iGEM 2025 — Team keystone

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![A screenshot of a calendar  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5739/notebook/1-1.webp)

**Date: Feb 18th-25th, 2025**

Main Goal: Kickoff Meeting

Participants: Aveline Guo, Cherry Li, Emily Li, Ethan Liu, Howard Zhang, Judy Yu, Kevin Zheng, Kitty Yuan, Kitty Mai, Lily
Li, Nina Yang, Sunny Hu, Yvonne Li, Zihan Chen, Zoe Wang

Location: Keystone Academy

Content:

Section 1 – What you may expect in iGEM

* Tangible Outcomes in iGEM Competition: Competition deliverables, participation certificate, medals, prizes
* Intangible Outcomes in iGEM Competition: A rewarding learning experience
* Insights from Judging iGEM Projects

Section 2 – Human Practices: Responsibilities & Improvement

* Human Practices
* Case Sharing

Section 3 – Briefing iGEM Villages and Engineering

* Popular iGEM Competition Villages & Engineering Cycle
* Basic Biology Laboratory Skills
* Data Processing & Analysis
* Common Research Software

Section 4 – Entrepreneurial Ventures in iGEM Projects

* Entrepreneurship Requirements in iGEM Competition
* Top 1 Business Plan in 2023 iGEM (High School)

Section 5 – Tips for Initial Project Brainstorming

* Innovation and Practically
* iGEM Responsibility
* Compliance with Competition Rules and Meeting the Gold Medal Criteria

Section 6 – Synthetic Biology Basics

* Tangible Outcomes in iGEM Competition: Competition deliverables, participation certificate, medals, prizes
* Intangible Outcomes in iGEM Competition: A rewarding learning experience
* Insights from Judging iGEM Projects

Section 7– iGEM Villages Overview

* Environment
* Healthcare
* Advancements

Section 8– Representative Projects

* Environment – Bioremediation
* Environment – Food & Nutrition
* Healthcare – Therapeutics
* Healthcare – Diagnosis

Section 9 – Attributes of High-Impact iGEM Projects

Learning Materials:

iGEM website: <https://igem.org/>iGEM Website:
<https://igem.org/>

About Human Practices:
<https://responsibilityigem.org/human-practices/what-is-human-practices>

About Judging and Awards:
<https://competition.igem.org/judging/medals>

About Judging and Special Prizes:
<https://competition.igem.org/iudging/rubric>

Whitelist for Experiments:
<https://responsibility.igem.org/safety-policies/white-list>

<https://2024.igem.wiki/keystone>

https://2024.igem.wiki/gems-taiwan/ (Grand Prize Winner)

<https://mp.weixin.qq.com/s/xeuC-NExG99pte3zF7NnhA>

<https://mp.weixin.qq.com/s/cOx9WU9BgapJMv4opk7Fcw>

Notes:

In this lesson, we learnt that iGEM projects are organized into groups called “Villages”. These villages focus on solving
global problems in three areas: the environment, healthcare, and technology. As we learned high-impact projects usually deal
with a big problem in the real world, include strong work in the wet lab and dry lab, and show clear practical uses.

![A screenshot of a calendar  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5739/notebook/2-2.webp)

**Date: Mar 4th-25th, 2025**

Main Goal:

Literature Retrieval Guidance/Brainstorm 1

The Theoretical Foundation of Synthetic Biology

Basic Molecular Biology Experimental Operations 1

Participants: Aveline Guo, Cherry Li, Emily Li, Ethan Liu, Howard Zhang, Judy Yu, Kevin Zheng, Kitty Yuan, Kitty Mai, Lily
Li, Nina Yang, Sunny Hu, Yvonne Li, Zihan Chen, Zoe Wang

Location: Keystone Academy

**Content:**

Section 1 – Genetic Engineering Classic Route Analysis

Section 2 – Literature Reading, Brainstorm

Section 3–Experimental content

(1) Theoretical explanation 40 minutes

(2) Laboratory safety training 10 minutes

(3) Pipette usage 20 minutes

(4) Plasmid extraction 60 minutes

(5) Preparation of nucleic acid gel 20 minutes

(6) PCR amplification of DNA fragments 40 minutes

(7) Agarose gel electrophoresis 30 minutes

(8) Result analysis and explanation 10–20 minutes

(9) Cell Lysis

(10) Protein Purification

(11) Separation and Identification of Proteins

Section 4-Group Brainstorm Sharing

Hairball Buster – Emily Li + Judy Yu

Gout to Probiotics – Cherry Li + Sunny Hu

Sun Allergy – Yvonne Li

SpiceShield – Lily Li + Zoe Wang

![微信图片_20250930113904_51_34](https://static.igem.wiki/teams/5739/notebook/3-1.webp)![微信图片_20250930113905_52_34](https://static.igem.wiki/teams/5739/notebook/4-1.webp)

**![](https://static.igem.wiki/teams/5739/notebook/5-1.webp)**

**Date: April 1st-26th, 2025**

Main Goal: Brainstorm，logo

Participants: Aveline Guo, Cherry Li, Emily Li, Ethan Liu, Howard Zhang, Judy Yu, Kevin Zheng, Kitty Yuan, Kitty Mai, Lily
Li, Nina Yang, Sunny Hu, Yvonne Li, Zihan Chen, Zoe Wang

Location: Keystone Academy

Content:

Group Brainstorm Sharing (Final Round)

The Eco-Scent: Kitty Yuan + Nina Yang

Glucoguard – Type 1 Diabetes: Sophia Wu + Zihan Chen

Amyloidosis – Kevin Zheng + Howard Zhu

Bamboo & Algae as Carbon Sinks – Kitty Mai + Ethan Liu + Aveline Guo

SpiceShield – Lily Li + Zoe Wang

Review from Teachers

1. Mr. Kevin explains the voting rules

2. Each group has 2 minutes to campaign, briefly highlighting the proposal\'s key points; then each person casts 2 votes; the
votes are counted, and the winning proposal group is announced.

Section 1- Educational Activity Workshop

1. The types of media platforms and responsible persons have been determined, and the content for each platform team has
been finalized.

2. Educational activity plans for grades 1-3, grades 4-7, and the elderly have been completed.

Homework:

Judy, the leader of the children\'s group, and Howard, the leader of the elderly group, remind the students responsible for
each part to send the completed PPT, promotional pages, and card design files to the group tonight.

Students responsible for designing the logo, team name, and slogan must submit the finalized versions to the group by next
Saturday to facilitate registration on the online education platform and updates to subsequent content.

Section 2- Entrepreneurship Workshop

Work Division:

Market Size: Emily, Judy, Zoe

Unmet Needs: Aveline & Lily

STP: Zihan

Porter\'s Five Forces: Yvonne

SWOT: Sunny Hu, Kitty Yuan

PESTEL: Howard, Kevin

[Competitor Analysis](https://keystoneacademy-my.sharepoint.com/%3Aw%3A/r/personal/fangjia_yang_student_keystoneacademy_cn/_layouts/15/Doc.aspx?sourcedoc=%7B0ADB21E1-25A5-4EBC-907F-3613B5E3A9F3%7D&file=Competitive%20Analysis.docx&wdLOR=cD621D9D7-750B-9642-A6DD-76C0673AD5B3&fromShare=true&action=default&mobileredirect=true)
(Comparing Metrics): Cherry, Nina, Ethan Liu

7P Analysis: Emily, Judy, Zoe

Financial Forecast: Aveline & Lily

Risk Assessment: Zihan, Kitty Mai

![微信图片_20250930114323_54_34](https://static.igem.wiki/teams/5739/notebook/6.webp)![微信图片_20250930114414_55_34](https://static.igem.wiki/teams/5739/notebook/7.webp)

**Date: May 3rd-25th, 2025**

Main Goal:Education,human practice, Wetlab

Location: Keystone Academy

Participants: Aveline Guo, Cherry Li, Emily Li, Ethan Liu, Howard Zhang, Judy Yu, Kevin Zheng, Kitty Yuan, Kitty Mai, Lily
Li, Nina Yang, Sunny Hu, Yvonne Li, Zihan Chen, Zoe Wang

Main Goal: Interview Techniques and Human practices

Content:

Section 1

Ways to conduct interviews, emphasizing the importance of maintaining an objective stance, avoiding leading questions, and
paying attention to details during the interview process.

The steps of qualitative research, including identifying the research phenomenon, clarifying the research questions,
reviewing existing literature, designing interview outlines, conducting interviews, and writing research reports.

How to select appropriate interview types based on research objectives and nature and provided examples on how to design
interview questions and conduct interviews.

Section 2

Student Leader Election/Review of Educational Activities

Election for Coordinate team leaders:

Candidates - Howard, Aveline, Judy, Sunny, Zihan

Rules - Each candidate has 5 minutes to give a campaign speech, with the first 3 minutes being assisted by PPT, and the last
2 minutes being spent answering questions from classmates. After all candidates have finished their speeches and Q&A
sessions, immediately vote, count the votes, and announce the results.

Section 3

Results

Finalized future schedules and educational content of visiting the Kang Yu Xuan Elder’s Home.

Photos:

Section 4

Main Goal: Student Leader Election/Experimental Design Review

Participants: Aveline Guo, Cherry Li, Emily Li, Ethan Liu, Howard Zhang, Judy Yu, Kevin Zheng, Kitty Yuan, Kitty Mai, Lily
Li, Nina Yang, Sunny Hu, Yvonne Li, Zihan Chen, Zoe Wang

Location: Keystone Academy

Content:

Election for Wet Lab Leaders

Election for Dry Lab Leaders

Section 5 Review of Human Practice Activities

1. Reflection on Kang Yu Xuan Elder’s Home Visit

Discussed for improvements on the team’s experience educating elderly residents about Alzheimer’s Disease (AD)

Used a PowerPoint presentation to explain AD

Conducted interactive activities:

Memory card game

Coloring

Discussed the impact of these activities on engagement and understanding

2. Decision to Use Notion

Agreed to use Notion as the centralized tool for:

Taking notes

Organizing meeting content

Tracking progress

3. Planning for Neurologist Interview

Began coordinating a suitable time for an online interview with a neurologist

Ensured team availability and roles for the upcoming interview

![微信图片_20250930113903_50_34](https://static.igem.wiki/teams/5739/notebook/9.webp)![微信图片_20250930113902_49_34](https://static.igem.wiki/teams/5739/notebook/10.webp)

![](https://static.igem.wiki/teams/5739/notebook/june-1.webp)

**Date: June 3rd-17th, 2025**

Main Goal:

Review of Business Plan Development

Online Interview with a Neurologist on Diagnosis and Treatment of Alzheimer\'s Disease

Award Strategy Analysis - Specialization

Participants: Aveline Guo, Cherry Li, Emily Li, Ethan Liu, Howard Zhang, Judy Yu, Kevin Zheng, Kitty Yuan, Kitty Mai, Lily
Li, Nina Yang, Sunny Hu, Zihan Chen, Zoe Wang

Location: Keystone Academy

Content:

Section 1

1. Review HW previously assigned on April 26th. Revised and improved the section on “Unmet Needs” in the business plan.
2. Analyzed the current market strategies related to Alzheimer’s Disease (AD) and existing technologies
3. Began preparing for the upcoming interview (related to AD expert consultation)

Section 2

Discussion mainly focuses on the early screening and diagnosis of Alzheimer\'s disease:

Current early screening and diagnostic methods for Alzheimer\'s disease in China were introduced, including medical history
inquiry, symptom observation, neuropsychological tests, imaging examinations, etc.

Advantages and disadvantages of these methods were discussed, as well as possible future development directions such as
blood testing and urine testing.

How to improve the acceptance of Alzheimer\'s disease testing among the elderly, including strategies such as science
education and psychological counseling.

The possibility and challenges of incorporating Alzheimer\'s disease testing into public health programs were discussed,
including cost control, policy support, etc.

Section 3

Previous class content reviewed and introduced the process of using the Flex system to screen high protein nucleic acid
aptamers.

Explained the principle and application of Crispr technology and biosensors.

Discussed the optimization direction of the project, including model optimization. Emphasized the need to consider factors
such as design purpose, functionality, and carrier selection during the project process, and select appropriate sections for
modelling based on experimental results.

Reviewed the list of iGEM Special Prizes and discussed strategic alignment with our project by analyzing past winning cases
for Best Part Collection and Best Measurement.

Identified key criteria across special prize categories: coherence, documentation, functionality, reproducibility, and
usefulness.

![微信图片_2025-06-27_150647_320](https://static.igem.wiki/teams/5739/notebook/11.webp)![图片4](https://static.igem.wiki/teams/5739/notebook/12.webp)

**Date: June 22rd-30th, 2025**

Main Goal: Materials and Experiments

Participants: Aveline Guo, Cherry Li, Emily Li, Ethan Liu, Howard Zhang, Judy Yu, Kevin Zheng, Kitty Yuan, Kitty Mai, Lily
Li, Nina Yang, Sunny Hu, Zihan Chen, Zoe Wang

Location: China agricultural university

Content:

Preparation of culture medium

Gene synthesis and primer synthesis, material procurement

PCR amplification of the target gene

Transformation and verification

![微信图片_20250930115807_69_34](https://static.igem.wiki/teams/5739/notebook/13.webp)![微信图片_20250930115811_70_34](https://static.igem.wiki/teams/5739/notebook/14.webp)

![微信图片_20250930115811_70_34](https://static.igem.wiki/teams/5739/notebook/july.webp)

**Date: July 1st-31th, 2025**

Main Goal:

Task Allocation Clarification for the Educational Activities

Laboratory safety training and experiments

Participants: Aveline Guo, Cherry Li, Emily Li, Ethan Liu, Howard Zhang, Judy Yu, Kevin Zheng, Kitty Yuan, Kitty Mai, Lily
Li, Nina Yang, Sunny Hu, Yvonne Li, Zihan Chen, Zoe Wang

Location: China Agricultural University/China Academy of Chinese Medical Sciences

Content:

Section 1

Reviewed the lab design section of the project, focusing on SELEX and CRISPR-Cas12a technologies.

Clarified the daily schedule and structure of upcoming summer training activities.

Section 2

Youth education: Emily- Contact the YunAi organization, conduct education activities; Aveline, Lily - PPT+experiment design

On campus education: Aveline, Lily – Syn-bio week

Middle-aged education: Determine the time and location of the event (Aveline contact Jiangshan Fu community); Zoe, Judy.
Emil, Kitty Mai, Sunny - Prepare a PowerPoint presentation for middle-aged people to share AD and synthetic biology

Section 3

IHP: Howard - Send a questionnaire

Business plan: According to the previous division of labor, each group needs to send the completed parts to the group before
the training starts

Section 4

Prepare questions for the interview with Rashik Chand

Finalize the Survey Questions for “Alzheimer’s Disease Awareness and Detection Needs”

Prepare interview questions for 7.14th Interview with Mr. Wang

Brainstorm of the Team’s Wiki’s overall layout and style

Started working on the hand-drawn components

Establish social media accounts for the team [Dou Yin, Xiao Hong Shu etc.]

Location: China Agricultural University/China Academy of Chinese Medical Sciences

Section 4

Training on Safety Standards for Biological Laboratories [Need to Elaborate]

Afternoon – Howard, Kevin, Lily, Nina

Configuration of LB medium

PCR amplification of target genes

Plasmid extraction

Section 5

Conduct online interview with Rashik Chand

Send out the survey for “Alzheimer’s Disease Awareness and Detection Needs” on wenjuan.com

Goal: ≥100 responses

Conduct online interview Mr. Wang (a family member of an AD patient)

Establish future dates the educational activities on youth and middle-age; complete and finalize both educational PPT.

Prepare question for interview with Dr. Lu on July 15th

Section 6

Electrophoretic verification of the digested vector and target gene fragment

Gel-purified DNA fragment ligation into the vector and transformation of the recombinant construct containing the target
gene into E. coli

![微信图片_20250930115747_64_34](https://static.igem.wiki/teams/5739/notebook/15.webp)![微信图片_20250930120508_73_34](https://static.igem.wiki/teams/5739/notebook/16.webp)

Section 6

SDG Workshop

Conduct online interview with Dr. Lu (from Nanjing University of Traditional Chinese Medicine)

Prepare questions for interview with Jin Nuo Mei Biotechnology Co., Ltd on July 16th

Section 7

Screening of single colonies

Large-scale cultivation and expression induction

Section 8

Prepare questions for interview with Jin Nuo Mei Biotechnology Co., Ltd and rehearse the interview

Location: Building 16, Courtyard 25, Jinghai Fourth Road, Beijing Economic and Technological Development Zone

Visit and conduct off interview with Jin Nuo Mei Biotechnology Co., Ltd

Section 9

Protein extraction, purification, and identification

SDS-PAGE and Tau peptide immobilization

Section 10

Planning for content and distribution for Promotion Video and develop the script

Production and filming of promotion video, including drawing out the animations

Section 11

DNA library preparation

Initial SELEX screening – First Round

Section 12

Continue working on production of the promotion video

Prepare for the educational activity in the afternoon

Afternoon Group A– Emily, Judy, Zoe

Education Activity

Afternoon Group B – Howard, Zihan, Ethan, Nina

Continue working on the animations of the promotion video

Prepared Interview questions for the offline interview with Mr. Zhang on July 19th

Section 13

Positive SELEX screening round 2-7

Conduct interview with pharmaceutical and chemical investment expert – Mr. Zhang Antong about our business plan

Prepare interview question for the online interview with Dr. Hu on July 20th.

Revise business plan based on suggestions from the interview with Mr. Zhang

Section 14

Promotion video editing

Organize notes for Wiki

Work distribution for the defense presentation slides

Online interview Dr. Hu Chunyi

Continue revising business plan based on suggestions from the previous interviews

Section 15

Negative SELEX screening round 8 to 16.

Flow cytometric detection

ELISA verification of binding

Finalized work distribution for the defense presentation slides and Wiki content of the first summer training session.

Work on the defense presentation slides for the first summer training session.

Section 16

Defense presentation for the first summer training session. Content including:

Project Background,Project Goals,Methodology overview , Results, Human Practices,Safety

![Screenshot_2025-08-20_at_10.40.57](https://static.igem.wiki/teams/5739/notebook/17.webp)![b1bbfd4c3ab3620f7e2c4f8235ab41cf](https://static.igem.wiki/teams/5739/notebook/18.webp)![微信图片_20250930115754_67_34](https://static.igem.wiki/teams/5739/notebook/19.webp)![微信图片_20250930115748_65_34](https://static.igem.wiki/teams/5739/notebook/20.webp)

![微信图片_20250930115748_65_34](https://static.igem.wiki/teams/5739/notebook/august.webp)

**Date: August 1st-30th, 2025**

Location: China University of Technology and Keystone Academy

Participants: Aveline Guo, Cherry Li, Emily Li, Ethan Liu, Howard Zhang, Judy Yu, Kevin Zheng, Kitty Yuan, Kitty Mai, Lily
Li, Nina Yang, Sunny Hu, Yvonne Li, Zihan Chen, Zoe Wang

Content:

1. Second round of nucleic acid aptamer screening using the SELEX system
2. Measurement of fluorescence values by flow cytometry
3. Surface Plasmon Resonance (SPR) measurement
4. Enzyme-Linked Immunosorbent Assay (ELISA)
5. Wiki content writing, e.g., data and results organization, experimental documentation, etc.
6. UI motion design and visual design
7. Plasmid Construction of Cas12
8. Expression and Purification of Cas12 Protein
9. Preparation and Measurement of crRNADetermination of the Concentrations of ComDNA and the Tau/BD-tau Aptamer-dsDNA Complex
10. Quantitative Analysis of the Cas12a Reporting System
11. Educational Activities/SDGs
12. Educational Activities/Interview with the professor
13. Modeling and Hardware

![WechatIMG1948](https://static.igem.wiki/teams/5739/notebook/21.webp)![1a1ae4f38d31b95ad0ca8676d02e8907](https://static.igem.wiki/teams/5739/notebook/22.webp)![微信图片_20250930120448_72_34](https://static.igem.wiki/teams/5739/notebook/23.webp)![微信图片_20250930115617_56_34](https://static.igem.wiki/teams/5739/notebook/24.webp)

![微信图片_20250930115617_56_34](https://static.igem.wiki/teams/5739/notebook/sept.webp)

**Date:September 1st-27th, 2025**

Location: China University of Technology/Keystone Academy

Participants: Aveline Guo, Cherry Li, Emily Li, Ethan Liu, Howard Zhang, Judy Yu, Kevin Zheng, Kitty Yuan, Kitty Mai, Lily
Li, Nina Yang, Sunny Hu, Yvonne Li, Zihan Chen, Zoe Wang

Content:

1. Surface Plasmon Resonance (SPR) measurement
2. Enzyme-Linked Immunosorbent Assay (ELISA)
3. Wiki content writing, e.g., data and results organization, experimental documentation, BP etc.
4. Quantitative Analysis of the Cas12a Reporting System
5. Optimizing Modeling and Hardware

![微信图片_20250930115617_56_34](https://static.igem.wiki/teams/5739/notebook/oct.webp)

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{
"title": 'Overview',
"content": '

This comprehensive project design embodies a systematic and rigorous approach to investigating early screening strategies
for Alzheimer’s disease, with a specific focus on BD-tau as a target biomarker(Gonzalez-Ortiz etal ;2023).The methodology
encompasses several stages, first we are screening for high-affinity DNA aptamers targeting BD-tau through SELEX technology.
Then, using the nucleic acid aptamers screened by SELEX and integrating CRISPR-Cas12a signal amplification technology, an
aptamer-based biosensor is constructed to detect BD-tau protein levels in blood samples.This non-invasive detection method
is expected to become a highly effective tool for early diagnosis of AD, possessing significant clinical translational value
and application prospects.

Each of our engineering cycles comprehensively incorporates four key phases: design, build, functional test, and learn.
Furthermore, we continuously iterate and refine our approach based on experimental results, aiming to achieve the desired
outcomes. By establishing a robust platform for aptamer selection and validation, this project lays a foundational framework
for future development of point-of-care diagnostic devices and further exploration of nucleic acid-based biomarkers in
neurodegenerative disorders.

',
"children": []
},
{
"title": 'Cycle 1: Screening aptamers using SELEX (pET28a-BD-tau and pET28a-Tau)',
"content": '

**Design:**

**![phf6 (1) 图谱](https://static.igem.wiki/teams/5739/engineer/1.webp)![tau (1) 图谱](https://static.igem.wiki/teams/5739/engineer/2.webp)**

Figure 1. The map of BD-tau and Tau

The plasmid pET-28a(+) can be used for protein expression in E. coli. It characterized as high-Level expression and tight
regulation. The pET system utilizes the powerful bacteriophage T7 promoter, which drives extremely high levels of
transcription of the target gene in E. coli. Besides it has many Multiple Cloning Site (MCS), providing a range of unique
restriction enzyme sites for convenient insertion of the target gene, and the vector encodes an N-terminal 6xHis tag (and
optionally a C-terminal tag), which binds specifically to nickel ions (Ni²⁺). This allows for easy one-step purification
using Immobilized.

Phf6 is a sequence for BD-tau protein and tau is the sequence for tau protein. BD-tau has been proved that as a biomarker
for Alzheimer’s disease, has been more effective and accurate than traditional tau screenings(Teng etal, 2018; Guo etal,
2017). The difference between BD-tau and peripheral tau lies in the fact that peripheral tau contains an exon 4a insertion
between exons 4 and 5, whereas BD-tau lacks this insertion. Therefore, we utilized the polypeptide sequence of the PHF6
domain located at the exon 4/5 junction of BD-tau for SELEX-based aptamer screening(Kyalu etal ;2023). The DNA aptamer
designed to target specific sites of BD-Tau demonstrates both high sensitivity and specificity while effectively avoiding
interference from peripheral tau isoforms.

A DNA library, also known as a gene library, refers to a collection containing the complete or partial DNA sequences of an
organism. We screen this library for aptamers with high affinity to BD-tau. A single-stranded DNA (ssDNA) library with a
66-nucleotide random region flanked by fixed primer-binding sequences (e.g.,
5\'-CAGCACCGTCAACTGAAT-(N66)-GTGATGCGATGGAGATGT-3\'). The diversity of the library is approximately ~1.2×10¹⁶ unique
sequences.

**Build：**

![图片10](https://static.igem.wiki/teams/5739/engineer/3.webp)

Figure 2. Target gene PCR amplification diagram

We constructed pET28a-phf6 and pET28a-tau using restriction digestion and ligation. Agarose gel electrophoresis was
employed to separate and analyze the size of DNA fragments, thereby verifying the accuracy of gene amplification via PCR.
This technique utilizes an electric field to facilitate the migration of DNA fragments, with smaller fragments exhibiting
faster mobility. The size and purity of these fragments are assessed through staining and ultraviolet (UV) observation. By
referencing a molecular weight marker positioned on the left, the approximate base pair (bp) lengths of the bands can be
inferred. When compared to established reference values, the correct bands can be identified. For BD-Tau, the expected band
length is 102 bp, which corresponds to the observed band in the Figure 2. For Tau, the anticipated band length is 105 bp;
the first, second, and fourth bands align with this expectation, while the third band displays inconsistent intensities,
likely due to uneven gel loading. Consequently, the third band is deemed incorrect as it does not match the expected value
and is unsuitable for further experimentation. This analysis underscores the use of agarose gel electrophoresis in selecting
the appropriate plasmids for subsequent recovery and insertion into enzyme-digested vectors through T4 ligase.

![图片11](https://static.igem.wiki/teams/5739/engineer/4.webp)

Figure 3. Growth of plasmid (BD-Tau & Tau) on LB agar plates

Following restriction digestion and ligation, which involves the precise insertion of plasmids into specific sites on the
enzyme-digested vector to achieve effective integration of the target gene, the reconstructed plasmid was further cultured
using the spread plate method. After we have transferred the reconstructed plasmid into the E coli DH5α , we have done a
monoclonal colony verification. To verify successful plasmid uptake, we employed Kanamycin selection by culturing the
transformed bacteria on LB agar plates supplemented with Kanamycin, and an antibiotic for which resistance was encoded in
the plasmid. The Kanamycin should screen out all the E-coil that doesn’t has our plasmid. As we can see in the Figure 3,
both *E coli* DH5α has successfully cultivated by the LB agar plate, which indicates our plasmid transfer is
successful. Subsequently, the extracted plasmids were transformed into *E. coli*BL21, followed by
monoclonal verification.

![图片13](https://static.igem.wiki/teams/5739/engineer/5.webp)

Figure 4. Single colony PCR validation graph

Single colonies from each of the LB agar plates were selected and subjected to amplification PCR. Multiple samples were
collected from each plate to mitigate the impact of potential errors by ensuring redundancy. Subsequently, agarose gel
electrophoresis was performed on all samples obtained from the four plates. This technique was employed to separate and
analyze the DNA fragments by size, thereby verifying the accuracy of the genes amplified by PCR. By utilizing a molecular
weight marker, the approximate base pair (bp) lengths of the DNA bands were estimated and compared to predetermined values
to identify the correct bands. In Figure 4, the expected band length is 102 bp, and the colonies BD Tau-DH5α-3 and BD
Tau-BL21-3 are suitable for further experimentation. In Figure 4, the expected band length is 105 bp, and the colonies
Tau-DH5α-1 and Tau-BL21-4 are appropriate for subsequent experimentation. This analysis facilitates the identification of
colonies with the correct DNA bands, which can then be cultured overnight for further sequencing.

![图片12](https://static.igem.wiki/teams/5739/engineer/6.webp)

Figure 5. Sanger sequencing map

In our project, we employed DNA sequencing for the identification and characterization of our DNA samples. We utilized this
method to ascertain the complete nucleotide sequence of our reconstructed plasmids. This approach enabled us to verify both
the accuracy and integrity of the reconstructed plasmid. Most peaks were sharp and well-defined, particularly in the central
region of the sequence, suggesting minimal background noise and high signal clarity. Both reconstructed plasmids, BD Tau and
Tau, exhibited sequencing results that fell within the anticipated parameters, indicating no significant deviations. The
sequence alignment with the designed construct confirms the successful incorporation of the target gene into the plasmid.
The high quality of the sequencing data allows us to confidently verify the efficacy of our reconstructed plasmids and
demonstrates the absence of significant sequencing errors or contamination, as evidenced by the lack of ambiguous bases or
unexpected peaks in the figure. This level of accuracy is essential for subsequent applications, ensuring the plasmids\'
suitability for further experimental use. The accuracy of the sequencing results is crucial for the reliability of follow-up
studies, which is critical for ensuring the plasmid\'s applicability in subsequent experiments.

**Test：**

1. **SDS-PAGE analysis of protein expression in *E.Coli***

The harvested cells were lysed through sonication in a binding buffer composed of 20 mM Tris-HCl, 500 mM NaCl, and 20 mM
imidazole at pH 7.9. The soluble fraction was subsequently subjected to immobilized affinity chromatography utilizing Ni-NTA
resin. Stepwise elution with imidazole gradients ranging from 50 to 250 mM resulted in BD-tau with a purity exceeding 90%,
as verified by SDS-PAGE on a 15% gel.

![](https://static.igem.wiki/teams/5739/engineer/7-1.webp)

Figure 6. SDS-PAGE for the analysis of whole cell lysis and crude protein solution after centrifugation

SDS-PAGE was performed on plasmid-transformed *E. coli* BL21 to assess the initial protein expression levels. As
depicted in Figure 6, both the crude protein extracted from the cell lysate and the protein obtained from the centrifuged
supernatant revealed the presence of BD Tau (5.1 kDa) and Tau (5.2 kDa). The successfully expressed BD-tau and Tau proteins
can be used for SELEX screening.

**2. Aptamer Screening**

**2.1 RFU characterizes the SELEX process**

![图片18](https://static.igem.wiki/teams/5739/engineer/8.webp)

Figure 7. Flow symmetry concerning the product of various rounds of the SELEX process

Flow cytometry is a biological technique used for counting and sorting microscopic particles suspended in a fluid(Zuo etal,
2014). In our experiments, we first incubate Ni-NTA magnetic beads with His-tagged BD-tau protein to allow complex
formation. Subsequently, nucleic acid aptamers with high affinity can specifically bind to the BD-tau Ni-NTA magnetic beads.
The test sample contains four distinct components: Ni-NTA magnetic beads, BD-tau-conjugated Ni-NTA magnetic beads,
aptamer-bound BD-tau-Ni-NTA magnetic bead complexes, and free nucleic acid aptamers. Additionally, the nucleic acid aptamers
are 5\'-end labeled with a FAM fluorescent tag.

We employed flow cytometry to separate the four components in the experimental sample.The BD-tau-conjugated Ni-NTA magnetic
beads and unconjugated Ni-NTA beads can be distinguished and sorted based on their physical properties, but they yield no
RFU signal due to the absence of fluorescent labeling. Furthermore, free nucleic acid aptamers, being DNA molecules below
the detection threshold of conventional flow cytometry, cannot be directly detected by this method. RFU signals are
generated only when nucleic acid aptamers are complexed with BD-tau-Ni-NTA magnetic beads. The RFU value thereby serves as a
quantitative indicator of the relative extent of aptamer-bead binding.

A 66-mer random DNA library (5\'-GCCGGATCCGTCGAC-(N66)-GAAGCTTGGTACCGAGCTC-3\') underwent 17 iterative rounds of selection
against immobilized BD-tau (2 μg per round). The stringency of the selection process was progressively increased by reducing
the incubation time from 60 to 20 minutes, introducing competitive elution with 10 mM free BD-tau in round 8, and decreasing
the target concentration to 1 μg in the final rounds. Enriched pools were amplified using flanking primers containing
Illumina adapter sequences, facilitating next-generation sequencing (MiSeq, 2×150 bp) after round 17.

This study presents our findings on flow symmetry concerning the product of various rounds of the
SELEX process. RFU, or Relative Fluorescence Units, quantifies the intensity of the fluorescent signal emitted by the FAM
attached to the F primer during PCR. As illustrated in the figure 7, RFU values progressively increased from round 1 to
round 6, indicating a gradual enrichment of aptamers with binding affinity for BD-tau. Post round 6, we implemented reverse
screening to eliminate non-specific binders, which resulted in a noticeable decline in RFU. By round 12, after additional
rounds of stringent selection, there was a substantial decrease in RFU, suggesting that only a small pool of aptamers with
the highest affinity remained.Finally, the selected nucleic acid aptamers were sequenced to determine their precise
nucleotide sequences, thereby providing the essential molecular information for subsequent development steps.

**2.2 Molecular docking screening of nucleic acid aptamers**

From the 27 nucleic acid aptamers selected through the SELEX system, we employed molecular docking to predict the binding
efficacy between BD-tau and the aptamers, ultimately screening three aptamers with the highest affinity.The binding score
results from the molecular docking are presented in Table 1.

Table 1. Binding Affinity of Aptamer-1, Aptamer-2, and Aptamer-3 with BD-tau

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Protein2 | Binding Score (kcal/mol) | Contact Sites (protein1) | Contact Sites (DNA) | Combination  Type |
| Aptame-1 | -224.03 | ASN327, LYS331, HIS362, LYS369, THR373, LYS375, GLN351 | DC19, DT20, DT13, DA52, DA5, DT6, DT54 | Salt bridge,  Hydrogen bond,  Hydrophobic interaction |
| Aptame-2 | -210.46 | GLN351, ARG349, LYS353, SER356, LYS375, THR373 | DG12, DG8, DC46, DG16, DT22 | Salt bridge,  Hydrogen bond,  Hydrophobic interaction |
| Aptame-3 | -201.37 | LYS353, LYS369, THR373, HIS374, LYS375 | DC1, DC3, DG45, DC43, DA37 | Salt bridge,  Hydrogen bond,  Hydrophobic interaction |

The table 1 demonstrate that BD-tau exhibits binding affinity to Aptamer-1, Aptamer-2, and Aptamer-3, though no significant
differences were observed among them. This indicates limitations in the molecular docking results, necessitating further
experimental validation to determine the binding affinity of Aptamer-1, Aptamer-2, and Aptamer-3 for BD-tau.

**2.3 Characterization of binding affinity by SPR**

To identify the nucleic acid aptamer with the highest binding affinity for BD-tau from Aptamer-1, Aptamer-2, and Aptamer-3,
we employed Surface Plasmon Resonance (SPR) technology. Biotin exhibits high-affinity interaction with streptavidin.
Therefore, we injected the biotinylated target protein BD-tau (ligand) into specific flow cells of the SPR sensor chip
pre-coated with streptavidin (SA), enabling its specific and stable capture. When a high-affinity nucleic acid aptamer
approaches, it will bind to BD-tau.

The binding event between the two molecules will lead to an increase in response units (RU). By fitting the sensorgram data
with a 1:1 Langmuir binding model, the dissociation constant (Kd) value can be calculated. The Kd value serves as a
quantitative measure of the binding affinity between BD-tau and the nucleic acid aptamer.

We serially diluted Aptamer-1, Aptamer-2, and Aptamer-3 to graded concentrations (e.g., 0 μM, 5 μM, 10 μM, 30 μM, 100 μM,
150 μM) and sequentially injected them at a constant flow rate over the sensor chip surface immobilized with the target
protein to monitor the binding interactions.

![](https://static.igem.wiki/teams/5739/engineer/9.webp)

Figure 8. Affinity assessed by using surface plasmon resonance

The initial graph illustrates the relationship between aptamer concentration and the energy required to dissociate the
aptamer from its target protein. Five different concentrations of the aptamer were utilized for the SPR analysis.
Subsequently, the second graph re-plotted the data from the Figure 8 with the x-axis representing aptamer concentration, and
a logistic best-fit line was derived.

Table 2. Aptamer Sequences and Dissociation Constants

![图片10](https://static.igem.wiki/teams/5739/engineer/10.webp)

The Table 2 above illustrates the three optimal aptamers. The dissociation constant (Kd) represents the affinity for the
target protein, with this value being determined through the Surface Plasmon Resonance (SPR) method. A lower Kd value
indicates a higher affinity. The midpoint of this graph was identified as the dissociation constant (Kd) value. In our
analysis of various aptamers using SPR, we observed that a lower Kd value corresponds to a higher energy requirement for
dissociation, indicating greater affinity. Notably, the Aptamer-3 exhibited a Kd value of approximately 56.66, signifying
maybe affinity for the BD-tau protein.Based on the Kd values, we selected Aptamer-3 from the three nucleic acid aptamers as
having the highest affinity for BD-tau, and subsequently validated the specificity of Aptamer-3 for BD-tau using ELISA.

**3. Aptamer-3 Verification Assays**

We further validated the binding affinity of aptamer Aptamer-3 using ELISA. To simulate conditions in AD and non-AD
patients, artificial blood was spiked with varying concentrations of BD-tau and peripheral tau, thereby minimizing potential
interference from other factors. Specifically, the biotinylated nucleic acid aptamer Aptamer-3 was immobilized on ELISA
plates, followed by incubation with BD-tau protein and colorimetric detection. The measured absorbance quantitatively
reflected the relative concentration of BD-tau protein.

![图片121](https://static.igem.wiki/teams/5739/engineer/11.webp)

Figure 9. Quantitative binding analysis of BD-Tau aptamer in simulated plasma environments

In the figure 9A, the initial graph illustrates a positive correlation between the increased concentration of our Aptamer-3
and the bound BD-tau levels. The figure 9B demonstrates the results of an ELISA assay conducted to detect Alzheimer\'s
Disease (AD) within a simulated blood plasma environment. The data indicate that the ELISA assay consistently identified
elevated BD-tau levels in the plasma of the AD group, whereas the control group exhibited significantly lower BD-tau levels.
These findings suggest that the aptamer is capable of accurately detecting AD within a plasma environment.

**4. Machine Learning Model Simulation**

Utilizing expression level data from these three proteins (Tau,BD-tauand BD-tau+Tau) by Elisa date, we developed a machine
learning model to predict individual Alzheimer\'s Disease (AD) status. The results demonstrate the feasibility of this
approach. The Support Vector Machine (SVM) model, in particular, demonstrated superior performance, while the Random Forest
(RF) model requires further optimization. Additionally, we employed methods such as Out-of-Fold (OOF) prediction to
thoroughly evaluate the model\'s accuracy and reliability. For detailed modeling procedures, click here: <https://2025.igem.wiki/keystone/model>

**ROC Curve (Receiver Operating Characteristic Curve)**

This chart for evaluating the performance of a binary classification model shows how the model\'s performance changes under
different classification thresholds. The closer the curve is to the top-left corner, the better the model\'s performance.

The x-axis of the chart represents the False Positive Rate (FPR), also known as the fall-out rate, which indicates the
proportion of all actually negative instances that are incorrectly predicted as positive. The y-axis represents the True
Positive Rate (TPR), also known as recall or sensitivity, which indicates the proportion of all actually positive instances
that are correctly predicted as positive.

The AUC (Area Under Curve) is a key metric for measuring a model\'s ability to distinguish between classes. Its value ranges
from 0 to 1; a value closer to 1 indicates better model performance in distinguishing between the positive and negative
classes.

**4.1 Random Forest (RF) Model**

**![rf_cv_report_01](https://static.igem.wiki/teams/5739/engineer/12.webp)**

Figure 10. The ROC curve of Random forest model

The figure 10 indicate that the RF model achieved a mean AUC of 0.789 with a standard deviation of 0.131 under 5-fold
cross-validation, indicating that the model possesses reasonably good overall discriminative ability.

**4.2 Support Vector Machine (SVM) Model**

**![图片33](https://static.igem.wiki/teams/5739/engineer/13.webp)**

Figure 11. The ROC curve of SVM model

The figure 11 indicate that the SVM model achieved a mean AUC of 0.860 with a standard deviation of 0.119 under 5-fold
cross-validation, demonstrating relatively strong overall discriminative ability.

**1.3 Lasso Model**

**![图片34](https://static.igem.wiki/teams/5739/engineer/14.webp)**

Figure 12. The ROC curve of Lasso model

The figure 12 indicate that the Lasso model achieved a mean AUC of 0.844 with a standard deviation of 0.104 under 5-fold
cross-validation, demonstrating good and relatively consistent overall discriminative ability.

**ROC Curve Cross Comparison**

A comparative analysis of the three models reveals a performance hierarchy. The SVM model achieved the highest mean AUC
(0.860), indicating superior overall discriminative ability. It was followed closely by the Lasso model (0.844), which also
demonstrated the lowest standard deviation (0.104), signifying the most stable and reliable performance across
cross-validation folds. While the Random Forest (RF) model possessed reasonably good discriminative ability (AUC 0.789), its
higher standard deviation (0.131) points to greater performance variability, and its mean AUC was lower than the other two
models. In conclusion, the SVM model is the recommended choice for predictive power, though the Lasso model presents a
compelling alternative due to its consistency.

**Learn:**

In Cycle 1, we constructed pET-28a-BD-tau and pET-28a-Tau plasmids and successfully expressed both Tau and BD-tau proteins.
Meanwhile, a nucleic acid aptamer (Aptamer-3) with high affinity for BD-tau was screened via SELEX. Experimental data
further demonstrated that this aptamer can accurately detect BD-tau in a plasma environment.Simultaneously, the data
indicate that the nucleic acid aptamer can accurately detect BD-tau in artificial plasma. Additionally, the feasibility of
Alzheimer\'s disease prediction was assessed through Machine Learning Model Simulation.However, ELISA cannot precisely
quantify BD-tau concentrations, and this method carries the risk of false-positive results, making it difficult to
accurately distinguish Alzheimer\'s disease patients. Therefore, we have integrated CRISPR-Cas12a signal amplification
technology to construct a BD-tau aptamer-based biosensor for detecting BD-tau protein levels in blood samples. The
development of a simple, efficient, low-cost, and stable biosensing platform holds significant implications for Alzheimer\'s
disease detection

',
"children": []
},
{
"title": 'Cycle 2: BD-tau Aptamer-Based Biosensor (pET28a-cas12a)',
"content": '

**Design:**

The “magnetic beads-ComDNA/aptamer-dsDNA” biosensor consists of three components: streptavidin-coated magnetic beads, a
biotin-modified single stranded DNA (ssDNA) partially complementary to the aptamer sequence, and a hybrid strand formed by
the engineered aptamer linked to a Cas12a-activating double stranded DNA (dsDNA) sequence. The streptavidin-coated magnetic
beads have a diameter of 1 μm, with a theoretical loading capacity of 500 pmol of biotinylated single stranded
oligonucleotide (24 nt in length) per mg. The ComDNA is synthesized commercially and shares a complementary sequence of
10-15 bases in length with the engineered aptamer. The complex formed by the complementary pairing of ComDNA and the aptamer
is a commonly used method for constructing an aptamer switch.

In the absence of the analyte, the ComDNA binds to the aptamer, preventing the formation of the aptamer’s secondary
structure. When the analyte is present, the binding affinity between the analyte and the aptamer is stronder than the force
of complementarity between the ComDNA and the aptamer. This displaces the ComDNA, allowing the aptamer to form its secondary
structure. In the constructed magnetic bead-ConDNA/aptamer-dsDNA complex, this is manifested as the analyte binding to the
aptamer, causing the separation of the aptamer from the ComDNA anchored on the magnetic bead(figure 13).

![图片13](https://static.igem.wiki/teams/5739/engineer/15.webp)

Figure 13. The Operating Principle of the Magnetic Bead Biosensor

In this biosensor complex, the performance in responding to the analyte depends on the quality of the aptamer switch, which
is determined by its signal-to-noise ratio (SNR). This ration is defined as the signal intensity (amount of released
aptamer-dsDNA) in the presence of a high concentration of analyte divided by the background signal intensity (amount of
released aptamer-dsDNA) in the absence of the analyte. The SNR of the aptamer switch is influenced by two factors: first the
affinity of the aptamer, which is determined during the selection process and does not require further optimization;
furthermore, second the suitability of the complementary sequence between the ComDNA and the aptamer. In principle, the
ComDNA should bind to the aptamer as tightly as possible in the absence of the analyte(Xu et al., 2023).

The Cas12a reporter system consists of four key components: the Cas12a protein, a crRNA molecule, a single-stranded DNA
(ssDNA) reporter probe labeled with a fluorophore and a quencher at opposite ends, and a double-stranded DNA
(dsDNA)(Gonzalez-Ortiz etal, 2014).In this system, the crRNA forms a complex with the Cas12a protein, guiding it to
specifically recognize and cleave the target dsDNA (cis-cleavage). Upon completion of the targeted dsDNA cleavage, the
Cas12a protein undergoes a conformational change, activating its trans-cleavage activity, which enables non-specific
degradation of any surrounding ssDNA molecules. The nucleotide chain reporter probe, labeled with a fluorophore and a
quencher, is cleaved by activated Cas12a, resulting in the separation of the quencher from the fluorophore(figure 14). Upon
excitation light irradiation, fluorescence is emitted. The fluorescence intensity can be used to quantify the concentration
of BD-tau.

![图片14](https://static.igem.wiki/teams/5739/engineer/16-new.webp)

Figure 14. Schematic Diagram of the Sensor

**Build：**

**1. Construction of Plasmid pET-28a-Cas12a**

Since the primers already contained NheI and HindIII restriction sites, which correspond to the sites within the vector
pET28a. A 1% agarose gel electrophoresis can separate negatively charged nucleic acid fragments of different sizes in the
PCR product under an electric field. As shown in the figure, the target band has a size of 3753 bp, and a bright and wide
band can be clearly identified, indicating that we successfully amplified and separated the target fragment(Figure 16). The
weaker bands surrounding the target band may be due to nonspecific primer binding and abnormal amplification.

![图片3](https://static.igem.wiki/teams/5739/engineer/17.webp)

Figure 15. PCR amplification of Cas12a.

The ligated plasmid was subsequently amplified in cells through chemical transformation and antibiotic selection. After
mixing, heat-shock, ice bath, and recovery, the cells were centrifuged, most of the medium was discarded, and the pellet was
resuspended before plating onto LB agar plates containing kanamycin. The plates were incubated statically at 37 °C for 12
hours, and the results are shown in the figure 16.

![图片4](https://static.igem.wiki/teams/5739/engineer/18.webp)

Figure 16. Chemical transformation results of pET28a-LbCas12a

Next, PCR verification was performed on the colonies on the plate to ensure that the plasmids containing the target band had
successfully entered the cells and replicated. A certain number of single colonies were selected and dissolved in 10 μl of
sterile ddH₂O, and 1 μl of the solution was used for PCR verification. The results are shown in the figure 17.

![图片5](https://static.igem.wiki/teams/5739/engineer/19.webp)

Figure 17. Colony PCR results.

As shown in the figure 17, the target band size is 3753 bp, and a bright band can be clearly observed, indicating that the
desired gene fragment has been successfully transformed into the cells. However, to ensure that no potential mutations
occurred, this clone was sent to a gene service company for plasmid extraction and Sanger sequencing to verify the sequence
accuracy. The results are shown in the figure below.

![图片6](https://static.igem.wiki/teams/5739/engineer/20.webp)

Figure 18. Sequencing validation.

In the figure 18, the target sequence can be clearly detected in its entirety. Within the high-confidence regions (noting
that Sanger sequencing may show lower confidence at the ends, and a single reaction can only cover approximately 1000 bp,
which is why multiple sequences are joined end-to-end in the figure18), the sequencing quality is good, with well-resolved
peaks. No mutations are observed in the target fragment, indicating that the fragment has been successfully integrated into
the plasmid backbone and that the recombinant plasmid has been successfully constructed.

**2. crRNA Construction**

In vitro synthesis of crRNA is typically based on the recognition sequence of T7 RNA polymerase (T7 promoter). By
synthesizing one oligonucleotide containing the T7 promoter sequence and annealing it with another oligonucleotide
complementary to the target sequence, a double-stranded DNA transcription template is obtained, which serves as a template
for crRNA synthesis by T7 polymerase. The template DNA was generated by annealing oligo DNA, and the PCR product was
purified by agarose gel electrophoresis. The results are shown in the figure 19.

![图片8](https://static.igem.wiki/teams/5739/engineer/21.webp)

Figure 19. Oligo DNA validation

As shown in the figure 19, a clear target band appears at 59 bp, while the faint band at the bottom is due to primer dimers
formed by complementary primers. After obtaining the crRNA transcription template DNA, crRNA was synthesized using the
HiScribe T7 Quick High-Yield RNA Synthesis Kit. The resulting RNA was quantified, with a concentration of 60 ng/μl.

**Test：**

**1. Protein expression**

In the T7 expression system (pET28a), the target gene is placed under the control of a T7 promoter, and the host (BL21(DE3))
contains T7 RNA polymerase (or a T7 RNAP that can be induced by IPTG). Upon addition of the inducer (IPTG), expression of T7
RNAP is induced, which in turn drives high-level expression of the target protein in the cells. The resulting plasmid was
introduced into E. coli BL21(DE3) via chemical transformation, and the colony plates are shown in the figure 20.

![图片4](https://static.igem.wiki/teams/5739/engineer/22.webp)

Figure 20. Verification of BL21(DE3) chemical transformation results

Single colonies from the figure above were expanded in 600 ml of culture and induced with IPTG for overnight expression at
16°C. The cells were collected, washed, lysed, and centrifuged for subsequent purification. Ni²⁺ and other metal ions
exhibit high specificity and chelating ability toward His-tagged fusion proteins, allowing the fusion proteins to be
captured on a designated chromatographic medium while untagged proteins are washed away. Using this principle, the protein
was purified, and the purified fractions were analyzed by SDS-PAGE. The results are shown in the figure.

![图片7](https://static.igem.wiki/teams/5739/engineer/23.webp)

Figure 21. SDS-PAGE of Cas12a

The figure clearly displays the bands corresponding to the crude protein, wash fractions, and purified protein. Lanes E1–E5
represent the purified protein, showing a strong band at the target position (approximately 120 kDa) with very high
concentration and minimal contaminating proteins, indicating high efficiency of protein expression and purification. This
observation is further supported by the bands in the crude protein sample. In contrast, the wash fractions contain mostly
contaminating proteins and only trace amounts of the target protein.

**2. Characterization and optimization of biosensor performance**

**2.1 Screening ComDNA for the highest signal-to-noise ratio**

We constructed the biosensor using Aptamer-3. And we were constructed using five different sources of ComDNA and incubated
with BD-Tau at the same concentration. Following the experimental procedure, the released protein–aptamer-3–dsDNA complexes
were collected, and the dsDNA content was quantified by qPCR. The sample with the highest dsDNA content was identified,
thereby confirming the ComDNA with the highest signal-to-noise ratio. The results are shown in the figure 22.

![Melt Curve Plot-1](https://static.igem.wiki/teams/5739/engineer/24.webp)

Figure 22. qPCR verification of the ComDNA with the highest signal-to-noise ratio

As shown in figure 22, only ComDNA3 exhibited a relatively high signal-to-noise ratio, while the other samples showed no
significant signal in the test.

**2.2 Fluorescence intensity detection of BD-Tau protein at different concentrations**

An aptamer-3-based biosensor was constructed using ComDNA3, and we further incorporated the Cas12a cleavage module, enabling
the sensor to convert aptamer recognition events into amplified fluorescent signals, thereby establishing a complete
detection model. To evaluate its feasibility in practical applications, we designed a concentration gradient experiment
based on the physiological concentration range of BD-Tau protein in Alzheimer’s disease patients. Samples at different
concentrations were then added to the sensing system, and the changes in fluorescence signals were monitored in real time
using a microplate reader.

![图片13](https://static.igem.wiki/teams/5739/engineer/25.webp)

Figure 23. Fluorescence intensity detection of BD-Tau protein at different concentrations

The differences in signal between concentrations were not pronounced, and the sensitivity remains limited. This observation
suggests that there is still room for improvement in the detection performance of the current system, potentially due to
insufficient aptamer binding affinity or low efficiency of complex release.

**Learn：**

In cycle 2, the figure 23 showed that the differences in signal between concentrations were not pronounced. The potential
reasons for the experimental failure may primarily include the following two aspects:

1. The CRISPR-based BD-tau sensor requires high aptamer affinity, and the suboptimal performance is primarily attributed to
   the low binding affinity of the current Aptamer-3 toward BD-tau.
2. The underlying principle of BD-tau aptamer-based biosensor may proves unsuitable.

To investigate the causes of the failure, we implemented the following optimizations:

To investigate the low binding affinity of the nucleic acid aptamers, we consulted Professor Lu and subsequently optimized
the SELEX screening procedure and conditions. Specifically, we employed BD-tau for positive selection while simultaneously
introducing Tau for counter-selection, thereby eliminating nucleic acid aptamers that exhibit affinity for Tau.

To validate the feasibility of the BD-tau aptamer-based biosensor principle, we consulted Dr. Wang for professional advice.
Ultimately, we decided to utilize nucleic acid aptamers with high affinity for Tau protein by an earlier iGEM team, in order
to verify the functionality of the biosensor.

',
"children": []
},
{
"title": 'Cycle 3: Tau Protein Aptamer-Based Biosensor',
"content": '

**Design and Build**

The design and construction of Cas12a and crRNA are detailed in Cycle 2.

**Test**

**1. Aptamer Non-Interference Validation**

To validate the feasibility of the biosensor, we selected a Tau protein aptamer sequence (target: 2N4R-Tau) that was
previously characterized. To verify whether aptamer binding affects the spatial conformation of dsDNA and thereby influences
subsequent signal transduction efficiency, we designed a comparative experiment. In brief, one system contained only the
naked dsDNA fragment as a control, to test the activation capacity of Cas12a in the absence of aptamer interference. The
other system represented the complete sensor setup, including the aptamer–dsDNA complex and T-Tau protein, to evaluate
whether signal transduction remains effective when the aptamer participates and binds to the target protein. The results,
quantified by fluorescence microplate reader, are shown figure 24.

![图片15](https://static.igem.wiki/teams/5739/engineer/26.webp)

Figure 24. Validation of whether aptamer binding affects dsDNA-mediated Cas12a activation (T-Tau)

The figure 24 showed that when 30 μM tau protein was added to the system, the sensor’s response reached, or even slightly
exceeded, the signal level observed with naked dsDNA alone. This indicates that the binding of the aptamer to tau protein
did not negatively affect the conformation of dsDNA or its ability to activate Cas12a.

**2. Aptamer-Based Biosensor Optimization**

**2.1 Screening for ComDNA with Optimal Signal-to-Noise Ratio**

To further evaluate the binding efficacy of ComDNA to the Tau aptamer-dsDNA complex and ensure efficient dissociation of
ComDNA upon Tau protein. We designed five ComDNA variants (ComDNA1–ComDNA5) for systematic testing. Following incubation
with an excess of Tau protein, the released Tau aptamer-dsDNA complex was quantified using quantitative PCR (qPCR). A
control group, in which the Tau protein was replaced with an equivalent volume of water, was included to calculate the
signal-to-noise ratio. This allowed for the screening of the ComDNA sequence with the highest signal-to-noise ratio.

![](https://static.igem.wiki/teams/5739/engineer/27.webp)

Figure 25. Signal-to-noise ratio of ComDNA verified with T-Tau (Normalization Gene:actin )

The figure25 clearly indicate that under the positive control condition with T-Tau, the sensor generated a significant
fluorescent response, confirming the overall feasibility of the system. Additionally, in the comparative experiments among
different ComDNAs, ComDNA3 outperformed the other sequences, exhibiting a higher signal-to-noise ratio and a more stable
response.We have selected ComDNA3 as the linker to construct the biosensor.

**2.2 Determination of Tau Aptamer-dsDNA Complex Concentration**

Following the screening of the aptamer switch ComDNA3, it is necessary to determine the optimal concentration of the Tau
aptamer-dsDNA complex. Different concentrations of the Tau aptamer-dsDNA complex were used to construct the "magnetic
bead-biotinylated ComDNA-Tau aptamer-dsDNA" complex. The signal-to-noise ratios at different Tau aptamer-dsDNA
concentrations were evaluated to identify the optimal concentration.

![](https://static.igem.wiki/teams/5739/engineer/28.webp)

Figure 26. Determination of the concentration of the T-Tau aptamer–dsDNA complex

Figure 26 shows that the signal intensity increases with rising nucleic acid aptamer concentration, reaching a plateau
between 10 μM and 20 μM. The system demonstrated maximum response at 10 μM. and 20μM .Therefore, 10 μM is likely the optimal
concentration for the aptamer–dsDNA complex.

**2.3 Determination of Incubation Time for BD-tau with the Biosensor**

To determine the optimal incubation time between the target protein and the sensor for practical applications, we designed
and conducted a corresponding experiment. Under the same protein concentration, samples were incubated for different
durations, and the fluorescence signals at each time point were collected and measured to compare the trend of signal
intensity over time.

![](https://static.igem.wiki/teams/5739/engineer/29.webp)

Figure 27. Determination of the incubation time between T-Tau and the aptamer–dsDNA complex

The figure 27 showed that the fluorescence intensity reached its maximum after 40 minutes of incubation, and further
extension of the reaction time did not result in any significant increase in signal. This indicates that at 40 minutes, the
binding between the target protein and the sensor had essentially reached saturation, and the system had completed effective
signal transduction. Therefore, 40 minutes was determined to be the optimal incubation time for this sensor in practical
applications.

**3. Detection of Tau Protein Using an Aptamer-Based Biosensor**

**3.1 Protocol for Standard Curve Preparation**

Following the optimization of the above conditions—with ComDNA3 selected as the linker, the Tau aptamer–dsDNA complex
concentration set at 10 μM, and the incubation time determined as 40 minutes—we proceeded with the detection of Tau protein.

Based on the designed sensor structure, sensor units were constructed using T-Tau and its corresponding Tau-aptamer.
Gradient concentrations of T-Tau were added to generate a standard curve. The results are shown in the figure 28.

![图片14](https://static.igem.wiki/teams/5739/engineer/30.webp)

Figure 28. Standard curve of T-Tau

As shown in the figure 28, the fluorescence intensity exhibited a clear linear relationship with the target protein
concentration. This result indicates that the release of the aptamer–dsDNA and the subsequent activation of Cas12a
trans-cleavage are quantitatively dependent on the amount of protein present in the system. The observed linearity not only
demonstrates the sensitivity of the sensing module but also validates its reliability for quantitative detection. Such
linear correlation strongly supports that the system can be calibrated using a standard curve, thereby enabling accurate
concentration measurements in practical applications. These findings not only confirm the applicability of the sensor but
also highlight its potential for further optimization in real biological samples.

**3.2 Biosensor Specificity Evaluation**

Next, we evaluated the recognition specificity of this module, including T-Tau, bovine serum albumin (BSA), human serum
albumin (HSA), IgE, and IgG. The results are shown figure 29.

![图片14](https://static.igem.wiki/teams/5739/engineer/30-1-1.webp)

Figure 29. Protein specificity detection of T-Tau

The results showed that this module exhibited a significant response to T-Tau, while no detectable response was observed for
common interfering proteins in blood. This finding highlights the excellent specificity of the system, indicating that the
sensor can effectively distinguish the target protein from background proteins

**Learn**

We have constructed a nucleic acid aptamer-based biosensor for Tau protein, which enables specific recognition of Tau. The
aforementioned results demonstrate the theoretical feasibility of our aptamer-based biosensor. The observed low sensitivity
in detecting BD-tau protein may be attributed to insufficient binding affinity of the nucleic acid aptamer toward BD-tau.
Therefore, we optimized the SELEX screening procedure and conditions by employing BD-tau for positive selection while
simultaneously introducing Tau for counter-selection, thereby eliminating nucleic acid aptamers with affinity for Tau. We
subsequently performed a second round of SELEX screening to isolate nucleic acid aptamers specific for BD-tau.

',
"children": []
},
{
"title": 'Cycle 4: Screening for Aptamers SELEX',
"content": '

Design and Build

The design and construction of proteins BD-tau and Tau are described in Cycle 1.

Test

**1. SELEX Screen**

**1.1 RFU characterizes the SELEX process**

This is our flow symmetry result on the product of different round of SELEX process. RFU means the intensity of fluorescent
signal that released by RAM we placed on F primer in PCR. As depicted in the Figure 30, the RFU increased progressively from
round 1 to round 6, reflecting a gradual enrichment of aptamers with binding affinity for BD-tau. After round 6, we
initiated reverse screening to eliminate non-specific binders, resulting in a noticeable decline in RFU. By round 12,
following additional rounds of stringent selection, the RFU decreased substantially, indicating that only a small pool of
aptamers with the highest affinity remained.

![2-流式](https://static.igem.wiki/teams/5739/engineer/32.webp)

Figure 30. Flow symmetry concerning the product of various rounds of the SELEX process

**1.2 Screening for Aptamers Molecular Docking**

We employed 27 DNA sequences obtained from the SELEX system to predict their interactions with Tau and BD-Tau proteins
through molecular docking, aiming to screen nucleic acid aptamers with higher binding affinity. The PDB files of Tau and
BD-Tau proteins were acquired from AlphaFold. Both files were submitted to the HADDOCK 2.4 platform
(https://rascar.science.uu.nl/haddock2.4), which facilitates complex molecular docking and interaction analysis for
researchers. For detailed modeling procedures, click here: <https://2025.igem.wiki/keystone/model>

Table 3. Nucleic Acid Aptamer Sequences and Z-Scores

|  |  |  |  |
| --- | --- | --- | --- |
| Number | **Aptamer Sequences** | **T-tau Z-Score** | **BD-Tau Z-Score** |
| 1 | 5\' -TCACCTGAGACTTGACGATGGCATCACTCCCCCCCACCTATTACATCATCATAAATTGAGTGCTATCGTCTGTCCA - 3\' | **-1.1** | **-1.3** |
| **2** | 5\' - TCACCTGAGACTTGACGATGGCCTCCCCCTCACGCACTCTTCCGTTTCTTCTTATCTGAGTGCTATCGTCTGTCCA - 3\' | **-0.9** | **-1.2** |
| **3** | 5\' - TCACCTGAGACTTGACGATGGAACTCCCCCCACCATTATCAGCGCACCACCATTGTAGAGTGCTATCGTCTGTCCA - 3\' | **-1.2** | **-1.5** |
| **4** | 5\' - TCACCTGAGACTTGACGATGGTTTAACTCCCCCACGCCCCCCGCCAACCCATCTCCAGAGTGCTATCGTCTGTCCA - 3\' | **-2.5** | **-1.6** |
| **5** | 5\' - TCACCTGAGACTTGACGATGGTACGACGGCCCCCCGATTATGCGACTACTTGATTTGAGTGCTATCGTCTGTCCA - 3\' | **-1.5** | **-1.7** |
| **6** | 5\' - TCACCTGAGACTTGACGATGGTCAGAACGACGCGCCCCCCACCTCATTCATTATTTTGAGTGCTATCGTCTGTCCA - 3\' | **-1.7** | **-2.1** |
| **7** | 5\' - TCACCTGAGACTTGACGATGGTGACCACCCCCCACGCACACACACCTCTTCCATCCTGAGTGCTATCGTCTGTCCA - 3\' | **-1.7** | **-2.0** |
| ****8**** | **5\' - TCACCTGAGACTTGACGATGGCACTACCCCTCCCTACTAAGCACGGTATCTTGTACTGAGTGCTATCGTCTGTCCA - 3\'** | ****-1.2**** | ****-1.8**** |
| **9** | 5\' - TCACCTGAGACTTGACGATGGGAACAAACACCGCGACCACCCCCCCACTTAACTCCTGAGTGCTATCGTCTGTCCA - 3\' | **-2.1** | **-1.4** |
| **10** | 5\' - TCACCTGAGACTTGACGATGGCAATCCCCCCGACACCGAATCCTAAGCGAACAACGCGAGTGCTATCGTCTGTCCA - 3\' | **-1.4** | **-1.2** |
| **11** | 5\'- TCACCTGAGACTTGACGATGGACTCACAAACTCGAGCCACCCCCGACCCACACAACAGAGTGCTATCGTCTGTCCA - 3\' | **-1.7** | **-1.8** |
| **12** | 5\'- TCACCTGAGACTTGACGATGGTACTCCCCCCCAACCTAATAGCTCTTTACCCTCTGAGAGTGCTATCGTCTGTCCA - 3\' | **-1.5** | **-1.8** |
| **13** | 5\' - TCACCTGAGACTTGACGATGGCCGACTCCCCACCCTACATCGCAACATTGACTATTAGAGTGCTATCGTCTGTCCA - 3\' | **-1.7** | **-1.3** |
| ****14**** | **5\' - TCACCTGAGACTTGACGATGGTTCTACACTGCCCCCCCGACCCGCCAGACCAACCCAGAGTGCTATCGTCTGTCCA - 3\'** | ****-1.0**** | ****-2.1**** |
| **15** | 5\' - TCACCTGAGACTTGACGATGGCAATCCTCCGAGCTCCACCCACCCTTACTCAACATTGAGTGCTATCGTCTGTCCA - 3\' | **-2.6** | **-2.2** |
| ****16**** | **5\' - TCACCTGAGACTTGACGATGGCGCTACCCCCTAACTTCAACCCGCATTATTCTAGCTGAGTGCTATCGTCTGTCCA - 3** | ****-1.8**** | ****-2.3**** |
| **17** | 5\' - TCACCTGAGACTTGACGATGGTTACCGAACCCGACACCCCCGCCGACACCAGCCCCAGAGTGCTATCGTCTGTCCA - 3\' | **-2.2** | **-1.5** |
| **18** | 5\' - TCACCTGAGACTTGACGATGGCCCCCCCCCGCACCGCCTCATTCAGCATACTAATACGAGTGCTATCGTCTGTCCA - 3\' | **-1.9** | **-1.2** |
| **19** | 5\' - TCACCTGAGACTTGACGATGGCAACCACCCCCCCTGGCTACATCATATTCTTATCTTGAGTGCTATCGTCTGTCCA - 3\' | **-2.1** | **-1.3** |
| **20** | 5\' - TCACCTGAGACTTGACGATGGTTTCTTCGCCCCCCCCACACACTACACGTTTCTTCTGAGTGCTATCGTCTGTCCA - 3\' | **-2.2** | **-2.1** |
| **21** | 5\' - TGACTGATTTACGGAAGCTGAATAAGGACTGCTTAGGATTGCGATGATTCAGCT - 3\' | **-2.6** | **-1.5** |
| **22** | 5\' - TGACTGATTTACGGAAGTTACGGACGGATGTCAGTGGTATAGTAATCCGTAACT - 3\' | **-1.7** | **-1.5** |
| **23** | 5\' - TGACTGATTTACGGAAGCTGAATAAGGACTGCTTAGGATTGCGATGATTCAGCT - 3\' | **-1.4** | **-1.6** |
| **24** | 5\' - CGTAAATCAGTCAGAAGCTGAATAAGGACTGCTTAGGATTGCGATGATTCAGCT - 3\' | **-1.4** | **-1.2** |
| **25** | 5\' - GCGGAGCGTGGCAGG - 3\' | **-2.3** | **-1.4** |
| **26** | 5\' - CCTGCCACGCTCCGC - 3\' | **-2.3** | **-1.6** |
| **27** | 5\' - CTGAATCATCGCAATCCTAAGCAGTCCTTATTCAGAAAAAAAAAAAAAAAA - 3\' | **-2.1** | **-2.0** |

Based on the molecular docking results of all 27 DNA sequences listed in Table 2, the binding strength between the DNA
sequences and the target proteins was evaluated using Z-scores. A lower Z-score indicates a higher binding potential. We
specifically prioritized nucleic acid aptamers exhibiting strong binding affinity to BD-Tau while showing weak affinity to
Tau protein. This screening process identified three DNA sequences—Aptame-08, Aptame-14 and Aptame-16—with promising
docking results.

**1.3 Surface Plasmon Resonance (SPR) for Binding Affinity Characterization**

**![图片31](https://static.igem.wiki/teams/5739/engineer/33.webp)**

Figure 31. Affinity assessed by using surface plasmon resonance

**![图片32](https://static.igem.wiki/teams/5739/engineer/34.webp)**

Figure 32. Affinity assessed by using surface plasmon resonance

**![图片33](https://static.igem.wiki/teams/5739/engineer/35.webp)**

Figure 33. Affinity assessed by using surface plasmon resonance

To identify the nucleic acid aptamer with the highest binding affinity for BD-tau from Aptamer-08, Aptamer-14, and
Aptamer-16, we employed Surface Plasmon Resonance (SPR) technology. After we have our aptamer, we tested its affinity by
SPR. the first graph shows how the concentration of aptamer affect the amount of energy needed to separate our aptamer and
its target protein. It selected 5 different concentration of aptamer to do the SPR. The second graph then graphed out the
first graph with x-axis changed to the concentration of aptamer and derived a logistic best fit line. Then, we get the mid
point of the graph, thats our Kd value. As we tested other aptamers using the SPR, the lower the Kd value is, the higher
energy will be needed to seperate aptamer and target protein, which means higher affinity.

As shown in the figure 31，32 and 33, the Kd values for Aptamer-08, Aptamer-14, and Aptamer-16 are 11.32, 6.38, and 21.38,
respectively. Among them, Aptamer-14 demonstrates the lowest Kd value compared to Aptamer-08 and Aptamer-16, indicating it
possesses the highest binding affinity for BD-tau. Based on the Kd values, we selected Aptamer-14 as the nucleic acid
aptamer with the strongest affinity for BD-tau from the three candidates, and subsequently validated its specificity for
BD-tau using ELISA.

**2. Aptamer-14 Verification Assays**

We further validated the binding affinity of Aptamer-14 using ELISA. To simulate conditions in AD and non-AD patients,
artificial blood was spiked with varying concentrations of BD-tau and peripheral tau, thereby minimizing potential
interference from other factors. Specifically, the biotinylated nucleic acid aptamer Aptamer-14 was immobilized on ELISA
plates, followed by incubation with BD-tau protein and colorimetric detection. The measured absorbance quantitatively
reflected the relative concentration of BD-tau protein.

![Aptamer-14-Elisa](https://static.igem.wiki/teams/5739/engineer/36.webp)

Figure 34. Quantitative binding analysis of BD-Tau aptamer in simulated plasma environments

In Figure 34 above, the results demonstrate a correlative trend in the fluorescence concentration levels of BD-tau. It can be observed that in the artificially simulated plasma samples from the Alzheimer\'s disease (AD) group, all samples classified and tested via ELISA exhibited elevated BD-tau levels. This indicates that the aptamer remains capable of accurately detecting Alzheimer\'s disease in a plasma environment.

****Learn：****

In Cycle 4, the nucleic acid aptamers obtained from the second round of SELEX screening demonstrated significantly
enhanced binding affinity compared to those from the initial screening, validating the effectiveness of our SELEX
platform. This confirms that the SELEX technology can be widely applied to screen nucleic acid aptamers for other target
proteins. Furthermore, the results indicate that Aptamer-14 exhibits high-affinity interaction with BD-tau, making it
suitable for constructing a biosensor to detect BD-tau.

',
"children": []
},
{
"title": 'Cycle 5: BD-tau Aptamer14-Based Biosensor',
"content": '

**Design and Build**

The design and construction of Cas12a and crRNA are detailed in Cycle 2.

**Test：**

**1. Aptamer-14 Non-Interference Validation**

We formed a complex using Aptamer-14 and dsDNA to further validate whether the structure of the nucleic acid aptamer affects
Cas12a cleavage efficiency. Using 10 μM dsDNA as a positive control, Figure 35 demonstrates that the relative fluorescence
intensity increases with rising dsDNA concentration. The signal observed at 10 μM dsDNA was higher than that of the control
group, demonstrating that the incorporation of the aptamer does not compromise Cas12a-mediated cleavage efficiency.

**![dsDNA](https://static.igem.wiki/teams/5739/engineer/37.webp)**

Figure 35. Validation of whether aptamer binding affects dsDNA-mediated Cas12a activation (BD-Tau)

**2. Aptamer 14-Based Biosensor Optimization**

**2.1 Screening for ComDNA with Optimal Signal-to-Noise Ratio**

**Melt Curve**

This panel shows the raw melt curves for the different BD-Tau aptamer–ComDNA duplexes. On the x-axis is temperature (°C) and
on the y-axis is fluorescence (RFU). As the temperature increases, the double-stranded regions of each dsDNA gradually
denature into single strands, causing the dye that intercalates into double-stranded DNA to be released and the fluorescence
to decrease. The downward slopes and inflection points indicate the melting transition of each duplex. Curves with a
sharper, more defined drop represent more uniform and stable duplexes, while broader transitions suggest heterogeneous
structures or weaker binding.

**Melt Peak**

This plot displays the derivative of the melt curve (–dRFU/dT vs. temperature), which highlights the temperature at which
the most rapid loss of fluorescence occurs—effectively the melting temperature (Tm) peak. Each distinct peak corresponds to
a specific duplex population. A higher, sharper peak at a specific temperature indicates a stronger and more uniform duplex,
while multiple or broad peaks indicate heterogeneity. Comparing the Tm values across ComDNA candidates lets you identify
which constructs form the most stable and predictable complexes with the aptamer.

**Amplification Curves**

This panel shows real-time amplification curves (RFU vs. cycle number) for reactions containing the T-Tau aptamer–ComDNA
complexes. Each curve represents fluorescence accumulation as the reaction progresses. Early, steep rises indicate rapid
signal generation and efficient Cas12a activation, while late or shallow curves reflect slower or weaker responses. By
comparing the amplification profiles of different ComDNA candidates, you can gauge both sensitivity and kinetics—ideally you
want a construct that shows an early threshold cycle and high end-point fluorescence.

![Com1-5](https://static.igem.wiki/teams/5739/engineer/38.webp)

Figure 36. Signal-to-noise ratio of ComDNA verified with BD-Tau (Normalization Gene:actin )

The figure 36 clearly indicate that under the positive control condition with BD-Tau, the sensor generated a significant
fluorescent response, confirming the overall feasibility of the system.The relative fluorescence value of ComDNA3 was
significantly higher than that of other groups, indicating that ComDNA3 achieves the highest signal-to-noise ratio. These
experimental results are consistent with the findings presented in Cycle 3, confirming the selection of ComDNA3 for
biosensor fabrication.

**2.2 Determination of Tau Aptamer 14-dsDNA Complex Concentration**

![浓度](https://static.igem.wiki/teams/5739/engineer/39.webp)

Figure 37. Signal-to-noise ratio of ComDNA verified with BD-Tau (Normalization Gene:actin )

Figure 37 shows that the signal intensity increases with rising nucleic acid aptamer concentration. The system demonstrated
maximum response at 10 μM .

**2.3 Determination of Incubation Time for BD-tau with the Biosensor**

**![时间](https://static.igem.wiki/teams/5739/engineer/40.webp)**

Figure 38. Signal-to-noise ratio of ComDNA verified with BD-Tau (Normalization Gene:actin )

The figure 38 showed that the fluorescence intensity reached its maximum after 40 minutes of incubation, and further
extension of the reaction time did not result in any significant increase in signal. This indicates that at 40 minutes, the
binding between the target protein and the sensor had essentially reached saturation, and the system had completed effective
signal transduction. Therefore, 40 minutes was determined to be the optimal incubation time for this sensor in practical
applications.

**3.Detection of Tau Protein Using an Aptamer 14-Based Biosensor**

**3.1 Standard Curve**

Based on the optimized conditions described above, the biosensor has constructed using Aptamer-14. This linearity
means that within this concentration range the assay behaves quantitatively: every increase in protein concentration
produces a proportional increase in fluorescence. The slope of the line represents assay sensitivity; the steeper the
slope, the greater the response per unit of protein. The intercept near zero indicates very low background, suggesting
minimal non-specific activation of Cas12a. With this curve, unknown samples can be interpolated accurately by matching
their fluorescence to the line. Replicates plotted at each concentration also demonstrate reproducibility, with small
variation between points. Together, these data confirm that the BD-Tau aptamer 14–dsDNA–Cas12a system can be used not
just for detection but for quantitative measurement of BD-Tau protein over a defined range.

![标准曲线](https://static.igem.wiki/teams/5739/engineer/41.webp)

Figure 39. The Standard Curve of BD-tau

**3.2 Biosensor Specificity Evaluation**

![特异性](https://static.igem.wiki/teams/5739/engineer/42.webp)

Figure 40. Specificity Testing of BD-tau Protein

This figure compares relative fluorescence signals generated by the system when exposed to BD-Tau and several unrelated
proteins (bovine serum albumin, human serum albumin, immunoglobulin E, immunoglobulin G). Only the bar corresponding to
BD-Tau shows a strong signal; all other proteins produce minimal responses near baseline. This demonstrates that the
aptamer–dsDNA complex specifically recognizes BD-Tau and does not significantly bind or activate Cas12a in the presence of
abundant off-target proteins. The low background from serum albumins and immunoglobulins indicates the assay is likely to
remain specific even in complex biological samples such as blood or cerebrospinal fluid. The error bars are small,
indicating good reproducibility of these specificity measurements. These results validate the selectivity of the BD-Tau
detection system and support its potential application as a reliable biomarker assay.

**4. Microfluidic Detection of Alzheimer\'s Disease**

We attached platinum nanoparticles (PtNPs) to the 3\' end of the nucleic acid aptamer, while conjugating the aptamer to
complementary DNA (ComDNA). The other end of ComDNA was immobilized to streptavidin-coated magnetic beads via biotin linkage
(Li et al., 2016; Song et al., 2011). In the presence of BD-tau, the activated CRISPR-Cas12a system cleaves the substrate,
and the resulting supernatant is transferred to a hydrogen peroxide (H₂O₂) chamber. The PtNPs catalyze the decomposition of
H₂O₂ into water and oxygen, and the released oxygen propels the red-dyed silicone oil to move forward. The distance of
movement correlates with BD-tau concentration—higher levels result in greater displacement of the indicator (Figure 41).

Chip Architecture Design:

Sample Injection Zone: Artificial blood samples for loading and testing

Reaction Microchamber: Pre-loaded with H₂O₂ solution as catalytic reaction substrate

Signal Detection Zone: Filled with red-dyed silicone oil as visual flow indicator

This innovation provides crucial technical support for developing a new generation of portable rapid AD diagnostic devices,
effectively addressing the limitations of traditional laboratory methods such as complex procedures, long processing times,
and dependence on large-scale instruments.

**![图片123](https://static.igem.wiki/teams/5739/engineer/43.webp)**

Figure 41. Microfluidic Sensor Principle

We also developed a lab prototype for our TauTrack microfluidics diagnostic platform and tested the its functionality accordingly. Please note that due to issues of time and material (especially the difficulty and time-consuming nature of producing integrated platinum nanoprobes, we chose an alternative using aptamer–comDNA–PtNP. By doing so we can design and experiment with all core functions of the TauTrack “samplein, resultout” microﬂuidics platform comprised “onchip no human interference” without changing the chip device process.

How to Reconstitute？

A BD‐Tau aptamer is used to bind the PtNP.

The Aptamer contains a short "dock" sequence that will hybridize with a complementary DNA (comDNA).

Streptavidin‐coated magnetic beads are used to capture the biotinylated comDNA.
PtNPs are initially fixed to the beads through the aptamer‐comDNA duplex as a complete sensor.

How It Fits the Workflow on‐chip in Three Stages

Stage 1: Target identification and release

BD‐Tau in the sample will bind the aptamer to destabilize the duplex (assisted by a designed toehold) and displace the comDNA.

Results: The PtNP‐aptamer is released into solution and the comDNA remains on the bead. The level of released PtNPs increases as the BD‐Tau concentration rises.

Stage 2: Magnetic separation

A magnet placed externally immobilizes the beads (together with any PtNPs which are still tethered).

Only the supernatant containing truly released PtNPs can pass through to the next chamber.

Stage 3: Catalytic ampliﬁcation and readout

The released PtNPs cause decomposition of H2O2 to generate oxygen in a closed chamber.

The pressure which is produced pushes a colored ink slug; the distance it moves reﬂects the level of BD‐Tau.

**Representative Results Showcase:**

We have preliminarily validated the feasibility of using microfluidic technology for AD detection with Aptamer-14. The
figure shows that Line 4 and Line 5 exhibit slightly higher signals than Line 1, Line 2, and Line 3, demonstrating the
operational functionality of our microfluidic chip. However, further optimization is required in practical operation.

**![图片111](https://static.igem.wiki/teams/5739/engineer/44.webp)**

Figure 42. Prototype of TauTrack Microfluidics Diagnostic Platform of BD-tau

**Learn：**

Further screening of aptamers with higher affinity for BD-tau can be conducted to construct aptamer-based biosensors, while
additionally validating their sensitivity. The nucleic acid aptamer platform for BD-tau developed by our team can provide
technical support to other research groups, while also offering a sensitive and efficient method for the detection of
Alzheimer’s disease.

Our machine learning model requires further optimization, which can be achieved by incorporating larger datasets and adding
evaluation metrics to enhance prediction accuracy and reliability.

For our next steps, we can optimize the product by leveraging microfluidic technology to enable Alzheimer\'s disease
detection.

',
"children": []
},
{
"title": 'References',
"content": '

Li Y , Xuan J , Song Y ,et al.Nanoporous Glass Integrated in Volumetric Bar-Chart Chip for Point-of-Care Diagnostics of
Non-Small Cell Lung Cancer[J].Acs Nano, 2016:1640-1647.DOI:10.1021/acsnano.5b07357.

Gonzalez-Ortiz, F., Kirsebom, B. E., Contador, J., Tanley, J. E., Selnes, P., Gísladóttir, B., Pålhaugen, L., Suhr
Hemminghyth, M., Jarholm, J., Skogseth, R., Bråthen, G., Grøndtvedt, G., Bjørnerud, A., Tecelao, S., Waterloo, K., Aarsland,
D., Fernández-Lebrero, A., García-Escobar, G., Navalpotro-Gómez, I., … Blennow, K. (2024). Plasma brain-derived tau is an
amyloid-associated neurodegeneration biomarker in Alzheimer\'s disease. *Nature Communications, 15*(1),
2908. <https://doi.org/10.1038/s41467-024-47286-5>

Gonzalez-Ortiz, F., Turton, M., Kac, P. R., Smirnov, D., Premi, E., Ghidoni, R., Benussi, L., Cantoni, V., Saraceno, C.,
Rivolta, J., Ashton, N. J., Borroni, B., Galasko, D., Harrison, P., Zetterberg, H., Blennow, K., & Karikari, T. K.
(2023). Brain-derived tau: A novel blood-based biomarker for Alzheimer’s disease-type neurodegeneration. *Brain, 146*(3), 1152–1165. <https://doi.org/10.1093/brain/awac407>

Guo, T., Noble, W., & Hanger, D. P. (2017). Roles of tau protein in health and disease. *Acta Neuropathologica, 133*(5), 665–704. <https://doi.org/10.1007/s00401-017-1707-9>

Kyalu Ngoie Zola, N., Balty, C., Pyr Dit Ruys, S., Vanparys, A. A. T., Huyghe, N. D. G., Herinckx, G., Johanns, M., Boyer,
E., Kienlen-Campard, P., Rider, M. H., Vertommen, D., & Hanseeuw, B. J. (2023). Specific post-translational
modifications of soluble tau protein distinguishes Alzheimer\'s disease and primary tauopathies. *Nature Communications, 14*(1), 3706. <https://doi.org/10.1038/s41467-023-39328-1>

Scarano, S., Mariani, S., & Minunni, M. (2015). SPR-based affinity biosensors as innovative analytical devices. *Journal of Lightwave Technology, 33*(16), 3374–3384. <https://doi.org/10.1109/JLT.2015.2442997>

Song Y , Zhang Y , Bernard P E ,et al.Multiplexed volumetric bar-chart chip for point-of-care diagnostics[J].Nature
Communications, 2011, 3:1283.DOI:10.1038/ncomms2292.

Teng, I. T., Li, X., Yadikar, H. A., Yang, Z., Li, L., Lyu, Y., Pan, X., Wang, K. K., & Tan, W. (2018). Identification
and characterization of DNA aptamers specific for phosphorylation epitopes of tau protein. Journal of the American
Chemical Society, 140(43), 14314–14323. <https://doi.org/10.1021/jacs.8b08645>

Zuo, M. (2014). Selection of nucleic acid aptamers for prealbumin based on SELEX technology and application of an
aptamer-based magnetic beads sensor for detection of bacterial endotoxins [Doctoral dissertation, University of Science
and Technology of China].

',
"children": []
},
] %}
{% set info\_title = "Engineering" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Engineering{% endblock %}

{% extends "layout.html" %}
{% set info\_data = [
{
"title": 'Cycle 1: SELEX System',
"content": '',
"children": [
{
"title": '1. Plasmid Construction',
"content": '

****1.1** LB Preparation**

 Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| Agar powder | 3g | Gelling agent |
| Yeast extract | 5g | Sugar/starch source |
| Trypton | 10g | Protein & nitrogen source |
| NaCl | 10g | Adjusts salinity |
| Kanamycin | 300μL | Used for verifying plasmid transfer success later |

 Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Measuring cylinder | 1 | Volume 1000mL |
| Erlenmeyer flask | 1 | Volume 1000mL |
| Scale | 1 | Precision of 1mg |
| Spatula | 1 |  |
| Petri dish | 20 |  |
| Disinfecting pot | 1 |  |

LB Preparation:

1. Measure 700mL of ddH2O using the measuring cylinder
2. Weigh 7 g of tryptone, 3.5 g of yeast extract and 7 g of NaCl using the scale & spatula.
3. Add 7 g of tryptone, 3.5 g of yeast extract and 7 g of NaCl to ddH2O. This is the liquid LB broth.
4. Gel Preparation:
5. Measure 300mL of ddH2O using the measuring cylinder.
6. Weigh out 3g of tryptone, 1.5g of yeast extract, 3g of NaCl and 3g of agar powder using the scale &
   spatula.
7. Add 3g of tryptone, 1.5g of yeast extract, 3g of NaCl and 3g of agar powder to the 300mL of ddH2O. This is the LB gel
   which will be used for culturing the modified bacteria later.
8. Disinfection & Verification Preparation
9. Place both the broth LB and gel LB into a sterilizing pot under 121ºC for 2hr
10. Distribute the 300mL LB gel evenly among the 20 Petri dishes.
11. Add 300μL of kanamycin distributed equally across the Petri dishes. [1]

 Note：

[1] The pET-28a plasmid which will be used as a vector for the target genes later in the procedure encodes a gene which
offers kanamycin resistance. This helps verify transformation success, as the E.coli will only survive the kanamycin if
they have successfully received the gene for kanamycin resistance.

**1.2 Plasmid Extraction**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| LB containing *e.coli* | 15mL(3mL/trial) | This was cultured 24hr prior to the experiment |
| SP1 | ≥1250μL (250μL/trial) |  |
| SP2 | ≥1250μL (250μL/trial) |  |
| SP3 | ≥1750μL(350μL/trial) |  |
| Buffer solution | ≥2500μL(500μL/trial) |  |
| Wash solution | ≥5mL(1mL/trial) |  |
| Elution buffer | ≥375μL(75μL/trial) |  |
| Binding columns | 10(2/trial) |  |

  Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Centrifuge | 1 | Capacity for 8000rpm-12000rpm, holds 1.5mL microtubes |
| Pipette | ≥1 | Capacity between 10μL-100μL |
| Pipette | ≥1 | Capacity between 100μL-1000μL |
| Test tube | ≥1 | Capacity 15mL (adjust as needed; 3mL needed/trial) |
| EP tubes | 5(1/trial) | 3mL |
| EP tubes | 10(2/trial) | 1.5mL |
| Styrofoam box  with ice inside | 1 | Enzyme/DNA storage |

Experimental Procedure

E.coli Preparation:

1. Culture 3mL of *E. coli* over 24hrs
2. Extract 1.5mL using pipette of in-solution *E. coli* and transfer to EP tube
3. Place in centrifuge at 8000rpm for 1min
4. Remove EP tube from centrifuge
5. Remove supernatant from EP tube via pipette, leaving the *e.coli* deposit at the bottom [1]
6. Repeat steps 2-5 for remaining 1.5mL
7. Repeat steps 2-6 for all 5 EP tubes

Lysis:

8. Add 250μL of SP1 solution and mix [2]
9. Add 250μL of SP2, then gently turn the EP tubes 2-4 times over 2-3min [3]
10. Add 350μL of SP3, then gently turn the EP tubes 10-20 times. Strands of genetic material should be visible after this.
    [4]

Purifying Target Plasmid:

11. Place all EP tubes in centrifuge at 12000rpm for 10min. [5]
12. Remove EP tubes from centrifuge.
13. Prepare new EP tubes with binding columns.
14. Add 500 μL buffer solution to EP tube containing the binding column
15. Place the EP tube containing the binding column & buffer solution within the centrifuge at 12000rpm for 1min.
16. Remove EP tubes from centrifuge.
17. Dispose of the eluate.
18. Extract the supernatant from EP tubes and transfer to binding column attached to EP tube. Do not disturb the e.coli
    pellet at the bottom of the previous EP tube. [6]
19. Add 500μL wash solution to the binding column. [7]
20. Place EP tube & binding column into centrifuge under 9000 rpm for 30sec.
21. Discard the eluate.
22. Repeat steps 19-21 twice for each sample of *E.coli*
23. Repeat steps 19-22 for all 5 samples.
24. Place the binding column into a clean 1.5ml EP tube and add 75μl of elution buffer to the adsorption film in the center of
    the binding column.[8]
25. Place all EP tubes & binding columns into centrifuge under 9000 rpm for 1min
26. Remove the binding column from the EP tube and store the elution buffer in the styrofoam ice box.

 Note：

[1] This concentrates the *e.coli* into a pellet.

[2] SP1 acts as a buffer, providing a suitable environment for the *e.coli*

[3] SP2 lyses the *e.coli*, releasing the pET-28a plasmid

[4] SP3 neutralizes SP2 (which is alkaline)

[5] This separates the light pET-28a plasmids out from the heavier components of the e.coli cell.

[6] The supernatant contains the target plasmids and the pellet contains lysed e.coli remains

[7] The wash solution displaces unwanted pieces of genetic material without the His-tag

[8] The elution buffer displaces the target plasmids from the binding column.

**1.3 Target Protein DNA PCR**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| 2X mix | 50μL | Contains *thermus aquaticus* DNA polymerase, free BPs, Mg+ (sourced from Vazyme Biotech ) |
| BD-tau F primer (10μm/μL) | 1μL (1μL/trial) |  |
| BD-tau R primer (10μm/μL) | 1μL (1μL/trial) |  |
| BD-tau template | 1μL |  |
| Τ-tau F primer (10μm/μL) | 1μL (1μL/trial) |  |
| T-tau R primer (10 μm/μL) | 1μL (1μL/trial) |  |
| T-tau template | 1μL |  |
| ddH2O | D+844μL(22μL/PCR trial, 400μL/template dilution, D for diluting primers, varies) |  |

  Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| PCR Machine | 1 |  |
| Pipette | 1 | Capacity between 1μL-50μL |
| PCR tubes |  | Capacity 200μL |
| Styrofoam box with ice inside | 1 | Enzyme/DNA storage |

System Preparation:

1. Add a volume of ddH2O 10 times as specified by the label on the microtube containing the primer into said
   microtube to dilute it to a concentration of 10μm/μL. This figure varies for each primer. [1]
2. Add 400μL of ddH2O into the microtubes containing 4μg of template for BD-tau/T-tau and mix.
3. Add, in order, 22μL of ddH2O, 25μL of 2X mix, 1μL of both the F and R primer solutions [2] prepared during the
   1st step for the template for BD-tau/T-tau, in addition to 1μL of the template itself to the microtube
4. Repeat steps 1-3 for each both BD-tau and T-tau
5. Repeat steps 1-4 for all 6 sets of BD-tau and Τ-tau
6. Transfer all systems to a PCR machine
7. PCR cycle:
8. Preheat 95ºC for 5min [3]
9. Heat to 95ºC for 15sec [4]
10. Cool to 63ºC for 30sec [5]
11. Heat to 72ºC for 1min [6]
12. Repeat ii-iv 35 times
13. Hold temperature at 72ºC for 10min [7]
14. Cool to 12ºC for storage [8]

 Note：

[1] Each primer is of different length and thus requires a different volume of solvent to dilute to the target molarity. The
figure given on the tube is the volume of ddH2O necessary to dilute the primers to 100μm/μL.

[2] These solutions containing the primers have a primer concentration of 10μM/μL

[3] This denatures all strands of target DNA (breaks the H-bonds), allowing for primer attachment later. The temperature is
held here to ensure thorough denaturing.

[4] This temperature spike serves the same purpose as the 10-minute one and occurs for each cycle.

[5] This allows for primer attachment. The temperature varies depending on the chosen primer.

[6] This is the optimal functioning temperature for T. aquaticus polymerase, thus starting the replication
process.

[7] This ensures that the polymerization process is fully complete for all newly produced strands.

[8] Setting the temperature lower stabilizes the newly replicated strands of DNA.

**1.4 Gel Electrophoresis**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| 1X TAE powder | 1g (1g/1L of gel) | Working concentration 40mM Tris base, 20mM acetic acid, 1 mM EDTA, pH 8.0 |
| ddH2O | 100mL |  |
| Agarose powder | 1g (1g/1L of gel) |  |
| 10,000X YeaRed Nucleic Acid Gel stain | 10μL(10μL/block of gel) |  |
| 6X Loading buffer | 20μL(10μL/trial) | Contains bisphenol blue |
| BD-tau PCR System | 250 (50μL/trial) | See “Target Protein DNA PCR” for specifics |
| T-tau PCR System | 250 (50μL/trial) | See “Target Protein DNA PCR” for specifics |

   Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Agarose gel electrophoresis mold | 1 |  |
| Agarose gel electrophoresis mold comb | 1 | 13 holes total |
| Pipette | 1 | Capacity 1μL-50μL |
| Gel electrophoresis machine | 1 | Gel block oriented horizontally; DNA runs parallel to the ground, distinct from SDS-PAGE setup |
| Measuring cylinder | 1 | Capacity ≥ 100mL |
| Conical flask | 1 | Capacity ≥ 100mL |

Gel Block Preparation:

1. Measure 1 L ddH2O using the measuring cylinder and transfer to a conical flask.
2. Add 1 bag of TAE buffer (~18.4 g) to the ddH2O in step 1.
3. Add 1g of agarose powder to the ddH2O in step 1.
4. Mix the solution thoroughly by shaking the flask until all added powders are dissolved.
5. Pour the solution into the gel electrophoresis mold.
6. Place the comb into the gel electrophoresis mold and fix it in place in the designated slots. Wait for the gel to
   solidify.
7. System Preparation:
8. Add 10 μL of 6X loading buffer to the PCR system from the previous procedure.
9. Mix evenly.
10. Repeat steps 7-8 for all PCR systems across all trials.
11. Electrophoresis:
12. Place the gel block into the gel electrophoresis machine. Ensure that the side of the gel block with holes is placed
    opposite to the cathode.
13. Add the 1X TAE buffer to the electrophoresis setu[
14. Using the pipette, add 20μL of 2k marker to the topmost hole.
15. Add 50μL of the BD-tau/T-tau PCR system into the subsequent holes.
16. Repeat step 13 for both PCR systems.
17. Repeat steps 13-14 for all trials.
18. Set the gel electrophoresis machine to 150 V and allow it to run for 20-30min. Stop once the bisphenol blue in the loading
    buffer has reached near the end of the gel block.

**1.5 DNA Recovery From Gel**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes |
| Anhydrous ethanol | 1500μL(500μL/trial) |  |
| B2 buffer | 1800μL(600μL/trial) |  |
| Wash solution | 4500μL(1500μL/trial) |  |

  Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes |
| Water Bath | 1 |  |
| Gel transilluminator | 1 |  |
| Scalpel | 1 |  |
| Pipette | ≥1 | Capacity between 10μL-100μL |
| Pipette | ≥1 | Capacity between 100μL-1000μL |
| Centrifuge | 1 |  |

Preparation:

1. Check whether there is sediment in Buffer B2
2. Add 500μL of anhydrous ethanol to the wash solution
3. Adjust the water bath pot to 50ºC
4. Gel Liquification
5. Place the block of gel under the gel transilluminator. [1]
6. Cut out the gel block containing the target fragment from the agarose gel using the scalpel and weigh it. Record the
   mass
7. Transfer the gel containing the target DNA to an EPtube. Dispose of the rest of the gel block.
8. Add 600μL B2 buffer to the EP tube containing the target DNA to the agarose gel block and place the EP tube in a 50ºC
   water bath for 11 minutes. [2]
9. Transfer the solution from step 7 into the binding column, centrifuge at 8000rpm for 30 sec, then discard the eluate.
   [3]
10. DNA Extraction:
11. Add 500μL wash solution, then centrifuge the EP-tube-binding-column complex at 9000rpm for 30 seconds. Discard
    the eluate. [4]
12. Repeat step 9 once
13. Centrifuge the empty binding column [5] at 9000rpm for 1 minute
14. Place the binding column into a clean 1.5mL EP tube and add 30μl of elution buffer to the adsorption film in the center of
    the binding column.
15. Allow the elution buffer to sit at room temperature for 1 minute
16. Centrifuge the binding-column-test-tube complex at 9000rpm for 1 minute and preserve the DNA solution in the tube.
    [6]

 Note：

[1] This shows where the target DNA is in the gel. The DNA fluoresces due to the nucleic acid gel stain added in the
previous procedure.

[2] This liquifies the gel.

[3] The centrifugal force pushes the solution through the adsorption film in the binding columns, leaving the DNA attached
to the film.

[4] This washes the film and removes unwanted materials from the adsorption film.

[5] The binding columns still contain the BD-tau/T-tau DNA.

[6] This removes the DNA from the binding column and suspends it in the elution buffer.

**1.6 Purifying Gel Recovery Products**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity per Set | Notes & Parameters |
| B2 buffer | 600μL (200μL/trial) |  |
| Wash solution | 4500μL (1500μL/trial) |  |
| Elution buffer | 90μL (30μL/trial) |  |

  Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes |
| Water bath | 1 |  |
| Centrifuge | 1 | Can generate 8000rpm-9000rpm |
| Pipette | ≥1 | Capacity between 10μL-100μL |
| Pipette | ≥1 | Capacity between 100μL-1000μL |

Preparation:

1. Add 200μL of B2 buffer to microtube containing recovered DNA. Repeat for all 3 types of DNA subject to PCR.
2. Place the microtube containing the buffer & recovered DNA in a water bath set to 55ºC for 5 minutes.
3. Washing:
4. Transfer the buffer solution & recovered DNA to a binding column attached to a test tube. Repeat for all 3 types of
   DNA subject to PCR.
5. Place all microtubes into centrifuge at 8000rpm for 30sec.
6. Add 500μL wash solution to the binding column. Repeat for all 3 types of DNA subject to PCR.
7. Place all microtube-binding column complexes into centrifuge at 9000rpm for 30sec
8. Remove all microtube-binding column complexes from centrifuge.
9. Repeat steps 5-7 twice, then do steps 6&7 again once without adding more wash solution.
10. Extracting target DNA:
11. Add 30μL elution buffer to binding column.
12. Allow the elution buffer to sit for 1 minute inside the binding column.
13. Place all microtube-binding column complexes in the centrifuge at 9000rpm for 1min. Preserve the liquid in the
    microtube.

**1.7 Double Digestion**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| 10X Buffer | 15 μL (5 μL/trial) |  |
| T-tau DNA | 25 μL (25 μL/trial) | Recovered from gel in previous procedure |
| BD-Tau DNA | 25 μL (25 μL/trial) | Recovered from gel in previous procedure |
| pET-28a | 25 μL (25 μL/trial) | Recovered from gel in previous procedure |
| Hind III | 3 μL(1μL/trial) | Hind III is an endonuclease that recognizes the following sequence: 5\' A^AGCTT 3\' [1] |
| Nhe I | 3 μL(1μL/trial) | Nhe I is an endonuclease that recognizes the following sequence: 5\' G^CTAGC 3\' |

  Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Micropipettes (P10, P20, P200) | 1 set |  |
| Sterile pipette tips | 1 box |  |
| Microtubes | 3 | 1 per reaction + extra, capacity 200μL |
| Test tube rack | 1 |  |
| Microcentrifuge | 1 |  |
| Vortex mixer | 1 |  |
| Water bath | 1 | Maintain 37°C |
| Styrofoam box with ice inside | 1 | Enzyme/DNA storage |
| Pipette | 1 | Capacity between 1μL-20μL |

System Preparation:

1. Add 15 μL ddH2O, 10μL of the target type of DNA for double digestion, 5μL of the 10X buffer, 1 μL Hind III
   and 1 μL Nhe I to a PCR tube.
2. Repeat step 1 for both remaining types of DNA subject to double digestion (T-tau DNA/BD-Tau DNA/pET-28a).
3. Place all samples into microcentrifuge for 30sec.
4. Incubation:
5. Place all samples into test tube rack.
6. Keep these solutions in the water bath and allow to incubate for 2h at 37ºC.

**1.8 Ligation**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity per Trial | Notes & Parameters |
| 10X buffer | 2μL |  |
| T4 ligase: | 1μL |  |
| Cleaved BD-tau/T-tau DNA sequences | 3 μL |  |
| Cleaved pET-28a | 1μL |  |
| ddH2O | 13μL |  |
| PCR tubes | 3 |  |

Ligated Solution Preparation:

1. Set the water bath to 50ºC and wait for its temperature to reach 50ºC.
2. Place the PCR tubes containing the double digestion solution in the water bath set to 50ºC for 15min. [1]
3. System Preparation:
4. Add 13μL ddH2O, 3μL of the target DNA sequence, 2μL of the 10X buffer and 1μL of pET-28a to an EP tube.[2]
5. Repeat step 1 for the remaining sequences subject to ligation (BD-tau/T-tau).
6. Place all PCR tubes in microcentrifuge for 30sec
7. Incubate in PCR machine for 1 hour under 16ºC

Note：

[1] “^” represents the site of cleavage.

[2] This denatures Nhe I and Hind III.

**1.9 Heat shocking**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| BL21 culture |  |  |
| DH5a culture |  |  |
| PET-28a solution (T-tau) | 10μL (5μL/trial) |  |
| pET-28a solution (BD-tau) | 10μL (5μL/trial) |  |
| LB broth | ≥3600μL (900μL/sample) | The broth should be sterile and should not contain kanamycin. |
| CaCl2 |  |  |

  Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Cooler | 1 | Generates temperatures of -80ºC or lower |
| Water bath | 1 | Temperature range includes 42ºC |
| Petri dishes | ≥6 | Prepared during LB preparation step; 0.1% kanamycin |

Storage:

1. Store the competent E.coli in a fridge set to -80ºC.
2. Sample Preparation:
3. Add 5μL of pET-28a solution carrying the BD-tau/T-tau gene to the BL21/DH5α e.coli samples and gently mix.
4. Repeat step 2 three additional times to generate a total of four samples, representing all possible combinations of one E.
   coli strain with one gene (BL21 + BD-tau, BL21 + T-tau, DH5α + BD-tau, DH5α + T-tau). [1]
5. Heat Shocking & Culturing:
6. Place the microtubes containing the e.coli samples into a 42ºC water bath for 45sec. [2] Quickly transfer the samples to
   an ice bath to cool.
7. Add 900 μL of kanamycin-free LB broth each microtube. Mix evenly by repeatedly drawing in and dispensing the LB using
   the pipette.
8. Once the e.coli-LB solutions are evenly mixed, centrifuge at 8000rpm for 5min.
9. Remove 900 μL of supernatant from all microtubes.
10. Use the samples to inoculate petri dishes. Allow the E.coli colonies to develop for 12-16h at 37ºC.
11. Preparation for PCR:
12. Pick random colonies in the culture & split each sampled colony into 2 equal portions; one will be used for PCR later
    the other inoculate LB broth [3] which contains 0.1% kanamycin from the LB preparation step [4].

 Note:

[1] 2 strains were cultured to find a (relatively) optimal strain for expressing the target proteins.

[2] This sudden change in heat increases membrane permeability & makes the e.coli cells competent.

[3] The petri dishes will be used to verify transfer success later, and the e.coli in the broth will be used for protein
expression.

[4] The pET-28a plasmid which will be used as a vector for the target genes later in the procedure encodes a gene which
offers kanamycin resistance. This helps verify transformation success, as only e.coli which have received the
kanamycin-resistance gene encoded on the pET-28a plasmid will survive the kanamycin.

**2.0  PCR For Target Genes Inside E.coli**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity per Trial | Notes & Parameters |
| Buffer 2X | 50 μL (5μL /sample) |  |
| F-primer | 2 μL (0.2μL/sample) |  |
| R-primer | 2 μL (0.2μL/sample) |  |
| ddH2O | 46 μL (4.6μL/sample) |  |
| BL21 e.coli carrying BD-tau DNA | N/A [1] | Engineered for superior protein-expressing capabilities |
| BL21 e.coli carrying T-tau DNA | N/A | Engineered for superior protein-expressing capabilities |
| DH5a e.coli carrying T-tau DNA | N/A | Engineered for superior transformation efficiency |
| DH5a e.coli carrying T-tau DNA | N/A | Engineered for superior transformation efficiency |

   Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Cooler | 1 | Generates |
| Microtubes | 16 | 4 for each combination of DNA and e.coli strain |

Preparing the System:

1. Add 4.6T μLddH2O, 5.0T μLbuffer, 0.2T μL F primer and 0.2T μL R primer to a test tube, where T is the number of
   desired trials. In this experiment, T=10.
2. Separate the system equally into T 10 μL portions.
3. Gently scrape e.coli colonies off the petri dishes used to cultivate e.coli after the heat-shocking procedure using a
   pipette[1] .
4. Transfer some colonies to the microtubes containing the PCR solution. Swap the pipette tip between each culture.
   [2]
5. Repeat steps 4-5 for all cultures.
6. PCR:
7. Preheat to 95ºC for 5min
8. Heat to 95ºC for 15sec
9. Cool to 65ºC for 15sec
10. Heat to 72ºC for 10sec
11. Repeat steps 8-10 30 times
12. Hold temperature at 72ºC for 10min

 Note:

[1] The colonies were extracted by scraping the petri dishes with a pipette tip, as opposed to being added as a solution,
thus making their volume negligible.

[2] This prevents cross-contamination.

',
},
{
"title": '2. Protein induction and expression',
"content": '

**2.1 Monitoring *E.coli* Growth Curve**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| LB | 8mL (2mL/test tube) | Contains 50ng/mL K+ (kanamycin) |

   Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Shaker | 1 | The shaker is sealed and conditioned |
| Timer | 1 | Optional, a clock may be used instead |
| Spectrophotometer | 1 | 500μL cuvette recommended |
| Test tube | 4 | Capacity 15mL (2 samples/test tube) |
| Pipette | ≥1 | Capacity between 100μL-1000μL |

Sample Preparation:

1. Mingle 2mL LB (K+ with 50ng/ml concentration) with 50 μL E. coli cultured in the heat shocking step. Mix evenly
2. Place all tubes into 37˚C shaker.
3. Observation:
4. Return to record data for OD600 at different points for both proteins. (0.5, 1, 2, 4, 6, 8, 12 and 24h) [1]
5. OD 600 Protocol:
6. Remove the cuvette from the spectrophotometer & clean its exterior if needed.
7. Add 500μL of sterile LB to the spectrophotometer cuvette.
8. Place the cuvette back into the spectrophotometer
9. Measure the OD 600 value of the blank. [2]
10. Remove & empty the cuvette.
11. Transfer the culture to the cuvette and measure the absorbance.
12. Place the cuvette back into the spectrophotometer.
13. Measure the OD-600 of the sample.

* Repeat steps v-viii for all samples.
* Note:

[1] OD600 is an approach which measures the light absorbance of bacterial or yeast cultures at 600nm wavelength using a
spectrophotometer. As the curve requires infrequent, periodic measurements, this procedure can take place alongside other
procedures.

[2] This pares the spectrophotometer.

**2.2 Protein Extraction & Purification**

 Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| Non-denaturing lysis buffer | 5mL |  |
| Wash solution | 3mL (500μL/Wash) |  |
| Ice |  |  |

  Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Centrifuge | 1 | Can generate 1000rpm--10000rpm |
| Sonicator | 1 |  |
| Beaker | 1 |  |

Sonification Lysis Preparation:

1. Centrifuge the tubes containing the E. coli culture at 6000rpm and 4˚C for 15 minutes to concentrate the E. coli into a
   pellet.
2. Extract & discard the supernatant using a pipette.
3. Add 20mL non-denaturing lysis buffer to the pellet. [1]
4. Vortex the solution to mix thoroughly.
5. Sonification Lysis:
6. Uncap the test tube containing the E.coli culture and place it into a beaker containing ice. [2]
7. Place the beaker on the sonicator’s platform with the test tube’s opening positioned directly under the needle and
   raise the platform until the needle is touching the solution. Ensure that the needle is not touching the test tube walls.
   [3]
8. Sonicate at 300 W with 2 s on/2 s off cycles for 30 min.
9. Remove the test tube from the sonicator once sonication is complete.
10. Repeat steps 5-8 for T-tau.
11. Extracting Target Proteins:
12. Centrifuge for 30 minutes at 10000 rpm at 4˚C.
13. Extract the supernatant, transfer to an 15 mL test tube and place it on ice.
14. Extract 20 μL of the supernatant for SDS-PAGE later.
15. Add 1 mL his-tag purification resin to an EP tube.
16. Centrifuge at 1000rpm at 4˚C for 10 seconds. Discard the supernatant. [4]
17. Add 500 μL non-denaturing lysis buffer to the EP tube and centrifuge at 1000 rpm at 4˚C for 10 seconds. [5]
18. Repeat step 15 2 more times.
19. Add his-tag purification resin to test tubes containing BD-tau. Mix evenly.
20. Incubate for 60min at 4ºC on a shaker.
21. Add 10mL of BD-tau supernatant and His-tag purification resin into large binding columns. [6] Keep the bottom capped.
22. Allow protein solution containing BD-tau bound to his-tag purification resin to pass through the binding column. The resin
    beads should remain above the filter.
23. Add 500μL wash solution to the binding column and allow it to pass through the binding column film.
24. Collect 20μL of the eluate for sampling later. Store the rest.
25. Repeat steps 20-21 2 more times.
26. Add 500μL elution buffer to the binding column and allow it to pass through the binding column film.
27. Collect 20μL of the eluate for sampling later. Store the rest.
28. Repeat step 23-24 3 more times.
29. Collect the final eluate.

Repeat steps 10-26 for T-tau
 Note:

[1] This suspends the e.coli in preparation for lysis

[2] Sonication generates heat. The ice cools the sample to prevent heat-induced protein denaturation.

[3] The needle and test tube would damage each other if in contact for extended periods during operation.

[4] This leaves only the resin on the binding column film. The eluate is the storage solution for the resin.

[5] The non-denaturing lysis buffer is used to wash the resin

[6] This binding column contains the his-tag purification resin beads and is larger than the binding columns used in the EP
tubes.

**2.3 SDS-PAGE**

 Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| ddH2O |  |  |
| 30% Acr-Bis |  |  |
| Gel buffer A | 3.75 mL |  |
| Gel buffer B | 2 mL |  |
| Mixing cups |  |  |

 Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| SDS-PAGE | 1 |  |

Sample Preparation:

1. Add 10 μL protein sample and 10 μL 2X loading buffer to an EP tube.
2. Heat the microtube to 95˚C for 5 minutes.
3. Casting SDS-PAGE Gel:
4. Fix the gel casting cassette into the casting frames.
5. Prepare the 15% resolving gel by adding 3.6ml ddH2O, 7.5 mL 30% Acr-Bis, 3.75 mL Gel Buffer A, 0.15
   mL 10% APS and 0.009 mL TEMED to a mixing cup.
6. Pipette the resolving gel into the gel casting cassette.
7. Add anhydrous ethanol to the gel casting cassette until the ethanol level reaches the height of the shorter plate
8. Wait for 10-15min.
9. Decant the ethanol.
10. Prepare the 5% stacking gel by adding 1.33mL ddH2O, 0.67 mL 30% Acr-Bis, 2 mL Gel Buffer B, 0.04 mL 10%
    APS and 0.004 mL TEMED to a mixing cup.
11. Pipette the stacking gel into the gel casting cassette.
12. Insert the comb into the gel casting cassette.
13. Wait for 10-15min.
14. Carefully remove the combs from the gel casting cassettes.

Buffer Preparation:

1. Prepare a conical flask with capacity 1 L or greater.
2. Add 1 bag (approx. 18.4g) of tris-glycine SDS buffer into the conical flask.
3. Measure 1 L of water using a measuring cylinder.
4. Add the water to the conical flask.
5. Mix evenly until tris-glycine SDS buffer is fully dissolved.
6. SDS-PAGE Electrophoresis:
7. Transfer the cassettes to an SDS-PAGE gel electrophoresis tub.
8. Add to each gel block the following samples:
9. Markers for the first 2 cells. For the remaining 9 cells, add 10 μL CL, FT, W1, W2, W3, E1, E2, E3 and E4 in order.
10. Add buffer to the space between the cassettes within the tank until the space is completely full.
11. Place the cap onto the setup. Ensure that the anodes and cathodes are properly paired.
12. Plug the cap’s wires into an electrophoresis power supply. Ensure that the anodes and cathodes are properly paired.
13. Turn on the power supply. Set the voltage to 60 V for 35min. Then, set the voltage to 130 V for 40min.
14. Gel Dyeing:
15. Cut the stacking gel from the gel block using a scalpel and discard it.
16. Place the resolving gel block into a dyeing tub and add 20 mL of Coomassie brilliant blue dye to the tub.
17. Place the dyeing tub onto a shaker and allow to dye overnight.
18. Place the dyed gel block into an SDS-PAGE gel reader and document the results.

',
},
{
"title": '3. SELEX',
"content": '

**3.1 SELEX screening for nucleic acid aptamers**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| DNA Oligo Library | / | 1503.75 μM (This will vary based on library) |
| ddH2O | / | / |
| PBS | / | / |
| Streptavidin magnetic bead solution | / | / |
| EP tubes | / | / |
| NaOH | / | / |
| T-tau | 1.5ml | 5mg/ml |
| BD-tau | 1.5ml | 5mg/ml |
| Ni-NTA beads | 2ml |  |

 Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Pipette | / | / |
| Microcentrifuge | 1 | 1 |
| Shaker | 1 | 1 |
| Magnetic separation rack | 1 | 1 |
| Nanodrop | 1 | 1 |

SELEX screening for Table 1:

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | {1} | {2} | {3} | {4} | {5} | {6} | {7} |
| Round | DNA Library (Concentration, Volume) | Counter-selection (Bead Volume, Time) | Positive BD-tau Selection (Bead Volume, Time) | Elution Volume | Secondary T-tau Counter-selection (Bead Volume, Time) | Wash (Volume, Repetitions) | PCR Cycles |
| 1 | 40 µM,  500 µL | – | 25 µL × 2,  60 min | 100 µL | – | 500 µL × 2 | 20 |
| 2 | 300 nM,  500 µL | – | 25 µL × 2,  45 min | 300 µL | – | 500 µL × 2 | 14 |
| 3 | 300 nM,  500 µL | – | 25 µL × 2,  30 min | 300 µL | – | 500 µL × 2 | 14 |
| 4 | 250 nM,  500 µL | – | 50 µL × 2,  30 min | 300 µL | – | 500 µL × 2 | 14 |
| 5 | 250 nM,  500 µL | – | 50 µL × 2,  30 min | 300 µL | – | 500 µL × 3 | 12 |
| 6 | 250 nM,  500 µL | – | 50 µL × 2,  30 min | 500 µL | 400 µL,  30 min | 500 µL × 3 | 12 |
| 7 | 250 nM,  500 µL | – | 50 µL × 2,  30 min | 500 µL | 400 µL,  30 min | 1000 µL × 3 | 22 |
| 8 | 250 nM,  300 µL | 125–1000µL, 30 min | 50 µL × 2,  30 min | 300 µL | 400 µL,  30 min | 1000 µL × 3 | 26 |
| 9 | 250 nM,  300 µL | 400 µL,  30 min | 50 µL × 2,  30 min | 300 µL | 400 µL,  30 min | 1000 µL × 3 | 24 |
| 10 | 250 nM,  300 µL | 1000 µL,  30 min | 50 µL × 2,  30 min | 300 µL | 1000 µL,  30 min | 1000 µL × 3 | 10 |
| 11 | 250 nM,  300 µL | 1000 µL,  30 min | 50 µL × 2,  30 min | 300 µL | 1000 µL,  30 min | 1000 µL × 3 | 18 |
| 12 | 250 nM,  300 µL | 1000 µL,  30 min | 50 µL × 2,  30 min | 500 µL | 1000 µL,  30 min | 1000 µL × 3 | 16 |
| 13 | 250 nM,  300 µL | 1000 µL,  30 min | 50 µL × 2,  30 min | 500 µL | 1000 µL,  30 min | 1000 µL × 3 | 16 |
| 14 | 250 nM,  300 µL | 1000 µL,  30 min | 50 µL × 2,  30 min | 500 µL | 1000 µL,  30 min | 1000 µL × 5 | 20 |
| 15 | 250 nM,  300 µL | 1000 µL,  30 min | 50 µL × 2,  30 min | 500 µL | 1000 µL,  30 min | 1000 µL × 5 | 14 |
| 16 | 100 nM,  200 µL | 200 µL,  30 min | 50 µL × 2,  30 min | 500 µL | 200 µL,  30 min | 1000 µL × 5 | 12 |

Ni-NTA Bead Preparation:

1. Thoroughly resuspend the stored Ni-NTA beads by gently shaking the bottle and pipetting the storage solution.
2. Extract 20 μL Ni-NTA beads and transfer to a 1.5ml EP tube.
3. Add 500 μL PBS and vortex thoroughly. Use microcentrifuge -10000rpm for 10 seconds.
4. Place the microtubes in the magnetic separation rack and allow the beads to collect against the tube wall.
5. Carefully discard the supernatant without disturbing the beads.
6. Repeat steps 4-6 3 times for each tube. For efficiency’s sake, sufficient beads for all subsequent rounds were washed at
   once.
7. Bead Suspension & Incubation:
8. Suspend the beads in 100 μL of PBS after washing.
9. Add His-tag peptides at a concentration of 400 µg/mL for rounds 1–7, or 800 µg/mL for rounds 8–17.
10. Incubate the tubes by placing them in a 4˚C shaker for 16-18hr. Maintain 4ºC for storage.
11. DNA Library Preparation:
12. Heat the library to 95˚C for 3 minutes
13. Snap-cool for 5 minutes to form ssDNA.

SELEX contains 16 rounds of selection, divided into Positive Selection (Rounds 1-5), Denoising I (Rounds 6-7) and Denoising
II (Rounds 8-16). For each section, refer to its corresponding procedure. {n} is used to represent variable values. Refer to
the corresponding cell in Table 14.1 at coordinates (n, round number) for the specific value of a given trial.

Positive Selection (Rounds 1-5):

![Copy of SELEX Positive(1)](https://static.igem.wiki/teams/5739/experiment/1.webp)

1. Add {1} nM of DNA library and {3} μL of BD-tau Ni-NTA magnetic beads to ({1(volume)} - - {3}) μL of PBS buffer
   in an EP tube. For round 1, Calculate with the following equation: , where is the volume of the library needed, is the
   molar concentration of the library and is the desired concentration.
2. Allow library &BD-tau Ni-NTA magnetic beads to incubate for Table1 column {3} min.
3. Add Table1 column {6} μL ddH2O to the EP tubes containing the library and Ni-NTA magnetic beads.
4. Place the EP tubes into the magnetic separation rack. Carefully discard the supernatant without disturbing the BD-tau
   Ni-NTA magnetic beads.
5. Repeat steps 3-4 an additional Table1 column {6} times.
6. Add Table1 column {4} μL Dnase-free ddH2O to the EP tube. Heat the supernatant to 95˚C for 7 minutes.
7. Place the system in the microcentrifuge for 30sec.
8. Conduct {7} rounds of PCR on the sample.
9. 20μL streptavidin magnetic beads separation of ssDNA
10. Conduct a nanodrop test for the supernatant to determine its concentration.
11. Calculate for the next trial given by the following equation: , where is the volume of the library needed, is the
    concentration measured via nanodrop and is the desired concentration.

Denoising I (Rounds 6-7)

![SELEX 6-7(1)](https://static.igem.wiki/teams/5739/experiment/2.webp)

1. Add Table1 column{1} nM of DNA library and Table1 column{3} μL of BD-tau Ni-NTA magnetic beads to Table1 column
   ({1(volume)} - - {3}) μL of PBS buffer to an EP tube.
2. Allow the library & BD-tau Ni-NTA magnetic beads to incubate for Table1 column {3} min.
3. Add Table1 column {6} μL ddH2O to the EP tubes containing the library and BD-tau Ni-NTA magnetic beads.
4. Place the EP tubes into the magnetic separation rack. Carefully discard the supernatant without disturbing the BD-tau
   Ni-NTA magnetic beads.
5. Repeat steps 3-4 an additional Table1 column {6} times.
6. Add Table1 column {4} μL PBS to the EP tube. Heat the supernatant to 95˚C for 7 minutes.
7. Place the test tube on the magnetic separation rack.
8. Transfer all eluted supernatant and Table1 column {5} μL of Ni-NTA magnetic beadsbeads to a new test tube.
9. Allow the beads and library to incubate for 30min.
10. Place the test tube on the magnetic separation rack. Extract all supernatant and transfer to a new EP tube.
11. Heat the supernatant to 95˚C for 7 minutes. Place the system in the microcentrifuge for 30sec.
12. Conduct Table1 column {7} rounds of PCR on the sample.
13. **20**μL **streptavidin magnetic beads separation of ssDNA**
14. Add 500 μL ΝaΟΗ to the EP tubes to wash out dsDNA.
15. Conduct a nanodrop test for the supernatant to determine its concentration.
16. Calculate for the next trial given by the following equation: , where is the volume of the library needed, is the
    concentration measured via nanodrop and is the desired concentration.

Denoising II (Rounds 8-16)

![Copy of SELEX Negative (1)(1)](https://static.igem.wiki/teams/5739/experiment/3.webp)

1. Add Table 1 column {1} nM of DNA library and Table1 column {2} μL of unloaded BD-tau Ni-NTA magnetic beads to
   Table1 column ({1(volume)} - - {2}) μL of PBS buffer to an EP tube.
2. Allow the library & BD-tau Ni-NTA magnetic beads to incubate for Table1 column {2} min.
3. Extract the supernatant and transfer to a new EP tube.
4. Add extract the supernatant to Table1 column {3} μL of BD-tau Ni-NTA magnetic beads.
5. Allow the library & BD-tau Ni-NTA magnetic beads to incubate for Table1 column {3} min.
6. Add Table1 column {6} μL PBS to the EP tube containing the library and BD-tau Ni-NTA magnetic beads.
7. Place the microtubes into the magnetic separation rack. Carefully discard the supernatant without disturbing the BD-tau
   Ni-NTA magnetic beads.
8. Repeat steps 4-5 an additional Table1 column {6} times.
9. Add Table1 column {4} μL PBS to the EP tube. Heat the supernatant to 95˚C for 7 minutes.
10. Place the test tube on the magnetic separation rack.
11. Transfer all eluted supernatant and Table1 column {5} μL of T-tau Ni-NTA magnetic beads to a new test tube.
12. Allow the beads and library to incubate for 30min.
13. Place the test tube on the magnetic separation rack. Extract all supernatant and transfer to a new EP tube.
14. Heat the supernatant to 95˚C for 7 minutes.
15. Place the system in the microcentrifuge for 30sec.
16. Conduct {7} rounds of PCR on the sample.
17. Conduct a nanodrop test for the supernatant to determine its concentration.
18. Calculate for the next trial given by the following equation: , where is the volume of the library needed, is the
    concentration measured via nanodrop and is the desired concentration.

Note:

Prepare a 50 μL system for PCR:

Prime Star 2X buffer 25 μL, SELEX F&R Primer 1 μL+1 μL, Library 23 μL.

| Step | Temp | Time | # of cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 95°C | 90s |  |
| Denaturation | 95°C | 30 sec | Table 1 |
| Primer Annealing | 57°C | 30 sec |
| Extension | 72°C | 60 sec |
| Final Extension | 72°C | 3 min |  |

**3.2 Flow Cytometry**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| 1X phosphate-buffered saline solution | / | / |
| SELEX samples for T-tau and BD-tau from rounds 1, 4, 7 and 14 | 80μL total (20μL/sample) | / |

Equipment statistics:

|  |  |
| --- | --- |
| Equipment Name | Quantity |
| Test tube | 5 |
| Cap sieve | 5 |
| Flow cytometer | 1 |

 Process :

1. Add 2500μL phosphate-buffered saline solution to the extra test tube if needed.
2. Label a test tube with its respective trial number.
3. Gradually add 500μL of phosphate-buffered saline solution along with the PCR solution of the given SELEX cycle to the test
   tube designated for said round of SELEX. To ensure full transfer of the PCR solution, add some volume of
   phosphate-buffered saline solution to the PCR tube containing the SELEX library, repeatedly pipette the mixture, then
   extract it and transfer to the larger test tube. [1]
4. Repeat for all cycles and for 2 control samples containing unbound streptavidin magnetic beads and unbound aptamers
   respectively. For this experiment, the library from the 7th SELEX cycle was diluted to 2 separate concentrations to find
   an optimal concentration for the cytometry machine later in the procedure.
5. Pass all solutions through separate 35μm-pore-size sieves to prevent the sample from jamming the flow cytometry machine.
6. Add a control/blank solution of unbound streptavidin magnetic beads to the cytometry machine to test it.
7. Adjust the parameters of the flow cytometry machine:
8. Set a range of detection. This will appear as a polygon on Graph 1 whose vertices may be determined by clicking on their
   desired locations. All datapoints within this polygon are registered.
9. Adjust the voltage until a clustered, elliptical distribution starts showing on Graph 1. The voltage used was ~0.62 V.
10. Export all data. Repeat for all trials.

Graph 1 shows the size & structural complexity of the aptamer-bead complexes along the Χ and Y axes respectively.

Graph 2 uses data and axes identical to graph one, but accounts for chimeric formations of the aptamers.

Graph 3 shows the number of particles (Y-axis) fluorescing at a given intensity (X-axis)

Table 2:

|  |  |
| --- | --- |
| Graph Name | Contents & Parameters |
| (Graph 1) | X-coordinate equals particle size, Y-coordinate shows particle complexity. |
| (Graph 2 | X-coordinate equals particle size, Y-coordinate shows particle complexity, accounts for chimeric formations |
| (Graph 3) | Shows number of particles fluorescing at a given intensity. X-axis represents different intensities, Y-axis represents number of particles. |

**3.3 SPR( Surface Plasmon Resonance)**

A. Chip Surface Preparation and Immobilization

1. Obtain 5 mg/ml of the biotinylated target protein BD-tau from step 2.2.
2. Select a pre-coated streptavidin (SA) SPR chip (such as a CM5 chip).
3. Inject the biotinylated target protein BD-tau into a specific flow cell of the SA chip to allow specific and firm capture.

B. Binding and Dissociation Experiment

1. Dilute the nucleic acid aptamer with running buffer to concentrations of 0 μM, 5 μM, 10 μM, 30 μM, 100 μM, 100 μM, and 150
   μM.
2. Inject the series of diluted aptamer solutions in ascending order of concentration over the chip surface immobilized with
   the target protein at a constant flow rate, monitoring the binding process.
3. After injecting each concentration sample for a period (typically 1–5 minutes), switch to running buffer to monitor the
   dissociation process (typically 5–10 minutes).
4. Between each cycle, briefly inject (30–60 seconds) a regeneration solution (for protein-nucleic acid interactions, 1–5 M
   NaCl) to thoroughly wash away the bound aptamer and regenerate the chip surface.

**3.4 ELISA (Enzyme-Linked ImmunoSorbent Assay)**

Material statistics:

|  |  |
| --- | --- |
| Material Name | Notes & Parameters |
| BD-tau aptamer solution | 5 mg/mL |
| T-tau aptamer solution | 5 mg/mL |
| BSA solution | 2mg/ml |
| Salmine |  |
| 1XTBST | 45mL of Distilled water, 5 mL of TBS, 25μL of Tris buffer salt solution containing Tween 20 |
| Sealing buffer | / |
| BD-tau protein solution | 5 mg/mL |
| T-tau protein solution | 5 mg/mL |
| Streptavidin/Poly HRP |  |
| Aluminum foil | / |
| Synthetic blood | Contains H2O, important proteins, various electrolytes, O2, CO2, steroids, clotting agents, glucose, etc. |

Equipment statistics:

|  |  |
| --- | --- |
| Equipment Name | Quantity |
| ELISA board | 10 |
| Pipette | 500 |
| Scissors | 1 |
| Microplate reader | 1 |

Binding Aptamers to Plate:

|  |  |  |
| --- | --- | --- |
| {1} | {2} | {3} |
| Aptamer type bound to ELISA board | Introduced Solution | Number of Cells |
| BD-tau | Polyprotein | 60 |
| BD-tau | T-tau | 96 |
| BD-tau | BSA | 96 |
| T-tau | BD-tau | 93 |
| BD-tau | BD-tau solution + polyprotein + BSA | 96 |

1. Dilute the {1} aptamer solution with the coating solution (pH9.6) to 1μg/mL.
2. Add 100μL of the diluted aptamer solution to each well of the 96-well plate. Incubate overnight at 4°C.
3. Blocking & Washing:
4. Wash the plate 3 times by adding 200 μL 1X TBST to each well.
5. Add 200μL of sealing buffer to each well; Incubate at room temperature for 1 hour or at 37 ° C for 30 minutes.
6. Wash the plate 3 times by adding 200 μL 1X TBST to each well.
7. Sample Preparation & Addition:
8. Prepare standard curve {2} samples with concentrations 1, 2, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000 and 10,000 pg/mL.
9. Randomly generate 2 equal-sized sets of {2} sample concentrations, one below 100 pg/mL and the other above 100 pg/mL and
   below 10 ng/mL. (See Attachment 1 for c++ code for generating random concentrations).
10. Prepare H2O-based and artificial blood-based storage solutions containing {2}. The proteins are sourced from earlier in
    the procedure.
11. Prepare solutions of all randomly-generated concentrations. Use the H2O storage solution to prepare 25% of randomized
    sample concentrations and artificial blood for the remaining 75%.
12. Add 100 μL/well of standard curve protein solutions prepared earlier to the first 13 wells in the ELISA plate.
13. Add the remaining randomized samples prepared in step 9 to the subsequent wells. The number of samples may be adjusted.
    Reference {3} for the figures in the original experiment. Record the sample concentration in each well.
14. Incubation:
15. Incubate at room temperature for 2 hours (or overnight at 4 ° C).
16. Wash the plate three times with TBST.
17. Fittest incubation:
18. Add 100μL of biotin solution (500nM TBST+20 mg/mL biotin in PVP buffer+100 μg/mL salmine DNA) to each well. [1]
19. Incubate the plate at room temperature for 1 hour.
20. Signal Detection:
21. Wash the plate three times with TBST.
22. Incubation of enzyme-labeled secondary antibodies
23. Add 100 μL of Streptavidin Poly HRP to each well diluted at a ratio of 1:10,000 to TBST. [2]
24. Incubate at room temperature for 1 hour. Wrap the sample in aluminium foil.  [3]
25. Post-Incubation Washing:
26. Wash the board 5 times with TBST. [4]`
27. Color Development & Measurement:
28. Add 100μl of TMB chromogenic substrate to each well and react in the dark for 10-15 minutes.
29. Add 50μL/well of termination solution with concentration 2M.
30. Measure the absorbance at 450nm using an microplate reader (reference wavelength: 450-620nm).
31. Repeat the entire procedure for all 5 combinations of aptamers and introduced solutions.

Note:

[1] This helps the biotin attach to the aptamer

[2] This helps the streptavidin poly HRP attach to the biotin. The streptavidin poly HRP has a peak absorbance rating of
~450 nm, which will be measured using abosorbance in step 22.

[3] This prevents light-induced damage to the dyes.

[4] This reduces noise by washing residual proteins away.

',
},
]
},
{
"title": 'Cycle 2: Aptamer-based biosensor for BD tau protein',
"content": '',
"children": [
{
"title": '1. Plasmid Construction',
"content": '

strong>1 Extract the pET28a plasmid

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| LB containing *e.coli*(pET28a) | 15mL(3mL/trial) | This was cultured 24hr prior to the experiment |
| SP1 | ≥1250μL (250μL/trial) |  |
| SP2 | ≥1250μL (250μL/trial) |  |
| SP3 | ≥1750μL(350μL/trial) |  |
| Buffer solution | ≥2500μL(500μL/trial) |  |
| Wash solution | ≥5mL(1mL/trial) |  |
| Elution buffer | ≥375μL(75μL/trial) |  |
| Binding columns | 10(2/trial) |  |

  Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Centrifuge | 1 | Capacity for 8000rpm-12000rpm, holds 1.5mL microtubes |
| Pipette | ≥1 | Capacity between 10μL-100μL |
| Pipette | ≥1 | Capacity between 100μL-1000μL |
| Test tube | ≥1 | Capacity 15mL (adjust as needed; 3mL needed/trial) |
| EP tubes | 5(1/trial) | 3mL |
| EP tubes | 10(2/trial) | 1.5mL |
| Styrofoam box  with ice inside | 1 | Enzyme/DNA storage |

Experimental Procedure

E.coli Preparation:

1. Culture 3mL of *E. coli* pET28a over 24hrs
2. Extract 1.5mL using pipette of in-solution *E. coli* and transfer to EP tube
3. Place in centrifuge at 8000rpm for 1min
4. Remove EP tube from centrifuge
5. Remove supernatant from EP tube via pipette, leaving the *e.coli* deposit at the bottom [1]
6. Repeat steps 2-5 for remaining 1.5mL
7. Repeat steps 2-6 for all 5 EP tubes

Lysis:

1. Add 250μL of SP1 solution and mix [2]
2. Add 250μL of SP2, then gently turn the EP tubes 2-4 times over 2-3min [3]
3. Add 350μL of SP3, then gently turn the EP tubes 10-20 times. Strands of genetic material should be visible after this.
   [4]

Purifying Target Plasmid:

1. Place all EP tubes in centrifuge at 12000rpm for 10min. [5]
2. Remove EP tubes from centrifuge.
3. Prepare new EP tubes with binding columns.
4. Add 500 μL buffer solution to EP tube containing the binding column
5. Place the EP tube containing the binding column & buffer solution within the centrifuge at 12000rpm for 1min.
6. Remove EP tubes from centrifuge.
7. Dispose of the eluate.
8. Extract the supernatant from EP tubes and transfer to binding column attached to EP tube. Do not disturb the e.coli
   pellet at the bottom of the previous EP tube. [6]
9. Add 500μL wash solution to the binding column. [7]
10. Place EP tube & binding column into centrifuge under 9000 rpm for 30sec.
11. Discard the eluate.
12. Repeat steps 19-21 twice for each sample of *E.coli*
13. Repeat steps 19-22 for all 5 samples.
14. Place the binding column into a clean 1.5ml EP tube and add 75μl of elution buffer to the adsorption film in the center of
    the binding column.[8]
15. Place all EP tubes & binding columns into centrifuge under 9000 rpm for 1min
16. Remove the binding column from the EP tube and store the elution buffer in the styrofoam ice box.

 Note：

[1] This concentrates the *e.coli* into a pellet.

[2] SP1 acts as a buffer, providing a suitable environment for the *e.coli*

[3] SP2 lyses the *e.coli*, releasing the pET-28a plasmid

[4] SP3 neutralizes SP2 (which is alkaline)

[5] This separates the light pET-28a plasmids out from the heavier components of the e.coli cell.

[6] The supernatant contains the target plasmids and the pellet contains lysed e.coli remains

[7] The wash solution displaces unwanted pieces of genetic material without the His-tag

[8] The elution buffer displaces the target plasmids from the binding column.

**1.2 Target Protein DNA PCR**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| 2X mix | 50μL | Contains *thermus aquaticus* DNA polymerase, free BPs, Mg+ (sourced from Vazyme Biotech ) |
| Cas12a F primer (10μm/μL) | 5μL (1μL/trial) |  |
| Cas12a R primer (10μm/μL) | 5μL (1μL/trial) |  |
| Cas12a template(10μm/μL) | 5 μL |  |
| ddH2O | D+844μL(22μL/PCR trial, 400μL/template dilution, D for diluting primers, varies) |  |

  Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| PCR Machine | 1 |  |
| Pipette | 1 | Capacity between 1μL-50μL |
| PCR tubes |  | Capacity 200μL |
| Styrofoam box with ice inside | 1 | Enzyme/DNA storage |

System Preparation:

1. Add a volume of ddH2O 10 times as specified by the label on the microtube containing the primer into said
   microtube to dilute it to a concentration of 10μm/μL. This figure varies for each primer. [1]
2. Add 400μL of ddH2O into the microtubes containing 4μg of template for BD-tau/T-tau and mix.
3. Add, in order, 22μL of ddH2O, 25μL of 2X mix, 1μL of both the F and R primer solutions [2] prepared during the
   1st step for the template for BD-tau/T-tau, in addition to 1μL of the template itself to the microtube.
4. Repeat steps 1-3 for each both BD-tau and T-tau
5. Repeat steps 1-4 for all 6 sets of BD-tau and Τ-tau
6. Transfer all systems to a PCR machine
7. PCR cycle:
8. Preheat 95ºC for 5min [3]
9. Heat to 95ºC for 15sec [4]
10. Cool to 63ºC for 30sec [5]
11. Heat to 72ºC for 1min [6]
12. Repeat ii-iv 35 times
13. Hold temperature at 72ºC for 10min [7]
14. Cool to 12ºC for storage [8]

 Note：

[1] Each primer is of different length and thus requires a different volume of solvent to dilute to the target molarity. The
figure given on the tube is the volume of ddH2O necessary to dilute the primers to 100μm/μL.

[2] These solutions containing the primers have a primer concentration of 10μM/μL

[3] This denatures all strands of target DNA (breaks the H-bonds), allowing for primer attachment later. The temperature is
held here to ensure thorough denaturing.

[4] This temperature spike serves the same purpose as the 10-minute one and occurs for each cycle.

[5] This allows for primer attachment. The temperature varies depending on the chosen primer.

[6] This is the optimal functioning temperature for T. aquaticus polymerase, thus starting the replication
process.

[7] This ensures that the polymerization process is fully complete for all newly produced strands.

[8] Setting the temperature lower stabilizes the newly replicated strands of DNA.

**1.3 Gel Electrophoresis**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| 1X TAE powder | 1g (1g/1L of gel) | Working concentration 40mM Tris base, 20mM acetic acid, 1 mM EDTA, pH 8.0 |
| ddH2O | 100mL |  |
| Agarose powder | 1g (1g/1L of gel) |  |
| 10,000X YeaRed Nucleic Acid Gel stain | 10μL(10μL/block of gel) |  |
| 6X Loading buffer | 20μL(10μL/trial) | Contains bisphenol blue |
| Cas12a PCR System | 250 (50μL/trial) | See “Target Protein DNA PCR” for specifics |

   Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Agarose gel electrophoresis mold | 1 |  |
| Agarose gel electrophoresis mold comb | 1 | 13 holes total |
| Pipette | 1 | Capacity 1μL-50μL |
| Gel electrophoresis machine | 1 | Gel block oriented horizontally; DNA runs parallel to the ground, distinct from SDS-PAGE setup |
| Measuring cylinder | 1 | Capacity ≥ 100mL |
| Conical flask | 1 | Capacity ≥ 100mL |

Gel Block Preparation:

1. Measure 1 L ddH2O using the measuring cylinder and transfer to a conical flask.
2. Add 1 bag of TAE buffer (~18.4 g) to the ddH2O in step 1.
3. Add 1g of agarose powder to the ddH2O in step 1.
4. Mix the solution thoroughly by shaking the flask until all added powders are dissolved.
5. Pour the solution into the gel electrophoresis mold.
6. Place the comb into the gel electrophoresis mold and fix it in place in the designated slots. Wait for the gel to
   solidify.
7. System Preparation:
8. Add 10 μL of 6X loading buffer to the PCR system from the previous procedure.
9. Mix evenly.
10. Repeat steps 7-8 for all PCR systems across all trials.
11. Electrophoresis:
12. Place the gel block into the gel electrophoresis machine. Ensure that the side of the gel block with holes is placed
    opposite to the cathode.
13. Add the 1X TAE buffer to the electrophoresis setu[
14. Using the pipette, add 20μL of 2k marker to the topmost hole.
15. Add 50μL of the cas12a PCR system into the subsequent holes.
16. Repeat step 13 for both PCR systems.
17. Repeat steps 13-14 for all trials.
18. Set the gel electrophoresis machine to 150 V and allow it to run for 20-30min. Stop once the bisphenol blue in the loading
    buffer has reached near the end of the gel block.

**1.4 DNA Recovery From Gel of Cas12a**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes |
| Anhydrous ethanol | 1500μL(500μL/trial) |  |
| B2 buffer | 1800μL(600μL/trial) |  |
| Wash solution | 4500μL(1500μL/trial) |  |

  Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes |
| Water Bath | 1 |  |
| Gel transilluminator | 1 |  |
| Scalpel | 1 |  |
| Pipette | ≥1 | Capacity between 10μL-100μL |
| Pipette | ≥1 | Capacity between 100μL-1000μL |
| Centrifuge | 1 |  |

Preparation:

1. Check whether there is sediment in Buffer B2
2. Add 500μL of anhydrous ethanol to the wash solution
3. Adjust the water bath pot to 50ºC
4. Gel Liquification
5. Place the block of gel under the gel transilluminator. [1]
6. Cut out the gel block containing the target fragment from the agarose gel using the scalpel and weigh it. Record the
   mass
7. Transfer the gel containing the target DNA to an EPtube. Dispose of the rest of the gel block.
8. Add 600μL B2 buffer to the EP tube containing the target DNA to the agarose gel block and place the EP tube in a 50ºC
   water bath for 11 minutes. [2]
9. Transfer the solution from step 7 into the binding column, centrifuge at 8000rpm for 30 sec, then discard the eluate.
   [3]
10. DNA Extraction:
11. Add 500μL wash solution, then centrifuge the EP-tube-binding-column complex at 9000rpm for 30 seconds. Discard
    the eluate. [4]
12. Repeat step 9 once
13. Centrifuge the empty binding column [5] at 9000rpm for 1 minute
14. Place the binding column into a clean 1.5mL EP tube and add 30μl of elution buffer to the adsorption film in the center of
    the binding column.
15. Allow the elution buffer to sit at room temperature for 1 minute
16. Centrifuge the binding-column-test-tube complex at 9000rpm for 1 minute and preserve the DNA solution in the tube.
    [6]

 Note：

[1] This shows where the target DNA is in the gel. The DNA fluoresces due to the nucleic acid gel stain added in the
previous procedure.

[2] This liquifies the gel.

[3] The centrifugal force pushes the solution through the adsorption film in the binding columns, leaving the DNA attached
to the film.

[4] This washes the film and removes unwanted materials from the adsorption film.

[5] The binding columns still contain the BD-tau/T-tau DNA.

[6] This removes the DNA from the binding column and suspends it in the elution buffer.

**1.5 Double Digestion of pET28a and cas12a**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| 10X Buffer | 15 μL (5 μL/trial) |  |
| Cas12a DNA | 25 μL (25 μL/trial) | Recovered from gel in previous procedure |
| pET-28a | 25 μL (25 μL/trial) | Recovered from gel in previous procedure |
| Hind III | 3 μL(1μL/trial) | Hind III is an endonuclease that recognizes the following sequence: 5\' A^AGCTT 3\' [1] |
| Nhe I | 3 μL(1μL/trial) | Nhe I is an endonuclease that recognizes the following sequence: 5\' G^CTAGC 3\' |

  Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Micropipettes (P10, P20, P200) | 1 set |  |
| Sterile pipette tips | 1 box |  |
| Microtubes | 3 | 1 per reaction + extra, capacity 200μL |
| Test tube rack | 1 |  |
| Microcentrifuge | 1 |  |
| Vortex mixer | 1 |  |
| Water bath | 1 | Maintain 37°C |
| Styrofoam box with ice inside | 1 | Enzyme/DNA storage |
| Pipette | 1 | Capacity between 1μL-20μL |

System Preparation:

1. Add 15 μL ddH2O, 10μL of the target type of DNA for double digestion, 5μL of the 10X buffer, 1 μL Hind III
   and 1 μL Nhe I to a PCR tube.
2. Repeat step 1 for both remaining types of DNA subject to double digestion (Cas12a /pET-28a).
3. Place all samples into microcentrifuge for 30sec.
4. Incubation:
5. Place all samples into test tube rack.
6. Keep these solutions in the water bath and allow to incubate for 2h at 37ºC.

**1.6 Ligation** **of pET28a and cas12a**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity per Trial | Notes & Parameters |
| 10X buffer | 2μL |  |
| T4 ligase: | 1μL |  |
| Cleaved cas12a DNA sequences | 3 μL |  |
| Cleaved pET-28a | 1μL |  |
| ddH2O | 13μL |  |
| PCR tubes | 3 |  |

Ligated Solution Preparation:

1. Set the water bath to 50ºC and wait for its temperature to reach 50ºC.
2. Place the PCR tubes containing the double digestion solution in the water bath set to 50ºC for 15min. [1]
3. System Preparation:
4. Add 13μL ddH2O, 3μL of the target DNA sequence, 2μL of the 10X buffer and 1μL of pET-28a to an EP tube.[2]
5. Repeat step 1 for the remaining sequences subject to ligation (Cas12a).
6. Place all PCR tubes in microcentrifuge for 30sec
7. Incubate in PCR machine for 1 hour under 16ºC

Note：

[1] “^” represents the site of cleavage.

[2] This denatures Nhe I and Hind III.

**1.7 Heat shocking**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| BL21 culture |  |  |
| DH5a culture |  |  |
| PET-28a-cas12a | 10μL (5μL/trial) |  |
| LB broth | ≥3600μL (900μL/sample) | The broth should be sterile and should not contain kanamycin. |
| CaCl2 |  |  |

  Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Cooler | 1 | Generates temperatures of -80ºC or lower |
| Water bath | 1 | Temperature range includes 42ºC |
| Petri dishes | ≥6 | Prepared during LB preparation step; 0.1% kanamycin |

Storage:

1. Store the competent E.coli in a fridge set to -80ºC.
2. Sample Preparation:
3. Add 5μL of pET-28a solution carrying the cas12a gene to the BL21/DH5α e.coli samples and gently mix.
4. Repeat step 2 three additional times to generate a total of four samples, representing all possible combinations of one E.
   coli strain with one gene (BL21 + BD-tau, BL21 + T-tau, DH5α + BD-tau, DH5α + T-tau). [1]
5. Heat Shocking & Culturing:
6. Place the microtubes containing the e.coli samples into a 42ºC water bath for 45sec. [2] Quickly transfer the samples to
   an ice bath to cool.
7. Add 900 μL of kanamycin-free LB broth each microtube. Mix evenly by repeatedly drawing in and dispensing the LB using
   the pipette.
8. Once the e.coli-LB solutions are evenly mixed, centrifuge at 8000rpm for 5min.
9. Remove 900 μL of supernatant from all microtubes.
10. Use the samples to inoculate petri dishes. Allow the E.coli colonies to develop for 12-16h at 37ºC.
11. Preparation for PCR:
12. Pick random colonies in the culture & split each sampled colony into 2 equal portions; one will be used for PCR later
    the other inoculate LB broth [3] which contains 0.1% kanamycin from the LB preparation step [4].

 Note:

[1] 2 strains were cultured to find a (relatively) optimal strain for expressing the target proteins.

[2] This sudden change in heat increases membrane permeability & makes the e.coli cells competent.

[3] The petri dishes will be used to verify transfer success later, and the e.coli in the broth will be used for protein
expression.

[4] The pET-28a plasmid which will be used as a vector for the target genes later in the procedure encodes a gene which
offers kanamycin resistance. This helps verify transformation success, as only e.coli which have received the
kanamycin-resistance gene encoded on the pET-28a plasmid will survive the kanamycin.

**2.0  PCR For Target Genes Inside E.coli**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity per Trial | Notes & Parameters |
| Buffer 2X | 50 μL (5μL /sample) |  |
| F-primer | 2 μL (0.2μL/sample) |  |
| R-primer | 2 μL (0.2μL/sample) |  |
| ddH2O | 46 μL (4.6μL/sample) |  |
| BL21 e.coli carrying cas12a DNA | N/A [1] | Engineered for superior protein-expressing capabilities |
| DH5a e.coli carrying cas12a DNA | N/A | Engineered for superior transformation efficiency |

   Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Cooler | 1 | Generates |
| Microtubes | 16 | 4 for each combination of DNA and e.coli strain |

Preparing the System:

1. Add 4.6T μLddH2O, 5.0T μLbuffer, 0.2T μL F primer and 0.2T μL R primer to a test tube, where T is the number of
   desired trials. In this experiment, T=10.
2. Separate the system equally into T 10 μL portions.
3. Gently scrape e.coli colonies off the petri dishes used to cultivate e.coli after the heat-shocking procedure using a
   pipette[1] .
4. Transfer some colonies to the microtubes containing the PCR solution. Swap the pipette tip between each culture.
   [2]
5. Repeat steps 4-5 for all cultures.
6. PCR:
7. Preheat to 95ºC for 5min
8. Heat to 95ºC for 15sec
9. Cool to 65ºC for 15sec
10. Heat to 72ºC for 10sec
11. Repeat steps 8-10 30 times
12. Hold temperature at 72ºC for 10min

 Note:

[1] The colonies were extracted by scraping the petri dishes with a pipette tip, as opposed to being added as a solution,
thus making their volume negligible.

[2] This prevents cross-contamination.

',
},
{
"title": '2. Protein induction and expression',
"content": '

**2.1 Monitoring *E.coli* Growth Curve**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| LB | 8mL (2mL/test tube) | Contains 50ng/mL Kan+ (kanamycin) |

   Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Shaker | 1 | The shaker is sealed and conditioned |
| Timer | 1 | Optional, a clock may be used instead |
| Spectrophotometer | 1 | 500μL cuvette recommended |
| Test tube | 4 | Capacity 15mL (2 samples/test tube) |
| Pipette | ≥1 | Capacity between 100μL-1000μL |

Sample Preparation:

1. Mingle 2mL LB (K+ with 50ng/ml concentration) with 50 μL pET18a-cas12a E. coli cultured in the heat shocking step. Mix
   evenly
2. Place all tubes into 37˚C shaker.
3. Observation:
4. Return to record data for OD600 at different points for both proteins. (0.5, 1, 2, 4, 6, 8, 12 and 24h) [1]
5. OD 600 Protocol:
6. Remove the cuvette from the spectrophotometer & clean its exterior if needed.
7. Add 500μL of sterile LB to the spectrophotometer cuvette.
8. Place the cuvette back into the spectrophotometer
9. Measure the OD 600 value of the blank. [2]
10. Remove & empty the cuvette.
11. Transfer the culture to the cuvette and measure the absorbance.
12. Place the cuvette back into the spectrophotometer.
13. Measure the OD-600 of the sample.

* Repeat steps v-viii for all samples.
* Note:

[1] OD600 is an approach which measures the light absorbance of bacterial or yeast cultures at 600nm wavelength using a
spectrophotometer. As the curve requires infrequent, periodic measurements, this procedure can take place alongside other
procedures.

[2] This pares the spectrophotometer.

**2.2 Protein Extraction & Purification**

 Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| Non-denaturing lysis buffer | 5mL |  |
| Wash solution | 3mL (500μL/Wash) |  |
| Ice |  |  |

  Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Centrifuge | 1 | Can generate 1000rpm--10000rpm |
| Sonicator | 1 |  |
| Beaker | 1 |  |

Sonification Lysis Preparation:

1. Centrifuge the tubes containing the E. coli (pET28a-cas12a) culture at 6000rpm and 4˚C for 15 minutes to concentrate the
   E. coli into a pellet.
2. Extract & discard the supernatant using a pipette.
3. Add 20mL non-denaturing lysis buffer to the pellet. [1]
4. Vortex the solution to mix thoroughly.
5. Sonification Lysis:
6. Uncap the test tube containing the E.coli culture and place it into a beaker containing ice. [2]
7. Place the beaker on the sonicator’s platform with the test tube’s opening positioned directly under the needle and
   raise the platform until the needle is touching the solution. Ensure that the needle is not touching the test tube walls.
   [3]
8. Sonicate at 300 W with 2 s on/2 s off cycles for 30 min.
9. Remove the test tube from the sonicator once sonication is complete.
10. Repeat steps 5-8 for T-tau.
11. Extracting Target Proteins:
12. Centrifuge for 30 minutes at 10000 rpm at 4˚C.
13. Extract the supernatant, transfer to an 15 mL test tube and place it on ice.
14. Extract 20 μL of the supernatant for SDS-PAGE later.
15. Add 1 mL his-tag purification resin to an EP tube.
16. Centrifuge at 1000rpm at 4˚C for 10 seconds. Discard the supernatant. [4]
17. Add 500 μL non-denaturing lysis buffer to the EP tube and centrifuge at 1000 rpm at 4˚C for 10 seconds. [5]
18. Repeat step 15 2 more times.
19. Add his-tag purification resin to test tubes containing BD-tau. Mix evenly.
20. Incubate for 60min at 4ºC on a shaker.
21. Add 10mL of BD-tau supernatant and His-tag purification resin into large binding columns. [6] Keep the bottom capped.
22. Allow protein solution containing BD-tau bound to his-tag purification resin to pass through the binding column. The resin
    beads should remain above the filter.
23. Add 500μL wash solution to the binding column and allow it to pass through the binding column film.
24. Collect 20μL of the eluate for sampling later. Store the rest.
25. Repeat steps 20-21 2 more times.
26. Add 500μL elution buffer to the binding column and allow it to pass through the binding column film.
27. Collect 20μL of the eluate for sampling later. Store the rest.
28. Repeat step 23-24 3 more times.
29. Collect the final eluate.

Repeat steps 10-26 for T-tau
 Note:

[1] This suspends the e.coli in preparation for lysis

[2] Sonication generates heat. The ice cools the sample to prevent heat-induced protein denaturation.

[3] The needle and test tube would damage each other if in contact for extended periods during operation.

[4] This leaves only the resin on the binding column film. The eluate is the storage solution for the resin.

[5] The non-denaturing lysis buffer is used to wash the resin

[6] This binding column contains the his-tag purification resin beads and is larger than the binding columns used in the EP
tubes.

**2.3 SDS-PAGE**

 Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| ddH2O |  |  |
| 30% Acr-Bis |  |  |
| Gel buffer A | 3.75 mL |  |
| Gel buffer B | 2 mL |  |
| Mixing cups |  |  |

 Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
|  |  |  |

Sample Preparation:

1. Add 10 μL protein sample and 10 μL 2X loading buffer to an EP tube.
2. Heat the microtube to 95˚C for 5 minutes.
3. Casting SDS-PAGE Gel:
4. Fix the gel casting cassette into the casting frames.
5. Prepare the 15% resolving gel by adding 3.6ml ddH2O, 7.5 mL 30% Acr-Bis, 3.75 mL Gel Buffer A, 0.15
   mL 10% APS and 0.009 mL TEMED to a mixing cup.
6. Pipette the resolving gel into the gel casting cassette.
7. Add anhydrous ethanol to the gel casting cassette until the ethanol level reaches the height of the shorter plate
8. Wait for 10-15min.
9. Decant the ethanol.
10. Prepare the 5% stacking gel by adding 1.33mL ddH2O, 0.67 mL 30% Acr-Bis, 2 mL Gel Buffer B, 0.04 mL 10%
    APS and 0.004 mL TEMED to a mixing cup.
11. Pipette the stacking gel into the gel casting cassette.
12. Insert the comb into the gel casting cassette.
13. Wait for 10-15min.
14. Carefully remove the combs from the gel casting cassettes.

Buffer Preparation:

1. Prepare a conical flask with capacity 1 L or greater.
2. Add 1 bag (approx. 18.4g) of tris-glycine SDS buffer into the conical flask.
3. Measure 1 L of water using a measuring cylinder.
4. Add the water to the conical flask.
5. Mix evenly until tris-glycine SDS buffer is fully dissolved.
6. SDS-PAGE Electrophoresis:
7. Transfer the cassettes to an SDS-PAGE gel electrophoresis tub.
8. Add to each gel block the following samples:
9. Markers for the first 2 cells. For the remaining 9 cells, add 10 μL CL, FT, W1, W2, W3, E1, E2, E3 and E4 in order.
10. Add buffer to the space between the cassettes within the tank until the space is completely full.
11. Place the cap onto the setup. Ensure that the anodes and cathodes are properly paired.
12. Plug the cap’s wires into an electrophoresis power supply. Ensure that the anodes and cathodes are properly paired.
13. Turn on the power supply. Set the voltage to 60 V for 35min. Then, set the voltage to 130 V for 40min.
14. Gel Dyeing:
15. Cut the stacking gel from the gel block using a scalpel and discard it.
16. Place the resolving gel block into a dyeing tub and add 20 mL of Coomassie brilliant blue dye to the tub.
17. Place the dyeing tub onto a shaker and allow to dye overnight.

Place the dyed gel block into an SDS-PAGE gel reader and document the results.

',
},
{
"title": '3. crRNA preparation',
"content": '

**3.1 Preparation of crRNA**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| crRNA-T7-promoter-F | 300μL | 10 pM |
| crRNA-T7-R | 300μL | 10 pM |
| Vitro Transcription Kit | 1 | / |
| RNA Clean-up Kit | 1 | / |
| **RNase-free Water** | 50ml | / |

 Equipment statistics:

|  |  |  |
| --- | --- | --- |
| Equipment Name | Quantity | Notes & Parameters |
| Spectrophotometer | 1 | / |
| PCR | 1 | / |
| RNase-free Laminar Flow Cabinet | 1 | / |

Preparation Procedure:

1. According to the design, primers crRNA-T7-promoter-F and crRNA-T7-R, used for preparing the template DNA for crRNA
   synthesis, were synthesized by the company. The primers were dissolved in RNase-free water, and their concentrations were
   measured using a NanoDrop spectrophotometer and adjusted to 10 µM.
2. 9 µL of crRNA-T7-promoter-F, 9 µL of crRNA-T7-R, and 2 µL of 10x Taq DNA Polymerase PCR Buffer were aliquoted into a PCR
   tube. The mixture was vortexed and briefly centrifuged to collect the liquid at the bottom of the tube.
3. The tube was placed in a PCR machine. Step 1: Denaturation at 94 °C for 5 minutes. Step 2: 74 cycles were run with the
   temperature decreasing by 1 °C every 30 seconds, stopping at 20 °C. This yielded 20 µL of crRNA transcription template
   DNA.

After obtaining the crRNA transcription template DNA, the HiScribe T7 Quick High-Yield RNA Synthesis Kit was used to prepare
the crRNA. The specific steps are as follows:

1. 20 µL of crRNA transcription template DNA, 1 µL of RNase Inhibitor, 5 µL of RNA Polymerase Mix, and 25 µL of dNTP mixture
   were aliquoted into a PCR tube. The mixture was vortexed and briefly centrifuged.
2. The tube was placed in a PCR machine and incubated at 37 °C for 15 hours.
3. 1 µL of DNase was added, and incubation continued at 37 °C for 1 hour. After the reaction, 50 µL of pre-purified crRNA was
   obtained.

After transcription, the crRNA was purified using the RNA Clean & Concentrator TM-5 kit. The specific steps are as
follows:

1. The entire 50 µL of pre-purified crRNA was transferred to a 1.5 mL nuclease-free microcentrifuge tube. 100 µL of RNA
   Binding Buffer was added and mixed thoroughly, followed by the addition of 150 µL of 100% ethanol and thorough mixing.
2. The mixture was transferred to a silica spin column and centrifuged at 10,000 × g for 1 minute. The flow-through was
   discarded.
3. 400 µL of RNA Prep Buffer was added to the column, followed by centrifugation at 10,000 × g for 1 minute. The flow-through
   was discarded.
4. 700 µL of Wash Buffer was added to the column, followed by centrifugation at 10,000 × g for 1 minute. The flow-through was
   discarded. This washing step was repeated once.
5. The empty column was centrifuged at 10,000 × g for 2 minutes to remove residual ethanol. The column was then transferred
   to a new 1.5 mL nuclease-free microcentrifuge tube.
6. 50 µL of Nuclease-free Water was added directly to the silica membrane. After incubating for 2 minutes, the column was
   centrifuged at 10,000 × g for 2 minutes. The column was discarded, and the eluate (containing the purified crRNA) was
   collected, yielding 50 µL of purified crRNA.
7. The concentration of the purified crRNA was measured using a NanoDrop spectrophotometer and adjusted to 10 µM. After
   adjustment, the crRNA was aliquoted into PCR tubes, 20 µL per tube. The aliquots were stored at -80°C for future use.

(Note: Throughout the crRNA preparation experiment, nuclease-free consumables were used, and all procedures were performed
in an RNase-free environment.)

',
},
{
"title": '4. Aptamer-based biosensor',
"content": '

****4.1 Preparation of Aptamer-dsDNA****

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| Tau aptamer-F | 200μL | 10μmol |
| dsDNA-F | 200μL | 10μmol |
| dsDNA-R | 200μL | 10μmol |
| **Streptavidin magnetic beads** | 1ml | 1ml |
| ComDNA | 500μL | 10μmol |
| PBST | 500ml |  |

 Equipment statistics:

|  |  |
| --- | --- |
| Equipment Name | Quantity |
| NanoDrop | 1 |
| PCR | 1 |
| RNase-free Laminar Flow Cabinet | 1 |
| **Magnetic Rack** | 3 |

Preparation Procedure:

1. The corresponding customized aptamer (Tau aptamer-F) and the forward/reverse primers (dsDNA-F and dsDNA-R) for generating
   the Cas12a-activating dsDNA were synthesized by the company and adjusted to a concentration of 10 μmol.
2. The three components were mixed in a 1:1:1 ratio in a solution containing 1× Taq Buffer and thoroughly mixed.
3. The mixture was incubated at 95 °C for 5 minutes, followed by gradual cooling to room temperature at a rate of 2 °C per
   minute.
4. The resulting aptamer-dsDNA complex was quantified using a NanoDrop spectrophotometer, diluted to a final concentration of
   5 μM, and stored at -20°C for future use.

**4.2 Beads-ComDNA/Aptamer-dsDNA Complex**

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| Tau aptamer-F | 200μL | 10μmol |
| dsDNA-F | 200μL | 10μmol |
| dsDNA-R | 200μL | 10μmol |
| **Streptavidin magnetic beads** | 1ml | 1ml |
| ComDNA | 500μL | 10μmol |
| PBST | 500ml |  |

 Equipment statistics:

|  |  |
| --- | --- |
| Equipment Name | Quantity |
| NanoDrop | 1 |
| PCR | 1 |
| RNase-free Laminar Flow Cabinet | 1 |
| **Magnetic Rack** | 3 |

Preparation Procedure:

1. Take out the streptavidin magnetic beads stored at 4 °C and vortex thoroughly to ensure a homogeneous suspension.
2. Transfer 20 μL of the bead suspension to a nuclease-free 1.5 mL microcentrifuge tube. Place the tube on a magnetic rack
   for 1 minute to separate the beads from the storage solution. Carefully aspirate and discard the storage solution.
3. Add 200 μL of 1× PBST buffer, vortex to mix, then place the tube on the magnetic rack for 1 minute. Discard the
   supernatant. Repeat this washing step three times to thoroughly remove the NaN₃ preservative from the streptavidin
   magnetic bead storage solution.
4. After washing, add 20 μL of the pre-quantified, 15 μM biotin-modified ComDNA to the tube. Then add 1× PBST buffer to bring
   the total volume to 200 μL.
5. Vortex the mixture and then incubate on a rotator for 30 minutes to allow thorough binding between the ComDNA and the
   streptavidin on the beads.
6. After incubation, place the tube on the magnetic rack for magnetic separation. Aspirate and discard the supernatant to
   remove excess ComDNA that did not bind to the beads.
7. Add 200 μL of 1× PBST buffer, vortex to mix, place on the magnetic rack for 1 minute, and discard the supernatant. Repeat
   this washing step twice.
8. After washing, add 20 μL of the pre-prepared 5 μM aptamer-dsDNA complex to the tube. Add 1× PBST buffer to bring the total
   volume to 200 μL.
9. Vortex the mixture and then incubate on a rotator for 30 minutes to allow hybridization between the aptamer-dsDNA and the
   ComDNA on the beads.
10. After incubation, place the tube on the magnetic rack for magnetic separation. Aspirate and discard the supernatant to
    remove excess aptamer-dsDNA that did not bind.
11. Add 200 μL of 1× PBST buffer, vortex to mix, place on the magnetic rack for 1 minute, and discard the supernatant. Repeat
    this washing step three times.
12. After the final wash, add 900 μL of 1× PBST buffer to resuspend the "Beads-ComDNA/Aptamer-dsDNA" complex. Aliquot 18 μL of
    the suspension into individual PCR tubes for subsequent detection assays.

',
},
{
"title": '5. Functional Validation and Optimization ',
"content": '

**5.1 Biosensing platform optimization**

Assessment of the Aptamer\'s Effect on Cas12a Activation by dsDNA

Goal: It is first necessary to verify whether the dsDNA can still be cleaved by Cas12a after binding to the aptamer.

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| Tau aptamer-F | 200μL | 10μmol |
| dsDNA-F | 200μL | 10μmol |
| dsDNA-R | 200μL | 10μmol |
| **Streptavidin magnetic beads** | 1ml | 1ml |
| ComDNA | 500μL | 10μmol |
| PBST | 500ml |  |

 Equipment statistics:

|  |  |
| --- | --- |
| Equipment Name | Quantity |
| NanoDrop | 1 |
| PCR | 1 |
| RNase-free Laminar Flow Cabinet | 1 |
| **Magnetic Rack** | 3 |

Preparation Procedure:

1. Prepare Tau aptamer-dsDNA solutions at different concentrations (5, 10, 15, 20, 25, 30 μmol). Use a dsDNA solution (30
   μmol) as a positive control.
2. Add the three components in a 1:1:1 ratio to a solution containing 1× Taq Buffer and mix thoroughly.
3. Incubate the mixture at 95 °C for 5 minutes, then gradually cool to room temperature at a rate of 2 °C per minute.
4. Quantify the resulting aptamer-dsDNA using a NanoDrop spectrophotometer and store at –20 °C for future use.
5. Add the prepared samples to the Cas12a reaction system (e.g., a 2.5× Cas12a reporter assay system) and measure the
   time-dependent fluorescence intensity curves to confirm that the fluorescence intensity is positively correlated with the
   concentration of Tau/BD-tau aptamer-dsDNA.
   1. **Screening of ComDNA for the Highest Signal-to-Noise Ratio**

Goal: It is first necessary to verify whether the dsDNA can still be cleaved by Cas12a after binding to the aptamer.

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| Tau aptamer-F | 200μL | 10μmol |
| dsDNA-F | 200μL | 10μmol |
| dsDNA-R | 200μL | 10μmol |
| **Streptavidin magnetic beads** | 1ml | 1ml |
| ComDNA | 500μL | 10μmol |
| PBST | 500ml |  |
| qPCR-F | 10pM |  |
| qPCR-R | 10pM |  |
| 2XSYBR qPCR Mix | 5ml |  |
| Tau | / | 5mg/ml |
| BD-tau | / | 5mg/ml |

 Equipment statistics:

|  |  |
| --- | --- |
| Equipment Name | Quantity |
| NanoDrop | 1 |
| PCR | 1 |
| RNase-free Laminar Flow Cabinet | 1 |
| **Magnetic Rack** | 3 |
| **qPCR** | 1 |

Procedure:

1. Prepare five distinct ComDNA-Tau aptamer-dsDNA complexes according to the steps described in Section 4.2.
2. Add a 10 μmol excess of Tau protein/BD-tau and incubate at 4 °C for 60 minutes.
3. Searate the supernatant using a magnetic rack and store it at -80 °C.
4. Quantify the amount of released BD-tau/Tau aptamer-dsDNA complex using quantitative real-time PCR (qPCR). Use a control
   sample where an equivalent volume of water is added instead of Tau protein. Calculate the signal-to-noise ratio, using
   actin as the internal reference gene, to identify the ComDNA sequence yielding the highest signal-to-noise ratio.

**qPCR Reaction Setup:**

|  |  |
| --- | --- |
| Component | Volume |
| 2XSYBR qPCR Mix | 12.5 |
| Template | 7 |
| qPCR-F | 7 |
| qPCR-R | 7 |
| R-free H2O | 16.5 |

**qPCR ProgramqPCR Program:**

|  |  |  |
| --- | --- | --- |
| Temperature | Time | Cycle |
| 95°C | 2:00 |  |
| 95°C | 1:00 | 30 |
| 60°C | 0:30 |
| 72°C | 0:30 |
| 95°C | 0:15 |  |
| 60°C | 1:00 |  |
| 95°C | 0:15 |  |
| 60°C | 1:00 |  |

1. After adjusting the qPCR program, data were collected.
   1. **Determination of the Concentrations of ComDNA and the Tau/BD-tau Aptamer-dsDNA Complex**

Goal：The screening of aptamer-switched ComDNA requires the quantification of both the ComDNA and the Tau/BD-tau
aptamer-dsDNA complex.

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| Tau aptamer-F | 200μL | 10μmol |
| dsDNA-F | 200μL | 10μmol |
| dsDNA-R | 200μL | 10μmol |
| **Streptavidin magnetic beads** | 1ml | 1ml |
| ComDNA | 500μL | 10μmol |
| PBST | 500ml |  |
| qPCR-F | 10pM |  |
| qPCR-R | 10pM |  |
| 2XSYBR qPCR Mix | 5ml |  |
| Tau | / | 5mg/ml |
| BD-tau | / | 5mg/ml |

 Equipment statistics:

|  |  |
| --- | --- |
| Equipment Name | Quantity |
| NanoDrop | 1 |
| PCR | 1 |
| RNase-free Laminar Flow Cabinet | 1 |
| **Magnetic Rack** | 3 |
| **qPCR** | 1 |

Procedure:

1. The "beads-biotinylated ComDNA-Tau aptamer-dsDNA" complex was constructed by adding different concentrations (1, 2, 5, 10,
   20 μM) of ComDNA to the BD-tau/Tau aptamer-dsDNA, according to the protocol in Section 4.2.
2. Add a 10 μmol excess of Tau protein/BD-tau and incubate at 4 °C for 60 minutes.
3. Searate the supernatant using a magnetic rack and store it at -80 °C.
4. Quantify the amount of released BD-tau/Tau aptamer-dsDNA complex using quantitative real-time PCR (qPCR). Use a control
   sample where an equivalent volume of water is added instead of BD-tau/Tau protein. Calculate the signal-to-noise ratio,
   using actin as the internal reference gene.

**qPCR Reaction Setup:**

|  |  |
| --- | --- |
| Component | Volume |
| 2XSYBR qPCR Mix | 12.5 |
| Template | 7 |
| qPCR-F | 7 |
| qPCR-R | 7 |
| R-free H2O | 16.5 |

**qPCR ProgramqPCR Program:**

|  |  |  |
| --- | --- | --- |
| Temperature | Time | Cycle |
| 95°C | 2:00 |  |
| 95°C | 1:00 | 30 |
| 60°C | 0:30 |
| 72°C | 0:30 |
| 95°C | 0:15 |  |
| 60°C | 1:00 |  |
| 95°C | 0:15 |  |
| 60°C | 1:00 |  |

1. After adjusting the qPCR program, data were collected.

**5.3. Determination of the Incubation Time for BD-tau/Tau with the ComDNA-Aptamer-dsDNA Complex**

Goal: To optimize the incubation time of BD-tau/Tau protein with the sensor to ensure experimental success.

Procedure:

1. Using the ComDNA sequence and concentration screened in step 5.2, prepare the sensor according to the protocol in section
   4.2.
2. Add 10 μM BD-tau/Tau and incubate for different time periods (10, 20, 30, 40, 50, 60 min) to assess the release of the
   BD-tau/Tau aptamer-dsDNA complex.
3. Perform quantitative analysis using qPCR. The PCR protocol is as follows.

**qPCR Reaction Setup:**

|  |  |
| --- | --- |
| Component | Volume |
| 2XSYBR qPCR Mix | 12.5 |
| Template | 7 |
| qPCR-F | 7 |
| qPCR-R | 7 |
| R-free H2O | 16.5 |

**qPCR ProgramqPCR Program:**

|  |  |  |
| --- | --- | --- |
| Temperature | Time | Cycle |
| 95°C | 2:00 |  |
| 95°C | 1:00 | 30 |
| 60°C | 0:30 |
| 72°C | 0:30 |
| 95°C | 0:15 |  |
| 60°C | 1:00 |  |
| 95°C | 0:15 |  |
| 60°C | 1:00 |  |

**5.4 Quantitative Analysis of the Cas12a Reporting System**

Goal：After determining the concentrations of ComDNA and the Tau aptamer-dsDNA complex as well as the incubation time, the
complete sensor response and signal output procedure was performed to detect BD-tau/Tau protein.

Material statistics:

|  |  |  |
| --- | --- | --- |
| Material Name | Quantity | Notes & Parameters |
| Tau | / | 5mg/ml |
| BD-tau | / | 5mg/ml |
| Magnetic Beads-cDNA/Aptamer-dsDNA | 2ml |  |
| Cutsmart buffer | 2ml |  |
| Enzyme Inhibitor | 5ml |  |
| crRNA | 1ml | 10 μM |
| Cas12a protein | 1ml | 10 μM |
| ss-DNA |  | 10 μM |

 Equipment statistics:

|  |  |
| --- | --- |
| Equipment Name | Quantity |
| **Multimode Microplate Reader** | 1 |
| NanoDrop | 1 |

Procedure:

1. Prepare a stock solution of standard concentration using BD-tau/Tau protein, and then perform a series of twofold
   dilutions to obtain protein samples of varying concentrations.
2. Add 2 μL of each gradient concentration sample to 18 μL of the "Magnetic Beads-cDNA/Aptamer-dsDNA" complex solution. Mix
   well and incubate at room temperature for 40 minutes.
3. After incubation, perform magnetic separation for 1 minute, and then transfer 10 μL of the supernatant to a new PCR tube.
4. Take 10 μL each of the post-reaction supernatant from the "Magnetic Beads-cDNA/Aptamer-dsDNA" complex solution (or
   standard concentrations of Cas12a-activating dsDNA).
5. Add 2 μL of each sample to the bottom of the wells in a microplate.
6. In a 200 μL PCR tube, prepare the detection mixture by adding the following:

|  |  |
| --- | --- |
| Component | Volume |
| 10x CutSmart buffer | 10 μL |
| RNase inhibitor | 1 μL |
| 10 μM crRNA | 0.5 μL |
| 10 μM Cas12a protein | 0.5 μL |
| 10 μM ssDNA reporter probe | 5 μL |
| RNA-free H20 | 73 μL |

1. Vortex the mixture thoroughly to ensure homogeneity. Use a mini centrifuge to spin down any liquid on the tube walls.
2. Aliquot 9 μL of the detection reaction mixture into each pre-loaded well of the microplate. Dispense the liquid onto the
   left and right sidewalls of the wells.
3. Use a microplate centrifuge to spin the plate, ensuring the reaction mixture collects at the bottom of the wells.
4. Turn on the multimode microplate reader. Set the instrument to read every 1.5 minutes for a total duration of 60 minutes.
   Use an excitation wavelength of 520 nm and an emission wavelength of 480 nm.

**5.5 Evaluation of Sensor Specificity and Effectiveness**

Goal: For comparison, Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), Immunoglobulin E (IgE), and Immunoglobulin G
(IgG) can be selected for specificity detection. The fluorescence intensity is to be analyzed when the concentration of
these analogs is 10 μmol (consistent with the Tau protein concentration).

Procedure:

1. Take 10 μmol solutions of BSA, HSA, IgE, and IgG.
2. Add 2 μL of each protein solution to 18 μL of the "Magnetic Beads-cDNA/Aptamer-dsDNA" complex solution. Mix well and
   incubate at room temperature for 40 minutes.
3. After incubation, perform magnetic separation for 1 minute, and then transfer 10 μL of the supernatant to a new PCR tube.
4. Take 10 μL of the post-reaction supernatant from each "Magnetic Beads-cDNA/Aptamer-dsDNA" complex solution mixture.
5. Add 2 μL of each supernatant to the bottom of individual wells in a microplate.
6. In a 200 μL PCR tube, prepare the detection mixture by adding the following:

|  |  |
| --- | --- |
| Component | Volume |
| 10x CutSmart buffer | 10 μL |
| RNase inhibitor | 1 μL |
| 10 μM crRNA | 0.5 μL |
| 10 μM Cas12a protein | 0.5 μL |
| 10 μM ssDNA reporter probe | 5 μL |
| RNA-free H20 | 73 μL |

1. Vortex the mixture thoroughly and use a mini centrifuge to spin down the liquid collected on the tube walls.
2. Aliquot 9 μL of the detection reaction mixture into each pre-loaded well of the microplate. Dispense the liquid onto the
   left and right sidewalls of the wells.
3. Use a microplate centrifuge to spin the plate, ensuring the reaction mixture is centrifuged to the bottom of the wells.
4. Turn on the multimode microplate reader. Set the instrument to read every 1.5 minutes for a total duration of 60 minutes,
   using an excitation wavelength of 520 nm and an emission wavelength of 480 nm.

For cycle 3-5 the primary experiment steps are identical with those of cycle 1-2，so we do not duplicate the detailed steps here.

**Attachments 1:**

(1) ELISA Random Clinical Trial Generator

#include <iostream>

#include <iomanip>

#include <random>

#include <cstdlib>

#include <ctime>

#include <cmath>

**int** main(**int** argc, **const** **char** \* argv[]) {

**int** varimax, varimin, solutionvol=200;

**int** trialnum;

std::cout << "Insert the number of trials needed:" << std::endl;

std::cin >> trialnum;

std::cout << "Insert the maximum and minimum concentrations for any given trial starting with the smaller parameter:"
<< std::endl;

std::cin >> varimin >> varimax;

**double** trials [trialnum+1];

srand(time(0));

**for** (**int** i=1;i<=trialnum;++i){

**double** random = varimin + rand()%(varimax-varimin);

trials [i] = random;

**int** conplace=0;

**int** concentrations [trialnum+1];

**for** (**int** constor = trials [i]/5;constor>0;constor/=10){

++conplace;

}

concentrations[i] = pow(10,conplace);

**double** substrate = (trials[i]/5)\*2\*pow(10,2-conplace);

std::cout << trials [i] << " " << 5 \* concentrations [i] << " " << substrate << "
"<< solutionvol-substrate << std::endl;

//std::cout << conplace << std::endl;

//std::cout << 5\*concentrations [i] << std::endl;

}

**return** 0;

}

',
},
]
},
] %}
{% set info\_title = "Experiments" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Experiments{% endblock %}

{% extends "layout.html" %}
{% set info\_data = [
{
"title": 'Overview',
"content": '

This comprehensive project design embodies a systematic and rigorous approach to investigating early screening strategies
for Alzheimer’s disease, with a specific focus on BD-tau as a target biomarker(Gonzalez-Ortiz etal ;2023).The methodology
encompasses several stages, first we are screening for high-affinity DNA aptamers targeting BD-tau through SELEX technology.
Then, using the nucleic acid aptamers screened by SELEX and integrating CRISPR-Cas12a signal amplification technology, an
aptamer-based biosensor is constructed to detect BD-tau protein levels in blood samples.This non-invasive detection method
is expected to become a highly effective tool for early diagnosis of AD, possessing significant clinical translational value
and application prospects.By establishing a robust platform for aptamer selection and validation, this project lays a
foundational framework for future development of point-of-care diagnostic devices and further exploration of nucleic
acid-based biomarkers in neurodegenerative disorders.

',
"children": []
},
{
"title": 'Part 1. SELEX-1',
"content": '',
"children": [
{
"title": '1. Construction of pET28a-BD-tau and pET8a-tau plasmids.',
"content": '

![图片10](https://static.igem.wiki/teams/5739/result/1.webp)

Figure 1. Target gene PCR amplification diagram

We constructed pET28a-phf6 and pET28a-tau using restriction digestion and ligation. Agarose gel electrophoresis was
employed to separate and analyze the size of DNA fragments, thereby verifying the accuracy of gene amplification via PCR.
This technique utilizes an electric field to facilitate the migration of DNA fragments, with smaller fragments exhibiting
faster mobility. The size and purity of these fragments are assessed through staining and ultraviolet (UV) observation. By
referencing a molecular weight marker positioned on the left, the approximate base pair (bp) lengths of the bands can be
inferred. When compared to established reference values, the correct bands can be identified. For BD-Tau, the expected band
length is 102 bp, which corresponds to the observed band in the Figure 1. For Tau, the anticipated band length is 105 bp;
the first, second, and fourth bands align with this expectation, while the third band displays inconsistent intensities,
likely due to uneven gel loading. Consequently, the third band is deemed incorrect as it does not match the expected value
and is unsuitable for further experimentation. This analysis underscores the use of agarose gel electrophoresis in selecting
the appropriate plasmids for subsequent recovery and insertion into enzyme-digested vectors through T4 ligase.

![图片11](https://static.igem.wiki/teams/5739/result/2.webp)

Figure 2. Growth of plasmid (BD-Tau & Tau) on LB agar plates

Following restriction digestion and ligation, which involves the precise insertion of plasmids into specific sites on the
enzyme-digested vector to achieve effective integration of the target gene, the reconstructed plasmid was further cultured
using the spread plate method. After we have transferred the reconstructed plasmid into the E coli DH5α , we have done a
monoclonal colony verification. To verify successful plasmid uptake, we employed Kanamycin selection by culturing the
transformed bacteria on LB agar plates supplemented with Kanamycin, and an antibiotic for which resistance was encoded in
the plasmid. The Kanamycin should screen out all the E-coil that doesn’t has our plasmid. As we can see in the Figure 2,
both *E coli* DH5α has successfully cultivated by the LB agar plate, which indicates our plasmid transfer is
successful. Subsequently, the extracted plasmids were transformed into *E. coli*BL21, followed by
monoclonal verification.

![图片14](https://static.igem.wiki/teams/5739/result/3.webp)

Figure 3. Single colony PCR validation graph

Single colonies from each of the LB agar plates were selected and subjected to amplification PCR. Multiple samples were
collected from each plate to mitigate the impact of potential errors by ensuring redundancy. Subsequently, agarose gel
electrophoresis was performed on all samples obtained from the four plates. This technique was employed to separate and
analyze the DNA fragments by size, thereby verifying the accuracy of the genes amplified by PCR. By utilizing a molecular
weight marker, the approximate base pair (bp) lengths of the DNA bands were estimated and compared to predetermined values
to identify the correct bands. In Figure 3, the expected band length is 102 bp, and the colonies BD Tau-DH5α-3 and BD
Tau-BL21-3 are suitable for further experimentation. In Figure 3, the expected band length is 105 bp, and the colonies
Tau-DH5α-1 and Tau-BL21-4 are appropriate for subsequent experimentation. This analysis facilitates the identification of
colonies with the correct DNA bands, which can then be cultured overnight for further sequencing.

![图片12](https://static.igem.wiki/teams/5739/result/4.webp)

Figure 4. Sanger sequencing map

In our project, we employed DNA sequencing for the identification and characterization of our DNA samples. We utilized this
method to ascertain the complete nucleotide sequence of our reconstructed plasmids. This approach enabled us to verify both
the accuracy and integrity of the reconstructed plasmid. Most peaks were sharp and well-defined, particularly in the central
region of the sequence, suggesting minimal background noise and high signal clarity. Both reconstructed plasmids, BD Tau and
Tau, exhibited sequencing results that fell within the anticipated parameters, indicating no significant deviations. The
sequence alignment with the designed construct confirms the successful incorporation of the target gene into the plasmid.
The high quality of the sequencing data allows us to confidently verify the efficacy of our reconstructed plasmids and
demonstrates the absence of significant sequencing errors or contamination, as evidenced by the lack of ambiguous bases or
unexpected peaks in the figure 4. This level of accuracy is essential for subsequent applications, ensuring the plasmids\'
suitability for further experimental use. The accuracy of the sequencing results is crucial for the reliability of follow-up
studies, which is critical for ensuring the plasmid\'s applicability in subsequent experiments.

',
},
{
"title": '2. SDS-PAGE analysis of protein expression in E.Coli.',
"content": '

The harvested cells were lysed through sonication in a binding buffer composed of 20 mM Tris-HCl, 500 mM NaCl, and 20 mM
imidazole at pH 7.9. The soluble fraction was subsequently subjected to immobilized affinity chromatography utilizing Ni-NTA
resin. Stepwise elution with imidazole gradients ranging from 50 to 250 mM resulted in BD-tau with a purity exceeding 90%,
as verified by SDS-PAGE on a 15% gel.

![](https://static.igem.wiki/teams/5739/result/5-1.webp)

Figure 5. SDS-PAGE for the analysis of whole cell lysis and crude protein solution after centrifugation

SDS-PAGE was performed on plasmid-transformed *E. coli* BL21 to assess the initial protein expression levels. As
depicted in Figure 5, both the crude protein extracted from the cell lysate and the protein obtained from the centrifuged
supernatant revealed the presence of BD Tau (5.1 kDa) and Tau (5.2 kDa). The successfully expressed BD-tau and Tau proteins
can be used for SELEX screening.

',
},
{
"title": '3. RFU characterizes the SELEX process.',
"content": '

![图片18](https://static.igem.wiki/teams/5739/result/6.webp)

Figure 6. Flow symmetry concerning the product of various rounds of the SELEX process

Flow cytometry is a biological technique used for counting and sorting microscopic particles suspended in a fluid. In our
experiments, we first incubate Ni-NTA magnetic beads with His-tagged BD-tau protein to allow complex formation.
Subsequently, nucleic acid aptamers with high affinity can specifically bind to the BD-tau Ni-NTA magnetic beads. The test
sample contains four distinct components: Ni-NTA magnetic beads, BD-tau-conjugated Ni-NTA magnetic beads, aptamer-bound
BD-tau-Ni-NTA magnetic bead complexes, and free nucleic acid aptamers. Additionally, the nucleic acid aptamers are 5\'-end
labeled with a FAM fluorescent tag.

We employed flow cytometry to separate the four components in the experimental sample.The BD-tau-conjugated Ni-NTA magnetic
beads and unconjugated Ni-NTA beads can be distinguished and sorted based on their physical properties, but they yield no
RFU signal due to the absence of fluorescent labeling. Furthermore, free nucleic acid aptamers, being DNA molecules below
the detection threshold of conventional flow cytometry, cannot be directly detected by this method. RFU signals are
generated only when nucleic acid aptamers are complexed with BD-tau-Ni-NTA magnetic beads. The RFU value thereby serves as a
quantitative indicator of the relative extent of aptamer-bead binding.

A 66-mer random DNA library (5\'-GCCGGATCCGTCGAC-(N66)-GAAGCTTGGTACCGAGCTC-3\') underwent 17 iterative rounds of selection
against immobilized BD-tau (2 μg per round). The stringency of the selection process was progressively increased by reducing
the incubation time from 60 to 20 minutes, introducing competitive elution with 10 mM free BD-tau in round 8, and decreasing
the target concentration to 1 μg in the final rounds. Enriched pools were amplified using flanking primers containing
Illumina adapter sequences, facilitating next-generation sequencing (MiSeq, 2×150 bp) after round 17.

This study presents our findings on flow symmetry concerning the product of various rounds of the
SELEX process. RFU, or Relative Fluorescence Units, quantifies the intensity of the fluorescent signal emitted by the FAM
attached to the F primer during PCR. As illustrated in the figure 6, RFU values progressively increased from round 1 to
round 6, indicating a gradual enrichment of aptamers with binding affinity for BD-tau. Post round 6, we implemented reverse
screening to eliminate non-specific binders, which resulted in a noticeable decline in RFU. By round 12, after additional
rounds of stringent selection, there was a substantial decrease in RFU, suggesting that only a small pool of aptamers with
the highest affinity remained.Finally, the selected nucleic acid aptamers were sequenced to determine their precise
nucleotide sequences, thereby providing the essential molecular information for subsequent development steps.

',
},
{
"title": '4. Molecular docking screening of nucleic acid aptamers.',
"content": '

From the 27 nucleic acid aptamers selected through the SELEX system, we employed molecular docking to predict the binding
efficacy between BD-tau and the aptamers, ultimately screening three aptamers with the highest affinity.The binding score
results from the molecular docking are presented in Table 1.

Table 1. Binding Affinity of Aptamer-1, Aptamer-2, and Aptamer-3 with BD-tau

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Protein2 | Binding Score (kcal/mol) | Contact Sites (protein1) | Contact Sites (DNA) | Combination  Type |
| Aptame-1 | -224.03 | ASN327, LYS331, HIS362, LYS369, THR373, LYS375, GLN351 | DC19, DT20, DT13, DA52, DA5, DT6, DT54 | Salt bridge,  Hydrogen bond,  Hydrophobic interaction |
| Aptame-2 | -210.46 | GLN351, ARG349, LYS353, SER356, LYS375, THR373 | DG12, DG8, DC46, DG16, DT22 | Salt bridge,  Hydrogen bond,  Hydrophobic interaction |
| Aptame-3 | -201.37 | LYS353, LYS369, THR373, HIS374, LYS375 | DC1, DC3, DG45, DC43, DA37 | Salt bridge,  Hydrogen bond,  Hydrophobic interaction |

The table 1 demonstrate that BD-tau exhibits binding affinity to Aptamer-1, Aptamer-2, and Aptamer-3, though no significant
differences were observed among them. This indicates limitations in the molecular docking results, necessitating further
experimental validation to determine the binding affinity of Aptamer-1, Aptamer-2, and Aptamer-3 for BD-tau.

',
},
{
"title": '5. Characterization of binding affinity by SPR.',
"content": '

To identify the nucleic acid aptamer with the highest binding affinity for BD-tau from Aptamer-1, Aptamer-2, and Aptamer-3,
we employed Surface Plasmon Resonance (SPR) technology. Biotin exhibits high-affinity interaction with streptavidin.
Therefore, we injected the biotinylated target protein BD-tau (ligand) into specific flow cells of the SPR sensor chip
pre-coated with streptavidin (SA), enabling its specific and stable capture. When a high-affinity nucleic acid aptamer
approaches, it will bind to BD-tau.

The binding event between the two molecules will lead to an increase in response units (RU). By fitting the sensorgram data
with a 1:1 Langmuir binding model, the dissociation constant (Kd) value can be calculated. The Kd value serves as a
quantitative measure of the binding affinity between BD-tau and the nucleic acid aptamer.

We serially diluted Aptamer-1, Aptamer-2, and Aptamer-3 to graded concentrations (e.g., 0 μM, 5 μM, 10 μM, 30 μM, 100 μM,
150 μM) and sequentially injected them at a constant flow rate over the sensor chip surface immobilized with the target
protein to monitor the binding interactions.

![](https://static.igem.wiki/teams/5739/result/7.webp)

Figure 7. Affinity assessed by using surface plasmon resonance

The initial graph illustrates the relationship between aptamer concentration and the energy required to dissociate the
aptamer from its target protein. Five different concentrations of the aptamer were utilized for the SPR analysis.
Subsequently, the second graph re-plotted the data from the Figure 7 with the x-axis representing aptamer concentration, and
a logistic best-fit line was derived.

Table 2. Aptamer Sequences and Dissociation Constants

![图片10](https://static.igem.wiki/teams/5739/result/8.webp)

The Table 2 above illustrates the three optimal aptamers. The dissociation constant (Kd) represents the affinity for the
target protein, with this value being determined through the Surface Plasmon Resonance (SPR) method. A lower Kd value
indicates a higher affinity. The midpoint of this graph was identified as the dissociation constant (Kd) value. In our
analysis of various aptamers using SPR, we observed that a lower Kd value corresponds to a higher energy requirement for
dissociation, indicating greater affinity. Notably, the Aptamer-3 exhibited a Kd value of approximately 56.66, signifying
maybe affinity for the BD-tau protein.Based on the Kd values, we selected Aptamer-3 from the three nucleic acid aptamers as
having the highest affinity for BD-tau, and subsequently validated the specificity of Aptamer-3 for BD-tau using ELISA.

',
},
{
"title": '6. Aptamer-3 Verification Assays.',
"content": '

We further validated the binding affinity of aptamer Aptamer-3 using ELISA. To simulate conditions in AD and non-AD
patients, artificial blood was spiked with varying concentrations of BD-tau and peripheral tau, thereby minimizing potential
interference from other factors. Specifically, the biotinylated nucleic acid aptamer Aptamer-3 was immobilized on ELISA
plates, followed by incubation with BD-tau protein and colorimetric detection. The measured absorbance quantitatively
reflected the relative concentration of BD-tau protein.

![图片121](https://static.igem.wiki/teams/5739/result/9.webp)

Figure 8. Quantitative binding analysis of BD-Tau aptamer in simulated plasma environments

In the figure 8A, the initial graph illustrates a positive correlation between the increased concentration of our Aptamer-3
and the bound BD-tau levels. The figure 8B demonstrates the results of an ELISA assay conducted to detect Alzheimer\'s
Disease (AD) within a simulated blood plasma environment. The data indicate that the ELISA assay consistently identified
elevated BD-tau levels in the plasma of the AD group, whereas the control group exhibited significantly lower BD-tau levels.
These findings suggest that the aptamer is capable of accurately detecting AD within a plasma environment.

',
},
{
"title": '7. Machine Learning Model Simulation',
"content": '

Utilizing expression level data from these three proteins (Tau,BD-tauand BD-tau+Tau) by Elisa date, we developed a machine
learning model to predict individual Alzheimer\'s Disease (AD) status. The results demonstrate the feasibility of this
approach. The Support Vector Machine (SVM) model, in particular, demonstrated superior performance, while the Random Forest
(RF) model requires further optimization. Additionally, we employed methods such as Out-of-Fold (OOF) prediction to
thoroughly evaluate the model\'s accuracy and reliability. For detailed modeling procedures, click here: <https://2025.igem.wiki/keystone/model>

**ROC Curve (Receiver Operating Characteristic Curve)**

This chart for evaluating the performance of a binary classification model shows how the model\'s performance changes under
different classification thresholds. The closer the curve is to the top-left corner, the better the model\'s performance.

The x-axis of the chart represents the False Positive Rate (FPR), also known as the fall-out rate, which indicates the
proportion of all actually negative instances that are incorrectly predicted as positive. The y-axis represents the True
Positive Rate (TPR), also known as recall or sensitivity, which indicates the proportion of all actually positive instances
that are correctly predicted as positive.

The AUC (Area Under Curve) is a key metric for measuring a model\'s ability to distinguish between classes. Its value ranges
from 0 to 1; a value closer to 1 indicates better model performance in distinguishing between the positive and negative
classes.

**7.1 Random Forest (RF) Model**

**![rf_cv_report_01](https://static.igem.wiki/teams/5739/result/10.webp)**

Figure 9. The ROC curve of Random forest model

The figure 9 indicate that the RF model achieved a mean AUC of 0.789 with a standard deviation of 0.131 under 5-fold
cross-validation, indicating that the model possesses reasonably good overall discriminative ability.

**7.2 Support Vector Machine (SVM) Model**

**![图片33](https://static.igem.wiki/teams/5739/result/11.webp)**

Figure 10. The ROC curve of SVM model

The figure 10 indicate that the SVM model achieved a mean AUC of 0.860 with a standard deviation of 0.119 under 5-fold
cross-validation, demonstrating relatively strong overall discriminative ability.

**7.3 Lasso Model**

**![图片34](https://static.igem.wiki/teams/5739/result/12.webp)**

Figure 11. The ROC curve of Lasso model

The figure 12 indicate that the Lasso model achieved a mean AUC of 0.844 with a standard deviation of 0.104 under 5-fold
cross-validation, demonstrating good and relatively consistent overall discriminative ability.

**ROC Curve Cross Comparison**

A comparative analysis of the three models reveals a performance hierarchy. The SVM model achieved the highest mean AUC
(0.860), indicating superior overall discriminative ability. It was followed closely by the Lasso model (0.844), which also
demonstrated the lowest standard deviation (0.104), signifying the most stable and reliable performance across
cross-validation folds. While the Random Forest (RF) model possessed reasonably good discriminative ability (AUC 0.789), its
higher standard deviation (0.131) points to greater performance variability, and its mean AUC was lower than the other two
models. In conclusion, the SVM model is the recommended choice for predictive power, though the Lasso model presents a
compelling alternative due to its consistency.

',
},
]
},
{
"title": 'Part 2: BD-tau Aptamer 3-Based Biosensor',
"content": '',
"children": [
{
"title": '1. Construction of Plasmid pET-28a-Cas12a',
"content": '

Since the primers already contained NheI and HindIII restriction sites, which correspond to the sites within the vector
pET28a. A 1% agarose gel electrophoresis can separate negatively charged nucleic acid fragments of different sizes in the
PCR product under an electric field. As shown in the figure, the target band has a size of 3753 bp, and a bright and wide
band can be clearly identified, indicating that we successfully amplified and separated the target fragment(Figure 12). The
weaker bands surrounding the target band may be due to nonspecific primer binding and abnormal amplification.

![图片3](https://static.igem.wiki/teams/5739/result/13.webp)

Figure 12. PCR amplification of Cas12a.

The ligated plasmid was subsequently amplified in cells through chemical transformation and antibiotic selection. After
mixing, heat-shock, ice bath, and recovery, the cells were centrifuged, most of the medium was discarded, and the pellet was
resuspended before plating onto LB agar plates containing kanamycin. The plates were incubated statically at 37 °C for 12
hours, and the results are shown in the figure 13.

![图片4](https://static.igem.wiki/teams/5739/result/14.webp)

Figure 13. Chemical transformation results of pET28a-LbCas12a

Next, PCR verification was performed on the colonies on the plate to ensure that the plasmids containing the target band had
successfully entered the cells and replicated. A certain number of single colonies were selected and dissolved in 10 μl of
sterile ddH₂O, and 1 μl of the solution was used for PCR verification. The results are shown in the figure 14.

![图片5](https://static.igem.wiki/teams/5739/result/15.webp)

Figure 14. Colony PCR results.

As shown in the figure 15, the target band size is 3753 bp, and a bright band can be clearly observed, indicating that the
desired gene fragment has been successfully transformed into the cells. However, to ensure that no potential mutations
occurred, this clone was sent to a gene service company for plasmid extraction and Sanger sequencing to verify the sequence
accuracy. The results are shown in the figure below.

![图片6](https://static.igem.wiki/teams/5739/result/16.webp)

Figure 15. Sequencing validation.

In the figure 15, the target sequence can be clearly detected in its entirety. Within the high-confidence regions (noting
that Sanger sequencing may show lower confidence at the ends, and a single reaction can only cover approximately 1000 bp,
which is why multiple sequences are joined end-to-end in the figure18), the sequencing quality is good, with well-resolved
peaks. No mutations are observed in the target fragment, indicating that the fragment has been successfully integrated into
the plasmid backbone and that the recombinant plasmid has been successfully constructed.

',
},
{
"title": '2. crRNA Construction',
"content": '

In vitro synthesis of crRNA is typically based on the recognition sequence of T7 RNA polymerase (T7 promoter). By
synthesizing one oligonucleotide containing the T7 promoter sequence and annealing it with another oligonucleotide
complementary to the target sequence, a double-stranded DNA transcription template is obtained, which serves as a template
for crRNA synthesis by T7 polymerase. The template DNA was generated by annealing oligo DNA, and the PCR product was
purified by agarose gel electrophoresis. The results are shown in the figure 16.

![图片8](https://static.igem.wiki/teams/5739/result/17.webp)

Figure 16. Oligo DNA validation

As shown in the figure 16, a clear target band appears at 59 bp, while the faint band at the bottom is due to primer dimers
formed by complementary primers. After obtaining the crRNA transcription template DNA, crRNA was synthesized using the
HiScribe T7 Quick High-Yield RNA Synthesis Kit. The resulting RNA was quantified, with a concentration of 60 ng/μl.

',
},
{
"title": '3. Protein expression',
"content": '

In the T7 expression system (pET28a), the target gene is placed under the control of a T7 promoter, and the host (BL21(DE3))
contains T7 RNA polymerase (or a T7 RNAP that can be induced by IPTG). Upon addition of the inducer (IPTG), expression of T7
RNAP is induced, which in turn drives high-level expression of the target protein in the cells. The resulting plasmid was
introduced into E. coli BL21(DE3) via chemical transformation, and the colony plates are shown in the figure 17.

![图片4](https://static.igem.wiki/teams/5739/result/18.webp)

Figure 17. Verification of BL21(DE3) chemical transformation results

Single colonies from the figure above were expanded in 600 ml of culture and induced with IPTG for overnight expression at
16°C. The cells were collected, washed, lysed, and centrifuged for subsequent purification. Ni²⁺ and other metal ions
exhibit high specificity and chelating ability toward His-tagged fusion proteins, allowing the fusion proteins to be
captured on a designated chromatographic medium while untagged proteins are washed away. Using this principle, the protein
was purified, and the purified fractions were analyzed by SDS-PAGE. The results are shown in the figure.

![图片7](https://static.igem.wiki/teams/5739/result/19.webp)

Figure 18. SDS-PAGE of Cas12a

The figure clearly displays the bands corresponding to the crude protein, wash fractions, and purified protein. Lanes E1–E5
represent the purified protein, showing a strong band at the target position (approximately 120 kDa) with very high
concentration and minimal contaminating proteins, indicating high efficiency of protein expression and purification. This
observation is further supported by the bands in the crude protein sample. In contrast, the wash fractions contain mostly
contaminating proteins and only trace amounts of the target protein.

',
},
{
"title": '4. Characterization and optimization of biosensor performance',
"content": '

**4.1 Screening ComDNA for the highest signal-to-noise ratio**

We constructed the biosensor using Aptamer-3. And we were constructed using five different sources of ComDNA and incubated
with BD-Tau at the same concentration. Following the experimental procedure, the released protein–aptamer-3–dsDNA complexes
were collected, and the dsDNA content was quantified by qPCR. The sample with the highest dsDNA content was identified,
thereby confirming the ComDNA with the highest signal-to-noise ratio. The results are shown in the figure 19.

![Melt Curve Plot-1](https://static.igem.wiki/teams/5739/result/20.webp)

Figure 19. qPCR verification of the ComDNA with the highest signal-to-noise ratio

As shown in figure 19, only ComDNA3 exhibited a relatively high signal-to-noise ratio, while the other samples showed no
significant signal in the test.

**4.2 Fluorescence intensity detection of BD-Tau protein at different concentrations**

An aptamer-3-based biosensor was constructed using ComDNA3, and we further incorporated the Cas12a cleavage module, enabling
the sensor to convert aptamer recognition events into amplified fluorescent signals, thereby establishing a complete
detection model. To evaluate its feasibility in practical applications, we designed a concentration gradient experiment
based on the physiological concentration range of BD-Tau protein in Alzheimer’s disease patients. Samples at different
concentrations were then added to the sensing system, and the changes in fluorescence signals were monitored in real time
using a microplate reader.

![图片13](https://static.igem.wiki/teams/5739/result/21.webp)

Figure 20. Fluorescence intensity detection of BD-Tau protein at different concentrations

The differences in signal between concentrations were not pronounced, and the sensitivity remains limited. This observation
suggests that there is still room for improvement in the detection performance of the current system, potentially due to
insufficient aptamer binding affinity or low efficiency of complex release.

',
},
]
},
{
"title": 'Part 3: Tau Protein Aptamer-Based Biosensor',
"content": '',
"children": [
{
"title": '1. Aptamer Non-Interference Validation',
"content": '

To validate the feasibility of the biosensor, we selected a Tau protein aptamer sequence (target: 2N4R-Tau) that was
previously characterized. To verify whether aptamer binding affects the spatial conformation of dsDNA and thereby influences
subsequent signal transduction efficiency, we designed a comparative experiment. In brief, one system contained only the
naked dsDNA fragment as a control, to test the activation capacity of Cas12a in the absence of aptamer interference. The
other system represented the complete sensor setup, including the aptamer–dsDNA complex and T-Tau protein, to evaluate
whether signal transduction remains effective when the aptamer participates and binds to the target protein. The results,
quantified by fluorescence microplate reader, are shown figure 21.

![图片15](https://static.igem.wiki/teams/5739/result/22.webp)

Figure 21. Validation of whether aptamer binding affects dsDNA-mediated Cas12a activation (T-Tau)

The figure 21 showed that when 30 μM tau protein was added to the system, the sensor’s response reached, or even slightly
exceeded, the signal level observed with naked dsDNA alone. This indicates that the binding of the aptamer to tau protein
did not negatively affect the conformation of dsDNA or its ability to activate Cas12a.

',
},
{
"title": '2. Aptamer-Based Biosensor Optimization',
"content": '

**2.1 Screening for ComDNA with Optimal Signal-to-Noise Ratio**

To further evaluate the binding efficacy of ComDNA to the Tau aptamer-dsDNA complex and ensure efficient dissociation of
ComDNA upon Tau protein. We designed five ComDNA variants (ComDNA1–ComDNA5) for systematic testing. Following incubation
with an excess of Tau protein, the released Tau aptamer-dsDNA complex was quantified using quantitative PCR (qPCR). A
control group, in which the Tau protein was replaced with an equivalent volume of water, was included to calculate the
signal-to-noise ratio. This allowed for the screening of the ComDNA sequence with the highest signal-to-noise ratio.

![](https://static.igem.wiki/teams/5739/result/23.webp)

Figure 22. Signal-to-noise ratio of ComDNA verified with T-Tau (Normalization Gene:actin )

The figure 22 clearly indicate that under the positive control condition with T-Tau, the sensor generated a significant
fluorescent response, confirming the overall feasibility of the system. Additionally, in the comparative experiments among
different ComDNAs, ComDNA3 outperformed the other sequences, exhibiting a higher signal-to-noise ratio and a more stable
response.We have selected ComDNA3 as the linker to construct the biosensor.

**2.2 Determination of Tau Aptamer-dsDNA Complex Concentration**

Following the screening of the aptamer switch ComDNA3, it is necessary to determine the optimal concentration of the Tau
aptamer-dsDNA complex. Different concentrations of the Tau aptamer-dsDNA complex were used to construct the "magnetic
bead-biotinylated ComDNA-Tau aptamer-dsDNA" complex. The signal-to-noise ratios at different Tau aptamer-dsDNA
concentrations were evaluated to identify the optimal concentration.

![](https://static.igem.wiki/teams/5739/result/24-1.webp)

Figure 23. Determination of the concentration of the T-Tau aptamer–dsDNA complex

Figure 23 shows that the signal intensity increases with rising nucleic acid aptamer concentration, reaching a plateau
between 10 μM and 20 μM. The system demonstrated maximum response at 10 μM. and 20μM .Therefore, 10 μM is likely the optimal
concentration for the aptamer–dsDNA complex.

**2.3 Determination of Incubation Time for T-tau with the Biosensor**

To determine the optimal incubation time between the target protein and the sensor for practical applications, we designed
and conducted a corresponding experiment. Under the same protein concentration, samples were incubated for different
durations, and the fluorescence signals at each time point were collected and measured to compare the trend of signal
intensity over time.

![](https://static.igem.wiki/teams/5739/result/25.webp)

Figure 24. Determination of the incubation time between T-Tau and the aptamer–dsDNA complex

The figure 24 showed that the fluorescence intensity reached its maximum after 40 minutes of incubation, and further
extension of the reaction time did not result in any significant increase in signal. This indicates that at 40 minutes, the
binding between the target protein and the sensor had essentially reached saturation, and the system had completed effective
signal transduction. Therefore, 40 minutes was determined to be the optimal incubation time for this sensor in practical
applications.

',
},
{
"title": '3. Detection of Tau Protein Using an Aptamer-Based Biosensor',
"content": '

**3.1 Protocol for Standard Curve Preparation**

Following the optimization of the above conditions—with ComDNA3 selected as the linker, the Tau aptamer–dsDNA complex
concentration set at 10 μM, and the incubation time determined as 40 minutes—we proceeded with the detection of Tau protein.

Based on the designed sensor structure, sensor units were constructed using T-Tau and its corresponding Tau-aptamer.
Gradient concentrations of T-Tau were added to generate a standard curve. The results are shown in the figure 25.

![图片14](https://static.igem.wiki/teams/5739/result/26.webp)

Figure 25. Standard curve of T-Tau

As shown in the figure 25, the fluorescence intensity exhibited a clear linear relationship with the target protein
concentration. This result indicates that the release of the aptamer–dsDNA and the subsequent activation of Cas12a
trans-cleavage are quantitatively dependent on the amount of protein present in the system. The observed linearity not only
demonstrates the sensitivity of the sensing module but also validates its reliability for quantitative detection. Such
linear correlation strongly supports that the system can be calibrated using a standard curve, thereby enabling accurate
concentration measurements in practical applications. These findings not only confirm the applicability of the sensor but
also highlight its potential for further optimization in real biological samples.

**3.2 Biosensor Specificity Evaluation**

Next, we evaluated the recognition specificity of this module, including T-Tau, bovine serum albumin (BSA), human serum
albumin (HSA), IgE, and IgG. The results are shown figure 26.

![图片14](https://static.igem.wiki/teams/5739/result/27.webp)

Figure 26. Protein specificity detection of T-Tau

The results showed that this module exhibited a significant response to T-Tau, while no detectable response was observed for
common interfering proteins in blood. This finding highlights the excellent specificity of the system, indicating that the
sensor can effectively distinguish the target protein from background proteins

',
},
]
},
{
"title": 'Part 4: Screening for Aptamers SELEX',
"content": '',
"children": [
{
"title": '1. RFU characterizes the SELEX process',
"content": '

This is our flow symmetry result on the product of different round of SELEX process. RFU means the intensity of fluorescent
signal that released by RAM we placed on F primer in PCR. As depicted in the Figure 27, the RFU increased progressively from
round 1 to round 6, reflecting a gradual enrichment of aptamers with binding affinity for BD-tau. After round 6, we
initiated reverse screening to eliminate non-specific binders, resulting in a noticeable decline in RFU. By round 12,
following additional rounds of stringent selection, the RFU decreased substantially, indicating that only a small pool of
aptamers with the highest affinity remained.

![2-流式](https://static.igem.wiki/teams/5739/result/28.webp)

Figure 27.Flow symmetry concerning the product of various rounds of the SELEX process

',
},
{
"title": '2. Screening for Aptamers Molecular Docking',
"content": '

We employed 27 DNA sequences obtained from the SELEX system to predict their interactions with Tau and BD-Tau proteins
through molecular docking, aiming to screen nucleic acid aptamers with higher binding affinity. The PDB files of Tau and
BD-Tau proteins were acquired from AlphaFold. Both files were submitted to the HADDOCK 2.4 platform
(https://rascar.science.uu.nl/haddock2.4), which facilitates complex molecular docking and interaction analysis for
researchers. For detailed modeling procedures, click here: <https://2025.igem.wiki/keystone/model>

**Table 2. Nucleic Acid Aptamer Sequences and Z-Scores**

|  |  |  |  |
| --- | --- | --- | --- |
| Number | **Aptamer Sequences** | **T-tau Z-Score** | **BD-Tau Z-Score** |
| 1 | 5\' -TCACCTGAGACTTGACGATGGCATCACTCCCCCCCACCTATTACATCATCATAAATTGAGTGCTATCGTCTGTCCA - 3\' | **-1.1** | **-1.3** |
| **2** | 5\' - TCACCTGAGACTTGACGATGGCCTCCCCCTCACGCACTCTTCCGTTTCTTCTTATCTGAGTGCTATCGTCTGTCCA - 3\' | **-0.9** | **-1.2** |
| **3** | 5\' - TCACCTGAGACTTGACGATGGAACTCCCCCCACCATTATCAGCGCACCACCATTGTAGAGTGCTATCGTCTGTCCA - 3\' | **-1.2** | **-1.5** |
| **4** | 5\' - TCACCTGAGACTTGACGATGGTTTAACTCCCCCACGCCCCCCGCCAACCCATCTCCAGAGTGCTATCGTCTGTCCA - 3\' | **-2.5** | **-1.6** |
| **5** | 5\' - TCACCTGAGACTTGACGATGGTACGACGGCCCCCCGATTATGCGACTACTTGATTTGAGTGCTATCGTCTGTCCA - 3\' | **-1.5** | **-1.7** |
| **6** | 5\' - TCACCTGAGACTTGACGATGGTCAGAACGACGCGCCCCCCACCTCATTCATTATTTTGAGTGCTATCGTCTGTCCA - 3\' | **-1.7** | **-2.1** |
| **7** | 5\' - TCACCTGAGACTTGACGATGGTGACCACCCCCCACGCACACACACCTCTTCCATCCTGAGTGCTATCGTCTGTCCA - 3\' | **-1.7** | **-2.0** |
| ****8**** | **5\' - TCACCTGAGACTTGACGATGGCACTACCCCTCCCTACTAAGCACGGTATCTTGTACTGAGTGCTATCGTCTGTCCA - 3\'** | ****-1.2**** | ****-1.8**** |
| **9** | 5\' - TCACCTGAGACTTGACGATGGGAACAAACACCGCGACCACCCCCCCACTTAACTCCTGAGTGCTATCGTCTGTCCA - 3\' | **-2.1** | **-1.4** |
| **10** | 5\' - TCACCTGAGACTTGACGATGGCAATCCCCCCGACACCGAATCCTAAGCGAACAACGCGAGTGCTATCGTCTGTCCA - 3\' | **-1.4** | **-1.2** |
| **11** | 5\'- TCACCTGAGACTTGACGATGGACTCACAAACTCGAGCCACCCCCGACCCACACAACAGAGTGCTATCGTCTGTCCA - 3\' | **-1.7** | **-1.8** |
| **12** | 5\'- TCACCTGAGACTTGACGATGGTACTCCCCCCCAACCTAATAGCTCTTTACCCTCTGAGAGTGCTATCGTCTGTCCA - 3\' | **-1.5** | **-1.8** |
| **13** | 5\' - TCACCTGAGACTTGACGATGGCCGACTCCCCACCCTACATCGCAACATTGACTATTAGAGTGCTATCGTCTGTCCA - 3\' | **-1.7** | **-1.3** |
| ****14**** | **5\' - TCACCTGAGACTTGACGATGGTTCTACACTGCCCCCCCGACCCGCCAGACCAACCCAGAGTGCTATCGTCTGTCCA - 3\'** | ****-1.0**** | ****-2.1**** |
| **15** | 5\' - TCACCTGAGACTTGACGATGGCAATCCTCCGAGCTCCACCCACCCTTACTCAACATTGAGTGCTATCGTCTGTCCA - 3\' | **-2.6** | **-2.2** |
| ****16**** | **5\' - TCACCTGAGACTTGACGATGGCGCTACCCCCTAACTTCAACCCGCATTATTCTAGCTGAGTGCTATCGTCTGTCCA - 3** | ****-1.8**** | ****-2.3**** |
| **17** | 5\' - TCACCTGAGACTTGACGATGGTTACCGAACCCGACACCCCCGCCGACACCAGCCCCAGAGTGCTATCGTCTGTCCA - 3\' | **-2.2** | **-1.5** |
| **18** | 5\' - TCACCTGAGACTTGACGATGGCCCCCCCCCGCACCGCCTCATTCAGCATACTAATACGAGTGCTATCGTCTGTCCA - 3\' | **-1.9** | **-1.2** |
| **19** | 5\' - TCACCTGAGACTTGACGATGGCAACCACCCCCCCTGGCTACATCATATTCTTATCTTGAGTGCTATCGTCTGTCCA - 3\' | **-2.1** | **-1.3** |
| **20** | 5\' - TCACCTGAGACTTGACGATGGTTTCTTCGCCCCCCCCACACACTACACGTTTCTTCTGAGTGCTATCGTCTGTCCA - 3\' | **-2.2** | **-2.1** |
| **21** | 5\' - TGACTGATTTACGGAAGCTGAATAAGGACTGCTTAGGATTGCGATGATTCAGCT - 3\' | **-2.6** | **-1.5** |
| **22** | 5\' - TGACTGATTTACGGAAGTTACGGACGGATGTCAGTGGTATAGTAATCCGTAACT - 3\' | **-1.7** | **-1.5** |
| **23** | 5\' - TGACTGATTTACGGAAGCTGAATAAGGACTGCTTAGGATTGCGATGATTCAGCT - 3\' | **-1.4** | **-1.6** |
| **24** | 5\' - CGTAAATCAGTCAGAAGCTGAATAAGGACTGCTTAGGATTGCGATGATTCAGCT - 3\' | **-1.4** | **-1.2** |
| **25** | 5\' - GCGGAGCGTGGCAGG - 3\' | **-2.3** | **-1.4** |
| **26** | 5\' - CCTGCCACGCTCCGC - 3\' | **-2.3** | **-1.6** |
| **27** | 5\' - CTGAATCATCGCAATCCTAAGCAGTCCTTATTCAGAAAAAAAAAAAAAAAA - 3\' | **-2.1** | **-2.0** |

Based on the molecular docking results of all 27 DNA sequences listed in Table 2, the binding strength between the DNA
sequences and the target proteins was evaluated using Z-scores. A lower Z-score indicates a higher binding potential. We
specifically prioritized nucleic acid aptamers exhibiting strong binding affinity to BD-Tau while showing weak affinity to
Tau protein. This screening process identified three DNA sequences—Aptame-08, Aptame-14 and Aptame-16—with promising
docking results.

',
},
{
"title": '3. Surface Plasmon Resonance (SPR) for Binding Affinity Characterization',
"content": '

**![图片31](https://static.igem.wiki/teams/5739/result/29.webp)**

Figure 28. Affinity assessed by using surface plasmon resonance

**![图片32](https://static.igem.wiki/teams/5739/result/30.webp)**

Figure 29. Affinity assessed by using surface plasmon resonance

**![图片33](https://static.igem.wiki/teams/5739/result/31.webp)**

Figure 30. Affinity assessed by using surface plasmon resonance

To identify the nucleic acid aptamer with the highest binding affinity for BD-tau from Aptamer-08, Aptamer-14, and
Aptamer-16, we employed Surface Plasmon Resonance (SPR) technology. After we have our aptamer, we tested its affinity by
SPR. the first graph shows how the concentration of aptamer affect the amount of energy needed to separate our aptamer and
its target protein. It selected 5 different concentration of aptamer to do the SPR. The second graph then graphed out the
first graph with x-axis changed to the concentration of aptamer and derived a logistic best fit line. Then, we get the mid
point of the graph, thats our Kd value. As we tested other aptamers using the SPR, the lower the Kd value is, the higher
energy will be needed to seperate aptamer and target protein, which means higher affinity.

As shown in the figure 28, 29 and30, the Kd values for Aptamer-08, Aptamer-14, and Aptamer-16 are 11.32, 6.38, and 21.38,
respectively. Among them, Aptamer-14 demonstrates the lowest Kd value compared to Aptamer-08 and Aptamer-16, indicating it
possesses the highest binding affinity for BD-tau. Based on the Kd values, we selected Aptamer-14 as the nucleic acid
aptamer with the strongest affinity for BD-tau from the three candidates, and subsequently validated its specificity for
BD-tau using ELISA.

',
},
{
"title": '4. Aptamer-14 Verification Assays',
"content": '

We further validated the binding affinity of Aptamer-14 using ELISA. To simulate conditions in AD and non-AD patients,
artificial blood was spiked with varying concentrations of BD-tau and peripheral tau, thereby minimizing potential
interference from other factors. Specifically, the biotinylated nucleic acid aptamer Aptamer-14 was immobilized on ELISA
plates, followed by incubation with BD-tau protein and colorimetric detection. The measured absorbance quantitatively
reflected the relative concentration of BD-tau protein.

![Aptamer-14-Elisa](https://static.igem.wiki/teams/5739/result/32.webp)

Figure 31. Quantitative binding analysis of BD-Tau aptamer in simulated plasma environments

In the above figure 31, the first graph shows how the increase concentration of our aptamer has a positive correlatipn
with the binded BD-tau level. we can see for the AD group plasma we classified Elisa all tested out a high BD-tau level,
and control group has a significantly lower BD-tau level tested out. which shows aptamer can still accurately detect AD in
Plasma envrionment. the nucleic acid aptamers obtained from the second round of SELEX screening demonstrated significantly
enhanced binding affinity compared to those from the initial screening, validating the effectiveness of our SELEX
platform. This confirms that the SELEX technology can be widely applied to screen nucleic acid aptamers for other target
proteins. Furthermore, the results indicate that Aptamer-14 exhibits high-affinity interaction with BD-tau, making it
suitable for constructing a biosensor to detect BD-tau.

',
},
]
},
{
"title": 'Part 5: BD-tau Aptamer14-Based Biosensor',
"content": '',
"children": [
{
"title": '1. Aptamer-14 Non-Interference Validation',
"content": '

We formed a complex using Aptamer-14 and dsDNA to further validate whether the structure of the nucleic acid aptamer affects
Cas12a cleavage efficiency. Using 10 μM dsDNA as a positive control, Figure 32 demonstrates that the relative fluorescence
intensity increases with rising dsDNA concentration. The signal observed at 10 μM dsDNA was higher than that of the control
group, demonstrating that the incorporation of the aptamer does not compromise Cas12a-mediated cleavage efficiency.

**![dsDNA](https://static.igem.wiki/teams/5739/result/33.webp)**

Figure 32. Validation of whether aptamer binding affects dsDNA-mediated Cas12a activation (BD-Tau)

',
},
{
"title": '2. Aptamer 14-Based Biosensor Optimization',
"content": '

**2.1 Screening for ComDNA with Optimal Signal-to-Noise Ratio**

**Melt Curve**

This panel shows the raw melt curves for the different BD-Tau aptamer–ComDNA duplexes. On the x-axis is temperature (°C) and
on the y-axis is fluorescence (RFU). As the temperature increases, the double-stranded regions of each dsDNA gradually
denature into single strands, causing the dye that intercalates into double-stranded DNA to be released and the fluorescence
to decrease. The downward slopes and inflection points indicate the melting transition of each duplex. Curves with a
sharper, more defined drop represent more uniform and stable duplexes, while broader transitions suggest heterogeneous
structures or weaker binding.

**Melt Peak**

This plot displays the derivative of the melt curve (–dRFU/dT vs. temperature), which highlights the temperature at which
the most rapid loss of fluorescence occurs—effectively the melting temperature (Tm) peak. Each distinct peak corresponds to
a specific duplex population. A higher, sharper peak at a specific temperature indicates a stronger and more uniform duplex,
while multiple or broad peaks indicate heterogeneity. Comparing the Tm values across ComDNA candidates lets you identify
which constructs form the most stable and predictable complexes with the aptamer.

**Amplification Curves**

This panel shows real-time amplification curves (RFU vs. cycle number) for reactions containing the T-Tau aptamer–ComDNA
complexes. Each curve represents fluorescence accumulation as the reaction progresses. Early, steep rises indicate rapid
signal generation and efficient Cas12a activation, while late or shallow curves reflect slower or weaker responses. By
comparing the amplification profiles of different ComDNA candidates, you can gauge both sensitivity and kinetics—ideally you
want a construct that shows an early threshold cycle and high end-point fluorescence.

![Com1-5](https://static.igem.wiki/teams/5739/result/34.webp)

Figure 33. Signal-to-noise ratio of ComDNA verified with BD-Tau (Normalization Gene:actin )

The figure 33 clearly indicate that under the positive control condition with BD-Tau, the sensor generated a significant
fluorescent response, confirming the overall feasibility of the system.The relative fluorescence value of ComDNA3 was
significantly higher than that of other groups, indicating that ComDNA3 achieves the highest signal-to-noise ratio. These
experimental results are consistent with the findings presented in Cycle 3, confirming the selection of ComDNA3 for
biosensor fabrication.

**2.2 Determination of BD-tau Aptamer 14-dsDNA Complex Concentration**

![浓度](https://static.igem.wiki/teams/5739/result/35.webp)

Figure 34. Signal-to-noise ratio of ComDNA verified with BD-Tau (Normalization Gene:actin )

Figure 34 shows that the signal intensity increases with rising nucleic acid aptamer concentration. The system demonstrated
maximum response at 10 μM .

**2.3 Determination of Incubation Time for BD-tau with the Biosensor**

**![时间](https://static.igem.wiki/teams/5739/result/36.webp)**

Figure 35. Signal-to-noise ratio of ComDNA verified with BD-Tau (Normalization Gene:actin )

The figure 35 showed that the fluorescence intensity reached its maximum after 40 minutes of incubation, and further
extension of the reaction time did not result in any significant increase in signal. This indicates that at 40 minutes, the
binding between the target protein and the sensor had essentially reached saturation, and the system had completed effective
signal transduction. Therefore, 40 minutes was determined to be the optimal incubation time for this sensor in practical
applications.

',
},
{
"title": '3.Detection of Tau Protein Using an Aptamer 14-Based Biosensor',
"content": '

**3.1 Standard Curve**

Based on the optimized conditions described above, the biosensor has constructed using Aptamer-14. This linearity means that
within this concentration range the assay behaves quantitatively: every increase in protein concentration produces a
proportional increase in fluorescence. The slope of the line represents assay sensitivity; the steeper the slope, the
greater the response per unit of protein. The intercept near zero indicates very low background, suggesting minimal
non-specific activation of Cas12a. With this curve, unknown samples can be interpolated accurately by matching their
fluorescence to the line. Replicates plotted at each concentration also demonstrate reproducibility, with small variation
between points. Together, these data confirm that the BD-Tau aptamer 14–dsDNA–Cas12a system can be used not just for
detection but for quantitative measurement of BD-Tau protein over a defined range.

![标准曲线](https://static.igem.wiki/teams/5739/result/37.webp)

Figue 36.The Standard Curve of BD-tau

**3.2 Biosensor Specificity Evaluation**

![特异性](https://static.igem.wiki/teams/5739/result/38.webp)

Figure 37. Specificity Testing of BD-tau Protein

This figure compares relative fluorescence signals generated by the system when exposed to BD-Tau and several unrelated
proteins (bovine serum albumin, human serum albumin, immunoglobulin E, immunoglobulin G). Only the bar corresponding to
BD-Tau shows a strong signal; all other proteins produce minimal responses near baseline. This demonstrates that the
aptamer–dsDNA complex specifically recognizes BD-Tau and does not significantly bind or activate Cas12a in the presence of
abundant off-target proteins. The low background from serum albumins and immunoglobulins indicates the assay is likely to
remain specific even in complex biological samples such as blood or cerebrospinal fluid. The error bars are small,
indicating good reproducibility of these specificity measurements. These results validate the selectivity of the BD-Tau
detection system and support its potential application as a reliable biomarker assay.

',
},
{
"title": '4. Microfluidic Detection of Alzheimer\'s Disease',
"content": '

We attached platinum nanoparticles (PtNPs) to the 3\' end of the nucleic acid aptamer, while conjugating the aptamer to
complementary DNA (ComDNA). The other end of ComDNA was immobilized to streptavidin-coated magnetic beads via biotin linkage
(Li et al., 2016; Song et al., 2011). In the presence of BD-tau, the activated CRISPR-Cas12a system cleaves the substrate,
and the resulting supernatant is transferred to a hydrogen peroxide (H₂O₂) chamber. The PtNPs catalyze the decomposition of
H₂O₂ into water and oxygen, and the released oxygen propels the red-dyed silicone oil to move forward. The distance of
movement correlates with BD-tau concentration—higher levels result in greater displacement of the indicator (Figure 38).

Chip Architecture Design:

Sample Injection Zone: For loading serum/plasma samples to be tested

Reaction Microchamber: Pre-loaded with H₂O₂ solution as catalytic reaction substrate

Signal Detection Zone: Filled with red-dyed silicone oil as visual flow indicator

This innovation provides crucial technical support for developing a new generation of portable rapid AD diagnostic devices,
effectively addressing the limitations of traditional laboratory methods such as complex procedures, long processing times,
and dependence on large-scale instruments.

**![图片123](https://static.igem.wiki/teams/5739/result/39.webp)**

Figure 38.Microfluidic Sensor Principle

We also developed a lab prototype for our TauTrack microfluidics diagnostic platform and tested the its functionality accordingly. Please note that due to issues of time and material (especially the difficulty and time-consuming nature of producing integrated platinum nanoprobes, we chose an alternative using aptamer–comDNA–PtNP. By doing so we can design and experiment with all core functions of the TauTrack “samplein, resultout” microﬂuidics platform comprised “onchip no human interference” without changing the chip device process.

How to Reconstitute？

A BD‐Tau aptamer is used to bind the PtNP.

The Aptamer contains a short "dock" sequence that will hybridize with a complementary DNA (comDNA).

Streptavidin‐coated magnetic beads are used to capture the biotinylated comDNA.
PtNPs are initially fixed to the beads through the aptamer‐comDNA duplex as a complete sensor.

How It Fits the Workflow on‐chip in Three Stages

Stage 1: Target identification and release

BD‐Tau in the sample will bind the aptamer to destabilize the duplex (assisted by a designed toehold) and displace the comDNA.

Results: The PtNP‐aptamer is released into solution and the comDNA remains on the bead. The level of released PtNPs increases as the BD‐Tau concentration rises.

Stage 2: Magnetic separation

A magnet placed externally immobilizes the beads (together with any PtNPs which are still tethered).

Only the supernatant containing truly released PtNPs can pass through to the next chamber.

Stage 3: Catalytic ampliﬁcation and readout

The released PtNPs cause decomposition of H2O2 to generate oxygen in a closed chamber.

The pressure which is produced pushes a colored ink slug; the distance it moves reﬂects the level of BD‐Tau.

**Representative Results Showcase:**

We have preliminarily validated the feasibility of using microfluidic technology for AD detection with Aptamer-14. The
figure shows that Line 4 and Line 5 exhibit slightly higher signals than Line 1, Line 2, and Line 3, demonstrating the
operational functionality of our microfluidic chip. However, further optimization is required in practical operation.

**![图片111](https://static.igem.wiki/teams/5739/result/40.webp)**

Figure 39.Microfluidic Sensor result.

',
},
{
"title": '',
"content": '',
},
]
},
] %}
{% set info\_title = "Results" %}
{% block title %}Results{% endblock %}
{% block lead %}Results{% endblock %}