Project Overview

Motivation

Like all iGEM teams, QGEM 2015 sought out to solve a real-world problem with synthetic biology. Our project this year focused on the topics of organ donation and transplantation surgeries as well as the challenges surrounding these procedures. Each year, thousands are affected by the constant struggle to find organs for people in need, and we wanted to see if science could come to the rescue!

The demand for organs for transplantation has been steadily increasing as waiting lists become longer and longer. Over 1600 Canadians are added to the organ wait list every year.¹ Figure 1 illustrates how over the years the gap between the number of people on organ wait lists and the number of donors has been steadily increasing.

However, organ shortage appears to only to be one of the challenges to transplant medicine. To effectively tackle this problem, we had to understand the full picture. Along with the increasing demand for organs, we also face inadequate organ preservation methods, limiting the amount of time an organ can be stored for transplantation. This creates a very small window of time for officials to find a compatible recipient for the organ.³ Because of this limiting timespan, organ rejection and incompatibility generates another large problem in the organ transplant field. This leads to the loss of a significant number of organs that become non-viable after harvest.

The development of new organ storage and tissue preservation methods for transplantation has been a point of interest in research for over 50 years, and techniques including cryopreservation have greatly improved. Current storage methods involve bringing the organ down to cool temperatures, slowing cell metabolism and storing the organ for a certain amount of time. Storage involves the use of University of Wisconsin (UW) solution, a cold storage solution containing solutes at optimal osmotic concentrations. UW also uses metabolically inert substances to maintain this concentration which is ideal for organ storage. The kidney and liver for example, can be successfully preserved with UW for 24-36 hours. Other organs such as the heart and lungs can only be stored for about 6 hours.⁵

QGEM this year decided to help improve transplant medicine by working to solve the organ storage problem. We want to ameliorate preservation techniques by using a method that is already found and used effectively in nature by various organisms: antifreeze proteins.

The Ice Queen

This summer, our team chose to investigate AFPs and explore their potential application in cryopreservation. Our AFP of choice was the type III protein from the ocean pout fish. Our central project focused on optimizing antifreeze activity by anchoring multiple AFPs to a self-assembling protein scaffold. Our AFP-scaffold complex, affectionately termed the Ice Queen, sets out to increase the local concentration of AFPs, enabling more favourable interactions between proteins and ice crystal surfaces.⁶ These interactions slow down the ice recrystallization process by depressing the freezing point, a concept termed thermal hysteresis.

The Ice Queen employs a coiled-coil anchoring method; a high affinity, highly specific E/K coil system. This is a structural motif consisting of two engineered, heterodimeric alpha-helical coiled-coils. The interaction strength has been shown to be of similar strength to covalent interactions thus acting with minimal reversibility.⁷ The appeal of the specificity and strength of this system presented an attractive means of anchoring our Type III AFPs to the each of the scaffold subunits.

Icefinity

As an addition to our project this year, we decided to further explore the topic of AFPs and their variety of applications. Protein stability is of great importance when considering the use of AFPs in industry. Typical industrial processes often involve harsh conditions which will lead to denaturation (i.e. loss of function) for the protein. Therefore, in conjunction with improving the activity of the Type III AFP, we set out to increase the stability of the protein through circularization techniques.

Using Heidelberg’s intein biobrick BBa_K1362000, we performed the intein-splicing reaction with the AFP to generate a circular protein: Icefinity. Heidelberg and other research parties have shown an increase in thermostability after cyclizing any protein.^{8, 9} This summer, Circularization of the AFP has been a wonderful addition to our exploration of this fascinating protein. It also gave us the chance to make use of the open source iGEM registry and even improve upon Heidelberg’s construct. Read about our results here!

The QGEM team worked to develop Heidelberg’s intein biobrick by adding a T7 promoter to our submitted circular AFP construct. Inserting a promoter allows our biobrick to not only be a carrier, but also be an expression construct of our circular AFP. This biobrick part BBa_K1831000is described in more detail on our Parts Page and the iGEM registry.

References

1. Canadian Transplant Society. (2014).

2. "US Government Information on Organ and Tissue Donation and Transplantation". (2012). US Department of Health & Human Services.

3. "Heart Transplantation Programs Face Significant Challenges". (2015). TransMedics.

4. Image from the Wisconsin State Journal online: http://host.madison.com/news/local/health_med_fit/new-organ-preservation-technique-could-replace-uw-solution/article_8bf59f4e-80ff-5d82-b6ad-613657316713.html

5. Guibert et al. (2011). "Organ Preservation: Current Concepts and New Strategies for the Next Decade". Transfusion Medicine and Hemotherapy. 38(2): 125-142.

6. Stevens et al. (2015). "Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery". Bioconjugate Chemistry. [Epub ahead of print]

7. Tripet et al. (1996). "Engineering a de novo-designed coiled-coil heterodimerization domain for the rapid detections, purification and characterization of recombinantly expressed peptides and proteins". Protein Engineering. 9(11):1029-1042.

8. Jeffries et al. (2006). "Stabilization of a binary protein complex by intein-mediated cyclization". Protein Science. 15:2612-2618.

9. Iwai, H., and Pluckthun, A. (1999). "Circular L-lactamase: stability enhancement by cyclizing the backbone". FEBS. 459:166-172.

Judging Criteria

Bronze

• A list of acknowledgements can be found on our Attributions page, and our generous sponsors are described on our Sponsorship page.
• Our Parts page describes 4 new biobrick parts that have also been submitted to the registry.

Silver

• We have experimentally validated the expression of our circularized AFP and AFP-Ecoil BioBricks: Bba____ and Bba____. Both these parts have been submitted to the registry
• One of our Human Practices activities this summer was focused on exploring the intellectual property (IP) and patenting process for the Ice Queen. We looked into the feasibility of patenting our AFP-Scaffold complex; during our investigation we also learned a lot about the benefits and limitations of patenting IP. Read more about it here.

Gold

• We have improved the characterization of the Calgary 2013 Team's BioBricks; Bba__ and Bba__. As well, we also used Heidelberg's intein BioBrick for our own purposes and added a T7 promoter, allowing for direct expression of our protein without any subcloning.
• We also integrated the theme of education throughout our Human Practices Project; we organized a workshop for SHAD valley students, gave a public seminar on the applications our project, and participated in course development work. Take a look at our Human Practices page!