iGEM 2022 — Team toronto

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# Notebook

A quick summary of 2022 iGEM Toronto journey. Details can be found in the Experiment page

| Date | Description |
| --- | --- |
| 02.04.2022 - 07.08.2022 | Project planning and designing Compared different detection assays methods and selected LAMP Designed multiple sets of LAMP primers Ordered LAMP primers and reagents |
| 08.08.2022 - 14.08.2022 | Preparing reagents Resuspended primers and DNA Aliquoted reagents LAMP Experiments Preliminary primer set testing with NEB Warmstart colorimetric master mix |
| 15.08.2022 - 21.08.2022 | LAMP Experiments Testing LAMP Primers with Lucigen Master Mix |
| 22.08.2022 - 29.08.2022 | LAMP Experiments Testing LAMP Primers with Gel Electrophoresis verification Troubleshooting Gel Electrophoresis contamination |
| 29.08.2022 - 04.09.2022 | LAMP Experiments Testing Primer ratios |
| 05.09.2022 - 11.09.2022 | LAMP Experiments LAMP test on qPCR machine for melting curve analysis Freeze drying LAMP assay |
| 12.09.2022 - 02.10.2022 | Break from lab experiments |
| 03.10.2022 - 10.10.2022 | LAMP Experiments LAMP test on qPCR machine for melting curve analysis |

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# Engineering Success:

Our hardware team performed multiple design cycles on the device prototype. The general design cycle for all the parts usually consists of collecting data (for example, taking measurements and researching the material to use), making the initial prototype, testing the prototype with multiple users, assessing the prototype’s usability, and modifying the prototype based on the testing and user feedback. The threads fitting and mechanism designed for the DNA extraction tool and liquid handling process were our main contributions to achieving safe, functional, intuitive products for onsite PCR experiments.

We also demonstrated engineering success in the wetlab and dry lab team in primer design. Our prediction model is able to rank primers according to the criteria. Its accuracy validated by the wet lab as our primer sets with low confidence ranking have a failure in amplification. To build upon our engineering success, we can apply machine learning and build our model with a larger dataset.

Extraction Tool

Extraction Tool

| Iteration | Design and Build | Learnings from testing the Design |
| --- | --- | --- |
| 1st Generation (Fig. 1) | 3D-printed drill design has the conventional screwdriver mechanism. | - Able to drill out wood dust (granular) from the sapwood layer. - Hard to apply hand force and the drill moved around when forces was applied. |
| 2nd Generation (Fig. 1) | A drill handle (similar to the corkscrew) was added to the 1st Generation design which allowed users to apply more power when drilling. | - Able to apply force better and without moving the drill during the extraction process - Able to obtain wood dust form the sapwood layer, however the handle is too short. |
| 3rd Generation (Figure 3) | Same concept from 2nd Generation, however the drill handle size increases and finger slots were added | - Successfully obtain wood dust from the sapwood layer - The handle size and finger slots fit perfectly to grasp the drill - Able to apply force without moving the drill during the extraction process. |

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/screen-shot-2022-10-08-at-12-29-18-pm.png)](https://static.igem.wiki/teams/4479/wiki/engineering-success/screen-shot-2022-10-08-at-12-29-18-pm.png)

|  |  |  |
| --- | --- | --- |
| ![Snow](https://static.igem.wiki/teams/4479/wiki/engineering-success/untitled.png)  ![Forest](https://static.igem.wiki/teams/4479/wiki/engineering-success/untitled-1.png) | ![Mountains](https://static.igem.wiki/teams/4479/wiki/engineering-success/untitled-3.png) | |
| Figure 1: 1st Generation Extraction Tool | Figure 2: 2nd Generation Extraction Tool | Figure 3: 3rd Generation Extraction Tool |

Liquid Handling and Mixing:

To validate the liquid mixing mechanism we used a pipette to control the input and output amounts of water liquid. After collecting 6 sample points, we observed 14.3% error on average with standard deviation of 1.2%.

Liquid Handling and Mixing

| Iteration | Design and Build | Learnings from testing the design |
| --- | --- | --- |
| 1st Generation (Figure 4) | Using Scotch Yoke Mechanism, rotational motion translated to vertical movement. The user rotate the handle and move the cylinder up and down to crush and mix the liquid in the container. | The design could mix the liquid however it cannot perform grinding the sapwood to small pieces to extract the DNA. |
| 2nd Generation (Figure 5) | Similar to garden hose valve with an opening valve inside. | 3D printer could not print the design and it has no foil cutter. |
| 3rd Generation (Figure 6) | Foil cutter was added as a needle. Rubber bands and rubber band slots hold together the three-piece design. | Needle was not easily printable from 3D printer. |
| 5th Generation (Figure 8) | The connector tubes were redesigned for a smaller liquid container. The foil cutter’s size was redesigned as well. The rubber band design was removed | Dimensions were off. The rubber band design was removed because the tube held together well enough and the rubber band complicated the design. |
| 6th Generation (Figure 9) | The design (including the threads) now fits the liquid container. | Dimensions were off as shown in the figure 9. |
| 7th Generation (Figure 10) | A new cutter design was used because of the smaller liquid containers. | Too small to print. |
| 8th Generation - Current Design (Figure 11) | The cutter was enlarged. The aligning mechanism was also redesigned to make sure it was printed properly. | Worked successfully and printed properly |

Test results with current design - 8th Generation To validate the liquid mixing mechanism we used a pipette to control the input and output amounts of water liquid. After collecting 6 sample points, we observed 14.3% error on average with
standard deviation of 1.2%.

| Initial | Amount Added | Amount Final | Amount Error % |
| --- | --- | --- | --- |
| 100µL | 400µL | 440µL | 12% |
| 100µL | 400µL | 420µL | 16% |
| 100µL | 400µL | 430µL | 14% |
| 200µL | 300µL | 433µL | 14.4% |
| 200µL | 300µL | 427µL | 14.6% |
| 200µL | 300µL | 425µL | 15% |

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/screen-shot-2022-09-23-at-9-20-22-pm.png)](https://static.igem.wiki/teams/4479/wiki/engineering-success/screen-shot-2022-09-23-at-9-20-22-pm.png)

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/screen-shot-2022-09-23-at-9-20-30-pm.png)](https://static.igem.wiki/teams/4479/wiki/engineering-success/screen-shot-2022-09-23-at-9-20-30-pm.png)

Figure 4: First generation of the liquid mixing and handling design

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/june-30-no-2.png)](https://static.igem.wiki/teams/4479/wiki/engineering-success/june-30-no-2.png)

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/june-30.png)](https://static.igem.wiki/teams/4479/wiki/engineering-success/june-30.png)

Figure 5: Second Generation of the liquid mixing and handling design

![](https://static.igem.wiki/teams/4479/wiki/engineering-success/july-25-no-2.png)

Figure 6: Third Generation of the liquid mixing and handling design

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/aug-8-no-2.jpg)](https://static.igem.wiki/teams/4479/wiki/engineering-success/aug-8-no-2.jpg)

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/aug-8.png)](https://static.igem.wiki/teams/4479/wiki/engineering-success/aug-8.png)

Figure 7: Fourth Generation of the liquid mixing and handling design

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/aug-26-no-2.png)](https://static.igem.wiki/teams/4479/wiki/engineering-success/aug-26-no-2.png)

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/aug-26.jpg)](https://static.igem.wiki/teams/4479/wiki/engineering-success/aug-26.jpg)

Figure 8: Fifth Generation of the liquid mixing and handling design

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/sep-3-no-2.png)](https://static.igem.wiki/teams/4479/wiki/engineering-success/sep-3-no-2.png)

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/sep-3.jpg)](https://static.igem.wiki/teams/4479/wiki/engineering-success/sep-3.jpg)

Figure 9: Sixth Generation of the liquid mixing and handling design

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/sep-8-no-2.png)](https://static.igem.wiki/teams/4479/wiki/engineering-success/sep-8-no-2.png)

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/sep-8-no-3.png)](https://static.igem.wiki/teams/4479/wiki/engineering-success/sep-8-no-3.png)

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/sep-8.png)](https://static.igem.wiki/teams/4479/wiki/engineering-success/sep-8.png)

Figure 10: Seventh Generation of the liquid mixing and handling design

### Heating Mechanism:

Overall goal, Current Step, and Next Step:

Based on the requirement to perform the LAMP (PCR) reaction on site, we attempted to design a portable heating unit that can heat up to 71C (+ or -1C) consistently for 30 min. We currently have a prototype that is portable and can sustain
heat up to 100C. We also found a suitable thermistor that measures the temperature with a one-degree fluctuation. The main components we used included a PTC heating module, a 10k thermistor, an Arduino nano, a rotary encoder, and an OLED
screen for readings. For the next step, we plan to work on a consistent heating function for a low-cost and portable heating unit. We also aim to design a PID control algorithm for precision, a portable battery or alternative battery options
for forestry practitioners and researchers to work on site.

Based on the requirement to perform the LAMP (PCR) reaction on site, we attempted to design a portable heating unit that can heat up to 71C (+ or -1C) consistently for 30 min. We currently have a prototype that is portable and can sustain
heat up to 100C. We also found a suitable thermistor that measures the temperature with a one-degree fluctuation. The main components we used included a PTC heating module, a 10k thermistor, an Arduino nano, a rotary encoder, and an OLED
screen for readings. For the next step, we plan to work on a consistent heating function for a low-cost and portable heating unit. We also aim to design a PID control algorithm for precision, a portable battery or alternative battery options
for forestry practitioners and researchers to work on site.

### Heating Element

We tested Peltier, PTC, and coiled heaters and selected the PTC heater as it consumes the least amount of energy with room-efficient build for portable on-site heating purposes.

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/img-2142.jpg)](https://static.igem.wiki/teams/4479/wiki/engineering-success/img-2142.jpg)

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/screen-shot-2022-10-08-at-12-31-11-pm.png)](https://static.igem.wiki/teams/4479/wiki/engineering-success/screen-shot-2022-10-08-at-12-31-11-pm.png)

### Thermistor

We tested a 1K NTC thermistor, a 10K NTC thermistor, an IR temperature sensor, and a 5V 10A temperature control relay. The 10K NTC thermistor has the most accurate temperature reading since it employs contact sensing.

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/img-2217.jpg)](https://static.igem.wiki/teams/4479/wiki/engineering-success/img-2217.jpg)

Then, we tested with the temperature module. We needed more control of the interface and indicators for practitioners to understand.

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/img-2230.jpg)](https://static.igem.wiki/teams/4479/wiki/engineering-success/img-2230.jpg)

To address this issue, we designed a LCD screen that can display input reading.

We also have designed the heating plate made of aluminum for the LAMP reaction

[![](https://static.igem.wiki/teams/4479/wiki/engineering-success/img-3867.jpg)](https://static.igem.wiki/teams/4479/wiki/engineering-success/img-3867.jpg)

(Version 1.0 on the left and version 2.0 on the right)

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# Experimental Story

Detecting Bretziella fagacearum DNA on site
with colorimetric loop-mediated isothermal amplification (LAMP) method

#### Table of Contents

* [Selecting LAMP as the DNA amplification method](#select)
* [Determining target DNA](#targetdna)
* [Finding suitable primers](#primers)
* [Determining possible LAMP reaction reagents](#reagents)
  + [Addressing requirement 1: Easy visualization](#visual)
  + [Addressing requirement 2: Ensuring LAMP remains equipment free and deliverable in field settings](#free)
* [LAMP experimental workflow](#workflow)
  + [Summary of Experimental Plan](#summary)
  + [Step 1: Screen the primer sets through colorimetric LAMP reaction](#step1)
  + [Step 2: Perform gel electrophoresis to better validate if the reaction results are false positive or false negative](#step2)
  + [Step 3: Fluorescence LAMP experiment and melting curve analysis, combined with step 4: perform replicates to ensure experimental validity](#step3)
  + [Step 5: Test if the selected LAMP reaction still works well when it is freeze-dried](#step5)

## Selecting LAMP as the DNA amplification method

After multiple meetings with various stakeholder groups (see [Human Practice](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) wiki page),
the iGEM Toronto wetlab team determined the most important requirements
for their hypothetical diagnostic method:
detecting the oak wilt pathogen quickly (in ~1 hour or less)
and in the field with minimal lab equipment.
To achieve these broad requirements,
our diagnostic method must have strong diagnostic and analytical sensitivity and specificity. Additionally,
it would need to be portable to be easily brought into field environments (such as forests without electricity) where oak wilt resides.

Top of mind in the realm of sensitive and specific diagnostics were methods based
on the amplification of genomic material,
which in our case was oak wilt DNA.
While a variety of current amplification techniques existed,
they each had drawbacks that would inhibit their success in the detection of oak wilt DNA in the field.

Some well-known techniques are listed below (Zanoli, 2013):

- **Polymerase chain reaction (PCR):** while this reaction has strong analytical and diagnostic sensitivity and specificity, it requires the use of bulky and expensive thermocycler equipment for its denaturing, annealing, and extension steps
- **Nucleic acid sequence-based amplification (NASBA):**does not have high analytical sensitivity
- **Self-sustained sequence replication (3SR):**does not have high analytical sensitivity
- **Strand displacement amplification (SDA):**is costly; has large backgrounds on visualization methods

Given the need to create a fast, sensitive, specific, and portable diagnostic tool, the wetlab team decided that a newer DNA amplification method called loop-mediated isothermal amplification (LAMP) was a perfect candidate, since it overcame almost all of the drawbacks of the techniques above. Especially compared to PCR, thermal cycler is not necessary for LAMP.

This led to the team’s main project goal: develop a suitable LAMP reaction and process (with high viability and easy visualization) to amplify and detect oak wilt DNA. Ideally, our developed LAMP reaction would also meet the WHO’s ASSURED list of characteristics for diagnostic tests: high affordability, sensitivity, specificity, user friendliness, rapidity and robustness, being equipment-free, and deliverability.

## Determining target DNA

The drylab and wetlab teams worked together to find suitable regions of oak wilt DNA to target for amplification; this region would be conserved for all individuals in the oak wilt species, but non-conserved between similar fungal species. This would ensure that if any DNA were to be amplified by our suitable LAMP reaction, it would solely belong to oak wilt and not another fungus.

After BLASTing a variety of oak wilt and other fungal genomic fragments, the drylab and wetlab teams found multiple target regions that were suitable for LAMP. The two most suitable regions selected for experimentation were BT and MCM7
(see [Dry Lab wiki](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) for in-depth selection and analysis of primers).

## Finding suitable primers

Drylab used two online tools, Primer Explorer (PE) and GLAPD, to generate suitable primers for a LAMP reaction using the above target DNA regions as templates.

Drylab generated eight primer sets in total, with each set containing 4 primers: FIP (Forward inner primer), BIP (Backward inner primer), F3 (Forward primer), and B3 (Backward primer). The naming conventions of our candidate primer designs are as follows:

- Primers targeting the Beta-Tublin (BT) region: PE1, PE2, PE3, GLAPD1
- Primers targeting the Minichromosome Maintenance Complex Component (MCM7) region: PE1, PE2, GLAPD1, GLAPD2

Note: Primer explorer design (PE). Whole Genome Based LAMP Primer Design (GLAPD)

The wetlab focused on identifying successful pairs of LAMP primers generated by the dry lab

## Determining possible LAMP reaction reagents

#### Addressing requirement 1: Easy visualization

Because of the need to do in-field diagnostics of oak wilt, wetlab needed a way to visualize whether their LAMP reaction would be successful as the reaction proceeded. Colorimetric LAMP was the optimal solution for this requirement.

Colorimetric LAMP mechanism

The binding of nucleotides by a DNA polymerase to template DNA releases hydrogen ions, thus reducing the pH of the reaction solution – this could be easily visualized via the addition of an indicator to the reaction mix, indicating that a successful amplification of DNA must cause the reaction to change color! (Tanner, 2015)

Reagents used

The New England Biolabs (NEB) WarmStart® Colorimetric LAMP 2X Master Mix was used.

* Key features of NEB WarmStart Master Mix:

1. This master mix provides a low-buffer reaction solution containing a visible pH indicator
2. Expect color change from pink to yellow in 30 minutes after incubating the LAMP reaction at 65C
3. According to Dr. Nathan Tanner, the WarmStart Master Mix contains glycerol, which makes it incompatible for freeze-drying.

Purpose: Quick primer screening

If the mix turns from pink to yellow, wetlab could proceed with further testing (such as gel electrophoresis and specificity testing); if not, they would know that the reaction did not work (provided proper controls were in place) and should not proceed with further testing, saving us time, reagents, and money.

#### Addressing requirement 2: Ensuring LAMP remains equipment free and deliverable in field settings

Given the existing body of research on freeze-dried LAMP reactions and the recommendations by Dr. Pardee, the wetlab team decided that utilizing freeze-dried LAMP would be most suitable to using LAMP in the field. (Pardee, 2016) Because the colourimetric NEB LAMP master mix cannot be freeze-dried, iGEM Toronto experimented with

the LavaLAMP DNA Master Mix

in addition to NEB master mix.

* Key features of LavaLAMP:

1. Increased sensitivity and specificity
2. Can communicate with the manufacturer to customize the master mix for better performance
3. Freeze-dry compatible

Hence, wetlab developed the following combinations of LAMP experiments they could conduct given their primer sets, target DNA regions, and master mixes available.

## LAMP experimental workflows

Now that the dry lab had generated their candidate LAMP primers, the wet lab needed a way to experimentally determine the validity of these candidates as a diagnostic tool to succeed at amplifying oak wilt DNA.

#### Summary of Experimental Plan

| Step | Goal | Status |
| --- | --- | --- |
| 1 | Screen the primer sets through colorimetric LAMP reaction | Completed |
| 2 | Perform gel electrophoresis to better validate if the reaction results are false positive or false negative | Completed |
| 3 | Quantitatively compare the degree of amplification for the better primer sets | Completed |
| 4 | Replicate the above results to ensure experimental validity | Completed |
| 5 | Test if the selected LAMP reaction still works well when it is freeze-dried | Completed |
| 6 | Test if the selected LAMP reaction still works well when it is freeze-dried and when it is on paper | We did not experimentally demonstrate it because freeze dried paper-based LAMP assay is well-proven in literature (Wang, 2021) (Reboud, 2018) (Pardee, 2014). |
| 7 | Perform field tests (done in collaboration with all subteams) | We did not perform field tests because Michigan foresters notified us that spring/summer are the only seasons that can collect good samples to experiment with oak wilt disease. |

Details about what wet lab did can be found in the [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D) section.

Positive control: verified stock template DNA and primer set that work with the NEB master mix (We used the positive control provided by Lucigen)

Negative controls: consists of all LAMP reactions in a designated group WITHOUT template DNA. 1uL of water is added to replace the reaction’s template DNA.

## Step 1: Screen the primer sets through colorimetric LAMP reaction

### Materials and Protocols

The materials and protocols of the experiments are documented on the [Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) section.

### Combination of experiments

Each group represents a defined set of candidate LAMP experiments to be conducted together (to best compare results and adjust the protocols further). Because the color change of the reaction mix is not clear, the NEB protocol recommended users to run the reaction for an additional 10 minutes (NEB, 2016).

![step1 table](https://static.igem.wiki/teams/4479/wiki/step1-table.jpg)

Table 1: Groups of candidate LAMP experiments to be conducted by wetlab team (FIRST ROUND)

(Details in the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) section):
We hypothesize that primers designed for the MCM7 region are better at amplifying oak wilt DNA than the primers designed for the BT region because the primer set of PE2 of the MCM7 region had a slight color change.

### Learning from our experimental results

After reviewing these results, the wetlab team members met to discuss how to optimize our experimental protocol to yield better LAMP reaction products. The team had a few important realizations.

| Observation | Interpretation | Solution |
| --- | --- | --- |
| When we ran the thermal cycler for an extra 10 mins, the color change for PE2 was more visually obvious. | We excluded loop primers which are known to increase the speed of the LAMP reactions. | We let the reaction run for 60 minutes, as long as the negative control is still negative. |

Since the results with the NEB WarmStart Colorimetric master mix are not ideal, we experimented on Lucigen master mix to see if we can get more indicative results.

Lucigen master mix is colorless. Without access to fluorescence dye at the moment, we decided to visualize the results by gel electrophoresis.

## Step 2: Perform gel electrophoresis to better validate if the reaction results are false positive or false negative

### Materials and Protocols

The materials and protocols of the experiments are documented on the [Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) section.

### Combination of experiments

![step2 table](https://static.igem.wiki/teams/4479/wiki/step2-table.jpg)

Table 2: Groups of candidate LAMP experiments to be conducted by wetlab team (SECOND ROUND).

Note: 71 °C was in the middle of the Lucigen master mix recommended reaction temperature range (Lucigen);

### Quick Summary of Step 2

(Details in the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) section):

### Learning from our experimental results

After reviewing these results, the wetlab team members met to discuss how to optimize our experimental protocol to yield better LAMP reaction products. The team had a few important realizations.

| Observation | Interpretation | Solution |
| --- | --- | --- |
| Negative control shows a gel band that looks similar to the positive control. | In general, LAMP reactions are highly susceptible to contaminants in the lab. The major cause of contamination comes from our team opening the reaction tubes to perform gel electrophoresis. The DNA amplicons were released to the lab space as we opened the lab tubes. | To prevent such contamination, all surfaces and apparatus were wiped with 70% ethanol before reagents were mixed. Additionally, each LAMP reaction was prepared in a biosafety cabinet to prevent particulate matter or contents from the reaction/reagent tubes from entering any of the reaction mixtures. Furthermore, the wetlab LAMP reaction preparation protocol was designed to minimize opening and closing of tubes |

We performed group 1C and group 1D again with the proposed contamination controls in place. The controls could not fully eliminate the contamination issue.

We reached out to Jennifer and a graduate student in the Pardee lab, both had experience with LAMP. They shared that the only way we could avoid environmental contamination is by fully disinfecting the lab space or by changing the lab space. They also suggested that our lab reagents (master mix, primers, and/or DNA templates) could also be contaminated.

Therefore, iGEM Toronto immediately canceled any experimental plans related to gel electrophoresis and worked on finding another lab space that we could conduct our experiments in.

iGEM Toronto is thankful that the Pardee lab offered space and a clean NEB fluorescence master mix to continue our experiments at our most difficult time.

## Step 3: Fluorescence LAMP experiment and melting curve analysis, combined with step 4: perform replicates to ensure experimental validity

### Context and rationale:

In order to determine the most robust primer set from the candidates and to assess their amplification speed to determine which of them was best suited to in-field diagnostic use (this would ensure that diagnosis was rapid, which is a WHO ASSURED criteria), wetlab performed a fluorescence LAMP experiment using a qPCR machine; this allowed for a quantitative comparison of the degree of amplification with respect to time for each of the above primer sets.

Additionally, melting curve analysis was performed in tandem with the fluorescence LAMP experiment to verify if the amplification is targeted. When there is one single peak, it implies that there is only one single amplification product. By comparing the melting temperatures of the experimental sets, the wet lab can verify if the amplification is specific. When there is a shift in the melt peak of the same sample, it implies that there is off target amplification.

These data would aid wetlab in further decision-making regarding optimizations to the final LAMP reaction.

### Reagents

Clean NEB WarmStart® Fluorescent LAMP/RT-LAMP Kit with UDG (provided by Jennifer’s lab) was used for the fluorescence LAMP experiment to prevent contamination.

Primer stocks are also used to perform this experiment to prevent contamination.

All of our samples were run under 65 degree celsius with SYBR-Green fluorescent dye. Each tube of the test plate is 25 microliter with 12.5 microliter of NEB-WarmStart Master Mix 2X, 9 micro-liter of ddH2O, 1 microliter of customized concentration of DNA solution, and 2.5 microliter of customized primer mix.

### qPCR machine set-up:

The qPCR machine was programmed to undergo 60 cycles of amplification at 65 °C, each for 1 minute, followed by a series of heating and cooling for the melting curve analysis (see the figure below for details).

![step3 figure1](https://static.igem.wiki/teams/4479/wiki/step3-figure1.jpg)
![step3 figure2](https://static.igem.wiki/teams/4479/wiki/step3-figure2.jpg)
![step3 figure3](https://static.igem.wiki/teams/4479/wiki/step3-figure3.jpg)

### qPCR plate set-up:

The qPCR plate had 384 slots for reagents (see image above). To test all four primer sets for the MCM7 region (PE1, PE2, GLAPD1 and GLAPD2) and determine their limit of detection (LoD), wetlab employed the following dilution series of oak wilt target DNA (in ng/uL): 10, 5, 1, 0.5, 0.1. Three replicates for each dilution were performed (see Table 4 below).

![step3 table](https://static.igem.wiki/teams/4479/wiki/step3-table.jpg)

The wetlab team’s experimental results indicated that the above primer sets displayed logistic amplification above threshold levels, confirming their viability, with GLAPD1 displaying the most rapid amplification (see wetlab’s results page on the wiki).

## Step 5: Test if the selected LAMP reaction still works well when it is freeze-dried

### Motivation

LAMP reagents must be stored in a -20C fridge. Transportation of LAMP reagents in the forest would be a challenge. To achieve on-site detection of oak wilt, freeze-drying / lyophilization is a method to preserve our reagents, allowing our detection tool to be more portable.

### Materials and Protocols

Lucigen LavaLAMP master mix is used for this experiment because it can be freeze-dried. The freeze-dry experiment protocol is supplied by Jennifer and executed by Jennifer for our safety. We resuspended the freeze-dried reaction with ddH2O and measured the amount of fluorescence signal released during amplification using the qPCR machine.

### Combination of experiments

![step5 table](https://static.igem.wiki/teams/4479/wiki/step5-table.jpg)

### Quick Summary of Step 5

Target region MCM7 of the oak wilt DNA template was amplified by the PE1 primer set.

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# Results: Engineering Success of LAMP Primers for oak wilt

## Step 1: Screen the primer sets through colorimetric LAMP reaction

We first run the LAMP reaction for all sets with the NEB WarmStart Master
Mix, the amplifications are shown by the color change from pink to yellow.

Expected Results

Negative reactions are shown as pink around RGB (245,66,141)

Positive reactions are shown as yellow around RGB (245, 215, 66

## IGEM Toronto results

![](https://static.igem.wiki/teams/4479/wiki/wetlab-results/neb-mcm7-colour-30.jpg)

Fig 1. NEB LAMP kit on target region BT The primer ratio used is
FIP/BIP:F3/B3 = 8:1, and diluted to 1.6uM and 0.2uM in the mix. The
reaction also included two different target DNA concentrations, 2ng/ul,
and 10 ng/ul. The picture was taken at the 40 min mark.

![](https://static.igem.wiki/teams/4479/wiki/wetlab-results/neb-mcm7-colour-30.jpg)
![](https://static.igem.wiki/teams/4479/wiki/wetlab-results/neb-mcm7-colour-40.jpg )

Fig 2. NEB LAMP kit on target region MCM7 The primer ratio used is
FIP/BIP:F3/B3 = 8:1, and diluted to 1.6uM and 0.2uM in the mix. The
reaction also includes two different target DNA concentrations, 2ng/ul,
and 10 ng/ul. The left image was taken at the 30 min mark. The right
image was taken at the 40 min mark.

![](https://static.igem.wiki/teams/4479/wiki/wetlab-results/neb-mcm7-40.jpg)

Fig 3. NEB LAMP kit on target region MCM7 amplified by the primer set
PE2. The negative control, positive control, and the experimental result
are placed side by side to reflect the slight color change.

Using Google Colour Picker, the negative control RGB is (189,9,6). This
pink is darker than the expected result due to shadows. The positive
control RGB is (230,137,44). The yellow is not as bright yellow as the
expected result. The graduate student from Pardee's lab suggested that
is due to the heater we used in the lab does not have a heated lid to
keep the temperature even throughout the reaction tube. The experimental
result RGB is (235, 90, 12).

This experimental step does not give a clear result, although we could
see a slight color change on the PrimerExplorer 2 design for MCM7 (Fig
3). We believe this is not a strong evidence of success. It serves as a
direction indicator. We learnt that the pH dye changes color from pink
to yellow if and only if amplification takes place within the mix.

We hypothesize that primers designed for the MCM7 region are better at
amplifying oak wilt DNA than the primers designed for the BT region.

## Step 2: Perform gel electrophoresis to better validate if the reaction results are false positive or false negative

![](https://static.igem.wiki/teams/4479/wiki/wetlab-results/20220822-bt.png)

Fig 4A. Gel eletrophoresis result of the primer sets designed for the BT region. Lucigen Master Mix was used. This experiment was performed on 20220822

![](https://static.igem.wiki/teams/4479/wiki/wetlab-results/20220829-mcm7.png)

Fig 4B. MCM7, Lucigen, 20220829

To visualize the effectiveness of each primer set, we run gel electrophoresis on each of the LAMP products for the two target regions BT and MCM7, respectively (Fig 4. A, B). From Fig 4. A, primers designed for BT show no amplification at all, which is contrary to our expectation. In comparison, there are amplifications for MCM7 (Fig 4. B), especially for primer designer PE1. There are visible bands at 200 bp that match the target amplicon (215bp). This suggests that PE1 design for MCM7 is successful, while it could not be concluded only by the note, because:

1. gel electrophoresis with LAMP products inevitably produces smear and multiple off-target bands.
2. The
   gel electrophoresis does not validates quantitatively.
3. Reiteration of
   the experiment does not guarantee in replication of the same result.

We repeat in the gel electrophoresis experiment with changes in LAMP parameters. There are contamination issues found in the negative control group, and the experiment is set-backed by those unexplainable results. After consulting with specialists in the LAMP, Jennifer Doucet, we decided to use qPCR to quantitatively visualize our experimental result to gain a better understanding.

## Step 3: Quantitatively compare the degree of amplification for the better primer sets, combined with step 4: replicate the above results to ensure experimental validity

After a general test on each primer set and two different DNA regions, we
focus on amplifying the MCM7 DNA region because these primer sets showed
signs of success in previous experiments.

The context and rationale, reagents, qPCR machine set up and qPCR plate
set up are explained in the [Experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page.

Successful amplification of Oak Wilt MCM7 DNA region with all the primer
sets

\*\* Please note that the background noise of the amplification plot goes up to delta Rn = 5.00e+4

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/4479/wiki/wetlab-results/pe1-amp.png) Fig 5. Amplification plot of Oak Wilt PE1 LAMP Primers. The delta Rn reflects the LAMP fluorescence signal from SYBR Green minus the baseline signals by the machine. The plots of the same color are replicates. | Each primer set shows positive LAMP amplification on the MCM7 region for the following reasons:  The fluorescence signals of all samples pass the threshold in less than 60 minutes.   Issue:  The no template negative control also showed amplifications. Since we used a new master mix from the Pardee lab and stock primers, we can confirm that our stock DNA template has been contaminated. |
| ![](https://static.igem.wiki/teams/4479/wiki/wetlab-results/pe2-amp.png) Fig 6. Amplification plot of Oak Wilt PE2 LAMP Primers. The delta Rn reflects the LAMP fluorescence signal from SYBR Green minus the baseline signals by the machine. The plots of the same color are replicates. |
| ![](https://static.igem.wiki/teams/4479/wiki/wetlab-results/glapd1-amp.png) Fig 7. Amplification plot of Oak Wilt GLAPD1 LAMP Primers. The delta Rn reflects the LAMP fluorescence signal from SYBR Green minus the baseline signals by the machine. The plots of the same color are replicates. |
| ![](https://static.igem.wiki/teams/4479/wiki/wetlab-results/pe1-amp.png)Fig 8. Amplification plot of Oak Wilt GLAPD2 LAMP Primers. The delta Rn reflects the LAMP fluorescence signal from SYBR Green minus the baseline signals by the machine. The plots of the same color are replicates. |

## Comparing the Cq of each primer set

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Conc. of DNA template | 10ng/uL | 5ng/uL | 1ng/uL | 0.5ng/uL | 0.1ng/uL |  |
| Primer set | Mean Cq (cycles) and StDev (Note: 1 cycle = 1 minute) | | | | | Average |
| PE1 | 32.84+/-2.86 | 40.19+/-7.78 | 32.79+/-2.95 | 31.98+/-3.50 | 29.22+/-8.50 | 35 min |
| PE2 | 37.74+/-5.43 | 40.20+/-5.79 | 33.61+/-2.91 | 37.36+/-5.19 | 35.43+/-5.88 | 36 min |
| GLAPD1 | 24.65+/-2.06 | 25.64+/-1.62 | 25.02+/-1.91 | 29.60+/-4.07 | 35.17+/-0.41 | 27 min |
| GLAPD2 | 33.65+/-4.52 | 32.97+/-2.87 | 32.78+/-1.75 | 35.66+/-3.23 | 32.45+/-8.65 | 34 min |

![](https://static.igem.wiki/teams/4479/wiki/wetlab-results/10ul-amp.png)

Fig 9. Amplification plot of Oak Wilt LAMP Primers with 10ng/uL of DNA
template in the reaction. The delta Rn reflects the LAMP fluorescence
signal from SYBR Green minus the baseline signals by the machine. The
plots of the same color are replicates. The amplification curves of GLAPD1
look more similar to each other, compared to other primer sets.

Learnings

The GLAPD1 primer set took 27 cycles (approximate to 27 min based on the
qPCR machine set up) to reach the threshold line. The reaction efficiency
is higher than other primer sets. Three replicates were performed. The
standard deviations of the mean Cq for the GLAPD1 replicates are smaller
than the other primer sets. It also demonstrates consistency.