Our team has worked very hard to try and attain as many objectives as possible. Here we summarize all the medal criteria we achieved to meet.
We have described the attribution of each team member in the attributions page. The project-related work, experiments, and activities of each team member are described in detail. The contributions of external professionals (e.g. doctors and a clinical scientist) and local people (i.e. those who completed our surveys and a patient) who provided critical or useful advice are described under external attribution page. The attributions clearly presents how each team member took responsibility for their own areas of expertise or interest, and cooperated with others, allowing us to complete our project efficiently.
The project description is available at
description page.
Our project aims to decrease deaths caused by lung cancer. We initially found that limitations of current metastasis detection methods, leads to a low frequency of metastasis detection. Accordingly, our project aims to design and develop a device that is affordable and accessible for metastasis detection. We focus on capturing and labeling lung cancer circulating tumor cells (CTCs), optimized by a synthetic biology approach.
To make CTC detection affordable, we decided to replace antibody based CTC detection with DNA nanostructure-based CTC capture and synthetic protein based CTC-labeling. To generate the CTC-capturing DNA tetrahedron, we apply in vivo rolling circle replication in E.coli. to produce ssDNA for tetrahedron assembly.
To make the public aware of the danger of lung cancer, methods of cancer prevention, and the potential of synthetic biology for CTC detection, we held numerous educational events.
We believe our project can not only decrease the deaths caused by lung cancer metastasis by promoting CTC-detection, but also decrease cancer occurrence by raising public awareness of the problem.
The detailed contributions of our project are shown on the contribution page of the Wiki .
As our contribution in synthetic biology, we provided a new basic part which encodes the fluorescence protein mGreen Lantern (mGL), which is considerably brighter than the standard GFP proteins
(BBa_K4674000 and BBa_K4674001). To express the mGL fusion protein, we provide the part
BBa_K4674010.
We also provide basic parts expressing ssDNA binding protein
(BBa_K4674003) and rolling circle replication (RCR) initiation enzyme RepA
(BBa_K4674002). We also provide a composition part, a part set for expressing basic components and target cassette for RCR
(BBa_K4674011 and BBa_K4674012).
In hardware we provided the blueprints of hardware components for future application, including the main chamber, trident microchannel, microchannel box, and the tri-syringe holder.
The engineering success is described in detail on the engineering page of our Wiki.
Through several engineering cycles, we observed the gap between experimental design and the real world, which pushed us to overcome the emerging problems to optimize our DNA tetrahedron and the fusion protein recognizing circulating tumor cells (CTCs).
In the first generation of our DNA tetrahedron, we assembled four separate single standed DNAs (ssDNA) into a tetrahedron in a complementary manner. The result indicated that the separate ssDNAs produced too many polyhedron side products.
We then fused the separate ssDNAs into one tetrahedral ssDNA, and produced this tetrahedral ssDNA by rolling circle amplification (RCA). To separate the tetrahedral ssDNA concatemer into monomers, we introduced a cis-autosplicing sequence and induced splicing through adding zinc ions. The binding motifs of PBSII-Zif268 protein are also embedded in the tetrahedral ssDNA, which are responsible for anchoring the DNA tetrahedron on the main chamber.
After discussing with experts, we found that high concentration of tetrahedral ssDNA generated by RCA also form polyhedrons. Therefore, we decided to apply rolling circle replication (RCR) to produce circular tetrahedral ssDNA monomer in E. coli. The ssDNA binding protein was co-expressed to protect the circular tetrahedral ssDNA as well as facilitate purification.
Regarding CTC labeling proteins, we build several structures of mGreen Lantern (mGL) fluorescent protein fused with CTC recognizing peptide (C7) using different lengths of linker. The flexibility of C7 peptide in fusion protein with different lengths of linker was tested by modeling
(model page)
and protein docking. The results indicated the minimal length of the linker is 4 amino acids. Finally we confirm the mGL-4A-C7 fusion protein is functional by fluorescence microscope.
Through these engineering cycles, we optimized our tetrahedral ssDNA and the mGL-4A-C7 fusion protein.
The number of deaths caused by lung cancer is a major problem both globally and locally. Therefore, we initiated this CTC-FAST project to reduce the death number by monitoring the appearance of circulating tumor cells (CTCs) after surgery on the one hand, and through spreading the knowledge of prevention on the other hand as a complementary. Moreover, we involved both professionals and the public in
all stages
of the project.
To make sure the CTC-FAST project meets real needs, we first performed street surveys to determine what kind of product and price would be acceptable by the public. We then interviewed experts to make sure our project would meet the standards for clinical application. Finally, we talked with a patient and medical consultant to understand the patients' perspective.The clinical doctors and medical technologists suggested that we focus on monitoring metastasis after surgery, and reminded us of the issues of specificity and sensitivity. The patient and medical consultant helped us understand the problems in the real world.
To make the public aware of lung cancer, synthetic biology, and our device, we focused on the publicity of cancer prevention through education and outreach. To reach local people, we held community events for the elderly and synthetic biology camps for the young. To reach people at a distance, we conducted postcard campaigns and online surveys.By engaging with experts from various medical fields, we have been able to devise innovative experimental designs and develop tools that better align with societal needs, particularly the issue of high mortality rates associated with lung cancer.
Our best new basic part is: BBa_K4674001
We also provided other basic parts associated with RCR mediated circular ssDNA generation in vivo.
This part BBa_K4674001 encodes the mGreenLantern (mGL) fluorescence protein fused with the CTC recognition peptide. mGL is an improved type of green fluorescent protein that in our work serves as a labeling material for lung cancer CTCs.
According to the reference, The excitation and emission spectra of mGL are compatible with typical GFP filter sets, suggesting a general application of mGL protein. The mGL protein exhibits significantly higher cellular brightness than the standard eGFP. Accordingly, our experiment indicated that mGL is at least twofold brighter than eGFP when observed under the filter. Our experiment also confirmed the mGL-4A-C7 could label the CTC mimics, SKOV3 cell. Together, we believe that the part BBa_K4674001 can serve as an appropriate alternative to eGFP, which potentially leads to more efficient and clearer results in future fluorescence labeling experiments.
To automatically capture, label, and detect metastasis, we developed the CTC-FAST device, which is described in detail at
hardware page
. CTC-FAST is composed of three systems. The first system is the fluid propulsion system for propelling CTCs for capture, labeling, and detection. The second system is the microfluidic technology for cell alignment. The third system is the fluorescence detection system for detecting light emitted from fluorescence-labeled CTCs after laser excitation. These systems will be automatically controlled by software designed by our team.
We built this hardware and hope to contribute it to the iGEM community when other teams need similar automatic detection devices in the future.
We are committed to reducing the number of lung cancer deaths. To that goal, we actively went out of the laboratory to contact various stakeholders (doctors, experts, patients, and the public), and gained a lot of inspiration and valuable insights, enabling us to incorporate different aspects for our project. Simultaneously, we also delved into the commercial viability and future potential of CTC detection technology to test our innovative techniques and educational initiatives. Below we summarize how we incorporated this information into CTC-FAST, demonstrating our team's gradual progress toward our goal. More details can be found on our
human practices page.
The medical doctors and medical staff we interviewed provided information about their experience treating and curing lung cancer for our
clinical application.
They also revealed the shortcomings of current CTC detection－time consuming and unaffordable for most patients. Based on their experience, we were able to
understand
what the patients really need and accordingly
optimize our experiment and hardware.
By exchanging information
with advisors and other teams, we set priorities for our design, and prioritized the values of accessibility, affordability, and ease of use to provide an effective tool for postoperative patients. Finally, we
managed
to produce an affordable and accessible CTC detection method, which can have a positive impact on society.
In addition to the technical part of CTC-FAST, we also wanted to find out what else we can do to decrease the deaths caused by lung cancer. Through street interviews, we found that approximately 80% of the public is unaware of the severity or symptoms of lung cancer, not to mention the assay for CTC detection. As a result, we decided to
emphasize prevention,
raising the awareness of lung cancer severity and symptoms in the public through
education
as well as
outreach.
We organized teen camps, lectures for the elderly, in-school publicity campaigns, and postcard campaigns, striving to reach people of different ages through appropriate methods. Combining the approach of technological innovation and that of information spreading, we believe a
better world
with fewer people suffering from the deadly threat of lung cancer will come in the near future.
We are the CTC-FAST - We are going to Change The Century FAST.