Project Description

EAT
SHOOT
LEAVE

Our Promotion Video

Introduction

What problem are we dealing with?

Colorectal cancer (CRC) is a disease of the colon and rectum, which are parts of the digestive system. Unlike most cancers, colorectal cancer is often preventable with screening and has a survival rate is 95% when detected early. Therefore, CRC can be stopped! However, people are dying because of a lack of information. CRC is the third deadliest cancer in Belgium, around 7 new cases per month are diagnosed in Belgium. Every day 5.787 cases and 2.575 deaths are registered worldwide (Colemont, 2022). This can be avoided by raising awareness. The cancer starts off by cells that divide very fast until they form a polyp, and the danger of growth can be illustrated by the 5 stages of colorectal cancer:

  • 0 = earliest stage and is grown in situ (in mucosa).
  • 1 = it went through the mucosa and has invaded the muscular layer of the colon.
  • 2 = has grown into the outermost layers of the colon or rectum.
  • 3 = it spread to nymph node.
  • 4 = spread to more than 1 distant organ (such as the liver or lung).

Figure 1: Cases and deaths in Belgium
Figure 2: Polyp to cancer

The cancer takes 8 to 10 years to develop, but initially there are no symptoms. Hence, early detection is the key to reducing CRC related deaths. Therefore, screening every 10 years is recommended. If a close relative had or has CRC, the odds of getting the disease are higher. In this case it is advised to screen every 5 years. Since 15% of people that get the disease are younger than 50, screening should start from the age of 45. Before that, prevention starts with a healthy diet and lifestyle. In Belgium, detection of polyps is mainly done by a stool swab (FIT test) followed by colonoscopy. The latter is an invasive method, which many people are uncomfortable with. Next to this, stool tests, blood-based DNA tests and sigmoidoscopy are possible as detection methods, but these cannot remove polyps. Today only 52.3% percent of the Flemish population and less than 20% of the Wallonian population in Belgium participates in the screening program (Colemont, 2022). Following a positive diagnosis of CRC, the primary treatment methods include surgical resection, chemotherapy and radiationtherapy. Because of the invasiveness or non-specificity of these treatment options, quality of life is severely decreased during and after treatment. We hope to develop the foundation of a new treatment option as well as improve the prognosis of this disease. This is done by both developing the proof of concept for a non-invasive method as informing and educating about the risks.

Figure 3: Prevention start with healthy diet
Figure 4: Participants screening in Belgium

... And why?

Our project aims to facilitate the treatment of CRC by creating a bacterial biosensor that immediately transcribes a drug when colorectal cancer is detected. The biosensor would be administered easily via a pill. Our proof of concept is twofold: develop a concentration dependent response system and implement a killswitch. The response system is to identify and locate a CRC tumor, and the killswitch is needed to not allow genetically engineered bacteria to spread freely in the environment once it leaves the body.

We choose our project with the intention of helping with local problems while choosing a field we are passionate about. After looking through the main health related problems in Belgium, we considered biomedical and environmental applications. After countless evenings of brainstorming, we agreed that a biomedical application would be more interesting for all the team members. No matter the field or the nationality, the desire of improving (and possibly saving) lifes brought us together and pushed us forward.

References
  1. Colemont, L. (2022 September 13). Why campaign against colon cancer? [Keynote lecture]. Online.

Our Project

Our solution and its implementation are centered around creating a new, cheap, and easy to use treatment option that also overcomes the negative effects of other CRC treatment methods. The factors we focused on were:

1. Implications of invasive methods

2. Deterioration in quality of life due to lack of specificity


“Sometimes the easiest way to solve a problem is to stop participating in the problem.”- Jonathan mead

Our team tackled these problems with a different perspective. We focused on developing a dose-dependent biosensor that can be administered to the patient in the form of an innocuous pill. By using a biosensor in the form of a pill, this redirects the invasive drawbacks of surgery. This should lower the uncomfortable and anxious prejudices of having to do with localized treatment. Other biosensors have been developed, but these had low specificity and tunability, which we try to improve with our concept. Treatment without incisions prevents the healing period associated with surgery, thereby improving the quality-of-life post-treatment.

A biosensor is a compact device that measures specific molecules to inform us about health and disease by producing signals. The first ever developed biosensor was able to determine the amount of glucose in our blood, which is helpful for diabetes patients. Our biosensor would be able to detect the presence of colorectal cancer and could transcribe a drug, when it senses a high concentration of CRC specific biomarkers. To develop such a biosensor, we use a bacteria, of which we manipulate its plasmid to contain the right genes for recognition of the biomarker and transcription of a drug.

The developed biosensor can detect specific biomarkers that tackle our considered factor of specificity. Upon detection of the specific biomarkers, it releases a reporter which later could be replaced with a therapeutic molecule. Well, this does not sound that simple, nor does its development. Therefore, we split our solution and its implementation into three parts for a simplistic approach:

EAT → SHOOT → LEAVE

“Simplicity is the ultimate sophistication.” – Leonardo Da Vinci

EAT: the pill

The Eat part refers to the uptake of the pill. It goes through the intestine to the gut, as this is the place where colorectal cancer develops. This medical application should make use of bacteria that naturally inhabit the gut microbiome (like Lactobacillus spp.) to reduce the risk of disturbing the normal digestive process (Shreiner, 2015) and to ensure its survival while delivering the medication. However, the techniques for growth and manipulation of these species are still under development (Song et al, 2018). For our project, a proof of concept was done instead. E. coli was selected as a host organism. E. Coli plasmid is easy to extract and manipulate (Castiñeiras et al., 2018). It is also capable of replicating rapidly, which is convenient for our experiments.

Figure 5: Biosensor in pill
References
  1. Mangalea, M., & Borlee, B. (2022). The NarX-NarL two-component system regulates biofilm formation, natural product biosynthesis, and host-associated survival in Burkholderia pseudomallei. Scientific Reports, 12(1). doi: 10.1038/s41598-021-04053-6
  2. Selas Castiñeiras, T., Williams, S., Hitchcock, A., & Smith, D. (2018). E. coli strain engineering for the production of advanced biopharmaceutical products. FEMS Microbiology Letters, 365(15). doi: 10.1093/femsle/fny162
  3. Shreiner, A., Kao, J., & Young, V. (2015). The gut microbiome in health and in disease. Current Opinion In Gastroenterology, 31(1), 69-75. doi: 10.1097/mog.0000000000000139
  4. Song, X., Huang, H., Xiong, Z., Ai, L., & Yang, S. (2017). CRISPR-Cas9 D10A Nickase-Assisted Genome Editing in Lactobacillus casei. Applied And Environmental Microbiology, 83(22). doi: 10.1128/aem.01259-17

SHOOT: dose-dependent system

We implemented a dose-dependent system in our biosensor for detection. In the medical implementation of the system, the carrier bacteria would detect a colorectal cancer biomarker and release the therapeutic substance for the tumor treatment if the concentration exceeds a certain threshold. So only when enough biomarkers of the cancer are present, the drug will be transcribed, otherwise the bacteria will ignore the low concentration and do nothing while leaving the body. As this is a proof of concept, we do not work with cancer biomarkers, but use flavone naringenin as a stand-in for the cancer biomarkers. We did not identify a possible biomarker for CRC. The plasmid pSynSense2.5000 developed by De Paepe et al. (2018) is used to turn E. coli into a capable biosensor. The modified plasmid of E. coli leads to the creation of a cellular circuit that responds to the concentration of the biomarker naringenin in the medium. Taking advantage of the regulation capacities of the two-component nitrate sensing system NarX-NarL, it is possible to generate a genetic circuit that expresses a therapeutic protein from a specific threshold concentration of biomarker. To evaluate the expression of NarX during our project, the therapeutic substance release or transcription of the drug is replaced by the fluorescent protein mKATE, by this we are way able to see if our biosensor is working as it should. Fluorescence should only be visible if the concentration of the biomarker is higher than the threshold of the concentration biomarker.

Figure 6: E. coli with narengin dimerization

LEAVE: Kill-Switch

Beginning of the end...

To ensure the deactivation of the bacteria after leaving the body, a temperature regulated kill switch was implemented. When the bacteria are exposed to temperatures lower than body temperature (37 ºC), a cascade of toxins would shut down vital functions in the bacteria. When the bacteria is released into the environment, it will die. This ensures biosafety as no genetically engineered bacteria should be freely released in nature. To achieve this, a toxin-antitoxin mechanism was implemented and evaluated.

Figure 7: Biosensor leaving the body safely

Future prospects

Despite our efficient planning for the iGEM project (with most lab work during the summer), we were not able to fully realize the entire scope of the project. Although we were able to perform all experiments in E. coli, we also wanted to establish the results in Lactobacillus spp. to characterize our proof of concept thoroughly. Our main goal of developing a pill for CRC screening that patients can take safely still remains. However, a different type of research is needed to make it possible. Is important to be aware of the public perception of using genetically engineered bacteria for healthcare applications. An open discussion between the government, the patients and the scientists is necessary to provide sufficient information and to build trust. Some additional measures could be taken regarding safety, following future guidelines on genetically engineered bacteria. For now, we aim to prove that the concept of a non-invasive detection and treatment method with a higher specificity, feasibility and tunability is achievable. This could lead to the solution for making screening for CRC more accessible in the future.