iGEM 2022 — Team linkoping-sweden

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block image %} ![](https://static.igem.wiki/teams/4433/wiki/notebook.jpg) {% 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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# Spring

## Week 10-13

First group meeting was held and brainstorming of project ideas was started. The group was divided into focus groups.

## Week 14-16

The brainstorming was summarized into three different categories: Mosquito-borne disease, Cancer cells and Neurodegenerative diseases. The project was decided to be a “Tau detection kit” for neurodegenerative diseases. Research was made to find options for our detection method.

## Week 17-19

It was decided that the detection kit should aim to detect mTBI (mild Traumatic Brain Injury). Further investigations were made to get the outline of the project. A lot of contact with the PI's to plan the project together.

## Week 20-22

First meeting with an expert - Juewen Liu. Work on the protocols for the lab work was started and a list for inventories was started.

# June

## Week 23

Worked on the overall protocols for the lab work. It was decided that the modeling group will model the 3D-structure of the aptamers and its binding to tau-protein.

## Week 24

First IDT order was placed. It was determined that the modeling group should model the aptamers with and without linker. Finalized the overall protocols for the lab work.

## Week 25

Made preparations for the upcoming lab work. Stock solutions were made, E.coli was transformed and tau was expressed. The plasmids were also prepped and the purity of them was checked with an agarose gel.

## Week 26

E.coli culture was made and tau protein was purified. The synthetic tears were prepared with the necessary enzymes and substances. We also made an SDS-PAGE to check on the tau protein elutions and found some impurities. Finally the concentration was measured with spectrophotometry, which indicated a low protein concentration.

# July

## Week 27

A new overnight culture was made and incubated for a longer time than last week. After sonication, IMAC was performed and the concencentration was finally measured with spectrophotometry. This resulted in a higher yield of tau protein. A first try of the LAMP-setup was tested with the recognition-sequence, which resulted in a successful color change.

## Week 28

Protein dialysis was performed. Synthetic tear solutions with different tau concentrations were prepared. The LAMP-reaction was made with and without PCR cleanup using the recognition-sequence. A native gel was made as a control.

## Week 29

The tau-protein was firstly bound to non-magnetic Ni-beads, however no tau protein-band was observed on the SDS-PAGE. The protein was then bound to magnetic Ni-beads and aptamer 1 and 2 without linker were added in separate runs. The LAMP-reaction was then performed for the different supernatants which only indicated a small color change. Modeling concluded that aptamers with linker should bind the best.

## Week 30

A fluorescence scan was made to control the elutions and washing steps from the binding to the magnetic Ni-beads. A negative control was made for the LAMP-reaction and the LAMP-reaction was performed with aptamer 1 and 2, both with linker this time. Aptamer 1+linker showed a complete color change and aptamer 2+linker a minor color change. An agarose gel was made to confirm the results - this confirmed that aptamer 1+linker seems to bind the best.

# August

## Week 31

MST was run in three rounds. First to check if dye bound to tau, the second and third, to check the binding between tau and aptamer 1+linker at two different concentrations. The first run showed a high affinity but no results of binding were obtained in the second and third run. An ELISA assay was run with wells with monoclonal antibodies and tau bound directly to the plate. LAMP-reaction was performed in the wells which was successful, but also the blanks had a color change. The group hosted NiC (Nordic iGEM Conference).

## Week 32

Started a new ELISA assay run. The same results were obtained and it was tried out again with a harsher wash. Unfortunately the blanks still changed color. The aptamer was dissolved in BSA instead of water, unfortunately that did not improve the results either. A lot of trouble shooting was made with the PIs.

## Week 33

Lamp was tested on synthetic tears. Unfortunately even the synthetic tears which were supposed to be without tau protein turned color. Troubleshooting was made and it was decided to try with less aptamer concentration. The project promotion video was finalized.

## Week 34

Troubleshooting of the LAMP reaction was made. Including and excluding crucial parts from the reaction to see what could be the cause for the false positives. It was concluded that the primers seem to be the faulty component and a theory was made that the concentration might be too high in the reaction.

# Autumn

## Week 35-37

The group's focus was on finalizing texts for the wiki page from all focus groups.

## Week 38-40

LAMP-reaction was performed with lower primer concentrations. Unfortunately false positives were still an issue. The team continued on working on the wiki page and the final project presentation

GO TO TOP

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{% extends "layout.html" %}
{% block title %}Engineering success{% endblock %}
{% block image %} ![](https://static.igem.wiki/teams/4433/wiki/engineering.jpg) {% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

* [**Design**](#Design)
* [**Build**](#Build)
* [**Test**](#Test)
* [**Learn**](#Learn)

## How we followed the engineering cycle

## Design

We started with the idea of making a kit for detecting tau in tears to diagnose mild traumatic brain injury (mTBI). This led to us doing further research on mTBI and its biomarker. Noticeably, the tau protein was one of the biomarkers used to diagnose mTBI, and led to us thinking of a way to capture tau proteins in tears. However, before we proceeded to design our project, we conducted various interviews to a wide range of people to see if our kit would be an innovative enough idea, and how impactful our kit would become. Based on the interviews, we have received various inputs. For example, a health care worker implied that our kit would be cost efficient and time efficient (not only for the patient but for the healthcare worker as well), and it would be a kit that would provide a great impact on the public, and ice-hockey players. In another one of our interviews, a researcher suggested that we use aptamers in our project. This concept was new to most of the members, as aptamers was a relatively new concept. Thus, we conducted extensive research on aptamers as well as a new method called RCA-LAMP: a newly used method for amplification of nucleotide sequences.
Aptamers are chosen as the novelty to this project, as it is a single strand of oligonucleotides that can be modified to the specific binding of our desired target. This gave us the idea to modify the aptamer to bind to the tau protein so that we could be able to detect possible minor traumatic brain injuries. In order to do so, we used the method RCA-LAMP. This method uses a padlock probe, p53 sequence (which serves as a recognition site for the padlock probe); and in our kit, a linker. The linker is used to connect the aptamer to the p53 sequence in the padlock probe, and create some distance between them. However, this raised some concerns, ‘would the linker possibly interfere with the binding between aptamer and tau protein?’.
In order to verify whether an interference would occur if the linker is present and would disrupt the binding between the aptamer and tau protein, we modeled the structure of the aptamer and ran multiple simulations and programs to obtain the aptamer structure, and information regarding the binding between the aptamer and tau, along with the binding between the aptamer, tau, and linker. Based on the results, it is confirmed that the linker does not interfere with the binding between tau and aptamer.
After this confirmation, we continued to design protocols that we would follow during the wet lab, as well as risk assessments of possible chemicals that would be used. First, we planned a protocol for tau protein transfection, agarose gel, SDS-PAGE, IMAC, and LAMP.

## Build

We added a plasmid containing the coding sequences for antibiotic resistance against kanamycin, hexa-histidine tag in fusion with the tau protein, and GFP, into the E.coli colonies. This means that we can use antibiotics to find the transfected cells, and due to the presence of GFP, the fusion protein would have a visible green color. As the fusion protein would contain a hexa-histidine tag, we used the IMAC method for protein purification. This would allow the binding between the magnetic bead and hexa-histidine tag.
We then grew the bacteria in agar laden petri dishes containing the antibiotic Kanamycin to only get the transfected cells. The controlled variables are the petri dishes containing only the agar. After culturing the bacteria in wet culture, we lyzed them to acquire the proteins from the bacteria. The plasmid was sent off to a sequencing facility in Germany to verify our DNA sequence. We were later informed that the transfection occurred as we had hoped.
The proteins were run on an SDS-PAGE apparatus to verify the size. We used nickel beads that bound to the his tag to clean out them via IMAC. We also used a nanodrop to get the concentration of the protein. Additionally, synthetic tears were made to mimic human tears in order to obtain more reliable results that are applied to the real world. Synthetic tears were used to run with LAMP.

## Test

The aptamer-LAMP setup was tested with tau and tears. To look at the aptamer binding, we combined the tau+aptamer complex and ran it on an agarose gel, and compared it to just pure aptamer, as the bound complex would have a higher mass and migrate shorter if bound to the verified tau, compared to the tau alone. However, we noticed that in our results, we could not detect the aptamer. Thus, LAMP was run again but this time to see if this would occur again. After the repetition, it was noticed that the aptamer did not show within the SDS-PAGE. Thus, we reviewed our protocol again to see if the design of the protocol would require trouble-shooting. This was done every week to ensure that the results we obtained would be as accurate and precise as possible.

![](https://static.igem.wiki/teams/4433/wiki/engineering-cycle.png)

## Learn

Throughout the whole process, it has taught us to develop and hone our innovative skills, especially in the lab, as modification of the protocol would occur and we were required to figure out which part needs changing, and ‘why?’. So, every week the lab would discuss results that have been obtained from the lab. For example, the first week of lab, we had to culture the bacterias overnight. There was an issue that the tau protein was not present in the bacterias. Therefore, we analyzed the protocol to identify possible errors, and noticed that the incubation period could be a possible source of error, as it should have been left for longer. Thus, we cultured the bacteria again and incubated longer to see if there would be any effects. The new culture contained the tau protein that we expected.
Furthermore, during the last few weeks of the lab, we noticed that our kit started amplification in the negative control. Therefore, all components used in RCA-LAMP were dissected to pinpoint a possible problem. We theorized that our primer dimerization could be the culprit for there to be no binding between the tau protein and aptamer, and could be the reasons for the false positive results. Thus, we conducted the experiment with a series of primer concentrations to see if the results would change and if it would become negative. But to no avail, the results still remained positive. Hence, we continued to see if there were other components in our kit that were possibly ‘incorrect’ or the reason for these false positives. However, further experiments would be required but we are positive that this issue could be resolved.

GO TO TOP

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block image %}
![](https://static.igem.wiki/teams/4433/wiki/dsc-7908.jpg)
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{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## Lab protocols

Below, the protocols for all wet lab steps can be found.

LAMP p53

# LAMP p53

The recognition sequence is a p53 sequence taken from Marciniak et al. 2008 (see: [parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27Parts%27%29%20%7D%7D)), that we have added to our aptamer construct via a linker, and that is recognized by the padlock probe. We ran RCA-LAMP on the recognition sequence alone first to make sure that LAMP worked. RCA-LAMP consists of three main parts: phosphorylation of padlock probe, preparation of ligation mix, preparation of amplification mix.

#### Preparations

* Sterile water: Pour 250 mL deionized water into a 500 mL flask and autoclave it.
* 24 uM FIB primer: 10 uL of 100 uM FIB (stock solution) with 32 uL of water.
* 24 uM BIP primer: 10 uL of 100 uM BIP (stock solution) with 32 uL of water.
* 2.5 uM DNA sequence (aptamer/recognition sequence): 2 uL of 100 uM of DNA with 78 uL of water.

#### Protocol

1. Phosphorylation of padlock probe:
   1. Add in an eppendorf tube 3 uL of padlock probe.
   2. Add 5 uL of 10x T4 DNA ligase buffer.
   3. Add 1 uL of T4 polynucleotide kinase.
   4. Add 41 uL of water to make a 50 uL solution.
   5. Incubate for 30 min at 37°C, then for 20 min at 65°C.
2. Ligation mix:
   1. Add in an eppendorf tube 1 uL of 10x T4 DNA ligase buffer.
   2. Add 1 uL of diluted DNA sequence.
   3. Add 1.7 uL of phosphorylated linear padlock probe.
   4. Add 1 uL of T4 DNA ligase.
   5. Add 5.3 uL of water to make 10 uL mix.
   6. Incubate for 1 hour at room temperature.
   7. PCR DNA cleanup from New England Biolabs to clean the DNA in ligation mix from any impurities.
      1. Add 70 uL of binding cleanup buffer to the eppendorf tube.
      2. Transfer the 80 uL solution into a column and insert the column in a collection tube.
      3. Centrifuge the tube for 1 min. Discard flow-through and reinsert column into collection tube.
      4. Add 200 uL of wash buffer in column and centrifuge for 1 min. Discard is optional.
      5. Repeat the previous step. Discard flow-through and collection tube.
      6. Insert column into an eppendorf tube and add 6 uL of elution buffer. Incubate for 1 min before centrifuging. Discard column.
3. Amplification mix:
   1. Add into a PCR tube 20 uL of 2x WarmStart colorimetric LAMP master mix.
   2. Add 2 uL of diluted FIB and 2 uL of diluted BIP.
   3. Add 6 uL of water.
   4. Mix the 6 uL of purified ligation mix with amplification mix. Incubate for 1 h at 65°C.

LAMP - Magnetic Beads

# LAMP - Magnetic Beads

After monitoring that RCA-LAMP was successful, the next step was to see whether the aptamer would detect tau protein. To capture tau, we used magnetic nickel beads. Tau binds to the beads thanks to the fusion of a his-tag to the tau protein. Aptamers were added to tau in beads and then LAMP was performed.

#### Preparations

* Equilibration buffer: Make 1 L of equilibration buffer by adding 6,03 g of 50mM Tris, 1,36 g of 20 mM Imidazole and 17,53 g of 300 mM NaCl and then filling up the flask until the 1 L mark is reached. The elution buffer should have a pH of 8. Adjust pH if necessary.
* Elution buffer: Make 0,5 L of elution buffer by adding 3,02 g of 50 mM Tris, 17,02 g of 20 mM Imidazole and 8,77 g of 300 mM NaCl and then filling up the flask until the 500 mL mark is reached. The elution buffer should have a pH of 7. Adjust pH if necessary.
* Sterile water: Pour 250 ml deionized water into a 500 ml flask and autoclave it.
* A PCR DNA cleanup kit is needed.

#### Protocol

1. Tau binding to magnetic beads:
   1. Resuspend magnetic beads by vortexing and immediately pipette 50 uL into an eppendorf tube.
   2. Add 100 uL of dialysed tau and place the tube on an end-over-end shaker for 1 h.
   3. Place the tube on a magnetic rack and wait for 1 min before pipetting out and discarding supernatant.
   4. Wash beads with 500 uL of equilibration buffer (20 mM of imidazole). Add the buffer, place the tube in the magnetic rack for 1 min and discard the buffer.
   5. Add 1 uL of 100 uM aptamer to the beads and dilute with 200 uL of equilibration buffer.
   6. Place the tube on an end-over-end shaker for 1 h, then place it on the magnetic rack for 1 min.
   7. Discard the supernatant and wash with 500 uL equilibration buffer.
   8. Add 50 uL of elution buffer (200 uM imidazole), incubate for 1 min, place on magnetic rack for 1 min and collect eluate.
2. Ligation mix:
   1. Add in an eppendorf tube 1 uL of 10x T4 DNA ligase buffer.
   2. Add 5 uL of eluate.
   3. Add 1.7 uL of phosphorylated linear padlock probe.
   4. Add 1 uL of T4 DNA ligase.
   5. Add 1.3 uL of water to make 10 uL mix.
   6. Incubate for 1 h at room temperature.
   7. PCR DNA cleanup from New England Biolabs to clean the DNA in ligation mix from any impurities.
      1. Add 70 uL of binding cleanup buffer to the eppendorf tube.
      2. Transfer the 80 uL solution into a column and insert the column in a collection tube.
      3. Centrifuge the tube for 1 min. Discard flow-through and reinsert column into collection tube.
      4. Add 200 uL of wash buffer in column and centrifuge for 1 min. Discard is optional.
      5. Repeat the previous step. Discard flow-through and collection tube.
      6. Insert column into an eppendorf tube and add 6 uL of elution buffer. Incubate for 1 min before centrifuging. Discard column.
3. Amplification mix:
   1. Add into a PCR tube 20 uL of 2x WarmStart colorimetric LAMP master mix.
   2. Add 2 uL of diluted FIP and 2 uL of diluted BIP.
   3. Add 6 uL of water.
   4. Mix the 6 uL of purified ligation mix with amplification mix. Incubate for 1 h at 65°C.

LAMP - Antibodies

# LAMP - Antibodies

His-tag antibodies were used to capture tau in tears. High-binding 96-well plates were coated with antibodies, solution containing tau was added, aptamer was added and then LAMP was performed. To get reliable results triplicates of antibody + tau were prepared, as well as triplicates of just tau coating wells. Finally, blanks were also used.

#### Preparations

* PBS: 1 PBS tablet + 1 L deionized water.
* PBS-T: mix 300 mL pBS with 150 uL of Tween.
* 2% BSA: mix 0.024 g of BSA powder in 12 mL of PBS.

#### Protocol

1. Plate coating:
   1. Mix 120 uL of antibody with 2280 uL of buffer.
   2. Added 100 uL of the mix to 6 wells (triplicates + blanks) in a 96-well plate.
   3. Serial dilution of dialysed tau was done to get tau concentration of 1/16 \* 3.2 g/L = 0.2 g/L.
   4. Add 100 uL of diluted tau to 3 wells.
   5. Cover the plate with plastic and incubate overnight at 4°C.
2. Tau addition:
   1. Wash all wells 4 times with 100 uL PBS-T.
   2. Add 100 uL of 2% BSA per well.
   3. Cover with plastic and incubate for 1h at room temp.
   4. Repeat the serial dilution of tau and add 100 uL of 0.2 g/L tau to 3 of the wells coated with antibody.
   5. Cover with plastic and store in the fridge overnight.
3. Aptamer-linker addition:
   1. Wash 4x with 100 uL of PBS-T per well.
   2. Dilute 12.5 uL of 100 uM aptamer in 4987.5 uL of BSA.
   3. Add 100 uL of diluted aptamer to each well.
   4. Cover plate with plastic and incubate for 2h at room temp.
   5. Wash 4x with PBS-T.
4. Ligation mix:
   1. Mix in an eppendorf tube 10 uL of 10x T4 DNA ligase buffer, 17 uL of phosphorylated linear padlock probe, 10 uL of T4 DNA ligase, 13 uL of sterile water.
   2. Add 10 uL of mix to each well.
   3. Incubate for 1 hour at room temperature.
   4. No PCR DNA cleanup is performed.
5. Amplification mix:
   1. Mix in an eppendorf tube 200 uL of 2x WarmStart colorimetric LAMP master mix, 20 uL of diluted FIB, 20 uL of diluted BIP, 60 uL of sterile water.
   2. Add 30 uL of the mix to each well. Incubate for 1 hour at 65°C.

Agarose gel

# Agarose gel

To validate the purity and amplification of the plasmid an agarose gel was used. Agarose gel is used to separate macromolecules, such as DNA, based on size and charge. This was also done to verify the binding of aptamer to protein.

#### Preparations

* Add 50 mL of 10x TAE and 450 mL deionized water into a 500 mL flask.
* Add 1g of agarose powder into 100 mL of 1X TAE to make a 1% agarose gel for plasmid verification. For LAMP validation, 1.5 g agarose powder was added to 100 mL 1 x TAE to make a 1.5% agarose gel. Heat the solution in the microwave for 2 minutes until it is completely transparent.
* Before pouring the agar solution into the gel mould, let it cool down a little. Insert a comb and let the gel set for 20-30 min.

#### Protocol

1. Plasmid analysis
   1. Centrifuged the purified plasmid for a couple of seconds.
   2. Add 11uL of water and 4uL of 6x loading dye into a tube containing 5uL of the plasmid.
   3. Load the samples on the agarose gel and as a reference 5uL of a DNA ladder.
   4. Insert the gel in an electrophoresis cassette and add 1X TAE buffer until it covers the gel. Run the gel at 100 V for one hour.
   5. Stain the gel in ethidium bromide for 5-10 min.
   6. Rinse the gel in deionized water.
   7. Visualise the DNA bands by a UV machine at 302 nm.
   8. Take a picture.
2. LAMP verification
   1. Centrifuged the aptamer for a couple of seconds.
   2. Add 16uL of aptamer solution water and 4uL of loading dye into a tube.
   3. Load 5 uL of ladder and 20 uL of samples into the agarose gel for plasmid validation.
   4. Insert the gel in an electrophoresis cassette and add 1X TAE buffer until it covers the gel. Run the gel at 100 V for one hour.
   5. Stain the gel in ethidium bromide for 5-10 min.
   6. Rinse the gel in (a container of) deionized water.
   7. Visualise the aptamer bands by a UV machine at 302 nm.
   8. Take a picture.

MST

# MST

MST is a method to check the affinity between a fluorescent-labelled protein and a non-fluorescent ligand. Microscale thermophoresis is based on the detection of a temperature-induced change in fluorescence of a target as a function of the concentration of a non-fluorescent ligand. Interactions are detected by changes in molecular diffusion or fluorescence intensity. In this case, the target whose change in fluorescence is observed is the tau protein, which was labelled using Red-tris-NTA dye (interacting with the His-tagged tau protein), and the non-fluorescent ligand is the aptamer with linker.

#### Preparations

* PBS: 1 PBS tablet + 1 L deionized water.
* PBS-T: mix 300 mL PBS with 150 uL of Tween.
* 50 nM Dye: 2 uL of 5 uM Red-Tris NTA mixed with 198 uL of PBS-T.
* 100 nM Dye: 2 uL of 5 uM Red-Tris NTA mixed with 98 uL of PBS-T.
* His-tagged protein: make a 4 uM tau solution in PBS-T, volume 30 uL by mixing 2,6 uL of 45,3 uM tau in 27,4 uL PBS-T.
* His-tagged protein: make a 4 uM tau solution in PBS-T, volume 100 uL by mixing 8,8 uL of tau in 91,2 uL PBS-T.

#### Protocol

1. Affinity between dye and tau:
   1. Prepare 16 PCR tubes. Add 20 uL of 4uM tau solution in tube 1. In tubes 2-16, 10 uL of PBS-T.
   2. Transfer 10 uL of tau solution from tube 1 to tube 2 and mix by pipetting up and down. Continue with the dilution series by repeating the process for all tubes. Discard 10 uL from tube 16 to have the same volume in all.
   3. Add 10 uL of 50 nM dye to all tubes. Incubate for 30 min at room temp.
   4. Load capillaries with samples from each tube. Simply put the capillary in the tube until the solution has occupied its length.
   5. Run MST program and check results.
2. Protein labeling:
   1. Mix 90 uL of 4 uM tau with 90 uL of 100 nM dye in an eppendorf tube.
   2. Incubate for 30 min at room temperature.
   3. Centrifuge at 13400 g for 10 min.
3. Binding assay:
   1. Prepare a 2 uM aptamer solution by mixing 0,5 uL of 100 uM aptamer with 24,5 uL PBS-T.
   2. Prepare 16 PCR tubes. Add 20 uL of aptamer solution in tube 1. In tubes 2-16, 10 uL of PBS-T.
   3. Perform serial dilution, as done in step 1.2.
   4. Add 10 uL of RED-tris-NTA-labeled tau protein to each tube and mix by pipetting up and down.
   5. Load capillaries and run MST program.

SDS-PAGE

# SDS-PAGE

SDS-PAGE was used to verify tau protein expression and purity. The SDS method requires multiple components like gels, buffers and the gel electrophoresis apparatus.

#### Preparations

* SDS buffer: Prepare 1 L of Tris glycine SDS-buffer by diluting 10X stock solution to 1X with deionized water. Final concentrations are as follows: 25 mM Tris, 192 mM glycine and 0.1% SDS. Adjust pH to 8.3.
* Add two gels in the cassette holder. Test for leaks by adding a buffer in between the gels first. Repeat if using more than two. If only one gel is added, one blank needs to be added as well.
* Add the cassette holder into the SDS apparatus, and pour the SDS buffer all the way in between the gels and up to the demarcation line.
* Destaining solution: Prepare 1L of solution by adding 500 mL water, 400 mL methanol and 100 mL acetic acid into a plastic bottle.

#### Protocol

1. Run gel:
   1. Prepare samples in a fume hood by mixing 5uL of 4X SDS-buffer(containing gel-loading dye bromophenol blue) with 1uL of BME and 14 uL of sample solution.
   2. Heat samples on a heating block at 95°C for 5 min.
   3. Load 10 uL of samples and 3uL of ladder into the wells of the gel.
   4. Run the gels at 200 V until migration of the samples into the gel is observed, then increase the voltage to 300 V for 25 min or until the bands reach the bottom of the gel.
2. Staining the gel:
   1. Remove the gel from the holder and place it into a smaller container. Add deionized water carefully to the container. Carefully pour off the deionized water.
   2. Transfer to a fume hood.
   3. Add just enough Coomassie Brilliant Blue R-250 Dye staining solution to cover the gel and put the container on a shaker. Leave the container on the shaker. The shaker should be set to gently shake the samples.
   4. Destain the gel by pouring off the staining solution and by adding destaining liquid.
   5. Add tissue to the side of the container to soak up more stain.
   6. Change both tissue and destaining solution periodically until the gel is transparent and the protein bands show clear contrast to background.

\*Note that we also analyzed our gel using a fluorescence imager with a 480 nm excitation filter. This was possible because we expressed a tau-GFP fusion protein.

Synthetic Tears

# Synthetic tears

As using human tears could be limited, synthetic tears were made that closely resemble human tears. Synthetic tears with and without tau were made. Another advantage is that synthetic tears make it easier to quantify the results and troubleshoot experiments.

#### Preparations

* NaCl stock solution (1:10) 1,25 mol/L.
  + Add 1.825 g NaCl to 25 mL Water.
  + Mix and label the samples.
* CaCl2 stock solution (1:10) 14 mmol/L.
  + Add 0.075g CaCl2 to 50 mL water.
  + Mix and label the sample.
* MgCl2 stock (1:10) 20 mmol/L.
  + Add 0.0935 g MgCl2 to 50 mL water.
  + Mix and label the sample.
* Glucose stock solution (1:100) 20 mmol.
  + Add 0.9g glucose to 25 mL water.
  + Mix and label the sample.
* KCl stock solution (1:10) 0,16 mol/L.
  + Add 0,12g KCl to 50 mL water.
  + Mix and label the sample.
* All stock solutions can be stored in room temperature.

#### Protocol

1. Make the synthetic tears
   1. Add 5 mL NaCl stock solution.
   2. Add 5 mL CaCl2 stock solution.
   3. Add 5 mL MgCl2 stock solution.
   4. Add 5 mL KCl stock solution.
   5. Add 500 uL glucose stock solution.
   6. Add water up to 50 mL.
   7. Divide into 2 vials.
   8. Add 0.25 g of luophilized bovine serum albumin and 0.075 g of lyophilized hen egg white lysozyme to solution.
   9. Store unused stock solution at 4°C.
2. Add tau to the tears.
   1. Take 5 mL of finished tears to test tubes.
   2. Add tau from the stock solution to concentrations of 10 ng/mL, 5 ng/mL, 1 ng/mL, and leave one as is, and add deionized water.
   3. Store the solutions at 4°C.

Tau Production

# Tau production

Before we can use our tau protein it needs to be synthesised and purified first. The production of tau is divided into three stages namely plasmid transformation, tau expression and lastly purification. At some of the intermediate steps, samples were taken and frozen for troubleshooting if something went wrong in later steps. Two strains of E.coli competent cells were used. One for the the purpose of the production of tau (NEB BL21/DE3) and another strain that will later be used for plasmid preparation (NEB DH5-α).

#### Preparations

* LB medium: Make 2 batches of 1L LB medium by adding 20 g of LB-mix into a 1 L flask and top it up with deionized water until the 1 L mark is reached.
* LB agar: Make 1L of LB-agar by adding 15 g of agar powder to a 1 L flask and top it up with LB medium until the 1 L mark is reached.
* Kanamycin stock solution: Add 0,3 g of Kanamycin into a 10 mL volumetric flask and fill up to the 10 mL mark so that the final concentration is 30 mg/mL. The 10mL stock solution was aliquoted into 1,5 mL eppendorf tubes.
* Sterile Water: Pour 250mL deionized water into a 500 mL flask and autoclave it.
* 1 mM of IPTG.
* Elution buffer: Make 0,5 L of elution buffer by adding 3,02 g of 50 mM Tris, 17,02 g of 20 mM Imidazole and 8,77 g of 300 mM NaCl and then filling up the flask until the 500 mL mark is reached. The elution buffer should have a pH of 7. If the solution doesn’t have this specific pH it needs to be adjusted.
* Equilibration buffer: Make 1 L of equilibration buffer by adding 6,03 g of 50 mM Tris, 1,36 g of 20 mM Imidazole and 17,53 g of 300 mM NaCl and then filling up the flask until the 1 L mark is reached. The elution buffer should have a pH of 8. If the solution doesn’t have this specific pH it needs to be adjusted.

#### Protocol

1. Agar plates:
   1. Melt LB agar in the microwave until it is completely liquid.
   2. Pour LB agar into sterile petri dishes until the bottom is covered.
   3. Put the lids on halfway so that half of the petri dish is open, to allow it to set.
2. Plasmid transformation:
   1. Check the concentration of the tau plasmid by using a Nanodrop. Dilute the plasmid using sterile water to the wanted concentration if the plasmid is not already in the wanted concentration.
   2. Thaw two tubes of NEB DH5-ɑ competent E.coli cells and NEB BL21/DE3 competent E.coli cells on ice for 10 min. Pipette 1 uL of the plasmid mixture into the two tubes.
   3. Leave the tubes on ice for 30 min and then heat shock at 42°C for 10 seconds.
   4. Leave the tubes on ice for 5 min.
   5. Pipette 250 uL of room temperature SOC into each of the tubes.
   6. Incubate the tubes at 37°C for 1 hour with vigorous shaking at 250 rpm.
   7. Spread both of the strains on 6 agar plates each and put in 37°C incubator overnight.
3. Plasmid culturing and Tau expression:
   1. Prepare a preculture: add 20 mL of LB medium, 20 uL of kanamycin and 1 colony of BL21/DE3 into a 50 mL Falcon tube. Repeat this step for the DH5-ɑ strain. The remaining colonies on the agar plate can be sealed with Parafilm and stored at 4°C until further use.
   2. Incubate the tubes at 37°C overnight with vigorous shaking at 240 rpm. In the meantime, prepare and autoclave 1 L of LB-medium.
   3. Add 5 mL of glycerol into the falcon tube containing the DH5-ɑ cells and flash freeze it in liquid nitrogen for 2-4 seconds. Store the sample in -80°C.
   4. Put 1 mL kanamycin and 10 mL of preculture in the autoclaved LB medium.
   5. Take 1 mL of the culture and measure O.D. in O.D.-meter. This sample serves as blank.
   6. Divide the BL21/DE3 culture equally into two falcon tubes.
   7. Incubate for 90 min at 37°C at 90 rpm. Measure O.D. of 1 mL from each tube and compare with blank.
   8. Add 0.5 mL of 1 mM IPTG into each falcon tube.
   9. Incubate the tubes at 37°C for 4h or 16°C overnight at 90 rpm.
4. Tau Purification:
   1. Add into a PCR tube 20 uL of 2x WarmStart colorimetric LAMP master mix.
   2. Add 2 uL of diluted FIB and 2 uL of diluted BIP.
   3. Add 6 uL of water.
   4. Mix the 6 uL of purified ligation mix with amplification mix. Incubate for 1 hour at 65°C.
5. Protein dialysis:
   1. Prepare a 14 kDA membrane by cutting approximately 10 cm of the membrane and place it in a beaker filled with deionized water.
   2. Transfer the membrane into a plastic container and add two clamps on one side of the membrane.
   3. Open the membrane on the open side carefully and pippet the tau elution into the membrane.
   4. Add 2 clips to the open end of the membrane.
   5. Add the now filled membrane into a beaker containing dialysis buffer.
   6. Place the beaker at 4°C on a magnetic stirrer for 1 hour.
   7. Remove the dialysis buffer and replace it with TAE dialysis buffer containing Sodium Azide.
   8. Keep the beaker on a magnetic stirrer overnight.
   9. Remove the clips on one side of the membrane and pipette out the contents into a 15 mL Falcon tube.

Plasmid preparation

# Plasmid preparation

In order to check the purity and if the plasmid that is being used has the wanted construct plasmid preparation was done. The NEB DH5-α strain of E.coli competent cells were used for plasmid preparation.

#### Preparations

* Plasmid preparations should already have been done for the NEB DH5-α strain.
* A plasmid cleanup kit such as the Monarch® Plasmid Miniprep Kit from New England BioLabs is needed.
* LB medium: Make 1 L LB medium by adding 20 g of LB-mix into a 1L flask and top it up with deionized water until the 1 L mark is reached.
* Kanamycin: Thaw an already made aliquot of 30 mg/mL kanamycin until it is completely liquid.

#### Protocol

1. Bacterial growth
   1. Add 10 mL of LB-medium and 10 uL of Kanamycin as well as 2-3 colonies of DH5-α into a 50 mL Falcon Tube.
   2. Incubate the tube at 240 rpm at 37°C for 4-5 hours.
2. Plasmid preparation
   1. Pour 1 mL of the culture into a 1,5 mL eppendorf tube.
   2. Centrifuge the tube for 1 min and discard the supernatant.
   3. Use a plasmid cleanup kit such as the Monarch® Plasmid Miniprep Kit to isolate the plasmid.
   4. The plasmid can now be sent for sequencing.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block image %} ![](https://static.igem.wiki/teams/4433/wiki/bottles-below-min.jpg)
{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

* [**Tau expression**](#tauexpress)
* [**Recog. site - LAMP**](#p53)
* [**Magnetic beads**](#LAMP1)
* [**Synt. tears LAMP**](#tears-lamp)
* [**Synt. tears SDS**](#tears-sds)
* [**ELISA sandwich**](#ELISA)
* [**Troubleshooting**](#troubleshoot)
* [**MST**](#MST)

## Results

## Tau expression and purification

Tau protein is the central part of our project and, therefore, we first had to produce our wanted protein before we could use it for further experiments. We used a plasmid in which the tau sequence (human tau 0N4R with an N-terminal His-tag) was fused to enhanced green fluorescent protein (eGFP) sequence, resulting in His-tau-eGFP protein. This was helpful as tau could easily be visualised by the naked eye. As can be seen in figure 1, there was a definite green colour to our samples which confirmed the production of tau.

![](https://static.igem.wiki/teams/4433/wiki/figure-1a-results.png)

![](https://static.igem.wiki/teams/4433/wiki/figure-1b-results.png)

***Figure 1:** Cultures with Tau expressed samples (left) and pellets (right).*

After eluting tau from the nickel column through IMAC, we visualised it in an SDS-PAGE gel to check the identity and purity of the protein. We tested a sample of E. coli culture after tau induction but before cell sonication, the flowthrough and the wash from IMAC as controls, side by side with successive elutions of tau. The results can be seen in figure 2.

The wells contained the following:

1. Protein ladder
2. Tau Elution 5
3. Tau Elution 4
4. Tau Elution 3
5. Tau Elution 2
6. Tau Elution 1
7. Wash
8. Flowthrough
9. After induction
10. Protein ladder

![](https://static.igem.wiki/teams/4433/wiki/figure-2-results-fixed.png)

***Figure 2:** SDS- PAGE gel on different tau elutions. Expected molecular weight of the His-tau-eGFP protein (tau) was 70.6 kD.*

The results showed that there was no impurity in elution 4 and 5. However, the tau yield was small. Impurities are seen in elutions 1,2 and 3, while the yield is much bigger. The wash doesn’t seem to have many impurities. This means that the washing step wasn’t able to remove most of the impurities. However, it is also visible that the wash didn’t remove a lot of tau. The flowthrough had a lot of impurities and even had some tau. The sample from after induction shows a thin tau band, even though sonication hadn’t been performed yet at that point.
These results were also supported by a fluorescence scan performed on another unstained SDS-PAGE gel (figure 3).

Since the tau elutions contained impurities, it was decided to dialyse them.

![](https://static.igem.wiki/teams/4433/wiki/figure-3-results.png)

***Figure 3:** Fluorescence scan on SDS-PAGE gel.*

## Recognition sequence- LAMP

Recognition sequence is a p53 sequence taken from Marciniak et al. 2008 (see [Parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)), that we have added to our aptamer construct via a linker, and that is recognized by the padlock probe. We ran RCA-LAMP on the recognition sequence alone first to make sure that LAMP worked. RCA-LAMP consists of three main parts: phosphorylation of padlock probe, preparation of ligation mix, preparation of amplification mix.

![](https://static.igem.wiki/teams/4433/wiki/figure-4-results.png)

***Figure 4:** Positive result from LAMP using the recognition sequence (p53).*

There was one problem that was persisting throughout the samples. If the sample was removed from the PCR machine and amplification occurred and a pH change took place, the sample would turn back to its original pink colour. It was decided to run a PCR cleanup on the samples. After this cleanup step was incorporated the positive samples stayed the yellow colour after it was removed from the heat. In figure 5, it can be seen that a sample is turning back to its original pink colour.

![](https://static.igem.wiki/teams/4433/wiki/figure-5-results.png)

***Figure 5:** Positive yellow sample tuning back to its original pink colour.*

## LAMP detection of tau in magnetic beads

After seeing that RCA-LAMP is successful, the next step was to see whether the aptamer would detect tau. To capture tau, we used magnetic nickel beads (figure 6). Tau binds to the beads thanks to the his-tag. Aptamers were added to tau bound to beads and then LAMP was performed. This procedure was done for both aptamer 1 and 2.

![](https://static.igem.wiki/teams/4433/wiki/figure-6-results.png)

***Figure 6:** Eluate of tau and aptamer*

Both showed colour changes to different degrees, where aptamer 1 showed the most colour change. To verify the results, an agarose gel was run. Figure 7 shows the agarose gel, where well 1 contains aptamer 1, well 2 aptamer 2 and well 4 contains a DNA ladder. The rest of the wells are empty.
From the image it is clear that the colour change was a result of DNA amplification and not simply pH change. Moreover, LAMP performed with aptamer 1 has the biggest amount of amplification. Therefore, it was decided to continue the rest of the experiments with aptamer 1.

![](https://static.igem.wiki/teams/4433/wiki/figure-7-results.png)

***Figure 7:** Agarose gel showing amplification using different aptamers.*

Finally, the procedure was repeated again with a negative control. No tau was added to the magnetic beads and the rest of the steps remained unchanged. The reason for this was to make sure that aptamers didn’t bind to the magnetic beads. The results can be seen below in figure 8.

![](https://static.igem.wiki/teams/4433/wiki/figure-8-results.png)

***Figure 8:** Positive and negative control LAMP.*

## Synthetic tears with and without tau - LAMP

Synthetic tears were prepared to mimic human tears with some of the most basic electrolytes and proteins. Synthetic tears of different tau concentrations were tested on magnetic beads using LAMP.
Unfortunately, the results weren’t satisfactory. All synthetic tears (even the ones without any tau), had a colour change which was due to DNA amplification as confirmed by agarose gel results (figure 9 and 10).

![](https://static.igem.wiki/teams/4433/wiki/figure-9-results.png)

***Figure 9:** Agarose gel of LAMP from synthetic tears of different tau concentrations (from left to right: no tau, 10 ug/mL, 10 ng/mL, 5ng/mL, ladder).*

![](https://static.igem.wiki/teams/4433/wiki/figure-10-results.png)

***Figure 10:** Agarose gel of LAMP from synthetic tears without tau and (From left to right: ladder, synthetic tears with no tau).*

The first suspicion we had at seeing these results was that some contamination of the synthetic tears was possible. However, since the result is the same for the human tears, we also suspected that the aptamer might be too sensitive and that we needed to reduce its concentration.

## Synthetic tears - SDS

To see the difference in composition of human tears versus the synthetic tears with varying concentrations of tau, an SDS-PAGE was run. The samples loaded in the SDS gel wells contained:
Well 1 - ladder
Well 2 - synthetic tears 1 ng/mL tau
Well 3 - synthetic tears 2.5 ng/mL tau
Well 4 - synthetic tears 5 ng/mL tau
Well 5 - synthetic tears 10 ng/mL tau
Well 6 - synthetic tears 10 ug/mL tau
Well 7 - synthetic tears 0 ng/mL tau
Well 8 - synthetic tears 0 ng/mL tau

![](https://static.igem.wiki/teams/4433/wiki/figure-11-results.png)

***Figure 11:** SDS-PAGE of synthetic tears of varying tau concentrations.*

From the image above, the results show that there is definitely some contamination in the synthetic tears as they all appear to be the same even though two of them don’t contain any tau.

## Antibody-aptamer ELISA sandwich

His-tag antibodies were used to capture tau in tears. High-binding 96-well plates were coated with antibodies, solution containing tau was added, aptamer with linker was added and then LAMP was performed. To get reliable results triplicates of antibody + tau were prepared, as well as triplicates of just tau coating wells. Finally, blanks were also used. ELISA was run twice to verify results.
In the first run, LAMP was performed on 3 wells coated with antibody and 3 wells directly coated with tau (as a positive control) in the fourth row, as well as three blanks in the last row containing antibody, PBS buffer and LAMP mixes (figure 12.a). From the results in figure 12.a, it seemed that the aptamer in the blank wells was not completely washed off. Therefore, ELISA was repeated with a few changes: aptamer was diluted in BSA instead of water, washing was done with PBS-T instead of PBS. In the second run, LAMP was tested on the row with three blanks containing buffer and LAMP mix, and the column with two tau-coated wells (figure 12.b).

![](https://static.igem.wiki/teams/4433/wiki/figure-12a-results.png)

![](https://static.igem.wiki/teams/4433/wiki/figure-12b-results.png)

***Figure 12:** First ELISA run (a) and second run (b) with negative results.*

From the results seen above, both runs gave unsatisfactory results. While it’s a good thing that there’s a colour change in the wells containing tau, the blanks, which also changed colour even though they were not supposed to, indicate that the positive results are not reliable. For this reason, an agarose gel was run using the samples from the second run only. As can be seen from figure 13, there has been no amplification, meaning that the colour change was simply a false positive.

![](https://static.igem.wiki/teams/4433/wiki/figure-13-results.png)

***Figure 13:**  Agarose gel results of ELISA (from left to right: ladder, blank, tau-coated).*

## Troubleshoot of LAMP

Since LAMP results from synthetic tears, human tears and ELISA were unsatisfactory, a series of experiments were performed to verify that the detection method was working properly. These experiments consisted of repeating the protocol in “LAMP detection of tau in magnetic beads” while removing a component of the LAMP one at a time to see the effects each had. The results were similar for most of them. The following samples were in the wells left to right.

1. Normal, No Aptamer
2. No Padlock Probe, 0.0001uM Aptamer
3. No Padlock Probe, 0.001uM Aptamer
4. No ligase, 0.0001uM Aptamer
5. No ligase, 0.001uM Aptamer
6. Normal, 0.0001uM Aptamer
7. Normal, 0.001uM Aptamer
8. No FIP and BIP, 0.0001uM Aptamer
9. No FIP and BIP, 0.001uM Aptamer
10. No BIP, 0.0001uM Aptamer
11. No BIP, 0.001uM Aptamer
12. No FIP, 0.0001uM Aptamer
13. No FIP, 0.001uM Aptamer
14. DNA Ladder

![](https://static.igem.wiki/teams/4433/wiki/figure-14-results.png)

***Figure 14:**  Agarose gel results of Troubleshoot LAMP experiments*

The removal of each component resulted in amplification, as seen in figure 14, except for the cases where at least one of the primers was not present in the mix. Consequently, the problem with the detection kit generating false positives resided in the primers being faulty. This might be either a manufacturer’s defect or a problem with storage and handling.

## MST

MST is a method to check the affinity between a fluorescent-labelled protein and a non-fluorescent ligand. Microscale thermophoresis is based on the detection of a temperature-induced change in fluorescence of a target as a function of the concentration of a non-fluorescent ligand. Interactions are detected by changes in molecular diffusion or fluorescence intensity. In this case, the target whose change in fluorescence is observed is the tau protein, which was labelled using Red-tris-NTA dye (interacting with the His-tagged tau protein), and the non-fluorescent ligand is the aptamer.
Firstly, the affinity between tau and the dye was checked. The result was a dissociation constant (Kd) of 240 nM showing that we have successfully detected His-tagged tau protein.
After seeing that tau interacts strongly with the dye, the protocol was followed for the labelling of tau with the dye. Then MST was repeated for the labelled protein and aptamer. Unfortunately, the results were inconclusive and did not confirm any affinity between tau and the aptamer. The reasons may be that the fluorescence change of the RED-tris-NTA-tau protein interacting with the aptamer is not sufficient due to the dynamic properties of the intrinsically disordered tau protein.

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