Contribution

Overview

Here we report the contributions made by our team towards the iGEM competition. During our project we developed a shuttle vector for Komagataella phaffii, a protocol for zymosan labeling, a protocol for a binding assay and two scripts for the binding site prediction tool P2Rank. Additionally, we designed two protocols for school visits focusing on restriction enzymes and pamphlets regarding tips for the inclusion of visually impaired persons.


Shuttle Vector pSB3CYP_Zeo (BBa_K4675005)

Traditionally, most iGEM teams decide to work with Escherichia coli in their projects. Despite all its advantages it has serious shortcomings for heterologous protein expression and purification [1]​. Therefore, eukaryotic expression systems, such as yeast have received more and more attention [2]​. However, most iGEM teams still rely on E. coli as the sole chassis within their project. A reasonable explanation for this is the fact that a lack in iGEM teams working with yeasts also results in a low number of parts in the iGEM registry. And even if basic parts like plasmid backbones are available, they are often only designed for model organisms such as Saccharomyces cerevisiae.  As we wanted to expand the scope of organisms utilized by iGEM teams within their projects, we developed an RFC[1000] compatible shuttle vector system for the non-model yeast  Komagataella phaffii which is an excellent expression system for recombinant protein production, especially in terms of posttranslational modifications [3]​. The system is also well suited for correct folding and secretion of the desired protein [3]​ and we regard it as a great contribution for future iGEM projects. We believe with our adapted vector; more teams can work with such a versatile expression system. 

To this end, we build upon the pSB3CY yeast shuttle vector, designed by the iGEM Teams Münster and Dresden 2022, the iGEM Teams of WWU Münster and TU Dresden 2022 designed a level 2 shuttle vector for Saccharomyces cerevisiae, the so called pSB3CY_aeBlue (BBa_K4188001), which is a further development of the iGEM standard part pSB301 (Fig. 1). Due to the design of the plasmid the color of the transformed E. coli colonies changes, depending on the presence of a selection marker. If a selection marker gene is present, the colony shows a red color, and if not, the colonies appear in a dark blue color. The selection marker is interchangeable using the BsmBI type II restriction enzyme. The expression cassette can be inserted in the shuttle vector using standard iGEM parts and is compatible with the MoClo system. 

Figure 1 depicts the circular plasmid map of the shuttle vector pSB3CY_aeBlue. The vector is 6171 base pairs in length. Incorporated into the plasmid are the aeBlue gene flanked by restrictions sites for BsmBI, NotI and SpeI and the 2µ ori flanked by the restriction sites BsmBI, SpeI and BamHI. Furthermore, the p15A ori and a chloramphenicol resistance gene are present on the vector. At last, the mRFP1 gene is present on the vector, flanked by two SapI and BsaI restriction sites.
Figure 1: pSB3CY_aeBlue shuttle vector (BBa_K4188001), developed by WWU Münster and TU Dresden 2022. aeBlue is a chromoprotein, responsible for a blue color of the colonies

Since we are working with the yeast K. phaffii, we had to modify the shuttle vector to fit our expression system. In silico we redesigned the existing pSB3CY_aeBlue by exchanging the existing 2µ Ori with the K. phaffii specific PARS (BBa_J435251). By that we designed the pSB3CYP_aeblue (BBa_K4675005) plasmid (Fig. 2). As a selection marker we chose ScZeo-marker (BBa_J435280), a yeast antibiotic marker for bleomycin resistance. The vector created in this process was named pSB3CYP_Zeo (BBa_K4675009) (Fig. 3, Fig. 4). 

Figure 2 depicts the circular plasmid map of the pSB3CYP_aeBlue shuttle vector after the 2µ ori has been replaced with the PARS ori for Komagataella phaffii. The plasmid has a length of 5003 base pairs. As with the shuttle vector 2µ in the pSB3CY_aeBlue, the PARS is flanked by restriction sites for BsmBI, SpeI and BamHI Furthermore, the p15A ori and a chloramphenicol resistance gene are present on the vector. At last, the mRFP1 gene is present on the vector, flanked by two SapI and BsaI restriction sites.
Figure 2: pSB3CYP_aeBlue shuttle vector (BBa_K4675005), a further developed level 2 shuttle vector of pSB3CY_aeBlue shuttle vector (BBa_K4188001), created by WWU Münster and TU Dresden 2022. The origin of replication is exchanged for an ori of Komagataelle phaffii.  aeBlue is a chromoprotein, responsible for a blue color of the colonies.

To use the shuttle vector in vitro, we used the PARS and ScZeo-marker parts of iGEMs distribution kit and introduced them into the pSB3CY_His (BBa_K4188002) plasmid which was in stock at our lab. The exchange of the 2µ Ori with the PARS was realized by using the restriction enzymes BamHI and SpeI. The already existing histidine selection marker was replaced with the ScZeo-marker using SpeI and NotI restriction enzymes. After designing the pSB3CYP_Zeo plasmid, we were able to clone it into K. phaffii and grew yeast colonies on medium containing the antibiotic Zeocin®. This functioned as a control, proving that we had not only designed but also tested a functioning shuttle vector for K. phaffii. Making the pSB3CYP_Zeo plasmid available for future iGEM teams, will strongly facilitate the usage of the non-model yeast K. phaffii. We hope this contribution will elevate the adoption of novel chassis organisms within the iGEM community to expand synthetic biology beyond E. coli. 

Figure 3 depicts the circular plasmid map of the pSB3CYP_Zeo shuttle vector. The plasmid has a length of 4392 base pairs. Incorporated into the vector are the PARS ori and a zeocin resistance gene, replacing the 2µ ori and aeBlue of the pSB3CY_aeBlue shuttle vector. The NotI and SpeI restriction sites are flanking the zeocin resistance gene, the SpeI and BamHI restriction sites are flanking the PARS origin of replication. The BsmBI restriction sites were removed in the cloning process. Furthermore, the p15A ori and a chloramphenicol resistance gene are present on the vector. At last, the mRFP1 gene is present on the vector, flanked by the SapI and BsaI restriction sites.
Figure 3: pSB3CYP_Zeo shuttle vector (BBa_K4675005). Further developed pSB3CY shuttle vector with a Zeocin/Bleomycin resistance 

Figure 4 depicts Escherichia coli pSB3CYP_Zeo colonies growing on a LB + Chloramphenicol agar plate. The colonies are colored pink, showcasing that a selection marker is integraded into the vector.
Figure 4: Escherichia coli pSB3CYP_Zeo (BBa_K4675005) colonies on an LB + Chloramphenicol plate. The plasmid contains a selection marker, therefore the colonies show a pink color.

Vector pCTcon2 for Yeast Surface Displays

An essential part of our project was the investigation of the binding affinity of different Vitellogenin domains to zymosan. Additionally, we employed a directed evolution approach via error-prone PCR to obtain a binding-optimized variant. Therefore, we utilized a yeast surface display in the strain Saccharomyces cerevisiae EBY100. 

A common vector used for the presentation of different constructs in a yeast surface display (YSD) is the pCTCon2 provided by Addgene [7, 8]. The vector is suited for both Escherichia coli with the ampicillin resistance AmpR, and S. cerevisiae with the tryptophan selection marker TRP1. The most essential part of the vector is the strong Gal1 promoter. The promoter enables a precise induction of the construct display on the surface of yeasts [8]. The CAP-binding site enhances the binding affinity of the RNA polymerase to the promoter [9]. Another important part of the constructs is the Aga2p which encodes for the protein anchor of the constructs. To learn more about the molecular mechanism behind it, visit our results page. We introduced Aga2p with a length of 261 bp via a Gibson Assembly reaction into the empty vector alongside a flexible (G4S)3 linker, which aids in protein folding and function. To enable measurement of the constructs, two fusion epitopes, the cMyc-tag and the HA-tag, are put in the vector. Both tags can be used to bind labeled antibodies. Thereby, the display of the constructs can be quantified [10]. For our project, we tagged the cMyc-tag with the cMyc-Monoclonal Antibody (9E10) (ThermoFisher, # 910MYCL). We preferred the cMyc-tag over the HA-tag because it is likely to be more steric-free. Still, as an outlook for other teams, it is recommended to also target the HA-tag because its n-terminal position provides redundancy and the cMyc-tag is sterically inaccessible. The empty pCTcon2 vector in combination with the Aga2p and the fusion proteins is our contribution for other teams to easily perform a YSD themselves and bring their projects to the next step. 

Figure 4 depicts the circular plasmid map of the pSB3CYP_Zeo shuttle vector. The plasmid has a length of 4392 base pairs. Incorporated into the vector are the PARS ori and a zeocin resistance gene, replacing the 2µ ori and aeBlue of the pSB3CY_aeBlue shuttle vector. The NotI and SpeI restriction sites are flanking the zeocin resistance gene, the SpeI and BamHI restriction sites are flanking the PARS origin of replication. The BsmBI restriction sites were removed in the cloning process. Furthermore, the p15A ori and a chloramphenicol resistance gene are present on the vector. At last, the mRFP1 gene is present on the vector, flanked by the SapI and BsaI restriction sites.
Figure 4: The backbone vector pCTcon2, provided by Addgene, created by Dane Wittrup, expanded with the Aga2p.

Protocol for Zymosan Labelling

Many protocols are available for the labeling of smaller biomolecules like glucose or proteins, but not for polymers like zymosan. Zymosan labeling was an essential part in our project for the success of the designed experiments, leading us to develop a protocol for zymosan labeling.

PDF: Protocol for Zymosan Labeling


Protocol for Vitellogenin Binding Assay

Investigating the binding affinity of proteins is a key step in characterizing proteins. Our Team developed a cost-effective protocol for the testing of Apis mellifera's Vitellogenin binding affinity towards zymosan, which can be used by other teams to investigate Vitellogenins pathogen associated molecular pattern binding affinity.

PDF: Protocol for Vitellogenin Binding Assay


Software: P2Rank Scripts

Binding sites are essential parts of proteins for the binding of ligands and their catalytic functions, thus making their prediction a fundamental step in characterizing proteins [4]​. P2Rank is a tool often used for the prediction of binding sites, because it shows good prediction results and was developed having the aim of generating a user-friendly UI [5]​. For many proteins, such as the Apis mellifera Vitellogenin, there is no experimental data on their 3D structure available, requiring the simulation of protein conformation and their binding sites. This is a problem that could affect many future iGEM teams. 

Our Team used the P2Rank tool to predict the pathogen associated molecular pattern binding site of the Vitellogenin. While it is very user friendly compared to other available tools for this purpose, we found it to be lacking in two aspects: 

  • While it does provide a script for showing the predicted pockets in the visualization software Pymol, no such script is provided for USCF ChimeraX, a visualization tool which is also widely used. Unlike Pymol, ChimeraX is free of charge for academic use. 

  • The 2.4 release of P2Rank contains a model specialized for the prediction on AlphaFold predicted structures, but investigations have shown that one must consider the confidence metric by AlphaFold for those results to be valid [6]​. 

We created two tools in Python addressing these issues and decided to provide them here for other people to use. They’re available under https://gitlab.igem.org/2023/software-tools/unimuenster. 

The first tool takes the output from P2Rank and creates a ChimeraX script from it, allowing the user to easily visualize the results. By default, it uses colors which are specifically optimized for color vision deficiency. The user can specify distinct colors. 

For the second tool: AlphaFold provides a confidence metric together with its predictions called pLDDT. As mentioned above, this confidence metric should be considered when evaluating prediction results based on AlphaFold structures [6]​. Our tool calculates the mean pLDDT of the involved residues for every predicted pocket respectively and adds it to the result file from P2Rank. 

We believe our tools will be especially useful to iGEM teams looking to do binding site prediction in the future, as it facilitates easier usage of the program. 


School Visit Protocol

Education and knowledge transfer is an important part of the iGEM community, and many teams are conducting school visits. During our project we developed two school visit protocols, including tips and tricks for other teams planning to do school visits.

PDF: School Visit Protocol


Inclusivity Pamphlets

Visually impaired people face many barriers when approaching biology. Our aim was to improve accessibility and lay the foundation for future experiments and ideas. During the research for our project, we have gathered a lot of information regarding the inclusivity of blind and visually impaired people, along with possible implementations. In order to aid future iGEMers in the first steps of inclusivity, we have shared our insights by designing the pamphlets “First Steps to Inclusion” and “Inclusive Wiki Design”. The pamphlet “First Steps to Inclusion” contains advice on general matters regarding inclusivity of blind and visually impaired people in the areas of biology, lab work, and distribution of information. The pamphlet “Inclusive Wiki Design” deals with the creation of an inclusive wiki regarding potential implementations and designs. With these tips we hope to lay the foundation for future iGEM teams to better incorporate inclusivity and apply it to their projects. 

PDF: Pamphlet First Steps to Inclusion


PDF: Pamphlet Inclusive Wiki Design


References

  1. [1] G. L. Rosano and E. A. Ceccarelli, “Recombinant protein expression in Escherichia coli: advances and challenges,” Frontiers in microbiology, vol. 5, p. 172, 2014, doi: 10.3389/fmicb.2014.00172.
  2. [2] R. Baghban et al., “Yeast Expression Systems: Overview and Recent Advances,” Molecular biotechnology, vol. 61, no. 5, pp. 365–384, 2019, doi: 10.1007/s12033-019-00164-8.
  3. [3] M. Karbalaei, S. A. Rezaee, and H. Farsiani, “Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins,” Journal of cellular physiology, vol. 235, no. 9, pp. 5867–5881, 2020, doi: 10.1002/jcp.29583.
  4. [4] J. Kandel, H. Tayara, and K. T. Chong, “PUResNet: prediction of protein-ligand binding sites using deep residual neural network,” Journal of cheminformatics, vol. 13, no. 1, p. 65, 2021, doi: 10.1186/s13321-021-00547-7.
  5. [5] R. Krivák and D. Hoksza, “P2Rank: machine learning based tool for rapid and accurate prediction of ligand binding sites from protein structure,” Journal of cheminformatics, vol. 10, no. 1, p. 39, 2018, doi: 10.1186/s13321-018-0285-8.
  6. [6] M. Akdel et al., “A structural biology community assessment of AlphaFold2 applications,” Nature structural & molecular biology, vol. 29, no. 11, pp. 1056–1067, 2022, doi: 10.1038/s41594-022-00849-w.
  7. [7] pCTcon2 by Addgene. Available: https://www.addgene.org/41843/ (accessed: Oct. 9, 2023) 
  8. [8] Ginger Chao, Wai L. Lau, Benjamin J. Hackel, Stephen L. Sazinsky, Shaun M. Lippow, K. Dane Wittrup, “Isolating and engineering human antibodies using yeast surface display”, 2006, natureprotocols  
  9. [9] William S. Reznikoff, “The lactose operon-controlling elements: a complex paradigm”, (Sept. 1992) molecular microbiology Volume 6, Issue 17 Pages2419-2548 doi: 10.1111/j.1365-2958.1992. 
  10. [10] David W. Colby, Brenda A. Kellogg, Christilyn P. Graff, Yik A. Yeung, Jeffrey S. Swers, K. Dane Wittrup, “Engineering Antibody Affinity by Yeast Surface Display”, Methods in Enzymology: Protein Engineering Volume 388, 348–358., (2004) doi:10.1016/S0076-6879(04)88027-3      

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