iGEM 2020 — Team ShanghaiTech_China

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:ShanghaiTech China/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:ShanghaiTech China/Engineering

[

](https://static.igem.org/mediawiki/2020/2/21/T--ShanghaiTech_China--loading.mp4)

Header

[CESAR](https://2020.igem.org/Team%3AShanghaiTech_China)

* [HOME](https://2020.igem.org/Team%3AShanghaiTech_China)
* [TEAM](https://2020.igem.org/Team%3AShanghaiTech_China/Team)
  + [TEAM MEMBERS](https://2020.igem.org/Team%3AShanghaiTech_China/Team)
  + [COLLABORATION](https://2020.igem.org/Team%3AShanghaiTech_China/Collaborations)
  + [ATTRIBUTIONS](https://2020.igem.org/Team%3AShanghaiTech_China/Attributions)
* [PROJECT](https://2020.igem.org/Team%3AShanghaiTech_China/Description)
  + [DESCRIPTION](https://2020.igem.org/Team%3AShanghaiTech_China/Description)
  + [DESIGN](https://2020.igem.org/Team%3AShanghaiTech_China/Design)
  + [ENGINEERING SUCCESS](https://2020.igem.org/Team%3AShanghaiTech_China/Engineering)
  + [PROOF OF CONCEPT](https://2020.igem.org/Team%3AShanghaiTech_China/Proof_Of_Concept)
  + [PROPOSED IMPLEMENTATION](https://2020.igem.org/Team%3AShanghaiTech_China/Implementation)
  + [PROJECT MODELING](https://2020.igem.org/Team%3AShanghaiTech_China/Model)
  + [PROTOCOLS](https://2020.igem.org/Team%3AShanghaiTech_China/Experiments)
* [CONTRIBUTION](https://2020.igem.org/Team%3AShanghaiTech_China/Contribution)
  + [CONTRIBUTION](https://2020.igem.org/Team%3AShanghaiTech_China/Contribution)
  + [PART COLLECTION](https://2020.igem.org/Team%3AShanghaiTech_China/Parts)
  + [IMPROVEMENT](https://2020.igem.org/Team%3AShanghaiTech_China/Improvement)
* [HUMAN PRACTICES](https://2020.igem.org/Team%3AShanghaiTech_China/Human_Practices)
  + [INTEGRATED HUMAN PRACTICES](https://2020.igem.org/Team%3AShanghaiTech_China/Human_Practices)
  + [EDUCATION AND SCIENCE COMMUNICATION](https://2020.igem.org/Team%3AShanghaiTech_China/Education)
* [SAFETY](https://2020.igem.org/Team%3AShanghaiTech_China/Safety)
* [JUDGING FORM](https://igem.org/2020_Judging_Form?id=3454)
* [AWARDS](https://2020.igem.org/Team%3AShanghaiTech_China/Awards)

[![](https://static.igem.org/mediawiki/2020/9/92/T--ShanghaiTech_China--backtop2.png)](#top)

Engineering Success

Engineering Success

## Overview

Having paid a great effort to literature and experiment, we have engineered a brand new system combining aptamer and Cas12a to detect antibiotics, which features two key rules when designing the part. The data showed that the system worked very successfully.

To prevent and control the spread of antibiotic resistance, thousands of environmental inspectors in China are sent out to measure antibiotic residues in the environment, which is important for antibiotic-abuse surveillance. To make the process more convenient, we aim to develop a portable and rapid-response method to detect antibiotic residues on-site . After paying a great effort into the literature, we found that aptamer is a great tool to detect small molecules including antibiotics1. Aptamer for kanamycin, a kind of antibiotics, has been created and reported to have the strong binding activity 2,3. Aptamers are nucleic acids which are similar to antibodies but cheaper and more flexible. However, it has several other advantages, one of which is that they have the ability of switching their structures upon the recognition of the specific target. Using screening methods like SELEX, specific aptamers with strong binding activity will be sorted out. For example, kanamycin aptamer would form a G-quadruplex upon the binding of kanamycin.

Now we have the tool for antibiotic binding. But aptamer itself can not give out signals, how could we use it in antibiotic measuring? To achieve this, we need to combine aptamer with our Cas12a reporting module. We immediately head for the literature for inspiration. Luckily, we found a solution can probably be the bridge to combine aptamer and Cas12a-reporting module4. They found an oligonucleotide which could base pair with the aptamer. It is not until the existence of small molecules that the oligonucleotide can be recognized by Cas12a and give off signal. Inspired by this work, we set about developing a novel system targeting antibiotic residues combining these two powerful tool. Here is how it works.

![](https://static.igem.org/mediawiki/2020/0/03/T--ShanghaiTech_China--engineering.png)

Figure 1. Workflow of the system in the literature, adapted from [4].

We designed two aptamers containing the structure-switching sequence to lock another oligonucleotide called activator DNA. Since it is locked, it cannot activate Cas12a’s cleavage. But once aptamers have recognized antibiotics, the activators will be released and can then then serve as the target DNA to activate Cas12a. Then as we introduced in [Proof of Concept](https://2020.igem.org/Team%3AShanghaiTech_China/Proof_Of_Concept), the Cas12a will cleave reporter ssDNA and let the whole system emit fluorescence.

In fact, several considerations are involved in the designing process of aptamers and activator DNA since they are not exactly the same as the original aptamer. After referring to the literature4 and combining the knowledge we have learned, we summarized the following tips. First and foremost, the structure-switching sequences should be included in the aptamers. Secondly, additional sequences should be added at the front and end of the original aptamer to bind the activator DNA more tightly, which can help reduce leakage. Also, based on the knowledge we knew about Cas12a at that time, the activator should be a double-strand DNA with PAM sequence.

However, if the activator is double strand structure, it will be impossible for it to pair with aptamers. How to fix this problem? We returned to the article, and found that the author used single-strand DNA as activator! We did not know who was wrong there. But based on our experience of Cas12a, we did not believe that at first. So, we lay that over and turn to other possible solutions. After several weeks, we did not make any progress in this problem. At the time we almost give up this design, we found out that when Cas12a targets ssDNA, it actually does not need a PAM sequence5! This information dispelled our doubts and boosted our confidence in the design of the aptamers and the activator.

![](https://static.igem.org/mediawiki/2020/f/ff/T--ShanghaiTech_China--contribution1.jpg)

Figure 2. The design norm of locked activator and modifying aptamer.

So, we redesigned the element like Figure 2. It obeys the two rules we mentioned above. The structure-switching sequences should be included in the aptamers. And additional sequences should be added at the front and end of the original aptamer to bind the activator DNA more tightly, which can help reduce leakage. And we named it as Aptamer Sandwich. Unsurprisingly, the results turned out to be extremely good. We did orthogonal experiments(Table 1&2) concerning the concentrations of aptamer-activator DNA complex and kanamycin. We concluded that, as the concentration of kanamycin increased, the fluorescence intensity increased as well (Figure 3 a&b). Which means that fluorescence strength has positive relation with Kanamycin concentration. Another thing this result indicates that as Sandwich’s concentration rise, the fluorescence had reduced(Figure3 b). We hypothesized that this may be due to excessive activator may suppress reaction due to unbalance ratio. This suggests that appropriate Aptamer Sandwich concentration is important.

![](https://static.igem.org/mediawiki/2020/e/e2/T--ShanghaiTech_China--xrj.jpg)

Figure 3. The characterization results of CESAR-1 system:

(a) The fluorescence intensity over different concentrations of kanamycin and aptamer-sandwich complex

(b) The bar graph of fluorescence intensity at 30min

![](https://static.igem.org/mediawiki/2020/8/81/T--ShanghaiTech_China--cb.jpg)
![](https://static.igem.org/mediawiki/2020/c/c3/T--ShanghaiTech_China--cc.jpg)

Table1 & 2: orthogonal experiments’ condition Sandwich=Aptamer-Sandwich

To summarize, we designed a brand-new element for aptamer to combine with Cas12a detection system. We set out from our needs, created the designed based on literature information, and finally did experiment to successfully tested it. We are proud of our achievement, and it is a great pleasure that we can provide to future iGEM teams with our independently developed components!

Aptamer Sandwich links: [BBa\_K3454034](http://parts.igem.org/Part%3ABBa_K3454034), [BBa\_K3454035](http://parts.igem.org/Part%3ABBa_K3454035), [BBa\_K3454036](http://parts.igem.org/Part%3ABBa_K3454036).

### Reference

1 Dunn, M. R., Jimenez, R. M., & Chaput, J. C. (2017). Analysis of aptamer discovery and technology. Nature Reviews Chemistry, 1(10), 76.
[https://doi.org/10.1038/s41570-017-0076](%E2%80%9Chttps%3A//doi.org/10.1038/s41570-017-0076%E2%80%9D)

2 Ma, X., Qiao, S., Sun, H., Su, R., Sun, C., & Zhang, M. (2019). Development of structure-switching aptamers for kanamycin detection based on fluorescence resonance energy transfer. Frontiers in Chemistry, 7(FEB), 1–10.
[https://doi.org/10.3389/fchem.2019.00029](%E2%80%9Chttps%3A//doi.org/10.3389/fchem.2019.00029%E2%80%9D)

3 Xing, Y.-P., Liu, C., Zhou, X.-H., & Shi, H.-C. (2015). Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay. Scientific Reports, 5, 8125.
[https://doi.org/10.1038/srep08125](%E2%80%9Chttps%3A//doi.org/10.1038/srep08125%E2%80%9D)

4 Xiong, Y., Zhang, J., Yang, Z., Mou, Q., Ma, Y., Xiong, Y., & Lu, Y. (2020). Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid Targets. Journal of the American Chemical Society, 142(1), 207–213.
 [https://doi.org/10.1021/jacs.9b09211](%E2%80%9Chttps%3A//doi.org/10.1021/jacs.9b09211%E2%80%9D)

5 Kellner, M. J., Koob, J. G., & Gootenberg, J. S. (2019). SHERLOCK : nucleic acid detection with CRISPR nucleases. Nature Protocols, 14(October).
[https://doi.org/10.1038/s41596-019-0210-2](%E2%80%9Chttps%3A//doi.org/10.1038/s41596-019-0210-2%E2%80%9D)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:ShanghaiTech China/Experiments

[

](https://static.igem.org/mediawiki/2020/2/21/T--ShanghaiTech_China--loading.mp4)

Header

[CESAR](https://2020.igem.org/Team%3AShanghaiTech_China)

* [HOME](https://2020.igem.org/Team%3AShanghaiTech_China)
* [TEAM](https://2020.igem.org/Team%3AShanghaiTech_China/Team)
  + [TEAM MEMBERS](https://2020.igem.org/Team%3AShanghaiTech_China/Team)
  + [COLLABORATION](https://2020.igem.org/Team%3AShanghaiTech_China/Collaborations)
  + [ATTRIBUTIONS](https://2020.igem.org/Team%3AShanghaiTech_China/Attributions)
* [PROJECT](https://2020.igem.org/Team%3AShanghaiTech_China/Description)
  + [DESCRIPTION](https://2020.igem.org/Team%3AShanghaiTech_China/Description)
  + [DESIGN](https://2020.igem.org/Team%3AShanghaiTech_China/Design)
  + [ENGINEERING SUCCESS](https://2020.igem.org/Team%3AShanghaiTech_China/Engineering)
  + [PROOF OF CONCEPT](https://2020.igem.org/Team%3AShanghaiTech_China/Proof_Of_Concept)
  + [PROPOSED IMPLEMENTATION](https://2020.igem.org/Team%3AShanghaiTech_China/Implementation)
  + [PROJECT MODELING](https://2020.igem.org/Team%3AShanghaiTech_China/Model)
  + [PROTOCOLS](https://2020.igem.org/Team%3AShanghaiTech_China/Experiments)
* [CONTRIBUTION](https://2020.igem.org/Team%3AShanghaiTech_China/Contribution)
  + [CONTRIBUTION](https://2020.igem.org/Team%3AShanghaiTech_China/Contribution)
  + [PART COLLECTION](https://2020.igem.org/Team%3AShanghaiTech_China/Parts)
  + [IMPROVEMENT](https://2020.igem.org/Team%3AShanghaiTech_China/Improvement)
* [HUMAN PRACTICES](https://2020.igem.org/Team%3AShanghaiTech_China/Human_Practices)
  + [INTEGRATED HUMAN PRACTICES](https://2020.igem.org/Team%3AShanghaiTech_China/Human_Practices)
  + [EDUCATION AND SCIENCE COMMUNICATION](https://2020.igem.org/Team%3AShanghaiTech_China/Education)
* [SAFETY](https://2020.igem.org/Team%3AShanghaiTech_China/Safety)
* [JUDGING FORM](https://igem.org/2020_Judging_Form?id=3454)
* [AWARDS](https://2020.igem.org/Team%3AShanghaiTech_China/Awards)

[![](https://static.igem.org/mediawiki/2020/9/92/T--ShanghaiTech_China--backtop2.png)](#top)

Description

Protocols

# Plasmids contruction

## TOPO cloning:

Hieff Clone® Zero TOPO-Blunt Cloning Kit
[-Hieff Clone® Zero TOPO-Blunt Cloning Kit.pdf](https://static.igem.org/mediawiki/2020/b/bd/T--ShanghaiTech_China--p1.pdf)

## PCR:

PrimeSTAR® Max DNA Polymerase
 [-PrimeSTAR-Max-DNA-Polymerase—fast-and-high-fidelity-PCR.pdf](https://static.igem.org/mediawiki/2020/5/5a/T--ShanghaiTech_China--p2.pdf)

Tsingke Golden Mix
[-Tsingke Golden Mix.pdf](https://static.igem.org/mediawiki/2020/c/c9/T--ShanghaiTech_China--p3.pdf)

CWBIO 2×Es Taq MasterMix (Dye)
[-2×Es Taq MasterMix（Dye）.pdf](https://static.igem.org/mediawiki/2020/3/32/T--ShanghaiTech_China--p4.pdf)

# DNA purification

## DNA clean up:

TIANquick Midi Purification Kit
[-https://en.tiangen.com/content/details\_69\_5465.html](https://en.tiangen.com/content/details_69_5465.html)

## DNA gel purification:

TIANgel Purification Kit
[-TIANgel Purification Kit-200903.pdf](https://static.igem.org/mediawiki/2020/2/29/T--ShanghaiTech_China--p5.pdf)

## Plasmid extraction:

TIANprep Rapid Mini Plasmid Kit -DP105
[-TIANprep Rapid Mini Plasmid Kit-180103.pdf](https://static.igem.org/mediawiki/2020/c/c0/T--ShanghaiTech_China--p6.pdf)

# RNA preparation

## T7 in vitro transcription:

HiScribe™ T7 High Yield RNA Synthesis Kit
[-https://international.neb.com/products/e2040-hiscribe-t7-high-yield-rna-synthesis-kit#Protocols,%20Manuals%20&%20Usage](https://international.neb.com/products/e2040-hiscribe-t7-high-yield-rna-synthesis-kit#Protocols,%20Manuals%20&%20Usage)

## RNA purification:

crRNA preparation
[-crRNA preparation.pdf](https://static.igem.org/mediawiki/2020/6/6f/T--ShanghaiTech_China--p7.pdf)

# Cas12a preparation

Cas12a extraction and purification
[-Cas12a extraction and purification.pdf](https://static.igem.org/mediawiki/2020/2/23/T--ShanghaiTech_China--p8.pdf)

Protein SDS-PAGE electrophoresis protocol
[-Protein SDS-PAGE electrophoresis protocol.pdf](https://static.igem.org/mediawiki/2020/8/89/T--ShanghaiTech_China--ProteinSDS-PAGEelectrophoresiprotocol.pdf)

# Aptamer related materials

Aptamer-sensing protocol
[-Aptamer-sensing protocol.pdf](https://static.igem.org/mediawiki/2020/3/3d/T--ShanghaiTech_China--p9.pdf)

* [Plasmids contruction](#1)
* [DNA purification](#2)
* [RNA preparation](#3)
* [Cas12a preparation](#4)
* [Aptamer related materials:](#5)

![](https://static.igem.org/mediawiki/2020/f/ff/T--ShanghaiTech_China--but.png)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:ShanghaiTech China/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)