iGEM 2021 — Team NTU-Singapore

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NTU-Singapore/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)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NTU-Singapore/Engineering

* [Home](/Team%3ANTU-Singapore)
* [Project](/Team%3ANTU-Singapore)
  + [Project Description](/Team%3ANTU-Singapore/Description)
  + [Eng Success](/Team%3ANTU-Singapore/Engineering)
* [Team](/Team%3ANTU-Singapore)
  + [Our Team](/Team%3ANTU-Singapore/Attributions)
* [Collaboration](/Team%3ANTU-Singapore)
  + [Collaborations](/Team%3ANTU-Singapore/Collaborations)
  + [Partnership](/Team%3ANTU-Singapore/Partnership)
* [Parts](/Team%3ANTU-Singapore)
  + [Contribution](/Team%3ANTU-Singapore/Contribution)
  + [Improvement](/Team%3ANTU-Singapore/Improvement)
* [Human Practices](/Team%3ANTU-Singapore)
  + [Integrated Human Practices](/Team%3ANTU-Singapore/Human_Practices)
  + [Education and Communication](/Team%3ANTU-Singapore/Communication)
* [Implementation](/Team%3ANTU-Singapore)
  + [Proposed Implementation](/Team%3ANTU-Singapore/Implementation)
  + [Proof of Concept](/Team%3ANTU-Singapore/Proof_Of_Concept)
* [Entrepreneurship](/Team%3ANTU-Singapore)
  + [Business Plan](/Team%3ANTU-Singapore/Entrepreneurship)
* [Data](/Team%3ANTU-Singapore)
  + [Survey Data](/Team%3ANTU-Singapore/Data)

![](https://static.igem.org/mediawiki/2021/3/31/T--NTU-Singapore--EngSucc.png)

Design

First, we construct the guide RNA (gRNA) for BRAF by selecting the region of BRAF where the mutation occurs. Our designed gRNA has a spacer length of 20 nucleotides, which is effectively short but still performs optimally for detection. With our designed gRNA whose sequences complement the BRAF-V600E gene, we are able to test it on the BRAF gene for both wild type (BRAF V600) and mutant (BRAF E600).

Build

To obtain the DNA template for both wild type BRAF and mutant BRAF, we conducted PCR using ordered primers. After the PCR, we ran the PCR products through the agarose gel (gel electrophoresis) to select the desired DNA fragment that contains the sequence of interest. The DNA fragments are then extracted from the gel.

The guide RNA (gRNA) is synthesized by In vitro Transcription (IVT). The IVT product will then be purified before it can be used as gRNA. The RNP complex is obtained by adding the gRNA and the LbCas12a enzyme through incubation at 37°C for 30 minutes.

Test

To determine the optimum concentration of DNA for maximum detection, we carried out serial dilution from 2E11 to 2E0 to the DNA templates. After diluting, we used Loop-mediated isothermal amplification (LAMP) to amplify the diluted and non-diluted DNA templates.

To test the performance of LbCas12a with the BRAF DNA templates, we used a trans-cleavage reaction using a Fluorophore-Quencher (FQ) oligo. In an FQ reaction, when LbCas12a binds to a DNA, it will cleave the DNA. In addition to cleaving the DNA, due to its collateral ssDNase activity, it will cut the short TTATT linker between the fluorophore and the quencher, and fluorescence will emit.

Learn

After carrying out the FQ reaction, we obtained the data as shown below.

![](https://static.igem.org/mediawiki/2021/9/97/T--NTU-Singapore--brafv600efq.png)

Figure 1. Graph of normalized signal ratio Mutant to Wild-Type with 1X Lamp product (blue) and 50X diluted lamp product (orange) for BRAF-V600 (left) and BRAF-E600 (right) after undergoing LAMP and FQ reaction.

The figure shows that the signal ratio for 50X diluted LAMP product is higher for both wild type BRAF and mutated BRAF. From this data that we have obtained, we concluded that 50X dilution gives the optimum concentration of DNA for amplification with LAMP. We then proceeded to see if we are able to improve the Mutant/WT ratio but using an mutated version of LbCas12a.

[Click here to see!](https://2021.igem.org/Team%3ANTU-Singapore/Improvement)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NTU-Singapore/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)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NTU-Singapore/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)