iGEM 2024 — Team iisc-bengaluru

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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# Lab Notebook

Protein Expression
Protein Purification and Verification
Phosphorylation
Click Reaction and binding of Aptamers
Asymmetric PCR

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# Engineering Cycles

## Experiment: Predicting the structure of pTau with several combinations of phosphorylation sites

**DESIGN**: We can model a phosphorylation site (mostly Threonine or Serine, although Tyrosine also potentially phosphorylates) with a phosphomimetic amino acid (primarily Aspartic acid or Glutamic acid) which mimics the charge distribution or structure parameters. Thus, some Alzheimer’s marker phosphorylation can be incorporated into the protein by phosphomimetics to mimic the same conditions.

**BUILD**: We selected a few sites of relevance: 231T, 198S, 199S, 205S etc and replaced with Aspartic acid.

**TEST**: We obtained the structures using ChimeraX and AlphaFold by using energy minimisation option.

**LEARN**: The structures are completely different from the initial intrinsically disordered. The arising hydrophobicity can be correlated to the tendency of aggregation.

## Experiment: Preparation of competent BL21 DE3 E. coli cells

**DESIGN**: Proliferation of BL21 E. coli cells, and modifying the newly formed cells to make competent cells. Competent cells can take up plasmids and can express them.

**BUILD**: We used chemical method of producing competent cells. The preformed BL21 cells (brought from another lab) were propagated and then subjected to buffers containing RbCl, CaCl2, PIPES, etc. to make them competent to induce plasmid and express it.

**TEST**: Transformed plasmid, specific for expression in BL21 cells, and plated it on non-antibiotic LB agar plate. Control was setup, which involved transformation of original BL21 (from which it was propagated) with the same plasmid.

**LEARN**: We found that the original stock of BL21 had proper growth, which approved the functionality of the plasmid. But, unfortunately there was no growth in the prepared competent cell. This might have happened due to improper methodologies or slight difference in buffer. Utmost care needs to be taken during competent cell preparation. There is also a high chance of contamination, and it involves destruction of cells.

## Experiment: Transformation of Tau 441 plasmid into BL 21 DE3 E. coli.

**DESIGN**: Transformation of Tau/pET29b (Addgene #16316) plasmid into BL 21 E. coli using heat shock method.

**BUILD**: We modified some aspects of a general heat-shock method of plasmid transformation. The protocol we followed can be found in the protocols section.

**TEST**: We grew the primary culture of bacteria in Kanamycin antibody. The plasmid, which has KanR gene, expresses in Kanamycin antibody.

**LEARN**: We saw proper growth of Bacterial culture, which meant that the bacteria successfully took up the plasmid and could express it. Afterwards we also extracted protein from it, which again proves its success.

## Experiment: Expression and purification of Human Tau 441 protein.

**DESIGN**: After successful transformation, the bacterial culture can express the protein. The protein can be extracted and purified to obtain pure protein, free from contamination. This protein can be used further in testing and phosphorylation.

**BUILD**: The protein could be expressed using IPTG, in E. coli bacterial culture. We modified some aspects of a general protein purification. The protocol we followed can be found in the protocols section. We could use Ni-NTA gravity column method of purification, due to availability of His tag in our construct.

**TEST**: We ran SDS gel to obtain protein bands. We compared it to the standard protein marker. The protein, conjugated with His tag, needs to come at around 50kDa range.

**LEARN**: We could obtain a single protein band in the eluted fraction. We did find bands in the same range in Wash fraction and slightly in flow through, which concludes that this might not be the efficient method of purification, in terms of wastage. But we are sure that this is a fairly good method, in terms of the quality of protein obtained.

## Experiment: To show the presence of 231pTau and binding of aptamers to it, simultaneously.

**DESIGN**: Since the aptamers bind with the protein pTau, we expect a shift or smear of the protein bands when the protein incubated with the aptamer at optimal concentration is loaded on a native gel made with TBE. Thus, if we do a WB with the aptamer incubated protein, there will be an observable compared with the case of no aptamers.

**BUILD**: We improvised the standard protocol of EMSA, instead of using autoradiography we used visualisation of the protein using the antibody conjugate (pTau rabbit monoclonal and anti-rabbit hrp-conjugated).

**TEST**: We did Western blot with the protein incubated with the aptamers at 0.1 micromolar concentration.

**LEARN**: We found that although the smear is evident, there is a significant background. This is because of using skimmed milk as blocking agent, which contains casein. Casein is a phosphoprotein and the pTau antibody may have some non-specific binding tendency to Casein as an Ab selected for a phosphoprotein may have non-specific binding with phosphoproteins as the phosphorylated moiety is always either of Threonine, Serine or Tyrosine.

## Experiment: Using random mutagenesis in SELEX for quicker results.

**DESIGN**: SELEX relies on randomness, as the chances of an optimal sequence to bind with the target increases with large combinations. Thus, if the randomisation is done at a greater degree, in each round of SELEX on a partition of the eluded aptamers, the selection of Aptamers may be accomplished in exponentially lesser time. We found that the incorporation of non-conventional bases like dPTP and oxo-GTP can cause random point mutations at an incredible level of 10% after a few cycles of PCR followed by asymmetric PCR. We needed a high mutation rate due to small size of the aptamers.

**BUILD**: Since our Aptamers were already selected for the target by 17 cycles by the original authors, we planned to perform the same with a different sample DNA. However, on analysing the aptamers, we hypothesised that high G repeats may have been responsible for a better binding to the target. dPTP causes G biased transitions.

**TEST**: On performing the PCR, we couldn’t observe the bands of the PCR product in the gel.

**LEARN**: The reasons could be firstly, incorporation of non-conventional bases might slow down the action of normal Taq-polymerase. Secondly, the dPTP or oxo-GTP incorporated DNA may not be a perfect helix, as these bases exist in tautomeric form.

## Experiment: Click reaction of C8-Alkyne dCTP with [S, R, S]-AHPC-PEG1-Azide (VHL Ligand)

**DESIGN**: C8-Alkyne dCTP may undergo cycloaddition with VHL ligand, having an azide label. This product after HPLC purification can be incorporated into an aptamer by terminal transferase. The ligand can help localising the Aptamer around VHL E3 ubiquitin ligase, leading to polyubiquitination of the target.

**BUILD**: We set the protocols for accomplishing click reaction of C8-Alkyne dCTP with [S, R, S]-AHPC-PEG1-Azide (VHL Ligand). We also modelled the structure of the product.

**TEST**: We did the calculation of the radius of gyration and other aspects of the molecule by computer simulation.

**LEARN**: We got the radius of gyration to be 16.54652 Angstroms, which shows that the final molecule is highly coiled and thus, may have unexpected behavior.

## Experiment: Testing of software of previous iGEM team and troubleshooting of several bugs and errors.

**DESIGN**: Run MAWS on the VQIINK part of 2mz7 pdb (residues 275-280) and get an Aptamer Sequence.

**BUILD**: Perform the necessary improvements to the software as mentioned in the AptaLoop documentation. Create a GUI (tkinter based or web based, preferably web-based) of the software for the help of others who will use this tool. Understanding what sqm actually does, and what is exactly stored in it, along with the error messages shown by it.

**TEST**: Run the examples, to get an idea what is happening in each module. Chopped down 2MZ7 PDB to get the said hexapeptide (VQIINK). Added H atoms wherever was missing using PyMol to get a working.

**LEARN**: Residues more than 10 did not work. Infinite pause time error was occurring because of an os.waitpid() function. Duplicate atom warning was coming up. Understood which Aptamer sequence to work with given a whole pool is generated in output. Need to start building the part and integrate other tools, and also the GUI.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# Experiments

## Plasmid Extraction

**Aim:** To extract Tau plasmid from E. coli (BL21 strain) cells

**Materials Required:** HiMedia Miller Luria Bertani Agar, HiMedia Miller Luria Bertani Broth, E. coli BL21 cells containing Tau plasmid, Invitrogen's Plasmid Mini-Prep Kit, Kanamycin

#### Procedure:

##### Step 1: Streaking of bacteria containing tau plasmid on antibiotic medium

1. Weigh 4g of HiMedia Miller Luria Bertani Agar and transfer into an Erlenmeyer flask
2. Measure 100ml of distilled water and pour it into the Erlenmeyer flask containing LB agar.
3. Cover the mouth of the flask with a piece of aluminium foil
4. Gently place the flask in an autoclave bag and place it in the autoclave
5. Autoclave the medium at 121°C and 15 PSI for 30-60 minutes
6. Transfer the flask into a Laminar Air Flow. The following steps are performed in a Laminar Air Flow under sterile conditions
7. Add 100µl of 100ng/ml kanamycin into 100ml of medium
8. Pour the medium into 5-7 sterilised agar plates
9. Streak a small amount of BL21 cells containing tau plasmid onto the plate using sterilised inoculation loop or pipette tip
10. Incubate the plate in an incubator set at 37°c for 12-14 hours

##### Step 1: Streaking of bacteria containing tau plasmid on antibiotic medium

1. Weigh 2.5g of HiMedia Miller Luria Bertani Broth and transfer into an Erlenmeyer flask
2. Measure 100ml of distilled water and pour it into the Erlenmeyer flask containing LB agar.
3. Cover the mouth of the flask with a piece of aluminium foil
4. Gently place the flask in an autoclave bag and place it in the autoclave
5. Autoclave the medium at 121°C and 15 PSI for 30-60 minutes
6. Transfer the flask into a Laminar Air Flow. The following steps are performed in a Laminar Air Flow under sterile conditions
7. Pipette out 5ml of LB broth into a 15ml centrifuge tube
8. Add 5µl of 100ng/ml kanamycin to the broth in the centrifuge tube
9. Pick a small amount of BL21 cells from the parent stock using a pipette tip or an inoculation loop and add this inoculum to the broth to set up a primary culture. For successive primary cultures the inoculum was picked up from the among colonies formed on the kanamycin-LB plate.
10. Incubate the plate in an incubator set at 37°c for 12-14 hours

##### Step 3: Plasmid Extraction

1. Pellet down the cells in the primary culture by centrifugation at 3,500rpm for 15-20 minutes

Tau plasmid was isolated from BL21 cells by following the protocol provided in Invitrogen’s Plasmid Mini-Prep Kit. The protocol has been provided here for reference.

2. Resuspend the pelleted cells in 250 µL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain. Ensure RNase A has been added to the Resuspension Solution.
3. Add 250 µL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
4. Add 350 µL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.
5. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
6. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate. Close the bag with GeneJET Spin Columns tightly after each use!
7. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube. Do not add bleach to the flow-through
8. Optional: use this preliminary washing step only if EndA+ strains which have high level of nuclease activity are used. Wash the GeneJET spin column by adding 500 µL of Wash Solution I (#R1611, diluted with isopropanol) and centrifuge for 30-60 sec. Discard the flow-through. This step is essential to remove trace nuclease activity.
9. Add 500 µL of the Wash Solution (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
10. Repeat the wash procedure (step 8) using 500 µL of the Wash Solution.
11. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
12. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 µL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane
13. Discard the column and store the purified plasmid DNA at -20°C.

## Competent Cell Preparation

**Aim:** Preparation of chemically competent bacterial cells

**Materials Required:** HiMedia Miller Luria Bertani Agar, HiMedia Miller Luria Bertani Broth, RbCl, MnCl2.4H2O, CH3COOK, CaCl2, glycerol, MOPS, 0.45µ filter

#### Procedure:

##### Step 1: Streaking of bacteria containing tau plasmid on LB agar medium

1. Weigh 4g of HiMedia Miller Luria Bertani Agar and transfer into an Erlenmeyer flask
2. Measure 100ml of distilled water and pour it into the Erlenmeyer flask containing LB agar.
3. Cover the mouth of the flask with a piece of aluminium foil
4. Gently place the flask in an autoclave bag and place it in the autoclave
5. Autoclave the medium at 121°C and 15 PSI for 30-60 minutes
6. Transfer the flask into a Laminar Air Flow. The following steps are performed in a Laminar Air Flow under sterile conditions
7. Add 100µl of 100ng/ml kanamycin into 100ml of medium
8. Pour the medium into 5-7 sterilised agar plates
9. Streak 100µl of BL21(DE3) gold E. coli cells on a plate using sterilised inoculation loop or pipette tip
10. Incubate the plate in an incubator set at 37°c for 12-14 hours

##### Step 2: Setting up a primary culture 1

1. Weigh 2.5g of HiMedia Miller Luria Bertani Broth and transfer into an Erlenmeyer flask
2. Measure 100ml of distilled water and pour it into the Erlenmeyer flask containing LB agar.
3. Cover the mouth of the flask with a piece of aluminium foil
4. Gently place the flask in an autoclave bag and place it in the autoclave
5. Autoclave the medium at 121°C and 15 PSI for 30-60 minutes
6. Transfer the flask into a Laminar Air Flow. The following steps are performed in a Laminar Air Flow under sterile conditions
7. Pipette out 5ml of LB broth into a 15ml centrifuge tube
8. Add 5µl of 100ng/ml kanamycin to the broth in the centrifuge tube
9. Pick a colony from the plate and inoculate the LB medium in the centrifuge tube.
10. Incubate the primary culture in an incubator set at 37°c for 12-14 hours

##### Step 3: Setting up a secondary culture 1

1. Add 1ml of primary culture to 100ml of LB medium
2. Incubate the secondary culture at 37°C with shaking at 200rpm
3. Periodically check the OD of the culture using a spectrophotometer
4. When the OD reaches 0.4-0.6, shift the flask onto ice and leave it be for 20 minutes

##### Step 4: Preparation of buffer 1

1. Weigh/measure and transfer the following chemicals into a 500ml screwcap bottle

| Chemicals | Concentration in the buffer | Amount in the buffer (100ml) |
| --- | --- | --- |
| RbCl | 100mM | 1.208g |
| MnCl2·4H2O | 50mM | 0.990g |
| CH3COOK | 30mM | 0.294g |
| CaCl2 | 10mM | 0.110g |
| Glycerol | 15% | 15ml |

2. Make up most (97-98ml) of the volume with MilliQ water
3. Adjust the pH of the buffer using acetic acid until it reaches a value of about 5.8
4. Make up any remaining volume with MilliQ water

##### Step 5: Preparation of buffer 2

1. Weigh/measure and transfer the following chemicals into a 50ml centrifuge tube

| Chemicals | Concentration in the buffer | Amount in the buffer (100ml) |
| --- | --- | --- |
| PIPES | 10mM | 42mg |
| RbCl | 10mM | 24.2mg |
| CaCl2 | 75mM | 0.166g |
| Glycerol | 15% | 3ml |

2. Make up most (97-98ml) of the volume with MilliQ water
3. Adjust the pH of the buffer using acetic acid until it reaches a value of about 6.5
4. Make up any remaining volume with MilliQ water

##### Step 6: Preparing chemically competant cells

1. Centrifuge the secondary culture at 2700g and 4°C temperature for 15-20 minutes
2. Discard the supernatant
3. Resuspend the cells in the filter-sterilised buffer 1 in a 2:5 ratio of buffer: cell culture. Resuspension must be done by swirling on ice
4. Incubate the resuspended solution on ice
5. Centrifuge the resuspended solution at 2700g, at 4°C temperature for 15-20 minutes
6. Discard the supernatant, taking care not to dislodge the pellet.
7. Resuspend the pellet in filter sterilised buffer 2 in a ratio of 0.2:5 buffer: cell culture
8. Aliquote 100µl of resuspended solution in microcentrifuge tubes and preincubate on ice
9. Flash-freeze the solution in the microcentrifuge tube using liquid nitrogen
10. Store the competent cells at -80°C

## Transformation

**Aim:** Transformation of BL21 cells with tau plasmid

**Materials Required:** HiMedia Miller Luria Bertani Agar, Kanamycin (100mg/ml)

#### Procedure:

##### Step 1: Competent Cell Preparation

Follow the protocol for competent cell preparation

##### Step 2: Transformation

1. Incubate competent BL21 cells (100µl aliquote) and isolated tau plasmid DNA on ice (at about 4°C) for around 10 minutes
2. Add 10ng of plasmid DNA to the competent cells
3. Place the microcentrifuge tube containing competent cells and plasmid DNA in a water bath set at 42°C for 60-90 seconds to give them a heat shock
4. Incubate the cells on ice for 5 minutes
5. Add 1ml of LB broth to the cells
6. Incubate the cells at 37°C in an incubator with shaking at 200rpm for 40 minutes to 1 hour
7. Plate the cells on LB agar containing kanamycin (100µl of 100mg/ml kanamycin in 100ml LB agar)
8. Incubate the plate in an incubator set at 37°C for 14-16 hours
9. Streak a small amount of BL21 cells containing tau plasmid onto the plate using sterilised inoculation loop or pipette tip
10. Incubate the plate in an incubator set at 37°c for 12-14 hours

## Protein Purification

**Aim:** To induce expression of tau protein in tau plasmid containing BL21 cells and purify tau protein produced

**Materials Required:** LB medium, 100mg/ml kanamycin, NaH2PO4, Na2HPO4, Isopropyl ß-D-1-thiogalactopyranoside (IPTG), Ethylenediaminetetraacetic acid (EDTA), Beta-mercaptoethanol (BME), NaCl, Phenylmethylsulfonyl fluoride (PMSF), DNase, MgCl2, imidazole, NaOH solution, 0.45µ filter

#### Procedure:

##### Step 1: Setting up a primary culture of transformed tau plasmid containing BL21 cells

Carry out the following steps in a laminar air flow under sterile conditions

1. To 3ml of LB medium in a 15ml centrifuge tube, and 3µl of 100mg/ml kanamycin
2. Pick a single isolated colony from the plate having transformed tau plasmid containing BL21 cells and inoculate the LB medium to set up a primary culture
3. Incubate the primary culture overnight at 37°C in an incubator

##### Step 2: Setting up a secondary culture of transformed tau plasmid containing BL21 cells

Carry out the following steps in a laminar air flow under sterile conditions

1. To 100ml of LB medium, add 100µl of 100mg/ml kanamycin
2. Once the OD600 of the primary culture reaches a value of around 1.4 - 1.6, add 1ml of the primary culture to 100ml of antibiotic containing LB medium to set up secondary culture
3. Incubate the secondary culture at 37°C in an incubator
4. Periodically measure the OD600 of the culture

##### Step 3: IPTG induction of tau protein expression

1. Once the OD600 of the secondary culture reaches a value of around 0.8 – 1, add 100µl of 1M IPTG to the secondary culture to induce tau protein expression
2. Incubate the culture at 18°C in an incubator with shaking at 200rpm for 18-20 hours
3. Centrifuge the secondary culture at 3500rpm and a temperature of 4°C to obtain a cell pellet
4. Store the pellet obtained at -80°C

##### Step 4: Preparation of NaP buffer

1. Prepare NaP buffer by following the below recipe

| Components | Concentration in the buffer |
| --- | --- |
| NaH2PO4 | 24.59mM |
| Na2HPO4 | 75.41mM |

##### Step 5: Preparation of lysis buffer

1. Prepare lysis buffer, without PMSF, by following the recipe below

| Components | Concentration in the buffer |
| --- | --- |
| NaP | 50mM |
| BME | 5mM |
| EDTA | 1mM |
| NaCl | 150mM |
| PMSF | 1 |

##### Step 6: Lysis of IPTG induced cells by sonication and precipitation of DNA

1. Resuspend the pellet of IPTG induced cells in 2ml of lysis buffer
2. Add 1mM of PMSF to the suspension
3. Lyse the cells by sonication (50% amplitude, at 10 second intervals for 20 minutes)
4. Add DNase to the cell lysate at a concentration of 5µg per gram of cell pellet
5. Add MgCl2 to the lysate such that its final concentration in the lysate is 5mM
6. Incubate the lysate at room temperature for 40 minutes
7. Centrifuge this solution at 13000rpm for 30 minutes
8. Collect the supernatant in a separate microcentrifuge tube
9. Store the precipitate at 4°C. This will be used while running SDS PAGE.

##### Step 7: Preparation of equilibration buffer

1. Prepare equilibration buffer by following the recipe below

| Components | Concentration in the buffer |
| --- | --- |
| NaP | 50mM |
| BME | 5mM |
| NaCl | 150mM |

2. Adjust the pH of the buffer until it reaches a value of around 7

##### Step 8: Preparation of elution buffer

1. To a small volume of equilibration buffer, add imidazole such that its final concentration in the elution buffer is 500mM. This serves as the elution buffer

##### Step 9: Protein purification by Ni-NTA affinity chromatography

1. Syringe filter the obtained supernatant using 0.45µ filter
2. Clean a chromatography column using NaOH
3. Wash the column using 5-6 column volumes of water
4. Fill the column with Ni-NTA resin
5. Equilibrate the Ni-NTA column using 4-5 column volumes equilibration buffer
6. Pass the filtered supernatant through the Ni-NTA column
7. Collect the flow-through in a microcentrifuge tube. This will be used later while running SDS PAGE
8. Wash the column using 4-5 column volumes of equilibration buffer
9. Collect the filtrate in a microcentrifuge tube. This will be used later while running SDS PAGE
10. Elute the bound proteins by passing the elution buffer through the Ni-NTA column
11. Collect the eluted product in a microcentrifuge tube. This will be used later while running SDS PAGE. This fraction should contain the tau protein. This can be verified by running an SDS PAGE.

## SDS-Polyacrylamide Gel Electrophoresis

**Aim:** To visualise and identify tau protein by running SDS PAGE

**Materials Required:** 30% acrylamide mix, 1.5M Tris pH 8.8, 1.0M Tris pH 6.8, SDS, Ammonium persulphate (APS) , N,N,N′,N′ -Tetramethylethylenediamine (TEMED), Tris-HCL, glycerol, (Betamercaptoethanol) BME, bromophenol blue, isopropyl alcohol, Bio-Rad Mini PROTEAN Tetra Cell, Bio-Rad PowerPac system

#### Procedure:

##### Step 1: Preparation of resolving buffer

1. 1. Prepare 5ml of resolving buffer, without APS solution and TEMED, by following the recipe below

| Components | Volume |
| --- | --- |
| dH2O | 1.9 mL |
| 30% Acrylamide Mix | 1.7 mL |
| 1.5M Tris pH 8.8 | 1.3 mL |
| 10% SDS | 0.050 mL |
| 10% APS (Freshly Prepared Stock) | 0.050 mL |
| TEMED | 0.002 mL |

##### Step 2: Preparation the stacking buffer

1. Prepare 2ml of stacking buffer, without APS solution and TEMED, by following the recipe below

| Components | Volume |
| --- | --- |
| dH2O | 1.4 mL |
| 30% Acrylamide Mix | 0.33 mL |
| 1.0M Tris pH 8.8 | 0.25 mL |
| 10% SDS | 0.020 mL |
| 10% APS (Freshly Prepared Stock) | 0.020 mL |
| TEMED | 0.002 mL |

##### Step 3: Hand casting a polyacrylamide gel using Bio-Rad Mini PROTEAN Tetra Cell

1. Assemble the glass cassette sandwich provided in the Bio-Rad Mini PROTEAN Tetra Cell kit
2. Place a comb into the assembled gel sandwich. Use a marker to mark a point on the glass plate 1cm below the level of teeth of the comb. Remove the comb from the gel cassette
3. Add 0.5ml of resolving buffer into a microcentrifuge tube. To this, add 0.010ml of 10% APS and 0.004ml of TEMED. This constitutes a sealing gel.
4. Immediately pour the sealing gel into the gel cassette.
5. Allow the sealing gel to polymerise
6. Add APS and TEMED to the remaining resolving buffer
7. Immediately pour the resolving buffer into the cassette up to the level of the mark
8. Overlay the buffer with isopropyl alcohol
9. Allow the gel to polymerise for 40-60 minutes
10. Once the gel is polymerised, decant the isopropyl alcohol layer from the cassette
11. Rinse the top of the gel with distilled water and blot away any excess water using a filter paper
12. Add APS and TEMED to the stacking buffer
13. Immediately pour the stacking buffer into the gel cassette
14. Before allowing the gel to polymerise, insert the comb into the gel cassette vertically, making sure no air bubbles are trapped beneath its teeth
15. Allow the gel to polymerise for 30-45 minutes

##### Step 4: Preparation of Loading Buffer

1. Prepare 2X loading buffer by following the recipe below

| Components | Amount |
| --- | --- |
| Tris-HCl | 1 mL |
| SDS | 0.2 g |
| Glycerol | 1 mL |
| BME | 0.5 mL |
| Bromophenol | 0.001 g |

##### Step 5: Preparation of protein samples

1. Add 100µl of samples collected at different stages of protein purification to separate microcentrifuge tubes
2. Add 100µl of 2X Loading buffer to each of the microcentrifuge tubes
3. Heat the contents of each microcentrifuge tube to 98°C for 2-3 minutes using a dry bath
4. Centrifuge each of the samples, using the mini spin

##### Step 6: Preparing the running buffer

1. Add 900ml of distilled water to 100ml of 10X TGS buffer to prepare 1X TGS buffer

##### Step 7: Loading the samples and running the SDS PAGE

1. Place the gel cassette in the Bio-Rad Mini PROTEAN Tetra Vertical Electrophoresis Cell by making use of the Electrode assembly and Bio-Rad Buffer Dam.
2. Fill the chamber with 1X TGS buffer
3. Lift the comb out of the gel cassette taking care not to damage any of the wells
4. Into a well at one end of the edge add 4µl of Promega’s Broad Range Protein Molecular Weight Markers
5. Load 20µl of each sample into separate wells
6. Connect the Electrophoresis Cell to Bio-Rad PowerPac system and apply a potential difference of 150V across the gel
7. Let the electrophoresis run for 60 minutes or until the dye front reach the end of the gel

## Bradford Assay

**Aim:** To determine the concentration of protein extracted and purified using Bradford Assay

**Materials Required:** Bovine serum albumin (BSA), Bradford reagent

#### Procedure:

##### Step 1: Preparation of standard solutions of bovine serum albumin (BSA)

1. Prepare a 1mg/ml stock solution of BSA by dissolving 1mg of BSA in 1ml of distilled water
2. Using the stock solution, prepare standard solutions of the following concentrations by performing serial dilutions: 100µg/ml, 80 µg/ml, 60 µg/ml, 40 µg/ml, 20 µg/ml, 10 µg/ml.

| Concentration (µg/ml) | Volume of BSA solution | Volume of distilled water(µl) |
| --- | --- | --- |
| 100 | 100µl of stock soln. | 900 |
| 80 | 800µl of soln. 1 | 200 |
| 60 | 750µl of soln. 2 | 250 |
| 40 | 667µl of soln. 3 | 333 |
| 20 | 500µl of soln. 4 | 500 |
| 10 | 500µl of soln. 5 | 500 |

##### Step 2: Plotting a standard curve

1. To 100µl of 100µg/ml standard BSA solution taken in a cuvette, add 900µl of Bradford reagent.
2. Incubate the solution for 5 minutes.
3. Measure the absorbance of the solution at 595nm using a spectrophotometer. Record the absorbance.
4. Repeat the above steps for all standard solutions and record their absorbance at 595nm.
5. Draw a standard curve by plotting the concentration of standard solutions on the X-axis and corresponding absorbance at 595nm on the Y-axis.

##### Step 3: Measuring the concentration of tau protein

1. Make a 1:5 dilute solution of tau protein by adding 400µl of distilled water to 100µl of tau protein in a microcentrifuge tube.
2. To 100µl of this solution taken in a cuvette, add 900µl of Bradford reagent.
3. Incubate the solution for 5 minutes.
4. Measure the absorbance of the solution at 595nm using a spectrophotometer. Record the absorbance.
5. On the standard curve, draw a line perpendicular to the X-axis, intersecting the Y-axis at the absorbance of the tau protein solution.
6. From the point of intersection of this line with the curve, drop a perpendicular to the X-axis and record the intercept of this line with the X-axis.
7. The recorded intercept gives the concentration of a 1:5 dilute solution of the tau sample. Multiply this concentration by 5 to obtain the concentration of purified tau protein.

## Phosphorylation of protein

**Aim:** To phosphorylate purified tau protein

**Note: The following protocol has been optimised for 2.86mM (as measured by Bradford Assay) of purified tau protein**

**Materials Required:** Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 50mM MgCl2 solution, 10mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES buffer), Promega’s r-ATP (10mM), GSK-3β enzyme, Laemmli buffer

#### Procedure:

##### Step 1: Preparation of Phosphorylation buffer

1. Prepare 1ml of phosphorylation buffer by following the recipe below

| Components | Concentration (mM) | Amount |
| --- | --- | --- |
| EGTA | 0.625 | 0.0002375g |
| MgCl₂ soln. (50mM) | 0.6 | 12µl |
| HEPES (10mM) | 5 | 500µl |
| H₂O | - | 282µl |
| ATP (10mM) | 0.4 | 4µl |
| GSK-3β | - | 2µl |

##### Step 2: Phosphorylation of purified tau protein

1. Add 200µl of 2.86Mm (as measured by Bradford assay) tau protein
2. Incubate the mixture in a water bath set at 30°C for 22 hours

##### Step 3: Stopping the phosphorylation reaction

1. After 22 hours of incubation, to 1ml of the mixture, add 1ml of Laemmli buffer.
2. Boil the mixture for 5 minutes to stop the phosphorylation reaction.
3. Store the mixture at room temperature.

## Click Reaction

**Aim:** Click reaction of C8-Alkyne dCTP with [S, R, S]-AHPC-PEG1-Azide (VHL Ligand)

**Materials Required:** HEPES Buffer (10 mM, pH 7.8), CuSO4, Sodium L-ascorbate, C8-Alkyne dCTP, [S, R, S]-AHPC-PEG1-Azide

#### Procedure:

1. Prepare 75µl of solution of 100nmol [S, R, S]-AHPC-PEG1-Azide and 50nmol C8-Alkyne dCTP in HEPES buffer.
2. Initiate the reaction by adding CuSO4 and sodium L-ascorbate such that the final concentration of these components in solution is 472 µM and 18.3 mM, respectively.
3. Shake the mixture at 150rpm for 3 hours at 37°C.
4. Store the product at room temperature.

## Electrophoretic Mobility Shift Assay

**Aim:** To verify aptamer binding to 231-phosphorylated tau by performing an electrophoretic mobility shift assay

**Materials Required:** 30% Acrylamide solution, Tris-HCl, 10% APS solution, TEMED, Phospho-Tau (7ng/ml in Laemmli buffer and phosphorylation buffer, DNA aptamer, nuclease free water, polyvinylidene fluoride (PVDF) membrane, Semi-Dry Transfer Cell (Tran Blot SD BioRad), TBE Buffer, TBS Buffer, skimmed milk powder, Anti-231pTau Ab (Rabbit monoclonal from ThermoFisher), Anti-Rabbit HRP-conjugated Ab, X-ray plates, ECL substrate, 5M NaCl, 1M KCl, 1M MgCl2, 0.5M EDTA, 1M DTT, 10mg/ml BSA, Laemmli Buffer, Ponceau stain (optional)

#### Procedure:

##### Step 1: Preparing the binding buffer

1. Prepare 1ml of 4X Binding buffer by following the recipe below

| Components | Volume (in µl) |
| --- | --- |
| 1M Tris-HCl pH 7.5 | 50 |
| 5M NaCl | 10 |
| 1M KCl | 200 |
| 1M MgCl2 | 5 |
| 0.5M EDTA pH 8.0 | 10 |
| 1M DTT | 5 |
| 10mg/ml BSA | 25 |
| ddH2O | 695 |

##### Step 2: Hand casting a native PAGE gel

1. Hand cast a native PAGE gel by following the first 3 steps of the SDS PAGE protocol, without adding SDS solution to the resolving and stacking buffers

##### Step 3: Pre-running the gel

1. Place the gel cassette in the Bio-Rad Mini PROTEAN Tetra Vertical Electrophoresis Cell using the Electrode assembly and Bio-Rad Buffer Dam.
2. Fill the chamber with 1X TBE buffer.
3. Lift the comb out of the gel cassette, taking care not to damage any of the wells.
4. Connect the Electrophoresis Cell to the Bio-Rad PowerPac system and apply a potential difference of 150V across the gel.
5. Pre-run the gel for 20 minutes.

##### Step 4: Setting up binding reactions

1. Set 20µl binding reaction in PCR tubes having the following composition

| Components | Volume (in µl) |
| --- | --- |
| 4X Binding Buffer | 5 |
| Phosphorylated Tau Protein | 10 |
| DNA Aptamer | 1 |
| Nuclease Free Water | 4 |

2. Set up a control reaction for each sample of phosphorylated tau having the same composition as the binding reaction but substituting the DNA aptamer with nuclease free water.
3. Incubate for 30 minutes in the dark at room temperature

##### Step 5: Running a native PAGE

1. After incubation, add 15µl Laemmli buffer to each sample.
2. Into a well at one end of the edge, add 4µl of Promega’s Broad Range Protein Molecular Weight Markers.
3. Load 20µl of each sample into a separate well.
4. Connect the Electrophoresis Cell to the Bio-Rad PowerPac system and apply a potential difference of 150V across the gel.
5. Let the electrophoresis run for 60 minutes or until the dye front reaches the end of the gel.

##### Step 6: Blot transfer

1. Remove the polyacrylamide gel from the gel cassette.
2. Blot transfer the proteins in the gel onto the PVDF membrane at 10V overnight.
3. Stain the membrane with Ponceau stain to check for the transfer of protein from gel to membrane.
4. Wash off Ponceau stain by multiple water washes.

##### Step 7: Blocking for prevention of non-specific binding of antibodies

1. Block various potential binding sites on the membrane by incubating it with 1% skimmed milk for 1 hour.
2. Wash the membrane with TBS twice for 5 minutes each.

##### Step 8: Primary antibody incubation

1. Incubate the membrane with 1:2000 dilution anti-231pTau antibody for 6 hours at 4°C.
2. Wash the membrane with TBS thrice for 5 minutes each.

##### Step 9: Secondary antibody incubation

1. Incubate the membrane with 1:2000 dilution anti-Rabbit HRP-conjugated antibody overnight at 4°C.
2. Wash the membrane with TBS twice for 5 minutes each.
3. Give the membrane a final wash with TBS for 2 hours.

##### Step 10: Visualisation of the blot

1. Pour ECL substrate onto the membrane and absorb any excess.
2. Visualise in a dark room.
3. Record the localisation of chemiluminescence by allowing for a short exposure.
4. Develop the X-ray plate for final visualisation.

# Lab Results

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# Lab Results

### Further Plans

Once our aptamers our generated, along with our work with Targeted Protein Delivery, we wish to parallely check out some ideas.

Diagnosis: By conjugating our aptamer with a light sensitive molecule, we hope to achieve for a tool which can help in diagnosis. Since reports have shown that p231 Tau can play a very important role in preclinical diagnosis of Alzheimer's, we would hope to develop this into an assay that can streamline the development of further diagnosis tools on CSF/plasma samples. This will help in a better understanding of the disease in a person in conjunction with other tests.

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