iGEM 2025 — Team mit-mahe

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Proper documentation is a necessity for scientific research. It ensures
reproducibility and accountability and facilitates analysis and long-term
integrity. This section serves as a chronologically compiled record of all
wet lab experiments performed.

This page has a handy filter feature so that any individual category of
experiments can be viewed exclusively.

## Wet Lab Training

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## Wet Lab Basic Training Assessment

### 17th March

1. All wet lab members passed the basic lab training assessment
   conducted by our primary investigator, Dr. Ritu Raval.
2. A lab familiarization session was held where we visited the lab &
   made an inventory of chemicals, reagents, and glassware.

## Wet Lab Basic Training

Basic molecular experiments were studied, performed, and analyzed.
The goal of this training session was to acclimate all the wet lab
members to the lab work environment and engage in troubleshooting as
and when required.

## Competent cell preparation and Transformation

### 18th March to 21st May

Day 0: *E. coli* DH5α culture from glycerol stock was revived
by plating a parent plate, which served as our source for the
primary culture. All required glassware were autoclaved.

Day 1: The primary culture was kept overnight for incubation, and
the acid-salt buffers were made.

Day 2: The secondary culture was inoculated. Confirmatory tests,
like Indole tests and Gram staining, were carried out at every step
to eliminate contamination. The competent cells were prepared, and
aliquots were stored at -20°C.

Day 3: The competent cells were thawed and incubated with pET22b
plasmids of 43.8 μg/mL concentration, and were transformed. The
cells were then plated on ampicillin plates.

![Fig A. Transformed E. coli DH5α (pET22b) plates with the control](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/wet-lab-training/a-transformed-ecoli-dh5-pet22b-plates-with-the-control.webp)

Fig A. Transformed *E. coli* DH5α (pET22b) plates with the
control.

Hence, *E. coli* DH5α cells were successfully made competent
and transformed with pET22b.

## SDS-PAGE

### 21st April

Sodium dodecyl sulphate polyacrylamide agarose gel electrophoresis
(SDS-PAGE) was one of the experiments performed in the basic
training period to learn the skills of gel making and, familiarize
with protein extraction and purification protocols.

The following gel percentages were used, given by Ms. Atheena Ma’am,
who guided us through the experiment.

Table 1. SDS - PAGE gel percentages

Constituents | Resolving Gel | | Stacking | Resolving Gel | | Stacking ||  | 12.5% | 15% | 4% | 12.5% | 15% | 4% |
| MilliQ Water | 2.303 mL | 1.467 mL | 3.505 mL | 1.151 mL | 0.733 mL | 1.752 mL |
| 30.8% Acrylamide | 4.159 mL | 5 mL | 0.739 mL | 2.079 mL | 2.5 mL | 0.369 mL |
| 1 M Tris: pH-8.8 | 3.327 mL | 3.323 mL | - | 1.663 mL | 1.661 mL | - |
| 0.5 M Tris: pH-6.8 | - | - | 0.649 mL | - | - | 0.324 mL |
| 10% SDS | 100 µL | 100 µL | 50 µL | 50 µL | 50 µL | 25 µL |
| 10% APS | 100 µL | 100 µL | 50 µL | 50 µL | 50 µL | 25 µL |
| TEMED | 10 µL | 10 µL | 6 µL | 5 µL | 5 µL | 3 µL |
|  | **For 2 gels** | | | **For 1 gel** | | |

![Fig B. SDS PAGE gel](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/wet-lab-training/b-sds-page-gel.webp)

Fig B. SDS PAGE gel

* The ladder was loaded in the first lane with a 10 to 245 kDa size
  range.
* Lane 1 is protein samples lysed from *E. coli* DH5α.
* Lane 2 is a protein of 70 kDa weight, extracted from
  *Candida albicans*, given by Ms. Atheena Ma’am.

Therefore, protein separation, purification, and analysis were
successfully conducted.

## Colony PCR

### 22nd April

From the transformed plate, one *E. coli* DH5α colony was
suspended in 10 µL of water. We used Takara Star 16S RNA primers for
the same. The reaction mixture contents were used as follows:

Table 2. PCR Master Mix contents

| Reagent | Volume (in µL) |
| --- | --- |
| Buffer | 12.5 (2X) |
| DNTPs | 2 |
| Forward primer | 1.5 |
| Reverse primer | 1.5 |
| Template | 1 |
| Taq polymerase | 0.25 |
| Water | 6.25 |

![Fig C. Amplified DNA run on 1% agarose gel with ladder. ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/wet-lab-training/c-amplified-dna-run-on-1-agarose-gel-with-ladder.webp)

Fig C. Amplified DNA run on 1% agarose gel with ladder.

* A 1kB DNA ladder was loaded into lane L.
* The amplified DNA was observed as a bright band in lane 1.

Hence, colony PCR from the transformed *E. coli* colonies was
performed, and the DNA was amplified.

## Plasmid Isolation

### 22nd May

1. From the transformed *E. coli* DH5α plates, one colony was
   resuspended in an Eppendorf tube with 30 µL of water, and the
   pET22b plasmid was isolated.
2. The QIAprep Spin Miniprep Kit – Plasmid Purification was used
   (QIAGEN, n.d.).
3. The symbols marked with a circle in the protocol were performed.

![Fig D. Spectrophotometer measurement of plasmid concentration using a nanocuvette  ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/wet-lab-training/d-spectrophotometer-measurement-of-plasmid-concentration-using-a-nanocuvette.webp)

Fig D. Spectrophotometer measurement of plasmid concentration
using a nanocuvette.

* The pET22b plasmid used during transformation was at a
  concentration of 43.8 μg/mL. As observed above, 42.2 μg/mL was
  isolated. These readings were taken using a nanocuvette with a
  spectrophotometer.
* The A260/280 ratio for DNA must be ideally ~1.8 to be considered
  pure. We obtained 1.78. The A260/230 ratio is supposed to be
  within 2.0 and 2.2. We obtained a lower value of 1.49, which can
  be attributed to phenol contamination or residual salts.
* Therefore, pET22b plasmids have been successfully isolated from
  *E. coli* DH5α culture and were stored at -20°C.

### References:

QIAGEN. (n.d.). QIAprep Spin Miniprep Kit | Plasmid DNA Isolation.
Retrieved October 6, 2025

<https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/plasmid-dna/qiaprep-spin-miniprep-kit>

### Week 1 - 25th July to 1st August

## *P. capsici* Subculturing 1

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## *P. capsici* Subculturing 1

### 25th July

#### Preparation of Media Plates for Subculturing

To subculture the *Phytophthora capsici* received from the
Indian Institute of Spices Research, potato dextrose agar (PDA) and
carrot agar were prepared in autoclaved glassware.

#### Preparation of Carrot Agar:

1. 20 g of carrot was blended and ground using a mortar and pestle.
2. 60 mL of distilled water was added to the carrot puree in a
   beaker.
3. The mixture was then boiled in a microwave.
4. The froth that developed on top was discarded to get a clear
   solution.
5. The mixture was then filtered through a muslin cloth, resulting in
   a clear, slightly orange solution.
6. The filtrate’s volume was then made up to 100 mL in a measuring
   flask using distilled water.
7. The carrot solution was combined with 1.8 g of agar in a conical
   flask.

#### Preparation of PDA:

2.4 g potato dextrose broth powder was combined with 100 mL
distilled water and 1.8 g agar in a conical flask.

The prepared nutrient media were autoclaved along with the cork
borer.

![Fig 1. Carrot agar media and PDA before autoclaving](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week1/1-carrot-agar-media-and-pda-before-autoclaving.webp)

Fig 1. Carrot agar media and PDA before autoclaving

### 26th July

#### Culturing *P. capsici* using Carrot Agar and PDA

1. The laminar air flow (LAF) chamber was cleaned, and the apparatus
   required for *P. capsici* culturing (disposable inoculation
   loops, agar plates, cork borer, etc.) was placed under the UV.
2. Four plates of carrot agar and PDA were made, of which three
   plates of each medium were designated for culturing. The cork
   borer technique was practiced on the additional PDA plate that was
   prepared.
3. Disks were cut from the parent culture using the cork borer,
   ensuring that they were not positioned too close to each other due
   to the disrupted hyphal network near the cut regions. The cut
   disks were then lifted using disposable inoculation loops.
4. Each disk was inverted before being placed on the agar plate to
   ensure that the mycelium was in contact with the agar. Some disks
   were difficult to remove due to the dense mycelial network.
5. One plate of each medium was also used to check the colony
   diameter from the centrally placed disk.
6. Two plates each of PDA and carrot agar were utilized for
   subcultures. Two disks were positioned at the periphery of the
   plates, ensuring ample separation from the edges. The procedure
   was conducted under the observation and guidance of the PI.
7. The plates were then initially placed in a cupboard, following
   which they were positioned in a dark incubator at 28°C, incubated
   upright (lid on top) to prevent the disks from losing contact with
   the agar.

![Fig 2. Phytophthora capsici plate received from IISR](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week1/	2-phytophthora-capsici-plate-received-from-iisr.webp)

Fig 2. *Phytophthora capsici* plate received from IISR

![Fig 3. Close-up picture of the mycelial plug](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week1/	3-close-up-picture-of-the-mycelial-plug.webp)

Fig 3. Close-up picture of the mycelial plug

## *P. capsici* Growth Curve 1 (26th July - 1st August)

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## Colony Diameter Measurement (Growth Curve)

### 27th July (Day 1)

A divider and ruler was used to measure the diameter of the growing
colony.

Plug diameter: 8 mm

#### Growth Curve PDA Day 1 (24 hours)

Y axis: 2.3 cm

X axis: 2.55 cm

Final reading: 1.625 cm

#### Growth Curve Carrot Agar Day 1 (24 hours)

Y axis: 3.6 cm

X axis: 3.6 cm

Final reading: 2.8 cm

The decontaminated cork borer was soaked in soap solution and
cleaned with distilled water. It was then autoclaved for future use.

#### Conclusion:

The growth of the culture was observed to be more rapid on carrot
agar compared to PDA.

### 28th July (Day 2)

Plug diameter: 8mm

#### Growth Curve PDA Day 2 (48 hours)

Y axis: 4.3 cm

X axis: 4.1 cm

Final reading: 3.4 cm

#### Growth Curve Carrot Agar Day 2 (48 hours)

Y axis: 6.2 cm

X axis: 6.3 cm

Final reading: 5.45 cm

### 29th July (Day 3)

Plug diameter: 8 mm

#### Growth Curve PDA Day 3 (72 hours)

Y axis: 5.95 cm

X axis: 6.1 cm

Final reading: 5.225 cm

#### Growth Curve Carrot Agar Day 3 (72 hours)

Y axis: 8.2 cm

X axis: 8.2 cm

Final reading: 7.4 cm

### 30th July (Day 4)

Plug diameter: 8 mm

#### Growth Curve PDA Day 4 (96 hours)

Y axis: 7.8 cm

X axis: 7.7 cm

Final reading: 6.97 cm

#### Growth Curve Carrot Agar Day 4 (96 hours)

Y axis: 8.2 cm

X axis: 8.2 cm

Final reading: 7.4 cm

### 31st July (Day 5)

Plug diameter: 8 mm

#### Growth Curve PDA Day 5 (120 hours)

Y axis: 7.8 cm

X axis: 7.7 cm

Final reading: 6.95 cm

#### Growth Curve Carrot Agar Day 5 (120 hours)

Y axis: 8.2 cm

X axis: 8.2 cm

Final reading: 7.4 cm

### 1st August (Day 6)

#### Growth Curve PDA Day 6 (144 hours)

Y axis: 7.8 cm

X axis: 7.7 cm

Final reading: 6.95 cm

#### Growth Curve Carrot Agar Day 6 (144 hours)

Y axis: 8.2 cm

X axis: 8.2 cm

Final reading: 7.5 cm

## Lactophenol Blue Staining

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## Lactophenol Blue Staining

### 26th July

#### Lactophenol blue staining:

1. A microscope slide was labelled, and a drop of lactophenol blue
   stain was placed at its center.
2. A small sample of the culture was carefully collected using a
   disposable inoculation loop, ensuring that the sporangium was not
   damaged. It was then spread onto the stain.
3. A cover slip was gently placed over the stained sample to prevent
   the formation of air bubbles.
4. The slide was then observed under a microscope at 10x and 40x
   magnifications.
5. Hyphae were observed under 40x magnification.

![Fig 4. P. capsici hyphae observed under 40x](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week1/4-p-capsici-hyphae-observed-under-40x.webp)

Fig 4. *P. capsici* hyphae observed under 40x

### 29th July

![Fig 5. P. capsici observed under 40x](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week1/5-p-capsici-observed-under-40x.webp)

Fig 5. *P. capsici* observed under 40x

## *P. capsici* Water Storage

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## Water Storage for *P. capsici*

### 29th July

#### *P. capsici* water storage:

1. ⁠1 mL of autoclaved sterile water was added to a 2 mL Eppendorf
   tube.
2. 4 fungal plugs were added to each tube.
3. The tubes were then parafilmed and stored at 15-28°C.

## Testing Chitosan Solubility

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## Chitosan nanoparticle production

### 30th July

#### Chitosan nanoparticle production preparation

1. The low molecular weight chitosan (Sigma Aldrich 448869-50G) was
   tested for its solubility.
2. Two samples of 4 mg chitosan in 1 mL of distilled water and 1%
   acetic acid solution were prepared. They were subjected to
   vortexing for 3 minutes.
3. The chitosan in the 1% acetic acid solution completely dissolved,
   whereas the water sample still had suspended chitosan particles.
4. To produce nanoparticles, a 2.4 mg/mL of Sodium Tripolyphosphate
   (TPP) solution was prepared and allowed to drop from a burette, to
   achieve the ideal flow rate of 60 mL/hr. The duration between two
   consecutive drops to achieve this was determined to be 6-7
   seconds.
5. The volume of chitosan nanoparticles to be prepared was adjusted
   by determining the magnetic bead size and the container in which
   the nanoparticles would be prepared (30-40 mL in 100 mL beakers).

![Fig 6. Chitosan in 1% acetic acid](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week1/6-chitosan-in-1-acetic-acid.webp)

Fig 6. Chitosan in 1% acetic acid

![Fig 7. Chitosan in sterile water (did not dissolve)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week1/7-chitosan-in-sterile-water-did-not-dissolve.webp)

Fig 7. Chitosan in sterile water (did not dissolve)

#### Conclusions:

The chitosan solution had better dissolution in 1% acetic acid
than in distilled water. To achieve the desired flow rate, the
duration between two drops must be between 6-7 seconds.

## Chitosan Nanoparticle Production 1

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## Chitosan Nanoparticle Production

### 31st July

1. To prepare 4 mg/mL of chitosan solution, 120 mg of chitosan was
   added to 30 mL of 1% acetic acid solution.
2. The chitosan solution was subjected to stirring using a magnetic
   stirrer at its maximum stable speed. On observing the lack of
   dissolution after a few hours, it was subjected to overnight
   stirring.
3. 30 mL of 2.4 mg/mL of TPP solution was prepared. The solution was
   passed through a 0.22 μm PVDF filter and kept ready for usage.

### 1st August

1. 30 mL of 0.4% chitosan solution was made the previous day;
   however, the volume had reduced to 20 mL upon overnight stirring,
   due to which the solution was brought up to 30 mL by the addition
   of 1% acetic acid solution.
2. The solution was then subjected to magnetic stirring. Syringe
   filtration was attempted; however, the process could not be
   performed due to the following reasons:
   * Solution was highly viscous.
   * A 0.22 μm pore size filter was used, which increased the risk
     of clogging.After 1 hour of syringe filtration, less than 5 mL of the filtrate
   was obtained.
3. The solution was subjected to filtration using a syringe pump.
   This did not work since:
   * The solution was too viscous.
   * The volume of the solution to be filtered was very low.
4. On the recommendation of the PI, 1 mL of acetic acid was added to
   the solution and magnetic stirring was carried out.
5. The solution was filtered using regular filter paper, but the
   process was unsuccessful as the solution was absorbed into the
   filter paper.
6. As Whatman filter paper was not available, the chitosan-acetic
   acid solution was stored in a 50 mL Falcon tube.
7. 30 mL of 0.1% chitosan-acetic acid solution was prepared and left
   for stirring overnight.

#### Conclusions:

Chitosan solution is highly viscous and cannot be filtered
effectively using syringe filtration.

### Week 2 - 2nd August to 8th August

## Chitosan Nanoparticle Production 2

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## Chitosan Nanoparticle Production

### 2nd August

1. 0.4% and 0.1% chitosan solutions were prepared in 1% acetic acid
   and stirred overnight.
2. The volume of both solutions was brought up to 30 mL by adding 1%
   acetic acid. The adjustment was necessary because the volume had
   decreased from 30 mL to less than 20 mL due to evaporation during
   stirring, as the solutions were not covered.
3. The 0.1% chitosan solution was filtered using filter paper. Owing
   to the viscosity of the 0.4% solution, an additional 1mL of acetic
   acid was added to it to reduce the viscosity.
4. The TPP solution was freshly prepared and filter sterilized using
   a 0.22 μm PVDF filter.
5. Using a burette, 6mL of TPP solution was added dropwise to 20 mL
   each of the 0.1% and 0.4% chitosan solutions under constant
   stirring at maximum stable speed. This ensured a 1:3 ratio of TPP
   to chitosan.
6. It was observed that the flow rate in the burette reduced as time
   passed. An attempt was made to ensure that the duration between
   two drops was 6–10 seconds.
7. Both solutions were allowed to stir for an additional 10 minutes
   post the addition of TPP. It was observed that the chitosan
   solution went from clear to opalescent, indicating the presence of
   nanoparticles.
8. 2 mL of each solution was analyzed at the Manipal Institute of
   Applied Physics, using a Particle Size Analyzer (Horiba Scientific
   Nano Partica SZ-100).

#### Results:

#### 0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 141.7 nm

PI: 0.428

Zeta Potential (mean): 9.9 mV

#### 0.4% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined

PI: Couldn’t be determined

Zeta Potential (mean): 26 mV

#### Conclusions:

The hydrodynamic radius of the 0.1% nanoparticle solution was
ideal, but the zeta potential was significantly low. The 0.4%
solution’s particle size was much higher than expected, due to the
formation of aggregates and insufficient filtration. Its Zeta
potential was also much lower than expected.

The Zeta potential could be increased by altering the amount of
acetic acid used, whereas the stirring duration would optimise the
nanoparticle size, along with proper filtration of the chitosan
solution.

![Fig 8. Chitosan solutions before and after the addition of TPP (left to right)(1)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week2/8-chitosan-solutions-before-and-after-the-addition-of-tpp-left-to-right-1.webp)
![Fig 8. Chitosan solutions before and after the addition of TPP (left to right)(2)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week2/8-chitosan-solutions-before-and-after-the-addition-of-tpp-left-to-right-1.webp)

Fig 8. Chitosan solutions before and after the addition of TPP
(left to right)

![Fig 18. Experimental setup for ionic gelation](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week2/9-experimental-setup-for-ionic-gelation.webp)

Fig 9. Experimental setup for ionic gelation

## *P. capsici* Growth Curve 1 (2nd August - 8th August)

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## Colony Diameter Measurement (Growth Curve)

### 2nd August (Day 7)

#### Growth Curve PDA Day 7 (168 hours)

Y axis: 8.3 cm

X axis: 8.3 cm

Final reading: 7.5 cm

#### Growth Curve Carrot Agar Day 7 (168 hours)

Y axis: 8.2 cm

X axis: 8.2 cm

Final reading: 7.4 cm

### 3rd August (Day 8)

#### Growth Curve PDA Day 8 (192 hours)

Y axis: 8.3 cm

X axis: 8.3 cm

Final reading: 7.5 cm

#### Growth Curve Carrot Agar Day 8 (192 hours)

Y axis: 8.2 cm

X axis: 8.2 cm

Final reading: 7.4 cm

### 4th August (Day 9)

#### Growth Curve PDA Day 9 (216 hours)

Y axis: 8.3 cm

X axis: 8.3 cm

Final reading: 7.5 cm

#### Growth Curve Carrot Agar Day 9 (216 hours)

Y axis: 8.2 cm

X axis: 8.2 cm

Final reading: 7.4 cm

### 5th August (Day 10)

#### Growth Curve PDA Day 10 (240 hours)

Y axis: 8.3 cm

X axis: 8.3 cm

Final reading: 8.2 cm

#### Growth Curve Carrot Agar Day 10 (240 hours)

Y axis: 8.2 cm

X axis: 8.2 cm

Final reading: 7.4 cm

#### Conclusions:

Over the course of the ten days, it was observed that the
*P. capsici* showed faster growth on the carrot agar plate
as compared to the potato dextrose agar plate. The carrot agar
plate showed uniform growth and was identical to the original
*P. capsici* plate we had received from IISR, Kozhikode.

## Chitosan Nanoparticle Production 3

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## Chitosan Nanoparticle Production

### 3rd August

1. 30 mL of 0.4% chitosan solution was prepared in 1% acetic acid
   solution. The solution was subjected to overnight stirring.

### 4th August

1. The 0.4% chitosan solution kept for stirring overnight was
   opalescent, with fine suspended particles observed. This was due
   to improper cleaning of the beakers, which might have contained
   residual TPP solution.
2. A fresh 0.4% chitosan solution was prepared and kept for overnight
   stirring.
3. As seen in Fig 10., the chitosan solution became foamy and opaque
   after stirring overnight.

![Fig 10. Chitosan solution after magnetic stirring overnight](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week2/10-chitosan-solution-after-magnetic-stirring-overnight-it-was-very-foamy-and-opaque.webp)

Fig 10. Chitosan solution after magnetic stirring overnight

## Chitosan Nanoparticle Production 4

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## Chitosan Nanoparticle Production

### 5th August

1. The overnight 0.4% chitosan solution was observed to be foamy the
   next morning, and therefore passed through filter paper.
2. TPP solution was freshly prepared and passed through a 0.22 μm
   PVDF filter.
3. 6 mL of TPP solution was added to 20 mL of the 0.4% chitosan
   solution using a burette. On average, the time between drops was
   adjusted to 7–10 seconds.
4. The solution was allowed to stir throughout the process, and for
   an additional 25 minutes post the addition of TPP.
5. The samples were tested using the Particle Size Analyzer.

#### Results:

#### 0.4% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined

PI: Couldn’t be determined

Zeta Potential (mean): 1 mV

Subsequently, two chitosan solutions (0.1% chitosan in 1% acetic
acid and 0.2% chitosan in 1% acetic acid) were prepared to further
optimize the concentrations for nanoparticle production.

#### Conclusions:

The 0.4% chitosan solution was ruled out for nanoparticle
production, as aggregation was inferred from the Particle Size
Analyzer results.

## Chitosan Nanoparticle Production 5

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## Chitosan Nanoparticle Production

### 7th August

1. The overnight 0.1% chitosan solutions were filtered.
2. A 2.4 mg/mL TPP solution was prepared and filtered using a 0.22 μm
   PVDF filter.
3. A glass column with precise flow control was used to maintain a
   constant flow rate of 7.2 seconds between TPP drops. The 1:2 and
   1:3 TPP to chitosan solutions were prepared by adding 7.5 mL and 5
   mL of TPP to 15 mL of chitosan solutions, respectively.
   ![  Fig 11. Glass column setup to achieve the ideal TPP flow rate](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week2/11-glass-column-setup-to-achieve-the-ideal-tpp-flow-rate.webp)

   Fig 11. Glass column setup to achieve the ideal TPP flow rate
4. Both solutions were subjected to magnetic stirring throughout the
   process. They were allowed to stir for an additional 20 minutes
   post-TPP addition.
5. The samples were tested using the Particle Size Analyzer.

#### Results:

#### 0.1% Chitosan Solution in 1% Acetic Acid (1:2 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined

PI: Couldn’t be determined

Zeta Potential (mean): 16.3 mV

#### 0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 1808.0 nm

PI: 2.515

Zeta Potential (mean): 9.8 mV

![ Fig 12. The chitosan nanoparticle solutions that were subjected to analysis](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week2/12-the-chitosan-nanoparticle-solutions-that-were-subjected-to-analysis.webp)

Fig 12. The chitosan nanoparticle solutions that were subjected to
analysis

Subsequently, two 30 mL 0.1% chitosan solutions of 1.22% and 0.8%
acetic acid were prepared. The solutions were stirred overnight.

#### Conclusions:

The non-ideal values for polydispersity, hydrodynamic radius, and
zeta potential could be due to insufficient mixing during
nanoparticle formation. A fine suspension observed in the
solutions indicates the same. The solutions must be subjected to
sonication after the addition of TPP.

## Chitosan Nanoparticle Production 6

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## Chitosan Nanoparticle Production

### 8th August

1. The overnight chitosan solutions were passed through filter paper.
   ![Fig 13. Foamy chitosan solution before filtering](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week2/13-foamy-chitosan-solution-before-filtering.webp)

   Fig 13. Foamy chitosan solution before filtering
2. A 2.4 mg/mL TPP solution was prepared and filtered through a 0.22
   μm PVDF filter.
3. The addition of TPP was done using the glass column, with a
   constant flow rate of 8 seconds between drops. 6 mL of TPP was
   added to each of the 20 mL chitosan solutions to make 1:3
   nanoparticle solutions.
4. The solutions were stirred throughout the process. They were
   allowed to spin for 20 minutes post-TPP addition.
5. The samples were tested using the Particle Size Analyzer.

#### Results:

#### 0.1% Chitosan Solution in 0.8% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined

PI: Couldn’t be determined

Zeta Potential (mean): 14.4 mV

#### 0.1% Chitosan Solution in 1.22 % Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined

PI: Couldn’t be determined

Zeta Potential (mean): 16.5 mV

Subsequently, two chitosan solutions (0.1% chitosan in 1% acetic
acid) were prepared. The volume was increased to 40 mL to account
for losses during overnight stirring.

#### Conclusions:

A fine suspension was observed yet again in the solutions,
indicating insufficient mixing during ionic gelation. This
confirms that sonication of the sample for a period of 10 minutes
(10 seconds ON/OFF) at 50% amplitude is necessary.

## *P. capsici* Subculturing 2

+

✖

## *P. capsici* Subculturing 2

### 8th August

1. The LAF and apparatus (inoculation loops, carrot agar plates, cork
   borer, etc) were cleaned and sterilized through UV treatment.
2. Mycelial discs from the primary culture were made and inverted
   onto 3 fresh carrot agar plates, using the cork borer and
   inoculation loops. These plates were used to study the growth of
   the culture.
3. One plate was set with 2 mycelial discs to serve as the
   subculture.
4. The plates were then placed in the dark incubator at 28°C.

### Week 3 - 9th to 15th August

## Chitosan Nanoparticle Production 7

+

✖

## Chitosan Nanoparticle Production

### 9th August

1. Chitosan nanoparticles were prepared using 0.1% and 0.2% chitosan
   solutions, keeping the acetic acid concentration constant, and a
   chitosan to TPP ratio of 1:3.
2. The overnight stirred chitosan solutions of 0.1% and 0.2% were
   used. The 0.1% solution was less foamy.
3. 2.4 mg/mL of TPP solution was prepared. The solution was filtered
   using a 0.22 μm PVDF filter. A total volume of 40 mL of chitosan
   in acetic acid was subjected to magnetic stirring, and 30 mL was
   measured to make the nanoparticles and account for any losses
   during filtration.
4. Using the glass cylinder with a valve, a constant flow rate of 8
   seconds was maintained between consecutive drops. Both solutions
   were stirred using separate magnetic stirrers, and after adding
   TPP, they were allowed to spin for an additional 20 minutes.
5. The samples were then sonicated for 10 minutes with 50% amplitude
   and a 10-second ON/OFF pulse cycle.

#### Results:

#### 0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 146.5 nm
PI: 0.287
Zeta Potential (mean): 13.5 mV

#### 0.2% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined
PI: Couldn’t be determined
Zeta Potential (mean): 21.1 mV

#### 0.1% Chitosan solution in 0.8% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 148.1 nm
PI: 0.471
Zeta Potential (mean): 15.1 mV

#### 0.1% Chitosan solution in 1.22% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 113.3 nm
PI: 0.376
Zeta Potential (mean): 19.7 mV

#### Conclusions:

A fine suspension of particles was observed in both solutions,
indicating insufficient mixing.

The chitosan solutions must be subjected to sonication for 10
minutes, with 10 seconds ON/OFF off pulse at 60% amplitude.

The nanoparticle samples made on 8th August were also
subjected to sonication with the same parameters before
characterization.

## *P. capsici* Growth Curve 2

+

✖

## Colony Diameter Measurement (Growth Curve)

### 9th August (Day 1)

#### Growth Curve Carrot Agar Plate 1 (24 hours)

Y axis: 1.7 cm
X axis: 2.05 cm
Final reading: 1.875 cm

#### Growth Curve Carrot Agar Plate 2 (24 hours)

Y axis: 2.35 cm
X axis: 2.45 cm
Final reading: 2.4 cm

#### Growth Curve Carrot Agar Plate 3 (24 hours)

Y axis: 2.55 cm
X axis: 2.4 cm

Final reading: 2.475 cm

### 10th August (Day 2)

#### Growth Curve Carrot Agar Plate 1 (48 hours)

Y axis: 4.4 cm
X axis: 4.9 cm

Final reading: 3.85 cm

#### Growth Curve Carrot Agar Plate 2 (48 hours)

Y axis: 5.4 cm
X axis: 4.8 cm

Final reading: 4.3 cm

#### Growth Curve Carrot Agar Plate 3 (48 hours)

Y axis: 5.2 cm
X axis: 4.5 cm

Final reading: 4.05 cm

### 11th August (Day 3)

#### Growth Curve Carrot Agar Plate 1 (72 hours)

Y axis: 7.4 cm
X axis: 7.35 cm

Final reading: 6.575 cm

#### Growth Curve Carrot Agar Plate 2 (72 hours)

Y axis: 7.8 cm
X axis: 7.4 cm

Final reading: 6.8 cm

#### Growth Curve Carrot Agar Plate 3 (72 hours)

Plate 3 was observed to contain visible contamination. The plate was
decontaminated and discarded.

## Detached Leaf Assay 1

+

✖

## Detached Leaf Assay

### 12th August

1. Three plastic, transparent boxes were bought from the store and
   were wiped thoroughly with 70% ethanol.
2. Only the bottom of the boxes were lined with moistened cotton to
   ensure that the sides were still see-through.
3. *Piper nigrum* Paniyur 1 variety leaves were taken from the
   campus. The third leaf was taken precisely from the growing tip to
   ensure uniform maturity. These leaves were cleaned with 70%
   ethanol and placed flat on the moistened cotton.
4. The subcultures used in all cases were 68 hours old and were grown
   in carrot agar plates.
5. There were six leaves plucked, and the assay was performed in
   duplicates. One was for the control, where only the leaf and the
   oomycete were placed. The second one was to test the effect of 1%
   acetic acid alone.
6. The third was to test the 0.1% chitosan in 1% acetic acid solution
   (1:3 TPP to CS) nanoparticles.
7. A sterile one-time use needle was heat-flamed and used to poke
   10-20 holes across the centre of the leaf. The needle was then
   discarded.
8. A 5 mm mycelial disk from subculture was placed at the centre so
   that the mat made contact with the leaf surface. Two leaves were
   inoculated in this manner, serving as the control.
9. For the second set, 1 mL of 1% acetic acid solution was sprayed on
   two leaves using a spray bottle, which was sterilized earlier.
   Then the same inoculum was made.
10. For the third set, 1 mL of 0.1% chitosan in 1% acetic acid with
    1:3 TPP to CS nanoparticle solution was sprayed on two leaves. The
    leaf was then inoculated.
11. The boxes were sealed tightly and placed in the sunlight. This is
    called the Cabin Sequester Method. The lesions were then observed
    and measured periodically.

### 13th August

#### Observation:

The lesions on the detached leaf assay performed yesterday were
observed. The boxes had moisture in them as there was visible mist
on the inner surfaces.

There was a black spot underneath the plug on the control leaves,
specifically visible in the right one.

Visible mycelial growth was observed on the leaf treated with acetic
acid.

The leaf treated with chitosan nanoparticles (0.1% CS in 1% AA with
1:3 TPP to CS) remained the same compared to the time of
inoculation.

These zoospore induction petri dishes were observed, and the plugs
had absorbed the water.

### 15th August (48 hrs)

#### Control leaf (left) -

X axis 1.8 cm
Y axis 1.4 cm

#### Control leaf (right) -

X axis 0.9 cm
Y axis 1.3 cm

The leaves sprayed with 1% acetic acid and 0.1% chitosan solution
showed no lesion formation. However, mycelial growth was observed on
the leaves sprayed with acetic acid.

![Fig 14. Control leaves(1)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week3/14-control-leaves-1.webp)
![Fig 14. Control leaves(2)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week3/14-control-leaves-2.webp)

Fig 14. Control leaves

![Fig 15. Leaves sprayed with 1% Acetic acid ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week3/15-leaves-sprayed-with-1-acetic-acid.webp)

Fig 15. Leaves sprayed with 1% Acetic acid

![Fig 16. Leaves sprayed with 0.1% chitosan nanoparticle solution ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week3/16-leaves-sprayed-with-0-1-chitosan-nanoparticle-solution.webp)

Fig 16. Leaves sprayed with 0.1% chitosan nanoparticle solution

## *P. capsici* Sporulation 1

+

✖

## Zoospore Induction

### 12th August

1. Two autoclaved petri dishes were filled halfway with autoclaved
   distilled water and kept with the lid facing upward.
2. Ten-fifteen 5 mm mycelial disks were cut on a subculture plate
   approximately 68 hrs old.
3. These plugs were suspended in the petri dish with water so that
   the mat faced upwards and the agar chunk remained soaked in the
   water.
4. The plates were parafilmed carefully and placed for incubation
   with illumination at 26°C.

![Fig 17. Zoospore induction - Mycelial plugs kept for illuminated incubation.](http://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week3/17-zoospore-induction-mycelial-plugs-kept-for-illuminated-incubation.webp)

Fig 17. Zoospore induction - Mycelial plugs kept for illuminated
incubation.

### 14th August

The previously made 0.1% chitosan in 1% acetic acid solution (1:3
TPP to chitosan ratio) was filtered.

The two sporulation plates kept for 48 hrs of light incubation were
given a cold shock treatment by keeping them in the 4°C fridge for
30 minutes.

The zoospore solution from these plates was used to make six
Eppendorf vials:

1. Two tubes contained the zoospore solution (1 mL pipetted out,
   which was later aliquoted to 100 μL)
2. Two tubes contained zoospore solution + 1% acetic acid solution (1
   mL zoospore solution + 0.5 mL acetic acid)
3. Two tubes contained zoospore solution + the 0.1% chitosan
   nanoparticle solution (1 mL zoospore solution + 0.5 mL chitosan NP
   solution)

100 μL of Methylene blue dye was added to the vials containing only
the 100 μL zoospore solution & the solutions were observed using a
haemocytometer.

![Fig 18. Observation of the zoospore solution under a microscope](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week3/18-observation-of-zoospore-solution-under-a-microscope.webp)

Fig 18. Observation of the zoospore solution under a microscope

#### Conclusions:

The amount of methylene blue added was too much to visualize the
staining properly. We were advised to add a drop or two to the 100
μL of zoospore solution.

## Chitosan Nanoparticle Production 8

+

✖

## Chitosan Nanoparticle Preparation

### 12th August

Re-sonication of CSNP samples from 9th and 10th
August and their characterization.

1. The CSNP samples prepared earlier, which were sonicated once
   already at 50% amplitude with a 10-second ON/OFF pulse for 20
   minutes, were to be subjected to the same process again.
2. The parameters were tweaked this time to be 60% amplitude, 10
   seconds ON/OFF for 20 minutes. These were sent for
   characterization using a Particle Size Analyzer.

#### Results:

#### 0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 146.5 nm
PI: 0.287
Zeta Potential (mean): 12.2 mV

#### 0.2% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined
PI: Couldn’t be determined
Zeta Potential (mean): 21.1 mV

#### 0.1% Chitosan solution in 0.8 % Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 148.1 nm
PI: 0.471
Zeta Potential (mean): 15.1 mV

#### 0.1% Chitosan solution in 1.22 % Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 113.3 nm
PI: 0.376
Zeta Potential (mean): 19.7 mV

#### Conclusions:

Though the hydrodynamic radius seemed to be around the expected
range, the Zeta potential values weren’t as anticipated, even
after sonication. It was then established that the pH should be
measured after every step to gauge the stability.

## Chitosan Nanoparticle Production 9

+

✖

## Chitosan Nanoparticle Production

### 12th August

1. Two solutions of 30 mL of 0.1% chitosan in 1% acetic acid were
   made. They were magnetically stirred for 30 minutes.
2. Following this, they were sonicated at 60% amplitude with a
   10-second ON/OFF pulse for 20 minutes.
3. These were then stored at 4°C.

### Week 4 - 16th to 22nd August

## Detached Leaf Assay 1

+

✖

## Detached Leaf Assay

### 16th August (72 hrs)

#### Control leaf (left) -

X axis: 1.9 cm
y axis: 2.15 cm

#### Control leaf (right) -

X axis: 0.9 cm
Y axis: 1.5 cm

![Fig 19. Control leaves (1)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week4/19-control-leaves-1.webp)
![Fig 19. Control leaves (2)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week4/19-control-leaves-2.webp)

Fig 19. Control leaves

![Fig 20. Leaves sprayed with 1% Acetic acid](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week4/20-leaves-sprayed-with-1-acetic-acid.webp)

Fig 20. Leaves sprayed with 1% Acetic acid

![Fig 21. Leaves sprayed with 0.1% chitosan nanoparticle solution ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week4/21-leaves-sprayed-with-0-1-chitosan-nanoparticle-solution.webp)

Fig 21. Leaves sprayed with 0.1% chitosan nanoparticle solution

### 17th August (96 hrs)

#### Control leaf (left) -

X axis: 2.3 cm
Y axis: 2.95 cm

#### Control leaf (right) -

X axis: 1.1 cm
Y axis: 1.5 cm

![Fig 22. Control leaves (1)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week4/22-control-leaves-1.webp)
![Fig 22. Control leaves (2)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week4/22-control-leaves-2.webp)

Fig 22. Control leaves

![Fig 23. Leaves sprayed with 1% Acetic acid](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week4/23-leaves-sprayed-with-1-acetic-acid.webp)

Fig 23. Leaves sprayed with 1% Acetic acid

![Fig 24. Leaves sprayed with 0.1% chitosan nanoparticle solution](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week4/24-leaves-sprayed-with-0-1-chitosan-nanoparticle-solution.webp)

Fig 24. Leaves sprayed with 0.1% chitosan nanoparticle solution

## Chitosan Nanoparticle Production 9

+

✖

## Chitosan Nanoparticle Production

### 19th August

1. Two solutions of 0.1% chitosan in 1% acetic acid were prepared
   earlier and kept. The TPP solution was also prepared beforehand.
2. The chitosan solutions were sonicated for 10 minutes with a 10
   seconds ON/OFF pulse at 60% amplitude.
3. The chitosan solution's pH was measured after the same. It was
   found to be 2.71. While loading one of the samples into the pocket
   pH meter, the pipette was contaminated with an unknown liquid,
   which was also accidentally introduced into the sample. Due to
   this unfortunate event, we discarded one of the two chitosan
   solutions. Following this, the viable sample was filtered.
4. The pH of TPP was also measured, and it turned out to be 8.53.
   Post which the TPP was syringe filtered.
5. The burette was rinsed with TPP, and 10 mL was filled. A beaker
   with 30 mL chitosan solution (1:3 TPP to chitosan ratio) was spun
   at the maximum stable flow rate using a magnetic stirrer. TPP was
   added to the periphery at the highest possible flow rate.
6. The solution was allowed to stir for the next 20 minutes. This was
   then sonicated with the same parameters as earlier.
7. The samples were then sent for characterization using a Particle
   Size Analyzer.

#### Results:

#### 0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined
PI: Couldn’t be determined
Zeta Potential (mean): 1.9 mV

#### Conclusions:

It has been established that the ideal flow rate for TPP addition
is 6-8 seconds, and the magnetic stirring has to be on the maximum
possible limit with the highest vortex effect, ensuring no
splashing.

The reason for the unsuccessful nanoparticle production was the
rapid flow rate at which TPP was added.

After discussing with our PI, Dr. Ritu Raval, we were advised to
produce another nanoparticle in duplicates the following day (0.1%
CS, 1% acetic acid, 1:3 TPP to CS).

## Chitosan Nanoparticle Production 10

+

✖

## Chitosan Nanoparticle Production

### 20th August

1. Two 0.1% chitosan solutions were made in 1% acetic acid.
2. The solutions underwent magnetic stirring for 30 minutes and were
   sonicated for 10 minutes each, with a 10-second ON/OFF pulse at
   60% amplitude. The TPP solution was freshly prepared as well.
3. The pH of the acetic acid, TPP, and chitosan solutions were
   measured using a pocket pH meter and were found to be 1.51, 9.18,
   and 2.23, respectively.
4. The solutions were filtered afterwards, and 10 mL of the TPP
   solution was loaded onto the burette. 30 mL of the chitosan
   solution was placed on a magnetic stirrer below, to obtain the
   desired 1:3 TPP to chitosan ratio.
5. The TPP was added dropwise (7-second interval between drops),
   after which the solution was stirred for 20 minutes. One of the
   samples was lost at this stage due to human error.
6. The remaining sample was then sonicated at the same parameters and
   sent for characterization using a Particle Size Analyzer.

#### Results:

#### 0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): Couldn’t be determined
PI: Couldn’t be determined
Zeta Potential (mean): 10.2 mV

#### Conclusions:

Upon consulting our PI regarding the results, several
optimizations to the protocol were suggested. The pH of the
chitosan solution was to be adjusted to between 4 and 4.5, using
1N NaOH. An additional centrifugation and decanting step was
suggested post-nanoparticle formation to obtain the ideal zeta
potential.

## Chitosan Nanoparticle Production 11

+

✖

## Chitosan Nanoparticle Production

### 21st August

1. A 1% acetic acid solution was prepared, with a pH of 2.63. On
   preparing two 40 mL, 0.1% chitosan solutions, which underwent
   magnetic stirring (Sample 1 using Trishul magnetic stirrer, Sample
   2 using Eltech magnetic stirrer) for 30 minutes, their pH was
   adjusted from 2.63 to 4.01. This was done by adding 2150 μL of 1 N
   NaOH to 35 mL of the chitosan solution.
2. The solutions underwent sonication (5 minutes, 10 seconds ON/OFF
   pulse, 40% amplitude) at optimized parameters.
3. The TPP and chitosan solutions were filtered and loaded onto the
   burette and beaker. The TPP was added to the chitosan solutions
   dropwise in a 1:3 ratio of TPP to chitosan solution, at the ideal
   flow rate of 7 seconds between drops. Following this, the
   nanoparticle solutions were stirred for 20 minutes.
4. After nanoparticle formation, the solutions underwent sonication
   for 5 minutes at 40% amplitude with a 10 seconds ON/OFF pulse
   cycle.
5. The solutions underwent centrifugation for 5 minutes at 1000 rpm
   at 26°C. The solutions were then decanted using a glass pipette
   while preserving the Falcon tube's orientation from the
   centrifuge.
6. These solutions were then characterized using a Particle Size
   Analyzer.

#### Results:

#### Sample 1- 0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 267.2 nm
PI: 0.363
Zeta Potential (mean): 80.4 mV

#### Sample 2- 0.1% Chitosan Solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 78.5 nm
PI: 0.237
Zeta Potential (mean): 10.3 mV

#### Conclusions:

Upon consulting our PI regarding the results, specific
optimizations to the protocol were suggested. After adding TPP to
the chitosan solution, the centrifugation must be 8 minutes rather
than 5 to adjust the size. Based on the results, we were advised
to use the Eltech magnetic stirrer for future nanoparticle
preparations.

### Week 5 - 23rd to 29th August

## *P. capsici* Growth Curve 3

+

✖

## Colony Diameter Measurement (Growth Curve)

### 25th August

Carrot agar media was prepared by combining 20 g of grated carrot
and 1.8 g agar to observe the growth of *P. capsici*.

Potato dextrose agar was also prepared using 2.4 g of potato
dextrose broth and 1.8 g of agar, to compare the growth on the two
media.

Carrot agar and PDA were poured into 4 petri dishes, and were left
in the 4°C fridge.

### 26th August

The plates were placed in the LAF. The *P. capsici* culture
inoculated on 8th August was used to make the mycelial plugs, with
the help of an 8 mm cork borer. The plugs were placed mycelium with
the mycelium facing down on the plates.

Two PDA plates were inoculated with a single plug at the centre for
growth measurements. The same was done for two carrot agar plates.

Additionally, a carrot agar plate was prepared with two mycelial
plugs on opposite sides. This would serve as the fresh subculture
plate.

The plates were then parafilmed and incubated at 28°C.

![Fig 25. Preparation of fresh media plates for the subculture and growth curve ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/25-preparation-of-fresh-media-plates-for-the-subculture-and-growth-curve.webp)

Fig 25. Preparation of fresh media plates for the subculture and
growth curve

### 27th August (Day 1)

#### Growth Curve Carrot Agar Plate 1 Day 1 (24 hrs)

Y axis: 3.45 cm
X axis: 3.5 cm
Final reading: 2.675 cm

#### Growth Curve Carrot Agar Plate 2 Day 1 (24 hrs)

Y axis: 4 cm
X axis: 3.35 cm
Final Reading: 2.825 cm

#### Growth Curve PDA Plate 1 Day 1 (24 hrs)

Y axis: 2.15 cm
X axis: 2.1 cm
Final Reading: 1.325 cm

#### Growth Curve PDA Plate 2 Day 1 (24 hrs)

Y axis: 2.3 cm
X axis: 2.1 cm
Final Reading: 1.4 cm

![Fig 26. Carrot agar plates 1 and 2 (24 hrs old)(1)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/26-carrot-agar-plates-1-and-2-24-hours-old-1.webp)![Fig 26. Carrot agar plates 1 and 2 (24 hrs old)(2)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/26-carrot-agar-plates-1-and-2-24-hours-old-2.webp)

Fig 26. Carrot agar plates 1 and 2 (24 hrs old)

![ Fig 27. PDA plates 1 and 2 (24 hrs old)(1)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/27-pda-plates-1-and-2-24-hours-old-1.webp)![ Fig 27. PDA plates 1 and 2 (24 hrs old)(2)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/27-pda-plates-1-and-2-24-hours-old-2.webp)

Fig 27. PDA plates 1 and 2 (24 hrs old)

### 28th August (Day 2)

#### Growth Curve Carrot Agar Plate 1 Day 2 (48 hrs)

Y axis: 5 cm
X axis: 4.7 cm
Final reading: 4.05 cm

#### Growth Curve Carrot Agar Plate 2 Day 2 (48 hrs)

Y axis: 5.3 cm
X axis: 4.9 cm
Final Reading: 4.3 cm

#### Growth Curve PDA Plate 1 Day 2 (48 hrs)

Y axis: 3.2 cm
X axis: 3.3 cm
Final Reading: 2.45 cm

#### Growth Curve PDA Plate 2 Day 2 (48 hrs)

Y axis: 3.2 cm
X axis: 3.1 cm
Final Reading: 2.35 cm

![Fig 28. Carrot agar plates 1 and 2 (48 hrs old)(1)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/28-carrot-agar-plates-1-and-2-48-hours-old-1.webp)![Fig 28. Carrot agar plates 1 and 2 (48 hrs old)(2)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/28-carrot-agar-plates-1-and-2-48-hours-old-2.webp)

Fig 28. Carrot agar plates 1 and 2 (48 hrs old)

![Fig 29. PDA plates 1 and 2 (48 hrs old)(1)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/29-pda-plates-1-and-2-48-hour-old-1.webp)![Fig 29. PDA plates 1 and 2 (48 hrs old)(2)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/29-pda-plates-1-and-2-48-hour-old-2.webp)

Fig 29. PDA plates 1 and 2 (48 hrs old)

### 29th August (Day 3)

#### Growth Curve Carrot Agar Plate 1 Day 3 (72 hrs)

Y axis: 6.4 cm
X axis: 6.5 cm
Final reading: 5.65 cm

#### Growth Curve Carrot Agar Plate 2 Day 3 (72 hrs)

Y axis: 6.9 cm
X axis: 6.0 cm
Final Reading: 5.65 cm

#### Growth Curve PDA Plate 1 Day 3 (72 hrs)

Y axis: 4.6 cm
X axis: 4.4 cm

Final Reading: 3.7 cm

#### Growth Curve PDA Plate 2 Day 3 (72 hrs)

Y axis: 4.7 cm
X axis: 4.5 cm

Final Reading: 3.8 cm

![Fig 30. Carrot agar plates 1 and 2 (72 hrs old)(1)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/30-carrot-agar-plates-1-and-2-72-hours-old-1.webp)![Fig 30. Carrot agar plates 1 and 2 (72 hrs old)(2)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/30-carrot-agar-plates-1-and-2-72-hours-old-2.webp)

Fig 30. Carrot agar plates 1 and 2 (72 hrs old)

![Fig 31. PDA plates 1 and 2 (72 hrs old)(1)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/31-pda-plates-1-and-2-72-hours-old-1.webp)![Fig 31. PDA plates 1 and 2 (72 hrs old)(2)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/31-pda-plates-1-and-2-72-hours-old-2.webp)

Fig 31. PDA plates 1 and 2 (72 hrs old)

## Chitosan Nanoparticle Production 12

+

✖

## Chitosan Nanoparticle Production

### 23rd August

1. Two 0.1% chitosan solutions were prepared in 40 mL of 1% acetic
   acid. Their pH was adjusted from 2.63 to 4.01. This was done by
   the addition of 2150 μL of 1 N NaOH. Prior to adding the NaOH to
   the chitosan solutions, an estimate of volume was acquired by
   directly adding NaOH to acetic acid.
2. The solutions underwent magnetic stirring for 30 minutes and
   sonication for 5 minutes at a 10 seconds ON/OFF pulse cycle at 40%
   amplitude.
3. The 2.4 mg/mL TPP and chitosan solutions were filtered and loaded
   onto the burette and beaker, respectively. The TPP was added
   dropwise after adjusting the flow rate to 7 seconds between drops,
   following which the nanoparticle solution was stirred for 20
   minutes.
4. A second round of ultrasonication was performed for 5 minutes at a
   10 seconds ON/OFF pulse cycle at 40% amplitude.
5. After this, the 2 solutions underwent centrifugation for 5 minutes
   at 1000 rpm at 26°C. The supernatant was collected while
   preserving their orientation as they would be in the centrifuge
   (to prevent disturbing the invisible pellet of macroparticles)
   using a glass pipette. These solutions were characterized using a
   particle size analyzer.

![Fig 32.  pH of the final nanoparticle solution](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week5/32-ph-of-the-final-nanoparticle-solution.webp)

Fig 32. pH of the final nanoparticle solution

#### Results:

#### A. 0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 68.7 nm
PI: 0.186
Zeta Potential (mean): 88.5 mV

#### B. 0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Hydrodynamic Radius (Z avg): 527.1 nm
PI: 0.316
Zeta Potential (mean): 46.3 mV

#### Conclusions:

The zeta potential obtained is ideal, but the size has to be
adjusted. To tackle this, we shall use a single magnetic stirrer
throughout to maintain uniformity. Additionally, the sonication
time post-nanoparticle formation should be increased to 8 minutes.

## Chitosan Nanoparticle Production 13

+

✖

## Chitosan Nanoparticle Production

### 28th August

1. 1% acetic acid solution was prepared, with the pH being 2.69. On
   preparing a 40 mL, 0.1% chitosan solution, which underwent
   magnetic stirring (using the Eltech stirrer) for 30 minutes, its
   pH was adjusted from 2.94 to 4.00. This was done by the addition
   of 1900 μL of 1 N NaOH to 35 mL of the chitosan solution.
2. The solution underwent sonication for 5 minutes, 10 seconds
   ON/OFF, 40% amplitude.
3. The TPP and chitosan solutions were filtered and loaded onto the
   burette and beaker, respectively. The TPP was added to the
   chitosan solution dropwise in a 1:3 ratio of TPP to chitosan
   solution, at the ideal flow rate of 7 seconds between drops,
   following which the nanoparticle solution was stirred for 20
   minutes.
4. Post nanoparticle formation, the solution then underwent
   sonication for 8 minutes at 40% amplitude with a pulse of 10
   seconds ON/OFF.
5. The solution underwent centrifugation for 5 minutes at 1000 rpm at
   26°C. It was then decanted using a glass pipette while preserving
   the Falcon tube's orientation out of the centrifuge. This solution
   was then characterized using a particle size analyzer.

#### Results:

#### 0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Zeta Potential: 21.7 mV
PI: 0.303
Z-average: 154.1 nm

#### Conclusions:

The zeta potential obtained is lower than ideal, but the size and
PI are optimal. To tackle this, the pH of the chitosan solution
must be brought slightly higher than 4, and this is to be tried
again.

## Chitosan Nanoparticle Production 14

+

✖

## Chitosan Nanoparticle Production

### 29th August

1. A 1% acetic acid solution was prepared, with a pH of 2.7. On
   preparing a 40 mL, 0.1% chitosan solution, which underwent
   magnetic stirring using an Eltech magnetic stirrer for 30 minutes.
   The pH of the solution was adjusted from 2.87 to 4.09 by adding
   1800 μL of 1 N NaOH to 35 mL chitosan solution.
2. The solution underwent sonication (5 minutes, 10 seconds ON/OFF,
   40% amplitude) at optimized parameters.
3. The TPP and chitosan solutions were filtered and loaded onto the
   burette and beaker. The TPP was added to the chitosan solutions
   dropwise in a 1:3 ratio of TPP to chitosan solution, at the ideal
   flow rate of 7 seconds between drops, following which the
   nanoparticle solutions were stirred for 20 minutes.
4. Due to a lack of time, the sample could not be sonicated,
   centrifuged, and further sent for characterization.
5. The sample was stored at 4°C for further processing the next day.

### Week 6 - 30th August to 5th September

## Chitosan Nanoparticle Production 14

+

✖

## Chitosan Nanoparticle Production

### 30th August

1. The sample made on the previous day was sonicated for 8 minutes at
   40% amplitude with a pulse of 10 seconds ON/OFF.
2. The solution underwent centrifugation for 5 minutes at 1000 rpm at
   26°C. It was then decanted using a glass pipette while preserving
   the Falcon tube's orientation out of the centrifuge. This solution
   was then characterized using a Particle Size Analyzer.

#### Results:

#### 0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Zeta Potential: 58.0 mV
PI: 0.336
Z-average: 107.0 nm

#### Conclusions:

The zeta potential, PI, and Z-avg were favourable. We had to
replicate the nanoparticle run using DEPC-treated RNase-free water
to see if there was any effect on the nanoparticle production.

## *P. capsici* Sporulation 2

+

✖

## Zoospore Induction

### 2nd September

The zoospore plates kept in the water medium for 48 hrs were
cold-shocked for 30 minutes. 1 mL of both samples was collected in
sterile Eppendorf tubes. The samples were visualized under 40x, and
not much was observed. A few drops of methylene blue were added to
each tube, and the same was visualized. Small globule-like cells
were observed to be wobbling around.

![Fig 33. P. capsici burst sporangium (dark blue), zoospores (small blue dots) stained in methylene blue under 40x](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week6/33-p-capsici-bursted-sporangium-dark-blue-zoospores-small-blue-dots-stained-in-methylene-blue-under-40x.webp)

Fig 33. *P. capsici* burst sporangium (dark blue), zoospores
(small blue dots) stained in methylene blue under 40x

#### Observation:

The same samples were loaded onto a Neubauer hemocytometer and
observed. The table below represents the number of zoospores present
in each cell of the hemocytometer when one of the 5x5 center cells
was viewed under 40X.

Table 1. Hemocytometer readings

| Column/Row | (1) | (2) | (3) | (4) |
| --- | --- | --- | --- | --- |
| (1) | 5 | 8 | 9 | 11 |
| (2) | 9 | 13 | 8 | 12 |
| (3) | 12 | 8 | 9 | 9 |
| (4) | 9 | 9 | 9 | 14 |

#### Calculations:

It was calculated that there were 3.85 × 107 zoospores
per mL of the zoospore solution.

## Detached Leaf Assay 2

+

✖

## Detached Leaf Assay

### 2nd September

#### Observation:

The detached leaf assay lesion growth readings were taken, and there
were lesions developed in the chitosan nanoparticle-treated pepper
leaves as well. Troubleshooting was done, and unfortunately, we had
put the leaves under UV radiation, which potentially had damaged the
leaves. This led us to the realization that the leaves should only
be cleaned with 70% ethanol.

## Preparation of DEPC Treated Water

+

✖

## Preparation of DEPC Treated Water

### 3rd September

1. ⁠999 mL of distilled water was added to a 1 L Borosil reagent
   bottle.
2. ⁠In the fume hood, using a glass pipette, 1 mL of DEPC solution
   (Sigma Aldrich) was carefully added to the Borosil reagent bottle.
   Note: Proper precautions were being taken while working with DEPC,
   which include using a fume hood, lab coat, gloves, mask, and
   protective goggles.
3. ⁠The DEPC solution was left in a shaking incubator at 28°C until
   the DEPC globules were completely dissolved, which took over 2
   hours.
4. ⁠The DEPC solution was stored in a dark cupboard to be used the
   next morning. The glass pipette used was placed in an autoclavable
   bag to be autoclaved the next day.

![Fig 34. DEPC globules](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week6/34-depc-globules.webp)

Fig 34. DEPC globules

## Chitosan Nanoparticle Production 15

+

✖

## Chitosan Nanoparticle Production

### 4th September

1. A 0.1% chitosan solution was prepared in 1% Acetic acid and
   underwent magnetic stirring for 30 minutes. The pH of the solution
   was adjusted from 2.73 to 4.01.
2. The solution was then sonicated for 5 minutes at 10-second ON/OFF
   pulse and 40% amplitude.
3. 30 mL of the chitosan solution was filtered into a beaker, after
   which 10 mL of TPP was filter sterilized and added dropwise using
   a burette at the ideal flow rate (7s between drops). Following the
   addition of TPP, the solution was stirred for 20 minutes.
4. The nanoparticle solution was then sonicated at room temperature
   for 5 minutes at 1000 rpm.
5. The resulting solution of pH 6.09 was decanted carefully and
   characterized using a Particle Size Analyzer.

#### Results:

#### 0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Zeta potential: 169 mV
Z-Average size: 95.1 nm
PI: 0.454

#### Conclusions:

The size, zeta potential, and PI obtained for the sample are all
satisfactory. The results obtained now have to be reproducible for
another nanoparticle sample.

## Detached Leaf Assay 3

+

✖

## Preparation of carrot agar plates and inoculation of *P. capsici*

### 4th September (Carrot agar plates preparation)

1. 20 g of carrot was blended using a mortar and pestle.
2. 60 mL of distilled water was added to the carrot puree in a
   beaker. The mixture was then boiled in a microwave.
3. The froth was discarded, after which the mixture was filtered
   through a muslin cloth, resulting in a clear, slightly orange
   solution.
4. The filtrate’s volume was then made up to 100 mL in a measuring
   flask using distilled water.
5. The carrot solution was combined with 1.8 g of agar in a conical
   flask, which was autoclaved and poured onto petri dishes.

### 5th September (Inoculation of *P. capsici*)

1. The LAF and apparatus (inoculation loops, carrot agar plates, cork
   borer, etc) were cleaned and sterilized through UV treatment.
2. ⁠Mycelial discs from the previously made subculture plates were
   made and inverted onto 2 fresh carrot agar plates, using an 8 mm
   cork borer and an inoculation loop. Each plate was set with 2
   mycelial discs.
3. The plates were then placed in the dark incubator at 28°C.

### Week 7 - 6th to 12th September

## Chitosan Nanoparticle Production 16

+

✖

## Chitosan Nanoparticle Production

### 6th September

1. A 0.1% chitosan solution was prepared in 1% acetic acid, which was
   stirred for 30 minutes. The pH of the solution was 2.91, which was
   not adjusted further (to optimize the pH of the final
   nanoparticles).
2. The solution underwent sonication for 5 minutes, 10-second ON/OFF
   pulse at 40% amplitude.
3. The chitosan and TPP solutions were filtered and loaded onto the
   beaker and burette, respectively. As done previously, 10 mL of TPP
   was added dropwise to 30 mL of chitosan solution at the ideal flow
   rate (7s between drops). Following this, the nanoparticle solution
   was stirred for 20 minutes.
4. This solution was then sonicated at the same parameters for 8
   minutes, following which it was centrifuged at room temperature
   for 5 minutes at 1000 rpm.
5. The resulting solution was decanted carefully and characterized
   using a Particle Size Analyzer. The final pH of the chitosan
   nanoparticle solution was 5.9.

#### Results:

#### 0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Zeta Potential: 184.5 mV
Z-Average size: 267.5 nm
PI: 0.488

#### Conclusions:

Although the pH of the nanoparticle solution was optimized and the
zeta potential obtained was encouraging, the size of the
nanoparticles was too large to use for siRNA encapsulation.

## Chitosan Nanoparticle Production 17

+

✖

## Chitosan Nanoparticle Production

### 9th September

1. The previous nanoparticle run with RNase-free water and an
   RNase-free water rinsed apparatus did not give us ideal results.
   The final pH of the nanoparticle solution was 5.9, without any pH
   adjustments using 1 N NaOH.
2. Upon discussion with our PI, we decided to add NaOH to get the
   chitosan solution's pH to 4.
3. Apart from this additional step, the same protocol was followed as
   done previously.

### 10th September

The chitosan nanoparticle solution prepared the previous day was
sonicated for two minutes at 40% amplitude with a 10-second ON/OFF
pulse. The sample was thereafter centrifuged at 1000 rpm for 5
minutes at 26°C and decanted.

#### Results:

Z- avg: 130.1 nm
PI: 0.516
Zeta Potential: 89.0 mV

#### Conclusions:

The Z-avg, PI, and zeta potential were ideal. The zeta potential
was lower than the last nanoparticle iteration, probably due to
characterizing the nanoparticle 24 hours after its preparation.

## *P. capsici* Sporulation 3

+

✖

## Zoospore Induction

### 8th September

1. Two autoclaved petri dishes were filled halfway with autoclaved
   distilled water and kept upright.
2. ⁠Ten 8 mm mycelial disks were cut on a 72 hours old culture plate.
3. The mycelial plugs were then suspended in the sterile water in the
   petri dish with the mycelium side facing upwards and agar side
   submerged.
4. The plates were parafilmed carefully and placed for incubation
   with illumination at 26°C.

## *P. capsici* Sporulation

### 10th September

1. ⁠The two sporulation plates kept for 48 hours of light incubation
   were given a cold shock treatment by keeping them in 4°C for 30
   minutes.
2. Six Eppendorf tubes were prepared for testing.
   * Two tubes served as negative controls and each received 1 mL
     of zoospore suspension from the two plates, respectively.
   * In two additional tubes containing 500 µL of zoospore
     suspension, 250 µL of 1% acetic acid was added.
   * To the remaining two tubes, each containing 500 µL of zoospore
     suspension, 250 µL of 0.1% chitosan nanoparticle was added.
3. ⁠The sample tubes, along with other necessary requirements, were
   taken to MCBR (Manipal Centre for Biotherapeutics Research) to be
   visualized using the upright trinocular phase contrast microscope.
4. A few drops of methylene blue was added to each sample tube. 10 μL
   of each sample was pipetted on clean glass slides and visualized
   at 40x and 100x.

![Fig 35. P. capsici zoospores observed at 100x using the upright trinocular phase contrast microscope ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week7/35-p-capsici-zoospores-observed-at-100x-using-the-upright-trinocular-phase-contrast-microscope.webp)

Fig 35. *P. capsici* zoospores observed at 100x using the
upright trinocular phase contrast microscope

Vid 1. Zoospores visualized at 100x without any treatment

Vid 2. Zoospores visualized at 100x with treatment of 1% acetic
acid

Vid 3. Zoospores visualized at 100x with treatment of chitosan
nanoparticle solution

#### Conclusions:

There was a slight change in the motility of the
nanoparticle-treated zoospores compared to the control, whereas
the acetic acid-treated sample was similar to the control. We were
advised by our PI to use a Neubauer chamber haemocytometer to
gauge and measure the effect on the motility of the zoospores
between the control and treated samples by measuring the time it
takes for a zoospore to move from one grid to the other.

## Detached Leaf Assay 3

+

✖

## Detached Leaf Assay Lesion Observations

### 10th September (24 hrs)

#### Untreated leaf left: (no lesions, but had brown spots towards the ends)

X axis: 0.0 cm
Y axis: 0.0 cm

#### Untreated leaf right: (no lesions, but had brown spots towards the ends)

X axis: 0.0 cm
Y axis: 0.0 cm

#### Control leaf left:

X axis: 1.3 cm
Y axis: 1.9 cm

#### Control leaf right:

X axis: 1.1 cm
Y axis: 1.7 cm

#### Acetic acid leaf left:

X axis: 0.8 cm
Y axis: 0.9 cm

#### Acetic acid leaf right:

X axis: 0.7 cm
Y axis: 0.7 cm

#### Chitosan nanoparticle leaf left:

X axis: 0.9 cm
Y axis: 0.7 cm

#### Chitosan nanoparticle leaf right:

X axis: 0.8 cm
Y axis: 0.1 cm

![Fig 36. P. nigrum leaves without P. capsici- Negative control](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week7/36-p-nigrum-leaves-without-p-capsici-negative-control.webp)

Fig 36. *P. nigrum* leaves without *P. capsici*-
Negative control

![Fig 37. Untreated P. nigrum leaves- Control](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week7/37-untreated-p-nigrum-leaves-control.webp)

Fig 37. Untreated *P. nigrum* leaves - Control

![Fig 38. Leaves sprayed with 1% Acetic acid](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week7/38-leaves-sprayed-with-1-acetic-acid.webp)

Fig 38. Leaves sprayed with 1% Acetic acid

![Fig 39. Leaves sprayed with 0.1% chitosan nanoparticle solution](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week7/39-leaves-sprayed-with-1-chitosan-nanoparticle-solution.webp)

Fig 39. Leaves sprayed with 0.1% chitosan nanoparticle solution

### 11th September (48 hrs)

#### Untreated leaf left: (no lesions, but had brown spots towards the ends)

X axis: 0.0 cm
Y axis: 0.0 cm

#### Untreated leaf right: (no lesions, but had brown spots towards the ends)

X axis: 0.0 cm
Y axis: 0.0 cm

#### Control leaf left:

X axis: 2.3 cm
Y axis: 4.3 cm

#### Control leaf right:

X axis: 3.2 cm
Y axis: 5.6 cm

#### Acetic acid leaf left:

X axis: 1.9 cm
Y axis: 2.3 cm

#### Acetic acid leaf right:

X axis: 1.4 cm
Y axis: 1.5 cm

#### Chitosan nanoparticle leaf left:

X axis: 1.4 cm
Y axis: 1.7 cm

#### Chitosan nanoparticle leaf right:

X axis: 1.3 cm
Y axis: 1.6 cm

![Fig 40. P. nigrum leaves without P. capsici- Negative control](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week7/40-p-nigrum-leaves-without-p-capsici-negative-control.webp)

Fig 40. *P. nigrum* leaves without *P. capsici* -
Negative control

![Fig 41. Untreated P. nigrum leaves- Control](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week7/41-untreated-p-nigrum-leaves-control.webp)

Fig 41. Untreated *P. nigrum* leaves - Control

![Fig 42. Leaves sprayed with 1% Acetic acid](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week7/42-leaves-sprayed-with-1-acetic-acid.webp)

Fig 42. Leaves sprayed with 1% Acetic acid

![Fig 43. Leaves sprayed with 0.1% chitosan nanoparticle solution](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week7/43-leaves-sprayed-with-01-chitosan-nanoparticle-solution.webp)

Fig 43. Leaves sprayed with 0.1% chitosan nanoparticle solution

### 12th September (72 hrs)

#### Untreated leaf left: (Contaminated)

X axis: 1.3 cm
Y axis: 0.8 cm

#### Untreated leaf right: (Contaminated)

X axis: 1.85 cm
Y axis: 2.5 cm

#### Control leaf left:

X axis: 7.9 cm
Y axis: 4.2 cm

#### Control leaf right:

X axis: 8.8 cm
Y axis: 6.4 cm

#### Acetic acid leaf left:

X axis: 4.7 cm
Y axis: 5.8 cm

#### Acetic acid leaf right:

X axis: 5.1 cm
Y axis: 8.6 cm

#### Chitosan nanoparticle leaf left:

X axis: 2.1 cm
Y axis: 3.4 cm

#### Chitosan nanoparticle leaf right:

X axis: 1.9 cm
Y axis: 2.8 cm

![Fig 44. P. nigrum leaves without P. capsici- Negative control ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week7/44-p-nigrum-leaves-without-p-capsici-negative-control.webp)

Fig 44. *P. nigrum* leaves without *P. capsici* -
Negative control

![Fig 45. Untreated P. nigrum leaves- Control ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week7/45-untreated-p-nigrum-leaves-control.webp)

Fig 45. Untreated *P. nigrum* leaves - Control

![Fig 46. Leaves sprayed with 1% Acetic acid](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week7/46-leaves-sprayed-with-1-acetic-acid.webp)

Fig 46. Leaves sprayed with 1% Acetic acid

![Fig 47. Leaves sprayed with 0.1% chitosan nanoparticle solution](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week7/47-leaves-sprayed-with-0-1-chitosan-nanoparticle-solution.webp)

Fig 47. Leaves sprayed with 0.1% chitosan nanoparticle solution

### Week 8 - 13th to 19th September

## Detached Leaf Assay 3

+

✖

## Detached Leaf Assay Lesion Observations

### 13th September (96 hrs)

#### Untreated leaf left:

X axis: 6.6 cm
Y axis: 10.5 cm

#### Untreated leaf right:

X axis: 3.1 cm
Y axis: 7.3 cm

#### Control leaf left:

X axis: 4.6 cm
Y axis: 8.4 cm

#### Control leaf right:

X axis: 8 cm
Y axis: 9.5 cm

#### Acetic acid leaf left:

X axis: 7.4 cm
Y axis: 11.2 cm

#### Acetic acid leaf right:

X axis: 8.5 cm
Y axis: 11.0 cm

#### Chitosan nanoparticle leaf left:

X axis: 3.9 cm
Y axis: 5.8 cm

#### Chitosan nanoparticle leaf right:

X axis: 2.9 cm
Y axis: 3.7 cm

![Fig 48. P. nigrum leaves without P. capsici- Negative control ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week8/48-p-nigrum-leaves-without-p-capsici-negative-control.webp)

Fig 48. *P. nigrum* leaves without *P. capsici* -
Negative control

![Fig 49. Untreated P. nigrum leaves- Control ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week8/49-untreated-p-nigrum-leaves-control.webp)

Fig 49. Untreated *P. nigrum* leaves - Control

![Fig 50. Leaves sprayed with 1% Acetic acid ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week8/50-leaves-sprayed-with-1-acetic-acid.webp)

Fig 50. Leaves sprayed with 1% Acetic acid

![Fig 51. Leaves sprayed with 0.1% chitosan nanoparticle solution ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week8/51-leaves-sprayed-with-01-chitosan-nanoparticle-solution.webp)

Fig 51. Leaves sprayed with 0.1% chitosan nanoparticle solution

### 14th September (120 hrs)

#### Untreated leaf left:

X axis: 6.9 cm
Y axis: 9.1 cm

#### Untreated leaf right:

X axis: 8.1 cm
Y axis: 12.5 cm

#### Control leaf left:

X axis: 5.6 cm
Y axis: 9.2 cm

#### Control leaf right:

X axis: 8.0 cm (whole leaf)
Y axis: 9.5 cm (whole leaf)

#### Acetic acid leaf left:

X axis: 8.4 cm (whole leaf)
Y axis: 11.2 cm (whole leaf)

#### Acetic acid leaf right:

X axis: 8.5 cm (whole leaf)
Y axis: 11.0 cm (whole leaf )

#### Chitosan nanoparticle leaf left:

X axis: 5.4 cm
Y axis: 8.0 cm

#### Chitosan nanoparticle leaf right:

X axis: 3.7 cm
Y axis: 6.2 cm

![Fig 52. P. nigrum leaves without P. capsici- Negative control ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week8/52-p-nigrum-leaves-without-p-capsici-negative-control.webp)

Fig 52. *P. nigrum* leaves without *P. capsici* -
Negative control

![Fig 53. Untreated P. nigrum leaves- Control ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week8/53-untreated-p-nigrum-leaves-control.webp)

Fig 53. Untreated *P. nigrum* leaves - Control

![Fig 54. Leaves sprayed with 1% Acetic acid](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week8/54-leaves-sprayed-with-1-acetic-acid.webp)

Fig 54. Leaves sprayed with 1% Acetic acid

![Fig 55. Leaves sprayed with 0.1% chitosan nanoparticle solution ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week8/55-leaves-sprayed-with-01-chitosan-nanoparticle-solution.webp)

Fig 55. Leaves sprayed with 0.1% chitosan nanoparticle solution

### 15th September (144 hrs)

#### Untreated leaf left:

X axis: 6.9 cm
Y axis: 9.1 cm

#### Untreated leaf right:

X axis: 10.1 cm
Y axis: 12.5 cm

#### Control leaf left:

X axis: 6.1 cm
Y axis: 9.2 cm

#### Control leaf right:

X axis: 8.0 cm
Y axis: 9.5 cm

#### Acetic acid leaf left:

X axis: 8.4 cm
Y axis: 11.2 cm

#### Acetic acid leaf right:

X axis: 8.5
Y axis: 11.0 cm

#### Chitosan nanoparticle leaf left:

X axis: 8.4 cm
Y axis: 11.2 cm

#### Chitosan nanoparticle leaf right:

X axis: 7.0 cm
Y axis: 9.2 cm

![Fig 56. P. nigrum leaves without P. capsici - Negative 							control](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week8/56-p-nigrum-leaves-without-p-capsici-negative-control.webp)

Fig 56. *P. nigrum* leaves without *P. capsici* -
Negative control

![Fig 57. Untreated P. nigrum leaves- Control ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week8/57-untreated-p-nigrum-leaves-control.webp)

Fig 57. Untreated *P. nigrum* leaves - Control

![Fig 58. Leaves sprayed with 1% Acetic acid](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week8/58-leaves-sprayed-with-1-acetic-acid.webp)

Fig 58. Leaves sprayed with 1% Acetic acid

![Fig 59. Leaves sprayed with 0.1% chitosan nanoparticle solution ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week8/59-leaves-sprayed-with-01-chitosan-nanoparticle-solution.webp)

Fig 59. Leaves sprayed with 0.1% chitosan nanoparticle solution

#### Conclusions:

Over the course of 6 days, the lesions formed were observed. It
was seen that the leaves treated with the chitosan nanoparticle
solution showed the slowest lesion formation and lowest infection
rates. In contrast, leaves treated with acetic acid and water had
rapid lesion growth. The control leaves did get infected, but the
infection pattern observed initially did not correlate with
*P. capsici*; However, the infection eventually spread to
cover the entire leaf. To avoid such contamination, extra measures
were taken to ensure sterility is maintained.

### Week 9 - 20th to 26th September

## Cytotoxicity Assay

+

✖

## siRNA Resuspension and Cytotoxicity Assay

### 22nd September

1 mL of diethyl pyrocarbonate (DEPC) was added to 999 mL of
distilled water to prepare DEPC water. This was carried out in the
fume hood, wearing gloves, a mask, a lab coat, and protective gear
like goggles. The solution was left in the shaker for 2-3 hours to
ensure complete dissolution. This solution was then stored for the
treating apparatus that would come in contact with the siRNA.

### 23rd September

The DEPC solution prepared the previous day was used to soak the
pipette tips and boxes, Falcon tubes, and Eppendorf vials that would
be used to resuspend the siRNA. The apparatus was left to soak till
bubbles appeared, indicating the effective action of the DEPC
solution.

All of the apparatus was then autoclaved to ensure they were RNase
and DEPC-free.

### 24th September

#### siRNA Resuspension:

1. Calculations for various dilution concentrations were made
   beforehand for the resuspension of siRNA. We began resuspending
   the better candidate, Candidate 1, which was verified to have a
   greater silencing efficiency against *P. capsici* through our
   Dry Lab tests.
2. The supplier's stock was first spun down, and the rim was wiped
   with a tissue soaked in ethanol.
3. ⁠1.5 mL Eppendorf tubes treated with DEPC were autoclaved, dried,
   and labelled.
4. Since the siRNA was fluorescence-tagged, all steps were carried
   out in the laminar airflow chamber with the lights off. Any tubes
   that contained siRNA were maintained on ice throughout.
   Nuclease-free water was used, and any apparatus that came in
   contact with the siRNA was DEPC-treated, including the tips and
   tip boxes. Utmost sterility was maintained throughout the
   procedure.
5. As per the supplier's data sheet, 57.8 μL of nuclease-free water
   was added to the master stock to obtain 57.8 μL of 100 μM stock.
   This was vortexed for 5 seconds and spun down again.
6. The 100 μM stock was further diluted into the following: 1 μM, 100
   nM, 50 nM, and 1.44 nM.
7. The same was divided into aliquots as such: 5 tubes that contained
   10 μL of 100 μM siRNA, four tubes that contained 20 μL of 1 μM
   siRNA, five tubes of 20 μL of 100 nM siRNA, two tubes of 25 μL of
   50 nM siRNA, and one tube of 30 μL of 1.44 nM siRNA.
8. The standard C1V1=C2V2
   formula was used to obtain the nuclease-free water value.
9. ⁠Post this, 10 μL of all concentrations of siRNA were collected
   separately for a cytotoxicity assay to determine the effect of
   various concentrations of siRNA on *P. nigrum* leaves.
10. ⁠Finally, all these Eppendorf tubes were sealed tightly with
    parafilm, wrapped with aluminium foil, labelled appropriately, and
    stored in -20°C.

![Fig 60. Resuspension of siRNA](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week9/60-resuspension-of-sirna.webp)

Fig 60. Resuspension of siRNA

#### Cytotoxicity Assay:

1. ⁠Plastic containers were cleaned with 70% ethanol, lined up with a
   layer of cotton, and wetted with autoclaved distilled water so
   that no excess water was drained out while tilting the boxes. This
   was placed under UV rays for 20 minutes to eliminate any other
   form of contamination.
2. *Piper nigrum* Panniyur 1 variety leaves were cleaned with
   70% ethanol and placed on the wet cotton layer. Pin pricks were
   made in a 4x4 pattern using autoclaved toothpicks.
3. ⁠There was a negative control leaf, which was the one with no
   treatment, and other controls: leaves with pin pricks and no water
   added additionally, and leaves with pin pricks that were applied
   with 10 μL of water.
4. All concentrations, from the highest, 100 μM, to the least, 1.44
   nM, were applied onto the leaves to test if there were any adverse
   effects for the siRNA.
5. Hence, the leaves were treated with 10 μL each of 100 μM, 1 μM,
   100 nM, 50 nM, and 1.44 nM, and all the boxes, including the
   control, were labelled.
6. All pipette tips, tip boxes, and any apparatus used were
   DEPC-treated, and utmost sterility was maintained.
7. The boxes with the leaves, both control and siRNA-treated, were
   placed in a place with appropriate light.
8. Observations were taken every 24 hours for any necrosis on the
   leaf's surface.

### 25th September (24 hrs)

![Fig 61. Negative control: Leaf with no pinpricks, leaf with pinpricks, and leaf with pinpricks treated with water (left to right)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week9/61-negative-control-leaf-with-no-pinpricks-leaf-with-pinpricks-and-leaf-with-pinpricks-treated-with-water-left-to-right.webp)

Fig 61. Negative control: Leaf with no pinpricks, leaf with
pinpricks, and leaf with pinpricks treated with water (left to
right)

![Fig 62. Leaves treated with 50 nM siRNA and 1.44 nM siRNA (left to right)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week9/62-leaves-treated-with-50-nm-sirna-and-44-nm-sirna-left-to-right.webp)

Fig 62. Leaves treated with 50 nM siRNA and 1.44 nM siRNA (left to
right)

![Fig 63. Leaves treated with 1 μM siRNA and 100 nM siRNA (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week9/63-leaves-treated-with-1-m-sirna-and-100-nm-sirna-left-to-right.webp)

Fig 63. Leaves treated with 1 μM siRNA and 100 nM siRNA (left to
right)

![Fig 64. Leaf treated with 100 μM siRNA](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week9/64-leaf-treated-with-100-m-sirna.webp)

Fig 64. Leaf treated with 100 μM siRNA

### 26th September (48 hrs)

![Fig 65. Negative control: Leaf with no pinpricks, leaf with pinpricks, and leaf with pinpricks treated with water (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week9/65-negative-control-leaf-with-no-pinpricks-leaf-with-pinpricks-and-leaf-with-pinpricks-treated-with-water-left-to-right.webp)

Fig 65. Negative control: Leaf with no pinpricks, leaf with
pinpricks, and leaf with pinpricks treated with water (left to
right)

![Fig 66. Leaves treated with 50 nM siRNA and 1.44 nM siRNA (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week9/66-leaves-treated-with-50-nm-sirna-and-44-nm-sirna-left-to-right.webp)

Fig 66. Leaves treated with 50 nM siRNA and 1.44 nM siRNA (left to
right)

![Fig 67. Leaves treated with 1 μM siRNA and 100 nM siRNA (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week9/67-leaves-treated-with-1-m-sirna-and-100-nm-sirna-left-to-right.webp)

Fig 67. Leaves treated with 1 μM siRNA and 100 nM siRNA (left to
right)

![Fig 68. Leaf treated with 100 μM siRNA ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week9/68-leaf-treated-with-100-m-sirna.webp)

Fig 68. Leaf treated with 100 μM siRNA

#### Observation:

It was observed that none of the leaves showed any signs of
necrosis, even with the 100 μM siRNA (our highest available
concentration- master stock) treated leaf, proving our siRNA is 100%
safe to *P. nigrum*.

## Detached Leaf Assay 4

+

✖

## Detached Leaf Assay Preparation

### 24th September

#### Carrot agar plates preparation:

1. ⁠Four carrot agar plates were made using 20 g of grated carrot and
   1.8% agar.
2. The media was autoclaved, and the plates were poured and labelled.

#### Inoculation of *P. capsici*:

1. ⁠The LAF and apparatus (inoculation loops, carrot agar plates,
   cork borer, etc) were cleaned and sterilized through UV treatment.
2. ⁠Mycelial discs from the previously made subculture plates were
   made and inverted onto 2 fresh carrot agar plates, using an 8 mm
   cork borer and an inoculation loop. Each plate was set with 2
   mycelial discs.
3. ⁠The plates were then placed in the dark incubator at 28°C.

### 5th September (Inoculation of *P. capsici*)

1. The LAF and apparatus (inoculation loops, carrot agar plates, cork
   borer, etc) were cleaned and sterilized through UV treatment.
2. ⁠Mycelial discs from the previously made subculture plates were
   made and inverted onto 2 fresh carrot agar plates, using an 8 mm
   cork borer and an inoculation loop. Each plate was set with 2
   mycelial discs.
3. The plates were then placed in the dark incubator at 28°C.

## Nanoformulation Production 1

+

✖

## Nanoformulation Production

### 25th September

1. We realised that 1.44 nM was too low of a concentration to be
   effective against *P. capsici*.
2. Through further literature review, it was understood that 2.88 μM
   would be an effective concentration of siRNA, considering only 10%
   of it would be encapsulated. Our PI recommended we use 15 μL of
   100 μM siRNA.
3. We first attempted a nanoparticle run without the addition of
   siRNA by reducing the volumes of the chitosan solution and TPP to
   see if we could achieve ideal results. This would have helped us
   gauge the volume of 100 μM siRNA required if the nanoparticle
   production were to be successful.
4. ⁠Hence, a nanoparticle solution was made with our optimized
   protocol, where the final solution had 20 mL of 0.1% chitosan
   solution and 6.667 mL of 2.4 mg/ml TPP.
5. It was characterized the following day using a Particle Size
   Analyzer.
6. Preparation for the nanoformulation production for the following
   day was carried out simultaneously. All the apparatuses were
   treated with DEPC water by soaking them until bubbles appeared.
   This roughly took about 4 hours. The apparatuses were dried in the
   hot air oven and autoclaved for use the next day.

### 26th September

The nanoparticles produced the previous day were sonicated for 2
minutes at 10-second ON/OFF pulse and 40% amplitude. The pH of the
final solution was observed to be 4.23. This sample was
characterized using a Particle Size Analyzer.

#### Results:

#### 0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio):

Zeta Potential: 20.6 mV
Z-Average size: 82.0 nm
PI: 0.426

#### Conclusions:

The size and polydispersity of the nanoparticles are favourable.
However, the zeta potential was lower than required for siRNA
encapsulation. As a result, the standard volumes of chitosan and
TPP were to be used for the encapsulation of siRNA.

### Week 10 - 27th September to 5th October

## Cytotoxicity Assay

+

✖

## Cytotoxicity Assay

### 27nd September (72 hrs)

![Fig 69. Negative control: Leaf with no pinpricks, leaf with pinpricks, and leaf with pinpricks treated with water (left to right)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/69-negative-control-leaf-with-no-pinpricks-leaf-with-pinpricks-and-leaf-with-pinpricks-treated-with-water-left-to-right.webp)

Fig 69. Negative control: Leaf with no pinpricks, leaf with
pinpricks, and leaf with pinpricks treated with water (left to
right)

![Fig 70. Leaves treated with 50 nM siRNA and 1.44 nM siRNA (left to right)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/70-leaves-treated-with-50-nm-sirna-and-1-44-nm-sirna-left-to-right.webp)

Fig 70. Leaves treated with 50 nM siRNA and 1.44 nM siRNA (left to
right)

![Fig 71. Leaves treated with 1 μM siRNA and 100 nM siRNA (left to right)](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/71-leaves-treated-with-1-m-sirna-and-100-nm-sirna-left-to-right.webp)

Fig 71. Leaves treated with 1 μM siRNA and 100 nM siRNA (left to
right)

![Fig 72. Leaf treated with 100 μM siRNA](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/72-leaf-treated-with-100-m-sirna.webp)

Fig 72. Leaf treated with 100 μM siRNA

## Nanoformulation Production 1

+

✖

## Nanoformulation Production

### 27th September

1. The nanoformulation (siRNA encapsulated within chitosan
   nanoparticles) was produced using our optimized chitosan
   nanoparticle production protocol, with an additional step of
   adding 15 μL of 100 μM siRNA solution to 10 mL of the TPP
   solution.
2. All glassware and plasticware were treated with DEPC solution,
   dried, and autoclaved. All the reagents and solutions were
   prepared using RNase-free water. Measures were taken to make the
   experimental setup as RNase-free as possible.
3. The pH of the chitosan solution was adjusted to 4 using 1 N NaOH.
4. The final nanoformulation was sonicated for 8 minutes at 40%
   amplitude with a 10-second ON/OFF pulse, following which it was
   centrifuged at room temperature for 5 minutes at 1000 rpm.
5. The resulting solution was decanted carefully and characterized
   using a Particle Size Analyzer.

#### Results:

#### 0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio) with encapsulated siRNA:

Zeta Potential: -4.0 mV
Z-Average size: 6410.3 nm
PI: 2.318

#### Conclusions:

The negative zeta potential indicated inefficient encapsulation of
the siRNA within the chitosan nanoparticles. The polydispersity
index was relatively high, possibly due to pH variation because of
the DEPC-treated apparatus and RNase-free water used during
preparation. The hydrodynamic radius was also observed to be very
large. To tackle these issues, we were advised by our PI, Dr. Ritu
Raval, to omit the adjustment of pH of the chitosan nanoparticle
solution, simply rinse all glassware and plasticware with
RNase-free water before use, and increase the sonication time of
the nanoformulation from 8 minutes to 10 minutes.

## Gel Retardation Assay 1

+

✖

## Gel Retardation Assay

### 28th September

1. 1 L of 1X TBE (Tris-Borate-EDTA) buffer was prepared from a 20X
   TBE buffer solution (50 mL of 20X TBE buffer was added to 950 mL
   of Milli-Q water). The buffer was added to the buffer tank.
2. 35 mL of 2% Low EEO agarose gel was prepared using 1X TBE buffer.
   The agarose-TBE solution was microwaved until boiling was
   achieved, and the low EEO agarose had completely dissolved.
3. Once the solution had cooled slightly, 1.16 µL of SYBR Safe was
   added, and the gel was poured into the gel casting tray with the
   comb. A sterile micropipette tip was used to push away any bubbles
   formed on the gel surface. Once the gel had set, it was placed in
   the buffer tank, and the comb was carefully removed.
4. The samples to be loaded onto the gel were prepared in an aseptic
   and RNase-free environment, using RNase-free plasticware. To each
   sample (except the DNA ladder), 2 µL of RNA loading buffer was
   added. The samples were spun using a microcentrifuge and then
   loaded onto the gel.

   The samples were loaded in the following order.
   1. 10 µL of 1 kb DNA ladder
   2. 10 µL of 50 nM siRNA
   3. 10 µL of nanoformulation (prepared on 27th
      September)
   4. 10 µL of nanoformulation treated with 0.5 mg/mL RNase (2 µL of
      RNase was added to 10 µL of the nanoformulation, and the
      sample was incubated for 5 minutes at 56°C)
   5. 10 µL of chitosan nanoparticle (prepared on 10th
      September)
   6. 10 µL of pre-decanted nanoformulation
5. The gel was allowed to run at 55 V for 2 hours, and checked in
   intervals.
6. The gel was visualized under a UV transilluminator.

#### Results:

![Fig 73. Gel run results. L - 1 kb DNA ladder, 1- 50 nM siRNA, 2- nanoformulation, 3- nanoformulation treated with 0.5 mg/mL RNase, 4- chitosan nanoparticle, 5- pre-decanted nanoformulation ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/73-gel-run-results-l-1-kb-dna-ladder.webp)

Fig 73. Gel run results. L - 1 kb DNA ladder, 1- 50 nM siRNA, 2-
nanoformulation, 3- nanoformulation treated with 0.5 mg/mL RNase,
4- chitosan nanoparticle, 5- pre-decanted nanoformulation

#### Conclusions:

The gel run was unsuccessful. No siRNA was visualized in the free
siRNA-loaded well or the nanoformulation-loaded wells. We decided
to perform the experiment again, this time with a better
nanoformulation.

## Nanoformulation Production 2

+

✖

## Nanoformulation Production

### 29th September

1. The nanoformulation (siRNA encapsulated within chitosan
   nanoparticles) was produced using our optimized chitosan
   nanoparticle production protocol, with an additional step of
   adding 15 μL of 100 μM siRNA solution to 10 mL of the TPP
   solution. The addition of TPP-siRNA solution and the steps
   following that were done in low light conditions to avoid
   quenching of our 6-FAM-tagged siRNA.
2. All glassware and plasticware were rinsed in RNase-free water and
   dried. All reagents and solutions were prepared using RNase-free
   water. Measures were taken to make the experimental setup as
   RNase-free as possible.
3. The pH of the chitosan solution was not adjusted to 4 using 1 N
   NaOH.
4. The final nanoformulation was sonicated for 10 minutes at 40%
   amplitude with a 10-second ON/OFF pulse, following which it was
   centrifuged at room temperature for 5 minutes at 1000 rpm. The pH
   of the final siRNA encapsulated nanoformulation solution was 3.24.
5. The resulting solution was decanted carefully and characterized
   using a Particle Size Analyzer.

#### Results:

#### 0.1% Chitosan solution in 1% Acetic Acid (1:3 TPP to Chitosan ratio) with encapsulated siRNA:

Zeta Potential: 35.8 mV
Z-Average size: 401.7 nm
PI: 0.509

#### Conclusions:

The nanoformulation produced was close to ideal and had a good
zeta potential, polydispersity index, and z-average values. We
further experimented with the nanoformulation to calculate the
entrapment efficiency of the siRNA within the chitosan
nanoparticles.

## Gel Retardation Assay 2

+

✖

## Gel Retardation Assay

### 29th September

1. The same 1X TBE buffer as prepared the previous day was used for
   the gel run.
2. 35 mL of 2% Low EEO agarose gel was prepared using 1X TBE buffer.
   The agarose-TBE solution was microwaved until boiling was
   achieved, and the low EEO agarose had completely dissolved.
3. Once the solution had cooled slightly, 1.16 µL of SYBR Safe was
   added, and the gel was poured into the gel casting tray with the
   comb. A sterile micropipette tip was used to push away any bubbles
   formed on the gel surface. Once the gel had set, it was placed in
   the buffer tank, and the comb was carefully removed.
4. The samples to be loaded onto the gel were prepared in an aseptic
   and RNase-free environment, using RNase-free plasticware. To each
   sample (except the DNA ladder), 2 µL of RNA loading buffer was
   added. The samples were spun using a microcentrifuge and then
   loaded onto the gel.

   The samples were loaded in the following order.
   1. 10 µL of 1 kb DNA ladder
   2. 10 µL of 50 nM siRNA
   3. 10 µL of nanoformulation
   4. 10 µL of nanoformulation treated with 0.5 mg/mL RNase (2 µL of
      RNase was added to 10 µL of the nanoformulation, the sample
      was incubated for 5 minutes at 56°C)
   5. 10 µL of chitosan nanoparticle (prepared on 10th September)
5. The gel was allowed to run at 55 V for 2 hours, and checked in
   intervals.
6. The gel was visualized under a UV transilluminator.

#### Results:

![Fig 74. Gel run results. L - 1 kb DNA ladder, 1- 50 nM siRNA, 2- nanoformulation, 3- nanoformulation treated with 0.5 mg/mL RNase, 4- chitosan nanoparticle ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/74-gel-run-results-l-1-kb-dna-ladder.webp)

Fig 74. Gel run results. L - 1 kb DNA ladder, 1- 50 nM siRNA, 2-
nanoformulation, 3- nanoformulation treated with 0.5 mg/mL RNase,
4- chitosan nanoparticle

#### Conclusions:

The gel run was unsuccessful. No siRNA was visualized in the free
siRNA-loaded well or the nanoformulation-loaded wells. Upon
troubleshooting with our iGEM mentor, Sabyasachi Banerjee, we were
advised to use autoclaved Milli-Q water for the preparation of the
TBE buffer and use RNaseZap liberally to ensure the
experimentation setup, glassware, and plasticware were RNase-free
for our next run.

## Entrapment Efficiency

+

✖

## Entrapment Efficiency of siRNA within Chitosan Nanoparticles

### 29th September

1. We centrifuged the siRNA-loaded chitosan nanoparticle sample that
   was prepared the same day at 11,000 rpm and 4°C for 37 minutes.
2. The supernatant was carefully removed from the siRNA-loaded CS-TPP
   nanoparticles. To measure the absorbance of the sample, a
   nanocuvette was cleaned using 70% ethanol.
3. 2 μL of 1% acetic acid was loaded as a blank in the nanocuvette,
   and absorbance was measured at 260 nm using a UV-Vis
   spectrophotometer.
4. 2 μL of the supernatant was loaded into the nanocuvette, and
   absorbance was measured at 260 nm using the UV-Vis
   spectrophotometer (we use the Eppendorf BioSpectrometer). We
   ensured an RNase-free environment was maintained throughout the
   experiment, and cleaned the nanocuvette with 70% ethanol between
   uses.
5. The entrapment efficiency was calculated using this formula:

   Entrapment Efficiency (%) =

   Csample − Csupernatant
   Csample
   × 100

   Csample (20.778 μg/mL) is the concentration of
   siRNA added, and Csupernatant (2.2 μg/mL) is the siRNA
   concentration in the supernatant.

![Fig 75. Absorbance readings of the nanoformulation  ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/75-absorbance-readings-of-the-nanoformulation.webp)

Fig 75. Absorbance readings of the nanoformulation

#### Results:

The entrapment efficiency was found to be 89.41%, indicating a high
percentage of siRNA encapsulated within the chitosan nanoparticles.

## Detached Leaf Assay 4

+

✖

## Detached Leaf Assay

### 27th September

1. ⁠The LAF and apparatus were cleaned and sterilized through UV
   treatment.
2. Mycelial discs from the previously made subculture plates were
   made and inverted onto fresh treated black pepper placed in
   plastic boxes lined with moist cotton, using a 5 mm cork borer and
   an inoculation loop.
3. Before inoculation of the leaves with mycelial discs, the leaves
   were treated with our nanoformulation, chitosan nanoparticles, and
   other controls were prepared.

   The prepared black pepper leaf samples were as follows:
   1. Untreated leaf
   2. Untreated leaf with pinpricks
   3. Leaf treated with 10 µL of water
   4. Leaf treated with 10 µL of acetic acid
   5. Leaf treated with 10 µL of chitosan nanoparticle solution
      (prepared on 10th September)
   6. Leaf treated with 10 µL of nanoformulation (prepared the same
      day)
   7. Leaf treated with 5 µL of 100 µM siRNA
   8. Leaf treated with 5 µL of 1 µM siRNA
   9. Leaf treated with 10 µL of 100 µM siRNA (prophylactic
      treatment - 72 hrs old treated leaf from cytotoxicity assay)
   10. Leaf treated with 10 µL of 1 µM siRNA (prophylactic treatment
       - 72 hrs old treated leaf from cytotoxicity assay)
   11. Leaf treated with 10 µL of 100 nM siRNA (prophylactic
       treatment - 72 hrs old treated leaf from cytotoxicity assay)
4. The boxes were then placed in a spot with moderate sunlight at
   room temperature.
5. Measurements for lesions were taken every 24 hours.

### 28th September (24 hrs)

![Fig 76. Untreated leaves - unpricked and pricked ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/76-untreated-leaves-unpricked-and-pricked.webp)

Fig 76. Untreated leaves - unpricked and pricked

![Fig 77. Leaves treated with water and acetic acid (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/77-leaves-treated-with-water-and-acetic-acid-left-to-right.webp)

Fig 77. Leaves treated with water and acetic acid (left to right)

![Fig 78. Leaves treated with chitosan nanoparticles and nanoformulation (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/78-leaves-treated-with-chitosan-nanoparticles-and-nanoformulation-left-to-right.webp)

Fig 78. Leaves treated with chitosan nanoparticles and
nanoformulation (left to right)

![Fig 79. Leaves treated with 100 µM siRNA and 1 µM siRNA (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/79-leaves-treated-with-100m-sirna-and-1m-sirna-left-to-right.webp)

Fig 79. Leaves treated with 100 µM siRNA and 1 µM siRNA (left to
right)

![Fig 80. Prophylactic treatment- leaves treated with 1 μM siRNA and 100 nM siRNA (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/80-prophylactic-treatment-leaves-treated-with-1m-sirna.webp)

Fig 80. Prophylactic treatment - leaves treated with 1 μM siRNA
and 100 nM siRNA (left to right)

![Fig 81. Prophylactic treatment- leaf treated with 100 μM siRNA ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/81-prophylactic-treatment-leaf-treated-with-100m.webp)

Fig 81. Prophylactic treatment- leaf treated with 100 μM siRNA

### 29th September (48 hrs)

#### Untreated leaf (no lesions seen)

#### Untreated leaf with pin pricks (no lesions seen)

#### Leaf treated with water:

X axis: 1.9 cm
Y axis 3.1 cm

#### Leaf treated with acetic acid:

X axis: 1.5 cm
Y axis: 1.7 cm

#### Leaf treated with chitosan nanoparticle:

X axis: 1.5 cm
Y axis: 1.8 cm

#### Leaf treated with nanoformulation:

X axis: 1.8 cm
Y axis: 2.6 cm

#### Leaf treated with free sirna 100 µM:

X axis: 1.1 cm
Y axis: 1.5 cm

#### Leaf treated with free sirna 1 µM:

X axis: 1.6 cm
Y axis: 1.7 cm

#### Prophylactic treatment leaf 1 µM:

X axis: 0.0 cm
Y axis: 0.0 cm

#### Prophylactic treatment leaf 100 nM

X axis: 0.9 cm
Y axis: 1.1 cm

#### Prophylactic treatment leaf 100 µM

X axis: 0.6 cm
Y axis: 0.8 cm

![Fig 82. Untreated leaves - unpricked and pricked  ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/82-untreated-leaves-unpricked-and-pricked.webp)

Fig 82. Untreated leaves - unpricked and pricked

![Fig 83. Leaves treated with water and acetic acid (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/83-leaves-treated-with-chitosan-nanoparticles-and-nanoformulation-left-to-right.webp)

Fig 83. Leaves treated with water and acetic acid (left to right)

![Fig 84. Leaves treated with chitosan nanoparticles and nanoformulation (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/84-leaves-treated-with-water-and-acetic-acid-left-to-right.webp)

Fig 84. Leaves treated with chitosan nanoparticles and
nanoformulation (left to right)

![Fig 85. Leaves treated with 100 µM siRNA and 1 µM siRNA (left to right). ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/85-leaves-treated-with-100m-sirna-and-1m-sirna-left-to-right.webp)

85. Leaves treated with 100 µM siRNA and 1 µM siRNA (left to
right)

![Fig 86. Prophylactic treatment- leaves treated with 1 μM siRNA and 100 nM siRNA (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/86-prophylactic-treatment-leaves-treated-with-1m-sirna.webp)

Fig 86. Prophylactic treatment - leaves treated with 1 μM siRNA
and 100 nM siRNA (left to right)

![Fig 87. Prophylactic treatment- leaf treated with 100 μM siRNA ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/87-prophylactic-treatment-leaf-treated-with-100m.webp)

Fig 87. Prophylactic treatment - leaf treated with 100 μM siRNA

### 30th September (72 hrs)

#### Untreated leaf (no lesions seen)

#### Untreated leaf with pin pricks (no lesions seen)

#### Leaf treated with water:

X axis: 2.3 cm
Y axis: 3.8 cm

#### Leaf treated with acetic acid:

X axis: 1.7 cm
Y axis: 2.8 cm

#### Leaf treated with chitosan nanoparticle:

X axis: 2.2 cm
Y axis: 2.9 cm

#### Leaf treated with nanoformulation:

X axis: 2.9 cm
Y axis: 3.9 cm

#### Leaf treated with free siRNA 100 µM:

X axis: 1.1 cm
Y axis: 1.9 cm

#### Leaf treated with free siRNA 1 µM:

X axis: 1.8 cm
Y axis: 2.5 cm

#### Prophylactic treatment leaf 1 µM:

X axis: 0.0 cm
Y axis: 0.0 cm

#### Prophylactic treatment leaf 100 nM

X axis: 0.9 cm
Y axis: 1.1 cm

#### Prophylactic treatment leaf 100 µM

X axis: 1.1 cm
Y axis: 1.0 cm

![Fig 88. Untreated leaves - unpricked and pricked  ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/88-untreated-leaves-unpricked-and-pricked.webp)

Fig 88. Untreated leaves - unpricked and pricked

![Fig 89. Leaves treated with water and acetic acid (left to right)  ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/89-leaves-treated-with-water-and-acetic-acid-left-to-right.webp)

Fig 89. Leaves treated with water and acetic acid (left to right)

![Fig 90. Leaves treated with chitosan nanoparticles and nanoformulation (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/90-leaves-treated-with-chitosan-nanoparticles-and-nanoformulation-left-to-right.webp)

Fig 90. Leaves treated with chitosan nanoparticles and
nanoformulation (left to right)

![Fig 91. Leaves treated with 100 µM siRNA and 1 µM siRNA (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/91-leaves-treated-with-100m-sirna-and-1m-sirna-left-to-right.webp)

Fig 91. Leaves treated with 100 µM siRNA and 1 µM siRNA (left to
right)

![Fig 92. Prophylactic treatment- leaves treated with 1 μM siRNA and 100 nM siRNA (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/92-prophylactic-treatment-leaves-treated-with-1m-sirna.webp)

Fig 92. Prophylactic treatment- leaves treated with 1 μM siRNA and
100 nM siRNA (left to right)

![Fig 93. Prophylactic treatment- leaf treated with 100 μM siRNA  ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/93-prophylactic-treatment-leaf-treated-with-100m.webp)

Fig 93. Prophylactic treatment- leaf treated with 100 μM siRNA

## Gel Retardation Assay 3

+

✖

## Gel Retardation Assay

### 3rd October

1. 1 L of 1X TBE (Tris-Borate-EDTA) buffer was prepared from a 20X
   TBE buffer solution (50 mL of 20X TBE buffer was added to 950 mL
   of autoclaved Milli-Q water). The buffer was added to the buffer
   tank.
2. 35 mL of 4% Low EEO agarose gel was prepared using 1X TBE buffer.
   The agarose-TBE solution was microwaved until boiling was
   achieved, and the low EEO agarose had completely dissolved.
3. Once the solution had cooled slightly, 1.16 µL of SYBR Safe was
   added, and the gel was poured into the gel casting tray with the
   comb. A sterile micropipette tip was used to push away any bubbles
   formed on the gel surface. Once the gel had set, it was placed in
   the buffer tank, and the comb was carefully removed.
4. The samples to be loaded onto the gel were prepared in an aseptic
   and RNase-free environment, using RNase-free plasticware. To each
   sample (except the DNA ladder), 2 µL of RNA loading buffer was
   added. The samples were spun using a microcentrifuge and then
   loaded onto the gel.

   The samples were loaded in the following order.
   1. 10 µL of 1 kb DNA ladder
   2. 10 µL of 50 nM siRNA
   3. 10 µL of 100 nM siRNA
   4. 10 µL of nanoformulation (prepared on 29th
      September)
   5. 10 µL of nanoformulation treated with 0.5 mg/mL RNase (2 µL of
      RNase was added to 10 µL of the nanoformulation, the sample
      was incubated for 5 minutes at 56°C)
   6. 10 µL of chitosan nanoparticle (prepared on 10th
      September)
5. The gel was allowed to run at 55 V for 2 hours, and checked in
   intervals
6. The gel was visualized under a UV transilluminator

#### Results:

![Fig 94. Gel run results ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/94-gel-run-results.webp)

Fig 94. Gel run results

#### Conclusions:

The gel run was successful. A faint band of siRNA was observed in
the 100 nM siRNA-loaded well. No siRNA was visualized in the
nanoformulation and chitosan nanoparticle-loaded wells. The 50 nM
siRNA loaded well showed no bands due to the low concentration of
the siRNA.

## SEM Analysis

+

✖

## Scanning Electron Microscopy (SEM)

### 30th September

1. The chitosan nanoparticle (prepared on 10th September )
   and nanoformulation samples were first sonicated for 2 minutes at
   40% amplitude with a 10-second ON/OFF pulse.
2. Glass coverslips were wiped with 70% ethanol.
3. 10 μL of each sample was smeared on individual glass cover slips
   and left to dry completely.
4. The samples were taken to the Central Analytical and
   Instrumentation Facility (CAIF), Manipal, for analysis, but were
   not analyzed due to issues with the gold sputter coater.

### 3rd October

1. The samples prepared on 30th September were
   successfully gold sputtered and taped on glass slides using carbon
   tape.
2. The chitosan nanoparticle and nanoformulation samples were then
   analyzed using the scanning electron microscope (Oxford EDS(X-act)
   scanning electron microscope).

#### Results:

![Fig 95. Chitosan nanoparticles visualized at 10,000x magnification](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/95-chitosan-nanoparticles-visualized-at-10000x-magnification.webp)

Fig 95. Chitosan nanoparticles visualized at 10,000x magnification

![Fig 96. Chitosan nanoparticles visualized at 250,000x magnification ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/96-chitosan-nanoparticles-visualized-at-250000x-magnification.webp)

Fig 96. Chitosan nanoparticles visualized at 250,000x
magnification

![Fig 97. Chitosan nanoparticles visualized at 350,000x magnification ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/97-chitosan-nanoparticles-visualized-at-350000x-magnification.webp)

Fig 97. Chitosan nanoparticles visualized at 350,000x
magnification

![Fig 98. Nanoformulation (prepared on 27th September) visualized at 5000x magnification ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/98-nanoformulation-prepared-on-27th-september-visualized-at-5000x-magnification.webp)

Fig 98. Nanoformulation (prepared on 27th September)
visualized at 5000x magnification

![Fig 99. Nanoformulation (prepared on 27th September) visualized at 200,000x magnification ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/99-nanoformulation-prepared-on-27th-september-visualized-at-20000x-magnification.webp)

Fig 99. Nanoformulation (prepared on 27th September)
visualized at 200,000x magnification

![Fig 100. Nanoformulation (prepared on 27th September) visualized at 250,000x magnification ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/100-nanoformulation-prepared-on-27th-september-visualized-at-250000x-magnification.webp)

Fig 100. Nanoformulation (prepared on 27th September)
visualized at 250,000x magnification

![Fig 101. Nanoformulation (prepared on 29th September) visualized at 6500x magnification ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/101-nanoformulation-prepared-on-29th-september-visualized-at-6500x-magnification.webp)

Fig 101. Nanoformulation (prepared on 29th September)
visualized at 6500x magnification

![Fig 102. Nanoformulation (prepared on 29th September) visualized at 100000x magnification ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/102-nanoformulation-prepared-on-29th-september-visualized-at-100000x-magnification.webp)

Fig 102. Nanoformulation (prepared on 29th September)
visualized at 100000x magnification

![Fig 103. Nanoformulation (prepared on 29th September) visualized at 150000x magnification ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/103-nanoformulation-prepared-on-29th-september-visualized-at-150000x-magnification.webp)

Fig 103. Nanoformulation (prepared on 29th September)
visualized at 150000x magnification

![Fig 104. Nanoformulation (prepared on 29th September) visualized at 350000x magnification ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/104-nanoformulation-prepared-on-29th-september-visualized-at-350000x-magnification.webp)

Fig 104. Nanoformulation (prepared on 29th September)
visualized at 350000x magnification

The SEM analysis of our samples showed us the size and morphology of
our chitosan nanoparticles and nanoformulations prepared. At
magnifications higher than 100000x, the chitosan nanoparticles and
nanoformulation appear as spherical beads in clusters.

It is worth noting that the sample solutions were not prepared the
same day as the SEM analysis and that there was a three-day delay
between the preparation of the samples on the glass coverslip and
the SEM analysis (the samples were stored in a desiccator during
this period to preserve them).

For our future SEM analysis, to obtain better imaging where the
nanoparticles are not in clusters but are spaced out, we were
advised by Dr. Archana Mahadev Rao, to dilute the sample and let the
sample simply air dry on the glass coverslip.

## *P. capsici* Sporulation 4

+

✖

## *P. capsici* Sporulation

### 2nd October

1. ⁠Two autoclaved petri dishes were filled with 15 mL of autoclaved
   distilled water and kept upright.
2. Ten 8 mm mycelial disks were cut from a
   *P. capsici* subculture plate.
3. ⁠The mycelial plugs were then suspended in the sterile water in
   the petri dish with the mycelium side facing upwards and the agar
   side submerged.
4. ⁠The plates were parafilmed carefully and placed for 48 hr
   incubation with illumination at 26°C.

### 4th October

1. ⁠The two sporulation plates kept for 48 hours of light incubation
   were given a cold shock treatment by keeping them at 4°C for 30
   minutes. This released the zoospores into the solution.
2. Six RNase-free PCR tubes were prepared with samples for testing.
   The samples were as follows:
   1. 10 µL of the zoospore solution.
   2. 4 µL of 1% acetic acid was added to 6 µL of the zoospore
      solution.
   3. 4 µL of chitosan nanoparticle solution (prepared on 10th
      September) was added to 6 µL of the zoospore solution.
   4. 4 µL of 7.23 µM of siRNA was added to 6 µL of the zoospore
      solution.
   5. 4 µL of 100 µM siRNA was added to 6 µL of the zoospore
      solution.
   6. 4 µL of the nanoformulation solution (prepared on 29th
      September) was added to 6 µL of the zoospore solution.
3. ⁠The sample tubes (incubated for 0.5 hours) and other necessary
   requirements were taken to MCBR (Manipal Centre for
   Biotherapeutics Research) to be visualized using the upright
   trinocular phase contrast microscope.
4. 10 μL of each sample was pipetted on clean glass slides and
   visualized at 40x and 100x.

Vid 4. Zoospores visualized at 100x without any treatment

Vid 5. Zoospores visualized at 100x with treatment of 1% acetic
acid

Vid 6. Zoospores visualized at 100x with treatment of chitosan
nanoparticle solution

Vid 7. Zoospores visualized at 100x with treatment of 7.23 µM
siRNA

Vid 8. Zoospores visualized at 100x with treatment of 100 µM siRNA

Vid 9. Zoospores visualized at 100x with treatment of
nanoformulation solution

#### Conclusions:

Zoospore movement was observed in the untreated, 1% acetic acid,
and chitosan nanoparticle solution-treated zoospore samples. For
these samples, at 100x magnification, it was observed that the
zoospores were motile and vigorously moving around the field.

The zoospore samples treated with 7.23 µM siRNA, 100 µM siRNA, and
the nanoformulation showed total reduction in zoospore motility.
At 100x magnification, throughout the sample at various fields of
view, it was observed that all zoospores had lost their motility.
This proves the silencing effect of the bZIP gene by the free
siRNA and siRNA encapsulated within the nanoformulation.

## Fluorescence Microscopy

+

✖

## Fluorescence Microscopy

### 4th October

1. The two previously made sporulation plates, kept for 48 hours of
   light incubation, were given a cold shock treatment by keeping
   them at 4°C for 30 minutes. This enabled the release of the
   zoospores from the sporangia into the solution.
2. Zoospore samples for testing were prepared in RNase-free PCR
   tubes. Since the 6-FAM-tagged fluorescent siRNA would get quenched
   easily when exposed to light, darkness was maintained when
   preparing the samples. The samples were as follows:
   1. 4 µL of 7.23 µM of siRNA was added to 6 µL of the zoospore
      solution.
   2. 4 µL of 100 µM siRNA was added to 6 µL of the zoospore
      solution.
   3. 4 µL of the nanoformulation solution (prepared on 29th
      September) was added to 6 µL of the zoospore solution.
3. The samples were incubated in the dark for 1.5 hours at room
   temperature, giving the siRNA enough time to be taken up by the
   zoospores.
4. The samples were pipetted onto clean glass slides, and a cover
   slip was placed on without air bubbles.
5. The samples were then visualized using a fluorescence microscope
   at 10x, 40x, 60x, and 100x magnification at FITC wavelength
   (excitation wavelength around 490-495 nm, and 6-FAM has 498 nm
   excitation wavelength and 515 nm emission wavelength) of the
   fluorescence microscope (we used an Olympus IX 73 Fluorescent
   Microscope).

![Fig 105. Sample treated with our nanoformulation. 6-FAM tagged siRNA visualized within P. capsici zoospores (round green structures) visualized at 100x magnification. The fluorescence of the zoospores indicated the successful release of the siRNA from the nanoformulation ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/105-sample-treated-with-our-nanoformulation-6-fam-tagged-sirna.webp)

Fig 105. Sample treated with our nanoformulation. 6-FAM tagged
siRNA visualized within *P. capsici* zoospores (round green
structures) visualized at 100x magnification. The fluorescence of
the zoospores indicated the successful release of the siRNA from
the nanoformulation

#### Conclusions:

Successful internalization of the 6-FAM tagged siRNA was
visualized at 100x magnification within
*P. capsici* zoospore, as indicated in the image captured.

## Detached Leaf Assay 5

+

✖

## Detached Leaf Assay with Treated Zoospores Alongside Mycelial Plugs

### 27th September

1. The LAF and apparatus (inoculation loops, carrot agar plates, cork
   borer, etc) were cleaned and sterilized through UV treatment.
2. ⁠Mycelial discs from the previously made subculture plates were
   made and inverted onto 2 fresh carrot agar plates, using an 8 mm
   cork borer and an inoculation loop. Each plate was set with two
   mycelial discs.
3. ⁠The plates were then placed in the dark incubator at 28°C.

### 30th September

1. Two autoclaved petri dishes were filled with 15 mL of autoclaved
   distilled water and kept upright.
2. Ten 8 mm mycelial disks were cut from a
   *P. capsici* subculture plate.
3. ⁠The mycelial plugs were then suspended in the sterile water in
   the petri dish with the mycelium side facing upwards and the agar
   side submerged.
4. ⁠The plates were parafilmed carefully and placed for 48 hrs
   incubation with illumination at 26°C.

### 2nd October

1. The two sporulation plates kept for 48 hrs of light incubation
   were given a cold shock treatment by keeping them at 4°C for 30
   minutes. This released the zoospores into the solution.
2. The LAF and apparatus were cleaned and sterilized through UV
   treatment.
3. The zoospore solution was aliquoted into tubes and was treated
   with our siRNA, chitosan nanoparticles, and nanoformulation.
4. Before inoculating the leaves with the treated zoospore samples,
   the leaves were pricked and placed in plastic boxes lined with
   moist cotton. The zoospore samples were carefully pipetted on the
   pricked area of the leaf surface.
   The prepared black pepper
   leaf samples were as follows:
   1. Untreated leaf with just pricks.
   2. Leaf treated with 10 µL of water.
   3. Leaf treated with 10 µL of zoopore solution treated with
      acetic acid solution (2:1 ratio).
   4. Two leaves treated with 10 µL of zoopore solution.
   5. Two leaves treated with 10 µL of zoopore solution treated with
      chitosan nanoparticle solution (prepared on 10th
      September in a 2:1 ratio)
   6. Two leaves treated with 10 µL of zoopore solution treated with
      nanoformulation (prepared on 29th September in a
      2:1 ratio)
   7. Two leaves treated with 10 µL of zoopore solution, treated
      with 5 µL of 1 µM siRNA.
5. Mycelial discs from the previously made subculture plates were
   made and inverted onto fresh treated black pepper leaves placed in
   plastic boxes lined with moist cotton, using a 5 mm cork borer and
   an inoculation loop.
6. Before inoculating the leaves with mycelial discs, the leaves were
   treated with our latest nanoformulation and chitosan
   nanoparticles.

   The prepared black pepper leaf samples were as follows:
   1. Leaf treated with 10 µL of chitosan nanoparticle solution
      (prepared on 10th September)
   2. Leaf treated with 10 µL of nanoformulation (prepared on 29th
      September)
7. ⁠The boxes were then placed in a spot with moderate sunlight at
   room temperature.
8. Measurements for lesions were taken every 24 hrs.

### 3rd October

No lesions were observed on any of the leaves.

![Fig 106. Untreated leaves -pricked  ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/106-untreated-leaves-pricked.webp)

Fig 106. Untreated leaves - pricked

![Fig 107. Leaves treated with water and acetic acid (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/107-leaves-treated-with-water-and-acetic-acid.webp)

Fig 107. Leaves treated with water and acetic acid (left to right)

![Fig 108. Leaves treated with zoospore suspension  ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/108-leaves-treated-with-zoospore-suspension.webp)

Fig 108. Leaves treated with zoospore suspension

![Fig 109. Leaves treated with zoopores were treated with chitosan nanoparticles](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/109-leaves-treated-with-zoopores-treated-with-chitosan-nanoparticles.webp)

Fig 109. Leaves treated with zoopores were treated with chitosan
nanoparticles

![Fig 110. Leaves treated with zoopores were treated with the nanoformulation ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/110-leaves-treated-with-zoopores-were-treated-with-the-nanoformulation.webp)

Fig 110. Leaves treated with zoopores were treated with the
nanoformulation

![Fig 111. Leaves treated with zoopores treated with 1 µM siRNA  ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/111-leaves-treated-with-zoopores-treated-with-1m-sirna.webp)

Fig 111. Leaves treated with zoopores were treated with 1 µM siRNA

![Fig 112. Leaves treated with chitosan nanoparticles  ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/112-leaves-treated-with-chitosan-nanoparticles.webp)

Fig 112. Leaves treated with chitosan nanoparticles

![Fig 113. Leaves treated with the nanoformulation solution  ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/113-leaves-treated-with-the-nanoformulation-solution.webp)

Fig 113. Leaves treated with the nanoformulation solution

### 4th October (48 hrs)

#### Untreated leaf (pricked):

No lesion seen

#### Just a water-treated leaf:

No lesion seen

#### Zoospore-treated leaf:

No lesion seen

#### Zoospore acetic acid-treated leaf:

No lesion seen

#### Zoospore Nanoparticle-treated leaf:

No lesion seen

#### Zoospore Nanoformulation-treated leaf:

No lesion seen

#### Zoospore-free siRNA-treated leaf:

No lesion seen

#### P. capsici plug and nanoformulation-treated leaf:

Left leaf (negligible):
X axis: 0.0 cm
Y axis: 1.2 cm

Right leaf:
X axis: 1.1 cm
Y axis: 1.3 cm

#### *P. capsici* plug and chitosan nanoparticle-treated leaf:

Left leaf:
X axis: 1.4 cm
Y axis: 1.3 cm

Right leaf:
X axis: 1.2 cm
Y axis: 1.3 cm

![Fig 114. Leaves treated with water and acetic acid (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/114-leaves-treated-with-water-and-acetic-acid.webp)

Fig 114. Leaves treated with water and acetic acid (left to right)

![Fig 115. Leaves treated with zoospore suspension ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/115-leaves-treated-with-zoospore-suspension.webp)

Fig 115. Leaves treated with zoospore suspension

![Fig 116. Leaves treated with zoopores were treated with chitosan nanoparticles](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/116-leaves-treated-with-zoopores-treated-with-chitosan-nanoparticles.webp)

Fig 116. Leaves treated with zoopores were treated with chitosan
nanoparticles

![Fig 117. Leaves treated with zoopores were treated with the nanoformulation](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/117-leaves-treated-with-zoopores-were-treated-with-the-nanoformulation.webp)

Fig 117. Leaves treated with zoopores were treated with the
nanoformulation

![Fig 118. Leaves treated with zoopores treated with one µM siRNA](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/118-leaves-treated-with-zoopores-treated-with-1m-sirna.webp)

Fig 118. Leaves treated with zoopores were treated with one µM
siRNA

![Fig 119. Leaves treated with chitosan nanoparticles  ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/119-leaves-treated-with-chitosan-nanoparticles.webp)

Fig 119. Leaves treated with chitosan nanoparticles

![Fig 120. Leaves treated with the nanoformulation solution](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/120-leaves-treated-with-the-nanoformulation-solution.webp)

Fig 120. Leaves treated with the nanoformulation solution

### 5th October (72 hrs)

#### Untreated leaf (pricked):

No lesion seen

#### Just a water-treated leaf:

No lesion seen

#### Zoospore-treated leaf:

No lesion seen

#### Zoospore acetic acid-treated leaf:

No lesion seen

#### Zoospore Nanoparticle-treated leaf:

No lesion seen

#### Zoospore Nanoformulation-treated leaf:

No lesion seen

#### Zoospore-free siRNA-treated leaf:

No lesion seen

#### *P. capsici* plug and nanoformulation-treated leaf:

Left leaf (negligible):
X axis: 1.3 cm
Y axis: 1.4 cm

Right leaf:
X axis: 1.7 cm
Y axis: 2.1 cm

#### *P. capsici* plug and chitosan nanoparticle-treated leaf:

Left leaf:
X axis: 2.2 cm
Y axis: 2.1 cm

Right leaf:
X axis: 1.6 cm
Y axis: 2.1 cm

![Fig 121. Leaves treated with water and acetic acid (left to right) ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/121-leaves-treated-with-water-and-acetic-acid.webp)

Fig 121. Leaves treated with water and acetic acid (left to right)

![Fig 122. Leaves treated with zoospore suspension ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/122-leaves-treated-with-zoospore-suspension.webp)

Fig 122. Leaves treated with zoospore suspension

![Fig 123. Leaves treated with zoopores treated with chitosan nanoparticles  ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/123-leaves-treated-with-zoopores-treated-with-chitosan-nanoparticles.webp)

Fig 123. Leaves treated with zoopores were treated with chitosan
nanoparticles

![Fig 124. Leaves treated with zoopores were treated with the nanoformulation](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/124-leaves-treated-with-zoopores-were-treated-with-the-nanoformulation.webp)

Fig 124. Leaves treated with zoopores were treated with the
nanoformulation

![Fig 125. Leaves treated with zoopores treated with 1 µM siRNA ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/125-leaves-treated-with-zoopores-treated-with-1m-sirna.webp)

Fig 125. Leaves treated with zoopores were treated with 1 µM siRNA

![Fig 126. Leaves treated with chitosan nanoparticles ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/126-leaves-treated-with-chitosan-nanoparticles.webp)

Fig 126. Leaves treated with chitosan nanoparticles

![Fig 127. Leaves treated with the nanoformulation solution ](https://static.igem.wiki/teams/5645/wiki-images/pages/notebook/week10/127-leaves-treated-with-the-nanoformulation-solution.webp)

Fig 127. Leaves treated with the nanoformulation solution

#### Conclusions:

Over three days, visible necrosis (blackening of the leaves) was
observed in the leaves treated with just the zoospore solution and
zoospores treated with 1% acetic acid solution. The leaf treated
with just water also showed visible necrosis due to contamination
from the leaf with zoopores treated with 1% acetic acid solution
(both leaves were placed in the same box). The leaves treated with
zoospores with chitosan nanoparticle, 1 µM siRNA, and the
nanoformulation showed minimal to no visible necrosis on the leaf
surface, indicating a successful qualitative assessment of the
chitosan nanoparticle, 1 µM siRNA, and the nanoformulation on
*P. capsici* zoopores.

The leaves treated with the chitosan nanoparticles and the
nanoformulation, upon which mycelial plugs were placed, showed
lesion formation. It was observed that the leaves treated with the
nanoformulation had smaller lesions compared to those treated with
the chitosan nanoparticle.

### Week 11 - 15th to 19th October

## SEM Analysis

+

✖

## Field Emission Scanning Electron Microscopy (FESEM) Post Wiki Freeze

### 15th October

1. The chitosan nanoparticle (prepared on September 10th)
   and nanoformulation sample (prepared on September 29th)
   were first sonicated for 2 minutes at 40% amplitude with a
   10-second ON/OFF pulse.
2. Glass coverslips were wiped with 70% ethanol.
3. The chitosan nanoparticle and nanoformulation solutions were
   diluted upto a 1:10 ratio (1 μL of nanoformulation/chitosan
   nanoparticle to 9 μL of DEPC-treated water).
4. 5 μL and 10 μL of each sample were pipetted onto individual glass
   coverslips and left to dry completely in the laminar airflow
   chamber.
5. The samples were kept in an ice box and taken to the Central
   Research Facility at the National Institute of Technology,
   Karnataka, Surathkal.

### 17th October

The samples prepared on October 15th were analyzed using
a field-emission scanning electron microscope (7610FPLUS, Jeol,
Japan).

#### Results:

![Fig 128. Chitosan nanoparticles visualized at 100,000x magnification (10 μL sample)](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig128-10ulofcsnp1-wikithaw.avif)

Fig 128. Chitosan nanoparticles visualized at 100,000x
magnification (10 μL sample)

![Fig 129. Chitosan nanoparticles visualized at 100,000x magnification (5 μL sample)](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig129-5ulofcsnp-wikithaw.avif)

Fig 129. Chitosan nanoparticles visualized at 100,000x
magnification (5 μL sample)

![Fig 130. Nanoformulation (prepared on 29th September) visualized at 100,00x magnification (10 μL sample) ](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig130-10ulnanoformulation-wikithaw.avif)

Fig 130. Nanoformulation (prepared on 29th September)
visualized at 100,000x magnification (10 μL sample)

![Fig 131. Nanoformulation (prepared on 29th September) visualized at 100,00x magnification (5 μL sample) ](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig131-5ulnf-wikithaw.avif)

Fig 131. Nanoformulation (prepared on 29th September)
visualized at 100,000x magnification (5 μL sample)

The SEM analysis of our samples revealed the size and morphology of
the chitosan nanoparticles and nanoformulation prepared.

Upon observing the image of the 5 μL and 10 μL chitosan nanoparticle
and nanoformulation samples, it was concluded that the sample volume
difference primarily affected the particle density and its
distribution on the coverslip, rather than the individual particle
morphology. Higher-volume samples showed more particles per field of
view. However, these samples may also exhibit more aggregation due
to particle crowding during the drying process.

It is worth noting that the sample solutions were not prepared on
the same day as the SEM analysis and that a three-day delay occurred
between the preparation of the samples on glass coverslips and the
FESEM analysis (the samples were stored in a desiccator during this
period to preserve them).

## Detached Leaf Assay 6

+

✖

## Detached Leaf Assay 6 - Effect of Nanoformulation after 3 weeks

A detached leaf assay was performed after the wiki freeze to test
the efficacy of the nanoformulation following 3 weeks of
preparation, and the percentage inhibition was compared to that of
the fresh sample.

The prepared black pepper leaf samples were as follows:

1. Untreated leaf with just pricks, leaf treated with 10 µL of water
2. Positive control (*P. capsici* alone, treated with 10 µL of
   water)
3. 10 µL of Acetic acid control
4. 10 µL of the nanoparticle solution
5. 10 µL of the nanoformulation

Mycelial discs from the previously made subculture plates were made
and inverted onto freshly treated black pepper leaves placed in
plastic boxes lined with moist cotton, using a 5 mm cork borer and
an inoculation loop.

Lesion growth was observed across the next three days.

### 17th October (24 hrs)

#### Untreated leaves (no lesions seen)

#### Positive Control:

X axis: 0 cm
Y axis: 0 cm

#### Leaf treated with acetic acid:

X axis: 1.0 cm
Y axis: 0.9 cm

#### Leaf treated with nanoparticles:

X axis: 0 cm
Y axis: 0 cm

#### Leaf treated with nanoformulation:

X axis: 0 cm
Y axis: 0 cm

![Fig 132. Untreated leaves](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig132-untreatedleaves-wikithaw.avif)

Fig 132. Untreated leaves

![Fig 133. Leaf with P. capsici alone- Positive control ](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig133-leaf-capsici-wikithaw.avif)

Fig 133. Leaf with *P. capsici* alone- Positive control

![Fig 134. Leaf treated with Acetic acid ](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig134-leaf-aa-wikithaw.avif)

Fig 134. Leaf treated with Acetic acid

![Fig 135. Leaf treated with chitosan nanoparticles](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig135-leaf-csnp-wikithaw.avif)

Fig 135. Leaf treated with chitosan nanoparticles

![Fig 136. Leaf treated with the nanoformulation](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig136-leaf-nf-wikithaw.avif)

Fig 136. Leaf treated with the nanoformulation

### 18th October (48 hours)

#### Untreated leaves (no lesions seen)

#### Positive Control:

X axis: 0.9 cm
Y axis: 0.8 cm

#### Leaf treated with acetic acid:

X axis: 1.0 cm
Y axis: 0.9 cm

#### Leaf treated with nanoparticles:

X axis: 0.8 cm
Y axis: 0.8 cm

#### Leaf treated with nanoformulation:

X axis: 0.4 cm
Y axis: 0.7 cm

![Fig 137. Untreated leaves](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig137-untreatedleaves-wikithaw.avif)

Fig 137. Untreated leaves

![Fig 138. Leaf with P. capsici alone- Positive control ](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig138-leaf-capsici-wikithaw.avif)

Fig 138. Leaf with *P. capsici* alone - Positive control

![Fig 139. Leaf treated with Acetic acid  ](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig139-leaf-aa-wikithaw.avif)

Fig 139. Leaf treated with Acetic acid

![Fig 140. Leaf treated with chitosan nanoparticles ](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig140-leaf-csnp-wikithaw.avif)

Fig 140. Leaf treated with chitosan nanoparticles

![Fig 141. Leaf treated with the nanoformulation ](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig141-leaf-nf-wikithaw.avif)

Fig 141. Leaf treated with the nanoformulation

### 19th October (72 hours)

#### Untreated leaves (no lesions seen)

#### Positive Control:

X axis: 1.5 cm
Y axis: 1.6 cm

#### Leaf treated with acetic acid:

X axis: 1.0 cm
Y axis: 1.0 cm

#### Leaf treated with nanoparticles:

X axis: 0.9 cm
Y axis: 0.9 cm

#### Leaf treated with nanoformulation:

X axis: 0.9 cm
Y axis: 0.8 cm

![Fig 142. Untreated leaves](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig142-untreatedleaves-wikithaw.avif)

Fig 142. Untreated leaves

![](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig143-leaf-capsici-wikithaw.avif)

Fig 143. Leaf with *P. capsici* alone - Positive control

![Fig 144. Leaf treated with Acetic acid  ](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig144-leaf-aa-wikithaw.avif)

Fig 144. Leaf treated with Acetic acid

![Fig 145. Leaf treated with chitosan nanoparticles](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig145-leaf-csnp-wikithaw.avif)

Fig 145. Leaf treated with chitosan nanoparticles

![Fig 146. Leaf treated with the nanoformulation](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/fig146-leaf-nf-wikithaw.avif)

Fig 146. Leaf treated with the nanoformulation

![](https://static.igem.wiki/teams/5645/wiki-images/ihp-and-notebook-icons/filter-icon.webp)

*P. capsici*

Chitosan Nanoparticle

Nanoformulation

Detached Leaf Assay

Fluorescence Microscopy

Cytotoxicity Assay
Clear Filter

{% endblock %}

{% extends "minimal-layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% set next\_page\_url = url\_for('pages', page='results') %}
{% block next\_page\_name %}Results{% endblock %}
{% block custom\_styling %}
{% endblock %}
{% block custom\_javascript %}

{% endblock %}
{% block page\_content %}

LEARN

TEST

DESIGN

BUILD

The Design-Build-Test-Learn (DBTL) cycle was foundational to the development of our project.
While developing each component, we incorporated learnings from every experimental outcome
towards building more fruitful procedures.

This process allowed us to continually improve the design and functionality of our system,
while also reframing our approach to scientific problem solving.

DBTL was much more than an engineering framework in our work, as it guided our way of
thinking and experimenting as young researchers in a foundational manner.

## Project DBTL

#### Iteration 1

+

✖

## Project Iteration 1

LEARN

TEST

DESIGN

BUILD

### Design

We surveyed literature on genes involved in the pathogenicity
and survival of *Phytophthora* spp. and shortlisted targets
with roles in virulence and signaling. Since several genes were
initially annotated only in related *Phytophthora* species,
their orthologous sequences were retrieved, and a BLAST search
was conducted against the *P. capsici* genome to confirm
the presence of homologs. However, preliminary testing revealed
high conservation and extensive off-target risks, making many of
these targets unsuitable. To refine our approach, we used the
transcriptomic data of *P. capsici* to validate gene
expression during infection, which led us to bZIP, a
transcription factor critical for pathogenicity.

### Build

The complete mRNA transcript was retrieved from curated
databases, and siRNA candidates were generated using siDirect
and siRNAPred. These were then filtered using established design
rules such as URA criteria, GC content distribution, and
nucleotide preferences at key positions.

The shortlisted siRNA candidates underwent rigorous structural
validation to ensure functional stability and specificity.
Target mRNA secondary structures were predicted using RNAfold to
confirm binding accessibility, while DuplexFold and MaxExpect
evaluated duplex stability and removed unstable constructs. To
further strengthen our process, off-target risks were assessed
through BLAST searches against the NCBI RefSeq RNA, RefSeq
Select RNA, and CORE nucleotide databases. Sequences showing
significant similarity to humans, black pepper (*P. nigrum*), or other local crops were excluded. After two full
Design–Build–Test–Learn iterations, four siRNA candidates were
finalized as stable, target-specific, and URA-compliant.

Building on these computationally validated candidates,
experimentation was designed through iHP, and literature reviews
were conducted for potential experiments to gauge the bZIP’s
pathogenicity, efficacy, and delivery strategies. The chitosan
nanoparticles were synthesized and optimized in the lab for our
application. The siRNA was then encapsulated in the nanoparticle
to function as the implementable nanoformulation.

### Test

Characterization of every nanoparticle and nanoformulation was
done using the Particle Size Analyzer. Encapsulation was
validated visually using gel retardation assays, and entrapment
efficiencies were calculated. A cytotoxicity assay was performed
using naked siRNA (ranging from 1.5 nM to 100 μM) on
*Piper nigrum* leaves.

Fluorescence microscopy helped visualize the uptake and
internalization of the siRNA, thereby helping us test the
release mechanism of the nanoparticle. Detached Leaf Assays were
performed both prophylactically and therapeutically, with
various controls, to observe the direct effect of the
nanoformulation, naked siRNA, and chitosan nanoparticles on the
leaves.

### Learn

The development of the nanoformulation was optimized through
several runs. However, complete encapsulation of the siRNA was
not observed, attributed to pH, fluctuation in flow rate, and
stirring speed. The siRNA showed 0% cytotoxicity towards the
plant at all concentrations. The detached leaf assays directly
showed a reduction in lesion formation on application of the
solution. This helped us measure the efficacy of our solution.
Complimenting these were the motility of the zoospores, which
were observed to be less upon the application of our solution.
The siRNA design process was lengthy and iterative, prompting us
to develop a pipeline integrating sequence retrieval, candidate
generation, URA (unique read alignments) based filtering, and
structural validation.

#### Iteration 2

+

✖

## Project Iteration 2

LEARN

TEST

DESIGN

BUILD

### Design

We developed an siRNA design automation pipeline that mimics the
Design-Build-Test-Learn (DBTL) cycle 1 by integrating multiple
siRNA Design software through a Selenium-based automation bot.
This system accepts a target gene as the initial input,
processes it through each software in a sequence, and passes the
output to the next stage, enabling end-to-end automated design
and evaluation of siRNA sequences.

![]()

### Build

Since siRNA is often delivered via various nanoparticle carriers
such as chitosan and lipids, we built a predictive model to
select the most suitable siRNA candidates based on their
interaction profiles with these nanoparticles. Using docking
simulation data, we trained a machine learning model to predict
the interaction of siRNA molecules with their nanoparticle
carriers. This addition helps tailor siRNA design not only for
gene silencing efficiency but also for optimal compatibility and
stability with delivery vehicles used in therapeutics and other
applications.

### Test

These optimized siRNA–nanoparticle pairs will undergo validation
through transcriptomic analysis and RT-qPCR to measure gene
silencing efficacy and specificity. In parallel, we are
exploring cell-based siRNA synthesis to enable upscaling, while
discussions have also begun for controlled field trials to test
the formulated solution under real-world conditions.

### Learn

The results of transcriptomic profiling and RT-qPCR will provide
feedback on silencing efficiency and off-target effects, feeding
back into the pipeline to refine future designs. The RT-qPCR
tests will also help learn the specificity and efficacy by
quantifying bZIP1 reduction, aiding in the measurement of the
RNAi system. Cell-based siRNA synthesis will allow us to bring
our solution to more farmers, upgrading the process. The
controlled field trials will be used to draw comparisons based
on the environment and help learn the other biosafety
considerations. This allows us to display the robustness of the
solution in the target environment and gauge real-world
efficacy.

## Dry Lab

### siRNA Design

#### Iteration 1

+

✖

## siRNA Design Iteration 1

LEARN

TEST

DESIGN

BUILD

### Design

Through a literature review, we identified multiple papers
discussing genes responsible for pathogenicity and survival of
*Phytophthora* spp., and we shortlisted a set of genes with
significant roles; silencing these targets could interfere with
critical signaling pathways essential for infection.

### Build

We began by examining genes across the *Phytophthora* genus
to identify those implicated in virulence and signaling. If a
candidate gene was not directly annotated in *P. capsici*,
the corresponding orthologous sequence from other Phytophthora
species was retrieved and subjected to BLAST analysis against
the *P. capsici* genome. This approach confirmed the
presence of homologous genes in *P. capsici* and ensured
that the sequence data used for further analysis were accurate,
complete, and directly relevant to the pathogen.

### Test

To evaluate whether the genes were suitable targets, the
full-length gene sequence was subjected to BLAST searches
against multiple reference databases. This was done to check the
level of sequence conservation and detect potential similarities
with homologous genes in non-target organisms. This step was
crucial because excessive conservation across species (crops or
beneficial plants) increases the risk of unintended off-target
silencing.

### Learn

The results we obtained showed too many off-target effects. Our
advisor suggested additional screening to check for
transcriptomic validation in *Phytophthora capsici* of the
gene targets that were selected from other
*Phytophthora* spp.

Table 1. Summary of potential gene targets

Gene | Pros | Cons | Results and learning || bZIP1 (Basic Leucine Zipper TF) ([Blanco & Judelson, 2005](https://doi.org/10.1111/j.1365-2958.2005.04575.x)) | Silencing *Pibzp1* in *P. infestans* abolished infection.  *Pc* ortholog sequence available. | Family conserved, but the specific virulence role was not clear. | **Advanced**  Strong virulence evidence  High-value target. |
| *PcCHS* (Chitin Synthase)  ([Cheng et al., 2019](https://doi.org/10.1111/1462-2920.14744)) | *PcCHS* knockout reduced growth, spore release, and virulence.  *Pc* sequence available. | Conserved enzyme family, but risk of fungal off-targets. | **Advanced**  Robust *Pc* evidence  Requires careful design. |
| GPA1 (G-protein α)  ([Latijnhouwers et al., 2003](https://doi.org/10.1046/j.1365-2958.2003.03893.x)) | Critical for motility/chemotaxis in *Ps*/*Pi*; single copy, essential. | Highly conserved across eukaryotes  Broad off-targets  *Pc* sequence context is uncertain. | **Eliminated**  Conservation makes it unsuitable. |
| *PcRXLR* effectors (family)  ([Cheng et al., 2022](https://doi.org/10.1094/mpmi-12-21-0295-r)) | Key virulence effectors: RNAi against RXLR1/4 reduces infection; *Pc* transcripts are available. | A very large multigene family, which leads to a redundancy problem | **Eliminated**  Redundancy lowers the impact of targeting single members. |
| *PcNLPs* (e.g., *PcNLP6*)  ([Park et al., 2023](https://doi.org/10.1186/s13765-023-00768-4)) | Encode necrosis-inducing proteins; dsRNA reduces infection and transcript levels. | A multigene family with overlapping roles, which leads to functional redundancy  This may mask the silencing of one gene. | **Eliminated**  Redundancy limits effectiveness unless multiple family members are targeted simultaneously. |
| *PiCAT2* (Catalase)  ([Wang et al., 2020](https://doi.org/10.1111/mpp.12907)) | In *Pi*, it affects growth, reproduction, stress tolerance, and virulence. | Very high off-target hits in diverse species  *Pc* link unclear. | **Eliminated**  Extensive off-targets,  Weak *Pc* evidence. |
| *PiCDC14* (Cdc14 Phosphatase)  ([Fong & Judelson, 2003](https://doi.org/10.1046/j.1365-2958.2003.03735.x)) | In *Pi*, required for sporulation and development. | Stage-specific function  Weak infection link in *Pc*. | **Eliminated**  Not directly tied to pathogenicity. |
| *PsMAPK7* (MAPK7)  ([Gao et al., 2014](https://doi.org/10.1111/mpp.12163)) | In *Ps*, required for stress tolerance, germination, and infection. | MAPKs are highly conserved, but no *Pc* data. | **Eliminated**  Conservation and species gap. |
| *PsMPK1* (SLT2-type MAPK)  ([Li et al., 2014](https://doi.org/10.1016/j.fgb.2014.01.003)) | In *Ps*, silencing abolished pathogenicity. | Strong conservation with plants/fungi, but off-target risk. | **Eliminated**  Unsuitable for specific silencing. |
| *PcCutinases* (e.g., *PcCut*, *PiCut3*)  ([Muñoz & Bailey, 1998](https://doi.org/10.1007/s002940050330)) | Early penetration factors:  dsRNA against cutinases impaired growth and pathogenicity. | A multigene family with overlapping roles causes a redundancy problem. | **Eliminated**  Silencing a single gene was unlikely to yield a meaningful effect. |

Blanco, F. A., & Judelson, H. S. (2005). A bZIP transcription
factor from *Phytophthora* interacts with a protein kinase
and is required for zoospore motility and plant infection.
*Molecular Microbiology, 56*(3), 638–648.
<https://doi.org/10.1111/j.1365-2958.2005.04575.x>

Cheng, W., Lin, M., Qiu, M., Kong, L., Xu, Y., Li, Y., Wang, Y.,
Ye, W., Dong, S., He, S., & Wang, Y. (2019). Chitin synthase is
involved in vegetative growth, asexual reproduction, and
pathogenesis of *Phytophthora capsici* and
*Phytophthora sojae*.
*Environmental Microbiology, 21*(12), 4537–4547.
<https://doi.org/10.1111/1462-2920.14744>

Cheng, W., Lin, M., Chu, M., Xiang, G., Guo, J., Jiang, Y.,
Guan, D., & He, S. (2022). RNAi-Based Gene Silencing of RXLR
Effectors Protects Plants Against the Oomycete Pathogen
*Phytophthora capsici*.
*Molecular Plant-Microbe Interactions, 35*(6), 440–449.
https://doi.org/10.1094/mpmi-12-21-0295-r

Fong, A. M. V. A., & Judelson, H. S. (2003). Cell cycle
regulator Cdc14 is expressed during sporulation but not hyphal
growth in the fungus‐like oomycete
*Phytophthora infestans*.
*Molecular Microbiology, 50*(2), 487–494.
<https://doi.org/10.1046/j.1365-2958.2003.03735.x>

Gao, J., Cao, M., Ye, W., Li, H., Kong, L., Zheng, X., & Wang,
Y. (2014). PsMPK7, a stress‐associated mitogen‐activated protein
kinase (MAPK) in *Phytophthora sojae*, is required for
stress tolerance, reactive oxygenated species detoxification,
cyst germination, sexual reproduction and infection of soybean.
*Molecular Plant Pathology, 16*(1), 61–70.
<https://doi.org/10.1111/mpp.12163>

Latijnhouwers, M., Ligterink, W., Vleeshouwers, V. G., Van West,
P., & Govers, F. (2003). A Gα subunit controls zoospore motility
and virulence in the potato late blight pathogen
*Phytophthora infestans*.
*Molecular Microbiology, 51*(4), 925–936.
<https://doi.org/10.1046/j.1365-2958.2003.03893.x>

Li, A., Zhang, M., Wang, Y., Li, D., Liu, X., Tao, K., Ye, W., &
Wang, Y. (2014). PsMPK1, an SLT2-type mitogen-activated protein
kinase, is required for hyphal growth, zoosporogenesis, cell
wall integrity, and pathogenicity in *Phytophthora sojae*.
*Fungal Genetics and Biology, 65*, 14–24.
<https://doi.org/10.1016/j.fgb.2014.01.003>

Muñoz, C. I., & Bailey, A. M. (1998). A cutinase-encoding gene
from *Phytophthora capsici* isolated by
differential-display RT-PCR. *Current Genetics, 33*(3),
225–230.
<https://doi.org/10.1007/s002940050330>

Park, M., Kweon, Y., Lee, D., & Shin, C. (2023). Suppression of
*Phytophthora capsici* using double-stranded RNAs targeting
NLP effector genes in *Nicotiana benthamiana*.
*Applied Biological Chemistry, 66*(1).
<https://doi.org/10.1186/s13765-023-00768-4>

Wang, T., Wang, X., Zhu, X., He, Q., & Guo, L. (2020). A proper
PiCAT2 level is critical for sporulation, sporangium function,
and pathogenicity of *Phytophthora infestans*.
*Molecular Plant Pathology, 21*(4), 460–474.
<https://doi.org/10.1111/mpp.12907>

#### Iteration 2

+

✖

## siRNA Design Iteration 2

LEARN

TEST

DESIGN

BUILD

### Design

The learnings accumulated thus far allowed us to identify bZIP,
a transcription factor implicated in the virulence of
*P. capsici*, for siRNA targeting. The complete mRNA
sequence was retrieved from curated databases.

![Fig 1. A flowchart depicting the siRNA design mechanism](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/1-flowchart-depicting-sirna-design.webp)

Fig 1. A flowchart depicting the siRNA design mechanism

### Build

Multiple siRNA prediction tools, including siDirect and
siRNAPred, were used to generate an initial pool of candidate
sequences. Design rules such as the URA criteria (low
seed-target duplex stability, A/U enrichment in positions 15–19,
and absence of internal repeats), GC content distribution, and
nucleotide preferences at specific positions were applied to
refine the pool.

### Test

The shortlisted siRNA candidates were subjected to structural
validation. Secondary structure predictions of the target mRNA
were performed using RNAfold, ensuring that the candidate
binding sites were located in accessible regions. Duplex
stability of the siRNA molecules was assessed using DuplexFold
and MaxExpect, which enabled the elimination of unstable
constructs.

Potential off-target effects were evaluated through BLAST
searches against the NCBI RefSeq RNA, RefSeq Select RNA, and
CORE nucleotide databases. Any siRNA showing significant
similarity to sequences in humans, black pepper (*P. nigrum*), or crops grown in close proximity was excluded.

### Learn

The results were analyzed to identify recurrent features of the
most promising siRNAs, such as AU-rich seed regions and stable
duplex formation. Insights from this cycle were integrated into
the next design iteration, resulting in refined and more
reliable siRNA candidates.

This iterative process ensured that the siRNA design was robust,
reproducible, and systematically validated.

## Software

### siUltimate

#### Iteration 1

+

✖

## siUltimate Iteration 1

LEARN

TEST

DESIGN

BUILD

### Design

The objective of building siUltimate was to develop an
end-to-end pipeline for siRNA design by integrating the existing
siRNA design software, significantly reducing the time required
for it.

### Build

#### Softwares Used

The initial designs of siUltimate involved automating the use of
three websites: siDirect, siRNApred, and DuplexFold. siDirect
and siRNApred are used to design siRNAs, while DuplexFold
predicts the secondary structure of a given siRNA. The TP53
tumor suppressor gene was chosen as an initial test sequence, as
it returned good results on all three of these softwares when
used with default settings.

#### Browser Automation

Selenium, a Python framework for browser automation, was used to
automate the interaction with these websites. Selenium allows
simulating interactions with websites in a human-like manner,
and elements can be selected using CSS selectors. It was used to
input mRNA sequences, change settings, and get results from the
websites.

#### Data Formatting

The resulting data from each website was converted into a pandas
dataframe, as it made it much easier to work with in code.
Formatting the siDirect output required splitting the guide
strand and passenger strand columns.

![Fig 2. First 8 formatted siDirect results for iteration 1](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/2-pipeline-dbtl1-sidirect-results.webp)

Fig 2. First 8 formatted siDirect results for iteration 1

siRNApred had a longer formatting process, requiring formatting
of the position and sequence columns. The extra hyphens (-) and
the text 3’ and 5’ had to be removed from all of the values in
the sequence column, and the data in the position column also
required removal of extra hyphens.

![Fig 3.  Formatted siRNApred results for iteration 1](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/3-pipeline-dbtl1-sirnapredresults.webp)

Fig 3. Formatted siRNApred results for iteration 1

#### Pooling of Results

The common siRNA sequences from the siDirect and siRNApred
output data were retrieved. This new set of siRNAs was ranked
according to the following parameters in a decreasing order of
priority:

1. siRNAs following the Ui-Tei rules (these rules act as a
   filter for siRNAs, and the siRNAs that do not follow them
   are ranked very low).
2. siRNAs following the Reynolds and Amarzguioui rules (these
   rules act as a way of ranking siRNAs, and siRNAs that follow
   these, in addition to the Ui-Tei rules, are the best).
3. The siRNApred score (this was the lowest priority of
   ranking, and it served as a tiebreaker for siRNAs ranked
   equally by the previous two parameters).

The top 15 siRNA sequences were chosen to continue on to the
next round of the pipeline.

![Fig 4.  Pooled siRNA results for iteration 1](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/4-pipeline-dbtl1-pooledsirnas.webp)

Fig 4. Pooled siRNA results for iteration 1

#### Filtering via DuplexFold

DuplexFold was used to predict the secondary structure of the
siRNAs. It outputted a CT (connect table) file, which was then
analyzed by the software. It checked whether the secondary
structure of the siRNA had a misregister, which would indicate
an unstable secondary structure. siRNAs failing this check were
eliminated from the data, and the final list of siRNAs was the
output of the pipeline.

![Fig 5.  Final list of siRNAs for TP53 gene outputted by siUltimate](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/5-pipeline-dbtl1-final-output.webp)

Fig 5. Final list of siRNAs for TP53 gene outputted by
siUltimate

### Test

The pipeline was tested with the TP53 gene sequence, and it
worked as expected. Following this, it was tested with the bZIP
gene, which returned no results.

### Learn

The bZIP gene returned no results as siDirect did not provide
any siRNAs for this gene. This was because the default settings
of siDirect have a very high siRNA screening threshold, which
was not met by the siRNAs for the bZIP gene. Fallback settings
needed to be added for these cases, progressively lowering the
threshold if the number of siRNAs outputted by siDirect was too
small.

#### Iteration 2

+

✖

## Adding Progressive Fallback Settings

LEARN

TEST

DESIGN

BUILD

### Design

Two levels of fallback settings for siDirect were selected:

1. Unchecking the “Hide less-specific siRNAs” checkbox: This
   filter was not important for our use case, as we were not
   designing siRNAs for humans, and this option checks for
   siRNA specificity in humans.
2. Increasing the seed duplex stability max temperature to
   30°C: This setting is important for reducing the number of
   off-target effects of the siRNA. siRNAs with a higher seed
   duplex stability temperature will have more off-target
   effects.

### Build

The browser automation code was modified to use these fallback
settings for siDirect. If the output did not have enough siRNAs,
the next fallback setting would be used.

### Test

![Fig 6. First 8 formatted siDirect results for iteration 2](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/6-pipeline-dbtl2-sidirect-results.webp)

Fig 6. First 8 formatted siDirect results for iteration 2

![Fig 7. Formatted siRNApred results for iteration 2](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/7-pipeline-dbtl2-sirnapredresults.webp)

Fig 7. Formatted siRNApred results for iteration 2

![Fig 8. Pooled siRNAs for iteration 2](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/8-pipeline-dbtl2-pooledsirnas.webp)

Fig 8. Pooled siRNAs for iteration 2

![Fig 9. Final list of siRNAs outputted for the bZIP gene by siUltimate](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/9-pipeline-dbtl2-final-output.webp)

Fig 9. Final list of siRNAs outputted for the bZIP gene by
siUltimate

The software returned good results for the bZIP gene. These
matched up with the siRNAs designed by the team earlier. The
software, while functional, was written entirely in a Jupyter
Notebook. This was a convenient interface for development, but
did not provide a good user interface for a non-tech-savvy user.

### Learn

It was important for siUltimate to have a graphical user
interface so that using it would be easier for researchers.

#### Iteration 3

+

✖

## Building the User Interface

LEARN

TEST

DESIGN

BUILD

### Design

The backend of the software was created using FastAPI, a Python
library that allows for quick and easy creation of simple
backends. The frontend used Jinja templates, as they allowed for
easy display of dynamic content.

### Build

#### Backend

The backend of the software was built with 6 endpoints:

* / (root) - Served the homepage, where the user could input
  the target mRNA sequence.
* /submit - Received the form input, created a new job,
  started the pipeline, and returned the job ID.
* /status - Served the status page. Returned the current
  status of a job when given its ID.
* /output - Served the output page. Returned the output of a
  completed job when given its ID.
* /output/json - Returned the output siRNA list in JSON
  format.
* /output/csv - Returned the output siRNA list in CSV format
  for data export/download.

#### Frontend

The frontend of the software was built with 3 webpages:

* **Home**: This was where the user could enter an input
  sequence to submit a job. On submission, it would display
  the job ID. In addition, buttons to check the status of the
  submitted job and to submit another job would replace the
  submit button.
* **Status**: It would show the current status of a job.
* **Output**: It would show the output list of siRNAs,
  sorted in order of score.

### Test

![Fig 10. The home page of the user interface](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/10-pipeline-dbtl3-homepage.webp)

Fig 10. The home page of the user interface

![Fig 11. The home page after submitting a job](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/11-pipeline-dbtl3-submitted.webp)

Fig 11. The home page after submitting a job

![Fig 12. The status page showing the current status of the job](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/12-pipeline-dbtl3-status.webp)

Fig 12. The status page showing the current status of the
job

![Fig 13. The status page on completion of the job](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/13-pipeline-dbtl3-completed.webp)

Fig 13. The status page on completion of the job

![Fig 14. The output page](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/14-pipeline-dbtl3-results.webp)

Fig 14. The output page

The software worked as expected, returning the same results for
the bZIP gene as in the previous iteration, with an improved
user experience.

### Learn

The software worked well with a good user experience. It,
however, lacked any API endpoints for other software to directly
integrate it into their workflows, and it was designed to be
easy to build and run locally. It could not be deployed as an
online website in its current state.

### S.E.N.S.E.

#### Iteration 1

+

✖

## S.E.N.S.E. Iteration 1

LEARN

TEST

DESIGN

BUILD

### Design

Our initial designs of the stability model were based on data
collected through docking and molecular simulations that
involved “Docking with Attracting Cavities” in Swiss Dock.

The initial design involved implementing and comparing three
machine learning algorithms- Lasso regression, Random Forest,
and XGBoost to predict SP-dG values from molecular docking.

### Build

Hyperparameter tuning was employed to enhance the performance of
Random Forest and XGBoost using cross-validation.

### Test

The testing phase revealed that despite tuning efforts, the
Lasso model was significantly better than both tree-based
methods, achieving an exceptional cross-validated RMSE of 0.0025
(+-0.0029) and R-squared of 0.9998 (+-0.0006). The Random Forest
and XGBoost models exhibited poor generalization with negative
R-squared values, indicating that they performed worse than a
simple mean predictor. The Lasso equation identified 'Nonpolar'
(coefficient: 0.7453) as the dominant feature, followed by
'Inter' (0.1773) and 'Polar15' (0.0910).

### Learn

From the SHAP analysis of the initial Lasso model, we got an
outlier at index 62 with unusually high 'Nonpolar' values, which
was identified as potentially skewing the model's performance.

![Fig 15. SHAP values to indicate the high parameter value for iteration 1](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/15-shap-values-iteration-1.webp)

Fig 15. SHAP values to indicate the high parameter value for
iteration 1

#### Iteration 2

+

✖

## S.E.N.S.E. Iteration 2

LEARN

TEST

DESIGN

BUILD

### Design

The same data as before was also fed into this second iteration.

The redesign phase focused on improving data quality by removing
this problematic data point.

### Build

Built the refined Lasso model on the cleaned dataset.

### Test

Testing showed remarkable improvement with near-perfect
performance, achieving an RMSE of 0.0013 (+-0.0004) and
R-squared of 1.0000 (+-0.0000). The updated model equation
showed more balanced feature contributions: SP-dG\_scaled =
(0.4640 × Nonpolar) + (0.4075 × Inter) + (0.1521 × Polar15),
with 'Nonpolar' remaining the most influential feature but with
reduced dominance compared to the first iteration.

### Learn

Suggests better model stability and generalizability after the
outlier removal process.The Swissdock data was found to be less
than ideal.

![Fig 16. SHAP values to indicate the high parameter value for iteration 2](https://static.igem.wiki/teams/5645/wiki-images/pages/engineering/16-shap-values-iteration-2.webp)

Fig 16. SHAP values to indicate the high parameter value for
iteration 2

#### Iteration 3

+

✖

## S.E.N.S.E. Iteration 3

LEARN

TEST

DESIGN

BUILD

### Design

The data collected came from docking results generated using
Glide in Maestro and HDOCK. For this iteration, our design aimed
to predict docking values for an unknown sequence. To do this,
we merged docking data with siRNA sequences and used Lasso
regression to predict binding stability.

### Build

We hit roadblocks: identifier columns had mismatched data types,
preventing merges, the code had indentation errors, and the
target variable kept disappearing during data transformations.

### Test

Testing showed that the 'Ligand RMSD' column was being dropped
during merge operations and when scaling features.

### Learn

We learned to explicitly track column preservation, format
identifiers consistently between datasets, and restructure the
preprocessing to handle the target variable separately from
features. After corrections over hours of debugging, the final
iteration successfully integrated everything- proper data
merging, sequence encoding, feature combination, while keeping
the target intact, and a functional Lasso model with prediction
capabilities for screening new sequences. The model trained on
H-Dock data was found to have better accuracy than the model
trained on Glide data. Hence, we decided to use H-Dock for data
collection and validation of the model.

#### Iteration 4

+

✖

## S.E.N.S.E. Iteration 4

LEARN

TEST

DESIGN

BUILD

### Design

The goal was to predict docking scores for siRNA-nanoparticle
complexes using only sequence information and nanoparticle type.
Two datasets (Lipid: 230 sequences, Chitosan: 220 sequences)
were combined. The approach involved encoding sequences into 217
numerical features: 215 from positional one-hot encoding of
43-nucleotide padded sequences, 18 from composition metrics
(GC/AU content, dinucleotide frequencies, entropy), and 2 for
nanoparticle type.

### Build

The datasets were merged with standardized column names, and
nanoparticle type labels were added. Feature engineering created
the full 217-feature set, and data was split 80/20 for training
and testing with standard scaling applied. All three models were
trained: Lasso with cross-validated regularization, Random
Forest with 200 trees, and XGBoost with gradient boosting.

### Test

XGBoost achieved the best performance, followed by Random Forest
with Lasso slightly behind. Lasso's feature selection reduced
217 features to 4 critical ones: nanoparticle type (coefficient
+68.42), AU content (+37.09), GC content (+30.94), and CG
dinucleotide frequency (+1.52). However, with new sequences, all
models returned nearly identical values for different sequences.
This indicated that they weren't capturing enough
sequence-specific variance to distinguish between inputs.

### Learn

Non-linear models significantly outperformed. The feature
selection validated biological intuition - nanoparticle
chemistry dominates binding behavior, followed by composition
metrics that affect RNA structure and flexibility.

The minimal contribution from positional encoding suggests
overall composition matters more than nucleotide order. The
prediction uniformity problem revealed that having many features
doesn't guarantee good discrimination if they don't capture
meaningful differences between sequences.

## Wet Lab

### Chitosan Nanoparticle (CSNP) Production

#### Iteration 1

+

✖

## Standardizing the Production Protocol

LEARN

TEST

DESIGN

BUILD

### Design

To establish the ionic gelation protocol in our lab, such that
it produces the ideal nanoparticle carriers. We first optimized
the experimental setup, concentrations, ratios, and volumes to
be used by running many iterations and varying individual
parameters.

### Build

Over each iteration of nanoparticle production (through ionic
gelation), we were able to test the following:

1. Chitosan solubility was tested in vials containing acetic
   acid and water.
2. The flow rate of TPP (on a burette) to be added to the
   stirring chitosan was estimated.
3. Two chitosan solutions of 0.4% and 0.1% concentrations were
   crosslinked with a 2.4 mg/mL TPP solution in a 3:1 ratio.
4. Nanoparticles with two chitosan: TPP ratios were made to
   verify whether the 3:1 ratio obtained from the literature is
   ideal.
5. Chitosan solutions were also prepared in 0.8% and 1.22%
   acetic acid solutions to gauge the effect on the
   nanoparticles.

### Test

Every nanoparticle sample was subjected to a Particle Size
Analyzer (Horiba Scientific Nano Partica SZ-100).

The parameters we focused on were:

1. Hydrodynamic radius: Effectively, the size of the
   nanoparticles. Ideally, it would be in the range of 100-200
   nm.
2. Zeta potential: A sufficient value is essential to allow the
   nanoparticle to penetrate the wall of P. capsici. Its value
   must be over 30 mV.
3. Polydispersity index: Refers to the distribution of weights
   in a sample. This value should not exceed 0.5.

### Learn

These initial runs allowed us to fine-tune the protocol and
finalize fundamental production parameters. The key learnings
were:

1. Chitosan solution must be prepared in 1% acetic acid, as
   complete solubility is observed on stirring.
2. The TPP crosslinker must be added to chitosan at an interval
   of 7 seconds between consecutive drops, at a 1:3 ratio.
3. The concentration of chitosan solution must be 0.1% (1
   mg/mL), as higher concentrations cause aggregation (evident
   from higher nanoparticle sizes).
4. Most samples showed fine suspensions or sizes as high as
   1808 nm. This indicated that sonication might be necessary
   for homogenization.

#### Iteration 2

+

✖

## Optimizing Nanoparticle Size

LEARN

TEST

DESIGN

BUILD

### Design

We realized that sonication of the sample is necessary to bring
down the size of the particles and prevent clumping. The goal
here was to focus on the nanoparticle size and obtain ideal
values for the same.

### Build

Every sample was then sonicated after the addition of TPP at
40-60% amplitude and a 10-second ON/OFF pulse for 8 minutes. All
other parameters previously optimized were kept constant. A
second sonication of 5 minutes for the initial 0.1% chitosan
solution was introduced to reduce the size of the particles.

### Test

On running the samples using a Particle Size Analyzer, we
observed a significant reduction in size. Most samples were now
showing sizes in the range of 100-200 nm (such as 148 nm, 113
nm, 146 nm).

### Learn

Although the hydrodynamic radius has been optimized, the zeta
potential remains lower than required. On consulting our PI, we
established that the pH of the solution should be measured at
every step to understand the stability. She recommended that the
pH of the initial chitosan solution also be adjusted to 4.0
using 1N NaOH. An additional centrifugation and decanting step
was also added post the nanoparticle sonication, to obtain the
ideal zeta potential.

#### Iteration 3

+

✖

## Addressing the Zeta Potential

LEARN

TEST

DESIGN

BUILD

### Design

The goal was now to increase the zeta potential of the particles
while retaining their size and low polydispersity. Measuring the
pH of all solutions and adjusting when necessary was essential.
The centrifugation of the nanoparticle sample was also
introduced to optimize the zeta values.

### Build

Adding on to the protocol optimized thus far, the pH of
chitosan, TPP, and nanoparticle solutions was measured. As the
ideal pH was 4.0, adjustments were made using 1N NaOH to bring
it to this value. The sonicated nanoparticle solutions were also
centrifuged for 5 minutes at 1000 rpm at 27°C. The supernatant
was collected thereafter and served as the final nanoparticle
sample to be analyzed.

### Test

On carrying out iterations with the new modifications, we
observed that the zeta potential of the samples was
significantly higher than earlier. Some of the samples showed
values close to 100 mV, with the average zeta being well over
the required range.

### Learn

Through these runs, we were able to produce nanoparticles with
sizes in the range of 100-200 nm, zeta potential of over 30 mV,
having a polydispersity of under 0.5. Although there were
certain outlier samples, the majority produced using this
optimized process showed a favourable combination for the
intended purpose.

#### Iteration 4

+

✖

## Preparation for siRNA Encapsulation

LEARN

TEST

DESIGN

BUILD

### Design

To prepare for the integration of siRNA into this process, we
conducted nanoparticle runs while using diethyl pyrocarbonate
(DEPC) and RNase-free water throughout the process. The
objective was to observe any potential effect these
modifications would have on the nanoparticles.

### Build

All glassware and apparatus used in the process were first
treated with DEPC water. The acetic acid, chitosan, and TPP
solutions were also prepared using RNase-free water. This
ensured that the entire setup was RNase-free and, therefore,
safe to encapsulate the siRNA inside the nanoparticle. The
protocol followed was the same, otherwise.

### Test

These nanoparticle samples maintained a satisfactory combination
of size (82 nm & 130 nm), zeta potential (89 mV and 169 mV), and
polydispersity (between 0.4-0.5).

### Learn

These final runs allowed us to prepare for the encapsulation of
siRNA while producing the nanoparticles. The result of all the
optimizations made thus far allowed us to safely and effectively
integrate siRNA into the process.

### Nanoformulation Production

#### Iteration 1

+

✖

## Nanoformulation Production Iteration 1

LEARN

TEST

DESIGN

BUILD

### Design

Having successfully produced nanoparticles with ideal
characteristics for siRNA delivery and reproduced these results
while maintaining RNase-free conditions, we sought to
incorporate the siRNA to build our working nanoformulation.

### Build

All glassware and apparatus were soaked in DEPC-treated water
for 3 hours and then autoclaved to mitigate any RNase
contamination. The pH of the chitosan solution was adjusted to
4.0, as done previously. 15 μL of 100 μM concentration siRNA was
combined with the tripolyphosphate crosslinker, which was then
added to the stirring chitosan solution.

### Test

The following results were obtained from the sample:

Hydrodynamic radius (Z-avg): 6410.3 nm
Zeta potential: -4.0 mV
Polydispersity index: 2.318

### Learn

Given that these results are a stark contrast to the other
iterations run so far, we learnt that extended treatment with
DEPC combined with the pH adjustment of the chitosan solution
could have shot up the final pH, which would affect the
stability, zeta potential, and size of the nanoparticles.
Therefore, we were recommended to simply rinse the apparatus
with RNase-free water and follow the same protocol without the
pH adjustment.

#### Iteration 2

+

✖

## Nanoformulation Production 2

LEARN

TEST

DESIGN

BUILD

### Design

The second run implemented the changes recommended to us by our
PI, Dr. Ritu Raval, to observe if these modifications were able
to encapsulate the siRNA and help produce a nanoformulation with
suitable characteristics.

### Build

The apparatus was simply rinsed with RNase-free water and dried,
as recommended. All solutions were also made with RNase-free
water, as previously done. The pH of the chitosan solution was
also not adjusted. Lastly, the nanoformulation sample was
sonicated for a longer period of 10 minutes.

### Test

The following were the Particle Size Analyzer results obtained
for the sample:

Hydrodynamic radius (Z-avg): 401.7 nm
Zeta potential: 35.8 mV
Polydispersity index: 0.509

The pH of the final nanoformulation was observed to be 3.24.

### Learn

The changes made in this run significantly improved every
parameter of the nanoparticles. Considering that the siRNA had
been encapsulated, an increase in particle size was a natural
and expected outcome. Moreover, the zeta potential and
polydispersity were extremely favourable for an encapsulated
sample. Therefore, we successfully produced our nanoformulation
with ideal characteristics. This sample could now be used in
other experiments to verify its effectiveness.

### Detached Leaf Assay

#### Iteration 1

+

✖

## Detached Leaf Assay Iteration 1

LEARN

TEST

DESIGN

BUILD

### Design

We aimed to understand the efficacy of our chitosan
nanoparticles by testing them directly against
*Phytophthora capsici*'s infection on
*Piper nigrum* leaves of the Panniyur-1 variety. We set up
negative and positive controls (untreated leaves and leaves
sprayed with water and acetic acid) to compare the lesions of
each. iHP sessions with Dr. Biju Narayanan helped design this
experiment.

### Build

*Piper nigrum* leaves of the same maturity were first
sprayed with water, 1% acetic acid, and two different
nanoparticle solutions (one sample had a better size and zeta
potential). Following this, 5 mm mycelial plugs were placed on
the leaves such that the hyphae came in contact with the leaf
tissue. Lesion growth on the leaf was measured over 3 days
post-infection.

### Test

Lesions on the positive controls grew substantially over the 3
days and became very large by the end. The size of the lesion
was larger in the case of water than acetic acid. However, the
leaves sprayed with the chitosan nanoparticles displayed the
least lesion growth. Moreover, the lesion on the nanoparticle
sample with the better size and zeta potential was comparatively
less.

### Learn

As expected, the chitosan nanoparticles significantly reduced
lesion growth on the *P. nigrum* leaves. This can be
attributed to their antifungal properties, which would produce
an even greater overall effect when combined with the siRNA.
Furthermore, nanoparticles with zeta potential and size values
closer to the ideal have a greater effect against
*P. capsici* infection, thereby validating our process.

#### Iteration 2

+

✖

## Detached Leaf Assay Iteration 2

LEARN

TEST

DESIGN

BUILD

### Design

The same experiment was repeated using various concentrations of
naked siRNA, in a prophylactic and therapeutic manner. The
effect of the nanoformulation was also tested on the leaves.

### Build

Leaves were sprayed with 10 µL of 1 µM and 100 nM concentrations
of siRNA and infected three days later with a mycelial plug
(prophylactic treatment). A set of leaves was also treated
simultaneously with infection to simulate therapeutic
application. Two nanoformulations were also tested to assess
their effectiveness.

### Test

The leaves sprayed with siRNA in a therapeutic manner developed
a lesion over 72 hours, although much smaller than the positive
control. However, the leaves sprayed with 1 µM siRNA
prophylactically did not develop lesions at all throughout the
period. Lesion development on the optimized nanoformulation
(zeta potential of 35.8 mV and 401.7 nm size) was absent as
well.

### Learn

We observed that prophylactic treatment of the leaves curbed
infection to a much greater degree. This validates the
preventative implementation we have designed. Although
application of any concentration of siRNA/nanoformulation
reduced infection compared to the positive controls, the effect
was more pronounced in the case of prophylactic 1 µM siRNA and
the optimized nanoformulation. Therefore, an effective
concentration of siRNA that curbs infection has been estimated.
Moreover, the nanoformulation showed near complete silencing of
infection, which indicates that the complex is stable, and the
siRNA is being released successfully to carry out gene
silencing.

A side-by-side comparison of these treatments can be found on
our
[measurement page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27measurement%27%29%20%7D%7D).

### *P. capsici* Zoospore Testing

#### Iteration 1

+

✖

## *P. capsici* Zoospore Testing Iteration 1

LEARN

TEST

DESIGN

BUILD

### Design

As the siRNA is designed to silence the bZIP gene (which
regulates zoospore motility) in *P. capsici* zoospores,
isolating and treating pathogenic zoospores with the siRNA and
observing its effect is another way of validating the gene
silencing activity of the siRNA. Since the siRNA is encapsulated
in chitosan nanoparticles, we first treated zoospores with the
nanoparticles to observe the effect.

### Build

Mycelial plugs from 72-hour-old *P. capsici* subcultures
were placed in autoclaved distilled water and incubated under
illumination at 25°C for 48 hours. The solution was then cold
shocked at 4°C for 30 minutes to facilitate the release of
motile zoospores from the sporangium. The zoospore solution was
then aliquoted into 500 μL in each tube, and 250 μL of chitosan
nanoparticle solution and 1% acetic acid were added to each
tube. The samples were lightly stained with methylene blue dye
and visualized at 40x and 100x using a Trinocular Upright Phase
Contrast microscope.

### Test

On two occasions, motile zoospores were successfully observed.
Using a Neubauer chamber the number of zoopores per mL of water
was 7.85 x 107. The zoospores were visualized and
treated with acetic acid (our chitosan nanoparticle solution was
suspended in the same), and our chitosan nanoparticle solution
during the next iteration. The acetic acid did not show any
change in zoospore motility, whereas those treated with the
chitosan nanoparticle showed a change in overall motility.

### Learn

Having understood that the chitosan nanoparticles affect the
zoospores, to precisely gauge the effect of the treated
zoospores, in our next iteration, we used a Neubauer chamber to
calculate the time it took for a zoospore to move from one part
of the grid to another.

#### Iteration 2

+

✖

## *P. capsici* Zoospore Testing Iteration 2

LEARN

TEST

DESIGN

BUILD

### Design

Zoospores were treated with varying concentrations of the siRNA
as well as the nanoformulation to observe their effect on
zoospore motility.

### Build

Zoospores were isolated from 72-hour-old cultures as done
earlier. 6 µL of zoospore samples were treated with 4 µL of 7.23
µM and 100 µM siRNA, as well as the nanoformulation. The samples
were incubated for 30 minutes and visualized at 100x over
multiple fields of view using a Trinocular upright phase
contrast microscope.

### Test

Untreated zoospore samples retained their motility. The same was
observed with acetic acid and nanoparticle treatments.

However, zoospores treated with the siRNA and nanoformulation
showed no observable motility over 20 seconds.

### Learn

Visible motility in the untreated, acetic acid, and
nanoparticle-treated zoospores indicated that infection had not
entirely been curbed. The complete reduction in motility on
siRNA and nanoformulation treatment conclusively verifies gene
silencing in the pathogenic zoospores. The result also validates
the encapsulation and release mechanisms of the
siRNA-nanoparticle complex.

{% endblock %}

{% extends "minimal-layout.html" %}
{% block title %}Experiments{% endblock %}
{% set next\_page\_url = url\_for('pages', page='notebook') %}
{% block next\_page\_name %}Notebook{% endblock %}
{% block custom\_styling %}
{% endblock %}
{% block custom\_javascript %}
{% endblock %} {% block page\_content %}

The experiments page contains all the final protocols used by the team for wet lab
experimentation. From preparation of carrot agar to encapsulation of siRNA, the protocols
give a step by step understanding of how wet lab conducted the experiments. Each protocol
includes the aim of the experiment, the materials required, the procedure for conducting the
experiment, along with any relevant references for the same. Each protocol has been
optimized and curated through literature reviews, iHP sessions and careful troubleshooting.

![Fig 1. Overview of Wet Lab Experiments](https://static.igem.wiki/teams/5645/wiki-images/pages/experiments/1-overview-of-wet-lab-experiments.webp)

Fig 1. Overview of wet lab experiments

## ![icon](https://static.igem.wiki/teams/5645/wiki-images/ihp-and-notebook-icons/water-in-capsici.webp) Carrot Agar Preparation

+

✖

## Carrot Agar Preparation

### Aim

To prepare carrot agar media for the growth of fungal cultures.

### Materials Required

1. Fresh carrots
2. Blender
3. Beaker
4. Muslin cloth
5. Agar powder
6. Vegetable peeler
7. Sterile water
8. Autoclave

### Procedure

1. Take 200 g of carrots, wash and peel them.
2. Dice/chop the carrots and blend them.
3. Add 750 mL of sterile water to a beaker and pour the puree from the blender
   into the beaker.
4. Let the puree boil till the solution becomes clear. Discard any scum that
   floats to the top.
5. Sieve the cooled solution through a muslin cloth and add sterile water until
   the final volume is 1L.
6. Add 1.8% w/v (18 g) agar powder to the solution.
7. Autoclave the solution and pour it into petri plates.

## ![icon](https://static.igem.wiki/teams/5645/wiki-images/ihp-and-notebook-icons/petridish.webp) *Phytophthora capsici* Subculturing

+

✖

## *Phytophthora capsici* Subculturing

### Aim

Subculturing of *Phytophthora capsici* on carrot media petri plates.

### Materials Required

1. *Phytophthora capsici* culture
2. Carrot agar petri plates
3. Laminar Hood
4. Inoculation loop
5. Forceps
6. Parafilm
7. 8 mm Cork borer
8. 70% Ethanol

### Procedure

Protocol adopted from
[Mohsan et al. (2017b)](https://doi.org/10.7324/jabb.2017.50407).

1. Prepare fresh carrot agar petri plates using carrot extract, agar, and
   sterile distilled water. Autoclave and pour it onto petri dishes; allow to
   solidify.
2. In a sterile laminar airflow chamber, sterilize the cork borer (dip the
   cutting edge in 70% ethanol, dry it properly, pass it through the flame, and
   let it cool). Take the fresh
   *Phytophthora capsici* culture (we sourced our culture from ICAR-Indian
   Institute of Spices Research, Kozhikode). Hold the cork borer vertically
   over the growing end of the fungal colony & press it firmly into the agar.
   Twist slightly to cut a circular plug. Sterilize the cork borer in between
   uses using the flame.
3. Transfer the plugs onto a fresh carrot agar plate (use forceps and an
   inoculation loop to aid the transfer); place the plugs with the mycelium
   side facing down. Parafilm the plate.
4. Incubate plates in the dark at 28°C.
5. After 4 to 5 days, mycelia would have colonized the whole plate, appearing
   as a mat, off-white in color.

### References

Mohsan, M., Ali, S., Shahbaz, M. U., Saeed, S., Burhan, M., & Shahbaz, M.
(2017). In vitro efficacy of different growth media and crude plant extracts
against mycelial growth of *Phytophthora capsici*.
*Journal of Applied Biology & Biotechnology*.
<https://doi.org/10.7324/jabb.2017.50407>

## ![icon ](https://static.igem.wiki/teams/5645/wiki-images/ihp-and-notebook-icons/growth-curve.webp) Colony Diameter Measurement *P. capsici*

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## Colony Diameter Measurement *P. capsici*

### Aim

To measure and visualize the growth of *Phytophthora capsici* by measuring
colony diameter over time.

### Materials Required

1. *P. capsici* culture
2. Carrot agar petri plates
3. Cork borer (8 mm diameter)
4. Forceps
5. Inoculation loop
6. 70% ethanol
7. Parafilm
8. Ruler/Vernier caliper

### Procedure

Protocol adopted from ([Lu et al., 2011c](https://doi.org/10.1094/pdis-05-11-0372);
[Santos et al., 2023d](https://doi.org/10.3390/jof9030360);
[Z. Wang et al., 2009c)](https://doi.org/10.1128/aem.00620-09).

1. Prepare fresh carrot agar petri plates using carrot extract, agar, and
   sterile distilled water. Autoclave and pour onto petri dishes, allowing them
   to solidify.
2. In a sterile laminar airflow chamber, sterilize the cork borer (dip the
   cutting edge in 70% ethanol, let it dry completely; then pass it through
   flame and let it cool). Take the fresh fungal culture. Hold the cork borer
   vertically over the growing end of the fungal colony & press it firmly into
   the agar. Twist slightly to cut a circular plug. Sterilize the cork borer in
   between uses using the flame.
3. Transfer a single plug onto a fresh carrot agar plate (use forceps and an
   inoculation loop to aid its transfer); place the plug with the mycelium side
   facing down in the center. Parafilm the plate. Prepare replicates to
   validate.
4. Incubate plates in the dark at 28°C.
5. Remove plates from incubation at set intervals (every 24/48 hrs). Measure
   the diameter of the colony in two perpendicular directions using the
   ruler/vernier caliper. Take the average and subtract the initial 8 mm
   diameter to get the reading. Place the plates back into incubation.
6. Plot a curve with the radial growth in mm (Y-axis) and time (X-axis).

### References

Lu, X. H., Hausbeck, M. K., Liu, X. L., & Hao, J. J. (2011b). Wild Type
Sensitivity and Mutation Analysis for Resistance Risk to Fluopicolide in
*Phytophthora capsici*. *Plant Disease, 95*(12), 1535–1541.
<https://doi.org/10.1094/pdis-05-11-0372>

Santos, M., Diánez, F., Sánchez-Montesinos, B., Huertas, V., Moreno-Gavira, A.,
García, B. E., Garrido-Cárdenas, J. A., & Gea, F. J. (2023c). Biocontrol of
Diseases Caused by
*Phytophthora capsici* and *P. parasitica* in Pepper Plants.
*Journal of Fungi, 9*(3), 360.
<https://doi.org/10.3390/jof9030360>

Wang, Z., Langston, D. B., Csinos, A. S., Gitaitis, R. D., Walcott, R. R., & Ji,
P. (2009b). Development of an Improved Isolation Approach and Simple Sequence
Repeat Markers To Characterize
*Phytophthora capsici* Populations in Irrigation Ponds in Southern Georgia.
*Applied and Environmental Microbiology, 75*(17), 5467–5473.
<https://doi.org/10.1128/aem.00620-09>

## ![icon](https://static.igem.wiki/teams/5645/wiki-images/ihp-and-notebook-icons/dropper.webp) Lactophenol Blue Staining

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## Lactophenol Blue Staining

### Aim

To observe the morphology of *Phytophthora capsici*.

### Materials Required

1. *P. capsici* culture
2. Lactophenol Cotton Blue stain
3. Forceps
4. Glass slides
5. Cover slips
6. Microscope

### Procedure

1. Place a drop of lactophenol cotton blue on a clean slide.
2. Using a sterile inoculation loop, suspend some
   *P. capsici* mycelia in the lactophenol cotton blue on the slide.
3. Cover the slide with a cover slip without any air bubbles.
4. Observe the slide at 10x and 40x with a microscope (we used a Cilika Tab Pro
   microscope).

## ![icon](https://static.igem.wiki/teams/5645/wiki-images/ihp-and-notebook-icons/leaf.webp) Detached Leaf Assay

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## Detached Leaf Assay

### Aim

To observe the infection of *Phytophthora capsici* on
*Piper nigrum* leaves and test our nanoformulation on the same with
established controls.

### Materials Required

1. Plastic boxes
2. Cotton
3. Autoclaved distilled H2O
4. Inoculation loop
5. Nichrome wires
6. Toothpicks
7. Spray bottles
8. 1% acetic acid
9. Chitosan Nanoparticles
10. Nanoformulation (siRNA encapsulated in the chitosan nanoparticles)
11. Leaves of *Piper nigrum* (Black Pepper)
12. *Phytophthora capsici* subculture, 72 hrs old
13. Forceps
14. 5 mm cork borers
15. Gloves
16. Mask
17. Lab coat
18. Tissues
19. 70% ethanol
20. Parafilm

### Procedure

The protocol was adopted from
[Paul et al. (2019b)](https://doi.org/10.1016/j.pmpp.2019.101436), and a few modifications were made.

1. Pluck leaves of *Piper nigrum* (we took the Panniyur 1 variety or any
   other strongly susceptible species) that are of the same maturity, colour,
   and size. Ideally, the leaf should be the third from the growing tip. Ensure
   they are clear of infection, yellowing, and spotting on both sides.
2. Clean the leaves thoroughly under water and gently dry using tissue papers.
   Wipe with 70% ethanol as well.
3. Airtight plastic boxes are sterilized using 70% ethanol and tissues to
   prevent contamination. They are then lined with a layer of cotton on the
   base, keeping the container walls clear for easier visualization.
4. Autoclaved distilled water is poured over the cotton lining until thoroughly
   soaked. The excess water is drained by tilting the container.
5. Label the boxes as per the experiments with the necessary controls.
6. Place the plastic containers, an inoculation loop, nichrome wires, a few
   toothpicks, spray bottles filled with the treatment solution, forceps, and
   5mm cork borers within the Laminar Air Flow hood, post cleaning with 70%
   ethanol under ultraviolet radiation for 20 minutes. Ensure GLP is followed;
   all personnel should wear lab shoes, lab coats, masks, and gloves.
7. After the UV treatment, gently arrange the cleaned leaves in the boxes and
   press them flat.
8. Using toothpicks, prick marks are to be made uniformly across all leaves at
   the centre. A 4x4 pattern was followed in all of them.
9. For the control set of leaves, autoclaved distilled water is to be sprayed
   and allowed to dry completely before inoculation.
10. For the 1% acetic acid control (because the nanoparticles and the
    nanoformulation are dissolved in 1% acetic acid), the leaves are sprayed
    with 1% acetic acid, and the leaf is allowed to dry.
11. The same applies to the nanoparticle control and the nanoformulation test.
    Spray equal volumes onto both the leaves and allow them to dry before
    introducing the oomycete.
12. A 72 hrs old *Phytophthora capsici* subculture is to be taken, and 5 mm
    cork borers (Heat flame before use and let it cool completely) are used to
    punch out mycelial plugs from the periphery of the subculture in the fungal
    mat.
13. Carefully, using the inoculation loop (preferably with a new nichrome wire
    to avoid cross-contamination), and the forceps (heat flame before use and
    let it cool thoroughly), pick out a mycelial disk and place one at the
    centre of each leaf where the pricks were made, such that the mycelial side
    touches the leaf.
14. Parafilm the subculture after taking out the disks and place them back in
    the dark incubator at 25°C.
15. Heat the inoculation loop thoroughly before and after use and ensure it is
    cooled down properly to prevent causing any damage to the mycelia. Dispose
    of the nichrome wire after use.
16. Sterilize the LAF with 70% ethanol post-use.
17. Seal the boxes tightly and place them in a spot with moderate sunlight at
    room temperature.
18. Lesion readings are to be taken daily, wherein the perpendicular (Y-axis)
    and the parallel (X-axis) lesion lengths are measured and noted. Take the
    average of the two values and subtract 5 mm (the diameter of the mycelial
    plug). Keep a track of the readings until the leaves are completely
    blackened.
19. Ensure to wear gloves and a mask whilst taking every reading. Sterilize
    hands with 70% ethanol before and after measuring the lesion growth size.
    Minimize the duration for which the boxes are kept open.

### References

Paul, B. B., Mathew, D., Beena, S., & Shylaja. (2019). Comparative transcriptome
analysis reveals the signal proteins and defence genes conferring foot rot (*Phytophthora capsici*
sp. nov.) resistance in black pepper (*Piper nigrum* L.).
*Physiological and Molecular Plant Pathology, 108*, 101436.
<https://doi.org/10.1016/j.pmpp.2019.101436>

## ![icon](https://static.igem.wiki/teams/5645/wiki-images/ihp-and-notebook-icons/cytotoxicity-assay.webp) Cytotoxicity Assay

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## Cytotoxicity Assay

### Aim

To determine if varying concentrations of bZIP1 siRNA used for testing show
cytotoxic effects on black pepper (*Piper nigrum*) leaves.

### Materials Required

1. Plastic boxes
2. Cotton
3. Autoclaved distilled H₂O
4. Varying concentrations of siRNA in nuclease-free water.
5. Leaves of *Piper nigrum* (Black Pepper)
6. Gloves
7. Mask
8. Lab coat
9. Tissues
10. 70% ethanol
11. RNase free 10 μL micropipette tips

### Procedure

1. Pluck leaves of *Piper nigrum* (we took the Panniyur 1 variety) are of
   the same maturity, colour, and size. Ideally, the leaf should be the third
   from the growing tip. Ensure they are clear of infection, yellowing, and
   spotting on both sides.
2. Clean the leaves thoroughly under water and gently dry using tissue paper.
   Wipe with 70% ethanol as well.
3. Airtight plastic boxes are sterilized using 70% ethanol and tissues to
   prevent contamination. They are then lined with a layer of cotton on the
   base, keeping the container walls clear for easier visualization.
4. Autoclaved distilled water is poured over the cotton lining until thoroughly
   soaked. The excess water is drained by tilting the container. Place the
   containers under ultraviolet light for 20 minutes.
5. After the UV treatment, gently arrange the cleaned leaves in the boxes and
   press them flat.
6. Using toothpicks, pricks are to be made uniformly across all leaves at the
   centre. A 4x4 pattern was followed in all of them.
7. Label the boxes as per the siRNA concentrations and necessary controls.
8. In an RNase-free environment, prepare siRNA of varying concentrations (We
   sourced our siRNA from Juniper Life Sciences ) in RNase-free Eppendorf tubes
   (Eg: 100 μM, 1 μM, 100 nM, 50 nM, etc).
9. In a laminar airflow chamber, carefully pipette 10 μL of the siRNA solution
   on the pricked area of each leaf. Ideally, duplicates of each leaf with a
   particular siRNA concentration should be made.
10. Seal the boxes tightly and place them in a spot with moderate sunlight at
    room temperature. Observe every 24 hrs.
11. Sterilize the LAF with 70% ethanol post-use.

## ![icon](https://static.igem.wiki/teams/5645/wiki-images/ihp-and-notebook-icons/zoospore.webp) *P. capsici* Sporulation

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## *P. capsici* Sporulation

### Aim

To produce and observe zoospores of *Phytophthora capsici* and test our
Nanoformulation on the same with established controls.

### Materials Required

1. Hemocytometer
2. Sterilized petri plates
3. Autoclaved distilled H₂O
4. Inoculation loop
5. Nichrome wires
6. Cooling incubator with illumination
7. Glass slides
8. 1% acetic acid
9. Chitosan Nanoparticles
10. Nanoformulation (siRNA encapsulated in the chitosan nanoparticles)
11. Glass coverslip
12. *Phytophthora capsici* subculture, 72 hrs old
13. Forceps
14. 5 mm cork borers
15. Gloves
16. Mask
17. Lab coat
18. Tissues
19. 70% ethanol
20. Parafilm
21. Micropipettes
22. Micropipette tips
23. 1.5 mL Eppendorf tubes
24. Methylene blue dye

### Procedure

1. Place an inoculation loop, nichrome wires, 5 mm cork borers, autoclaved
   distilled water, and petri plates within the Laminar Air Flow hood, post
   cleaning with 70% ethanol under ultraviolet radiation for 20 minutes. Ensure
   GLP is followed; all personnel should wear lab shoes, lab coats, masks, and
   gloves.
2. 72 hrs old *Phytophthora capsici* subcultures cultured on carrot agar
   medium are to be taken, and 5 mm cork borers (Heat flame before use and let
   it cool completely) are used to punch out several mycelial plugs from the
   periphery of the subculture in the fungal mat.
3. The zoospore suspension was prepared by carefully using the inoculation loop
   and the forceps to pick out the mycelial discs and placing 10 of them with
   the mycelial side facing upwards (the discs must be slightly immersed in
   water) in each sterile petri plate containing autoclaved sterile distilled
   water (half-filled with water). Double-parafilm the plates thereafter.
4. The plates were incubated at 25°C under continuous illumination for 48 hours
   to induce sporangial production.
5. Parafilm the subculture plates after removing the disks and place them back
   in the dark incubator at 25°C.
6. After 48 hrs, the suspension in the petri plates was cold shocked at 4°C for
   30 minutes to facilitate the release of zoospores.
7. Pipette out the zoospore solution and prepare the samples to be tested.
   * Tubes containing the zoospore solution- Negative control (1 mL
     pipetted out from each plate)
   * Tubes containing zoospore solution + 1% acetic acid solution (500 μL
     zoospore solution from each plate + 250 μL acetic acid)
   * Tubes containing the zoospore solution + the 0.1% chitosan
     nanoparticle solution (500 μL zoospore solution from each plate, 250
     μL chitosan NP solution)
   * Tubes containing the zoospore solution + the 0.1% siRNA encapsulated
     chitosan nanoparticle solution (500 μL zoospore solution from each
     plate, 250 μL siRNA chitosan NP solution- the nanoformulation)
8. Add a few drops of Methylene blue to each sample tube. Pipette out 10 μL of
   each sample on a clean glass slide and place a clean coverslip on top.
   Visualize at 10x, 40x, and 100x (we used the Olympus CX-43 upright
   trinocular phase contrast microscope ).
9. To measure changes in zoospore motility and get the number of zoospores per
   mL of the solution, pipette the samples carefully on a hemocytometer by
   carefully placing the tip at the edge of the slide such that the liquid
   flows below the coverslip by capillary action onto the etched platform.

   To measure changes in mobility, use the hemocytometer and measure the time
   it takes for a zoospore to move from one side of a square in the grid to the
   other. To count the number of cells per mL:
   1. Count the number of cells in each square.
   2. Count the number of cells in at least 10 squares (10-20 cells per
      square).
   3. Repeat and count at least 6 times to obtain minimal variation.

![Fig 2. P. capsici sporulation flowchart](https://static.igem.wiki/teams/5645/wiki-images/pages/experiments/2-p-capsici-sporulation.webp)

Fig 2. *P. capsici* sporulation flowchart

## ![icon](https://static.igem.wiki/teams/5645/wiki-images/ihp-and-notebook-icons/nanoparticle.webp) Chitosan nanoparticle preparation - Ionic Gelation

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## Chitosan nanoparticle preparation - Ionic Gelation

### Aim

Synthesis of chitosan nanoparticles using TPP crosslinker by ionic gelation.

### Materials Required

1. Chitosan (Molecular Weight ≈ 50,000-190,000 Da)
2. Sodium Tripolyphosphate (TPP)
3. Acetic acid (1% v/v solution)
4. Sterile distilled water
5. Magnetic stirrer
6. 1 N NaOH solution
7. PVDF syringe filters (pore size 0.22 μm)
8. Centrifuge
9. Beakers/flasks
10. Pipettes/measuring cylinders

### Procedure

Protocol adopted from ([De Carvalho et al., 2019b](https://doi.org/10.1016/j.msec.2019.109885);
[Oh et al., 2019b](https://doi.org/10.3390/agronomy9010021)) and optimized for our experimentation.

1. Prepare 1% Acetic acid (1 mL in 100 mL of distilled water). Calibrate the pH
   meter and measure the pH of the solution (should be 2.6-2.8).
2. Prepare 50 mL of 1 N NaOH solution (weigh 2 g of NaOH pellets and add
   sterile distilled water up to 50 mL). Simultaneously, turn on the ice
   machine.
3. Dissolve the low molecular weight chitosan powder at a concentration of 0.1%
   (w/v) in 1% (v/v) acetic acid (0.04 g in 40 mL of 1% Acetic acid solution).
4. Rinse the magnetic bead in distilled water. Stir the solution in a magnetic
   stirrer (Eltek MS 205) at maximum stable speed for 30 minutes to ensure
   complete dissolution.
5. Dissolve sodium tripolyphosphate (TPP) at a concentration of 0.24% (w/v) in
   sterile distilled water (0.072 g in 30 mL of sterile water).
6. Adjust the pH of the chitosan solution to 4 using 1 N NaOH solution. To do
   this,
   1. Check the pH of the chitosan solution by taking 5 mL in a Falcon
      tube.
   2. Determine an estimate of the volume of 1 N NaOH to be added to the
      solution by adjusting the pH of 1% acetic acid of the same volume.
   3. Accordingly, add the appropriate volume of NaOH to the chitosan
      solution and check the final pH (using 5 mL of the remaining
      solution).
7. Sonicate the chitosan solution at 40% amplitude, 10-sec ON/OFF pulse for 5
   minutes. Wipe the probe with 70% ethanol before and after use.
8. Filter the chitosan solution through regular filter paper into a beaker to
   remove any undissolved particles.
9. Syringe filter the TPP solution using a 0.22 μm PVDF filter into a Falcon
   tube until the volume is 10 mL.
10. Rinse the burette with distilled water and residual filtered TPP solution.
    Establish the optimal flow rate of roughly 7s between consecutive drops
    using the same. Once fixed, suspend the magnetic bead in the chitosan
    solution and begin stirring.
11. Load 10 mL of the TPP solution onto the burette and add drop by drop to 30
    mL of the chitosan solution. Ensure the drops fall at the periphery of the
    vortex.
12. Once 10 mL of the TPP solution has been added, stir the nanoparticle
    solution for another 20 minutes. Transfer the solution to a 50 mL Falcon
    tube.
13. Sonicate the nanoparticle solution at 40% amplitude, 10-sec ON/OFF pulse for
    8 minutes.
14. Centrifuge (we used the Eppendorf centrifuge 5804 R) the nanoparticle
    solution at 1000 rpm for 5 minutes at room temperature. Carefully place the
    glass pipette on the side of the Falcon tube that faced inwards during
    centrifugation. Ensure the orientation of the falcon tube is maintained
    throughout the process. Decant up to 20 mL of the solution into a Falcon
    tube. This serves as the nanoparticle solution.
15. Characterize the samples using a particle size analyzer and scanning
    electron microscope (we used the Horiba Scientific nanopartica nano particle
    analyzer SZ-100 and VO MA18 with Oxford EDS(X-act) scanning electron
    microscope). Store this solution at 4°C.

### References

De Carvalho, F. G., Magalhães, T. C., Teixeira, N. M., Gondim, B. L. C., Carlo,
H. L., Santos, R. L. D., De Oliveira, A. R., & Denadai, Â. M. L. (2019).
Synthesis and characterization of TPP/chitosan nanoparticles: Colloidal
mechanism of reaction and antifungal effect on *C. albicans* biofilm
formation. *Materials Science and Engineering C*, 104, 109885.
<https://doi.org/10.1016/j.msec.2019.109885>

Oh, J.-W., Chun, S. C., & Chandrasekaran, M. (2019). Preparation and In Vitro
Characterization of Chitosan Nanoparticles and Their Broad-Spectrum Antifungal
Action Compared to Antibacterial Activities against Phytopathogens of Tomato.
*Agronomy, 9*(1), 21.
<https://doi.org/10.3390/agronomy9010021>

## ![icon](https://static.igem.wiki/teams/5645/wiki-images/ihp-and-notebook-icons/sirna-in-nanoparticle.webp) siRNA Encapsulation in Chitosan Nanoparticles

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## siRNA Encapsulation in Chitosan Nanoparticles

### Aim

Encapsulation of siRNA in chitosan nanoparticles using the ionic gelation
method.

### Materials Required

1. Chitosan (Molecular Weight ≈ 50,000-190,000 Da)
2. siRNA of desired volume and concentration
3. Sodium Tripolyphosphate (TPP)
4. Acetic acid (1% v/v solution)
5. RNase-free water
6. Magnetic stirrer
7. PVDF syringe filters (pore size 0.22 μm)
8. Centrifuge
9. Beakers/flasks
10. Pipettes/measuring cylinders

### Procedure

Protocol adopted from ([Jarudilokkul et al., 2011b](https://doi.org/10.1007/s11814-010-0485-z);
[Katas & Alpar, 2006d](https://doi.org/10.1016/j.jconrel.2006.07.021);
[Raja et al., 2015c](https://doi.org/10.1371/journal.pone.0128963)).

1. Ensure all glassware, plasticware, and reagents prepared are RNase-free.
2. Prepare 1% Acetic acid (1 mL in 99 mL of RNase-free water). Calibrate the pH
   meter and measure the pH of the solution.
3. Dissolve the low molecular weight chitosan powder at a concentration of 0.1%
   (w/v) in 1% (v/v) acetic acid (0.04 g in 40 mL of 1% Acetic acid solution).
4. Rinse the magnetic bead in RNase-free water. Stir the solution in a magnetic
   stirrer (Eltek MS 205) at maximum stable speed for 30 minutes to ensure
   complete dissolution.
5. Dissolve sodium tripolyphosphate (TPP) at a concentration of 0.24% (w/v) in
   RNase-free water (0.072 g in 30 mL of sterile water).
6. Sonicate the chitosan solution at 40% amplitude, 10-sec ON/OFF pulse for 5
   minutes. Wipe the probe with 70% ethanol before and after use.
7. Filter the chitosan solution through normal filter paper into a beaker to
   remove undissolved particles.
8. Syringe filter the TPP solution using a 0.22 μm PVDF filter into a Falcon
   tube until the volume is 10 mL.
9. Add the desired siRNA concentration resuspended in nuclease-free water to
   the TPP solution in a laminar air flow chamber. We added 15 μL of 100 μM
   siRNA to 10 mL of TPP (giving a 1:666 ratio of siRNA to TPP). Ensure
   RNase-free tips are used and all surfaces are RNase-free.
10. Rinse the burette with RNase-free water and residual filtered TPP solution.
    Establish the optimal flow rate of roughly 7s between consecutive drops
    using the same. Once optimized, suspend the magnetic bead in the chitosan
    solution and begin stirring.
11. Load 10 mL of the siRNA-TPP solution onto the burette and add drop by drop
    to 30 mL of the chitosan solution. Ensure the drops fall at the periphery of
    the vortex.
12. Once 10 mL of the siRNA-TPP solution has been added, stir the nanoparticle
    solution for another 20 minutes. Transfer the solution to a 50 mL Falcon
    tube.
13. Sonicate the encapsulated siRNA solution at 40% amplitude, 10-sec ON/OFF
    pulse for 10 minutes.
14. Centrifuge the encapsulated siRNA solution at 1000 rpm for 5 minutes at room
    temperature. Ensure the orientation of the falcon tube is maintained
    throughout the process. Carefully place the glass pipette on the side of the
    Falcon tube that faced inwards during centrifugation. Decant up to 20 mL of
    the solution into a Falcon tube. Store at 4°C.

![Fig 3. siRNA encapsulation in chitosan nanoparticles flowchart](https://static.igem.wiki/teams/5645/wiki-images/pages/experiments/3-sirna-encapsulation.webp)

Fig 3. siRNA encapsulation in chitosan nanoparticles flowchart

### References

Jarudilokkul, S., Tongthammachat, A., & Boonamnuayvittaya, V. (2011).
Preparation of chitosan nanoparticles for encapsulation and release of protein.
*Korean Journal of Chemical Engineering, 28*(5), 1247–1251.
<https://doi.org/10.1007/s11814-010-0485-z>

Katas, H., & Alpar, H. O. (2006b). Development and characterisation of chitosan
nanoparticles for siRNA delivery.
*Journal of Controlled Release, 115*(2), 216–225.
<https://doi.org/10.1016/j.jconrel.2006.07.021>

Raja, M. A. G., Katas, H., & Wen, T. J. (2015b). Stability, Intracellular
Delivery, and Release of siRNA from Chitosan Nanoparticles Using Different
Cross-Linkers. *PLoS ONE, 10*(6), e0128963.
<https://doi.org/10.1371/journal.pone.0128963>

## ![icon](https://static.igem.wiki/teams/5645/wiki-images/ihp-and-notebook-icons/eta.webp) Entrapment efficiency

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## Entrapment efficiency

### Aim

To calculate the entrapment efficiency of siRNA in chitosan nanoparticles using
a UV-vis spectrophotometer.

### Materials Required

1. Centrifuge
2. UV-vis spectrophotometer
3. RNase-free water
4. Nanocuvette
5. Encapsulated siRNA in chitosan nanoparticle (suspended in 1% acetic acid)
6. 70% ethanol

### Procedure

Protocol adopted from ([Katas & Alpar, 2006e](https://doi.org/10.1016/j.jconrel.2006.07.021);
[Raja et al., 2015d](https://doi.org/10.1371/journal.pone.0128963)).

1. Centrifuge the siRNA-loaded chitosan nanoparticle sample at 11,000 rpm and
   4°C for 37 minutes.
2. Carefully remove the supernatant from the siRNA-loaded CS-TPP nanoparticles.
   Clean the nanocuvette using 70% ethanol.
3. Load 2 μL of 1% acetic acid as a blank in the nanocuvette and measure
   absorbance at 260 nm using a UV-vis spectrophotometer.
4. Load 2 μL of the supernatant into the nanocuvette and measure absorbance at
   260 nm using a UV-vis spectrophotometer (we use the Eppendorf
   BioSpectrometer). Ensure an RNase-free environment is maintained throughout
   the experiment, and clean the nanocuvette with 70% ethanol between uses.
5. Calculate the entrapment efficiency using the formula:

   Entrapment Efficiency (%) =

   Csample − Csupernatant
   Csample
   × 100

   Csample is the concentration of siRNA added, and Csupernatant
   is the siRNA concentration in the supernatant.

### References

Katas, H., & Alpar, H. O. (2006). Development and characterisation of chitosan
nanoparticles for siRNA delivery.
*Journal of Controlled Release, 115*(2), 216–225.
<https://doi.org/10.1016/j.jconrel.2006.07.021>

Raja, M. A. G., Katas, H., & Wen, T. J. (2015b). Stability, Intracellular
Delivery, and Release of siRNA from Chitosan Nanoparticles Using Different
Cross-Linkers. *PLoS ONE, 10*(6), e0128963.
<https://doi.org/10.1371/journal.pone.0128963>

## ![icon](https://static.igem.wiki/teams/5645/wiki-images/ihp-and-notebook-icons/gel.webp) Gel Retardation Assay

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## Gel Retardation Assay

### Aim

To determine if encapsulation of the siRNA in chitosan nanoparticles has been
successfully achieved by visualizing on a gel.

### Materials Required

1. Encapsulated siRNA chitosan nanoparticle solution
2. Naked siRNA
3. Chitosan nanoparticle solution
4. RNase
5. Low EEO Agarose
6. 1x TBE Buffer
7. DNA Ladder
8. RNA Loading Dye
9. SyberSAFE
10. RNase-free tips and PCR tubes
11. Autoclaved Milli-Q water
12. Microwave oven
13. Gel electrophoresis comb
14. Gel Casting Tray
15. Conical flask
16. Graduated cylinder
17. UV transilluminator
18. Gel electrophoresis unit

Prepare the buffer and gel with Autoclaved Milli-Q water. Store all reagents
(except siRNA, chitosan nanoparticle solution, encapsulated siRNA chitosan
nanoparticle solution, RNA loading dye) at room temperature.

### Procedure

Protocol adopted from ([Helling et al., 1974](https://doi.org/10.1128/jvi.14.5.1235-1244.1974);
[Katas & Alpar, 2006f](https://doi.org/10.1016/j.jconrel.2006.07.021);
[Rio et al., 2010](https://cshprotocols.cshlp.org/content/2010/6/pdb.prot5445.short);
[Scott et al., 2003](https://link.springer.com/protocol/10.1385/0-89603-258-2%3A339)).

1. Make 1x TBE Buffer by diluting the 50x TBE buffer in autoclaved Milli-Q
   water. For 1 L of 1x TBE buffer, add 20 mL 50x TBE buffer to 980 mL of
   autoclaved Milli-Q water.
2. Prepare 2% agarose gel (35 mL): Weigh 0.7 g Low EEO agarose powder, and add
   35 mL of 1x TBE buffer. After that, boil the agarose mixture in a microwave
   oven to melt it. Cool the solution to approximately 50–60 °C, add 1.16 μL of
   SybrSAFE, and mix well. Pour the gel into the gel casting tray with the comb
   attached. Using an RNase-free pipette tip, gently push aside any floating
   bubbles to the top.
3. Once the gel is solidified, place it in the 1x TBE buffer poured into the
   gel electrophoresis unit. Carefully remove the comb, ensuring no tears in
   the wells.
4. In an aseptic and RNase-free environment, prepare the samples for loading.
   Pipette 10 μL of each sample in RNase-free PCR tubes: chitosan nanoparticle
   solution, encapsulated siRNA in CSNP, naked siRNA, and siRNA in CSNP treated
   with RNase (2 μL of 0.5 mg/mL RNase, incubated for 5 minutes at 56 °C). A
   1:6 dilution of loading dye was added to each well (2 μL in each well).
5. Spin down the samples for a few seconds and load them into the gel. Load 10
   μL RNA ladder as well.
6. Carry out the electrophoresis at a voltage of 55 V for 2 hrs, checking the
   gel in intervals.
7. Visualize the siRNA bands under a UV transilluminator at a wavelength of 365
   nm.

### References

Helling, R. B., Goodman, H. M., & Boyer, H. W. (1974). Analysis of endonuclease
R· Eco RI fragments of DNA from lambdoid bacteriophages and other viruses by
agarose-gel electrophoresis.
*Journal of virology, 14*(5), 1235-1244.
<https://doi.org/10.1128/jvi.14.5.1235-1244.1974>

Katas, H., & Alpar, H. O. (2006c). Development and characterisation of chitosan
nanoparticles for siRNA delivery.
*Journal of Controlled Release, 115*(2), 216–225.
<https://doi.org/10.1016/j.jconrel.2006.07.021>

Rio, D. C., Ares, M., Hannon, G. J., & Nilsen, T. W. (2010). Nondenaturing
agarose gel electrophoresis of RNA.
*Cold Spring Harbor Protocols, 2010*(6), pdb-prot5445.
<https://cshprotocols.cshlp.org/content/2010/6/pdb.prot5445.short>

Scott, V., Clark, A. R., & Docherty, K. (1994). The gel retardation assay.
*Protocols for gene analysis*, 339-347.
[https://link.springer.com/protocol/10.1385/0-89603-258-2:339](https://link.springer.com/protocol/10.1385/0-89603-258-2%3A339%20)

## ![icon](https://static.igem.wiki/teams/5645/wiki-images/ihp-and-notebook-icons/firefly.webp) Fluorescence Microscopy

+

✖

## Fluorescence Microscopy

### Aim

To visualize the internalization of the 6-FAM-tagged siRNA within
*Phytophthora capsici* zoospores.

### Materials Required

1. *Phytophthora capsici* zoospores in autoclaved distilled water.
2. 6-FAM tagged siRNA
3. RNase-free Eppendorf tubes
4. RNase-free micropipette tips
5. Micropipette
6. Glass slides
7. Methylene blue
8. Hemocytometer
9. siRNA encapsulated in CSNP
10. 100 ng/mL siRNA
11. Nuclease-free water
12. Fluorescence microscope

### Procedure

Protocol adopted from ([Cheng et al., 2022c](https://doi.org/10.1094/mpmi-12-21-0295-r%20);
[M. Wang et al., 2016b](https://doi.org/10.1038/nplants.2016.151%20)).

1. Cold shock the 48 hrs incubated zoospore solution for 30 minutes at 4°C.
   Pipette 250 μL of the solution into a 1.5 mL Eppendorf tube and add 1 or 2
   drops of Methylene blue dye. Calculate the concentration of the zoospores
   per mL of solution using a hemocytometer. Dilute the zoospore suspension to
   the desired concentration.
2. Prepare the zoospore samples for testing. Add 4 μL of 100 ng/mL siRNA to 6
   μL of zoospore solution of 105 spores per mL to get a final siRNA
   concentration of 40 ng/mL. Prepare zoospore samples treated with siRNA
   encapsulated in CSNP. Use 10 μL of 105
   spores per mL as a negative control.
3. Incubate the samples in the dark for 0.5 hrs to 4 hrs at room temperature,
   giving the siRNA enough time to be taken up by the zoospores.
4. Pipette the samples onto clean glass slides and place a cover slip without
   any air bubbles.
5. Visualize the samples using a fluorescence microscope at 10x, 40x, and 100x
   magnification at 488 nm wavelength (6-FAM has an excitation maximum that
   closely matches the 488 nm spectral line of the argon-ion laser) of the
   fluorescence microscope (we used an Olympus IX 73 Fluorescent Microscope).

### References

Cheng, W., Lin, M., Chu, M., Xiang, G., Guo, J., Jiang, Y., Guan, D., & He, S.
(2022b). RNAi-Based Gene Silencing of RXLR Effectors Protects Plants Against the
Oomycete Pathogen *Phytophthora capsici*.
*Molecular Plant-Microbe Interactions, 35*(6), 440–449.
<https://doi.org/10.1094/mpmi-12-21-0295-r>

Wang, M., Weiberg, A., Lin, F., Thomma, B. P. H. J., Huang, H., & Jin, H.
(2016). Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs
confer plant protection. *Nature Plants, 2*(10).
<https://doi.org/10.1038/nplants.2016.151>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% set next\_page\_url = url\_for('pages', page='implementation') %}
{% block next\_page\_name %}Implementation{% endblock %}
{% block page\_content %}

Our goal this year was to establish a preventative, sustainable, and
efficacious biocontrol strategy for pepper foot rot. This process involved the
integration of multiple components that are individually effective into a
system that is superior to current methods of treatment. Furthermore, every
element of this system has been tested extensively against the pathogen,
*P. capsici*, to ensure it performs its respective functions. By
demonstrating the effectiveness of this particular system and the siRNA design
pipeline and model, we hope to make it easier for future researchers to
implement similar systems for the many infections that plague the agricultural
economy.

## Designing the siRNA

After a thorough review process for identifying effective gene targets in
*P. capsici* and an assessment of the off-target effects, secondary
structures, and stability of the siRNA-mRNA duplex, we identified a set of
viable candidates, two of which were to be used for the experimentation
process (Figure 1). We ensured these sequences adhered to standards such
as the Ui-Tei, Reynolds, and Amarzguioui design rules.

[⤓ Download Final siRNA Candidates](%7B%7B%20url_for%28%27static%27%2C%20filename%20%3D%20%27assets/final-sirna-candidates.xlsx%27%29%20%7D%7D)

![Duplexfold result for siRNA Candidate 1](https://static.igem.wiki/teams/5645/wiki-images/pages/results/1a-results-new.webp)
![Maxexpect result for siRNA Candidate 1](https://static.igem.wiki/teams/5645/wiki-images/pages/results/1b-results-new.webp)
![Duplexfold result for siRNA Candidate 2](https://static.igem.wiki/teams/5645/wiki-images/pages/results/1c-results-new.webp)
![Maxexpect result for siRNA Candidate 2](https://static.igem.wiki/teams/5645/wiki-images/pages/results/1d-results-new.webp)

Fig 1. DuplexFold and MaxExpect results for siRNA candidates 1 and 2,
respectively

DuplexFold: Predicts how well the siRNA guide strand binds to its target
mRNA. The figure shows a stable helix with strong base pairing and low
free energy value, indicating a stable interaction– an essential factor
for effective silencing. This result also tells us that the siRNA duplex
itself is structurally stable.

MaxExpect: Predicts the target mRNA’s most likely secondary structure. The
paired sections and loops demonstrate the accessibility of bases for siRNA
binding. Decreased free energy values indicate a steady and realistic
folding pattern.

## *P. capsici* Growth Measurements in Potato Dextrose Agar and Carrot Agar

To ensure that the cultures used for our experiments are grown under ideal
conditions, a comparison was drawn between potato dextrose agar (PDA) and
carrot agar (Figures 3 and 2). We observed that a steady and quick
increase in growth was observed in carrot agar in comparison to PDA ([Chemeltorit et al., 2017](https://doi.org/10.1007/s10658-016-0988-5)). It took 4-5 days for the fungal mat to cover the plate with an ideal
growth pattern.

![Fig 2. Colony Diameter Measurement for P. capsici on PDA](https://static.igem.wiki/teams/5645/wiki-images/pages/results/2-p-capsici-on-pda-and-carrot-new.webp)

Fig 2. Colony Diameter Measurement for *P. capsici* on PDA vs
Carrot Agar

![Fig 3. Colony Diameter Measurement for P. capsici on Carrot Agar](https://static.igem.wiki/teams/5645/wiki-images/pages/results/3a-p-capsici-plate-carrot-new.webp)
![Fig 3. Colony Diameter Measurement for P. capsici on Carrot Agar](https://static.igem.wiki/teams/5645/wiki-images/pages/results/3b-p-capsici-pda-new.webp)

Fig 3. *P. capsici* on Carrot Agar and PDA at 48hrs

## Lactophenol Blue Staining

Lactophenol blue staining allowed us to get a closer look at the
morphology of
*Phytophthora capsici*. Singular hyphae were observed under 40x
magnification (Figure 4).

![P. capsici in carrot agar after 48 hours](https://static.igem.wiki/teams/5645/wiki-images/pages/results/4-lactophenol-blue.webp)

Fig 4. *P. capsici* hyphae observed

## Chitosan Nanoparticle Production

After designing the siRNA that performs the gene silencing, we aimed to
produce and optimize the characteristics of our delivery system - the
chitosan nanoparticles, focusing mainly on their size, zeta potential, and
polydispersity. Many iterations of this process were carried out, varying
parameters such as chitosan and acetic acide concentrations, TPP to
chitosan ratios, and homogenization until we obtained the ideal carrier
for our siRNA.

By the end of this process, we were able to obtain an ideal combination of
parameters for our nanoparticles. The zeta potential was observed to be 89
mV, with the size being 130 nm, along with a polydispersity index of 0.516
(Figures 7 and 8).

More details on the effect of parameters on nanoparticle characteristics
are discussed in the
[measurement page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27measurement%27%29%20%7D%7D).

![Fig 5. P. capsici hyphae observed](https://static.igem.wiki/teams/5645/wiki-images/pages/results/5-size-distribution-new.webp)

Fig 5. Size distribution for optimized nanoparticles

![Fig 6. Size distribution for optimized nanoparticles](https://static.igem.wiki/teams/5645/wiki-images/pages/results/6-zeta-potential-new.webp)

Fig 6. Zeta potential distribution for optimized nanoparticles

## The Nanoformulation

The nanoformulation is the culmination of these components into one robust
system. It integrates the specificity of the siRNA with the enhanced
stability, uptake, and antifungal activity provided by the nanoparticle.

We produced our nanoformulation following the ionic gelation protocol
optimized thus far, and analyzed the parameters using a Particle Size
Analyzer. We were able to obtain a complex with a zeta potential of 35.8
mV, having a size of 401.7 nm with a 0.509 polydispersity index (Figures 7
and 8). Based on these close to ideal parameters, we were able to gauge
that the nanoformulation could effectively be implemented for gene
silencing.

![Fig 10. Size distribution for siRNA nanoformulation](https://static.igem.wiki/teams/5645/wiki-images/pages/results/7-size-nf-new.webp)

Fig 7. Size distribution for siRNA nanoformulation

![Fig 11. Zeta potential distribution for siRNA nanoformulation](https://static.igem.wiki/teams/5645/wiki-images/pages/results/8-zeta-potential-nf-new.webp)

Fig 8. Zeta potential distribution for siRNA nanoformulation

## Detached Leaf Assay

To understand the first-hand effect of our system and its individual
components on
*P. capsici*, we conducted a series of assays involving
*P. nigrum* leaves (of Panniyur-1 variety). Mature leaves that were
sprayed with the nanoparticle, naked siRNA, and nanoformulation were
inoculated with a mycelial plug of *P. capsici*. We established a
series of controls, such as untreated leaves and leaves sprayed with water
and acetic acid, to gauge the difference in lesions.

We first observed that the lesion growth was significantly reduced in the
case of the chitosan nanoparticle (Figure 9). The same was true for the
nanoformulation we produced. Moreover, on applying 10 μL of naked siRNA
(of 100 nM and 1 μM concentration) and placing a mycelial plug on the leaf
3 days later, we found that there was no lesion at all in the case of 1 μM
siRNA compared to the controls, indicating a complete stoppage of
infection (Figure 10).

This prophylactic treatment is akin to how our solution will be
implemented in pepper fields. The result visually shows the measurable
decrease in infection, and also proves that each component is successfully
performing its function in curbing the infection.

Table 1. Details of the chitosan nanoparticle and nanoformulation
solutions as used in the graphs

| Name | Date prepared | Polydispersivity Index | Hydrodynamic Radius | Zeta Potential |
| --- | --- | --- | --- | --- |
| Nanoparticle 1 | 4th September | 0.454 | 95.1 nm | 169.0 mV |
| Nanoparticle 2 | 6th September | 0.488 | 267.5 nm | 184.5 mV |
| Nanoparticle 3 | 10th September | 0.516 | 130.1 nm | 89.0 mV |
| Nanoformulation 1 | 27th September | 2.318 | 6410.3 nm | -4.0 mV |
| Nanoformulation 2 | 29th September | 0.509 | 401.7 nm | 35.8 mV |

![Fig 9. Detached leaf assay: Effect of nanoparticle 1 and other controls](https://static.igem.wiki/teams/5645/wiki-images/pages/results/9-effectof-nanoparticle-1-new.webp)

Fig 9. Detached leaf assay: Effect of nanoparticle 1 and other controls

![Fig 10. 1-way ANOVA test on the effect of nanoparticle 1 among other controls at 72 hours](https://static.igem.wiki/teams/5645/wiki-images/pages/results/10-np-effect-at-72hrs-new.webp)

Fig 10. 1-way ANOVA test on the effect of nanoparticle 1 among other
controls at 72 hrs

![Fig 11.  Detached leaf assay: Effect of Prophylactic and Therapeutic treatment with other controls](https://static.igem.wiki/teams/5645/wiki-images/pages/results/11-effect-of-p-and-t-new.webp)

Fig 11. Detached leaf assay: Effect of Prophylactic and Therapeutic
treatment with other controls

![Fig 12. 1-way ANOVA test on the effect of Prophylactic 1 μM siRNA among other controls at 72 hrs](https://static.igem.wiki/teams/5645/wiki-images/pages/results/12-effect-of-p-72hrs-new.webp)

Fig 12. 1-way ANOVA test on the effect of Prophylactic 1 μM siRNA among
other controls at 72 hrs

![Fig 13.  Detached leaf assay: Effect of Nanoformulation among other controls](https://static.igem.wiki/teams/5645/wiki-images/pages/results/13-effect-of-nf-new.webp)

Fig 13. Detached leaf assay: Effect of Nanoformulation among other
controls

![Fig 14. 1-way ANOVA test on the effect of Nanoformulation 2 among other controls at 72 hrs](https://static.igem.wiki/teams/5645/wiki-images/pages/results/14-effect-of-nf-2-at-72hrs-new.webp)

Fig 14. 1-way ANOVA test on the effect of Nanoformulation 2 among other
controls at 72 hrs

## Detached Leaf Assay (Post Wiki Freeze)

Mature leaves were sprayed with nanoparticles and nanoformulation. A
mycelial plug of *P. capsici* was placed over them. A set of controls
included untreated leaves, leaves with only pricks, and leaves sprayed
with a mixture of acetic acid and water. Lesion growth in these leaves was
then measured.

Over a three-day period, it was observed that the lesion growth was
minimal when 10 μL of the nanoformulation was applied. Although the
application of just the chitosan nanoparticles also reduced lesion growth,
the results were comparable to those obtained with acetic acid. These
results are consistent with previous DLA findings. It indicates that the
nanoformulation, produced with 15 μL of 100 μM siRNA, has remained stable
over a three-week period and continues to be the most effective at
reducing lesion growth.

![Fig 15. 1-way ANOVA test on the effect of Nanoformulation 2 among other controls at 72 hrs after 3 weeks](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/15-1-anova-test-wiki-thaw.avif)

Fig 15. 1-way ANOVA test on the effect of Nanoformulation 2 among other
controls at 72 hrs after 3 weeks

## Evaluating the Cytotoxicity of siRNA

To establish the safe concentration range of the siRNA, and ensure that
the reduction in infection is due to the intended mechanism (and not due
to toxicity from the siRNA itself), a cytotoxicity assay was carried out
wherein 10 μL of bZIP1 target gene’s siRNA (at concentrations 1.5 nM, 50
nM, 100 nM, 1 μM and 100 μM) was applied on
*P. nigrum*
leaves to observe any unintended effects. No such effect was observed over
a duration of 3 days.

## SEM Analysis

SEM analysis provided us with key insights into the morphology and size of
our nanoparticles and nanoformulations. At magnifications higher than
100000x, the chitosan nanoparticles and nanoformulation appeared as
spherical beads in clusters (Figures 17, 20, and 24). Variations in size
and shape were observed between the samples, due to the time period
between their production and characterization.

![Fig 16. Chitosan nanoparticles visualized at 10,000x magnification.
 ](https://static.igem.wiki/teams/5645/wiki-images/pages/results/15-chitosan-nanopartic-new.webp)

Fig 16. Chitosan nanoparticles visualized at 10,000x magnification.

![Fig 17. Chitosan nanoparticles visualized at 250,000x magnification.
 ](https://static.igem.wiki/teams/5645/wiki-images/pages/results/16-chitosan-nanoparticle-new.webp)

Fig 17. Chitosan nanoparticles visualized at 250,000x magnification.

![Fig 18. Chitosan nanoparticles visualized at 350,000x magnification.
 ](https://static.igem.wiki/teams/5645/wiki-images/pages/results/17-chitosan-nanopart-new.webp)

Fig 18. Chitosan nanoparticles visualized at 350,000x magnification.

![Fig 19. Nanoformulation (prepared on 27th September) visualized at 5000x magnification.
 ](https://static.igem.wiki/teams/5645/wiki-images/pages/results/18-nanoformulations-new.webp)

Fig 19. Nanoformulation (prepared on 27th September)
visualized at 5000x magnification.

![Fig 20. Nanoformulation (prepared on 27th September) visualized at 200,000x magnification.
 ](https://static.igem.wiki/teams/5645/wiki-images/pages/results/19-nanoformulations-200000x-new.webp)

Fig 20. Nanoformulation (prepared on 27th September)
visualized at 200,000x magnification.

![Fig 21. Nanoformulation (prepared on 27th September) visualized at 250,000x magnification.
 ](https://static.igem.wiki/teams/5645/wiki-images/pages/results/20-nanoformulations-visualised-at-250-000-new.webp)

Fig 21. Nanoformulation (prepared on 27th September)
visualized at 250,000x magnification.

![Fig 22. Nanoformulation (prepared on 29th September) visualized at 6500x magnification.
 ](https://static.igem.wiki/teams/5645/wiki-images/pages/results/21-nanoforumulations-visualised-at-6500x-new.webp)

Fig 22. Nanoformulation (prepared on 29th September)
visualized at 6500x magnification.

![Fig 23. Nanoformulation (prepared on 29th September) visualized at 100000x magnification.
 ](https://static.igem.wiki/teams/5645/wiki-images/pages/results/22-nanoformulations-visualised-at-100000x-new.webp)

Fig 23. Nanoformulation (prepared on 29th September)
visualized at 100000x magnification.

![Fig 24. Nanoformulation (prepared on 29th September) visualized at 150000x magnification
 ](https://static.igem.wiki/teams/5645/wiki-images/pages/results/23-nanoformulations-visualised-at-150000x-new.webp)

Fig 24. Nanoformulation (prepared on 29th September)
visualized at 150000x magnification

![Fig 25. Nanoformulation (prepared on 29th September) visualized at 350000x magnification
 ](https://static.igem.wiki/teams/5645/wiki-images/pages/results/24-nanoformulatiojs-visualised-at-350000x-new.webp)

Fig 25. Nanoformulation (prepared on 29th September)
visualized at 350000x magnification

## SEM analysis (Post Wiki Freeze)

The SEM analysis shows that siRNA encapsulation alters the morphology of
the chitosan nanoparticles. All the nanoparticle and nanoformulation
samples appeared as spherical beads in clusters. The nanoparticles appear
uniform and well-dispersed, whereas the nanoformulation samples exhibit
surface irregularity, aggregation, and the formation of cavity-like
structures, which indicate successful encapsulation. The 10 µL sample
exhibits a denser and more compact distribution with more clustering,
whereas the 5 µL sample has less aggregation with clear particle
boundaries. Overall, the changes in the surface texture validate the
loading of siRNA into the chitosan nanoparticles.

![Fig 26. 10 μL of chitosan nanoparticles visualized at 100,000x magnification ](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/26-10ul-of-csnp-1-wiki-thaw.avif)

Fig 26. 10 μL of chitosan nanoparticles visualized at 100,000x
magnification

![Fig 27. 5 μL of Chitosan nanoparticles visualized at 100,000x magnification ](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/27-5ul-of-csnp-wiki-thaw.avif)

Fig 27. 5 μL of chitosan nanoparticles visualized at 100,000x
magnification

![Fig 28. 10 μL of nanoformulation (prepared on 29th September) visualized at 100,00x magnification ](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/28-10ul-nanoformulation-wiki-thaw.avif)

Fig 28. 10 μL of nanoformulation (prepared on 29th September)
visualized at 100,00x magnification

![Fig 29.  5 μL of nanoformulation (prepared on 29th September) visualized at 100,00x magnification ](https://static.igem.wiki/teams/5645/wiki-images/pages/wiki-thaw/29-5u-lnf-wiki-thaw.avif)

Fig 29. 5 μL of nanoformulation (prepared on 29th September)
visualized at 100,00x magnification

## Entrapment Efficiency

The calculation of entrapment efficiency gives the percentage of siRNA
successfully encapsulated within the nanoparticles. A high encapsulation
efficiency is imperative to the effective release of siRNA within
*P.capsici*, and to also confer stability to the complex in the soil
environment.

The encapsulation efficiency was calculated using the formula:

Entrapment Efficiency (%) =

Csample − Csupernatant
Csample
× 100

Csample (20.778 μg/mL) was the concentration of siRNA added,
and Csupernatant
(2.2 μg/mL) was the siRNA concentration in the supernatant.

The entrapment efficiency was found to be 89.41%, indicating a high
percentage of siRNA encapsulation.

## Gel Retardation Assay

A gel retardation assay was performed to visualize the encapsulation of
the siRNA within the nanoparticle ([Katas et al., 2006](https://doi.org/10.1016/j.jconrel.2006.07.021)).

We ran our samples on a 4% agarose gel for 2 hours. Positive
(RNase-treated nanoformulation, nanoparticle) and negative (free siRNA)
controls were established. The ladder used was a 1kb DNA ladder from
GeneDireX.

Upon visualization of the gel under a UV transilluminator it was observed
that the 100 nM siRNA was observed as a faint band due to its low
concentration. No bands were observed for the nanoformulation,
nanoformulation treated with RNase and the chitosan nanoparticle.
Successful encapsulation was indicated by the entrapment of the siRNA in
the well.

![Fig 30. Visualization of the gel under a UV transilluminator](https://static.igem.wiki/teams/5645/wiki-images/pages/results/25-visualisation-of-the-gel-under-a-uv-new.webp)

Fig 30. Visualization of the gel under a UV transilluminator

## *P. capsici* Sporulation

Effective silencing of the bZIP gene within *P. capsici* zoospores
causes a reduction in zoospore motility ([Blanco & Judelson, 2005](https://doi.org/10.1111/j.1365-2958.2005.04575.x)).

To visualize this confirmatory effect, zoospores were isolated from
*P. capsici* cultures and treated with the following:

* 1% acetic acid
* Chitosan nanoparticle solution
* 7.23 µM siRNA (total siRNA in the sample was 40 ng/ml) ([Cheng et al., 2022](https://doi.org/10.1094/mpmi-12-21-0295-r))
* 100 µM siRNA
* Nanoformulation (prepared on 29th September)

The samples were incubated for 30 minutes and visualized at 100x
magnification using an Olympus CX-43 upright trinocular phase contrast
microscope.

Vid 1. Zoospores visualized at 100x without any treatment

Vid 2. Zoospores visualized at 100x with treatment of 1% acetic acid

Vid 3. Zoospores visualized at 100x with treatment of chitosan
nanoparticle solution

Vid 4. Zoospores visualized at 100x with treatment of 7.23 µM siRNA

Vid 5. Zoospores visualized at 100x with treatment of 100 µM siRNA

Vid 6. Zoospores visualized at 100x with treatment of nanoformulation
solution

Motile zoospores were observed in the untreated, 1% acetic acid, and
nanoparticle-treated samples. Zoospore samples treated with 7.23 µM siRNA,
100 µM siRNA, and the nanoformulation showed total reduction in zoospore
motility.

The observed effect over zoopore motility confirmed gene silencing by the
siRNA and nanoformulation.

## Fluorescence Microscopy

To further visualize the uptake of siRNA within
*P. capsici* zoospores, the siRNA was tagged with 6-FAM, a
fluorescent dye. The absorption and emission wavelengths are 495 nm and
517 nm, respectively.

Zoospore samples were incubated with naked siRNA and the nanoformulation
for 90 minutes, following which they were visualized using an Olympus IX
73 Fluorescent Microscope at 40x, 60x, and 100x.

Fluorescent structures were clearly visible at 100x magnification, which
were comparable to the zoospores observed through phase contrast
microscopy. This indicated successful internalization of the siRNA within
the zoospores, as well their intended release from the nanoparticle
complex.

![Fig 26. Left - P. capsici zoospores visualized at 100x magnification using phase contrast microscopy 
     Right - Internalization of the 6-FAM tagged siRNA within P. capsici zoospores, visualized using fluorescence microscopy 
 ](https://static.igem.wiki/teams/5645/wiki-images/pages/results/26a-p-casici-zoospores-new.webp)
![Fig 26. Left - P. capsici zoospores visualized at 100x magnification using phase contrast microscopy 
     Right - Internalization of the 6-FAM tagged siRNA within P. capsici zoospores, visualized using fluorescence microscopy ](https://static.igem.wiki/teams/5645/wiki-images/pages/results/26b-internalization-of-the-5-fam-new.webp)

Fig 31. Left - *P. capsici* zoospores visualized at 100x
magnification using phase contrast microscopy
Right -
Internalization of the 6-FAM tagged siRNA within
*P. capsici* zoospores, visualized using fluorescence microscopy

## The Software Model

Our software model works as a screening tool that evaluates the binding
between an siRNA sequence and the chosen nanoparticle.

The XGBoost outperformed Lasso and Random Forest by RMSE of 33.30 and an
R² value of 0.7459, explaining approximately 75% of the variance in the
docking scores. The model extracted 217 features based on the nucleotide
sequence, such as position, GC Content and etc, allowed to be attributed
when a new sequence is inputted. The model resulted in generating docking
scores for the new sequences that had a maximum error of ± 40.

Table 2. The error calculation with the docking scores performed through
HDOCK and the predicted scores of our model

| Seq. No. | Sequence (Both 5' → 3') | Docking Score for Chitosan | Docking Score for Lipid | Error Calc for Chitosan | Error Calc for Lipid |
| --- | --- | --- | --- | --- | --- |
| 1. | GGUACUACAACUAGUGUGACU  UCACACUAGUUGUAGUACCAG | -215.51 | -114.75 | +36 | -20 |
| 2. | CAGAGCUGAAGGAUAUGAAGA  UUCAUAUCCUUCAGCUCUGUG | -246.64 | -137.74 | +11 | -4 |
| 3. | GGAGACAGCGACAAGUUGUCG  ACAACUUGUCGCUGUCUCCAA | -215.24 | -106.34 | +39 | -31 |
| 4. | CCCGAGUGCGGUUUCACUAGG  UAGUGAAACCGCACUCGGGUG | -292.31 | -158.95 | -38 | +21 |
| 5. | GGGAGAGACUGGUGAUGAAGG  UUCAUCACCAGUCUCUCCCCC | -281.82 | -151.31 | -27 | +14 |

![Fig 32. Software output ](https://static.igem.wiki/teams/5645/wiki-images/pages/results/27-predicted-scores-using-model-new.webp)

Fig 32. Predicted Docking Scores using our software model

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