Poster: Worldshaper-Shanghai

Team Achievement

This is the poster of Team Worldshaper-Shanghai 2020. This year, our project is focusing on early prostate cancer diagnosis, developing a non-invasive, convenient, accessible method for rapid screening of early prostate cancer. As followed is the summary of our Achievements in this iGEM season.

Non-invasive technique for early stage prostate cancer diagnosis

Presented by: Team Worldshaper-Shanghai 2020

Team Roster

PIs: Shan Dong, Xixi Song
Instructors: Jie Hong, Haoyan Chen, Baoqing Xuan
Advisors: Ruyi Shi, Songlin Cai
Team members: Marnie Wang, TianZe Zhao, Ruining Guo, Yixuan Yang, Yishan Mao, Wenxin Wu, Nidixin Zhang, Zhiqian Wang, Jiaxin Tan, Tianyi Huang, Tiancheng Liu, Hanzhang Liang

Abstract

The most common clinical diagnosis nowadays for prostate cancer is digital rectal examination，serum PSA test and prostate biopsy, which, to some extend, is invasive or can cause physical discomfort. The goal of Worldshaper-Shanghai 2020 is to develop a new non-invasive technique for early stage prostate cancer diagnosis, mainly by expressing the color variation of the tested urine since urine test could be the simplest, painless and rapid test method for prostate cancer diagnosis.Our equipment design combined the technology of reverse transcription, gene amplification (PCR/RPA) and RNA hairpin structure (Toehold switch), to detect the specificity of RNA biomarkers of prostate cancer in the urine (PCA3 and KLK3), the final red fluorescence protein output will be report as visible results, so as to achieve the purpose of our visual diagnosis of prostate cancer.

Introduction & Inspiration

Prostate cancer is an extremely common cancer in elderly males that is curable at its early stage of development and progresses slowly but is hard to cure in its later stage (shown below). Due to the lack of obvious early symptoms and privacy reasons, the low diagnosis rate increases the difficulty of treatment and affects the prognosis of patients.

(Source of Information: National Cancer Institute, NCI,
https://seer.cancer.gov/statfacts/html/prost.html)

Currently there is no perfect method to detect prostate cancer (shown below), we are therefore in drastic want for newer non-invasive testing equipments that bypass all the aforementioned drawbacks.

(Source of Information: National Cancer Institute, NCI
https://www.cancer.gov/types/prostate/patient/prostate-treatment-pdq#section/all)

Methodology

Urine Sample

Urine test could be a simple, painless and rapid test method for prostate cancer, Various types of prostate biomarkers are present in the urine.

Biomarkers

We chose two new biomarkers of prostate cancer called PCA3 and KLK3 for diagnosis through related academic works and the GEPIA2 website. The expression levels of these two kinds of RNA in prostate cancer cells are much higher than those in normal cells.

References: Eskra J N, et al., Prostate Cancer and Prostatic Diseases 2019
References: Tang, Z. et al., Nucleic Acids Res 2017

Our detecting means

Two tools
1) Gene specific primers for detecting of biomarkers
2) Toehold switch for reporting visible test result
The concept behind our device is quite simple: first, all the RNA present in the urine sample is collected; the RNA is then reverse transcribed into cDNA, allowing for gene amplification to be carried out; The amplified products are transcribed and translated with toehold switch system in vitro. The success of the reaction can be judged by the fluorescence after the reaction.

Results and Conclusions

Primer Selection

To improve the efficiency of gene amplification, we selected the most appropriate pair of each gene by using PCR amplification. After finding the best pair for each gene, we added T7 promoter sequence and trigger sequence to primers. The results from PCR showed that the primers with overhang could also successfully amplify KLK3 and PCA3 without significantly affecting the efficiency.

Verification of Toehold Switch

PCA3 transcript and toehold plasmid were used for validation of in vitro transcription and translation. The results were as anticipated: samples containing PCA3 products and toehold plasmids showed brilliant fluorescence.

Proof and Modeling

We evaluated our method through synthetic urine:

1) A standard curve is plotted to determine the effective interval of copies of PCA3 in urine for the detection, the fluorescence intensity increased linearly from 200 copies to 800 copies, indicating that the concentration of PCA3 transcripts in this range can be considered as an effective identification range of our method.
2) The receiver operating characteristics (ROC) curve was developed to verify the reliability of our method, the AUC (area under curve) value is calculated to be 82.4%, indicating that our testing is quite reliable.

Human Practices

Integrated Human Practices

Project improvements
Urine Ethics, Sample Collection
Implementation
Use in community hospitals or physical examination centers
Removed common equipment
Public Engagement
Education about the significance of prostate cancer and its testing

Proposed Implementation

Advantages:

Discernible color results
Reagents stored in tubes
Save costs

Standard protocol:

Future Work

RPA testing

Prevent primer dimer and non-specific amplification

Improvements on kits design

Evaluate the sensitivity and reliability of the kit
Develop diagnostic criteria
Kit storage and supplier

Acknowledgements

We sincerely thank all those who have contributed!

Special thanks:

General Support:
Hangzhou Sipu Education and Technology, Co., Ltd.
Shanghai Cancer Institute

Lab Support:
JRDUN Biotechnogy(shanghai) Co.,Ltd.

Video Support:
Shanghai Wings Culture Communicationstion Co.,Ltd.

Human Practices Support:
Physician Hou, Shanghai Changhai Hospital
Professor Chu, Shanghai Gongli Hospital
Doctor Lu, Shanghai East hospital
Professor Huang, Zhejiang Sci-Tech University
Zhejiang Association for Science and Technology
Shanghai World Foreign Language Academy

Partnership:
OUC-China 2020 iGEM Team

Team:Worldshaper-Shanghai/Poster