Our team theme is “For Better Life and Better World”. We are developing an immunotherapeutic technique that combines genetic engineering of bacteria with chemical nano-immunomodulators for triple negative breast cancer.

Our integrated human practice includes social investigation, science communication, educational program, and social service. Social investigation is based on the epidemiological survey that is aimed to discover the causative factors of breast cancer as well as the current status of medication, disease management and policy. Science communications with clinical doctor as well as experts in synthetic biology, microbiology, instrumentation, public health, and artificial intelligence have help us improve the design and implementation of the project. The communication with enterprise CEO inspires us how to make an idea into a product that can serve the society. In addition to lectures, they participated in our consulting seminar and gave us suggestions. In order to seek collaborations and establish connections with other igem teams, we made a trip to Hainan Conference. Since the Hainan trip, we have established collaborations with 5 teams including Peking 2023, Peking HSC 2023, BNUZH-China 2023, Tsinghua 2023, and JLU-NBBMS 2023 that are also working on microbial therapy. We had 3 online project seminars and 1 online science popularization seminar together. There are two parts in our educational programs. Part one is self- education to team members including brainstorm seminars, safety training, synthetic biology training and freshmen lab show). Part two is public education to the society including field lectures in town or villages communities as well as stage lectures on woman’s day and mother’s day. We provide social services through a WeChat public account called “The Lanterns（兰子菽）” that was established for scientific popularization of the research and development of breast cancer. It is expected to raise public awareness and provide the society with related fundamental knowledges and resources. Our six IGEM teams have co-written a handbook for microbial mediated tumor therapy in synthetic biology. This jointed effort helps people to understand the fundamental principle and research progress. In order to serve those people living in rural villages, a portable device was designed for daily self-checking of breast health. Figure 1 shows that all activities of our human practices are integrated as a whole in accordance with national and institutional regulation and guidelines on lab safety, biosafety, ethics and norms.

Figure 1. The integrated human practice designed in the project

There are millions of people diagnosed with breast cancer worldwide. It has become one of the major global public health challenges. We have launched an online epidemiological questionnaire that was open and publicly available (Figure 1) in order to raise public awareness for early detection and prevention of breast cancer. The epidemiological survey is designed to discover causative factors that may be associated with the incidence, prevalence and prognosis of breast cancer over a population. From March 18 to September 10, 2023, we received a total of 337 valid questionnaires that covers 17 provinces or regions of China. Based on the collected data in 2023 and the method we established in 2022, two major risk factors related to breast cancer including behavioral and reproductive factors have been identified. Compared with the 2022 survey, additional psychological factors such as anxiety and environmental exposure as well as family history of breast diseases in addition to breast cancer are considered in 2023 survey.

Figure 1. Epidemiological questionnaire on breast diseases

Breast is made of lobules and ducts responsible for the production and transportation of milk, as well as connective tissues consisting of fibrous and fatty tissues that surround and hold everything together. ¹ There are different types of breast cancer that are initiated by the complex interactions of genetic makeup and the environment.² Breast cancer usually begin inside the milk ducts and/or the milk-producing lobules of the breast.³ The earliest in situ form is not life-threatening.3 But invasive cancerous cells can spread into nearby breast tissue or metastasize to lymph nodes and even distant organs, leading to the formation of solid lumps or masses that may be fatal.⁴ Notably, the global incidence of breast cancer is continuously increasing in regions that had low rates of the disease before.⁵ While some risks have been assessed, many are still under investigation.⁶ Family history has been usually considered as a high risk. However, some diagnosed people do not have a known family history of the disease.⁷

Possible early symptoms of breast cancer are summarized as follows.^8-10 The advancement of techniques have revolutionized various electronic devices for health screening and disease management with machine learning and automatic recognition that allow early detection and precision medicine.^11-15

• Local paroxysmal dull, throbbing, or piercing pain in breast or shoulder and back, especially for postmenopausal women.
• Palpable and movable lumps, masses or thickening that feels different from the surrounding tissue.
• Changes in the size and appearance of the breast, such as nipple retraction.
• Changes in the skin over the breast, such as dimpling, redness, pitting, orange peel like skin, and enlarged pores.
• A newly inverted nipple or nipple discharge.
• Asymmetric, protrusions, or edema nipple and eczema like changes.
• Peeling, scaling, crusting or flaking of the pigmented area of skin surrounding the nipple (areola) or breast skin
• Regional lymph node enlargement.

It has been recognized that the occurrence of breast cancer is caused by the accumulation of genetic mutations as well as epigenetic factors such as environment pollution, personal lifestyle and habbits.^16-19 Various state-of-the-art techniques have been developed for the diagnosis of breast cancer.^20-26 Although such equipment can offer precise measurement, they are not always accessible for individual daily self-check. Early detection of breast cancer demands the development of small-scale portable devices that can be afforded by most people.^27-37 Recent advancement in analytical techniques has even moved towards molecular classification in addition to conventional clinicopathologic features or routine biomarkers.^38-42 The newly discovered genetic, proteomic and metabolic biomarkers has improved treatment strategies of breast cancer.^43-47

A categorization method was used in the epidemiological survey that is based on early symptoms.⁴⁸ Age, gender, educational level, family history of breast diseases, behavioral and reproductive factors have been taken into the consideration. Environmental exposure and psychological states are also considered in 2023 survey. Validation of risk factors should be conducted in future follow-up investigations.^49-52 Table 1 lists the cross-sectional design⁵³ of the questionnaire for the collection and analysis of relevant data in order to investigate the current incidence and prevalence of breast cancer in a subset population in China.

The relationship among variable factors has been determined at first. Because the questionnaire was designed as binary or non-binary questions, for example, whether has a high fat diet, we adopt the phi correlation coefficient analysis to demonstrate the associations. The total number of the respondents was represented as N. For the i-th respondent, we define the random variables as shown in Table 2 and two generalized random vectors X_i and U_i in equation (1), which represents the binary and non-binary variables, respectively.

When only binary random variables are considered, variables are processed in an iterative sequence in order to find the relationships among random variable pairs. For each pair consisting of 2 random variables, for instance, X_i and X_j, there are 4 possible cases labeled in alphabetical order as shown in Table 3.

The phi coefficient introduced by Udny Yule⁵⁴ was used to determine the correlation between two binary variables, ranging from -1 to 1 that represents the strongest negative or positive correlations, respectively. The phi coefficient close to 0 indicates weak or no correlation. The number of respondents in each case is denoted as n_j,j=A,B,C,D and shown in equation (2) and Table 4.

When only binary random variables are considered, variables are processed in an iterative sequence in order to find the relationships among random variable pairs. For each pair consisting of 2 random variables, for instance, X_i and X_j, there are 4 possible cases labeled in alphabetical order as shown in Table 3.

The formula for the phi coefficient is defined as

Through iterative computation, we obtain the result shown in Figure 2.

Figure 2. Plot of the phi coefficients with random variables.

Suffering from breast diseases and suffering from breast cancer produces the largest positive phi coefficient, indicating those people who have breast diseases tend to have breast cancer. The second and third-largest phi coefficients are associated with breast tenderness with lumps or estrogen medication or birth control pills with coefficient 0.361 and 0.257, respectively. The forth-largest phi coefficient is associated with smoking and drinking with a phi coefficient 0.212. It means those people who possess the habit of drinking alcohol are also likely to smoke cigarettes. Moreover, the random variables X₁ and X₈ show a negative correlation with a phi coefficient equal to -0.313, indicating women are more likely to have physical exercise than men. The phi coefficients of the other random variable pairs are less than 0.2 in absolute value, meaning these binary variables can be considered as linearly irrelevant

Table 5 lists the biologically collated variables that are intrinsically correlated in biology such as gender and reproductive factors. These variables should be justified before we start to elucidate the unknown association between binary random variables and non-binary random variables,

We used the mathematical tool introduced by M. Baak, R. Koopman, H. Snoek, and S. Klous that serves as an extended and amended measure of Pearson’s correlation coefficient.⁵⁵ There are r rows and k columns in the contingency table and the cells are represented as C_ij. The total case number is defined as N=r∙k. For each random variable pair denoted by x and y, their probability distribution function ρ is defined as equation (4).

Herein, , denote the average value and σ_x,σ_y stand for the standard deviation, respectively. The ρ represents the Pearson correlation coefficient. We denote the observed value and the expected value of cell (i,j) by O_ij, E_ij respectively. The equation of Pearson’s X² test is defined as equation (5).

Using the definition of the probability density function, the probability of cell (i,j) is defined as

By assigning the value

Substitute to Formula (5), we obtain

Because the observed value O_ij is constructed independently on true observed values, an effective number of freedom n_f and the number of observed empty cells n_e are introduced to treat statistical noises.

We define the pedestal statistics χ_p² and χ_max² as follows.

In which c is added to exclude the outliers. To amend the Formula (8) by extending for case ρ=0 and case ρ=1, we obtain the formula (12).

The correlation coefficient ϕ_K can be calculated as follow.

The independent factors are identified through above analysis. To tackle with the interaction among correlated factors, we combine their effect into an overall effect-equivalent random variable. We define a new set of variables in Table 6.

The logistic regression method is applied to model the relationship between breast cancer status and the linear combination of potential factors. Define D={1,2,⋯,M}, E={0,1,⋯,M}, then

In which p is the probability for an individual to have breast cancer and {V_k} is the set of potential factors, and β_k is the weight for the k-th potential factor respectively. Using logit transformation, we convert the non-linear relationship between p and V_k into a linear relationship between logit(p) and V_k.

To obtain the most likely value of β_i, we use the maximum likelihood method56

Where y_k is the observed breast cancer variable for the k-th potential factor. Taking the partial derivative for each β_i and assigning them to 0, we obtain the following set of equations, in which V_nk represents the value of V_n of k-th factor.

The result of {β_i} is shown in Table 7. When β increases, the probability of having breast cancer p increases.

It is shown the accuracy of modeling results are highly limited by the biased sampling population. A representative biased result is the coefficient of education background (V₂) calculated as 0.317, which means the high education background causes high risk of breast cancer. This result is due to the fact that our questionnaire was mainly collected from people with high education level. Environmental exposure, smoking, family history and diet do not show strong positive association with breast cancer probably because of the problem of sampling size. Staying up, anxiety, alcohol consumption and pregnancy termination are positively identified as behavioral and reproductive risk factors related with breast cancer.

We have established a mathematic model for the interpretation of the epidemiological survey of breast cancer by using online questionnaires. The phi coefficient and amended Pearson coefficient are applied to investigate the relationship of binary and non-binary variables as well as the factors associated with the occurrence of breast cancer. It is revealed that behavioral and reproductive factors including sleep duration, anxiety, alcohol consumption and pregnancy termination are associated with the occurrence of breast cancer. However, the accuracy of our model is limited by the size of sampling population and follow-up investigation is needed to validate the risk factors.

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In order to meet with other IGEMers in China, six of our Guangxi-U-China team 2023 members have attended the 10^th IGEMers Communication Conference in Hainan from July 8-July 11, 2023 (Figure 2). We made a stage presentation and a poster show herein. In the conference, we have also listened to the presentations from other teams. Importantly, we made new friends and learned new knowledges in the conferences.

Figure 2. Attend the 10^th IGEMers communication conference in Hainan

Since this field trip, we have made collaborations with other 5 teams including Peking 2023, PekingHSC 2023, BNUZH-China 2023, Tsinghua 2023, and JLU-NBBMS 2023 that are working on microbial therapy for cancer. On July 27, Aug 9, and Sep 10, 2023, we had three online project seminars in which we discussed the technical progresses (Figure 3).

Figure 3. Jointed online project seminars by six teams

On Aug 5, 2023, we had an online consulting seminar with those experts. Bo Zhang, Luping Zhu, Yuqi Liu, and Xiaoxiong Deng gave presentations on bacteria engineering, safety, human practice and mathematic modeling, respectively. Professors Jianmin Wu, Lin Zhang, Bifeng Yuan, Xiaoyun Liu, Ping Wang, and Zuogong Yue gave comments and suggestions on our research progress.

Figure 4. Online project consulting seminar

Our team members are majored in life science and technology, computer science and technology, mechanistic engineering, animal science and technology, mathematics, arts, artificial intelligence, journalism and communications of with different education background. On Mar. 3, April 1, and June 25, 2023, training lectures on safety, norms and ethics were provided to team member (Figure 5).

Figure 5. Training of team members on lab safety, norms and ethics

Synthetic biology is a rapidly developing field that is not taught in regular classes of most universities. The emergency of synthetic biology is supported by a number of enabling technologies developed in multidisciplinary fields. Team members were trained for fundamental principles and basic experimental protocols of synthetic biology. Because a portable device designed for regular daily self-checking of breast health is arranged, training of instrumentation was also provided on March 18, 2023 (Figure 6).

Figure 6. Training of synthetic biology and instrumentation

We have regular brainstorm team seminars in which team members report their literature studies and experimental progresses. Any problems and experimental details associated with the project are discussed in brainstorm seminars that can continuously push forward the project. Team members also learn from regular literature studies. Through regular literature report, team members not only understand science frontiers but also the design of project, helpful experimental protocols. In 2023, we have had 12 brainstorm meetings and 38 presentations have been given so far (Figure 6).

Figure 7. Brainstorming team meetings

On important holidays in 2023 including “3.8 Women’s Day” and “Mother’s Day”, we gave public health lectures that provide people with fundamental knowledges of breast cancer and prevention strategies. We hope to raise public awareness for early detection and prevention. Women’s day lecture open to the public was arranged in university circular theater. Mother’s day lecture in Tencent Meeting was online open to the public (Figure 8).

Figure 8. Online or stage public educational lectures

In August and September of 2023, team members held public education lectures in urban communities or rural counties in four provinces or regions of China (including Guangxi Zhuang Autonomous Region, Hunan Province, Heilongjiang Province and Xinjiang Uygur Autonomous Region). The lecture focuses on the early symptoms, life style and environmental factors of breast cancer (Figure 9)

Figure 9. Public educational lectures in city communities and rural counties

On Sep 17, 2023, we held a jointed online science popularization seminar to first year freshmen in which six teams from different university participated, including Peking 2023, PekingHSC 2023, BNUZH-China 2023, Tsinghua 2023, and JLU-NBBMS 2023 teams. Representative team members from the six teams gave presentations. After the lectures, team members took new students to have a tour of the scientific lab (Figure 10). In Sep 12, 2023, team member provided a class presentation to the freshmen majored in life science and technology. These lectures cover the fundamental synthetic biology and the landscape of emerging therapy for cancer. We hope that our activities can inspire the new students in the university (Figure 10).

Figure 10. Public science popularization and scientific lab show to freshmen

Our public health WeChat account named as The Lanterns (兰子菽) was established in March of 2022. It provides social services for people who need help for understanding fundamental knowledges, diagnostic techniques and instruments, medication options and management policy of breast cancer. In 2023, 10 articles written by the team members are posted on the public health WeChat account (Figure 11).

Figure 11. The Lanterns public health WeChat account and posted articles

Our team members Xuemei Dong, Xinxi Liu and Fengyu Jiang have worked with team members from Peking 2023, PekingHSC 2023, BNUZH-China 2023, Tsinghua 2023, and JLU-NBBMS 2023 teams. We co-authored a handbook for microbial therapy including research background of biotherapy, engineered bacteria chassis for tumor treatment, ethic thinking and human practice (Figure 12). There are both English and Chinese versions and they are accessible for the public as required.

Figure 12. The handbook of microbial therapy co-authored by members from collaborated 6 teams

As we know, the treatment of breast cancer may be highly effective if it can be detected early. However, the state-of-the-art instruments are not always accessible for some people, in particular for those living in rural areas or urban people who are very busy everyday. We developed a portable device that is affordable for individuals to do daily self-checking and physical therapy at home (Figure 13). It is based on A-type ultrasound and near infrared irradiation. Currently, it is under testing and proof-of-principle demonstration. Related software has been developed (click for software download).

Figure 13. The design and building of a portable device

Figure 14. Project improvement and social contribution of our human practice

Our project has been continuously improved through various activities of our human practices. In this multidisciplinary project, we have collaborated with lots of people in different research fields and clinical practices. We learn not only basic sciences but also the thinking ways from experts, which broaden our visions and inspire our spirit. The communications with other teams help the establishment of friendship and provide opportunities to work together and share resources. The jointed efforts have been moving towards a better life and better world (Figure 14).