gBlocks® Gene Fragments facilitate development of IOD Band for detection of tumor migration
In 2015, for the first time, a team from the Czech Republic entered the iGEM (International Genetically Engineered Machine) competition. Each year, multidisciplinary teams made up of undergraduates, or graduate students, or high school students, from institutions around the world, work through the summer to research, design, and construct genetically engineered systems that will provide a positive contribution to their communities and society at large.
For this first Czech Republic team, members were brought together by the Georgiev Lab (www.ccy.zcu.cz)—perhaps the only laboratory in the country using synthetic biology methods, and quaintly called “The Bio Lab”, as there are no other biology laboratories at the university—from 2 universities, Charles University (Prague) and University of West Bohemia (Pilsen). Most of the students had an engineering background, with a few biologists and medical students completing the group of 9 undergraduate participants. An excerpt from the team’s official proclamation reads, “We strive to make synthetic biology more comprehensible and bearable to engineers. In a country where synthetic biology still lacks footing, we are outsiders in all of our respective departments.”
Integrated DNA Technologies offered all 2015 iGEM teams up to 20 kb each of free, synthetic double-stranded DNA fragments—gBlocks® Gene Fragments. These custom sequences simplify or even supplant cloning, saving scientists valuable time in construct assembly. 184 teams took advantage of this offer, expediting their projects. Daniel Georgiev, the Principle Investigator mentoring the team, noted, “The gBlocks Gene Fragments have been really nice to work with, because you can start with native sequences and design all the bells and whistles you need, such as restriction sites, Kozak sequences, and promoters. Then they are quickly synthesized for you, making a lot of projects possible downstream. And they really facilitated this year’s iGEM project, for example in construction of the receptors. I believe we used all 20 kb of the gBlocks Gene Fragments IDT offered.”
Project: Monitoring tumor cell mobility as a diagnostic
For their project, Team Czech Republic chose to develop the “IOD band”—a general diagnostic test for early detection and mapping of tumor cell mobility. Contained primary tumors often present no symptoms. When discovered early, they can often be safely removed without subsequent chemotherapy. When left untreated, primary tumors can spread to other parts of the body through the lymphatic or blood circulatory systems. Over time, the tumor cells can invade compatible organs, and secondary tumors called metastases or “mets” may develop. While there is still the possibility for effective treatment of early mets, later stage mets are usually associated with terminal diagnoses. Indeed, tumor mobility resulting in metastasis increases the likelihood of a terminal diagnosis by up to 99%. And, tumor mobility is incredibly difficult to diagnose due to the rarity of circulating tumor cells (CTCs) and their surface marker complexity.
The goal of the IOD band was to make CTC identification easy. Its main components are reprogrammed yeast cells, called Input Output Diploids, or IODs. As a single iGEM project, the IOD band seemed like an impossible goal, as there were no precedents for many of the system parts. The team was also young, and had very few resources. However, the students were able to successfully execute the project by simplifying the system as much as possible, dividing the system into modules that a few members each worked on, and including redundancy in the design.
Input Output Diploids (IODs)—yeast cells programmed for antigen detection and intercellular communication
IODs use antigen recognition and intercellular communication to create a logic network that can identify even a single cell carrying the targeted marker among a background of millions. CTC identification triggers a global response, resulting in IOD initiated clumping at levels visible to the eye (Figure 1). This outcome can be obtained in a test tube in just hours, significantly shortening what normally requires days when using current procedures.
Figure 1. Principles of the IOD diagnostic test. Yeast cells were genetically engineered to recognize specific tumor markers on other cells. Marker recognition initiates an intercellular signal detected by other yeast cells, causing them to aggregate, and resulting in a visible clump around the target cell.
The marker detection and signally pathways were designed to be modular so that they could be easily adapted by other researchers in their own experiments. Each IOD design includes 2 modular inputs and a single modular output. The inputs are 1) “locational tags”—single chain variable fragments (scFVs) displayed on the surface of the cell wall to enable IOD localization to areas presenting the scFV ligand (e.g,, tumor cell surface antigens), and 2) yeast pheromones detected by a receptor and signaling pathway. These inputs result in localized cell-to-cell communication. Pheromones serve as the IOD outputs. A modified signaling pathway transcription factor connects the inputs and outputs, such that a single IOD simply detects a pheromone signal and generates an aggregation signal in response.
To present a proof of concept for this invention at the September iGEM Jamboree competition, the team created diploid yeast cells expressing a set of pheromones and receptors (a scientific first) that directed the cells to use signal transmission to essentially perform logic operations on cell surface markers. The students then expressed a set of location tags that recognize common tumor surface markers and agglutinate cells. Together these functions result in a signal switch activated at encounter of each CTC candidate.
Modular and easy to adapt for different applications
IODs are amenable to modular engineering as they comprise only a few genes, and the intracellular biochemical reactions that connect the inputs to the outputs are inherently isolated from other organisms by the cell membrane. The team showed that the diagnostic test can be engineered by anyone, thanks to an easy assembly feature. IOD variants can be assembled from libraries of input and output haploids, without cloning. The haploid parts differ in their input receptors, location tags, and output pheromones. In a simple one day procedure, a researcher can assemble the desired inputs and outputs, creating a diploid yeast cell through sexual conjugation.
“Our team envisioned a ‘tumor marker body atlas’. A large number of tumor cell lines, as well as healthy tissues of common secondary sites, already have well-documented surface marker profiles. We analyzed a dataset of over 700 surface markers comprising common receptors, transmembrane proteins, and channel families to identify usable members with confirmed antibodies. We then linked each cell line to a unique combination of up to 3 markers represented by a simple 6 or 9 digit barcode. From this, we designed ‘The IOD Band’, a marker profile probe that locates cells with target marker combinations in samples of peripheral blood,” explained team member, Martin Cienciala.
Awards and validation encourage product development
The IOD band presents a completely novel solution for localizing tumor cells and characterizing their mobility, a task that up until now has been either impossible or has required days and expensive lab equipment. With sufficient development, the diagnostic test would likely take less than an hour and cost less than 1USD. Hence, it could be used for noninvasive preventive screening as well as patient monitoring.
In September 2015, most of the Czech Republic team members attended the International iGEM Jamboree, the culmination of the iGEM competition, held in Boston, MA, USA. Not expecting to win, they were sitting in the balcony, furthest away from the podium, when the Czech Republic was announced as one of the finalist teams. Team members Hynek Kasl and Veronika Kolejakova then had to rush to the stage for the immediate finalist presentations, given in front of all 2800 participants as well as the judges. If being named finalists for this first-year team wasn’t recognition enough, Team Czech Republic was further awarded Undergraduate Grand Prize 1st Runners Up. The team also won Best Undergrad Health and Medicine Project, Best Undergrad Software Tool, and Best Undergrad Model.
The team has since received a lot of media attention and even a visit by their Prime Minister. This validation of their achievements has team members focused on continuing the project. And it is their full intention to explore the business potential of the invention, within the cancer profiling market (estimated to exceed 35 billion USD by 2018) and within a wider bioengineering market. Learn more about the Czech Republic Team’s project on their iGEM Wiki site, http://2015.igem.org/Team:Czech_Republic.
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iGEM teams engineer success with gBlocks® Gene Fragments—By August, 2015, this year’s iGEM teams had made significant progress on their projects. We spoke with some of the teams to find out how the competition was going, and how they were using gBlocks Gene Fragments.
iGEM students engineer biological tools for a better world—Projects from 2 of the prize-winning 2013 iGEM teams show how non-standard natural and synthetic amino acids can be used in 1) peptide synthesis, and 2) tuberculosis monitoring and treatment. Both projects make use of gBlocks® Gene Fragments to speed construct assembly.
The gene construction revolution—See how use of high-quality, custom dsDNA fragments as a starting material allows you to turn what might otherwise be multi-step cloning assemblies into simpler reactions. You can often just order the entire target sequence ready for cloning or other uses.
Codon optimization tool makes synthetic genes easy—Use the IDT Codon Optimization Tool to simplify designing synthetic genes and gBlocks® Gene Fragments for expression in a variety of organisms.
IDT has improved the efficiency of CRISPR-Cas9 genome editing—Webinar summary: Learn how research conducted at IDT led to the development of a potent new set of CRISPR-Cas9 genome editing tools.
Author: Ellen Prediger, PhD, is a senior scientific writer at IDT
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