iGEM 2021 — Team NYU_Abu_Dhabi

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# Team:NYU Abu Dhabi/Notebook

[![](https://static.igem.org/mediawiki/2021/5/59/T--NYU_Abu_Dhabi--chytritectlogo.png)](https://2021.igem.org/Team%3ANYU_Abu_Dhabi)

Project

[Description](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Description)

[Design](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Design)

[Results](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Results)

[Engineering](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Engineering)

[Implementation](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Implementation)

Lab

[Protocols](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Protocols)

[Notebook](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Notebook)

[Safety](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Safety)

[Parts](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Parts)

[Proof of Concept](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Proof_Of_Concept)

[Entrepreneurship](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Entrepreneurship)

Human Practices

[Human Practices](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Human_Practices)

[Education](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Education)

[Communication](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Communication)

Team

[Our Team](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Team)

[Attributions](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Attributions)

[Collaborations](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Collaborations)

Judging

[Hardware](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Hardware)

[Inclusion](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Inclusivity)

[Sustainable Development](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Sustainable)

[Parts Contribution](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Contribution)

[Medals](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Medals)

N O T E B O O K

![](https://static.igem.org/mediawiki/2021/1/10/T--NYU_Abu_Dhabi--Photo_Lab2.jpeg)

*Take a look at our lab notebook to see the progression of our biology wet lab experiments!*

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

# Team:NYU Abu Dhabi/Engineering

[![](https://static.igem.org/mediawiki/2021/5/59/T--NYU_Abu_Dhabi--chytritectlogo.png)](https://2021.igem.org/Team%3ANYU_Abu_Dhabi)

Project

[Description](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Description)

[Design](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Design)

[Results](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Results)

[Engineering](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Engineering)

[Implementation](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Implementation)

Lab

[Protocols](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Protocols)

[Notebook](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Notebook)

[Safety](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Safety)

[Parts](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Parts)

[Proof of Concept](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Proof_Of_Concept)

[Entrepreneurship](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Entrepreneurship)

Human Practices

[Human Practices](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Human_Practices)

[Education](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Education)

[Communication](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Communication)

Team

[Our Team](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Team)

[Attributions](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Attributions)

[Collaborations](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Collaborations)

Judging

[Hardware](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Hardware)

[Inclusion](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Inclusivity)

[Sustainable Development](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Sustainable)

[Parts Contribution](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Contribution)

[Medals](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Medals)

E N G I N E E R I N G S U C C E S S

![](https://static.igem.org/mediawiki/2021/5/57/T--NYU_Abu_Dhabi--Photo_Tubes.jpeg)

[Introduction](#section1)
[Design](#section2)
[Biology Methodology](#section3)
[Engineering Methodology](#section4)
[Build](#section5)
[Test](#section6)
[Learn](#section7)

## Introduction

Our Engineering Success submission revolves around our efforts to improve the efficiency of target DNA extraction in our study while following the guidelines of the Engineering Design Cycle (Design → Build → Test → Learn→ Design...). For this section of our study, our experimentation focuses on determining the efficiency of our prototype microfluidic chip in extracting DNA from Bsal bacterial cultures.

## Design

First, a design for an experiment to test the extraction efficiency was laid out. Inoculations of Batrachochytrium salamandrivorans (Bsal) were mixed and the Optical Density(OD) was measured on the spectrophotometer (OD=0.842A) Converted OD to cell number (Number of cells/ml = 6.74E-08). Then, the Bsal mixture (24 ml) was separated into 6 different tubes of 4 ml for 5 tubes and 2ml for 1 tube, each tube with the same number of bacterial cells per milliliter.

Three tubes were sent to the biology team for DNA extraction using a QIAprep Spin Miniprep Kit, a current tool in the market used to extract high-quality DNA at large concentrations. The 3 other tubes were sent to the engineering team where our prototype microfluidic chips were used to extract DNA from the Bsal Samples. Both final concentrations were obtained and compared. The experiments were modeled as shown below:

## Biology Methodology

1. Miniprep of inoculations was performed (3 Bsal tubes of 4 ml each) which resulted in 50 μL from each tube. (Note: optional LyseBlue reagent was not added.)
2. Following the QIAprep Spin Miniprep Kit protocol, all centrifugation steps were carried out at 13000rpm in a table-top microcentrifuge. The bacterial overnight culture was centrifuged at 7830 rpm for 3 minutes at room temp. The liquid broth was removed so that only the pellet was left in the tube.
3. The pellet was resuspended in 250 µl Buffer P1 and was transferred into a microcentrifuge tube. 250 µl of Buffer P2 was added and the tube was inverted 6 times to mix the solution. 350µl of Buffer N3 was added and mixed immediately by inverting the tube 6 times.
4. The resulting solution was then centrifuged for 10 minutes at 13000rpm. 800µl of supernatant was applied to the QIAprep 2.0 spin column, centrifuged for 30 seconds, and the flow-through was discarded.
5. The QIAprep 2.0 spin column was washed by adding 0.5 ml of Buffer PB, centrifuged for 30 seconds, and had the flow-through discarded. The QIAprep 2.0 spin column was washed again by adding 0.75 ml of Buffer PE, centrifuged for 30 seconds, and the flow-through was discarded.
6. The QIAprep spin column was then transferred to the collection tube and centrifuged for 1 minute to remove residual wash buffer. It was then placed in a clean 1.5 ml microcentrifuge tube.
7. The DNA was eluted with 50 µl of Buffer EB to the center of the spin column and was left to stand for 1 minute then centrifuged for 1 minute.
8. 2µl of each sample was used on the Nanodrop 2000 Spectrophotometer to measure the concentration of the extracted DNA.

## Engineering Methodology

1. First, a prototype microfluidic chip was designed and built using a Silhouette Cameo cutting machine.
2. The chip was then passed through a plasma purification chamber to remove all impurities on the surfaces of the chip. To functionalize the chip, a solution of 60 µl of the APTES stock and 240 µl of distilled DNA-free water was then passed at 100 µl/min through the chip, but not allowed to flow out.
3. The microfluidic chip was then left to rest for one hour at a temperature of 65 C. After this, the chip was removed and the APTES stock solution was rinsed out using 900 µl of distilled DNA-free water flowing at 300 µl/min. Functionalization of the chip served the purpose of improving the binding capability of DNA to the inner walls of the chip.
4. The extraction process began after the functionalization of the chip. 3 tubes, each filled with 400 µl of Bsal sample, were centrifuged at 8000 rpm for 5 minutes. The bacterial broth was removed and 400 µl of Tris-EDTA buffer was added to each tube to lyse the bacterial cells and separate DNA from the dead cells.
5. 300 µl of this DNA solution from each tube was allowed to flow through the functionalized microfluidic chip at 100 µl/min. The flow was stopped once the fluid had reached the end of the microfluidic chip. The openings of the chip were then closed off to prevent contamination from air particles and the DNA samples were then left to incubate at room temperature for 20 minutes.
6. After this incubation period, the waste liquid in the chip was washed out using Phosphate Buffered Saline (PBS) solution, while the DNA clung to the inner surface of the microfluidic chip.
7. Sodium bicarbonate (NaHCO3) was then used to extract the target DNA into a thoroughly cleaned syringe at a rate of 100 µl/min.
8. The concentration of the extracted DNA was then measured using the Nanodrop 2000 Spectrophotometer and compared against the concentrations derived from the QIAprep Spin Miniprep Kit.

## Build

The build of the experiment can be seen in the video below, walking us through the process:

#### Test

Using the Nanodrop 2000 Spectrophotometer, the respective DNA concentrations from both tests were recovered.

#### Table 1: DNA Extraction Results from QIAprep Spin Miniprep Kit protocol

| Sample | Concentration (ng/µl.) |
| --- | --- |
| Bsal | 106.0 |
| Bsal | 116.17 |
| Bsal | 127.0 |

#### Table 2: DNA Extraction Results from Prototype Microfluidic Chip

| Sample | Concentration (ng/µl.) |
| --- | --- |
| Bsal | 6.5 |
| Bsal | 7.1 |
| Bsal | 7.9 |

The average concentration from the QIAprep Spin Miniprep Kit protocol was observed to be 116.6 ng/µl. The average concentration from the Prototype Microfluidic Chip was observed to be 7.2 ng/µl.

## Learn

### Successful approach

From the test results, it is observed that improvements can be made in our project. Such improvements are shown below.

1. The extraction medium can be changed from Sodium bicarbonate. Another possible alternative is Tris-EDTA Buffer which has the attribute to attract DNA from lysed cells.
2. Literature review suggests that DMP (dimethyl pimelimidate) can be used as a linker in the DNA solution to strengthen the ability of the target DNA to cling to the inner walls of the microfluidic chip. As such, tests with the addition of DMP (dimethyl pimelimidate) have commenced.

### Unsuccessful approach

After our initial results, we took inspiration from the EPFL 2019 igem team and tried to create a microneedle patch to perform extraction of DNA. We created the following patch using their dimensions and 3D printed it using a BMF S240 3D printer. Next, this was used to create a PDMS mold in which Polyvinyl alcohol (PVA) solution was added to attempt to create a microneedle patch. We were not able to proceed with this approach as the microneedles were not able to extract DNA from a meat-like material.

![Trulli](https://static.igem.org/mediawiki/2021/1/1f/T--NYU_Abu_Dhabi--MM.png)

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

# Team:NYU Abu Dhabi/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:NYU Abu Dhabi/Results

[![](https://static.igem.org/mediawiki/2021/5/59/T--NYU_Abu_Dhabi--chytritectlogo.png)](https://2021.igem.org/Team%3ANYU_Abu_Dhabi)

Project

[Description](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Description)

[Design](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Design)

[Results](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Results)

[Engineering](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Engineering)

[Implementation](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Implementation)

Lab

[Protocols](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Protocols)

[Notebook](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Notebook)

[Safety](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Safety)

[Parts](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Parts)

[Proof of Concept](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Proof_Of_Concept)

[Entrepreneurship](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Entrepreneurship)

Human Practices

[Human Practices](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Human_Practices)

[Education](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Education)

[Communication](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Communication)

Team

[Our Team](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Team)

[Attributions](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Attributions)

[Collaborations](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Collaborations)

Judging

[Hardware](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Hardware)

[Inclusion](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Inclusivity)

[Sustainable Development](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Sustainable)

[Parts Contribution](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Contribution)

[Medals](https://2021.igem.org/Team%3ANYU_Abu_Dhabi/Medals)

# R E S U L T S

[Overview](#overview)
[DNA extraction](#DNA extraction)
[Concentrator](#Concentrator)
[Future directions](#Future directions)

## Overview

This page provides a summary of our results and will be divided into 2 major sections: extraction and concentration. We will provide a comparison between using Chyritect and lab methods and highlight key advantages of our device. Finally, we will list the next few steps we intend to take to integrate the device and make it a fully-functional, independent diagnostic device. After discussions with various stakeholders, we focused our efforts on reducing the turnaround time while increasing the device sensitivity as much as possible.

## DNA Extraction

Our first set of experiments were to test the extraction of the inoculated Bd and Bsal plasmids DNA from E.Coli using the Surface chemistry method that we developed. We then compared these values with the concentration of DNA extracted using the standard QIAprep Spin Miniprep Kit. The following are the results obtained:

![Trulli](https://static.igem.org/mediawiki/2021/d/da/T--NYU_Abu_Dhabi--Bleh.png)

Table 1: DNA Extraction Results from QIAprep Spin Miniprep Kit protocol

![Trulli](https://static.igem.org/mediawiki/2021/1/17/T--NYU_Abu_Dhabi--Capture.png)

Table 2: DNA Extraction Results from Prototype Microfluidic Chip

The average concentration from the QIAprep Spin Miniprep Kit protocol was observed to be 116.6 ng/µl. The average concentration from the Prototype Microfluidic Chip was observed to be 7.2 ng/µl. While both process took approximately the same amount of time (30 mins), it is worth noting that the QIAprep spin Miniprep kit involves numerous steps with lab components like a centrifuge and multiple different buffers while the extraction microfluidic chip minimizes human intervention greatly.
While the difference in the amount of DNA extracted was significantly lower in the microfluidic component, we have since come to realize that it is still sufficient for downstream detection using the electrokinetic concentration method.

We were determined to increase the extraction efficiency so after a deep dive into existing literature we found that DMP (dimethyl pimelimidate) can be used as a linker in the DNA solution to strengthen the ability of the target DNA to cling to the inner walls of the microfluidic chip. Additionally, we learnt that Tris EDTA (TE) Buffer can be used to dissolve the DNA that clings to the walls in the final extraction step.

The following tables show the subsequent experiments we designed to increase the efficiency of extraction of DNA.

*Table 1: The represented data of the first trial of checking the efficiency of the DNA extraction through Nanodrop2000.*

![Trulli](https://static.igem.org/mediawiki/2021/7/7c/T--NYU_Abu_Dhabi--1.png)

![Trulli](https://static.igem.org/mediawiki/2021/4/4a/T--NYU_Abu_Dhabi--2.png)

*Figure 1: Graph representing the extraction efficiency rate according to the time of incubation.*

*Table 2: The represented data of the first trial of checking the efficiency of the DNA extraction after the addition of DMP linker through Nanodrop2000.*

![Trulli](https://static.igem.org/mediawiki/2021/5/54/T--NYU_Abu_Dhabi--3.png)

![Trulli](https://static.igem.org/mediawiki/2021/1/12/T--NYU_Abu_Dhabi--4.png)

*Figure 2: Graph representing the extraction efficiency rate according to the time of incubation (TE Buffer/DMP Linker).*

![Trulli](https://static.igem.org/mediawiki/2021/5/5f/T--NYU_Abu_Dhabi--5.png)

*Figure 3: Graph representing the difference in between the extraction efficiency depending on the addition of TE Buffer/DMP Linker, according to the time of incubation.*

Therefore we were able to optimize our extraction methods to increase efficiency and reduce the time taken to 30 mins after which the DNA solution is passed on to the concentrator chip for detection. In this process we also adopted the Engineering Design Cycle (Design → Build → Test → Learn→ Design...) to help us create a framework to work with.

## Concentrator Chip

We envision the extracted DNA to be passed into the concentrator chip where it will use electrokinetic concentration to focus the target DNA into a small region where it can interact with CRISPR Cas12a molecules and produce a fluorescent signal. In this set of experiments, we first tested the chip with a high concentration of Bd (136.5 ng/ul) and recorded the fluorescent signal and compared it with a control sample. The 2 graphs below show the results of these tests.

![Trulli](https://static.igem.org/mediawiki/2021/d/db/T--NYU_Abu_Dhabi--6.png)

![Trulli](https://static.igem.org/mediawiki/2021/3/3d/T--NYU_Abu_Dhabi--7.png)

*Figure 5: A visual representation of the fluorescence of a positive sample compared to the control test.*

In both of the above graphs there is a clear increase in the fluorescence intensity after 200 seconds of adding CRISPR reagents. In a lab setting this result takes 35 minutes to confirm the presence of Bd using CRISPR reagents. This could be attributed to the sensitivity of the optical microscope used in the lab once the DNA is concentrated but we envision our device to use photoelectronics to detect a change in fluorescence intensity.

Next, we decided to check the device's limit of detection and compare that to lab values for the same. The Biology team found the limit of detection in a lab setting to be 103 copies/ul (Figure 5). We used 50 ul aliquots of the same sample and ran tests on the concentrator chip and measured fluorescence intensity as a function of time and compared the values to the control sample. (Figure 6).

![Trulli](https://static.igem.org/mediawiki/2021/9/96/T--NYU_Abu_Dhabi--CRISPR.png)

![Trulli](https://static.igem.org/mediawiki/2021/d/d3/T--NYU_Abu_Dhabi--Graph.png)

The above results clearly indicate an increase in fluorescence intensity for all samples as low as 1 copy/ul. It was however not possible to make a quantifiable model for increase in fluorescence intensity as a function of concentration of target DNA as there was a significant amount of fluctuation between the different concentrations. We didn’t further explore this as Dr. Penner had mentioned that he did not need quantitative data. To make more reliable conclusions, we intend to repeat the above tests and compare the results.This test however shows the limit of detection to be as low as 1 copy/ul which makes electrokinetic concentration 1000 times more sensitive than present lab techniques.

## Future Directions

To complete the device, we intend to use photoelectronics to sense the subtle changes in emitted fluorescence intensity however the final module is still being constructed. In the meantime we intend to use a portable microscope along with the concentrator chip and the extraction module to accurately detect for the presence of Bd and Bsal in a sample.