iGEM 2021 — Team GCGS_China

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# Team:GCGS China/Notebook

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# Team:GCGS China/Engineering

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* [Home](https://2021.igem.org/Team%3AGCGS_China)
* Project
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  [+ Description](https://2021.igem.org/Team%3AGCGS_China/Description)
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  [+ Engineering](https://2021.igem.org/Team%3AGCGS_China/Engineering)
  [+ contribution](https://2021.igem.org/Team%3AGCGS_China/Contribution)
  [+ Implementation](https://2021.igem.org/Team%3AGCGS_China/Implementation)
* Achievement
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  [+ Partnership](https://2021.igem.org/Team%3AGCGS_China/Partnership)
  [+ Hardware](https://2021.igem.org/Team%3AGCGS_China/Hardware)
  [+ Measurement](https://2021.igem.org/Team%3AGCGS_China/Measurement)
* [Model](https://2021.igem.org/Team%3AGCGS_China/Model)
* Human Practices
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  [+ Integrated human practices](https://2021.igem.org/Team%3AGCGS_China/Human_Practices)
  [+ communication](https://2021.igem.org/Team%3AGCGS_China/Communication)
* Team
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  [+ Team Members](https://2021.igem.org/Team%3AGCGS_China/Team)
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* Lab
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  [+ Safety](https://2021.igem.org/Team%3AGCGS_China/Safety)
  [+ Measurement](https://2021.igem.org/Team%3AGCGS_China/Measurement)

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# Engineering success

## Introduction

To verify the functionality of two 78 bp aptamers targeting C4-HSL and 3O-C12-HSL as mentioned in the literature(Zhao et al., 2013), we used the SG fluorescence method as well as the FAM fluorescence quenching method.

### 1. SYBR GreenⅠ （SG）fluorescence method

#### Design

This method has been used several times as a label-free test and is based on the principle that SG acts as a DNA fluorescent dye that does not carry a fluorescent signal of its own (Abraham et al., 2018). When bound to the aptamer, it fluoresces because it is embedded in the secondary structure of the DNA. Thus when aptamer is bound to the SG dye, green fluorescence is produced when stimulated by excitation light（with the excitation Wavelength 485 nm and emission wavelength 535 nm). However, when the target small molecule is added to the system, the specific binding of the small molecule to the aptamer results in competition with the SG for the binding site, and as a result, the fluorescence value of the system decreases(figure 1).
 ![](https://static.igem.org/mediawiki/2021/0/0a/T--GCGS_China--sgi_.jpg)

Fig 1.SYBR Green I fluorescence-based aptasensing scheme

(McKeague et al.,2014)

The sequences of the original aptamers were as follows:

* 3O-C12-HSL-apt1:
  5'-GCAATGGTACGGTACTTCCCGGGGCCCGCTTCTGGTGCGGTGTACTAGTGACCGCAAAAGTGCACGCTACTTTGCTAA-3’
* C4-HSL-apt1:
  5'-GCAATGGTACGGTACTTCCCGGGGCCCGCTTCTGGTGCGGTGTACTAGTGACCGCAAAAGTGCACGCTACTTTGCTAA-3’

#### Analysis and learn

The ability of the 78 bp (base pairs) long aptamer to carry out fluorescence quenching was not effective（figure 2）; we began to discover why. It was later considered that even though the small molecule was bound to the long aptamer sequence, the constant region at both ends would not be affected by the small molecule and would still bind to the SG dye and produce fluorescence, which may be the reason for our un-ideal experiment result. Therefore, we removed the constant regions from two ends of the 78bp long aptamer. After further design and refinement, the new re-designed aptamer is 35bp long ( McKeague et al., 2014; Sarpong & Datta, 2012; Yi et al., 2019).

![](https://static.igem.org/mediawiki/2021/8/8a/T--GCGS_China--hy_.png)

Fig 2.The fluorescence quenching effect by C4-HSL(left) and 3O-C12-HSL(right）using 78bp aptamers

#### Redesign and build

To prove that our redesigned sequences are correct and usable, we synthesized new aptamers with only intermediate functional sequences according to the modified sequences, which are as follows（[BBa\_K3938001](http://parts.igem.org/Part%3ABBa_K3938001) [BBa\_K3938002](http://parts.igem.org/Part%3ABBa_K3938002)）：

* 3O-C12-HSL-apt3:5'-CGGGGCCCGCTTCTGGTGCGGTGTACTAGTGACCG-3'
* C4-HSL -apt3:5'-CGCTGCCCCCTGTCCTGGGTTAGCTAGCGAGAGCG-3'

The 78bp and 35bp aptamers reacted with SG fluorescence separately and the 78bp aptamer was found to have a higher fluorescence value than the 35bp sequence. This verified that the constant region itself also caused the SG to fluoresce(figure 3).

![](https://static.igem.org/mediawiki/2021/5/59/T--GCGS_China--yingguang_mijidu_.jpg)

Fig 3.SG-bound fluorescence values for different lengths of aptamer

(where C4 represents aptamer of C4-HSL and C12 represents aptamer of 3O-C12-HSL)

#### Test

Next, we tested the fluorescence intensity of small molecules (C4-HSL, C12-HSL) at concentrations of 0nM, 200nM, 400nM, 600nM, 800nM, 1000nM.The experimental results further demonstrated the superiority of the modified aptamers. Regardless of the concentration of small molecules, the SG fluorescent dye was successfully competed off by the small molecules bound to the aptamer, resulting in a significant decrease in fluorescence intensity(figure 4).

![](https://static.igem.org/mediawiki/2021/7/7d/T--GCGS_China--engin_test_zuo_1_.jpg) ![](https://static.igem.org/mediawiki/2021/b/b6/T--GCGS_China--engin_test_you_1_.jpg) |

Fig 4. The fluorescence quenching effect by C4-HSL(left) and 3O-C12-HSL(right）using 35bp aptamers

### 2. FAM labeled aptamer-based fluorescence structure switching assay

#### Design

Besides the SG fluorescence method, we also used another structure switching fluorescence to verify the aptamers can specifically bind to C4-HSL and 3O-C12-HSL. Aptamers with FAM-labeled at 5' end were designed and anti-aptamers which were complementary with the sequence close to the 5′ end of the aptamer were also designed with black hole quencher(BHQ1) labeled at 3' end. In the absence of AHL molecules, the anti-aptamers would be hybridized with the aptamers, causing fluorescence quenching. In the presence of AHL molecules, the small molecules would bind to aptamers, competing with anti-aptamers, thus leading to a fluorescence recovery(Li et al., 2018).

![](https://static.igem.org/mediawiki/2021/1/12/T--GCGS_China--FAM_.jpg)

Fig 5. The mechanism of aptamer based structure switching fluorescence assay

The 78bp aptamer was first tested for anti-aptamer length in this project to confirm the appropriate sequence length. The length of the complementary sequence was set to 100%, 50%, 33% of the original length of the aptamer respectively.

(Note: The aptamer for this procedure was derived from the same literature as the aptamer in the SG experiment, which screened three aptamers for C4-HSL and 3O-C12-HSL respectively. Apt1 was validated to have the smallest kd value among the documents, so all apt1 was used in the later experiments, and because of the similarity of these aptamers (because of the high similarity of these aptamers, the same idea can be used in later designs).

* 1.3O-C12-HSL-apt2：5'-FAM-GCAATGGTACGGTACTTCCCGCCGCCGCGTTCTCATCGCGTGATGTAGTGTCCGCAAAAGTGCACGCTACTTTGCTAA-3'
* 2.C12-anti1： 5'-TTAGCAAAGTAGCGTGCACTTTTGCGGACACTACATCACGCGATGAGAACGCGGCGGCGGGAAGTACCGTACCATTGC-BHQ-3'
* 3.C12-anti2： 5'-TTAGCAAAGTAGCGTGCACTTTTGCGGACACTACATCAC-BHQ-3'
* 4.C12-anti3： 5'-TTAGCAAAGTAGCGTGCACTTTTGCG-BHQ-3'* 4.C4-HSL-apt2: 5'-FAM-GCAATGGTACGGTACTTCCCGGGGCGGGCTGTCATGCCCATCCTACCGTGACCGCAAAAGTGCACGCTACTTTGCTAA-3'
  * 5.C4-anti1: 5'TTAGCAAAGTAGCGTGCACTTTTGCGGTCACGGTAGGATGGGCATGACAGCCCGCCCCGGGAAGTACCGTACCATTGC-BHQ-3'
  * 6.C4-anti2: 5'-TTAGCAAAGTAGCGTGCACTTTTGCGGTCACGGTAGGAT-BHQ-3'
  * 7.C4-anti3: 5'-TTAGCAAAGTAGCGTGCACTTTTGCG-BHQ-3'

### Learn and redesign

After experimental measurements, it was found that when the complementary sequence was half of the aptamer sequence, the whole system showed a more obvious fluorescence quenching effect and re-brightening. Based on the results above and the SG fluorescence method, we decided to use the aptamers without constant regions and anti-aptamers having half the length of the aptamers. The design would make the aptamer synthesis more simple and accurate, in addition leading to the better feature of aptamers. Thus we anticipated the results would show a significant fluorescence quenching caused by anti-aptamers and fluorescence recovery caused by AHL binding.

Build and test

We redesigned the aptamers and anti-aptamers, the sequence were synthed and as follows:

* 3O-C12-HSL apt4: 5'-FAM-CGGGGCCCGCTTCTGGTGCGGTGTACTAGTGACCG-3'
* C12-anti4 5'-CACCAGAAGCGGGCCCCG-BHQ-3'
* C4-HSL apt4: 5'-FAM-CGCTGCCCCCTGTCCTGGGTTAGCTAGCGAGAGCG-3'
* C4-anti4: 5'-CCAGGACAGGGGGCAGCG-BHQ-3'

The apt-4 for C4-HSL and 3O-C12-HSL were FAM modified apt2 for C4-HSL and 3O-C12-HSL respectively.

Using this sequence for FAM experiments, the results of the experiments appear to be better quenched and re-lit, as shown below (C4-HSL results on the left, 3O-C12-HSL results on the right（figure 6).

![](https://static.igem.org/mediawiki/2021/9/90/T--GCGS_China--build_1_.jpg) ![](https://static.igem.org/mediawiki/2021/5/5c/T--GCGS_China--build_and_test_.jpg) |

Fig 6. The fluorescence qunenching and recovery of FAM modified aptamers caused by anti-aptamers and AHL

### References:

* Abraham, K.M., et al., In Vitro Selection and Characterization of a Single-Stranded DNA Aptamer Against the Herbicide Atrazine. ACS Omega, 2018. 3(10): p. 13576-13583.
* Li, Y., L. Sun, and Q. Zhao, Development of aptamer fluorescent switch assay for aflatoxin B1 by using fluorescein-labeled aptamer and black hole quencher 1-labeled complementary DNA. Anal Bioanal Chem, 2018. 410(24): p. 6269-6277
* McKeague, M., et al., Selection and characterization of a novel DNA aptamer for label-free fluorescence biosensing of ochratoxin A. Toxins (Basel), 2014. 6(8): p. 2435-52.
* Sarpong, K. and B. Datta, Nucleic-Acid-binding chromophores as efficient indicators of aptamer-target interactions. J Nucleic Acids, 2012. 2012: p. 247280.
* Yi, H., et al., Fluorometric determination for ofloxacin by using an aptamer and SYBR Green I. Mikrochim Acta, 2019. 186(10): p. 668.
* Zhao, Z.G., et al., Screening and anti-virulent study of N-acyl homoserine lactones DNA aptamers against Pseudomonas aeruginosa quorum sensing. Biotechnology and Bioprocess Engineering, 2013. 18(2): p. 406-412.

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# Team:GCGS China/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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# Team:GCGS 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.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)