Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer deaths worldwide [1]. Early diagnosis is crucial for proper treatment of CRC. If CRC is detected early, the 5-year survival rate is 91%. However, late-stage detection drastically drops this number to 14% [1–3]. This emphasizes the need for an early detection method for CRC.

CRC is a disease in which cells in the colon or rectum grow uncontrollably. The amount of cancer growth has been subdivided into stages. An overview of the different CRC stages can be found in the table below [2,4].

There are several risk factors that increase the chance of developing CRC. Some of these risk factors are part of people’s lifestyle and can thus be changed. Smoking, alcohol consumption and lack of regular exercise have all been found to be CRC risk factors [1,3]. Another changeable factor is diet. Especially the Western diet, with a high intake of red and processed meat, sweets, and refined grains, is associated with a higher risk for CRC. A diet rich in fibres is thought to have the opposite effect [5].

Apart from changeable risk factors, there are also unchangeable factors like hereditary risks or age. Hereditary risk factors are for instance a family history of CRC or genetic defects like familial adenomatous polyposis. Other gut-related diseases like Irritable Bowel Syndrome are also risk factors for CRC [2]. Another hereditary risk factor is ethnic background. Alaskan Natives and African Americans have for instance been found to have an increased chance of developing CRC [3]. A big risk factor for CRC is age, wherein people above the age of 50 are in the risk group. 90% of CRC patients are over the age of 50. However, the disease is often more aggressive in younger patients [4].

Screening tests for CRC can be divided into two main groups: stool-based tests and visual exams. Stool-based tests like the Faecal Immunochemical Test (FIT) or the guaiac-based test, screen the stool for human blood. The FIT is the most common CRC screening method. Other stool-based tests are based on DNA, which check the DNA in the stool for DNA mutations often found in cancers and polyps.

When these tests are positive, a follow-up visual exam is needed to confirm the presence of CRC and to remove it if necessary. Visual exams check for abnormal structures in the colon and often have to be performed by a trained professional. Two examples of visual exams are a colonoscopy and a flexible sigmoidoscopy. With these, a tube with a camera (a colonoscope) is inserted into the colon through the anus. A flexible sigmoidoscopy is less invasive than a colonoscopy, but only allows to check the lower part of the colon, while a colonoscopy allows screening of the entire colon. Another visual exam is the virtual colonoscopy, where 3D images are made of the colon using CT-scans and X-rays [2][6]. Every test discussed here has its benefits and limitations, an overview of which can be seen in the table below.

As mentioned above, people above the age of 50 belong to a risk group for developing CRC. In the Netherlands, people above the age of 55 get an invitation to the Dutch CRC screening programme once every two years. The test used in the Dutch screening programme is the FIT described before. Unfortunately, the FIT has limited accuracy.

The total number of microorganisms present in the human body is known as the human microbiome. The intestines account for 95% of the microbiome, of which most microbes reside in the colon, where it is believed that they outnumber the cells in the human body. The intestinal microbiome is involved in lots of different functions, modulating the structure, biochemistry, and immunology of the gut [1,2]. A healthy microbiome is not easy to define since its composition varies enormously between individuals. Nevertheless, some traits are known to indicate the microbiome is in a good shape, such as having a great diversity of bacterial species [1]. The anomalous distribution of gut microbiota is called dysbiosis and has negative effects on the intestinal function. Many different diseases are tightly related to dysbiosis, such as obesity, degenerative neurologic disorders, and colorectal cancer.

We had to carefully select an organism for our project based on several factors. Since it will perform its function in the colon, the strain must be capable of surviving in that environment. Moreover, it must be safe for human consumption and not destabilise the microbiome. Finally, it must be genetically accessible. After an extensive literature research, we chose Escherichia coli Nissle 1917 (EcN) as the chassis of our project.

EcN is a gram-negative bacterium that Dr. Alfred Nissle isolated from the stool sample of a German soldier who was immune to a specific type of diarrhoea during World War I, in 1917. Since then, it has been widely used and distributed in many countries because of its probiotic properties [3]. Probiotics are defined as living microorganisms that confer positive health effects in the consumer when the dose is adequate. For that reason, most probiotics, including EcN, have the generally recognized as safe (GRAS) status, meaning that they are considered safe for human use [4].

EcN is a strain of the model organism E. coli (E. coli) and hence they share a big part of their genetic background, suggesting that they have many common characteristics. For instance, they grow fast, they are easy to cultivate in the laboratory and there are many genetic engineering tools available. Nevertheless, EcN is regarded as safer than other widely used E. coli strains [5]. Unlike other E. coli strains, such as BL21 or MG1655, EcN can be engineered to prevent genotoxicity. This can be achieved by preventing colibactin activation [6]. In addition, many engineering approaches have already been performed in this strain, including plasmid transformation and genetic modification, among others [7].