10.1016/j.jbiotec.2020.12.013

E. Modeling

  1. Yang, Jianyi et al. “The I-TASSER Suite: protein structure and function prediction.” Nature methods vol. 12,1 (2015): 7-8. doi:10.1038/nmeth.3213
  2. Zheng, Wei et al. “I-TASSER gateway: A protein structure and function prediction server powered by XSEDE.” Future generations computer systems : FGCS vol. 99 (2019): 73-85. doi:10.1016/j.future.2019.04.011
  3. Tam, Benjamin et al. “Combining Ramachandran plot and molecular dynamics simulation for structural-based variant classification: Using TP53 variants as model.” Computational and structural biotechnology journal vol. 18 4033-4039. 2 Dec. 2020, doi:10.1016/j.csbj.2020.11.041
  4. Gopalakrishnan, K et al. “Ramachandran plot on the web (2.0).” Protein and peptide letters vol. 14,7 (2007): 669-71. doi:10.2174/092986607781483912