Resuspension

We started our wet lab steps by resuspending Fragment-1, Fragment-2, and Fragment-3 to amplify them using a PCR reaction. The Fragments carry different genetic parts that will be used in our project according to the following figures:

FIG (1): showing TSLP role as a central epithelial “alarmin” driving multiple asthma phenotypes.

Resuspension protocol:

Material:

DNA fragments.
Distilled water.

Methods:

The DNA fragment locations were detected from the 96-well plate provided by Twist Bioscience.

Fragment-1 → A2
Fragment-2 → H1
Fragment-3 → D2

The tip of the pipette penetrated the cover of the multiwell plate to avoid cross-contamination.
The lyophilized fragments were diluted with 100 µL of distilled water.
We ensured that each lyophilized tube was well sealed.
Then, the lyophilized tube was mixed properly with the distilled water by shaking the tube forcefully until the pellet at the bottom was no longer visible.
The tube was spun down to collect the residuals on the wall of the tube.
100 µL of the medium was transferred to a sterile 1.5 mL Eppendorf tube. Then, the original tube was stored at -20°C until use (short-term storage).
Finally, each Eppendorf tube was labeled.

FIG (1): showing TSLP role as a central epithelial “alarmin” driving multiple asthma phenotypes.

PCR :

We used PCR to amplify our genetic fragments.

Material:

DNA templates (Fragment-1, Fragment-2, Fragment-3).
Primers compatible with each fragment.
Mastermix.
Distilled water.

Methods:

The lyophilized primers were resuspended by adding the following amount of distilled water to the lyophilized primers:

Primer	concentrations	Stock solution
Fragment-1 Forward	24 nmol	240 µL
Fragment-1 Reverse	24.9 nmol	250 µL
Fragment-2 Forward	27.1nmol	270 µL
Fragment-2 Reverse	23.1 nmol	230 µL
Fragment-3 Forward	22.4 nmol	224 µL
Fragment-3 Reverse	20.1 nmol	200 µL

Fragment-1 → A2
Fragment-2 → H1
Fragment-3 → D2

Then, we prepared the working solution of each fragment by adding 10 µL of its stock solution to 90 µL of distilled water for a total of 100 µL for each primer. Then we mixed it properly.
Aiming for a final pure volume of 50 µL, we added 25 µL of Master mix (2x concentration) for 3 PCR tubes.

Then, we added 2 µL of the forward and reverse primers’ working solution for each fragment to the master mix. Subsequently, we mixed them properly.
Finally, we added 0.5 µL of the template fragment.

PCR tube (50 µL)	Forward primer (working solution)	Reverse primer (working solution)	Master mix	DNA Template	Distilled water
Fragment-1	2 µL	2 µL	25 µL	0.5 µL	20.5 µL
Fragment-2	2 µL	2 µL	25 µL	0.5 µL	20.5 µL
Fragment-3	2 µL	2 µL	25 µL	0.5 µL	20.5 µL

FIG (1): showing TSLP role as a central epithelial “alarmin” driving multiple asthma phenotypes.

We put the final tubes into the PCR, and we set our program as follows:

Annealing temperature: 55 °C.
Elongation time: 1 min.

FIG (1): showing TSLP role as a central epithelial “alarmin” driving multiple asthma phenotypes.

Gel electrophoresis :

Material:

Agarose powder.
TAE buffer.
Ethidium bromide.
Loading dye.
PCR products.

Methods:

Gel preparation

Purpose: To create a solid, porous matrix through which DNA fragments can travel.

Ideally, the gel concentration should be 1.5 g of agarose powder in 100 ml of TAE buffer in a microwavable flask. To prepare our 50 ml tray volume. We had added 0.75 agarose powder to the 50 ml TAE buffer.
Subsequently, it was mixed properly and heated in the microwave for 1 min until the agarose was completely dissolved.
The agarose solution was left to cool down to about 50 °C for about 5 minutes, allowing it to solidify without warping the gel tray.

1.5 µL of Ethidium bromide was added to the agarose. Ethidium bromide allows us to visualize the DNA bands under ultraviolet (UV) light.
The gel was poured into a tray with a well comb (agarose was poured slowly to avoid bubble formation), which created wells for loading the samples.
The gel was allowed to solidify completely at room temperature for 20-30 minutes before use.

Sample loading and electrophoresis

Purpose: To introduce the DNA samples into the gel and use an electric current to separate the fragments.

Once solidified, the agarose gel was placed in an electrophoresis box filled with TAE buffer. This buffer conducts electricity and maintains a stable pH, allowing the negatively charged DNA to migrate through the gel.
Then, the gel box was filled with ethidium bromide until the gel was covered.
Then, a molecular weight ladder, containing DNA fragments of known sizes, was loaded into the first lane of the gel. It serves as a reference to estimate the sizes of the PCR products.
2 µL of the loading dye was mixed with 5 µL of each of the PCR products before loading.

11-

Then,5 µL of the samples were loaded carefully into the remaining wells.

The electric current at 80 -150 V was applied to the gel electrophoresis, with the negative electrode near the wells. This causes the negatively charged DNA fragments to move toward the positive electrode. Smaller fragments travel faster and farther than larger ones.
The run was stopped when the loading dye had moved approximately 75-80% of the way down the gel, which indicates sufficient separation of the DNA fragment (the run time was about 1-1.5h).
After the run time, the electrodes were disconnected from the power source, and then the gel was carefully removed from the gel box.

Visualization and interpretation of results

Purpose: To observe the separated DNA fragments and analyze the results.

Finally, we transferred the gel into a UV light device to visualize our fragments and interpret the results by comparing the position of the PCR product bands to the bands of the molecular weight ladder.

FIG (1): showing TSLP role as a central epithelial “alarmin” driving multiple asthma phenotypes.

Transformation :

We transformed the TagBFPV2 plasmid into Jm109 competent cells to test the functionality of M13.

Material:

DNA vector.
Competent cells.
Culture media.
Ampicillin.

The JM109 competent cells were taken out of -80°C and thawed on ice (approximately 20-30 mins).
The agar plates that contain the antibiotic were removed from storage at 4°C and allowed to warm up at room temperature.
The lyophilized DNA had been resuspended by adding 20 µL of distilled water to the DNA. Then, we mixed them properly.
5 µL of the resuspended DNA was added to 20-50 μL Jm109 competent cells in a Falcon tube. Then, it was gently mixed by flicking the bottom of the tube with your finger a few times.
The competent cells/DNA mixture was incubated on ice for 10 min.
Each of the transformation tubes was transferred to a hot path for 45 seconds, as the bottom 1/2 to 2/3 of the tube was placed in the path.
Then, the tubes were turned back on ice for 2 minutes.
250-1,000 μl LB media was added to the transformed bacteria, and it was left to grow on the shaker for 30 minutes.
Then, all the transformed bacteria were plated onto a 10 cm LB agar plate containing 5 µL of Ampicillin and were added to a liquid medium.
Finally, we put our mixture on the media, and it was left on the shaker at 37°C overnight.

PCR :

We used PCR to amplify fragments 1,2 again to get proper amounts and amplify Fragment 3 gradually to get the optimum annealing temperature..

Material:

DDNA templates (Fragment-1, Fragment-2, Fragment-3).
Primers compatible with each fragment.
Mastermix.
Distilled water.

Methods:

FIG (1): showing TSLP role as a central epithelial “alarmin” driving multiple asthma phenotypes.

We made another PCR reaction for fragments 1 and 2 with the same protocol.
Moreover, we ran a gradual PCR reaction for fragment 3 on 6 different annealing temperatures due to the gradient bands shown yesterday.

Also, we ran a PCR reaction for plasmid (1-5d) with an elongation time of 1 min and an annealing temperature of 48°C to test the functionality of M13 primer variants (3 forward and 2 reverse) with a total of 6 probabilities.

Primer	Code
M-13 Forward-1	1
M-13 Reverse-1	2
M-13 Reverse-2	3
M-13 Forward-2	4
M-13 Forward-3	5

Forward primers we had was tested with the two reverse primers.

Forward primer	Reverse primer
M-13 Forward-1 (1)	M-13 Reverse-1 (2)
M-13 Forward-1 (1)	M-13 Reverse-2 (3)
M-13 Forward-2 (4)	M-13 Reverse-1 (2)
M-13 Forward-2 (4)	M-13 Reverse-2 (3)
M-13 Forward-3 (5)	M-13 Reverse-1 (2)
M-13 Forward-3 (5)	M-13 Reverse-2 (3)

FIG (1): showing TSLP role as a central epithelial “alarmin” driving multiple asthma phenotypes.

Glycerol Stock :

FIG (1): showing TSLP role as a central epithelial “alarmin” driving multiple asthma phenotypes.

After the steps of Overnight Liquid Culture Inoculation.
We took two portions of the actively growing overnight culture. This ensured a backup in case one stock is contaminated or lost.
We took 2 glycerol stocks for our 1-5d transformed bacterial growth, each stock contained 500 μL of each overnight culture mixed with 500 μL of 50% glycerol in a 2 mL screw top tube.
The solution was mixed gently by inverting the tube several times, ensuring the glycerol was evenly distributed among the bacteria.
The prepared glycerol stock was snap frozen by placing the tubes into a liquid nitrogen bath. This rapid freezing technique helps prevent the formation of large, destructive ice crystals.
After snap-freezing, the stock was transferred to a -80°C freezer for long-term storage. Where it can remain viable for many years.

Plasmid Extraction :

We extracted our plasmid from the cells through three main steps: lysis, binding, and extraction.

FIG (1): showing TSLP role as a central epithelial “alarmin” driving multiple asthma phenotypes.

Methods:

Step (1): Lysis

We started by lysing the bacterial cells.
200 µL of P1 buffer (resuspension buffer) was added to rehydrate the bacterial pellet, which often contains RNase A to degrade cellular RNA. Then the P1 buffer was mixed by pipetting.
50 µL of P2 buffer (alkaline lysis buffer) was added to disrupt the cell membrane and denature proteins and DNA, including the bacterial chromosome and the plasmid. Then the P2 was mixed by inverting.
The P2 buffer was left for 5 minutes to allow complete lysis of the cell membrane.
350 µL of N3 buffer was added to neutralize the pH, leading to precipitation of the large chromosomal DNA and denatured protein, while the small, supercoiled plasmid DNA remains in solution. Then the N3 buffer was mixed by inverting.

Step (2): Binding and washing

The culture medium was centrifuged for 4 minutes to pellet the precipitated chromosomal DNA and cellular debris, leaving the plasmid DNA in the supernatant.
The upper layer was transferred to a spin column to allow the plasmid DNA to bind to the column's silica membrane in the presence of the high salt concentration from the N3 buffer.
Subsequently, the spin column was centrifuged, which forced the liquid (flow-through) containing cellular waste and other impurities through the column, leaving the plasmid DNA bound to the membrane.
500 µL of AW1 and 500 µL of AW2 (wash buffers) were added to remove any residual salts from the first wash.
We let the mixture dry for 2 minutes, which allows for the removal of all traces of the wash buffers, especially the alcohol, which can interfere with subsequent steps.

Visualization and interpretation of results

Finally, we eluted the plasmids with nuclease-free water from the column.

DNA Extraction :

We were going to purify two samples from Fragments 1 and 2 ( the result of PCR today and yesterday) and one sample of Fragment 3 ( annealing temperature of 60 °C).

FIG (1): showing TSLP role as a central epithelial “alarmin” driving multiple asthma phenotypes.

Methods:

Step 1: Binding

200 µL of RLT (RNA lysis buffer) was added to allow binding of DNA to the silica membrane in the spin column. Then it was mixed by pipetting.
40 µL of isopropanol was added, which promotes the binding of nucleic acids to the silica membrane by helping to dehydrate the DNA. Then it was mixed by inverting.
The mixture was poured into a spin column.
Then, the mixture was left for 5 minutes to allow the binding of the DNA to the column's membrane.

Step (2): washing

The mixture was centrifuged for 30 seconds, which allowed the removal of the unbound primers, enzymes, and salts.
Then, the mixture was washed with 500 µL of AW2 (wash buffer), which removed any remaining salts and impurities.
Then, the mixture was centrifuged to spin the buffer through the column and discard the flow-through.
Subsequently, we left it dry for 2 minutes. This ensured that all residual ethanol from the wash buffer was removed from the column.

Visualization and interpretation of results

The mixture was eluted with 50 µL water and placed in a new Eppendorf, which facilitated the release of the DNA from the silica membrane.
Then, a new Eppendorf tube was used to collect the clean DNA.
Then, we waited for 3 minutes, which allowed the water to fully rehydrate the DNA and dissociate it from the membrane, increasing the final yield.
Finally, the eluted DNA was centrifuged for 3 minutes.

Gel Electrophoresis :

We performed gel electrophoresis for all today's PCR products, as follows:

1- Gel electrophoresis for the gradual PCR amplification of Fragment-3, and the amplified Fragments-1,2:

2- Gel electrophoresis for the 1-d5 plasmid PCR product, which was performed using different M-13 primer variants to find the viable pair, and for the purified Fragments 1, 2, and 3 from yesterday’s and today’s reactions:

Digestion :

Purpose: To cut each of our fragments (1, 2, 3), which carry different genetic parts and will be used in our project. In this step, we cut each fragment to isolate each genetic part and use it in our project. These genetic parts are:

Fragment 1

Part name	Restriction 1	Restriction 2	Buffer
1- P11 promotor	Ecor1	Sac1	R2-1
2- Antitoxin promotor	Sac1	Kpn1	R2-1
3- SV40 Poly A signal	Kpn1	BamH	R1-1
4- Trrn B terminator	BamH	Xbal	R2-1
5- RBS	Xbal	Sel1	R3-1/span>
6- T7 promotor	Sel1	Pst1	R3-1
7- P-kat A promoter	Pst1	Hind 3	R2-1

Fragment 2

Part name	Restriction 1	Restriction 2	Buffer
1- P32 promoter + lac R operon	Sac1	Kpn1	R2-1
2- Pkat A + Rep repressor	Kpn1	Pst1	R1-1

Fragment 3

Part name	Restriction 1	Restriction 2	Buffer
1- Pkat + operator	Hind3	Xbal	R2-1
2- T7 + SVTR kozakr	Xbal	Ecor1	R2-1
3- P170-Cp promotor	Ecor1	Sac1	R2-1
4- P32 promoter with lac operon	Sac1	Kpn1	R2-1

● Our genetic parts were digested using different pairs of restriction enzymes. The restriction enzymes were used in specific amounts with various elements, ensuring that each part has different "sticky ends" on each side for directional cloning.

● The vector was cut with the same pair of restriction enzymes used for a specific genetic part to make it compatible with each Part.

● We ran the digested vectors and PCR products on PCR, specifically the digestion protocol, which consists of 2 stages:

Reaction setup: A specific mix for each reaction as follows:

Parts reaction	Volume	Vector reaction	Volume
1- PCR product (part)	5 μL	Vector	2 μL
2- Restriction 1	1 μL	Restriction 1	1 μL
3- Restriction 2	1 μL	Restriction 2	1 μL
4- Buffer	2 μL	Buffer	2 μL
5- H2O	11 μL	H2O	11 μL

Incubation and inactivation::

● Stage (1) Heat activation step to provide the optimal temperature (37 °C for 1 hr) for most restriction enzymes to cleave DNA efficiently.

● Stage (2) Heat-inactivation step to denature and kill the restriction enzymes at 85 °C, preventing them from cutting the DNA during the next ligation step.

Ligation:

Purpose: To ligate the cut genetic part (insert) with its corresponding vector, creating a new circular recombinant plasmid in a pure volume of 20 μL.

Methods:

1- We added 5 μL of the digested PCR product to 10 μL of the digested corresponding vector with 2 μL of the buffer ligase, 1 μL ligase enzyme, and 1 μL ATP.

2- The final component ratio was 2:1 of vector to insert volume (10 µL vector, 5 µL insert).

3- Finally, this mixture was incubated at room temperature for 1 hour, which is the optimal temperature to form phosphodiester bonds, sealing the nicks in the DNA backbone.

Transformation :

Purpose: To introduce the ligated plasmids into Jm109 competent cells to test the viability of our ligated plasmid. According to the following protocol:

Material:

- DNA vector.

- Competent cells.

- Culture media.

- Ampicillin.

Methods:

1- 5 μL of the ligated plasmids was added to Jm109 competent cells.

2- Then, the heat shock protocol was performed to increase the efficiency of DNA uptake by bacterial cells as follows:

● The mixture was left on the ice for 10 min, as the cold temperature stabilizes the cell membranes, making them more receptive to the plasmid DNA.

● Then, the mixture was exposed to heat shock by being transferred to a hot bath for 45 seconds. This creates a thermal imbalance across the cell membrane, allowing the DNA to enter.

● Then, they were turned back to the ice for 2 minutes, allowing for the recovery and stabilization of the bacterial membranes.

3- 250 μL of LB media was added to each of the 13 tubes, as it provides nutrients for the cells to recover and begin expressing the genes on the newly acquired plasmid.

4- Then, they were left on the shaker for 1 hour, ensuring proper aeration for the cells as they recover and begin expressing the antibiotic resistance gene on the plasmid.

5- We melted the solid media.

6- Then, 100 μL of Ampicillin and 50 μLof X-gal were added to the media, as the Ampicillin selects the bacteria that successfully took the plasmid, while the X-gal is used to differentiate between recombinant and non-recombinant plasmids.

7- The media was poured on the plates.

8- Then, 100 μLof Jm109 competent cells were added to the plates containing the selection media.

9- We spread the cells using glass beads.

10- Finally, the plates were incubated at 37 °C overnight, allowing the transformed bacteria to grow into colonies.

Transformation :

We repeat the transformation of the ligated parts of fragments 1, 2, and 3.

● N.B.The results of the previous transformation aren’t reliable, as the length of the PCR products wasn’t similar to the length of our parts.

Material:

- DNA vector.

- Competent cells.

- Culture media.

- Ampicillin.

Methods:

1- 5 μL of the ligated plasmids was added to Jm109 competent cells.

2- Then, the heat shock protocol was performed to increase the efficiency of DNA uptake by bacterial cells as follows:

● The mixture was left on the ice for 10 min, as the cold temperature stabilizes the cell membranes, making them more receptive to the plasmid DNA.

● Then, the mixture was exposed to heat shock by being transferred to a hot bath for 45 seconds. This creates a thermal imbalance across the cell membrane, allowing the DNA to enter.

● Then, they were turned back to the ice for 2 minutes, allowing for the recovery and stabilization of the bacterial membranes.

3- 250 μL of LB media was added to each of the 13 tubes, as it provides nutrients for the cells to recover and begin expressing the genes on the newly acquired plasmid.

4- Then, they were left on the shaker for 1 hour, ensuring proper aeration for the cells as they recover and begin expressing the antibiotic resistance gene on the plasmid.

5- We melted the solid media.

7- The media was poured on the plates.

8- Then, 100 μLof Jm109 competent cells were added to the plates containing the selection media.

9- We spread the cells using glass beads.

10- Finally, the plates were incubated at 37 °C overnight, allowing the transformed bacteria to grow into colonies.