iGEM 2023 — Team iiser-tvm

{% extends "layout.html" %}
{% block title %}{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

Welcome to IISER-TVM'S Lab Notebook Wiki! This digital notebook serves as a comprehensive record of experiments, observations, and research conducted in our laboratory. Whether you're a member of our team or an external collaborator, this wiki is designed to promote transparency and document our scientific journey. Feel free to explore this guide, we hope it will help you understand the significance of documentation and how to effectively utilize your lab notebook in your research endeavors.

##### WEEK 1

**Saturday - 8/7/2023**

*Making TE buffer for resuspension of IDT orders.*

Required final concentrations:

EDTA 0.1mM
Tris HCl- 10mM

#This EDTA concentration is for oligo storage.

First step, making 100 ml of 1M tris HCl and 0.5M EDTA

###### 1M Tris HCl

* Weigh out 12.11 g Tris(Tris buffer) and add to a 100 mL beaker
* Measure out 80 mL of distilled water and add to the beaker
* Add a magnetic flea and place it on a magnetic stirring plate to mix the solution.
* Add a pH meter to the solution to observe the pH
* Slowly add concentrated hydrochloric acid (HCl) solution and make up to pH 8.

###### 0.5M EDTA

* Weigh out 18.61 g EDTA disodium salt, dihydrate and add to a 100 mL beaker.
* Measure out 80 mL of distilled water and add to the beaker.
* Add a magnetic bead and place it on a magnetic stirring plate to mix the solution. The EDTA salt will not go into solution until the pH reaches 8.0.
* Add a pH meter to the solution to observe the pH
* To dissolve the salt, add sodium hydroxide (NaOH) pellets to the solution. Add a few pellets at a time and wait until the pellets have fully dissolved before adding more. It may take around 2 g (way more) of NaOH pellets before the pH is at 8.0.
* once close to 8 make a solution using pellets of naoh and slowly add the solution dropwise.

**To make the TE buffer**

* Measure out 1 mL 1M Tris-Cl (pH 8.0) and add to a 100 mL beaker
* Measure out 0.02 mL 0.5M EDTA (pH 8.0) and add to the beaker.
* Top up the solution to 100 mL by adding distilled water.
* Place the lid on the bottle and invert a few times to mix.
* To sterilise, autoclave the solution on a liquid cycle (20 min at 15 psi).
* Autoclaved everything and kept it at room temperature
(to do- label everything with new labels and store)

**Tuesday - 7/8/2023**

* Prepared IDT order google sheet: IDT order and resuspension details

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/spreadsheet-1.png)

* We got advice from Nishana ma'am and Swathi ma'am to use TE buffer to resuspend the oligos obtained from IDT

* TE buffer prep:

+ required final concentrations-

EDTA 0.1mM

Tris HCl- 10mM

###**This EDTA concentration is for oligo storage**

FIRST step, making 100 ml of 1M tris HCl and 0.5M EDTA

1M Tris HCl

Weigh out 12.11 g Tris(Tris buffer) and add to a 100 mL beaker

Measure out 80 mL of distilled water and add to the beaker

Add a magnetic flea and place on a magnetic stirring plate to mix the solution.

Add a pH meter into the solution to observe the pH

Slowly add concentrated hydrochloric acid (HCl) solution and make upto pH 8.

0.5M EDTA

Weigh out 18.61 g EDTA disodium salt, dihydrate and add to a 100 mL beaker.

Measure out 80 mL distilled water and add to the beaker.

Add a magnetic bead and place on a magnetic stirring plate to mix the solution. The EDTA salt will not go into solution until the pH reaches 8.0.

Add a pH meter into the solution to observe the pH.

To dissolve the salt, add sodium hydroxide (NaOH) pellets to the solution. Add a few pellets at a time and wait until the pellets have fully dissolved before adding more. It may take around 2 g(waay more) of NaOH pellets before the pH is at 8.0.

once close to 8 make a solution using pellets of naoh and slowly add the solution dropwise.

To make the TE buffer

Measure out 1 mL [1M Tris-Cl (pH 8.0)](https://toptipbio.com/tris-hcl-recipe/) and add to a 100 mL beaker

Measure out 0.02 mL [0.5M EDTA (pH 8.0)](https://toptipbio.com/0-5m-edta-ph-8-0-recipe/) and add to the beaker.

Top up the solution to 100 mL by adding distilled water.

Place the lid on the bottle and invert a few times to mix.

To sterilise, autoclave the solution on a liquid cycle (20 min at 15 psi).

Autoclaved everything and kept at room temperature:

* The prepared TE buffer had a pH of 8.7

**Tuesday - 7/11/2023**

* **Resuspension of oligos:**

+ We resuspended FRET aptamer cortisol (108288884), cDNA cortisol 1 (108288888), and cDNA cortisol 2 (108288889)

+ Oligo resuspension protocol:

* First, briefly centrifuge the tubes before opening.

* Resuspend in the prepared 1X TE buffer(10mM Tris HCl & 0.1mM EDTA), The tris Hcl acts as a buffer for the medium maintaining constant pH. The EDTA prevents the nuclear digestion of DNA.

* Prepare the recommended 100μM stock.

* If the aggregates are found after the addition of TE buffer, heat the mixture to 55 degree celsius for 1 to 5 mins

* Vortex thoroughly then briefly centrifuge

* If there are residual remains that are still found then run the mixture through Sephadex G-50 column

+ Using the above protocol, we prepared the stock of 100μM for each of the above mentioned oligos.

+ To prepare 10μM working concentration we used dilution calculator:

- Add 10μl of 100μM stock concentration in 90μl of TE buffer solution to get a final volume of 100μl of 10μM working concentration

+ To prepare 0.1μM and 0.5μM aliquot of FRET aptamer cortisol

- Add 2μl of 10μM working solution in 198μl of milliQ to get 200μl of 0.1 μM of FRET aptamer cortisol.

- Add 10μl of 10μM working solution in 190μl of milliQ to get 200μl of 0.5 μM of FRET aptamer cortisol.

* Measuring the fluorescence intensity of the 0.1μM and 0.5μM aliquots using a spectrofluorometer ( Fluorolog QM)

+ The FRET aptamer cortisol didn't show any considerable fluorescence for 0.1μM concentration, but a fluorescence peak was seen at 0.5μM concentration.

* Fluorolog Protocol:

+ We have a 200μl cuvette .

+ First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

+ Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl MilliQ in the cuvette to take the base reading.

+ Wipe the cuvette with tissue and place it in the spectrofluorometer.

+ Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.

+ Run the protocol.

+ Empty the cuvette and wash thoroughly with MilliQ.

+ Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

+ After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

**Wednesday - 7/12/2023**

* **Resuspension of oligos:**

+ We resuspended FRET aptamer cortisol (108288884), cDNA cortisol 1 (108288888), and cDNA cortisol 2 (108288889)

+ Oligo resuspension protocol:

* First, briefly centrifuge the tubes before opening.

* Resuspend in the prepared 1X TE buffer(10mM Tris HCl & 0.1mM EDTA), The tris HCl acts as a buffer for the medium maintaining constant pH. The EDTA prevents the nuclear digestion of DNA.

* Prepare the recommended 100μM stock.

* If the aggregates are found after the addition of TE buffer, heat the mixture to 55 degree celsius for 1 to 5 mins

* Vortex thoroughly then briefly centrifuge

* If there are residual remains that are still found then run the mixture through Sephadex G-50 column

+ Using the above protocol, we prepared the stock of 100μM for each of the above mentioned oligos.

+ To prepare 10μM working concentration we used dilution calculator:

- Add 10μl of 100μM stock concentration in 90μl of TE buffer solution to get a final volume of 100μl of 10μM working concentration

+ To prepare 0.1μM and 0.5μM aliquot of FRET aptamer cortisol

- Add 2μl of 10μM working solution in 198μl of milliQ to get 200μl of 0.1 μM of FRET aptamer cortisol.

- Add 10μl of 10μM working solution in 190μl of milliQ to get 200μl of 0.5 μM of FRET aptamer cortisol.

* Measuring the fluorescence intensity of the 0.1μM and 0.5μM aliquots using a spectrofluorometer ( Fluorolog QM)

+ The FRET aptamer cortisol didn't show any considerable fluorescence for 0.1μM concentration, but a fluorescence peak was seen at 0.5μM concentration.

* Fluorolog Protocol:

+ We have a 200μl cuvette .

+ First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

+ Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.

+ Wipe the cuvette with tissue and place it in the spectrofluorometer.

+ Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.

+ Run the protocol.

+ Empty the cuvette and wash thoroughly with MilliQ.

+ Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

+ After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

**Thrusday - 7/13/2023**

* While measuring fluorescence intensity, we got a peak for 0.5 μM FRET aptamer cortisol. So next, we wanted to see if 0.25μM conc will also give a peak.

* Made a 400μl 0.25μM FRET aptamer cortisol aliquot using 10μM working solution.

+ Take 10μl of 10μM FRET aptamer cortisol and add 390μl of MilliQ.

* Measured the fluorescence intensity of 0.25μM FRET aptamer cortisol using fluorolog and got fluorescence peak.

* Fluorolog Protocol:

+ Turn on the FluoroLog fluorometer and allow it to warm up for at least 15 minutes.

+ Attach the cuvette adaptor to the fluorometer.

+ We have a 200μl cuvette .

+ We need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

+ Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.

+ Wipe the cuvette with kim-tech wipes and place it in the spectrofluorometer.

+ Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 490nm.

+ Run the protocol.

+ Empty the cuvette and wash thoroughly with MilliQ.

+ Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

+ After taking all readings, thoroughly wash the cuvette with acetone, ethanol and MilliQ.

+ **Tips:**

- Make sure that the cuvette is clean and dry before using it.

- Use a solvent that is compatible with your sample.

- Fill the cuvette to the top to avoid bubbles.

- Make sure that the cuvette is properly seated in the cuvette adaptor and the fluorometer.

- Select the appropriate excitation and emission wavelengths for your sample.

- Set the scan speed and step size to match the requirements of your sample.

- Save the data to a computer for further analysis.

We decided to use 0.25μM FRET aptamer cortisol concentration constant for our experiments.

**Friday - 7/14/2023**

* **Incubation Time experiment for FRET aptamer cortisol and cDNA2 [1:1 concentration]**- to check the optimal time required for FRET aptamer cortisol and cDNA2 to bind at specific concentrations.

* Experiment Setup-

1) Take readings with 200μl MilliQ

2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.

3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.25μM cDNA2. Pipette mix it and measure change in fluorescencee every 10 mins for 90 min.

* Preparation of sample-

To make 200μl 0.25μM FRET aptamer cortisol, take 5μl of 10μM working concentration and 195μl MilliQ

To make 200μl 0.25μM cDNA2, take 5μl of 10μM working concentration and 195μl of MilliQ

* Fluorolog Protocol:

- We have a 200μl cuvette .

- First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

- Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.

- Wipe the cuvette with tissue and place it in the spectrofluorometer.

- Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.

- Run the protocol.

- Empty the cuvette and wash thoroughly with MilliQ.

- Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

- After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

.

##### WEEK 2

**Thursday - 13/7/2023**
*Resuspension in TE buffer and dilution in water(autoclaved)*

#LAF light off whenever using samples with fluorophore or quencher

###### miRNA 124-

* SEQ A - 11.9 nm - 119ul TE
* Seq- B - 38.3 - 383
* Seq C - 31.7 - 317
* miDNA 124 - 23.1 - 231

###### miRNA 132-

* SEQ A - 15.8 - 158
* Seq- B - 9.2 - 92
* miDNA 132 - 30.1 -301

###### Aliquot of 10 uM concentration and 200 ul volume made:-

* Take 20 ul of stock (100uM) and add 180 ul of autoclaved water.
* Cover the sequences having fluorophore and/or quencher with aluminium foil, and store in -20 degrees.

###### Building probe for 124

#using 10uM aliquot

* seq a = 15ul
* seq b= 15 ul
* seq c= 60 ul
* final conc = 1.667 uM
* final volume = 90 ul
* label = 124 probe, date- 13/7/2023
* Incubated at 48 degrees for 2 min and then at room temperature for 45 min.
* store in 4 degrees.

###### Make a 1 uM sample of the probe to load

* To get a 40 µL 1 µM solution, mix 24 µL of 1.667 µM oligo stock with 16 µL of water or TE buffer.

###### Making 2 uM solution for loading

* To make 20 µL 2 µM solution, mix 4 µL of 10 µM oligo stock with 16 µL of water or TE buffer.
  Made for 124 A, B And C.

###### Making Native PAGE for DNA gel

* 12 percent acrylamide bisacrylamide.
* To make TBE (100 ml)
* 10.8 g tris base
* 5.5 g boric acid
* 90 ml double-distilled H2O
* 4 ml 0.5 M EDTA solution (pH 8.0)
* To make poly acrylamide(19:1)
* 19 g acrylamide
* 1 g bisacrylamide
* 80 ml water
* stored in 4 degrees.

**Friday - 14/7/2023**

###### Making Native PAGE for DNA gel

* 12 percent acrylamide bisacrylamide.
* To make TBE (100 ml)
* 10.8 g tris base
* 5.5 g boric acid
* 90 ml double-distilled H2O
* 4 ml 0.5 M EDTA solution (pH 8.0)
* To make poly acrylamide(19:1)
* 19 g acrylamide
* 1 g bisacrylamide
* 80 ml water
* stored in 4 degrees.

Need 4 gel

Each

* Take 8 ml of the mixture.

* add 0.5 percentthe volume of APS = 0.5 percent of 8 = .04ml aps= 40 ul 10 percent APS

* TEMED- Take 1/10 of the volume of APS - 4ul (added 7)

add to a 15ml falcon , mix and pour fast.

wait 2 hour

**Made miDNA 132 aliquot - 10uM**

To get a **200 µL 10 µM** solution, mix **20 µL** of **100 µM** oligo stock with **180 µL** of water

**Made 124 probe + miDNA**

add 10ul 1uM probe and 2ul 10uM miDNA.

**Made 132 probe + miDNA**

add 13ul 1uM probe and 2ul 10uM miDNA.

To get a **30 µL 1 µM** solution, mix **18 µL** of **1.667 µM** oligo stock with **12 µL** of water or TE buffer.

**Make A+B for 124 and 132**

Add 8ul of 2uM A and B and incubate for 1 hour

Loading order- Labber (50bp), A, B, C, AB, probe, probe+miDNA

132- near glass

Run sample for 75 minutes at 55 v

Immerse the gel in syber safe for 30 min

store the used syber safe- labelled - used syber safe - in 50 ml flcon covered in aluminium foil

image

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/nanoprobe-2-16-7.jpg)

![]()

## inputs- use a 100 bp ladder just to show size

## remove gel and take image

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/nanoprobe-3-16-7.jpg)

![]()

##Increase conc of probe- load 15 ul each

**Saturday - 7/15/2023**

**Procured Low range DNA marker (50μg) from MR lab courtesy of our mentor Tejas.**

**Sunday - 7/16/2023**

* **NATIVE PAGE:**

* To run this Native gel we need to make the required concentration. of our oligos ie: the FRET aptamer and cDNA.

+ We decided to check the binding of the cDNA and aptamer, using the molar ratio of 1:3(aptamer : cDNA)

+ Sample prep

- cDNA 2 conc-- 9 microliters of 10 micromolar stock solution 21 micromolar (milliQ) for forming 30 microliter 3micromolar

- cDNA 2 conc-- 18 microliters of 10 micromolar stock solution 12 micromolar (milliQ) for forming 30 microliter 6micromolar

- FRET aptamer conc-- 12 microliters of 10 micromolar stock solution 48 micromolar (milliQ) for forming 60 microliter 2micromolar

- Incubate 30microlitre 2 micromolar FRET aptamer and 30 microlitre 6 micromolar cDNA for 60 min at 39°C to form the quantifier.

- Divided the quantifier volume in half and added cortisol (13:2) to the quantifier and incubate again for 1 hour at 39°C.

- Cortisol -- Made 0.2 mg/ml in 500 ml, then performed serial dilution to get 200ng/ml

Steps for serial dilution-- Label the tubes 1-7.

Add 100 microliters of the cortisol stock solution to tube 1.

Add 900 microliters of sterile water to tube 1.

Mix the contents of tube 1 thoroughly.

Transfer 100 microliters of the diluted cortisol solution from tube 1 to tube 2.

Add 900 microliters of sterile water to tube 2.

Mix the contents of tube 2 thoroughly.

Repeat steps 5-7 for tubes 3-6.

The final concentration of cortisol in tube 7 will be 200ng/ml.

- Add 10microlitre loading dye for 30microlitre sample.

Preparation of 12 percent acrylamide bisacrylamide.

To make TBE (100 ml)

+ 10.8 g tris base

+ 5.5 g boric acid

+ 90 ml double-distilled H2O

+ 4 ml 0.5 M EDTA solution (pH 8.0)

heated a bit

To make poly acrylamide(19:1)

+ 19 g acrylamide

+ 1 g bisacrylamide

+ 80 ml water

stored in 4 degree (B1 ABL)

**Friday - 7/14/2023**

Making APS ( 10 percent)

+ Weight 1 g of ammonium persulphate

+ add about 9.5 ml of sterile ddH2O

+ Vortex until fully dissolved

+ store at 4°C.

**Making gel mixture**

+ 30 ml polyacrylamide(19:1)

+ 10ml 10x TBE

+ 60 ml milliQ

Making the gel

+ Take 8 ml of the mixture.

+ add 0.5 percent the volume of APS = 0.5 percent of 8 = .04ml APS = 40 ul 10 percent APS

+ TEMED- Take 1/10 of the volume of APS - 4ul (added 7ul)

+ Wipe apparatus with ethanol(glass plate)

+ setup the apparatus(SDS page apparatus-biorad miniprotein tetra)

+ check if theres leakage using water

+ if no, remove water and add mixture

+ wait 2 hour

+ shift the gel to the machine.

+ add 1x TBE load sample

sample prep -

+ 15ul sample + 3ul loading dye

+ vortex and spindown

Loading order-

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Cortisol | Quantifier+ Cortisol | Quantifier | FRET Aptamer Cortisol | cDNA 2 cortisol |

+ The gel was run at 55V for 1 hour( check after 1 hr).

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/nanoprobe-1-14-7.jpg)

+ We did not get results- our cDNA ran out, either we need to run it at a higher gel concentration or for a shorter duration of time.

**Monday - 7/17/2023**

* **Incubation Time experiment for FRET aptamer cortisol and cDNA2 [1:3 concentration]**- to check the optimal time required for FRET aptamer cortisol and cDNA2 to bind at specific concentrations.

* Experiment Setup-

1) Take readings with 200μl MilliQ

2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.

3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.75μM cDNA2. Pipette mix it and measure change in fluorescencee every 10 mins for 90 min.

* Preparation of sample-

To make 200μl 0.25μM FRET aptamer cortisol, take 100μl of 0.5μM working concentration and 100μl MilliQ

To make 200μl 0.75μM cDNA2, take 15μl of 10μM working concentration and 185μl of MilliQ

* We received Serotonin, which is stored at 4°C.

**Tuesday - 7/18/2023**

* **Incubation Time experiment for FRET aptamer cortisol and cDNA2 [1:3 concentration] cont**- to check the optimal time required for FRET aptamer cortisol and cDNA2 to bind at specific concentrations.

+ Experiment Setup-

1) Take readings with 200μl MilliQ

2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.

3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.75μM cDNA2. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.

+ Preparation of sample- done on 7/17/2023

* **Incubation Time experiment for FRET aptamer cortisol and cDNA1 [1:3 concentration]**- to check the optimal time required for FRET aptamer cortisol and cDNA1 to bind at specific concentrations.

- Experiment Setup-

1) Take readings with 200μl MilliQ

2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.

3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.75μM cDNA1. Pipette mix it and measure the change in fluorescence every 10 mins for 120 min.

- Preparation of sample-

To make 200μl 0.25μM FRET aptamer cortisol, take 5μl of 10μM working concentration and 195μl MilliQ

To make 200μl 0.75μM cDNA1, take 15μl of 10μM working concentration and 185μl of MilliQ

* Fluorolog Protocol:

+ We have a 200μl cuvette .

+ First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

+ Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl MilliQ in the cuvette to take the base reading.

+ Wipe the cuvette with tissue and place it in the spectrofluorometer.

+ Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the absorbance peak was set at 495nm.

+ Run the protocol.

+ Empty the cuvette and wash thoroughly with MilliQ.

+ Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

+ After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

* NATIVE PAGE [Repeat]

* To run this Native gel we need to make the required concentration. of our oligos ie: the FRET aptamer and cDNA.

- We decided to check the binding of the cDNA and aptamer, using the molar ratio of 1:3(aptamer : cDNA)

- Sample prep

* cDNA 2 conc-- 3 microliters of 10 micromolar stock solution 12 micromolar (milliQ) for forming 30 microliter 2micromolar

* cDNA 2 conc-- 1 microliters of 100 micromolar stock solution 7 micromolar (milliQ) for forming 8 microliter 1micromolar

* cDNA 1 conc-- 3 microliters of 10 micromolar stock solution 12 micromolar (milliQ) for forming 30 microliter 2micromolar

* cDNA 1 conc-- 1 microliters of 100 micromolar stock solution 7 micromolar (milliQ) for forming 8 microliter 1micromolar

* FRET aptamer conc-- 6 microliters of 10 micromolar stock solution 9 micromolar (milliQ) for forming 15 microliter 4micromolar

* FRET aptamer conc-- 3 microliters of 10 micromolar stock solution 12 micromolar (milliQ) for forming 15 microliter 2micromolar

* Incubate 30microlitre 2 micromolar FRET aptamer and 30 microlitre 6 micromolar cDNA for 60 min at 39°C to form the quantifier.

* Divided the quantifier volume in half and added cortisol (13:2) to the quantifier and incubate again for 1 hour and 30 min at 39°C.

* Cortisol -- Made 0.2 mg/ml in 500 ml, then performed serial dilution to get 200ng/ml

Steps for serial dilution-- Label the tubes 1-7.

Add 100 microliters of the cortisol stock solution to tube 1.

Add 900 microliters of sterile water to tube 1.

Mix the contents of tube 1 thoroughly.

Transfer 100 microliters of the diluted cortisol solution from tube 1 to tube 2.

Add 900 microliters of sterile water to tube 2.

Mix the contents of tube 2 thoroughly.

Repeat steps 5-7 for tubes 3-6.

The final concentration of cortisol in tube 7 will be 200ng/ml.

* Add 10microlitre loading dye for 30microlitre sample.

Preparation of 12 percent acrylamide bisacrylamide.

To make TBE (100 ml)

- 10.8 g tris base

- 5.5 g boric acid

- 90 ml double-distilled H2O

- 4 ml 0.5 M EDTA solution (pH 8.0)

heated a bit

To make poly acrylamide(19:1)

- 19 g acrylamide

- 1 g bisacrylamide

- 80 ml water

stored in 4 degree (B1 ABL)

Making APS ( 10 percent)

- Weight 1 g of ammonium persulphate

- add about 9.5 ml of sterile ddH2O

- Vortex until fully dissolved

- store at 4°C.

**Making gel mixture**

- 30 ml polyacrylamide(19:1)

- 10ml 10x TBE

- 60 ml milliQ

Making the gel

- Take 8 ml of the mixture.

- add 0.5 percent the volume of APS = 0.5 percent of 8 = .04ml APS = 40 ul 10 percent APS

- TEMED- Take 1/10 of the volume of APS - 4ul (added 7ul)

- Wipe apparatus with ethanol(glass plate)

- setup the apparatus(SDS page apparatus-biorad miniprotein tetra)

- check if theres leakage using water

- if no, remove water and add mixture

- wait 2 hour

- shift the gel to the machine.

- add 1x TBE load sample

sample prep -

- 15ul sample + 3ul loading dye

- vortex and spindown

Loading order-

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| cDNA cortisol1 | cDNA cortisol2 | FRET aptamer cortisol | FRET Quantifier 1 | FRET Quantifier 2 | Quantifier 1 with cortisol | Quantifier 2 with cortisol |

- The gel was run at 55V for 1 hour ( check after 1 hr).

- We did not get results again.

**Friday - 7/21/2023**

* **Incubation Time experiment for FRET aptamer cortisol and cDNA1 [1:2 concentration]**- to check the optimal time required for FRET aptamer cortisol and cDNA1 to bind at specific concentrations.

* Experiment Setup-

1) Take readings with 200μl MilliQ

2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.

3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.5μM cDNA1. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.

* Preparation of sample-

To make 200μl 0.25μM FRET aptamer cortisol, take 5μl of 10μM working concentration and 195μl MilliQ.

To make 200μl 0.5μM cDNA1, take 10μl of 10μM working concentration and 190μl of MilliQ

* Fluorolog Protocol:

- We have a 200μl cuvette .

- First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

- Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.

- Wipe the cuvette with tissue and place it in the spectrofluorometer.

- Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.

- Run the protocol.

- Empty the cuvette and wash thoroughly with MilliQ.

- Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

- After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

.

##### WEEK 3

**Monday 7/17/2023**

**Make 124 probe**

##using 10uM aliquot

* seq a = 15ul

* seq b= 15 ul

* seq c= 60 ul

final conc = 1.667 uM

final volume = 90 ul

label = 132 probe 1.66uM, date

kept at 45 degree for 2 min and then at room temperature for 1 hour.

store in -4 degree.

**Make A+B**

##using 2uM aliquot

* seq a = 10 ul

* seq b= 10 ul

final conc = 1 uM

final volume = 20 ul

label = 124 AB, date

kept at 45 degree for 2 min and then at room temperature for 1 hour.

store in -4 degree.

**Make 20ul of 2uM A and B**

To get a **20 µL 2 µM** solution, mix **4 µL** of **10 µM** oligo stock with **16 µL** of water

**Tuesday 7/18/2023**

**To make 1uM 124 probe**

To get a **30 µL 1 µM** solution, mix **18 µL** of **1.667 µM** oligo stock with **12 µL** of water

**Make 124 probe + miDNA**

add 13ul 1uM probe and 2ul 10uM miDNA.

**Making APS (10 percent)**

* Weight 0.1 g of ammonium persulphate

* add about 0.95 ml of sterile ddH2O

* Vortex until fully dissolved

* store at 4°C.

**Made gel**

12 percent polyacrilamide solution- 8 ml

APS 10 percent- 40ul

TEMED - 7`ul

set for 2 hr

15ul sample- 3ul loading dye

run for 1 hour 55v

ladder used 100 BP

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/nanoprobe-4-20-7.jpg)

![]()

loading order- Ld, A,B,C,A+B, P,P+miDNA124.

Did not work

same result as the previous one.

**Wednesday 7/19/2023**

**Page for 124 - trial 4 (increase concentration of probe)**

**Make probe + miDNA 124**

15ul 1.66uM probe + 3ul 10uM miDNA

**Make 2uM A and B seq**

To get a 15 **µL 2 µM** solution, mix 3 **µL** of **10 µM** oligo stock with 12 **µL** of water

**Make A+B**

##using 2uM aliquot

* seq a = 10 ul

* seq b= 10 ul

incubate for 2 min in 45 degree and then for 1 hour in room temperature.

**Made gel**

12 percent polyacrilamide solution- 8 ml

APS 10 percent- 40ul

TEMED - 7`ul

set for 2 hr

15ul sample- 3ul loading dye

Run for 1 hour 55v

ladder used 100 BP

loading order- Ld, A,B,C,A+B, P,P+miDNA124.

run for 1 hour 55v

**Saturday - 7/22/2023**

* **Incubation Time experiment for FRET aptamer cortisol and cDNA2 [1:2 concentration]**- to check the optimal time required for FRET aptamer cortisol and cDNA2 to bind at specific concentrations.

* Experiment Setup-

1) Take readings with 200μl MilliQ

2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.

3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.5μM cDNA2. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.

* Preparation of sample-

To make 200μl 0.25μM FRET aptamer cortisol, take 5μl of 10μM working concentration and 195μl MilliQ.

To make 200μl 0.5μM cDNA2, take 10μl of 10μM working concentration and 190μl of MilliQ

* Fluorolog Protocol:

- We have a 200μl cuvette .

- First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

- Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.

- Wipe the cuvette with tissue and place it in the spectrofluorometer.

- Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.

- Run the protocol.

- Empty the cuvette and wash thoroughly with MilliQ.

- Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

- After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

**Monday - 7/24/2023**

* **Incubation Time experiment for FRET aptamer cortisol and cDNA2 and cDNA1 (1:4 concentration)** - to check the optimal time required for FRET aptamer cortisol with cDNA2 and cDNA1 to bind at specific concentrations.

+ Experiment Setup-

1) Take readings with 200μl MilliQ

2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.

3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 1μM cDNA2. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.

4)Take readings with 200μl MilliQ

5)Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.

6)Take 100μl 0.25μM FRET aptamer cortisol and 100μl 1μM cDNA1. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.

* Preparation of sample- [ for Incubation Time experiment for FRET aptamer cortisol and cDNA2 and cDNA1 (1:4 concentration) ]

To make 400μl 0.25μM FRET aptamer cortisol, take 10μl of 10μM working concentration and 390μl MilliQ.

To make 100μl 1μM cDNA1, take 10μl of 10μM working concentration and 90μl of MilliQ

To make 100μl 1μM cDNA2, take 10μl of 10μM working concentration and 90μl of MilliQ

* Fluorolog Protocol:

* We have a 200μl cuvette .

* First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

* Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.

* Wipe the cuvette with tissue and place it in the spectrofluorometer.

* Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.

* Run the protocol.

* Empty the cuvette and wash thoroughly with MilliQ.

* Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

* After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

.

##### WEEK 4

**Sunday - 7/23/2023**

**P+miDNA**

* 20 ul 1.66 uM probe + 5ul miDNA

**20ul 1.66 uM probe**

**30ul 2uM A and B**

* **30 µL 2 µM** solution, mix **6 µL** of **10 µM** oligo stock with **24 µL**

**10ul C 10uM**

**A+B**

* 10ul 2uM A and B

* 45 degree for 2 min and 1 hour in room temperature

**Making gel**

* 30 ml polyacrylamide(19:1)

* 10 ml 10x TBE

* 60 ml milliQ

**Making APS (10 percent)**

* Weight 0.1 g of ammonium persulphate

* add about 0.95 ml of sterile ddH2O

* Vortex until fully dissolved

* store at 4°C.

**make gel - 40ul aps, 7ul temed**

Add 3ul loading dye to all ,and load

55v 1 hour

**ladder10, a, b, c, ab, p ,p+mi**

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/nanoprobe-5-23-7.jpg)

![]()

**Monday - 7/24/2023**

![]()

**Monday - 8/4/2023**

* Autoclaved MilliQ

* We did resuspension of the following primers using TE buffer-

1) forward primer (108288891) - add 235μl of TE buffer

2) reverse primer (108288892) - add 296μl of TE buffer

3) gsα - we have 1000ng/μl, to make 10ng/μl, we add 100μl of TE buffer.

* Same Resuspension protocol was used.

+ Oligo resuspension protocol:

+ First, briefly centrifuge the tubes before opening.

+ Resuspend in the prepared 1X TE buffer(10mM Tris HCl & 0.1mM EDTA), The tris HCl acts as a buffer for the medium maintaining constant pH. The EDTA prevents the nuclear digestion of DNA.

+ Prepare the recommended 100μM stock.

+ If the aggregates are found after the addition of TE buffer, heat the mixture to 55 degree celsius for 1 to 5 mins

+ Vortex thoroughly then briefly centrifuge

+ If there are residual remains that are still found then run the mixture through Sephadex G-50 column

.

##### WEEK 5

**Friday - 8/11/2023**

PLATE READER STUFF

use water- 107 ul

100ul 0.5um probe +7um water

100ul 0.5um probe +7um midna

**200 µL 0.25 µM**
solution, mix
**30.1 µL**
of
**1.66 µM**
oligo stock with
**169.9 µL**

12 water

1a - water

1b - pbr

1c prb+dna

**Make 124 probe**

##using 10uM aliquot

* seq a = 15ul

* seq b= 15 ul

* seq c= 60 ul

final conc = 1.667 uM

final volume = 90 ul

label = 124 probe 1.66uM, date

kept at 45 degree for 2 min and then at room temperature for 1 hour.

store in -4 degree.

**Make 132 probe**

##using 10uM aliquot

* seq a = 15ul

* seq b= 15 ul

* seq c= 60 ul`

final conc = 1.667 uM

final volume = 90 ul

label = 132 probe 1.66uM, date

kept at 45 degree for 2 min and then at room temperature for 1 hour.

store in -4 degree.

To get a
**200 µL 0.25 µM**
solution, mix
**30.1 µL**
of
**1.66 µM**
oligo stock with
**169.9 µL**
of water.

**Monday - 8/7/2023**

9:30 am - Autoclaved Nuclease Free water

**Wednesday - 8/9/2023**

**RESUSPENSION OF DNA AND PRIMERS**

* To make 10ng/ul stock solution of gsα gene fragment, added 100 ul nuclease free water to TWIST tube containing 1000ng dried down DNA. Spin down, vortexed and stored in -20degree iGEM stock box

* To resuspend forward primers (23.5 nmol) - added 235 ul TE buffer. Spin down, vortexed and stored in -20 degree stock box. Final concentration - 100 uM

* To resuspend backward primers (29.6 nmol) - added 296 ul TE buffer. Spin down, vortexed and stored in -20 degree stock box. Final concentration - 100 uM

**Friday - 8/11/2023**

**PREPARATION OF WORKING STOCK**

* To make working stock of 100 ul of 0.2uM, added 0.2 uL of Forward primer stock to 99.8 uL milliQ

* To make working stock of 100 ul of 0.2uM, added 0.2 uL of Reverse primer stock to 99.8 uL milliQ

* To make working stock of 50 uL of 200 uM, added 0.25 ul of 10mM dNTPs to 49.75 ul milliQ

**Monday - 8/5/2023**

* We did Resuspension of the following oligos to prepare 100μM stock concentration.

1) FRET aptamer cortisol truncated- add 161μl of TE buffer.

2)cDNA truncated cortisol- add 383μl of TE buffer

* Same Resuspension protocol was used.

Oligo resuspension protocol:

* First, briefly centrifuge the tubes before opening.

* Resuspend in the prepared 1X TE buffer(10mM Tris HCl & 0.1mM EDTA), The tris HCl acts as a buffer for the medium maintaining constant pH. The EDTA prevents the nuclear digestion of DNA.

* Prepare the recommended 100μM stock.

* If the aggregates are found after the addition of TE buffer, heat the mixture to 55 degree celsius for 1 to 5 mins

* Vortex thoroughly then briefly centrifuge

* If there are residual remains that are still found, then run the mixture through Sephadex G-50 column

* We then prepared sample by performing dilutions using MilliQ.

1) To make 200μl 10μM FRET aptamer cortisol truncated, add 20μl of 100μM oligo and 180μl of MilliQ.

2) To make 200μl 0.25μM FRET aptamer cortisol truncated, add 5μl of 10μM oligo and 195μl of MilliQ.

3)To make 200μl 10μM cDNA truncated cortisol, add 20μl of 100μM oligo and 180μl of MilliQ.

4)To make 200μl 0.5μM cDNA cortisol truncated, add 10μl of 10μM oligo and 190μl of MilliQ.

* Incubation Time experiment for FRET aptamer cortisol truncated and cDNA truncated (1:2concentration) - to check the optimal time required for FRET aptamer cortisol truncated with cDNA trn to bind at specific concentrations.

* Experimental setup-

1) Take readings with 200μl MilliQ

2) Take 100μl 0.25μM FRET aptamer cortisol truncated and 100μl MilliQ. Pipette mix it and take reading.

3) Take 100μl 0.25μM FRET aptamer truncated cortisol and 100μl 0.5μM cDNA truncated cortisol. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.

* Fluorolog Protocol:

* We have a 200μl cuvette .

* First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

* Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.

* Wipe the cuvette with tissue and place it in the spectrofluorometer.

* Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.

* Run the protocol.

* Empty the cuvette and wash thoroughly with MilliQ.

* Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

* After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

**Thursday - 8/10/2023**

* Incubation Time experiment for FRET aptamer cortisol truncated and cDNA truncated (1:5concentration) - to check the optimal time required for FRET aptamer cortisol truncated with cDNA trn to bind at specific concentrations.

* Preparation of sample-

+ For FRET aptamertruncated 0.5 micro molar , add 20 microlitre of 10 micro molar stock in 190 microlitre milliQ to get 200microlitre sample.

+ For cdna truncated 2.5 micro molar , add 50 microlitre of 10 micro molar stock in 150 microlitre milliQ to get 200microlitre sample.

* Experimental setup-

1) Take readings with 200μl MilliQ

2) Take 100μl 0.5μM FRET aptamer cortisol truncated and 100μl MilliQ. Pipette mix it and take reading.

3) Take 100μl 0.5μM FRET aptamer truncated cortisol and 100μl 2.5μM cDNA truncated cortisol. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.

* Fluorolog Protocol:

+ We have a 200μl cuvette .

+ First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

+ Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl MilliQ in the cuvette to take the base reading.

+ Wipe the cuvette with tissue and place it in the spectrofluorometer.

+ Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.

+ Run the protocol.

+ Empty the cuvette and wash thoroughly with MilliQ.

+ Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

+ After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

**Friday - 8/11/2023**

* Incubation Time experiment for FRET aptamer cortisol and cDNA 1 (1:2 concentration) - to check the optimal time required for FRET aptamer cortisol with cDNA 1 to bind at specific concentrations.

* Preparation of sample-

- For FRET aptamer cortisol 0.25 micro molar , add 5 microlitre of 10 micro molar stock in 195 microlitre milliQ to get 200microlitre sample.

- For cdna 1 cortisol 0.5 micro molar , add 10 microlitre of 10 micro molar stock in 190 microlitre milliQ to get 200microlitre sample.

* Experimental setup-

1) Take readings with 200μl MilliQ

2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.

3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.5μM cDNA1 cortisol. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.

* Fluorolog Protocol:

+ We have a 200μl cuvette .

+ First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

+ Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl MilliQ in the cuvette to take the base reading.

+ Wipe the cuvette with tissue and place it in the spectrofluorometer.

+ Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.

+ Run the protocol.

+ Empty the cuvette and wash thoroughly with MilliQ.

+ Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

+ After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

##### WEEK 6

**Saturday - 8/12/2023**

**PCR AMPLIFICATION OF gsα GBLOCK**

|  |  |  |
| --- | --- | --- |
|  | Fnal concentration | Volume added |
| NEB Buffer |  | 2.5 uL |
| dNTPSs | 200uM | 0.5 uL |
| Forward primer | 0.2uM | 0.5 uL |
| Reverse primer | 0.2uM | 0.5 uL |
| Template DNA | 10ng/uL | 0.2 uL |
| NEB Taq polymerase |  | 0.125 uL |
| Nuclease free water |  | 20.675 uL |
| Total volume |  | 25 uL |

PCR protocol -saved under IGEMGSAP - ABL Thermocycler

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/pcr-amplification-of-gs-α-gblock.jpg)

![]()

Annealing temp - 57 degrees

**Sunday - 8/13/2023**

**AGAROSE GEL ELECTROPHORESIS**

To make a 2 percent gel (70 mL);

* 7 uL 10 X TBE in 63 uL milliQ for 60 mL 1X TBE

* Added 1.4 g low EEO agarose

* Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins

* Loaded 25uL sample + 5 uL loading dye

* Used two 1 kb ladder -

2.5 uL NEB ladder + 2 uL purple loading dye

5 ul Origin ladder (purple colour)

* Ran gel at 375 mA and 80 volts for 1 hour 30 mins

Gel imaged in the Gel Doc system - Filter 1

Saved under volume D > iGEM > iGEM 23 >GSAP pcr amp 1

**gsap pcr amp 1.scn**

No bands were observed.

**PCR AMPLIFICATION 2.0**

Used lab grade Taq polymerase and buffer from Satish lab

|  |  |  |
| --- | --- | --- |
| Buffer |  | 2.5 uL |
| dNTPSs | 200uM | 0.5 uL |
| Forward primer | 0.2uM | 0.5 uL |
| Reverse primer | 0.2uM | 0.5 uL |
| Template DNA | 10ng/uL | 0.2 uL |
| Taq polymerase |  | 0.5 uL |
| Nuclease free water |  | 22.8 uL |
| Total volume |  | 25 uL |

6:30 pm - Same PCR protocols in ABL Thermocycler

Ran for 2 h 18 mins

Stored in 4 degree

**Monday - 8/14/2023**

**AGAR GEL 2.0**

* Made 60 mL1x TBE buffer - 6mL TBE + 54 mL milliQ

* 0.6g low EEO agarose

* Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins

* Loaded 25uL sample + 5 uL loading dye

* 2.5 uL NEB ladder + 2 uL purple loading dye

* Ran gel at 375 mA and 80 volts for 1 hour 30 mins

NO BANDS OBSERVED.

**Wednesday - 8/16/2023**

Positive control (1.5kB) and primers procured from MR lab.

Annealing temp- 57 degrees

**GRADIENT PCR AMP 1.0**

Since discrepencies in calculated and given melting temperature

Made 5 tubes acc to PCR mixture 2.0 using lab grade Taq polymerase and buffer from MR lab labelled C,D,E,F,G

Made 2 tubes for positive control

* checked con

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| parts | Final con | Positive control A | Positive control B | C | D | E | F | G |
| Buffer |  | 1.25 uL | 2.5uL | 2.5 uL | 2.5 uL | 2.5 uL | 2.5 uL | 2.5 uL |
| dNTPSs | 200uM | 0.125 uL | 0.5uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL |
| Forward primer | 0.2uM | 0.125 uL | 0.5uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL |
| Reverse primer | 0.2uM | 0.125 uL | 0.5uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL |
| Template DNA | 10ng/uL | 0.2 uL | 0.2 | 0.2 uL | 0.2 uL | 0.2 uL | 0.2 uL | 0.2 uL |
| Taq polymerase |  | 0.125 uL | 0.5uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL |
| Nuclease free water |  | 13.05 uL | 22.8 uL | 22.8 uL | 22.8 uL | 22.8 uL | 22.8 uL | 22.8 uL |
| Total volume |  | 15 uL | 25 uL | 25 uL | 25 uL | 25 uL | 25 uL | 25 uL |
| Annealing temp |  | 57.1 | 57.1 | 57.1 | 58 | 61.2 | 62.5 | 63.1 |

**GRADIENT PCR 1.1**

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/gradient-pcr-1.jpg)

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/gradient-pcr-2.jpg)

**Thursday - 8/17/2023**

**GRADIENT PCR 1.1**

Added polymerase didn't have Mgcl2 - faulty polymerase.

After completing gradient pcr tried adding Mgcl2 to each tubes

Conc of Mgcl2 we had=25mM

Required conc of Mgcl2= 1.5mM

Quantity of each sample=25uL

To each tube( having 25uL sample) add:

* 1.8uL of mgcl2

* 3.2uL of nuclease free water.

Hence took 9uL Mgcl2(5 \* 1.8) and 16uL nuclease free water(5 \* 3.2), mixed them and added 5uL each in tubes B,C,D,E,F,G

To Sample A( Quantity= 15uL), add:

* .3uL Mgcl2

* 4.7uL nuclease free water

Ran gradient PCR again ( Temparature range-Gradient PCR 1.0)

Ran gel again

NO BANDS.( Adding Mgcl2 after PCR didn't work)

**Friday - 8/18/2023**

**GRADIENT PCR 2.0**

Taq polymerase with MgCl2 pre- added

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Parts | con | Positive control | H | I | J | K | L |
| Buffer |  | 2.5 uL | 2.5 uL | 2.5 uL | 2.5 uL | 2.5 uL | 2.5 uL |
| dNTPSs | 200uM | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL |
| Forward primer | 0.2uM | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL |
| Reverse primer | 0.2uM | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL |
| Template DNA | 10ng/uL | 0.5uL | 0.5uL | 0.5uL | 0.5uL | 0.5uL | 0.5uL |
| Taq polymerase |  | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL |
| Nuclease free water |  | 22.8 uL | 22.8 uL | 22.8 uL | 22.8 uL | 22.8 uL | 22.8 uL |
| Total volume |  | 25 uL | 25 uL | 25 uL | 25 uL | 25 uL | 25 uL |
| Annealing temp |  | 57.1 | 57.1 | 58 | 61.2 | 62.5 | 63.1 |

Same Mastercycler protocols as Gradient PCR 1.0

In addition, ran an extra PCR with the positive in ABL Thermocycler

|  |  |  |
| --- | --- | --- |
| parts | Con | M |
| Buffer |  | 2.5 uL |
| dNTPSs | 200uM | 1 uL |
| Forward primer | 0.2uM | 0.5 uL |
| Reverse primer | 0.2uM | 0.5 uL |
| Template DNA | 10ng/uL | 0.5uL |
| Taq polymerase |  | 0.5 uL |
| Nuclease free water |  | 19.5uL |
| Total volume |  | 25 uL |
| Annealing temp |  | 57 |

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/gradient-pcr-2-0.jpg)

**Ran AGAR GEL**

**Sunday - 8/13/2023**

* Incubation Time experiment for FRET aptamer cortisol and cDNA 1 (1:3 concentration) - to check the optimal time required for FRET aptamer cortisol with cDNA 1 to bind at specific concentrations.

* Preparation of sample-

+ For FRET aptamer0.25 micro molar , add 5 microlitre of 10 micro molar stock in 195 microlitre milliQ to get 200microlitre sample.

+ For cdna 1 0.75 micro molar , add 15 microlitre of 10 micro molar stock in 185 microlitre milliQ to get 200 microlitre sample.

* Experimental setup-

1) Take readings with 200μl MilliQ

2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.

3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.75μM cDNA1 cortisol. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.

+ Fluorolog Protocol:

+ We have a 200μl cuvette .

+ First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

+ Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl milliQ in the cuvette to take the base reading.

+ Wipe the cuvette with tissue and place it in the spectrofluorometer.

+ Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.

+ Run the protocol.

+ Empty the cuvette and wash thoroughly with MilliQ.

+ Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

+ After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

##### WEEK 7

**Saturday - 8/19/2023**

**Ran AGAR GEL**

* Made 60 mL1x TBE buffer - 6mL TBE + 54 mL milliQ

* 0.6g low EEO agarose

* Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins

* Loaded 25uL sample + 5 uL loading dye

* 2.5 uL NEB ladder + 2 uL purple loading dye

* Ran gel at 375 mA and 80 volts for 1 hour 30 mins

WE GOT THE BANDS

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/ran-agar-gel.png)

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/ran-agar-gel-1.png)

**ELUTION**

* Preheat heat block in 50 degrees

* Cut the gel and weigh it . For every 100mg take 200uL NT-1 ( Binding buffer )

Weight of gel= 600mg

NT-1 Buffer taken= 1200uL , in 1.5ml tube.

* Melted the gel (approx 15 min). Vortex it in every 5 min

* Took 2ml collecting tube and coloumn.Added 700uL of melted gel through sides of tube.

* Centrifuge it , 1000 rpm for 3 min

* After 3 min poured the solution in collecting tube back to coloumn

Repeat this thrice.

* After repeating thrice , stored the buffer in another tube in 4 degrees.

* Took 600uL wash buffer( NT-3) and added it into coloumn. Kept it undisturbed for 3 min

* After 3 min ,Centrifuge it in 2000rpm for 3 min

* After centrifuging discarded the wash buffer and repeated the step once more

* Then kept the tube in 13k rpm for 2 min - Dry spin

* Preheat 20uL of elution buffer in 50 degree

* Took 1.5ml autoclaved tube and added 10uL of elution buffer to coloumn

* Centrifuge it in 13k rpm for 2 min

* Added the remaining 10uL elution buffer to coloumn and centrifuge it in 13k rpm for 2 min

**NANODROP**

Got a conc of 6.1 ng/uL

**Monday - 8/21/2023**

**PCR AMPLIFICATION 3.0**

Did a final pcr

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | Final concentration |  |  | Volume added |
| NEB Buffer |  | 2.5 | 2.5 | 2.5 |
| dNTPs | 200uM | 1.5 | 1.5 | 1.5 |
| Forward primer | 0.2uM | 0.5 | 0.5 | 0.5 |
| Reverse primer | 0.2uM | 0.5 | 0.5 | 0.5 |
| Template DNA | 10ng/uL | 0.5 | 0.5 | 0.5 |
| NEB Taq polymerase |  | 0.5 | 0.5 | 0.5 |
| Nuclease free water |  |  |  |  |
| Total volume |  | 25 uL | 25 uL | 25 uL |

**Tuesday - 8/22/2023**

Made 500mL TBE buffer stock from 50X TBE

**Ran AGAR GEL**

* Made 60 mL1x TBE buffer - 6mL TBE + 54 mL milliQ

* 0.6g low EEO agarose

* Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins

* Loaded 25uL sample + 5 uL loading dye

* 2.5 uL NEB ladder + 2 uL purple loading dye

* Ran gel at 375 mA and 80 volts for 1 hour 30 mins

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/ran-agar-gel-5-9.png)

**ELUTION 1.0**

* Preheat heat block in 50 degrees

* Cut the gel and weigh it . For every 100mg take 200uL NT-1 ( Binding buffer )

Weight of gel= 406mg

NT-1 Buffer taken= 800uL , in 1.5ml tube.

* Melted the gel (approx 15 min). Vortex it in every 5 min

* Took 2ml collecting tube and coloumn.Added 700uL of melted gel through sides of tube.

* Centrifuge it , 1000 rpm for 3 min

* After 3 min poured the solution in collecting tube back to coloumn

Repeat this thrice.

* After repeating thrice , stored the buffer in another tube (Tube-1) in 4 degrees.

* Repeated the same steps with remaining NT-1 buffer( of 800uL)

Stored buffer in Tube-1 in 4 degrees.

* Took 600uL wash buffer( NT-3) and added it into coloumn. Kept it undisturbed for 3 min

* After 3 min ,Centrifuge it in 2000rpm for 3 min

* After centrifuging discarded the wash buffer and repeated the step once more

* Then kept the tube in 13k rpm for 2 min - Dry spin

* Preheat 20uL of elution buffer in 50 degree

* Took 1.5ml autoclaved tube and added 10uL of elution buffer to coloumn

* Centrifuge it in 13k rpm for 2 min

* Added the remaining 10uL elution buffer to coloumn and centrifuge it in 13k rpm for 2 min

**Wednesday - 8/23/2023**

**NANODROP**

Got a conc of 8.1 ng/uL

**RESTRICTION DIGESTION**

Restriction mixture

R1 containing DNA insert - Amplified gsα of 8.1ng / uL con

2. R2 containing Vector - pET19b 128ng/uL

|  |  |  |
| --- | --- | --- |
|  | R1 | R2 |
| To be cut | 15 uL- 121ng |  |
| Cutsmart buffer | 2 uL | 2 uL |
| BamH1-HF | 0.2 uL | 0.2 uL |
| Nde1 HF | 0.3 uL | 0.3 uL |
| Nuclease free water | 2.5 uL | 2.5 uL |
| Total volume | 20 uL | 20 uL |

Restriction enzymes;

* Keep on ice when not in the freezer - minimize exposure to temp above -20degree

* Should be the last component added to reaction

* Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube. Follow with a quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction

Incubated at 37 degree overnight

Stored in -20 degrees post incubation

##### WEEK 8

**Thursday- 31/8/2023**

**Making 0.5mg/ml solution of magnetic particle**

 Take 100ul and add 900ul water

0.001= 1 milligram/ml

 Take 500ul add 500ul water

0.5mg/ml

**TEM sample preparation (new batch)**

 Take 100 ul 0.5 mg/ml nanoparticle and add 400 ul 20 percent ethanol-water mixture.

take 6 u from this and drop it on a copper grid.

Let it dry in the open

Once dried, store in an Eppendorf tube.

**Friday - 1/9/2023**

**TEM ANALYSIS**

  Particle size still low but chose to continue with this.

**Sunday - 8/27/2023**

* Native page - repeat

* To run this Native gel we need to make the required concentration. of our oligos ie: the FRET aptamer and cDNA.

- We decided to check the binding of the cDNA and aptamer, using the molar ratio of 1:3(aptamer : cDNA)

- Sample prep

* cDNA 1 conc-- 9 microliters of 10 micromolar stock solution 21 micromolar (milliQ) for forming 30 microliter 3micromolar

* cDNA 1 conc-- 18 microliters of 10 micromolar stock solution 12 micromolar (milliQ) for forming 30 microliter 6micromolar

* FRET aptamer conc-- 12 microliters of 10 micromolar stock solution 48 micromolar (milliQ) for forming 60 microliter 2micromolar

* FRET aptamer conc-- 24 microliters of 10 micromolar stock solution 36 micromolar (milliQ) for forming 60 microliter 4micromolar

* Incubate 30microlitre 2 micromolar FRET aptamer and 30 microlitre 6 micromolar cDNA for 60 min at 39°C to form the quantifier.

* Divided the quantifier volume in half and added cortisol (13:2) to the quantifier and incubate again for 1 hour at 39°C.

* Cortisol -- Made 0.2 mg/ml in 500 ml, then performed serial dilution to get 200ng/ml

Steps for serial dilution-- Label the tubes 1-7.

Add 100 microliters of the cortisol stock solution to tube 1.

Add 900 microliters of sterile water to tube 1.

Mix the contents of tube 1 thoroughly.

Transfer 100 microliters of the diluted cortisol solution from tube 1 to tube 2.

Add 900 microliters of sterile water to tube 2.

Mix the contents of tube 2 thoroughly.

Repeat steps 5-7 for tubes 3-6.

The final concentration of cortisol in tube 7 will be 200ng/ml.

* Add 10microlitre loading dye for 30microlitre sample.

Preparation of 12 percent acrylamide bisacrylamide.

To make TBE (100 ml)

- 10.8 g tris base

- 5.5 g boric acid

- 90 ml double-distilled H2O

- 4 ml 0.5 M EDTA solution (pH 8.0)

heated a bit

To make poly acrylamide(19:1)

- 19 g acrylamide

- 1 g bisacrylamide

- 80 ml water

stored in 4 degree (B1 ABL)

Making APS ( 10 percent)

- Weight 1 g of ammonium persulphate

- add about 9.5 ml of sterile ddH2O

- Vortex until fully dissolved

- store at 4°C.

**Making gel mixture**

- 30 ml polyacrylamide(19:1)

- 10ml 10x TBE

- 60 ml milliQ

Making the gel

- Take 8 ml of the mixture.

- add 0.5 percent the volume of APS = 0.5 percent of 8 = .04ml APS = 40 ul 10 percent APS

- TEMED- Take 1/10 of the volume of APS - 4ul (added 7ul)

- Wipe apparatus with ethanol(glass plate)

- setup the apparatus(SDS page apparatus-biorad miniprotein tetra)

- check if theres leakage using water

- if no, remove water and add mixture

- wait 2 hour

- shift the gel to the machine.

- add 1x TBE load sample

sample prep -

- 15ul sample + 3ul loading dye

- vortex and spindown

Loading order-

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| ladder | FRET Aptamer | cDNA 1 | cortisol | quantifier | quantifier +cortisol |

- The gel was run at 55V for 1 hour( check after 1 hr).

- we still did not get good results, cdna ran out again.

##### WEEK 9

**Tuesday - 9/5/2023**

**making MES buffer (15 mM)**

 Measure 0.319g MES monohydrate

make upto 100 ml

 Adjust ph to 6 using 10 N NaOH (fast change in pH)(6.1)

 store in -20

10 N NaOH - Mix 4g pellet in 8 ml water.

**Friday - 9/8/2023**

**Running DLS - new batch**

To run DLS take 50 ul 0.5 mg nanoparticle solution and add 450 ul and vortex

Add the sample to the cuvette and run.

-21 mV - stable carboxyl groups proven

**Monday - 9/4/2023**

**RESTRICTION DIGESTION**

Restriction mixture

R1 containing DNA insert - Amplified gsα of 8.1ng / uL con

2. R2 containing Vector - pET19b 128ng/uL

|  |  |  |
| --- | --- | --- |
|  | R1 | R2 |
| To be cut | 15 uL- 121ng |  |
| Cutsmart buffer | 2 uL | 2 uL |
| BamH1-HF | 0.2 uL | 0.2 uL |
| Nde1 HF | 0.3 uL | 0.3 uL |
| Nuclease free water | 2.5 uL | 2.5 uL |
| Total volume | 20 uL | 20 uL |

Restriction enzymes;

* Keep on ice when not in the freezer - minimize exposure to temp above -20degree

* Should be the last component added to reaction

* Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube. Follow with a quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction

Incubated at 37 degree overnight

Stored in -20 degrees post incubation

**Tuesday - 9/5/2023**

**Ran AGAR gel**

* Made 60 mL1x TBE buffer - 6mL TBE + 54 mL milliQ

* 0.6g low EEO agarose

* Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins

* Loaded 25uL sample + 5 uL loading dye

* 2.5 uL NEB ladder + 2 uL purple loading dye

* Ran gel at 375 mA and 80 volts for 1 hour 30 mins

Band for plasmid was visible

Band for insert wasn't visible- INSERT CON INSUFFICIENT.

**ELUTION (Plasmid)**

Elution 1.0

Weight of gel= 260mg

Amount of NT 1 added=500uL

**NANODROP(Plasmid)**

Got a conc of 8ng/uL

**PCR**

To amplify insert

Optimizing for high yield - increasing primer concentration, inreasing number of cycles

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Buffer |  | 2.5 uL | 2.5 uL | 2.5 uL |
| dNTPSs | 200uM | 1 uL | 1 uL | 1 uL |
| Forward primer | 0.2uM | 1 uL | 1 uL | 1 uL |
| Reverse primer | 0.2uM | 1 uL | 1 uL | 1 uL |
| Template DNA | 10ng/uL | 0.5 uL | 0.5 uL | 0.5 uL |
| Taq polymerase |  | 0.5 uL | 0.5 uL | 0.5 uL |
| Nuclease free water |  | 18.5 uL | 18.5 uL | 18.5 uL |
| Total volume |  | 25 uL | 25 uL | 25 uL |

BAND NOT VISIBLE

**Wednesday - 9/6/2023**

**AMPLIFICATION PCR**

To amplify insert

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Buffer |  | 2.5uL |  |  |
| dNTPSs | 200uM | 1.5 uL |  |  |
| Forward primer | 0.2uM | 0.5 |  |  |
| Reverse primer | 0.2uM | 0.5 |  |  |
| Template DNA | 10ng/uL | 1uL |  |  |
| Taq polymerase |  | 0.5 uL |  |  |
| Nuclease free water |  | 18.5uL |  |  |
| Total volume |  | 25 uL |  |  |

**Ran AGAR gel**

* Made 50 mL 1x TBE buffer - 5mL TBE + 45 mL milliQ

* 0.5g low EEO agarose

* Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins

* Loaded 25uL sample + 5 uL loading dye

* 2.5 uL NEB ladder + 2 uL purple loading dye

* Ran gel at 375 mA and 80 volts for 1 hour 30 mins

**Thursday - 9/7/2023**

**ELUTION (insert)**

+ Elution 1.0 done

+ Weight of gel - 253mg

+ Amount of NT1 added 600uL

**NANODROP**

con -4.2 ng/uL

**LIGATION**

|  |  |  |
| --- | --- | --- |
|  | Conc | L1 |
| Inser | 4.2 ng/uL | 4.5uL |
| Vector | 8ng/uL | 8uL |
| T4 ligase |  | 1uL |
| T4 ligation buffer |  | 2uL |
| Nuclease fee water |  | 4.5uL |
| Total volume |  | 20uL |

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/ligation-6-9.png)

**LB Agar Plate Prep**

+ 0.9 g agar-agar power and 1.25g LB Broth and made it up to 50ml

+ Autoclaved at 121 degree for 15min

+ After autoclaving, aga was cooled to between 45°C and 50°C before pouring the plates to minimize the amount of condensation that forms

+ Added 50ul of 100mg/ml amp to the LB Agar before plating

+ 2 plates made and stored in 4 degree fridge

**Friday - 9/8/2023**

**Freshly prepared LB- growth media**

+ 0.25g LB broth in 10ml distilled H2O

+ Autoclaved at 121 degree for 15 min

**Transformation**

+ DH5 α comp cells obtained from -80 freezer and kept in ice for 15- 20 min

+ Keep an empty 2ml tube on ice

+ Take 100ul of comp cells into 2ml tubes, on ice

+ Add 5ul of plasmid DNA to comp cells. Don't mix. Flick it one time and tap on the counter 3-4 times

+ Incubate in ice for 30min.

+ Heat shock at 42 degree for 100sec and incubate in ice for 10 min.

+ Do no mix the sample at any of the above steps.

+ Add 800 ul LB into this mixture

+ Incubate in shaker at 37 degree 220 rpm for 1:30 hr

+ Pipette out 700 ul supernatent

+ Resuspend the cells in remaining 200ul and plate the entire sample

+ Plates kept for overnight incubation at 37 degree

( to be checked at 8:30 pm)

**Plating**

+ Plated 100 ul of comp cells and 200 ul of sample

+ Comp cells labelled - iGEM'23 gsα (1) negative control 8/9/23

+ IGEM'23 gsα (1) transformants 8/9/23

**Saturday - 9/2/2023**

* **Preparation of samples**- FRET aptamerCort truncated sensitivity experiment

* Different concentration of cortisol -:

10ng/ml - 5μl of 200ng/ml cort in 95μl Mq

50ng/ml- 25μl of 200ng/ml cort in 75μl Mq

100ng/ml- 50μl of 200ng/ml cort in 50μl Mq

150ng/ml-75μl of 200ng/ml cort in 15μl Mq

200ng/ml- take 100μl

250ng/ml-12.5μl of 0.02mg/ml cort in 87.5μl Mq

300ng/ml-15μl of 0.02mg/ml cort in 85μl Mq

350ng/ml-17.5μl of 0.02mg/ml cort in 82.5μl Mq

400ng/ml-20μl of 0.02mg/ml cort in 80μl Mq

* FRET aptamertruncated cortisol - 0.5μM 600μl- take 30μl of 10μM sample in 570μl Mq

* CDNA truncated cortisol- 1μM 600μl - take 60μl of 10μM sample in 540μl Mq

* We had three controls- Mq, only FRET aptamer, only cDNA

* We wanted the final volume of each well to be 150μl- so we took 50μl of FRET aptamer +50μl cDNA + 50μl cortisol of specific concentration

* **Order of samples in plate with the well number:**

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| MQ | FRET aptamer | cDNA | FRET aptamer +cDNA + 0ng/ml cortisol | FRET aptamer +cDNA + 10ng/ml cortisol | FRET aptamer +cDNA + 50 ng/ml cortisol | FRET aptamer +cDNA + 100ng/ml cortisol | FRET aptamer +cDNA + 150 ng/ml cortisol | FRET aptamer +cDNA + 200ng/ml cortisol | FRET aptamer +cDNA + 250 ng/ml cortisol | FRET aptamer +cDNA + 300ng/ml cortisol | FRET aptamer +cDNA + 350ng/ml cortisol | FRET aptamer +cDNA + 400ng/ml cortisol |
| A1 | B1 | C1 | H1 | D1 | E1 | F1 | G1 | A2 | B2 | C2 | D2 | E2 |

* Plate reader protocol for sensitivity experiments-

Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.

Add 100μl MilliQ as control in the first well.

50μl APTAMER + 50μl MilliQ was added and pipette mixed in the next well as second control.

50μl CDNA + 50μl MilliQ was added and pipette mixed in the next well as third control.

Add 50μl APTAMER + 50μl cDNA in the next well and pipette mix it. ( molar ratio of aptamer: cDNA is kept constant )

Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].

Keep the plate in the plate reader. Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.

After 90 min, take out the plate and add different concentrations of cortisol, from 0 to 400ng/ml, in consecutive wells.

Make all the wells upto 150μl.

Again take fluoroscence readings with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 60 minutes.

After 60 min, take out the plate.

Analyze the collected data and plot the graph.

**Monday - 9/4/2023**

* Aptasensor formation experiment for FRETserotonin aptamer and cDNA 1 (all concentrations) - to check the optimal time required for FRET aptamer serotonin with cDNA 1 to bind at specific concentrations.

+ Preparation of sample-

- For FRET aptamer0.25 micro molar , add 12.5 microlitre of 10 micro molar stock in 487.5 microlitre milliQ to get 500 microlitre sample.

- For different concentrations of cdna 1

| 0.25μM | 0.50μM | 0.75μM | 1μM | 1.25μM | Molar concentration |
| --- | --- | --- | --- | --- | --- |
| 1.875 | 3.75 | 5.625 | 7.5 | 9.375 | 10 micromolar cDNA sample |
| 73.175 | 71.25 | 69.375 | 67.5 | 65.625 | Milli Q |

Order of sample- It was added in the 5th column.

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| MQ | FRET aptamer serotonin | 1:1 | 1:2 | 1:3 | 1:4 | 1:5 |

* Plate reader protocol for aptasensor formation experiment-

Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.

Add 200μl MilliQ as control in the first well.

100μl APTAMER + 100μl MilliQ was added and pipette mixed in the next well as second control.

Add 100μl APTAMER + 100μl cDNA in the next well and pipette mix it.

Keep increasing concentrations of cDNA in each well. We used concentrations from 1:1 to 1:5 of aptamer:cDNA.

Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].

Keep the plate in the plate reader Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.

After 90 min, take out the plate.

Analyze the collected data and plot the graph.

**Tuesday - 9/5/2023**

* Aptasensor formation experiment for FRETserotonin aptamer and cDNA 2 (all concentrations) - to check the optimal time required for FRET aptamer serotonin with cDNA 2 to bind at specific concentrations.

+ Preparation of sample-

- For FRET aptamer0.25 micro molar , add 25 microlitre of 10 micro molar stock in 975 microlitre milliQ to get 1000 microlitre sample.

- For different concentrations of cdna 2

| 0.25μM | 0.50μM | 0.75μM | 1μM | 1.25μM | Molar concentration |
| --- | --- | --- | --- | --- | --- |
| 3.75 | 7.5 | 11.25 | 15 | 18.75 | 10 micromolar cDNA sample |
| 146.25 | 142.5 | 138.75 | 135 | 131.25 | Milli Q |

Order of sample- It was added in the 6th and 7th column replicates.

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| MQ | FRET aptamer serotonin | 1:1 | 1:2 | 1:3 | 1:4 | 1:5 |

* Plate reader protocol for aptasensor formation experiment-

Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.

Add 200μl MilliQ as control in the first well.

100μl APTAMER + 100μl MilliQ was added and pipette mixed in the next well as second control.

Add 100μl APTAMER + 100μl cDNA in the next well and pipette mix it.

Keep increasing concentrations of cDNA in each well. We used concentrations from 1:1 to 1:5 of aptamer:cDNA.

Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].

Keep the plate in the plate reader Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.

After 90 min, take out the plate.

Analyze the collected data and plot the graph.

* Aptasensor formation experiment for FRET cortisol aptamer and cDNA 1 and cDNA 2 (all concentrations) - to check the optimal time required for FRET aptamer cortisol with cDNA to bind at specific concentrations.

- Preparation of sample-

* For FRET aptamer0.25 micro molar , add 35 microlitre of 10 micro molar stock in 1365 microlitre milliQ to get 1400 microlitre sample.

* For different concentrations of cdna 2 (150microlitre) with replicate -

| 0.25μM | 0.50μM | 0.75μM | 1μM | 1.25μM | Molar concentration |
| --- | --- | --- | --- | --- | --- |
| 3.75 | 7.5 | 11.25 | 15 | 18.75 | 10 micromolar cDNA sample |
| 146.25 | 142.5 | 138.75 | 135 | 131.25 | Milli Q |

* For different concentrations of cdna 1 (75microlitre) -

| 0.25μM | 0.50μM | 0.75μM | 1μM | 1.25μM | Molar concentration |
| --- | --- | --- | --- | --- | --- |
| 1.875 | 3.75 | 5.625 | 7.5 | 9.375 | 10 micromolar cDNA sample |
| 73.175 | 71.25 | 69.375 | 67.5 | 65.625 | Milli Q |

Order of sample- It was added in the 1st (FRET cort+cDNA 1 ), 3rd(FRET cort +cDNA 2) and 5th (FRET cort+ cDNA 2 ) column.

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| MQ | FRET aptamer serotonin | 1:1 | 1:2 | 1:3 | 1:4 | 1:5 |

+ Plate reader protocol for aptasensor formation experiment-

Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.

Add 200μl MilliQ as control in the first well.

100μl APTAMER + 100μl MilliQ was added and pipette mixed in the next well as second control.

Add 100μl APTAMER + 100μl cDNA in the next well and pipette mix it.

Keep increasing concentrations of cDNA in each well. We used concentrations from 1:1 to 1:5 of aptamer:cDNA.

Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].

Keep the plate in the plate reader Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.

After 90 min, take out the plate.

Analyze the collected data and plot the graph.

**Wednesday - 9/6/2023**

* Aptasensor formation experiment for FRET cortisol aptamer and cDNA 1 (all concentrations) with replicates - to check the optimal time required for FRET aptamer cortisol with cDNA 1 to bind at specific concentrations.

+ Preparation of sample-

- For FRET aptamer0.25 micro molar , add 23.75 microlitre of 10 micro molar stock in 926.25 microlitre milliQ to get 950 microlitre sample.

- For different concentrations of cdna 2

| 0.25μM | 0.50μM | 0.75μM | 1μM | 1.25μM | Molar concentration |
| --- | --- | --- | --- | --- | --- |
| 3.75 | 7.5 | 11.25 | 15 | 18.75 | 10 micromolar cDNA sample |
| 146.25 | 142.5 | 138.75 | 135 | 131.25 | Milli Q |

Order of sample- It was added in the 10th and 12th column (replicates).

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| MQ | FRET aptamer serotonin | 1:1 | 1:2 | 1:3 | 1:4 | 1:5 |

* Plate reader protocol for aptasensor formation experiment-

Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.

Add 200μl MilliQ as control in the first well.

100μl APTAMER + 100μl MilliQ was added and pipette mixed in the next well as second control.

Add 100μl APTAMER + 100μl cDNA in the next well and pipette mix it.

Keep increasing concentrations of cDNA in each well. We used concentrations from 1:1 to 1:5 of aptamer:cDNA.

Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].

Keep the plate in the plate reader Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.

After 90 min, take out the plate.

Analyze the collected data and plot the graph.

**Friday - 9/8/2023**

* Aptasensor formation experiment for FRET truncated cortisol aptamer and cDNA 1 truncated (all concentrations), and serotonin aptamer with cDNA1 with replicates - to check the optimal time required for FRET aptamer with cDNA to bind at specific concentrations.

+ Preparation of sample-

- For FRET aptamertrn 0.5 micro molar , add 47.5 microlitre of 10 micro molar stock in 902.5microlitre milliQ to get 950 microlitre sample.

- For FRET aptamerser 0.25 micro molar , add 12.5 microlitre of 10 micro molar stock in 487.5microlitre milliQ to get 500 microlitre sample.

- For different concentrations of cdna 1 ser-

| 0.25μM | 0.50μM | 0.75μM | 1μM | 1.25μM | Molar concentration |
| --- | --- | --- | --- | --- | --- |
| 1.875 | 3.75 | 5.625 | 7.5 | 9.375 | 10 micromolar cDNA sample |
| 73.175 | 71.25 | 69.375 | 67.5 | 65.625 | Milli Q |

- For different concentrations of CDNA truncated- (with replicate)

| 0.25μM | 0.50μM | 0.75μM | 1μM | 1.25μM | Molar concentration |
| --- | --- | --- | --- | --- | --- |
| 3.75 | 7.5 | 11.25 | 15 | 18.75 | 10 micromolar cDNA sample |
| 146.25 | 142.5 | 138.75 | 135 | 131.25 | Milli Q |

Order of sample- It was added in the 1st and 3rd column ( FRET aptamer trn cDNA trn replicates), 5th column (FRET aptamer serotonin cDNA 1)

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| MQ | FRET aptamer serotonin | 1:1 | 1:2 | 1:3 | 1:4 | 1:5 |

* Plate reader protocol for aptasensor formation experiment-

Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.

Add 200μl MilliQ as control in the first well.

100μl APTAMER + 100μl MilliQ was added and pipette mixed in the next well as second control.

Add 100μl APTAMER + 100μl cDNA in the next well and pipette mix it.

Keep increasing concentrations of cDNA in each well. We used concentrations from 1:1 to 1:5 of aptamer:cDNA.

Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].

Keep the plate in the plate reader Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.

After 90 min, take out the plate.

Analyze the collected data and plot the graph.

##### WEEK 10

**Sunday - 10/9/2023**

**Making 50mg /ml particles-**

add 0.5g particles to 10 ml solution

**To make TE buffer with 5mM mg2+ conc** (using the TE buffer used for resuspension).

0.1 g MgCl2 into 100 ml TE (pH-8 )

**Make 132a and b and 124 a and b**

To get a 500 µL 200 nM solution, mix 10 µL of 10 µM oligo stock with 490 µL of water or TE buffer.

 for C To get a 1000 µL 600 nM solution, mix 60 µL of 10 µM oligo stock with 940 µL of water or TE buffer.

**make c working stock**

To get a 200 µL 10 µM solution, mix 20 µL of 100 µM oligo stock with 180 µL of water or TE buffer.

**Tuesday - 12/9/2023**

To get a 100 µL 0.5 µM solution of A,B, mix 5 µL of 10 µM oligo stock with 95 µL of water or TE buffer.

**attachment of sequence b to particle**

* 1 ml 50 mg magnetic nanoparticle + 1 ml MES wash (this step avoids agglomeration)

* absorb using magnet

* remove supernatant

* repeat wash

* absorb and remove supernatant

* add 100 ul mes and 100 ul EDS - blend for 30 min at room temperature

* remove supernatant

* add 200 ul dilute b (200nm conc)

* keep in mixer overnight at room temperature
* remove supernatant

* add 200 ul dilute a (200nm conc)

* add 200 ul dilute c (600nm conc)

* incubate for 1 hr

* remove supernatant

* add 50 ul TE buffer

* wash with it once, remove the supernatant

* add 50 ul TE buffer and it's done

**Saturday - 9/9/2023**

**LB AGAR PLATE PREP**

prepared and poured 10 new LB Agar plates

**TBE BUFFER STOCK PREPARED**

500 mL from 50X TBE

**Sunday - 9/10/2023**

2 colonies observed after 24 h incubation

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/2-colonies-observed-after-24-h-incubation.png)

**MASTERPLATE PREPARATION**

* Switched on the LAF and cleaned the working space with 70% Ethanol after sterilization.

* A colony on the transformation plate is gently touched with an autoclaved pipette tip and immediately

placed in contact with the surface of the new masterplate and spread over a small area. Even pressure is applied to the tip.

* The pipette tip is discarded into the pre prepared PCR tubes in order to run a colony PCR

* Master plates are labeled with date and colonies 1 & 2

**COLONY PCR**

* -Three mastermix tubes prepared and labelled - Control, C1 , C2

* Pipette tubes after plating colony 1 and 2 discarded into respective tubes. Control didn't have any pipette tubes.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | con | Control | C1 | C2 |
| NEB Buffer |  | 2.5 uL | 2.5 uL | 2.5 uL |
| dNTPSs | 200uM | 1.5uL | 1.5uL | 1.5uL |
| Forward primer | 0.2uM | 0.5 uL | 0.5 uL | 0.5 uL |
| Reverse primer | 0.2uM | 0.5 uL | 0.5 uL | 0.5 uL |
| Origin Taq polymerase |  | 0.5 uL | 0.5 uL | 0.5 uL |
| Nuclease free water |  | 19.5 uL | 19.5 uL | 19.5 uL |
| Total volume |  | 25 uL | 25 uL | 25 uL |

Thermocycler protocols saved under IGEMGSAP

**AGAR GEL ELECTROPHORESIS**

* Made 60 mL1x TBE buffer - 6mL TBE + 54 mL milliQ

* 0.6g low EEO agarose

* Melt for 1 min 30 secs, streaked with 2 uL EtBr, left to solidify for 45 mins

* Loaded 25uL sample + 5 uL loading dye

* 2.5 uL NEB ladder + 2 uL purple loading dye

* Ran gel at 90 v, 375 mA for 1 h 30 mins

No bands observed -TRANSFORMATION 1.0 didn't work.

**INSERT CHEMIDOC IMAGE**

**10 sep colony pcr.scn**

**COLONY PCR 2.0**

**Monday - 9/11/2023**

**COLONY PCR 2.0**

Did colony pcr again along with positive controls to check PCR conditions

+ cp 1- colony 1

+ cp2- colony 2

+ P(T)- Positive control from thejus

+ P(G)-Positive Control gsα

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  |  | CP1 | CP2 | P(G) | P(T) |
| NEB Buffer |  | 2.5 uL | 2.5 uL | 2.5 uL |  |
| dNTPSs | 200uM | 1.5 uL | 1.5 uL | 1.5 uL | 1.5 uL |
| Forward primer | 0.2uM | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL |
| Reverse primer | 0.2uM | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL |
| Origin Taq polymerase |  | 0.5 uL | 0.5 uL | 0.5 uL | 0.5 uL |
| Template |  | - | - | 0.2 uL | 0.5uL |
| Nuclease free water |  | 19.5 uL | 19.5 uL | 19.5 uL | 19.5 uL |
| Total volume |  | 25 uL | 25 uL | 25 uL | 25 uL |

Contamination was observed in prepared masterplates after 24 h - might be owing to improper working conditions in the LAF- faulty airflow

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/contamination-was-observed-in-prepared-masterplates-after-24-h.jpg)

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/contamination-was-observed-in-prepared-masterplates-after-24-h-1.jpg)

**Tuesday - 9/12/2023**

**TRANSFORMATION 1.1**

- Repeated transformation 1.0 procedure

- Used Comp cells from Tejas instead

- negative control=- 100ul of comp cells and transformants

**Wednesday - 9/13/2023**

Did **gsα PCR AMPLIFICATION**

* Prepared 5 tubes

Ran **AGAROSE GEL ELECTRPHORESIS**

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/agarose-gel-electrphoresis-12-9.png)

**ELUTION**- 3 tubes

gsα 1 - 5.6ng/uL

gsα 2- 5.86 ng/uL

gsα 3 - 7.14 ng/uL

**RESTRICTION DIGESTION**

* Prepared two tubes - one each containing insert and vector respectively. both are cut using the same restriction enzymes which are nde1 and bamh1

* For gsα - tube 3

* For vector - sandhya lab

* For restriction digestion and cutsmart buffer - natesh lab

|  |  |  |
| --- | --- | --- |
|  | INSERT gsα TUBE 3 | VECTOR PET 19B |
| To be cut | 19 uL - 135 ng | add around 150 ng of whatever con of vector they give and make total reaction upto 25uL |
| Cutsmart buffer | 2 uL | 2 uL |
| BamH1-HF | 0.2 uL | 0.2 uL |
| Nde1 HF | 0.3 uL | 0.3 uL |
| Nuclease free water | 3.5uL |  |
| Total volume | 25uL | 25uL |

Labelled the dry bath with sticky note, asking them not to turn off the machine.

**Thursday - 9/14/2023**

Restriction digestion finished

Ran **AGAROSE GEL ELECTROPHORESIS 1.0** gel - got bands

**ELUTION**

* 166 mg insert, added400 uL NT1

* 186mg vector, added 400 uL NT1

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/agarose-gel-electrphoresis-13-9.png)

**NANODROP**

* Insert - 6.48 ng/uL

* Vector- 12.17 ng/uL

Checked transformation plate after 24 hours - no colonies

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/hecked-transformation-plate-after-24-hours-no-colon15-9.png)

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/plate-14-9.png)

**Friday - 9/15/2023**

**LIGATION**

reaction set up at 4:45 am

Ligation finishes at 9 PM

|  |  |  |
| --- | --- | --- |
|  | L2 | Positive control P |
| Insert | 8uL (50ng) | - |
| Vector | 4.5uL (53ng) | 4,5uL |
| T4 buffer | 2 uL | 2uL |
| T4 Ligase | 1uL | 1uL |
| Nucelase free water | 4.5uL | 12.5uL |
| Total volume | 20uL | 20uL |

**LIGATION PCR**

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | L2a | L2b | INSERT | Vector |
| Ligation mixture ( 1:3 vector:insert ratio - | 1uL | 1uL | 0.5uL | 0.2uL |
| FP (4uM) | vector FP-- 1.25uL | insert FP - 1.25uL | 1.25 uL | 1.25 uL |
| RP (4uM) | insert RP - 1.25uL | vector RP-1.25uL | 1.25 uL | 1.25 uL |
| Taq polymerase | 0.5uL | 0.5uL | 0.5uL | 0.5uL |
| Taq pol buffer | 2.5uL | 2.5uL | 2.5uL | 2.5uL |
| dNTPS | 1uL | 1uL | 1uL | 1uL |
| Nuclease free water | 17.5 | 17.5 | 18 | 18.3 |
| Total volume | 25uL | 25uL | 25uL | 25uL |

* Got RF and FP from Sandhya ma'am lab ( Keerthana Chechi provided)

* RG 3 is forwards primer and RG 4 is reverse primer ( concentration 4uM )

**TRANSFORMATION 2.0**

Since transformation 1.1 didn't work, We changed the volume and plasmid dna from 5 to 2 ul and used comp cells from Tejas lab

**Sunday - 9/10/2023**

* **Preparation of samples**- sensitivity experiment- FRET aptamer serotonin and cDNA 1 and cDNA 2, FRET aptamer cortisol cDNA1 and FRET truncated aptamer cortisol cDNA truncated

* Different concentration of cortisol -: (\*5)

10ng/ml - 5μl of 200ng/ml cort in 95μl Mq

50ng/ml- 25μl of 200ng/ml cort in 75μl Mq

100ng/ml- 50μl of 200ng/ml cort in 50μl Mq

150ng/ml-75μl of 200ng/ml cort in 15μl Mq

200ng/ml- take 100μl

250ng/ml-12.5μl of 0.02mg/ml cort in 87.5μl Mq

300ng/ml-15μl of 0.02mg/ml cort in 85μl Mq

350ng/ml-17.5μl of 0.02mg/ml cort in 82.5μl Mq

400ng/ml-20μl of 0.02mg/ml cort in 80μl Mq

* Different concentration of serotonin -: (\*5)

10ng/ml - 5μl of 200ng/ml cort in 95μl Mq

50ng/ml- 25μl of 200ng/ml cort in 75μl Mq

100ng/ml- 50μl of 200ng/ml cort in 50μl Mq

150ng/ml-75μl of 200ng/ml cort in 15μl Mq

200ng/ml- take 100μl

250ng/ml-12.5μl of 0.02mg/ml cort in 87.5μl Mq

300ng/ml-15μl of 0.02mg/ml cort in 85μl Mq

350ng/ml-17.5μl of 0.02mg/ml cort in 82.5μl Mq

400ng/ml-20μl of 0.02mg/ml cort in 80μl Mq

* FRET aptamertruncated cortisol - 0.5μM 1700μl- take 85μl of 10μM sample in 1615μl Mq

* FRET aptamer cortisol - 0.25μM 1700μl- take 42.5μl of 10μM sample in 1657.5μl Mq

* FRET aptamer serotonin- 0.25μM 3400μl- take 85μl of 10μM sample in 3315μl Mq

* CDNA truncated cortisol- 1μM 1550μl - take 116.25μl of 10μM sample in 1433.75μl Mq

* CDNA1 cortisol- 0.75μM 1550μl - take 155μl of 10μM sample in 1345μl Mq

* CDNA1 serotonin - 0.75μM 1600μl - take 120μl of 10μM sample in 1480μl Mq

* We had three controls- Mq, only FRET aptamer, only cDNA

* We wanted the final volume of each well to be 150μl- so we took 50μl of FRET aptamer +50μl cDNA + 50μl cortisol of specific concentration

* **Order of samples in plate with the well number: ROW A and C ( FRET Ser and cDNA 1), ROW E and G(FRET Ser and cDNA 2), ROW B and D ( FRET Cort and cDNA1), ROW F and H ( FRET truncated cortisol and cDNA truncated)**

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| MQ | FRET aptamer | cDNA | FRET aptamer +cDNA + 0ng/ml cortisol | FRET aptamer +cDNA + 10ng/ml cortisol | FRET aptamer +cDNA + 50 ng/ml cortisol | FRET aptamer +cDNA + 100ng/ml cortisol | FRET aptamer +cDNA + 150 ng/ml cortisol | FRET aptamer +cDNA + 200ng/ml cortisol | FRET aptamer +cDNA + 250 ng/ml cortisol | FRET aptamer +cDNA + 300ng/ml cortisol | FRET aptamer +cDNA + 350ng/ml cortisol | FRET aptamer +cDNA + 400ng/ml cortisol |
| A1 | B1 | C1 | H1 | D1 | E1 | F1 | G1 | A2 | B2 | C2 | D2 | E2 |

* Plate reader protocol for sensitivity experiments-

Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.

Add 100μl MilliQ as control in the first well.

50μl APTAMER + 50μl MilliQ was added and pipette mixed in the next well as second control.

50μl CDNA + 50μl MilliQ was added and pipette mixed in the next well as third control.

Add 50μl APTAMER + 50μl cDNA in the next well and pipette mix it. ( molar ratio of aptamer: cDNA is kept constant )

Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].

Keep the plate in the plate reader. Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.

After 90 min, take out the plate and add different concentrations of cortisol, from 0 to 400ng/ml, in consecutive wells.

Make all the wells upto 150μl.

Again take fluoroscence readings with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 60 minutes.

After 60 min, take out the plate.

Analyze the collected data and plot the graph.

* **Incubation Time experiment for FRET aptamer cortisol and cDNA truncated (1:3 concentration)** - to check the optimal time required for FRET aptamer cortisol with cDNA truncated to bind at specific concentrations.

* Preparation of sample-

- For FRET aptamer cortisol 0.25 micro molar , add 5 microlitre of 10 micro molar stock in 195 microlitre milliQ to get 200microlitre sample.

- For cdna truncated cortisol 0.75 micro molar , add 7.5 microlitre of 10 micro molar stock in 92.5 microlitre milliQ to get 100microlitre sample.

* Experimental setup-

1) Take readings with 200μl MilliQ

2) Take 100μl 0.25μM FRET aptamer cortisol and 100μl MilliQ. Pipette mix it and take reading.

3) Take 100μl 0.25μM FRET aptamer cortisol and 100μl 0.5μM cDNAtruncated cortisol. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.

* Fluorolog Protocol:

+ We have a 200μl cuvette .

+ First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

+ Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl MilliQ in the cuvette to take the base reading.

+ Wipe the cuvette with tissue and place it in the spectrofluorometer.

+ Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.

+ Run the protocol.

+ Empty the cuvette and wash thoroughly with MilliQ.

+ Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

+ After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

**Wednesday - 9/13/2023**

Sensitivity of FRET aptamer Cortisol-

* **Preparation of samples**- FRET aptamerCort sensitivity experiment

* Different concentration of cortisol -:

10ng/ml - 5μl of 200ng/ml cort in 95μl Mq

50ng/ml- 25μl of 200ng/ml cort in 75μl Mq

100ng/ml- 50μl of 200ng/ml cort in 50μl Mq

150ng/ml-75μl of 200ng/ml cort in 15μl Mq

200ng/ml- take 100μl

250ng/ml-12.5μl of 0.02mg/ml cort in 87.5μl Mq

300ng/ml-15μl of 0.02mg/ml cort in 85μl Mq

350ng/ml-17.5μl of 0.02mg/ml cort in 82.5μl Mq

400ng/ml-20μl of 0.02mg/ml cort in 80μl Mq

* FRET aptamer cortisol - 0.25μM 1700μl- take 4.25μl of 100μM sample in 1695.75μl Mq

* CDNA1 cortisol- 0.75μM 1600μl - take 12μl of 100μM sample in 1588μl Mq

* We had two controls- Mq, only FRET aptamer

* We wanted the final volume of each well to be 150μl- so we took 50μl of FRET aptamer +50μl cDNA + 50μl cortisol of specific concentration

* **Order of samples in plate with the well number:**

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| MQ | FRET aptamer | cDNA | FRET aptamer +cDNA + 0ng/ml cortisol | FRET aptamer +cDNA + 10ng/ml cortisol | FRET aptamer +cDNA + 50 ng/ml cortisol | FRET aptamer +cDNA + 100ng/ml cortisol | FRET aptamer +cDNA + 150 ng/ml cortisol | FRET aptamer +cDNA + 200ng/ml cortisol | FRET aptamer +cDNA + 250 ng/ml cortisol | FRET aptamer +cDNA + 300ng/ml cortisol | FRET aptamer +cDNA + 350ng/ml cortisol | FRET aptamer +cDNA + 400ng/ml cortisol |
| A1 | B1 | C1 | D1 | E1 | F1 | G1 | H1 | A2 | B2 | C2 | D2 | E2 |

* Plate reader protocol for sensitivity experiments-

Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.

Add 100μl MilliQ as control in the first well.

50μl APTAMER + 50μl MilliQ was added and pipette mixed in the next well as second control.

50μl CDNA + 50μl MilliQ was added and pipette mixed in the next well as third control.

Add 50μl APTAMER + 50μl cDNA in the next well and pipette mix it. ( molar ratio of aptamer: cDNA is kept constant )

Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].

Keep the plate in the plate reader. Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.

After 90 min, take out the plate and add different concentrations of cortisol, from 0 to 400ng/ml, in consecutive wells.

Make all the wells upto 150μl.

Again take fluoroscence readings with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 60 minutes.

After 60 min, take out the plate.

Analyze the collected data and plot the graph.

**Thursday - 9/14/2023**

Biomarker mix experiment protocol-

* Plate reader protocol for biomarker mix experiments-

* Take a 96 well plate (We can add a maximum of 200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.

* Add 150μl MilliQ as control in the first well.

* Add 75μl APTAMER + 75μl cDNA in the next six wells and pipette mix it. ( molar ratio of aptamer: cDNA is kept constant )

* Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].

* Keep the plate in the plate reader. Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes (for formation of the quantifier)

* After 90 min, take out the plate and add different biomarkers ( 50μl - In order )

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| MQ | Quantifier | Quantifier+ Serotonin | Quantifier+ Cortisol | Quantifier+ miDNA 132 | Quantifier+ miDNA 124 | Mix |

* Mix- 12.5μl of each biomarker.

* Make the final volume of all the wells 200μl.

* Again take fluorescence readings with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 60 minutes.

* After 60 min, take out the plate.

* Analyze the collected data and plot the graph.

Experimental setup-

* ROW E and G for FRET aptamer serotonin and cDNA1 (with replicate)

**Friday - 9/15/2023**

Biomarker mix for FRET aptamerser and cDNA1 quantifier and FRET aptamertrn and cDNA trn quantifier

Experimental setup-

* ROW Band D for FRET aptamer serotonin and cDNA1 (with replicate)

* ROW F and H for FRET aptamer truncated cortisol and cDNA truncated (with replicate)

Biomarker mix experiment protocol-

* Plate reader protocol for biomarker mix experiments-

* Take a 96 well plate (We can add a maximum of 200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.

* Add 150μl MilliQ as control in the first well.

* Add 75μl APTAMER + 75μl cDNA in the next six wells and pipette mix it. ( molar ratio of aptamer: cDNA is kept constant )

* Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].

* Keep the plate in the plate reader. Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes (for formation of the quantifier)

* After 90 min, take out the plate and add different biomarkers ( 50μl - In order )

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| MQ | Quantifier | Quantifier+ Serotonin | Quantifier+ Cortisol | Quantifier+ miDNA 132 | Quantifier+ miDNA 124 | Mix |

* Mix- 12.5μl of each biomarker.

* Make the final volume of all the wells 200μl.

* Again take fluorescence readings with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 60 minutes.

* After 60 min, take out the plate.

* Analyze the collected data and plot the graph.

**Fluorolog FRET aptamer serotonin and cdna trn to check formation of aptasensor-**

* **Incubation Time experiment for FRET aptamer serotonin and cDNA truncated (1:3 concentration)** - to check the optimal time required for FRET aptamer serotonin with cDNA truncated to bind at specific concentrations.

* Preparation of sample-

- For FRET aptamer serotonin 0.25 micro molar , add 5 microlitre of 10 micro molar stock in 195 microlitre milliQ to get 200microlitre sample.

- For cdna truncated cortisol 0.75 micro molar , add 7.5 microlitre of 10 micro molar stock in 92.5 microlitre milliQ to get 100microlitre sample.

* Experimental setup-

1) Take readings with 200μl MilliQ

2) Take 100μl 0.25μM FRET aptamer serotonin and 100μl MilliQ. Pipette mix it and take reading.

3) Take 100μl 0.25μM FRET aptamer serotonin and 100μl 0.75μM cDNAtruncated cortisol. Pipette mix it and measure the change in fluorescence every 10 mins for 90 min.

* Fluorolog Protocol:

+ We have a 200μl cuvette .

+ First, we need to wash the cuvette. Wash the cuvette thoroughly first with milliQ, then with acetone and finally with ethanol.

+ Now, wash the cuvette with autoclaved Milli-Q once before taking 200μl MilliQ in the cuvette to take the base reading.

+ Wipe the cuvette with tissue and place it in the spectrofluorometer.

+ Set the protocol on the computer. We took a range scan to get emission readings between 495nm -700nm when the excitation peak was set at 495nm.

+ Run the protocol.

+ Empty the cuvette and wash thoroughly with MilliQ.

+ Add 200μl of sample in the cuvette. Wipe with a tissue and run the same protocol on the computer.

+ After taking all readings, wash the cuvette with acetone, ethanol and MilliQ thoroughly.

##### WEEK 11

**Tuesday - 9/19/2023**

Transformation plates observed after 24 h incubation

Colonies were observed in both plates

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/plate-19-9.png)

![](https://static.igem.wiki/teams/4693/wiki/notebook-images/plate-2-19-9.png)

##### WEEK 12

Exam Week :)

##### WEEK 13

**Monday- 2/10/2023**

Plate reader

c3 - te (108 ul)

c4 132 - (100p + 8 te)

c5 132 + dna (100p + 8 dna)

c6 124 - (100p + 8 te)

c7 - 124+dna (100p + 8 dna)

**Excitation scan**- 490 emission

**Building probe -**

**making more seq a and b of 200 nm**

To get a 1000 µL 200 nM solution, mix 20 µL of 10 µM oligo stock with 980 µL TE buffer

**for 600 nm C**

 To get a 1000 µL 600 nM solution, mix 60 µL of 10 µM oligo stock with 940 µL of TE buffer.

**Tuesday- 3/10/2023**

* 1 ml 1 mg/ml magnetic nanoparticle + 1 ml MES wash (this step avoids agglomeration)

* centrifuge, 8000 rpm for 5 min

* remove supernatant

* repeat wash (use a magnet instead of a centrifuge here)

* absorb and remove supernatant

* add 100 ul mes and 100 ul EDS

* -blend for 30 minutes at room temperature

* remove supernatant

* add 200 ul dilute b (200nm conc)

* Keep in mixer overnight at room temperature.

* remove supernatant

* add 200 ul dilute a (200nm conc)

* add 200 ul dilute c (600nm conc)

* incubate for 1 hr

* remove supernatant

* add 50 ul TE buffer

* was with it once, remove the supernatant

* add 50 ul TE buffer and it's done

 To make different conc

 500, 100, 50, 10 nm, 1 nm, 0.5nm, 0.1 nm, 0.01 nm = 10 pm

 500 µL 0.5 µM solution, mix 25 µL of 10 µM oligo stock with 475 µL of water or TE buffer.

* 500 µL 0.1 µM solution, mix 100 µL of 0.5 µM oligo stock with 400 µL of water or TE buffer
* 250 ul from 0.1 um+ 250 ul te = 50 nm
* 100 ul from 50nm and 400 ul te = 10 nm
* 50 ul 10nm 450 ul te = 1 nm
* 50 ul 1nm 450 ul te = 0.1 nm
* 50 ul 0.1nm 450 ul te = 0.01 nm = 10 pm

**Plate loading**

D3 - probe 132+ 75ul te, D4 - 500 nm , D5- 100 nm, D6 - 50nm, D7 - 10nm, D8- 1 nm, D9 - 0.1nm, D10 - 0.01 nm

E3 - probe 124 + 75 ul te, E4 - 500 nm , E5- 100 nm, E6 - 50nm, E7 - 10nm, E8- 1 nm, E9 - 0.1nm, E10 - 0.01 nm

in everything probe = 25 ul

midna = 75 ul

measurements to be taken at excitation - 490 nm

Emission - 500 - 600 nm (based on previous data)

**Wednesday - 10/4/2023**

ITC Experiment- [FRET aptamer serotonin 0.25μM + cDNA1 serotonin 1μM- 1:1 conc]

Sample preparation-

* FRET Apt Ser- for 200μl of 0.25μM - add 5μl of 10μM sample in 195μl MQ

* cDNA1 Ser- for 80μl of 1μM- add 8μl of 10μM sample in 72μl MQ

ITC Protocol-

* Prepare the sample and ligand solutions.

- The sample solution should contain the macromolecule whose binding affinity is being measured which in our case was the aptamer. The ligand solution should contain the molecule that is binding to the sample which for experiment was the cDNA. The concentrations of the sample and ligand solutions should be chosen carefully to ensure that the binding reaction is in the measurable range of the ITC instrument.

- The concentration we used was 0.25 micromolar and prepared 200 microliters of the aptamer to be inserted into the sample well, and 1 micromolar cDNA 80 microliter in volume was prepared to be titrated into the sample well.

+ Load the sample and ligand solutions into the ITC instrument. The ITC instrument has two cells: a sample cell and a reference cell. The sample solution is loaded into the sample cell and the ligand solution is loaded into the reference cell.

+ Equilibrate the sample and ligand solutions. The ITC instrument will equilibrate the sample and ligand solutions to the same temperature. This is important to ensure that the heat changes measured during the binding reaction are due only to the binding event.

+ Titrate the ligand solution into the sample cell. The ITC instrument will titrate the ligand solution into the sample cell in a series of small injections. The heat released or absorbed during each injection will be measured.

**Friday - 10/6/2023**

ITC Experiment- [FRET aptamer serotonin 0.25μM + cDNA2 serotonin 2μM- 1:2 conc]

Sample preparation-

* FRET Apt Ser- for 200μl of 0.25μM - add 5μl of 10μM sample in 195μl MQ

* cDNA2 Ser- for 80μl of 2μM- add 16μl of 10μM sample in 64μl MQ

ITC Protocol-

* Prepare the sample and ligand solutions.

- The sample solution should contain the macromolecule whose binding affinity is being measured which in our case was the aptamer. The ligand solution should contain the molecule that is binding to the sample which for experiment was the cDNA. The concentrations of the sample and ligand solutions should be chosen carefully to ensure that the binding reaction is in the measurable range of the ITC instrument.

- The concentration we used was 0.25 micromolar and prepared 200 microliters of the FRET aptamer ser to be inserted into the sample well, and 2 micromolar cDNA 2 80 microliter in volume was prepared to be titrated into the sample well.

+ Load the sample and ligand solutions into the ITC instrument. The ITC instrument has two cells: a sample cell and a reference cell. The sample solution is loaded into the sample cell and the ligand solution is loaded into the reference cell.

+ Equilibrate the sample and ligand solutions. The ITC instrument will equilibrate the sample and ligand solutions to the same temperature. This is important to ensure that the heat changes measured during the binding reaction are due only to the binding event.

+ Titrate the ligand solution into the sample cell. The ITC instrument will titrate the ligand solution into the sample cell in a series of small injections. The heat released or absorbed during each injection will be measured.

* **Aptasensor formation experiment** for FRET aptamer cortisol and cDNA 1 and cDNA2 (all concentrations), with replicates - to check the optimal time required for FRET aptamer with cDNA to bind at specific concentrations.

+ Preparation of sample-

- For FRET aptamercort 0.25 micro molar , add 6 microlitre of 100 micro molar stock in 2394microlitre TE buffer to get 2400microlitre sample.

- For different concentrations of cdna 1 and cDNA2 -

| 0.25μM | 0.50μM | 0.75μM | 1μM | 1.25μM | Molar concentration |
| --- | --- | --- | --- | --- | --- |
| 2.5 | 5 | 7.5 | 10 | 12.5 | 10 micromolar cDNA sample |
| 97.5 | 95 | 92.5 | 90 | 87.5 | TE buffer |

Order of sample- It was added in the 9th and 10thcolumn ( FRET aptamer cortisol and cDNA 1 with replicates), 11th and 12th column (FRET aptamer cortisol cDNA 2 with replicates)

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| MQ | FRET aptamer serotonin | 1:1 | 1:2 | 1:3 | 1:4 | 1:5 |

* Plate reader protocol for aptasensor formation experiment-

Take a 96 well plate (We can add a maximum of  200μl of sample in each well of the plate). We used the Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] for our experiments.

Add 200μl MilliQ as control in the first well.

100μl APTAMER + 100μl MilliQ was added and pipette mixed in the next well as second control.

Add 100μl APTAMER + 100μl cDNA in the next well and pipette mix it.

Keep increasing concentrations of cDNA in each well. We used concentrations from 1:1 to 1:5 of aptamer:cDNA.

Turn on the Tecan plate reader. [Infinite M plex TECAN plate reader].

Keep the plate in the plate reader Set the protocol to read fluorescence with excitation at 490 nm and emission at 520 nm. We took 4 readings per well at every 10 minute interval for 90 minutes.

After 90 min, take out the plate.

Analyze the collected data and plot the graph.

##### WEEK 14

**Sunday- 8/10/2023**

* 1 ml 1 mg/ml magnetic nanoparticle + 1 ml MES wash (this step avoids agglomeration)

* centrifuge, 8000 rpm for 5 min

* remove supernatant

* repeat wash (use a magnet instead of a centrifuge here)

* absorb and remove supernatant

* add 100 ul mes and 100 ul EDS

* -blend for 30 minutes at room temperature

* remove supernatant

* add 200 ul dilute b (200nm conc)

* Keep in mixer overnight at room temperature.

* remove supernatant

* add 200 ul dilute a (200nm conc)

* add 200 ul dilute c (600nm conc)

* incubate for 1 hr

* remove supernatant

* add 50 ul TE buffer

* was with it once, remove the supernatant

* add 50 ul TE buffer and it's done

Take four eppendorf tubes and label them as:

1. Probe 124 + TE Buffer (control)
2. Probe 124 + miDNA 132(10nM)
3. Probe 124 + miDNA 124(10nM)
4. Probe 124 + miDNA 124-2 (10nM)

Similarly prepare four eppendorf tubes for probe 132

* Add 25uL probe in each tube
* Add 75uL of of TE, miDNA 132, miDNA 124, miDNA 124-2, into respective labelled tubes at the time of loading
* Do the same for probes 132
* Take 100ul of the samples and pour it into the wells for plate reading

**Plate loading**

F3 - 132P + TE; F4 - 132P + miDNA 124; F5 - 132P + miDNA 132; F6 - 132P + miDNA 132-2

G3- 124P + TE; G4 - 124P + miDNA 124; F5- 124P + miDNA 132; G6 - 124P + miDNA 124-2

Measurement to be taken at an excitation of 490nm

Emission at 500-600nm range

**Saturday - 10/7/2023**

ITC Experiment- [FRET aptamer serotonin 0.25μM + serotonin ]

Sample preparation-

* FRET Apt Ser- for 200μl of 0.25μM - add 5μl of 10μM sample in 195μl MQ

ITC Protocol-

* Prepare the sample and ligand solutions.

- The sample solution should contain the macromolecule whose binding affinity is being measured which in our case was the aptamer. The ligand solution should contain the molecule that is binding to the sample which for experiment was the cDNA. The concentrations of the sample and ligand solutions should be chosen carefully to ensure that the binding reaction is in the measurable range of the ITC instrument.

- The concentration we used was 0.25 micromolar and prepared 200 microliters of the aptamer to be inserted into the sample well, and 2000ng/ml of serotonin 80 microliter in volume was prepared to be titrated into the sample well.

+ Load the sample and ligand solutions into the ITC instrument. The ITC instrument has two cells: a sample cell and a reference cell. The sample solution is loaded into the sample cell and the ligand solution is loaded into the reference cell.

+ Equilibrate the sample and ligand solutions. The ITC instrument will equilibrate the sample and ligand solutions to the same temperature. This is important to ensure that the heat changes measured during the binding reaction are due only to the binding event.

+ Titrate the ligand solution into the sample cell. The ITC instrument will titrate the ligand solution into the sample cell in a series of small injections. The heat released or absorbed during each injection will be measured.

**Sunday - 10/8/2023**

ITC Experiment- [FRET aptamer cortisol 0.25μM + cDNA1 cortisol 2μM- 1:2 conc]

Sample preparation-

* FRET Apt cort- for 200μl of 0.25μM - add 5μl of 10μM sample in 195μl MQ

* cDNA2 cort- for 80μl of 2μM- add 16μl of 10μM sample in 64μl MQ

ITC Protocol-

* Prepare the sample and ligand solutions.

- The sample solution should contain the macromolecule whose binding affinity is being measured which in our case was the aptamer. The ligand solution should contain the molecule that is binding to the sample which for experiment was the cDNA. The concentrations of the sample and ligand solutions should be chosen carefully to ensure that the binding reaction is in the measurable range of the ITC instrument.

- The concentration we used was 0.25 micromolar and prepared 200 microliters of the aptamer to be inserted into the sample well and 2 micromolar cDNA 80 microliter in volume was prepared to be titrated into the sample well.

+ Load the sample and ligand solutions into the ITC instrument. The ITC instrument has two cells: a sample cell and a reference cell. The sample solution is loaded into the sample cell and the ligand solution is loaded into the reference cell.

+ Equilibrate the sample and ligand solutions. The ITC instrument will equilibrate the sample and ligand solutions to the same temperature. This is important to ensure that the heat changes measured during the binding reaction are due only to the binding event.

+ Titrate the ligand solution into the sample cell. The ITC instrument will titrate the ligand solution into the sample cell in a series of small injections. The heat released or absorbed during each injection will be measured.

**Tuesday - 10/10/2023**

ITC Experiment- [FRET aptamer cortisol 0.25μM + cortisol 2000ng/ml]

Sample preparation-

* FRET Apt cort- for 200μl of 0.25μM - add 5μl of 10μM sample in 195μl MQ

ITC Protocol-

* Prepare the sample and ligand solutions.

- The sample solution should contain the macromolecule whose binding affinity is being measured which in our case was the aptamer. The ligand solution should contain the molecule that is binding to the sample which for experiment was the cDNA. The concentrations of the sample and ligand solutions should be chosen carefully to ensure that the binding reaction is in the measurable range of the ITC instrument.

- The concentration we used was 0.25 micromolar and prepared 200 microliters of the aptamer to be inserted into the sample well and 2000ng/ml 80 microliter in volume was prepared to be titrated into the sample well.

+ Load the sample and ligand solutions into the ITC instrument. The ITC instrument has two cells: a sample cell and a reference cell. The sample solution is loaded into the sample cell and the ligand solution is loaded into the reference cell.

+ Equilibrate the sample and ligand solutions. The ITC instrument will equilibrate the sample and ligand solutions to the same temperature. This is important to ensure that the heat changes measured during the binding reaction are due only to the binding event.

+ Titrate the ligand solution into the sample cell. The ITC instrument will titrate the ligand solution into the sample cell in a series of small injections. The heat released or absorbed during each injection will be measured.

[Aptamers](https://static.igem.wiki/teams/4693/wiki/wetlab/benchling/aptamer-lab-manual-benchling.pdf)

[Nanoprobes](https://static.igem.wiki/teams/4693/wiki/wetlab/benchling/nanoprobe-lab-manual-benchling.pdf)

[Gsα](https://static.igem.wiki/teams/4693/wiki/contributions/gs-alpha-lab-manual-benchling.pdf)

[Interlab](https://static.igem.wiki/teams/4693/wiki/wetlab/benchling/interlba-notebook-pdf-igem.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

---

# WETLAB

---

## Cortisol aptasensor formation and sensitivity

[Iteration 1](#!)
[Iteration 2](#!)
[Iteration 3](#!)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

## Formation using truncated aptamer and cDNA

---

# Design

---

The truncated aptamer sequence was referred to us by
Dr. Tarun Kumar Sharma from Gujarat Biotechnology University.
This aptamer is a 14-mer, which is claimed to conform and bind to
one of the prominent binding sites of the cortisol biomolecule.
The cDNA (complementary ssDNA) was designed to complement the half
length of the aptamer, making the cDNA 7bp long.
Given below are the possible conformations of the 14-mer aptamer
sequence obtained using DNA-fold software:

---

# Test

---

To check if the truncated aptamer and cDNA were binding,
a fluorescence-based test was performed using the flurolog equipment
(the working of which is mentioned on the experiment page).
The fluorescence-based tests were used to measure the optimum molar
ratio between the aptamer and the cDNA and the incubation time required
to form the aptasensor. To check the sensitivity of the truncated
aptasensor and to determine the efficiency and working of the aptasensor,
we performed plate reader experiments.

---

# Build

---

The aptamer sequence was modified with a 5' fluorophore attachment,
and the cDNA was modified with a quencher towards the 3' end.
The fluorophore and quencher pair were decided based on their
FRET efficiency. Our ideal choice for the fluorophore was
fluorescein-based FAM with an excitation peak at 498 nm and an
emission peak at 517 nm, and the quencher of choice was BHQ1,
which has an absorption range of 480 to 580 nm with maximum absorption
at 534 nm. The possible structure of the aptamer cDNA complex is
given below:

---

# Learn

---

We only observed a 10 per cent quenching from the above experimental
data in 90 minutes. As shown above, the optimum molar ratio of 1:2
(trunc aptamer: cDNA) was used for the sensitivity experiment.
The chosen aptasensor is not very sensitive within the range of
100 to 300 ng/ml cortisol, the physiological range in the human blood
serum. Therefore, there are better quantifiers for cortisol biomolecules
than the truncated aptasensor.

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

## Formation of cortisol aptasensor using 85 mer aptamer and cDNA (44mer)

---

# Learn

---

Due to the various challenges involved, and our need for knowledge
about modifying the aptamer sequence to achieve effective turn-on,
clickmer-based quantifiers were not a viable option for us.

---

# Design

---

* Aptamers are short, single-stranded DNA or RNA molecules that can
  bind to specific target molecules with high affinity and specificity.
  Aptamers can be modified with an alkyne group at one end to create a
  fluorescent aptasensor. This alkyne group can then be attached to an
  azide fluorescent dye using a copper-catalyzed azide-alkyne
  cycloaddition (CuAAC) reaction.
* When the aptamer binds to its target biomolecule, it undergoes a
  conformational change that exposes the azide group attached to the
  fluorescent dye. The CuAAC reaction can then bind the fluorescent
  dye to the aptamer. This results in a fluorescent signal that can
  be detected using a fluorometer or microplate reader.
* This method of creating a fluorescent aptasensor is called a
  "turn-on" mechanism because the fluorescence signal is only present
  when the aptamer is bound to its target biomolecule.
  This is in contrast to the "turn-off" mechanism, in which the
  fluorescence signal is detected when the aptamer is not attached to
  its target biomolecule.
* Turn-on mechanisms are generally more desirable for aptasensors
  as they are more sensitive and less prone to false positives.

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/dbtl-cycle-1-iteration-1/eng-pic-1.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/dbtl-cycle-1-iteration-1/eng-pic-4.png)

---

# Build

---

We modified our selected aptamers by adding an azide group using
Integrated DNA Technologies (IDT). To label the aptamers with the dye,
we purchased Cy3-alkyne dye to perform a copper-catalyzed azide-alkyne
cycloaddition (CuAAC) reaction.

---

# Test

---

We pitched this idea to multiple faculty members within our institute,
and Dr. Reji Varghese made us aware of the possible difficulties,
such as the efficiency of a click binding and also the difficulty in
modifying a specific nucleotide within an aptamer sequence that is
known to be attached to the binding site of the biomolecule.

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

## Formation of cortisol aptasensor using 85 mer aptamer and cDNA designed based on kD value.

---

# Learn

---

From the DBTL cycle, we learn that the ideal hybridisation time and
molar ratios show similar trends with the cDNA 1 85-mer aptamer complex;
hence, both cDNA 1 and cDNA 2 in combination with the 85-mer aptamer
can be used to form the ideal cortisol aptasensor.

---

# Design

---

Here, we used the same 85-mer aptamer mentioned in the previous iteration
but changed the design of our cDNA binding to the aptamer that forms the
aptasensor. This version of the cDNA was designed based on the kD value
of the aptamer; the cDNA pair compared to the kD value of the cortisol
binding with the 85-mer aptamer. As shown below, multiple complementary
sequences were designed to find a sequence with a higher kD value than
-16.1 that would not affect the binding and formation of the FRET system
designed. The chosen cDNA had a kD value of -10.3, and even though there
were multiple possible structures, the conformations didn't affect the
binding position of the cDNA, hence the interaction of the fluorophore
quencher pair.

---

# Build

---

The aptamer sequence was modified with a 5' fluorophore attachment,
and the cDNA was modified with a quencher towards the 3' end.
The fluorophore and quencher pair were decided on their FRET efficiency;
our ideal choice of the fluorophore was FAM, which is a fluorescein-based
fluorophore with an excitation peak at 498 nm and an emission peak at 517
nm, and the quencher of choice was BHQ1 which has an absorption range of
BHQ1 is from 480 to 580 nm with maximum absorption at 534 nm.
These sequences were then ordered from IDT for further testing.

---

# Test

---

To check if the 85-mer aptamer and the designed cDNA were binding,
a fluorescence-based test was performed using the flurolog equipment
(working is mentioned in the experiment page” link”) to confirm binding
between the aptamer and cDNA. We also conducted an isothermal titration
calorimetric experiment to obtain the kD and delta g value;
the fluorescence-based tests were used to measure the ideal molar ratio
between the aptamer and the cDNA and the incubation time required for the
formation of the aptasensor. To check the sensitivity of the obtained
aptasensor, we performed plate reader experiments to determine the
efficacy and detection limit of the aptasensor.

## Serotonin aptasensor formation and sensitivity

[Iteration 1](#!)
[Iteration 2](#!)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

## 44-mer serotonin aptamer and 20-mer cDNA

---

# Learn

---

The results above show that the hybridization of the cDNA and aptamer
causes an increase in fluorescence. In contrast, the interaction between
the fluorophore quencher pair should cause a decrease in fluorescence.
This could be because the partial binding of the cDNA occurred elsewhere
within the aptamer, causing a change in the conformation of the aptamer,
thereby exposing the fluorophore molecule. This theory would explain the
increase in fluorescence over time observed after the hybridization of
the cDNA with the aptamer.

---

# Design

---

The 44-mer aptamer was derived from the literature obtained from [r];
the possible conformations of the serotonin aptamer are given below.
The aptamer had a kd value of -30 nm and a delta g value of -10.62 kcal/mol.
This version of the cDNA was designed based on the kD value of the aptamer;
the cDNA pair compared to the kD value of the serotonin binding with the
44-mer aptamer. As shown below, multiple complementary sequences were
designed to find a sequence with a higher kD value than -30.1 that would
not affect the binding and formation of the designed FRET system.
The chosen cDNA had a kD value of -9.9 kcal/mol, and the possible
conformation didn’t affect the binding position of the cDNA,
hence the interaction of the fluorophore quencher pair.

---

# Build

---

The aptamer sequence was modified with a 5' fluorophore attachment,
and the cDNA was modified with a quencher towards the 3' end.
The fluorophore and quencher pair were decided on their FRET efficiency;
our ideal choice of the fluorophore was FAM, which is a fluorescein-based
fluorophore with an excitation peak at 498 nm and an emission peak at 517 nm,
and the quencher of choice was BHQ1 which has an absorption range of BHQ1 is
from 480 to 580 nm with maximum absorption at 534 nm.
These sequences were then ordered from IDT for further testing.
Attached below is the possible hybridised duplex structure acquired using the
duplex fold software
Attached below are the possible hybridised duplex structures acquired
using the duplex fold software: Link

<https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/DuplexFold/DuplexFold.html>

---

# Test

---

To check if the 44-mer serotonin aptamer and the designed cDNA were binding,
a fluorescence-based test was performed using the Teacan plate reader
equipment (working is mentioned in the experiment page” link”) to confirm
binding between the aptamer and cDNA. We also conducted an isothermal
titration calorimetric experiment to obtain the kD and delta g value;
the fluorescence-based tests were used to measure the ideal molar ratio
between the aptamer and the cDNA and the incubation time required for the
formation of the aptasensor. To check the sensitivity of the obtained
aptasensor, we performed plate reader experiments to determine the efficacy
and detection limit of the aptasensor.

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

## 44-mer serotonin aptamer and 20-mer cDNA

---

# Learn

---

The fluorescence-based test conducted using the plate reader gives us
an observable quenching of 30% on the formation of the aptasensor using
this cDNA, as seen with the cortisol aptasensor, which is a drastic
improvement from the previously noticed increase in fluorescence upon
hybridisation of the cDNA (12-mer) and the aptamer.
Sensitivity tests also show increased fluorescence intensity upon
binding with our Serotonin biomolecule. However, no more remarkable
perceivable fold change is observed compared to the previously formed
aptasensor.

---

# Design

---

Here, we used the same 44-mer aptamer mentioned in the previous iteration
but changed the design of our cDNA binding to the aptamer that forms the
aptasensor. Our team also designed a cDNA based on the inputs received
from Dr. Tarun Kumar Sharma from Gujarat Biotechnology University,
who suggested we design the cDNA to complement at least half the length
of the aptamer, so this version of our cDNA 1 was 20 bp long with delta
g value - (-29.6 kcal/mol).

---

# Build

---

The aptamer sequence was modified with a 5' fluorophore attachment,
and the cDNA was modified with a quencher towards the 3' end.
The fluorophore and quencher pair were decided on their FRET efficiency;
our ideal choice of the fluorophore was FAM, which is a fluorescein-based
fluorophore with an excitation peak at 498 nm and an emission peak at 517
nm, and the quencher of choice was BHQ1 which has an absorption range of
BHQ1 is from 480 to 580 nm with maximum absorption at 534 nm.
These sequences were then ordered from IDT for further testing.
Attached below is the possible hybridised duplex structure acquired
using the duplex fold software

---

# Test

---

To check if the 44-mer serotonin aptamer and the designed cDNA were
binding, a fluorescence-based test was performed using the Teacan plate
reader equipment (working is mentioned in the experiment page” link”)
to confirm binding between the aptamer and cDNA. We also conducted an
isothermal titration calorimetric experiment to obtain the kD and delta
g value; the fluorescence-based tests were used to measure the ideal
molar ratio between the aptamer and the cDNA and the incubation time
required for the formation of the aptasensor.
To check the sensitivity of the obtained aptasensor, we performed plate
reader experiments to determine the efficacy and detection limit of the
aptasensor.

---

# DRYLAB DBTL CYCLES

---

[Iteration 1](#!)
[Iteration 2](#!)
[Iteration 3](#!)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

---

# Learn

---

Due to the various challenges involved, and our lack of knowledge about how to modify the aptamer sequence to achieve effective turn-on, clickmer-based quantifiers are not a viable option at this time.

---

# Design

---

- Aptamers are short, single-stranded DNA or RNA molecules that can bind to specific target molecules with high affinity and specificity. To create a fluorescent aptasensor, aptamers can be modified with an alkyne group at one end. This alkyne group can then be attached to an azide fluorescent dye using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.
- When the aptamer binds to its target biomolecule, it undergoes a conformational change that exposes the azide group on the fluorescent dye. The CuAAC reaction can then occur, attaching the fluorescent dye to the aptamer. This results in a fluorescent signal that can be detected using a fluorometer or microplate reader.
- This method of creating a fluorescent aptasensor is called a "turn-on" mechanism because the fluorescence signal is only present when the aptamer is bound to its target biomolecule. This is in contrast to a "turn-off" mechanism, in which the fluorescence signal is present when the aptamer is not bound to its target biomolecule.
- Turn-on mechanisms are generally more desirable for aptasensors because they are more sensitive and less prone to false positives.

---

# Build

---

We used Integrated DNA Technologies (IDT) modifications to add an azide group to our selected aptamers. We planned to purchase Cy3-alkyne dye and perform a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction to label the aptamers with the dye.
<https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/DuplexFold/DuplexFold.html>

---

# Test

---

We pitched this idea to multiple faculty members within our institute and Dr. Reji Varghese made us aware of the possible difficulties such as the efficiency of a click binding and also the difficulty in modifying a certain nucleotide within an aptamer sequence that is known to be attached to the binding site of the biomolecule.

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

---

# Learn

---

We believe that the aptasensor method is a more practical choice for quantifying cortisol than clickmer-based methods, based on the following two factors:- Abundance of literature: There is a much larger body of literature available on aptasensors than on clickmer-based methods for cortisol quantification. This means that there are more well-established protocols and procedures available for aptasensor-based methods, and that there is a larger community of researchers who are familiar with and experienced in using these methods.
- Fewer theoretical problems and questions: Aptasensors are based on a well-established principle (FRET), and there are few theoretical problems or questions associated with their use. Clickmer-based methods, on the other hand, are a newer technology, and there are still some theoretical problems and questions that need to be addressed.

---

# Design

---

This mechanism works on the principle of Fluorescence resonance energy transfer (FRET) is a distance-dependent non-radiative transfer of energy from one fluorescent molecule (the donor) to another fluorescent molecule (the acceptor). It occurs when the two molecules are within close proximity (typically 10 nm or less) and their emission and excitation spectra overlap. We attach a fluorescent molecule (fluorophore) to the 5' end of the aptamer sequence and a quencher molecule to the 3' end of the complementary strand. When the cDNA and the aptamer bind to form a partial duplex structure, the fluorophore and the quencher molecule come close together, which quenches the fluorescence. This is our aptasensor complex. When the aptasensor complex interacts with the target biomolecule (cortisol), the cDNA strand is displaced. The free aptamer strand then changes conformation, allowing it to bind to the biomolecule. This causes an increase in fluorescence, which is quantified to measure the level of cortisol present. In other words, the aptasensor complex is like a switch. When the cDNA strand is bound to the aptamer, the switch is off. When the biomolecule binds to the aptamer, the switch is turned on. The fluorescence signal is directly proportional to the amount of biomolecule present, so we can use the fluorescence intensity to measure the biomolecule concentration.

---

# Build

---

The aptamer sequence was modified with a 5' fluorophore attachment, and the cDNA was modified with a quencher towards the 3’ end. The fluorophore and quencher pair were decided on their FRET efficiency; our ideal choice of the fluorophore was FAM, which is a fluorescein-based fluorophore with an excitation peak at 498 nm and an emission peak at 517 nm, and the quencher of choice was BHQ1 which has an absorption range of BHQ1 is from 480 to 580 nm with maximum absorption at 534 nm. These sequences were then ordered from IDT for further testing.

---

# Test

---

Various tests were performed for various types and concentration of aptasensors these test can be viewed in the results and proof of concept page.

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

---

# Learn

---

We realised why toehold switched would not be the ideal quantification method for miRNAs- It will have to include translatory machinery, which requires specific enzymes.
- An amplification step would be required to bring the concentration of miRNA to detectable levels, increasing the cost and complexity of the chip.
- Storage of RNA-based quantifiers would require specialised conditions.
- Enzymes used for translation must be freeze-dried and stored in the microchip.
- Leaky expression would still be present because of low levels of enzyme activity.
During this stage, we were doubtful about using Toehold switches. This is when we came across nanoprobes, which were DNA-based, did not require enzymes and had very good detection limits.[3]

---

# Design

---

Toehold switches are synthetic RNAs that mimic messenger RNAs. They contain a recognition sequence for a specific stimulus in the form of a specific “input” RNA and a recognition sequence that the protein-synthesizing machinery (ribosome) needs to bind to initiate the translation of a fused protein-coding sequence into its encoded protein product. In the absence of the “input” RNA, the toehold switch is kept in its OFF state by forming a hairpin structure that uses part of the “input” recognition sequence and the ribosome recognition sequence, which is kept inaccessible. The toehold switch is turned on when a stimulating “input” RNA binds to the toehold and induces the hairpin structure to open up, giving the ribosome access to its recognition sequence to start the synthesis of the encoded protein downstream [1]
Using a toehold switch, we intended to quantify our chosen miRNAs- miR124 and miR132. This could be made possible by tagging a GFP protein gene as the reporter sequence at the 3’ end of the toehold switch, which will get translated only in the presence of the trigger miRNA. Hence, the sample will show fluorescence in the presence of the respective miRNA, which could then be quantified to measure the amount of miRNAs in the sample.

---

# Build

---

- We were using the switchMI designer tool developed by the 2021 iGEM team UParis BME and NUPACK software to build the sequence of our toehold switches. [2]
- Superfolded GFP was chosen as the reporter sequence.
- We were able to generate a preliminary set of sequences based on this.

---

# Test

---

To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE.

To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE.
To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE.
To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE.
To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE.

---

# HARDWARE DBTL CYCLES

---

[Iteration 1](#!)
[Iteration 2](#!)
[Iteration 3](#!)
[Iteration 4](#!)
[Iteration 5](#!)
[Iteration 6](#!)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

---

# Learn

---

The led was able to detect higher intensities well but could not respond to lower levels of light. We would need a more sensitive unconventional but cheap sensor.

---

# Design

---

A container containing a source to mimic fluorescence (green which is expected from biological samples), a sensor to detect it and a self-contained microcontroller module for data collection and display

---

# Build

---

Box out of Cardboard, green led used as a source, used a photodiode for its high sensitivity to green light and fast response times and Arduino Uno as the microcontroller

---

# Test

---

Setup the container in a dark room with Arduino powered and outputting data to a laptop. Varied the voltage across the led in several small steps to achieve lowest possible levels of light via direct control from Arduino

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

---

# Learn

---

The phototransistor achieved saturation current production at moderate intensities of light but was more sensitive to lower levels of light compared to the photodiode. Signal was also amplified by using a large resistance (300kohm) in series with phototransistor. However, the detected emission was still visible to naked eye. We would need to make instrument more sensitive or amplify further.

---

# Design

---

A container containing a source to mimic fluorescence (green which is expected from biological samples), a sensor to detect it and a self-contained microcontroller module for data collection and display.

---

# Build

---

Box out of Cardboard, green led used as a source, used a phototransistor for its higher sensitivity to lower light and an Arduino Uno as the microcontroller

---

# Test

---

Setup the container in a dark room with Arduino powered and outputting data to a laptop. Varied the voltage across the led in several small steps to achieve lowest possible levels of light via direct control from Arduino.

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

---

# Learn

---

Photodarlington was much more sensitive to low levels of light. The sensor still picked up the light even after 1 million ohms ins series with led. However, reading was unreliable because of large fluctuation in signal. This was arising from the fact that Arduino uses Pulse Width Modulated signal to control led voltage and brightness, leading to rapid flickering. Increasing resistance (over 300kohm) in series with Darlington system did not reliably amplify signal anymore.

---

# Design

---

Similar design as earlier. Led was placed directly above the photodarlington ( sensor is minimally sensitive to light coming from +/- 5 degrees from directly vertically above it).

---

# Build

---

Box out of Cardboard, green led used as a source, used a photodarlington (in series with 300kohm resistor for amplification) for the highest sensitivity to lower light, and an Arduino Uno as the microcontroller.

---

# Test

---

Similar to above. 1 million ohms was placed in series with led to lower intensity of light as much as possible.

To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE.
To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE.
To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE.
To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE.

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

---

# Learn

---

The signal detected by the transistor was steady and showed good behavior. Detected amounts of light demonstrated nearly lower than naked eye detection. Detection was now limited by accuracy of ADC reading on Arduino, which is 10 microvolts. Laptops and devices were turned away or switched off as device was sensitive to even such minute light. Device was now ready to be tested with non-electronic signals.

---

# Design

---

Similar design as earlier. Led was placed directly above the photodarlington ( sensor is minimally sensitive to light coming from +/- 5 degrees from directly vertically above it).

---

# Build

---

Box out of Cardboard, green led used as a source, used a photodarlington (in series with 300kohm resistor for amplification) for the highest sensitivity to lower light, and an Arduino Uno as the microcontroller. External DC source used to drive led (with resistor in series to lower brightness)

---

# Test

---

Similar to above. 1 million ohms was placed in series with led to lower intensity of light as much as possible.

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

---

# Learn

---

Device performed exceptionally well. Reliably picked up readings until 4th order of dilution (10^-4 of stock concentration). 4th order and 5th order dilution were distinct; however, they were not reliably separated enough. Great care was needed for 3rd of dilution and below. Laptops were turned away from system and set at minimum brightness. All devices were moved off table and no physical contact with table was needed for most accurate readings. Any further noise signals were averaged out using an algorithm which gave effective readings every 20 seconds. To further improve sensitivity or accuracy, a voltage regulated DC source along with 16-bit external ADC (Arduinos is 10-bit) might be required. Now we pray biological samples are bright enough

---

# Design

---

Custom Led at 490nm (blue) driven by DC source (in series with 100 kohm resistor to avoid photobleaching) was used to excite PET chemical sample to mimic biological fluorescence, used a photodarlington (in series with 300 kohm resistor for amplification) as sensor, an Arduino Uno for data collection and display.

---

# Build

---

Interior of box was painted black to absorb any remnant blue light to reduce noise. Sensor was placed at 90 degrees and away from blue light source (sensor is minimally sensitive to light coming from +/- 5 degrees from directly vertically above it). Furthermore, A plane separated the blue light from the sensor to minimize undesirable signals. Cuvettes (or whatever they are called) were used to hold samples and were placed directly above sensor.

---

# Test

---

Interior of box was painted black to absorb any remnant blue light to reduce noise. Sensor was placed at 90 degrees and away from blue light source (sensor is minimally sensitive to light coming from +/- 5 degrees from directly vertically above it). Furthermore, A plane separated the blue light from the sensor to minimize undesirable signals. Cuvettes (or whatever they are called) were used to hold samples and were placed directly above sensor.

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/learn-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/design-act-copy.png)

![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/test-act-copy.png)
![](https://static.igem.wiki/teams/4693/wiki/assets/engineering/build-act-copy.png)

---

# Learn

---

All biological samples showed great distinction between stock and biomarker added scenarios. Detected fluorescence was close to or above 3rd order dilution readings from PET, making the setup less fragile to change while reliable reading data. Addition of nanoprobes further improved readings and gave better signals. Signals were stable. Development of ultra-sensitive, cheap fluorimeter, tailored o detect biomarker signals of green fluorescence was successfully developed.

---

# Design

---

Custom Led at 490nm (blue) driven by DC source (in series with 100 kohm resistor to avoid photobleaching) was used to excite wetlab biological samples, used a photodarlington (in series with 300 kohm resistor for amplification) as sensor, an Arduino Uno for data collection and display.

---

# Build

---

Interior of box was painted black to absorb any remnant blue light to reduce noise. Sensor was placed at 90 degrees and away from blue light source (sensor is minimally sensitive to light coming from +/- 5 degrees from directly vertically above it). Furthermore, A plane separated the blue light from the sensor to minimize undesirable signals. Cuvettes (or whatever they are called) were used to hold samples and were placed directly above sensor.

---

# Test

---

Biological samples at realistic concentrations and volumes were used. Readings for cases of without biomarker and with biomarker for all aptamers, mirna132 and mirna124 was done

---

# References

---

1. Tolentino, J. C., & Schmidt, S. L. (2018). DSM-5 Criteria and
   Depression Severity: Implications for Clinical Practice. Frontiers in psychiatry, 9, 450. doi.org/10.3389/fpsyt.2018.00450
   <https://doi.org/10.3389/fpsyt.2018.00450>
   (1 December 2011)- World Health Organisation.
2. <https://www.who.int/news-room/fact-sheets/detail/depression>

{% endblock %}

{% extends "layout.html" %}
{% block title %}{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

+ [Native PAGE](#head1)
+ [Dynamic Light Scattering (DLS)](#head2)
+ [Transmission Electron Microscopy (TEM)](#head3)
+ [Formation of Nanoprobe](#head4)
+ [Fluorescence-based Experiments using Microplate Reader](#head5)
+ [Fluorescence-based Experiments using Microplate Reader and FLUROLOG](#head6)
+ [Isothermal Titration Calorimetric Experiment](#head7)
+ [Polymerase Chain Reaction (PCR)](#subhead1)
+ [Agarose Gel Electrophoresis](#subhead2)
+ [Restriction Digestion](#head8)
+ [Ligation](#subhead3)
+ [Transformation](#subhead4)
+ [Plasmid Isolation](#head9)

## Native PAGE

A native PAGE (polyacrylamide gel electrophoresis) was chosen to verify that our complementary sequences were forming the probe and to demonstrate that our miDNA can bind to the probe. We used a 12 percentage gel, and the polyacrylamide solution used was in a 1:19 ratio of acrylamide to bisacrylamide. The 12 percentage gel was able to distinguish a 22 nucleotide difference in the sample.

The native PAGE ensures that the base pairing between the complementary strands remains intact during the electrophoresis, which is required to verify the formation of our probe. The experiment was performed using standard SDS PAGE equipment, but a longer gel is ideal.

[Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/native-page.pdf)

#### We conducted two experiments for the characterization of our carboxylated magnetic nanoparticles. Dynamic Light Scattering (DLS) The zeta potential analyzer is used to find if the nanoparticles were evenly distributed in the solution and to confirm the presence of stable carboxylic acid groups. This system is designed for the characterization of colloidal, nanoparticulate, and macromolecular samples. It can determine the zeta potential, which is a measure of the electrostatic potential that exists at the interface between a particle and the surrounding liquid or dispersion medium. The instrument works on the basis of Dynamic Light Scattering (DLS). It will detect the fluctuation in the intensity of scattered light over a time period, which is caused by the Brownian movement of particles and can calculate the zeta potential with this reading. The experiment requires the samples to be diluted and loaded into a cuvette The cuvette should be filled with around 500 microlitres of the aqueous solution of the particle. The cuvette must be carefully placed into the slot in the machine and the experiment can be run. A singular peak in the resulting graph would mean that the particles are well dispersed in the medium and a zeta potential value, lower than -20 mV will indicate that there are stable carboxyl groups present in the solution. This negative charge is formed because the carboxyl groups will lose a proton and become negatively charged when present in aqueous solution [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/dls.pdf) Transmission Electron Microscopy (TEM) To obtain high-resolution images of our nanoparticles for size determination, we conducted Transmission Electron Microscopy (TEM) studies. TEM is a microscopy technique that works by transmitting a beam of electrons through a thin specimen and capturing the transmitted electrons to create high-resolution images. The sample must be loaded on a copper grid and air-dried to remove all the excess water. This sample can be stored in a microcentrifuge tube at room temperature if needed. [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/tem-sample-preparation.pdf) Formation of Nanoprobe The binding of the probe to the carboxylated magnetic nanoparticle Our probe structure was divided into three different single-stranded DNA oligos, that would bind together by complementarity, to form the working probe. These three sequences were named A, B and C. Out of these structures, B contains an NH2 group that could be bound to the carboxyl (COOH) group present in the nanoparticle by an amide bond formed by EDC coupling reaction. This involves washing the nanoparticles with MES to prevent agglomeration, followed by treatment with EDC, which activates the carboxyl groups present in the nanoparticles. These activated carboxyl groups can now form an amide bond with sequence B. So, the required volume of sequence B is added to the activated carboxylated magnetic nanoparticles and incubated overnight at room temperature with constant shaking. After this step, the supernatant is removed and the required volume of sequences A and C are added and incubated for 1 hour. Now, The supernatant can be removed after pulling the particles down using a magnet and fresh medium can be added. * The cross-linkage will help to have a lower detection limit for the system. * Compared to the unattached state, there will be more probes present in a unit volume in this attached system. * Removing the supernatant after pulling the nanoprobes down using a magnet will help to remove excess unbound strands that have fluorophores attached. This is important to reduce the background noise and give better output. * These nanoprobes, after being dispersed in a media can be collected to one point using a magnet, which can help to concentrate the fluorescence to one region instead of being spread across the sample. This can also help to separate the nanoprobes from other particles that may be present in the sample that can physically block the fluorescence. [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/attachment-of-probe-to-nanoparticle.pdf) Nanoprobe fluorescence-based Experiments using Microplate Reader All the experiments mentioned below were performed on a Costar, black, flat-bottom plate. The readout was taken by performing an emission scan, with a constant excitation of 490 nm and emission range of 500 to 600 nm, with readings taken in 1 nm steps. - Fluorescence intensity change on adding the miDNA to the probe, before binding the probe to the nanoparticle. We conducted this experiment to see if the binding was indeed giving us a fluorescence change. The results from this experiment could be used as a comparison to show the effect of binding the probes to the nanoparticle - Fluorescence intensity change on adding the miDNA to the Nanoprobe (after attachment) This was performed to see if there was a considerable increase in fluorescence in the nanoprobe after miDNA was added. In the experiment, the amount of probe and total volume was kept constant. The control had an equal volume of the medium used for dilution of the miDNA as the volume of the miDNA sample. - Finding the detection limit of the nanoprobe This experiment was performed to see if our probes would be able to give a change in fluorescence intensity even at low levels of miDNA as the miRNA levels in the blood are in picomolar levels. We tested as low as 10 picomolar concentrations of miDNA. The volume of the probe and sample was kept constant and the concentration of the sample was varied. - Test for specificity We performed this experiment to see how specific our probe was as our probe should be able to differentiate between the target miRNA and other similar miRNA. For this, we ordered a miDNA with a difference in just two nucleotides, compared to our target miDNA. For the experiment, the amount of probe and total volume of solution was kept constant. We then made four samples for each probe, one with just media as the sample, one with a completely different miDNA as the sample, one with our target miDNA and one with the miDNA with just two nucleotide difference to the target miDNA. [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/plate-reader-experiments-protocol.pdf) Aptasensor fluorescence-based Experiments using Microplate Reader and FLUROLOG All the experiments mentioned below were performed on a Greiner 96 Flat Bottom Transparent Polystyrene Cat. No.: 655101/655161/655192 [GRE96ft.pdfx] plate. The FluoroLog fluorometer is a versatile instrument that can be used to measure the fluorescence of a wide variety of samples. It is important to follow the protocol carefully to ensure accurate and reproducible results. If you are using a 200 microliter cuvette, you will need to use a cuvette adaptor. The cuvette adaptor allows the smaller cuvette to be used in the fluorometer. - Finding the ideal incubation time for hybridisation of the aptasensor This experiment was designed to find the ideal time for the FRET system to show maximum quenching. FRET works by transferring energy from a donor fluorophore to an acceptor quencher when the two molecules are close together. In this experiment, a fluorophore was attached to the aptamer and a quencher was attached to the cDNA. When the aptamer and cDNA bind together to form a duplex, the fluorophore and quencher are brought close together, and FRET occurs. The fluorescence intensity was measured over time to determine the ideal time for maximum quenching to occur. In this experiment, the aptamer and cDNA were mixed together in specific proportions, such as 0.25 micromolar aptamer and 0.75 micromolar cDNA. The fluorescence intensity was then measured at the start of the experiment (time t=0) and every 10 minutes thereafter, until the fluorescence quenching did not change much over time. [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/formation-of-aptasensor-flurolog-protocol-docx.pdf) - Finding the ideal molar ratio for the formation of aptasensor This experiment was designed to determining the best ratio of aptamer to target molecule for the formation of an aptasensor.This experiment was performed on two different machines, a fluorometer and a microplate reader, to see if the results were similar. The goal was to determine if the trends observed were consistent across different machines, which would suggest that the results are reliable. [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/molar-ratio.pdf) - Finding the sensitivity of the aptasensor using various concentration so biomarker This experiment is a crucial test of the principle of the aptasensor, and it is designed to determine how sensitive the aptasensor is. The experiments were conducted on a microplate reader instead of a fluorometer because the researchers did not have enough reagents for the fluorometer. To conduct this experiment we select the most ideal incubation time and molar ratio for the formation the aptasensor and we take 150 microliter of the aptasensor and add 50 microliter of varying concentration of the biomarkers ranging from 10 ng/ml to 400 ng/ml mimicking the physiological range present in plasma. [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/sensitivity.pdf) Isothermal Titration Calorimetric Experiment Isothermal titration calorimetry (ITC) is a powerful technique for measuring the thermodynamics of biomolecular interactions, including the formation of DNA duplexes. It is a direct method that does not require any labeling or modification of the DNA strands. ITC is also very sensitive, allowing for the detection of even weak interactions. There are several reasons why ITC is useful for checking duplex formation between two DNA strands: - Direct measurement of thermodynamics: ITC directly measures the enthalpy and binding affinity of the interaction. This information can be used to assess the stability of the DNA duplex and to understand the forces that drive duplex formation. - Label-free detection: ITC does not require any labeling or modification of the DNA strands. This makes it a versatile technique that can be used to study a wide range of DNA duplexes, including those that are difficult or impossible to label. - High sensitivity: ITC is very sensitive, allowing for the detection of even weak interactions. This is important for studying DNA duplexes, which can be relatively weak, especially for short strands. The principle of isothermal titration calorimetry (ITC) is based on the direct measurement of the heat released or absorbed during a biomolecular binding event. ITC experiments are performed at a constant temperature, so the heat measured is directly proportional to the change in enthalpy of the binding reaction. ITC instruments typically consist of two cells: a sample cell and a reference cell. The sample cell contains the biomolecule of interest, while the reference cell contains a buffer solution. The two cells are enclosed in an adiabatic jacket, which prevents heat from being transferred to or from the environment. During an ITC experiment, a ligand solution is titrated into the sample cell. When the ligand binds to the biomolecule, heat is either released or absorbed. This heat is detected by the calorimeter and recorded as a thermogram. The thermogram is a plot of the heat released or absorbed as a function of the amount of ligand added. The thermogram can be analyzed to determine the following thermodynamic parameters of the binding reaction: - Binding affinity (K\_A): The strength of the interaction between the ligand and the biomolecule. - Enthalpy (ΔH): The amount of heat released or absorbed during the binding reaction. - Entropy (ΔS): The change in disorder of the system during the binding reaction. - Stoichiometry (n): The number of ligand molecules that bind to each biomolecule. [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/isothermal-titration-calorimetry-protocol.pdf) Polymerase Chain Reaction (PCR) PCR is used to amplify a desired DNA sequence.These are temperature influenced enzymatic reactions.It consists mainly of 3 steps. 1. Denaturation:Heating the sample at high temperature around 94-98°C.Denaturation step breaks the hydrogen bond between the double stranded DNA converting it into single stranded. 2. Annealing:After Denaturation the annealing temperature is reduced to a lower temperature around 50-65°C.During this step,the short primers bind/anneal to the complementary sequences in single stranded dna sequence. 3. Extension: Reaction temperature is raised to around 72°C.Heat stable DNA polymerase enzyme, Taq polymerase extends the primer by adding complementary nucleotide bases to single stranded DNA. All PCR reaction conducted by us were carried out in BIO-RAD T100 Thermal Cycler, except for Gradient PCR. It is very important to keep a sterile environment during the preparation of reaction mixtures. To confirm the functionality of our PCR , we included a positive control to amplify a gene fragment of similar size of the required gene. - Gradient PCR We ran a gradient PCR to find out the ideal annealing temperature of our primers. Gradient pcr allows to amplify multiples samples in different annealing temperature in a single run. By identifying the temperature that gives the highest yield of our desirable product we can find the optimal annealing temperature. It is similar to normal pcr amplification with respect to all other steps. The experiment was conducted in a Thermofischer SimpliAmp Thermal Cycler. - Ligation PCR Ligation PCR is performed to confirm the ligation reaction..It helps ensure that the ligation ha been completed without errors like deletions, re-arrangements or insertions.It aslo helps to understand the orientation of inserted fragment relative to vector. Forward primer and reverse primers are designed specifically for both insert and vector; one being specific to the vector while the other being specific to the insert. Analyzing the bands obtained upon running agarose gel electrophoresis, aforementioned assumptions can be confirmed. - Colony PCR Colony pcr is a rapid and cost effective method for screening a large number of bacterial clones. Here, we performed it to confirm that the host cells have taken up the recombinant DNA and to identify the required clonal colony. Selecting suitable bacteria colonies is very crucial for further steps of cloning. Primer set specific to the insert is added such that only those sequences, if present in the plasmid gets amplified. The bands obtained on the agar gel is further analyzed to ensure that size of amplified DNA fragment is close to insert size and thus confirm that the transformation has occurred. We also included a positive control to verify the performance of pcr reaction and check for contamination. [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/wetlab/gs-alpha-gradient-ligation-and-colony-pcr.pdf) Agarose Gel Electrophoresis Gel electrophoresis helps to separate the samples according to size in agarose gel.The samples are pipetted in to the wells of the gel after mixing with a loading dye (necessary for visualization). An electric current is applied to the Anode region causing the negatively charged dna to move towards the Cathode region. A molecular weight ladder is also loaded into a well along with the samples while running the gel. Obtained bands are compared against the bands of the ladder to determine size. The size is proportional to migration rate.Smaller fragments move quickly toward the bottom region. Ethidium Bromide is used to visualize the DNA in gel. The DNA sample takes up the dye as they migrate through the gel. Pale pink bands are observed on illumination with UV light once the electrophoresis is completed. We mainly performed Gel electrophoresis as a confirmation step for numerous experiments like PCR amplification reactions, restriction digestions, ligation reactions and transformations. All electrophoresis experiments were run on the Bio - Rad Horizontal Electrophoresis System. [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/wetlab/gs-alpha-agarose-gel-electrophoresis.pdf) Restriction Digestion During restriction digestion, specific restriction enzymes - usually endonucleases- cut the dna fragments at specific locations called recognition sites. In order to introduce our amplified insert into the vector pET19b, we need to create sticky ends on both insert and vector by restriction-digesting both DNA sequences using the same restriction enzymes. It is very crucial to select appropriate restriction enzymes for cutting both vector and insert. We used two different enzymes, one for each cutting each in order to decrease chances of non-specific cutting and ensure proper orientation of the insert in the vector. Restriction digestion was performed using BamH1 and Nde1, both high fidelity endonucleases.High fidelity(HF) enzymes have a higher accuracy rate.Appropriate incubation time was set, since excess incubation can lead to over digestion and formation of excessive DNA fragments. [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/wetlab/gsaplha-restriction-digestion.pdf) Ligation Ligation reaction is performed to join the compatible ends of the insert and vector after restriction digestion. After successful ligation, we obtain the recombinant DNA molecule - a circular plasmid- that can be transformed into host cells. We performed the reaction using T4 Ligase, a versatile ligase that ligates DNA fragments by catalyzing the formation of phosphodiester bonds between juxtaposed 5' phosphate and 3' hydroxyl termini in double-stranded DNA using ATP as a coenzyme. [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/wetlab/gsalpha-ligation.pdf) Transformation During bacterial transformation, bacteria takes up a naked DNA (foreign gene) from its environment.This is a horizontal gene transfer process. Not all bacteria can take up naked DNA. Bacteria is made artificially competent using chemical or electrical pulses.Transformation experiments are generally performed in a Laminar Air Flow(LAF) or Biosafety Cabinets with sterile equipments and reagents to reduce the risk of contamination. We are required to perform two transformations. - For amplification of recombinant DNA The first transformation is performed to introduce the recombinant DNA molecule into Dh5α strain of E.coli create in order to create multiple copies of the rDNA. Dh5α is known for its high tansformation efficiency. Since the natural competency of E. coli is very low or even nonexistent, the cells are made competent for transformation by heat shock. We also included a negative control for assessing the efficiency and specificity of transformation. - For expression of recombinant protein The second transformation is performed in order to transform the isolated plasmid into BL21 strain of E.Coli cells. BL21 is an E.coli strain commonly used for expression of recombinant protein due to its modified T7 RNA Polymerase system, high expression levels owing to high affinity of T7 RNA polymerase for it’s promoter and comparatively fast growth rate. [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/wetlab/gsalpha-transformation-into-bl21-cells-dh5-alpha-comp-cells-combined.pdf) Plasmid Isolation Alkaline lysis is used to separate plasmid DNA from other cell constituents,including genomic DNA. This is done to isolate the transformed pET19b plasmid which is then transformed into BL21 strain of E.coli for expression of protein. Alkali like 10N Naoh is added to the resuspended cells for cell lysing; a detergent like SDS is used to denature most of the cell protein. Cell debris is precipitated using potassium acetate which also helps in renaturing the double stranded plasmid DNA. This is then dissolved which is eluted after centrifuging. Final plasmid DNA precipitation is then carried out using ethanol/isopropanol. [Protocol](https://static.igem.wiki/teams/4693/wiki/assets/experiments/wetlab/gsalpha-plasmid-isolation.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

+ [Magnetic Nanoprobes](#head1)
+ [Aptasensors](#head2)
+ [Gsα Protein](#head3)

---

# Magnetic Nanoprobes

---

**CHARACTERISATION OF PROCURED NANOPARTICLES**

**Q) Do the magnetic nanoparticles have a stable carboxylic group, and do they disperse well in the medium?**

*Analysis of the zeta potential*
This experiment was done to verify that our nanoparticles had stable carboxyl groups.
After we received the graph from the zeta analyzer, we were able to see that

* The graph had a single peak: This showed that our particles were well dispersed in the medium. Well-dispersed particles will provide more accurate zeta potential values.
* The zeta potential was -21 millivolts. The negative zeta potential proved that the carboxyl groups were indeed present in the nanoparticles. The negative value is caused due to the deprotonation of the carboxyl group in aqueous solution.
* The negative value of less than -20 mV proves that the carboxyl groups are stable.

![](https://static.igem.wiki/teams/4693/wiki/wetlab/results-for-nanoprobe/zeta-potential-new.png)
*Figure 1 : Zeta potential value of the carboxylated magnetic nanoparticle*

**Q) What is the size of the nanoparticles?**

*TEM analysis:*
The TEM image given below shows that the nanoparticles we procured were, on average, about 12 nanometers in diameter.

![](https://static.igem.wiki/teams/4693/wiki/wetlab/results-for-nanoprobe/tem-new.png)
*Figure 2 : Transmission Electron Microscopy (TEM) image of the nanoparticle with 50 nm scale*

**CONFIRMATION OF PROBE FORMATION AND MIDNA BINDING**

**Q) Are the sequences actually binding to form the probe? If so, can the target miDNA bind to this probe?**

*Native PAGE:*
Native Polyacrylamide gel electrophoresis ensures that the structure of the probe remains intact. This experiment can verify if our probes are actually being formed and also if our miDNA is binding to the probe.
The bands in the native page given are in the following order from the left:
Sequence A (2), sequence B (3), sequence C (4), Sequence A+B (5), Sequence A+B+C (incubated for 1 hour (1:1:3 ratio) to form the probe) (6), Probe + miDNA (7)
Here,

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/native-page-132.png)
*Figure 3 : Gel (12%) image verifying probe formation and miDNA binding, for probe 132*

![](https://static.igem.wiki/teams/4693/wiki/wetlab/results-for-nanoprobe/native-page-124-1.png)
*Figure 3 : Gel (12%) image verifying probe formation and miDNA binding, for probe 124*

* The bands of sequence A and B are at the same level. These sequences have the same number of nucleotides.
* The A+B lane is higher than the A and B lanes, but the band is smeared. This is because A and B are only partially complementary, and all of them will not bind and form a stable structure.
* The lane which has A+B+C (which forms the probe) has just one band, which is higher than the A+B band, indicating that these three sequences are being bound together by complementarity and are forming a stable structure.
* Now, we can also see that the band in the lane of probe + miDNA is slightly higher than the band in the lane with just the probe. Indicating that the miDNA is indeed binding to the probe. The difference is only small because the miDNA is of just 22 nucleotide length.

**FLUORESCENCE-BASED EXPERIMENTS USING MICROPLATE READER.**

For all the following experiments,

* The percentage change in fluorescence intensity was calculated by
* The control used in all the experiments has an equal amount of probe and buffer without the miDNA present to make the volume constant.
* For all the experiments, the incubation time was 1 hour.

**Q) Did the miDNA binding actually cause the fluorophore quencher pair to move away?**
*Fluorescence intensity change upon adding the miDNA to the probe, before binding the probe to the nanoparticle.*
We tried both excitation and emission scans to see what the maximum change that we could detect was.
From these experiments, we could see that:

![](https://static.igem.wiki/teams/4693/wiki/wetlab/results-for-nanoprobe/132-probe-pre-particle-excitation-scan-page-0001.jpg)
*Figure 4 : graph representing percentage change in fluorescence intensity across different excitation wavelengths for 132 probes bound to miDNA 132*

![](https://static.igem.wiki/teams/4693/wiki/wetlab/results-for-nanoprobe/132-probe-pre-particle-emission.png)
*Figure 5 : graph representing percentage change in fluorescence intensity across different emission wavelengths for 132 probes bound to miDNA 132.*

![](https://static.igem.wiki/teams/4693/wiki/wetlab/results-for-nanoprobe/nanoprobe-124-pre-particle-excitation-scan-page-0001.jpg)
*Figure 6 : graph representing percentage change in fluorescence intensity across different excitation wavelengths for 124 probes bound to miDNA 124.*

* The maximum percentage change observed was 19 percentage.
* This was mainly because of the large background noise that was present as there was unbound A or B sequence, which had fluorophore attached to it present in the mixture. These could only be removed after binding the probes to the nanoparticle.
* It still showed that there was an increase in fluorescence intensity on the binding of miDNA in both 124 and 132 probes, proving that the binding of miDNA is causing the loop to open and separate the fluorophore quencher-pair

**Q) Did the binding of the probe to the magnetic nanoparticle to form the nanoprobe actually affect the readings?**

*Fluorescence intensity change on adding the miDNA to the Nanoprobe (after attachment)*
To test this, we first measured the fluorescence intensity change of the new nanoprobes.
We conducted an emission scan with a constant excitation of 490 nm and an emission range of 500 nm to 600 nm.

![](https://static.igem.wiki/teams/4693/wiki/wetlab/results-for-nanoprobe/percentage-change-in-fluorescence-intensity.png)
*Figure 7 : Figure showing the percentage change in fluorescence intensity across different emission wavelengths for nanoprobe 132 and nanoprobe 124 bound to their respective miDNA*

Here, we have a maximum percentage increase of 81 percentage.
Comparing the results before and after attachment of the probe to the nanoparticle to form the nanoprobe:

![](https://static.igem.wiki/teams/4693/wiki/wetlab/results-for-nanoprobe/maximum-percentage-increase-in-fluorescence.png)
*Figure 8 : Shows the maximum percentage increase in fluorescence observed before and after the probes were attached to the magnetic nanoparticle.*

This result clearly indicates that the nanoprobes give better fluorescence intensity change when compared to just probes.
The results for the detection limit and sensitivity experiments are provided in the [proof of concept page.](proof)
The future works planned for further development of our project can be found here

---

# Aptasensors

---

**Q) How did we confirm the hybridisation of the cDNA and the aptamer to form our aptasensor?**

*Native PAGE (14%)*

* The aim of this experiment was to detect the hybridised complex of cDNA 1 and the 85-mer aptamer.
* The protocol and the concentrations used are mentioned in the experiment and lab notebook page respectively.
* No clear indication of hybridisation could be derived from the native PAGE result, this could be due to the small size of our aptamer and cDNA. The experiment was carried out using a 16% gel and similar results were seen.
![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/18-8-cdna-2-native-page-big.png)
*Figure 9 : Native PAGE with cDNA 1 and 85-mer cortisol aptamer*
* The only band was observed for 85-mer aptamer plus cortisol ,we presume the cDNA 2 which is a 15-mer had run off the gel.
![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/23-8-cdna-1-2-native-page-big.png)
*Figure 10 : Native PAGE with both cDNA 1 and 2 and 85-mer cortisol aptamer*
* In this experiment, we increased the concentrations of the cDNA and the Quantifiers, as well as the percentage of the gel to 16%, in order to visualise the small fragments of cDNA, such as the cDNA 2 (15-mer). However, even with these modifications, we could only observe bands corresponding to the 85-mer aptamer.

*Isothermal Titration Calorimetry:*

* The aim of this experiment was to detect the hybridised complex of cDNA and aptamer.
* To check duplex formation between the 44-mer serotonin aptamer and 20-mer cDNA 1:

+ We prepared 0.25 micromolar concentration of the 44-mer serotonin aptamer and titrated it against 1 micromolar concentration of cDNA 1.The reference cell contained the same Milli-Q water that we used to prepare our working concentration.
+ 1.5 microliters of injection were given every 80 seconds for a total of 20 injections.

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/serotonin-aptamer-and-cdna-1-1mm-formation-itc-final-graph.png)
*Figure 11 : ITC final graph depecting serotonin aptamer and cDNA 1 serotoin binding*

* From the ITC result shown we can conclude that there is a strong association between cDNA 1 and the 44-mer serotonin aptamer. Given below are the obtained parameters from the ITC experiment:
![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/serotonin-aptamer-and-cdna-1-1mm-formation-parameters.png)
*Figure 12 : ITC serotonin aptamer and cDNA 1 parameters*

* To check duplex formation between the 85-mer cortisol aptamer and 40-mer cDNA 1:
  We used the same procedure as described above, and a detailed protocol is available on the [experiments page.<](experiments.html)/li>

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/cortisol-aptamer-and-cdna-1-2mm-formation-itc-final-graph.png)
*Figure 13 : ITC final graph 85-mer cortisol aptamer and cDNA 1 cortisol*

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/cortisol-aptamer-and-cdna-1-2mm-formation-parameters.png)
*Figure 14 : ITC 85-mer cortisol aptamer and cDNA 1 parameters*

In our experiments, we also observed a relationship between the cortisol cDNA 1 and the 85-mer aptamer. This relationship was evident in the shape of the graph, which became more sigmoid-shaped towards the end of the injections. This suggests that the two molecules are interacting with each other in a specific way.

We believe that this result could be strengthened by using a higher concentration of cDNA. This is because a higher concentration of cDNA would mean that more molecules would be available to bind to the aptamer. This would lead to more complete binding in fewer injections, and would therefore make the relationship between the two molecules more apparent.
To test this hypothesis, we would need to conduct further experiments. These experiments would involve using different concentrations of cDNA and measuring the binding between the cDNA and the aptamer. If our hypothesis is correct, we expect to see more complete binding in fewer injections when a higher concentration of cDNA is used.

* To check duplex formation between the 44-mer serotonin aptamer and 12-mer cDNA 2

+ We used the same procedure as described above, and a detailed protocol is available on the [experiments page.](experiments.html)
+ The results of the ITC experiment show that there is no obvious interaction between cDNA 2 and the 44-mer serotonin aptamer. This result is consistent with the results of the fluorescent-based hybridization experiments shown below.

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/serotonin-aptamer-and-cdna-2-1mm-formation-itc-final-graph.png)
*Figure 15 : ITC final graph 44-mer serotonin aptamer and cDNA 1 serotonin*

*Fluorescence based experiments:*

* We designed these experiments to detect the hybridization of the aptamer to the cDNA by observing the rate of quenching of the FRET signal. A successful binding event results in the quenching of the FRET signal.
* More details on the result can be found below.

**Q) How did we confirm the ideal molar ratio for the formation of our aptasensors?**

* Before we try to find the ideal molar ratio, we need to determine the lowest concentration of aptamer that is detectable in our fluorolog fluorospectrometer: The chosen starting concentration of 0.25 micromolar for the aptamer was derived by running various concentrations ranging from 0.1 micromolar to 0.5 micromolar concentrations,we wanted find the minimal concentration of aptamer that gives a considerable/detectable fluorescence level, attached below are the fluorescence emission scan ranging from 495 nm to 700 nm of the various molar concentrations:

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/0-1.png)
*Figure 16 : 0.1 Molar concentration of 85-mer aptamer cortisol*

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/0-25.png)
*Figure 17 : 0.25 Molar concentration of 85-mer aptamer cortisol*

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/0-5.png)
*Figure 18 : 0.5 Molar concentration of 85-mer aptamer cortisol*

|  |  |  |  |
| --- | --- | --- | --- |
| Concentration (micromolar) | 0.1 | 0.25 | 0.5 |
| Fluorescence obtained from fluorolog at 517 nm | 77490 | 689130 | 4852050 |

* We used the same starting concentration of 0.25 micromolar for the 85-mer aptamer and the 44-mer serotonin aptamer, but we used a starting concentration of 0.5 micromolar for the truncated cortisol aptamer. We determined this starting concentration using the same method as described above.
* Finding the ideal molar ratio is important for obtaining the most sensitive aptasensor. Now that we have determined the starting molar concentration for the aptamers, we need to determine the ideal molar ratio for forming our aptasensor. The ideal molar ratio can be measured by observing the difference in fluorescence intensity at t=0 (before binding) and t=60 mins (after binding).
* The ideal molar ratio will vary depending on the aptamer and target molecule being used. However, a good starting point is to use a molar ratio of 1:1. This means that there will be an equal number of aptamer molecules and target molecules in the solution. Once you have determined the ideal molar ratio, you can use it to prepare a solution for your aptasensor. This solution will be the most sensitive for detecting the target molecule.
![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/molar-ratio-cdna-1-cortisol-wmf.png)
*Figure 19 : Varying molar ratios of 85-mer aptamer and cDNA 1*
* For 85-mer cortisol aptamer and cDNA 1 the ideal molar ratio was observed to be 1:3 and 1:5 as seen from the graph
![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/molar-ratio-cdna-2-cortisol.png)
*Figure 20 : Varying molar ratio of 85-mer aptamer and cDNA 2*
* For the 85-mer cortisol aptamer and cDNA 2 the ideal molar ratio was observed to be 1:3
![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/molar-ratio-truncated-aptasensor.png)
*Figure 21 : Varying Molar ratios of truncated cortisol aptamer : cDNA*
* For the truncated aptamer and cDNA the ideal molar ratio was decided to be 1:2 because similar fluorescence differences were seen for 1:4 and 1:5.
![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/serotonin-cdna-1-molar-ratio.png)
*Figure 22 : Varying Molar ratios of 44-mer serotonin aptamer and cDNA 1*
* The ideal molar ratio of 44-mer serotonin aptamer to cDNA 1 serotonin was observed to be 1:3

**Q) How did we confirm the ideal hybridization time for formation of aptasensors?**

* The ideal hybridization time is determined by plotting the Ft/F0 values against time, using data from the fluorospectrometer and plate reader. This data can not only help us determine the ideal hybridization time, but it can also show us the quenching efficiency of the FRET system (fluorophore-quencher pair) for different molar ratios.
* Formation of cortisol aptasensor from 85-mer aptamer and cDNA 1
![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/cortisol-aptasensor-formation-cdna-1.png)
*Figure 23 : Cortisol aptasensor formation (85-mer aptamer & cDNA1) fluorolog data*

+ The data points in the graph were obtained from a Fluorolog fluorospectrometer. From the graph, we can estimate that the ideal hybridization time is around 50-60 minutes, because the graph plateaus around that time. This means that maximum quenching of the donor fluorophore is achieved within 30 minutes of the hybridization reaction.

- This data was also replicated using a 1:5 molar ratio of the 85-mer aptamer to cDNA 1 complex. From this, we can conclude that a 1:3 molar ratio is much more effective in forming the duplex due to the shorter hybridization time required to reach the plateau level of Ft/F0.

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/plate-reader-experiment-cdna-1-daa.png)
*Figure 24 : Cortisol aptasensor formation (85-mer aptamer & cDNA1) microplate reader data*

* This formation experiment of cortisol aptasensor with varying molar ratios ranging from 1:1 to 1:5 was performed using the microplate reader, The importance of this experiment is discussed in the measurements page. A major change in this experiment is the use of T.E. buffer to prepare the oligos aliquots instead of milliQ which was the resuspension media for the previous experiment. From this graph we were able to reproduce the ideal hybridisation time of 60 mins as seen from the fluorolog data. Upon examining the quenching outcomes, it is evident that the 1:3 molar ratio exhibits the highest quenching, approximately 45%. This finding aligns with our previous molar ratio results. The elevated quenching capability, rising from 30% as indicated in the fluorolog data to 45%, could potentially be attributed to the use of T.E buffer in lieu of milliQ. The need for using various controls in this experiment is discussed in the measurement page.

* Formation of cortisol aptasensor from 85-mer aptamer and cDNA 2:

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/cortisol-aptasensor-formation-cdna-2.png)
*Figure 25 : Cortisol aptasensor formation (85-mer aptamer & cDNA2) fluorolog data*
+ The graph shows the results of a fluorescence experiment to measure the hybridization time of an 85-mer aptamer to cDNA 2. The hybridization time is the amount of time it takes for the aptamer and cDNA 2 to bind to each other completely. The graph shows that the ideal hybridization time is around 50-60 minutes for both the 1:3 and 1:5 molar ratios. Maximum quenching of the fluorophore is achieved within 30 minutes of the hybridization reaction, for both the 1:3 and 1:5 molar ratios. This experiment shows that the ideal hybridization time is the same for both the 1:3 and 1:5 molar ratios. This suggests that the molar ratio of the aptamer to cDNA 2 does not significantly affect the hybridization time.
+ This graph shows the hybridisation time and the quenching capacity showing similar trends across both the cDNA that was designed for the 85-mer aptamer sequence.

* Formation of cortisol aptasensor from truncated aptamer and cDNA:

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/formation-of-truncated-cortisol-aptasensor-all-of-them.png)
*Figure 26 : Cortisol aptasensor formation (truncated 14-mer aptamer & cDNA2) fluorolog data*
+ From this graph we can see that no plateau has been reached even after running the experiment for 90 mins and the maximum quenching efficiency observed is around 12% which is seen by 1:2 molar ratio again aiding our result obtained from the molar ratio graph.

* Comparing the various possible cortisol aptasensors to determine the best cDNA aptamer pair:
![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/total-comparison-for-cortisol-aptasensor.png)
*Figure 27 : Comparison of cortisol aptasensor formation*

+ The engineering page provides a detailed explanation of the DBTL cycle used to develop this aptasensor. The graph shows that the truncated cDNA and aptamer complex does not produce a very efficient FRET system. This is likely because the truncated cDNA is shorter and has fewer binding sites for the aptamer. As a result, there are fewer FRET pairs formed, and the quenching efficiency is lower. Additionally, the graph shows that cDNA 1 and cDNA 2 produce similar results in terms of hybridization time and quenching efficiency, regardless of whether they are complexed with the 85-mer aptamer. This suggests that the binding affinity of the aptamer for cDNA 1 and cDNA 2 is similar. The overall results of this experiment suggest that the truncated cDNA and aptamer complex is not a very good candidate for developing an aptasensor. The quenching efficiency is too low, and the hybridization time is too long. cDNA 1 and cDNA 2 along with the 85-mer aptamer are better candidates for developing an aptasensor.

* Formation of serotonin aptasensor from 44-mer aptamer and serotonin cDNA 1:

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/serotonin-aptasensor-formation.png)
*Figure 28 : Serotonin aptasensor formation (44-mer serotonin aptamer & cDNA 1)*
+ We performed this experiment to determine the ideal molar ratio and hybridisation time for forming a serotonin aptasensor. We used a microplate reader to measure the fluorescence intensity of the aptasensor at different molar ratios of aptamer to cDNA. We performed multiple technical replicates and 3 experimental replicates to ensure the accuracy of our results. The ideal hybridization time for the serotonin aptasensor is observed to be around 50-60 minutes, which is similar to the ideal hybridization time for the cDNA cortisol aptamer complex. The quenching efficiency of the serotonin aptasensor is around 30% at a 1:3 molar ratio of serotonin to aptamer. This is consistent with the prediction that a 1:3 molar ratio is ideal for forming the serotonin aptasensor. The lowest quenching efficiency was observed at a 1:1 molar ratio of serotonin to aptamer, which was around 15%. The quenching data is supported by the ITC report, which shows that there is high affinity between the cDNA 1 and serotonin aptamer.

* Formation of serotonin aptasensor from 44-mer aptamer and serotonin cDNA 2:

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/hybridisation-time-for-formation-of-serotonin-aptasensor-cdna-2-1-1.png)

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/hybridisation-time-for-formation-of-serotonin-aptasensor-cdna-2-1-2.png)

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/hybridisation-time-for-formation-of-serotonin-aptasensor-cdna-2-1-3.png)

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/hybridisation-time-for-formation-of-serotonin-aptasensor-cdna-2-1-4.png)

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/hybridisation-time-for-formation-of-serotonin-aptasensor-cdna-2-1-5.png)

+ From the above graphs we can observe that there is an increase in fluorescence upon apparent hybridisation of the cDNA 2 and serotonin aptamer oligos for all molar ratios. This trend was also observed in the replicate data, which is plotted on the same graphs. Multiple technical replicates were also performed, as shown by the standard deviations plotted with both the first and replicate data points. This data is further justified by the ITC plot we obtained for cDNA 2 and serotonin aptamer which showed us that there was no significant association between cDNA 2 and serotonin aptamer.

**Q) How sensitive is our aptasensor?**

+ Sensitivity tests included using the ideal aptasensor determined by the previous experiments and measuring the increase in fluorescence obtained upon addition of varying concentration of our biomarker mimicking the human physiological value.
+ In conclusion we measured fold change with respect to aptasensor + 0ng/ml for the serotonin and cortisol aptasensor and for the serotonin aptamer, we could observe a linear relationship with increase in serotonin concentration but we found the cortisol aptasensor to show higher sensitivity with higher sensitivity from the range of 50ng/ml to 200 ng/ml which lies well in the range of human physiological serum levels of cortisol.
+ The analysis of the results will be discussed in length in our [proof of concept page](https://2023.igem.wiki/iiser-tvm/proof).

**Q) How specific is our aptasensor?**

+ To check the specificity of our aptasensor we conducted a biomarker mix test where we tested the specificity of our serotonin aptasensor by checking it’s rise in fluorescence upon addition of various different biomarkers other than serotonin and a mixture of all the biomarkers: the theory benign if our aptasensor is specific the increase in fluorescence upon addition of other biomarkers other than serotonin should show reduced increase in fluorescence compared to the increase obtained when bound to serotonin, this increase should also be visible when the aptasensor in exposed to the mixture of biomarkers.
+ In conclusion we observed that the serotonin aptasesnor showed a greater fluorescence difference when placed in addition with serotonin biomarker and the mixture of biomarker when compared to its difference shown in presence of other biomarkers. Hence this proves that our serotonin aptasensor is specific in nature.
+ The analysis of these results will be discussed in detail in the [proof of concept page](https://2023.igem.wiki/iiser-tvm/proof)

---

# Gsα Protein

---

Our Initial plans for Gsα protein included expression, purification and quantification by aptasensors. We intended to synthesize an aptamer for Gsα protein using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment ) method to select the aptamers that binds specific to the Gsα Protein.
Unfortunately, due to time and budget constraints we were unable to procure and work with the aptamers. The cloning experiments performed by us are documented here and our future protocols regarding purification and formation of aptasensors is further elaborated on the Implementation page.
The Gsα Gblock was procured through IDT.

**Q) How did we confirm the amplification of Gsα protein?**

We did a PCR reaction for the amplification of the Gsα Gblock. This PCR reaction was run at 57 degrees celsius and there was no band observed.
While consequent troubleshooting, the annealing temperature was manually calculated and obtained to be around 63 degrees celsius. In order to confirm the amplification of the Gsα Gblock, we ran a gradient PCR. The optimum temperature for amplification was found to be at 62.5 degrees celsius.

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/gsalpha-3.png)
*Figure 29 : Gradient PCR under Trans Illuminator*

* We did not visualize the ChemiDoc imaging system since we did not want to compromise the quality of our elute DNA. It was visualised in a UV transilluminator.
* Using the gradient function of the universal block, a gradient of 54 to 63°C was set. This was achieved by setting a 9 degree C gradient across 12 heating blocks. The following test parameters were selected: denaturation 95,30 s, annealing 55°C -63°C, 20 s, elongation 68°C, 150s, Taq-Polymerase 1.25units.
* Tubes H,I,J,K,L was annealed at 55.1, 57.5,60.5,62.4,63.1 degree celsius respectively. Positive control (1.5kB dna fragment) was annealed at 55.1 degrees.
* The bands were found to be most visible in lanes K and L. This implied that the correct annealing temperature lies in the range of 62-63 degrees.
* PCR amplification was performed again with the right annealing temperature

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/gsalpha-2.png)
*Figure 30 : PCR amplification of Gs alpha Gblock*

**Q) How did we confirm restriction digestion?**

After overnight restriction digestion, we ran an agar gel with the Gsα Gblock and PET 19b Plasmid. We used two positive controls, a gene fragment of 1.5kb and our amplified Gsα Gblock (1.2kB). However, while we observed bands for the restriction-digested insert and plasmid, we were not able to observe the band for positive control.

![](https://static.igem.wiki/teams/4693/wiki/wetlab/aptasensors/after-restriction-digestion.png)
*Figure 31 : Gel after restriction digestion*

* We did not visualize the ChemiDoc imaging system since we did not want to compromise the quality of our elute DNA. It was visualised in a UV transilluminator.
* Second band was observed to be around 1kb which confirms the formation of restriction digested insert of 1198bp size
* Third band is observed between 5kB and 6kB bands of the ladder which confirms the presence of restriction digested vector. An undigested plasmid would have given three different bands for nicked, linear and supercoiled. Since we observed only one band here, we assume that most of the pET19B(5.7kB) is succesfully restriction digested.
* Bands are not visible for the positive controls. This might be because

1. Compromised gel integrity on one half of the gel only - wells might have collapsed
2. Contamination of samples might hinder the migration of fragments during agarose gel electrophoresis.
3. Improper mixing of loading dye and sample

After restriction digestion we were able to get the fragments of insert and vector. So in order to introduce our desirable DNA fragment into our vector for cloning and transformation, ligation had to be done to join the insert and vector fragment. We set up a ligation reaction with a reverse and backward primer and a T4 DNA ligase. It did not yield a desirable result.
When further transformation experiments were carried out, we found 2 colonies. A master plate was made and a colony PCR was performed. This again did not work. The possible reasons for ligation PCR to not give desirable results could be due to the machine being faulty. Due to time constraint, we were unable to proceed forward with troubleshooting the protocol and further experiments for the Gsα Gblock.

{% endblock %}