1.1 How many people in the world get CRC?

Colorectal (also known as bowel) cancer is the 3rd most common cancer worldwide. It is the
3rd most common cancer in men and the 2nd most common cancer in women.

1.2 The number of cases by region and country

The striking variation in CRC globally reflects the large impact of lifestyle factors on cancer occurrence. Similarly,
wide differences within the United States in the prevalence of CRC risk factors, such as smoking and excess body weight,
and access to high‐quality

Xi Y, Xu P. Global colorectal cancer burden in 2020 and projections to 2040. Transl Oncol. 2021;14(10):101174.
doi:10.1016/j.tranon.2021.101174

1.3 how many people died because of CRC in the world?

0.94 million CRC caused deaths in 2020 worldwide, representing 10% of the global cancer
incidence (total 19.29 million new cases) and 9.4% of all cancer caused deaths (total 9.96 million deaths). CRC is the
third leading cause of cancer related deaths in both genders worldwide, with estimated 515,637 deaths among males and
419,536 deaths among females in 2020.

1.4 How many patients with colorectal cancer have survived within five years？

The 5-year relative survival rate for colorectal cancer in the United States is 65%. When diagnosed at the localized
stage, the survival rate is 91%, while at the regional stage, it is 73%. Late-stage diagnosis results in a 5-year
survival rate of 14%. However, patients with a limited number of tumors that can be surgically removed often have
improved survival rates. When considering colon cancer separately, the overall 5-year relative survival rate is 63%,
with localized stage survival at 91%, regional stage at 72%, and late-stage at 13%. For rectal cancer, the overall
5-year relative survival rate is 68%, with localized stage survival at 90%, regional stage at 74%, and late-stage at
17%.

The trends in CRC incidence and mortality can be categorized into three global categories.
Medium HDI nations (e.g., Brazil, Russia, China, Latin America, the Philippines, and the Baltics) have witnessed an
increase in both incidence and mortality due to economic transitions. High-HDI nations (e.g., Canada, UK, Denmark,
Singapore) have seen an increase in incidence but a drop in mortality due to improved treatment. Highest HDI nations
(e.g., US, Iceland, Japan, France) have experienced a decrease in both mortality and incidence due to successful
prevention and treatment efforts. However, CRC incidence has risen among those aged 20–49 years in the US, while overall
mortality rates have decreased, especially in the 75+ age group. The global burden of CRC is expected to increase by 60%
to over 2.2 million new cases and 1.1 million annual deaths by 2030.

Colorectal cancer (image: medicalnewstoday.com)

3.1the introductions of the traditional therapeutic methods

The recommended conventional treatment strategies for cancer typically involve surgical
removal of tumors, followed by radiotherapy using x-rays and/or chemotherapy. Surgery is most effective in early-stage
disease progression, while radiation therapy can cause damage to healthy cells and tissues. Although chemotherapy has
reduced morbidity and mortality, it also damages healthy cells, particularly those that are rapidly dividing and
growing. Drug resistance, a significant challenge in chemotherapy, occurs when cancer cells develop resistance to
anti-cancer drugs, primarily due to reduced drug uptake and increased drug efflux. Conventional chemotherapy has
limitations such as difficulty in selecting appropriate dosages, lack of specificity, rapid drug metabolism, and
significant side effects.

3.2 some therapeutic methods people now use

a. Stem cell therapy
b. Targeted therapy
c. Ablation therapy
d. Gene therapy
e. Natural antioxidants(the way we choose)

Introduction of E. coli Nissle 1917 for Genetic Engineering in Colorectal Cancer Treatment:
Escherichia coli Nissle 1917, a non-pathogenic strain of gut microbiota, is highly amenable to genetic engineering. It
offers convenience for introducing specific genes while being safe for human use due to its non-pathogenic nature and
low endotoxin production. These attributes make it an ideal candidate for our research project, focused on developing a
novel treatment approach for colorectal cancer.

Our project encompasses the design of three distinct systems: the Broccoli System, the
Curcumin System, and the Suicide System. I will proceed to provide individual introductions for each of these systems.

4.1 Sulforaphane production system

Cancer remains a global health challenge, necessitating innovative approaches for treatment
and prevention. One promising avenue of research involves leveraging the natural bioactive compound sulforaphane, known
for its anticancer properties. This compound has demonstrated the ability to inhibit cancer cell growth by inducing cell
cycle arrest (G2/M), up-regulating pro-apoptotic factors such as caspase 8, p21, p53, and Bax, while down-regulating
anti-apoptotic factors like Bcl-2 and Hsp90. However, the availability of sulforaphane in mammals is limited due to the
absence of myrosinase, the enzyme responsible for its conversion from glucosides. This deficiency has prompted
investigations into alternative means to enhance sulforaphane production and its potential anticancer efficacy.

4.1.1 Gene Wiring Map

4.1.2 Core Genes and Their Functions

4.1.2.1 Myrosinase

• Function: Myrosinase is a pivotal enzyme responsible for the conversion of glucosides to sulforaphane. It plays a central role in the release of sulforaphane from its precursor compounds in cruciferous vegetables.
• Source: Originally found in cruciferous plants, myrosinase is essential for the plant’s defense against herbivorous activity.

4.1.2.2Enzyme Reactions and Product Functions:

• Myrosinase Reaction: Myrosinase catalyzes the hydrolysis of glucosinolates to release sulforaphane. This reaction is essential for making sulforaphane bioavailable.
• Sulforaphane’s Role in Cancer Inhibition: Sulforaphane is known to exert anticancer effects through several mechanisms:
  • Cell Cycle Arrest (G2/M): Sulforaphane induces cell cycle arrest in G2/M phase, preventing uncontrolled cell proliferation.
  • Up-regulation of Pro-apoptotic Factors: It up-regulates pro-apoptotic factors like caspase 8, p21, p53, and Bax, promoting programmed cell death (apoptosis) in cancer cells.
  • Down-regulation of Anti-apoptotic Factors: Sulforaphane down-regulates anti-apoptotic factors such as Bcl-2 and Hsp90, further facilitating apoptosis.
• Enhancing Cancer Prevention: These actions collectively contribute to sulforaphane’s potential in cancer treatment and prevention by targeting multiple aspects of cancer cell growth and survival.

4.2 Curcumin Production System

Curcumin, derived from Curcuma longa, holds promise for colorectal cancer treatment due to
its anti-inflammatory and antioxidant properties. It inhibits tumor growth, modulates cancer-related molecular targets,
and enhances chemotherapy. Additionally, it shows potential for other health conditions. While more research is needed
for optimization, curcumin has diverse antitumor effects, including signaling pathway regulation, DNA repair modulation,
and enhanced chemotherapy effectiveness. Its anti-inflammatory properties reduce chronic inflammation linked to cancer
and improve antitumor immunity. Curcumin's antioxidant activity, neuroprotection, and detoxification benefits make it a
versatile compound.

Curcumin is synthesized through the phenylpropanoid pathway, involving the enzymes tyrosine aminonialase (TAL),
4-coumarate coenzyme A ligase (4CL), diketide-CoA synthase (DCS), and curcumin synthase (CURS). Among these, DCS and
CURS play crucial roles as key rate-limiting enzymes in curcumin biosynthesis.

To enhance the production of curcumin, we have positioned the two crucial rate-limiting enzymes, DCS and CURS,
downstream of the T7 promoter in the curcumin synthesis process.

4.3 Suicide system

Temperature-inducible promoters regulate gene transcription within a specific temperature
range, commonly used in biotechnology for temperature-sensitive gene expression control. They alter promoter activity in
response to temperature changes, modulating gene transcription. At low temperatures, the promoter is inhibited, reducing
gene activity, while high temperatures activate it. This sensitivity relies on specific regulatory elements and
temperature-sensitive factors.
We chose TcI42 promoter with an ideal activation threshold at 42°C, and its expression at 37°C is relatively low. In
Salmonella, the Tlp protein regulates virulence gene expression in warm hosts. Tcl is a temperature-sensitive mutant of
the lambda phage protein "cl," acting as a transcriptional repressor for lysogenic state maintenance.
By using Tcl42 as a promoter in a suicide system, households can safely produce sulforaphane and curcumin by adjusting
temperature, enabling domestic production of these compounds.

4.3.2 Suicide gene

The suicide gene induces programmed cell death or self-destruction under specific
conditions. It is combined with promoter sequences for precise gene expression control. It helps control the spread of
genetically modified organisms, ensuring environmental safety.
The suicide gene we used is SRRZ cleavage gene, consisting of perforin S, bacteriophage lysozyme R (transglycosylase),
and gene RZ.
The R gene produces a water-soluble transglycosylation enzyme that degrades the peptidoglycan cell wall.
The RZ gene produces an endopeptidase that cleaves peptidoglycan and crosslinks with the outer membrane, causing cell
wall rupture and release of intracellular substances.
The S gene alters plasma membrane permeability, allowing R and RZ enzymes to reach the cell wall, resulting in cell
lysis.
Both R and RZ gene products degrade the cell wall.
By using the lysing gene to break down cells, we ensure high biosecurity in the production process.

In summary, we have engineered a three-system therapeutic bacterium for treating colorectal cancer. Within the Broccoli
System, black mustard enzymes produce sulforaphane, while the Ginger System utilizes ferulic acid as a substrate to
produce curcumin. Both of these substances have significant potential in colorectal cancer treatment. Finally, our
designed suicide system, activated at 42 degrees Celsius, ensures self-destruction of the bacterial strain after
completing its tasks to prevent gene leakage.

Our target audience includes colorectal cancer patients, those who have recently undergone
surgical treatment and require post-operative care, and individuals at high risk of colorectal cancer.

To ensure biosafety throughout this process, we have implemented a suicide system and designed bacterial filters at the
hardware level.

In the practical application, users should start by purchasing broccoli from the market. They can then use our
homogenizer to break down the broccoli into juice. Next, pour the broccoli juice into the bag containing our engineered
bacterial freeze-dried powder through the funnel, seal the bag, and shake it vigorously to ensure thorough mixing of the
powder with the broccoli juice. Place the bag in our temperature-controlled machine (similar to a yogurt maker) and set
the temperature to 37 degrees Celsius for fermentation, which typically lasts 8-12 hours. During fermentation, the
temperature is briefly adjusted to 42 degrees Celsius to activate the suicide system. Once fermentation is complete,
open the bag and pour the fermented broccoli juice into our filtration device. Residues and engineered bacteria will be
trapped in the filter, leaving you with filtered liquid that can be consumed directly. (For more details, please refer
to the team's hardware page.)

This technology not only holds great promise for colorectal cancer treatment but also
demonstrates potential for future advancements in targeted cancer therapies and broader biotechnological applications.

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