iGEM 2021 — Team Stockholm

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Stockholm/Notebook

Notebook | iGEM Stockholm

* Team

  [Team](https://2021.igem.org/Team%3AStockholm/Team)[Attributions](https://2021.igem.org/Team%3AStockholm/Attributions)[Collaborations](https://2021.igem.org/Team%3AStockholm/Collaborations)[Partnership](https://2021.igem.org/Team%3AStockholm/Partnership)[Inclusivity](https://2021.igem.org/Team%3AStockholm/Inclusivity)[Sponsors](https://2021.igem.org/Team%3AStockholm/Sponsors)
* Project

  [Description](https://2021.igem.org/Team%3AStockholm/Description)[Contribution](https://2021.igem.org/Team%3AStockholm/Contribution)[Engineering](https://2021.igem.org/Team%3AStockholm/Engineering)[Experiments](https://2021.igem.org/Team%3AStockholm/Experiments)[Notebook](https://2021.igem.org/Team%3AStockholm/Notebook)[Proof Of Concept](https://2021.igem.org/Team%3AStockholm/Proof_Of_Concept)[Results](https://2021.igem.org/Team%3AStockholm/Results)
* [Model](https://2021.igem.org/Team%3AStockholm/Model)
* Human Practices

  [Human Practices](https://2021.igem.org/Team%3AStockholm/Human_Practices)[Implementation](https://2021.igem.org/Team%3AStockholm/Implementation)[Entrepreneurship](https://2021.igem.org/Team%3AStockholm/Entrepreneurship)[Communication](https://2021.igem.org/Team%3AStockholm/Communication)[Education](https://2021.igem.org/Team%3AStockholm/Education)
* [Safety](https://2021.igem.org/Team%3AStockholm/Safety)

[ ]

# Notebook

What did we do?

---

##### Contents

# Workflow

## June

* First PCDA synthesis using different concentrations for proof of concept. We tried to make a stock solution which did not work due to low solubility.
* Preparation of Brain Heart, Infusion, buffers, and solutions.

## July

* Inoculation of *C. acnes* and preparation of glycerol stocks.
* Buffer preparations.
* LTA-bead conjugation.
* Library preparation (resuspension).
* 1st round of SELEX.
  + No results, extensive troubleshooting and reruns.
  + Increased amount of PCR cycles, different template concentrations.
  + Noticed we had bought the wrong reverse primer, it had biotin on the wrong terminal.
* Another mistake that we noticed was that our protein A was stored at RT instead of the - 35 to - 25 degrees Celcius that is recommended on the package. According to instructors, this was nothing to be concerned about.
* A new PCDA solution was created with a concentration of 1 mM - the same concentration as is required for coupling. This solution became deep blue after polymerisation. This was subsequently used for all conjugation attempts.
* Aptamer coupling with PCDA was tested with different concentrations of PCDA in the coupling process, from a volume of 50 uL PCDA as suggested in the protocol to a maximum volume of 237.5 uL. A higher PCDA concentration seemed to provide a higher yield after dialysis, meaning that more PCDA vesicles were conjugated to the same amount of aptamers.
* Protein A coating of plates for protein A aptamer testing. The aptamer coupled to PCDA was tested in the protA wells for binding and colour change, which did not yield any colour change. Further investigation was needed.
* PCDA testing with heat. Heat was used to test if a colour change is possible: the PCDA solution did indeed turn to a bright orange when subjected to a burner. The PCDA was also tested with UV light, which did induce a colour change, although very slowly. The solution turned to a deep purple when left under UV light overnight.

## August

* Start of cell SELEX using *C. acnes* as targets.
* Protein A aptamer testing using affinity chromatography. A Mabselect column used for IgG purification was used to test the binding of our protein A aptamer. The aptamer solution ran through the column when the sample was applied, meaning that no binding took place. An attempt was made using magnetic protein A beads, with the same results. It is possible that the aptamer binds to a different part of protein A than is accessible in the column and on the beads.
* Finally, since none of the SELEX runs worked, and after extensive troubleshooting using different annealing temperatures, working concentrations and cycle numbers, we had a meeting with our advisor Dimitri. We concluded that the libraries required HPLC purification which we did not choose when ordering. Thus, a new library was ordered with HPLC purification.
* After consulting our supervisors about the PCR protocol, it was pointed out that our template concentration was excessively high in the PCR reaction. The consequence of this is that the DNA strands don't have the time and right conditions to denature for the PCR to work - they are too dense (3 000 ng vs. < 500 ng). Therefore, a PCR was done with "normal" concentrations, obtained from the polymerase's documentation. When reading the documentation, the dNTP concentration was too low (1 uM vs. 200 uM) and the primer concentration was too high (2.5 uM vs. 1 uM).
* Once the PCR was working and amplification bands were visible, we discovered that we had contamination since the NTC lane also displayed a band. Thus, extensive troubleshooting was done to figure the source of the contamination out. Tubes were prepared with one component switched for a new one in each. The gel showed that the dNTP tube was the source of the contamination.

## September

* A new SELEX amplification was performed, yielding no bands. Again, a new troubleshooting step had to be started, testing two different polymerases, two dNTP concentrations, as well as two annealing temperatures. Contamination was again present in the NTCs, so we used new polymerase and buffer, which worked.
* We performed our first working PCR amplification of our SELEX round 1 library, which yielded distinct bands. The next step was to separate the sense and antisense DNA strands, followed by desalting. This step was thought to be not working, but it turned out that the final concentration of aptamers was just very low. A final PCR was therefore done to check if aptamers were present in the end. Again, some troubleshooting was required since we had contamination, this time stemming from the primers.
* The protein A aptamer binding assay was designed for the fluorescein-tagged aptamer and using a plate reader. The first step was to make a calibration curve to see which aptamer concentrations are suitable for detection. After that, the binding assay was performed, showing that the aptamer binds to protein A.

## October

* A new SELEX round was started in order to test our troubleshooting guide and gained experience. This did not work since we got contamination in our PCRs, even after trying again two times with new aliquots of PCR reagents. Due to time constraints, no further attempts were performed and our focus moved on to testing the protein A aptamer and sending transformation samples for sequencing.
* A final protein A aptamer binding affinity test was run with the same method as before, showing no binding to protein A.

# BioBrick Engineering Workflow

## June

* Preparation of LB, LB-agar plates and stock solutions.
* The team learnt basic techniques: culturing bacteria in liquid LB and plating in LB-agar, E. coli transformation.

## July

* Cloning was started with the aim of practicing and creating a biobrick with protein A with a GFP attached. A plasmid from the 2019 distribution kit was used but it did not work, although the same plasmid from the 2021 distribution kit worked.
* The cloning strategy was designed: we decided to clone protein A and a reporter protein together in the same vector. AmilGFP (yellow chromoprotein) and GFP (green fluorescent protein) were chosen as potential reporter genes. Protein A, amilGFP and GFP BioBricks were obtained from the 2021 DNA Distribution Kit Plates. We chose several GFP BioBricks in case the transformation efficiency was not high in all of them.
  + Protein A: BBa\_K103003
  + GFP: BBa\_K608008, BBa\_K608010, BBa\_K608011
  + AmilGFP: BBa\_K1073024

The initial strategy was to digest both BioBricks (protein A and reporter protein) and ligate them together in a new open vector with Ampicillin resistance (PSB1A3).

* Several rounds of transformation, O/N culture, glycerol stock + miniprep were performed.

## August

* After several attempts we got a good plasmid prep and glycerol stocks for each plasmid.
* Digestion with the corresponding restriction enzymes was performed. Ligation mixtures containing of PSB1A3 + Protein A BB + reporter gene GFP(BBa\_K608011) or amilGFP (BBa\_K1073024) were set. We realised that we did not use Dpnl to degrade the RFP present in vector plasmid PSB1A3.
* Decided to simplify the cloning strategy: It is better if we deliver the construct in PSB1C3 rather than PSB1A3. The new strategy consists in digesting the Protein A BioBrick and then, ligate it in a linearized plasmid containing the reporter gene (GFP or amilGFP).
* Plasmids containing the reporter gene GFP(BBa\_K608011) or amilGFP (BBa\_K1073024) were digested with SpeI and PstI. Plasmids containing the Protein A BioBrick were digested with XbaI and PstI. Ligation reaction was set afterward.
* In order to be able to transform E. coli with the ligated construct we need to purify it from the gel. The ligation mixtures are not enough to do gel extraction.
* We decided to keep the cloning strategy but made slight changes: we decided to amplify Protein A BioBrick by PCR, using VF2 and VR standard primers. Then, digest the PCR product with EcoRI and XbaI. This digested product will be ligated with a higher amount of linearised plasmids containing the reporter gene GFP(BBa\_K608011) or amilGFP (BBa\_K1073024). This way we can perform gel extraction after ligation.
* PCR on Protein A BioBrick followed by a gel extraction (PCR clean-up was not an option, very small fragments) were performed.
* Digestion followed by gel extraction were performed in plasmids containing the reporter gene GFP(BBa\_K608011) or amilGFP (BBa\_K1073024). Optimisation of this step was needed.
* Ligation of Protein A + GFP and Protein A + amilGFP.
* TOP10 E. coli competent cells were transformed with the ligation mixtures. Glycerol stocks and minipreps from 3 colonies of each combination of Protein A + reporter gene were set.

## September

* The plasmid preps were sent for sequencing using VR and VF2 primers. The BioBricks sequences were correct but we realised we need to remove the STOP codons in between protein A CDS and reporter gene CDS.
* Quikchange Site-directed mutagenesis strategy was designed to change the STOP codons (TAA) for Ser (TCA). Protein A + Amil-GFP construct has 2 STOP codons in-between CDSs, but Protein A + GFP has 4 STOP codons and the CDS are not in-frame. Due to the time restriction, we decided to move on only with the Protein A + amilGFP construct. Primers for QuikChange mutagenesis were designed using Kozane.app.
* Quikchange protocol was designed taking into account the Aligent protocol and Phusion polymerase specificities. Since the annealing temperature is tricky to calculate (primers don't bind completely to the target sequence) we performed a gradient PCR using 3 different annealing temperatures: 55, 58 and 60 degrees Celsius.
* TOP10 E. coli competent cells were transformed using different volumes of Quickchange DNA mix: 5, 10 and 15uL.

## October

* Yellow colonies were only in 55 and 58 degrees Celsius plates. 8 colonies from the 55 degrees Celsius plate and 20 from the 58 degrees Celsius plate were used to set O/N cultures and glycerol stocks. Minipreps of 8 O/N cultures from 55 degrees Celsius and 8 O/N cultures from 58 degrees Celsius and the 16 samples were sent for sequencing.

# Lab Books

Feel free to read further into what we did in the lab in our lab books

[Stockholm Lab Book](https://static.igem.org/mediawiki/2021/f/fc/T--Stockholm--Lab-Book-Stockholm.docx)

[Cloning Lab Book](https://static.igem.org/mediawiki/2021/c/c9/T--Stockholm--Lab-Book-Cloning.docx)

# Protocols

## Synthetic Biology Lab Manual

[Lab Manual](https://static.igem.org/mediawiki/2021/0/02/T--Stockholm--Lab--Manual.pdf)

*Courtesy of Josefine Liljeruhm, Erik Gullberg and Anthony C. Forster, Uppsala University.*

## Selex

[DVS Aptamer Selection Protocol](https://static.igem.org/mediawiki/2021/6/6a/T--Stockholm--dvsapatmer.pdf)

[LTA Aptamer Selection Protocol](https://static.igem.org/mediawiki/2021/7/75/T--Stockholm--ltaapatmer.pdf)

## PCDA

[PCDA Synthesis](https://static.igem.org/mediawiki/2021/d/d5/T--Stockholm--PCDA-synthesis.pdf)

[PCDA Aptamer Conjugation](https://static.igem.org/mediawiki/2021/1/17/T--Stockholm--PCDA-aptamer.pdf)

[PCDA Concentration](https://static.igem.org/mediawiki/2021/c/c4/T--Stockholm--PCDA-concentration.pdf)

## Culture

[Ampicillin Solutions](https://static.igem.org/mediawiki/2021/f/f7/T--Stockholm--Ampicillin.pdf)

[CFU Determination](https://static.igem.org/mediawiki/2021/f/fc/T--Stockholm--cfu-protocol.pdf)

[Calibration Protocol](https://static.igem.org/mediawiki/2021/3/33/T--Stockholm--calibration-cloning.pdf)

The above protocol is an open access iGEM protocol by Paul Rutten from The University of Oxford et al.

## Cloning

[Monarch DNA Gel Extraction Kit Protocol](https://static.igem.org/mediawiki/2021/5/5e/T--Stockholm--monarch-DNA.pdf)

[Cloning and Transforming](https://static.igem.org/mediawiki/2021/5/55/T--Stockholm--cloningandtransforming.pdf)

[Gel Extraction](https://static.igem.org/mediawiki/2021/1/1b/T--Stockholm--Gel-extraction.pdf)

[QIAquick PCR Purification Kit](https://static.igem.org/mediawiki/2021/2/2d/T--Stockholm--QIA--PCR.pdf)

[QIAquick Gel Extraction Kit](https://static.igem.org/mediawiki/2021/b/b9/T--Stockholm--QIA--gel-extraction.pdf)

The above protocol is repeated in the Gel Extraction protocol, this is simply the official company card.

[![](https://static.igem.org/mediawiki/2021/b/bb/T--Stockholm--img--ki-whiteback-modified.png)](https://ki.se)[![](https://static.igem.org/mediawiki/2021/e/e2/T--Stockholm--img--kthwhite-modified.png)](https://kth.se)[![](https://static.igem.org/mediawiki/2021/d/da/T--Stockholm--img--stockholmuni-modified.png)](https://su.se)

Sample template built using the iGEM Wiki Starter Pack by BITS Goa.

Code released under the MIT license.

Based on [Bootstrap](https://getbootstrap.com) and themes [Flatly](https://bootswatch.com/flatly/) and [Darkly](https://bootswatch.com/darkly/) from [Bootswatch](https://bootswatch.com/).

Some content from the [iGEM Example Wiki](https://2020.igem.org/Team%3AExample). Images from [Unsplash](https://unsplash.com). Web fonts from [Google](https://fonts.google.com).

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Stockholm/Engineering

Engineering | iGEM Stockholm

* Team

  [Team](https://2021.igem.org/Team%3AStockholm/Team)[Attributions](https://2021.igem.org/Team%3AStockholm/Attributions)[Collaborations](https://2021.igem.org/Team%3AStockholm/Collaborations)[Partnership](https://2021.igem.org/Team%3AStockholm/Partnership)[Inclusivity](https://2021.igem.org/Team%3AStockholm/Inclusivity)[Sponsors](https://2021.igem.org/Team%3AStockholm/Sponsors)
* Project

  [Description](https://2021.igem.org/Team%3AStockholm/Description)[Contribution](https://2021.igem.org/Team%3AStockholm/Contribution)[Engineering](https://2021.igem.org/Team%3AStockholm/Engineering)[Experiments](https://2021.igem.org/Team%3AStockholm/Experiments)[Notebook](https://2021.igem.org/Team%3AStockholm/Notebook)[Proof Of Concept](https://2021.igem.org/Team%3AStockholm/Proof_Of_Concept)[Results](https://2021.igem.org/Team%3AStockholm/Results)
* [Model](https://2021.igem.org/Team%3AStockholm/Model)
* Human Practices

  [Human Practices](https://2021.igem.org/Team%3AStockholm/Human_Practices)[Implementation](https://2021.igem.org/Team%3AStockholm/Implementation)[Entrepreneurship](https://2021.igem.org/Team%3AStockholm/Entrepreneurship)[Communication](https://2021.igem.org/Team%3AStockholm/Communication)[Education](https://2021.igem.org/Team%3AStockholm/Education)
* [Safety](https://2021.igem.org/Team%3AStockholm/Safety)

[ ]

# Engineering

Here we present the engineering processes and how we tailored the engineering design cycle for the various experimental challenges in the project, MIKROSKIN.

---

##### Contents

# Overview of Engineering

At the heart of an iGEM project is the use of engineering principles in synthetic biology to find a solution to a problem. Typically, this is performed by engineering a biological system, e.g., a bacterial organism. With MIKROSKIN we decided not to use a biological system but explore another area of synthetic biology. Still, our project is centered around the engineering cycle with all its steps (Figure 1).

The cornerstone of our project is the production of a semi-quantitative rapid test for the skin microbial community. For our test we focused on two parts. First, we targeted the dominant species of *C. acnes* and all gram-positive skin bacteria by using SELEX and aptamer technology. Secondly, we created a colorimetric detection method using PCDA polymer to observe
aptamer binding to the ligand.

Here we present the engineering hub and how we tailored the different steps of the engineering design cycle for the various experimental challenges in our project: MIKROSKIN.

![The engineering cycle is composed of an initial research step and five recurring steps: Design, Build, Test, Learn, and Improve.](https://static.igem.org/mediawiki/2021/5/59/T--Stockholm--img--Engineering-cycle.png)

# Aptamer Production

## Research

The first step of the engineering cycle is intensive research. Every project idea needs to be backed up by previous research findings, which support the need, logic and feasibility of the project.

Our project idea focuses on the skin conditions acne vulgaris and dermatitis. Thus, the first step was to read upon both diseases. We scanned the literature on statistics around the diseases, their causes, diagnosis and treatment. While we started with two diseases, we soon expanded our research and included other diseases, their similarities and the way research on skin conditions is conducted. We concluded the first research phase with our project idea: developing a rapid test to detect skin diseases, by detecting imbalance in the skin microbiota.

## Design

After establishing our project idea - the creation of a rapid test - the design phase was initiated with several brainstorming sessions. During the sessions, we decided upon the important characteristics of our test and tried to come up with a design that fits best. We wanted the test to be easily accessible and as cost-effective as possible. Additionally, we aimed for a test that can be conducted without special equipment like sequencing. We started looking into molecules that would recognise and bind to specific targets, which we could base our test on.

Aptamers are small, single-stranded oligonucleotides that can assemble to form complex structures. These folded three-dimensional structures form interactions with small molecule targets which can be detected (1), as shown in Figure 2. The aptamer libraries are obtained using the Systematic Evolution of Ligands by Exponential enrichment (SELEX) technology. SELEX is a basic technique for the selection of aptamers in vitro against various targets, ranging from small molecules like ATP to proteins to even whole cells. The interactions between the aptamer and its target involve different intermolecular interactions including van der Waals forces, electrostatic interactions between charged groups, and three-dimensional shaping by hydrogen bonds.

Due to the ability of aptamers to recognise and bind specifically to targets, they are often compared to antibodies. The former, however, can be crafted to bind to multiple different targets while antibodies, which can only bind to immunogenic toxic targets. Aptamers are much smaller than antibodies, so they are able to fit into clefts and gaps on the surface of much larger target molecules that antibodies sometimes cannot reach. They are also more resilient against heat and organic solvents. These properties provide a huge market for aptamers to be used in, e.g, the field of diagnostics for targeting biomarkers, such as cancer, and in the drug delivery process.

Thus, we decided to base our test on aptamers.

![Schematic representation of aptamers mode of action.](https://static.igem.org/mediawiki/2021/d/de/T--Stockholm--img--Engineering.jpg)

Figure 2: Schematic representation of aptamers mode of action.

Next, we had to decide which targets we wanted to choose. We went back to our literature search and expanded it. This time, our search was focused on finding molecular targets involved in skin diseases, which could be used for aptamer selection.

Furthermore, we wanted the test to be semi-quantitative. We came up with the following strategy to be able to compare the detected amount of a target bacteria to all the skin bacteria in the sample.

We were aiming for the development of two aptamers: one targeting lipoteichoic acid (i.e. LTA), which is present on the cell wall of all gram-positive bacteria. Since gram-positive Actinobacteria are dominant on the skin (Figure 3), teichoic acid can be used for the quantification of the total bacteria in the skin microbiota.

The second aptamer should target a strain of *C. acnes*, which is dominant in acne-prone skin (4).

![Pie chart representing the diversity of bacteria in the skin microbiota. Modified from (3)](https://static.igem.org/mediawiki/2021/1/11/T--Stockholm--img--Skin-microbiota-proportion.png)

Figure 3: Pie chart representing the diversity of bacteria in the skin microbiota. Modified from (3)

Aptamers are developed by the cyclic method of SELEX (Systematic Evolution of Ligands by Exponential enrichment). SELEX starts with the incubation of an aptamer library with random sequences with the targets, *C. acnes* or LTA.

As a result, some aptamers will bind to the targets while unbound sequences will be removed by washing. The bound sequences are eluted from their target by using heat and are then amplified by error-prone PCR. This mutagenic PCR includes some modifications in the pre-selected aptamer sequences, creating a library of variants that will be used in the next round. This concludes the first round of SELEX, but in order to obtain the desired aptamers with high binding affinity, multiple SELEX rounds are necessary. SELEX is therefore a process of directed and targeted in vitro evolution.

## Building and Testing

The Build & Test step of our aptamer selection subproject started with the creation of our own cell SELEX protocol, for the production of the *C. acnes* aptamer, and in vitro SELEX protocol, for the production of the LTA aptamer. We have consulted with our advisor, Dimitri Van Simaeys, and referred to research papers to find the optimal protocols for aptamer selection.

In the following parts we will guide you through the different phases of the building and test steps of the engineering cycle. These phases are strongly connected to the learn and improve steps of the cycle, as each error identification required us to refine our ideas to move on with the project. Thus, each presented phase is a small engineering cycle in itself.

## Phase I - Ordering Primers

The beginning of the building phase was cumbersome. Understanding all specifications for the library as well as ordering the primer from the company (Integrated DNA Technologies (IDT)) was a challenge in itself. At first, we ordered the wrong sequence for our reverse primer.

This error led to unsuccessful PCR amplification of the SELEX library. All PCR products were checked by performing electrophoresis in a 2% agarose gel using TBE buffer. In various troubleshooting attempts we stumbled upon different common errors, such as forgetting to pipette the sample into PCR tubes. No amplification was visible in the form of a band, only primer dimers were detected (Figure 4). Primer dimers are potential byproducts of a PCR. They result from primers that have complementary sequences, which stick together. These dimers can be amplified in the PCR reaction, leading to intense bands with short base pair length.

![Primer dimer formation following PCR amplification. PCR products run in a 2% agarose gel. Lane 1 is Preamplified library, lane 2-7 PCR product with increasing cycle number from left to right, lane 8 non template control (NTC).](https://static.igem.org/mediawiki/2021/c/c7/T--Stockholm--img--Engineering-PCR-wrong-primer-final.png)

Figure 4: Primer dimer formation following PCR amplification. PCR products run in a 2% agarose gel. Lane 1 is Preamplified library, lane 2-7 PCR product with increasing cycle number from left to right, lane 8 non template control (NTC).

After a lot of troubleshooting, we started looking at our ordered material. Upon checking our primers for dimerisation potential, we recognised the major problem: We had ordered the complementary sequence to the 3' overhang in our library. However, instead of ordering 5'-3' we had ordered 3'-5', which made the primer useless.

This concluded a first engineering cycle. We designed our idea, built the PCR protocol and conducted it. We failed to achieve the expected results, but investigated why it failed and improved the protocol based on our reflections.

## Phase II - Primer Dimers

After having successfully identified and solved one major problem, we soon faced another one. Even when using the right primers we still had no library PCR amplification, only primer dimers. With the help of our instructors and advisors we evaluated the problems and again did several troubleshooting PCR attempts. While examining our protocol step by step, we realised that the annealing temperature should be reconsidered. The PCR annealing temperature needs to be adjusted according to the melting temperature (Tm) of the used primers, as it should be 3 to 5 degrees Celsius lower than the lowest Tm of the primers. As our primers had an annealing temperature of 55 degrees Celsius, we realised that our PCRs were performed using an annealing temperature, approximately 10 degrees Celsius higher than it should be.

This considered the use of annealing temperatures ranging from 50 to 60 degrees Celsius was tested. However, still no success in library amplification was achieved, as no band except the primer band was visible (Figure 5).

![2% agarose showing the library amplification products after PCR using different annealing temperatures.](https://static.igem.org/mediawiki/2021/0/04/T--Stockholm--img--Engineering-annealing-temperatures.png)

Figure 5: 2% agarose showing the library amplification products after PCR using different annealing temperatures.

## Phase III - Concentrations of the PCR Components

Next, we evaluated the concentrations of all used components of our PCR. We checked primer concentrations using the Nanodrop and determined that our diluted primer aliquotes did not have the desired concentration. Additionally, we checked our protocol and found out that we had used too high concentration of both primer and template in our PCR protocol. This can lead to decreased amplification due to spatial limitations.

All these troubleshooting steps finally resulted in a successful PCR amplification of our aptamer library. The bands in the agarose gel (Figure 6) correspond to the desired length of the initial library with about 80 nucleotides.

![2% agarose showing the library amplification products after PCR using different annealing temperatures.](https://static.igem.org/mediawiki/2021/7/7e/T--Stockholm--img--Engineering-PCR-success.jpg)

Figure 6: 2% agarose showing the library amplification products after PCR using different annealing temperatures.

## Phase IV - PCR Contamination

Unfortunately, after our successful PCR we faced another common problem when doing PCR: contamination. We experienced recurrent aptamer contamination in our Non-template control sample, meaning that some of the PCR components were contaminated with the template.

We identified the contaminated component by setting different PCR reactions without a template where one component at a time was changed for a fresh aliquot. This way, we could infer the culpable component by observing in which sample the contamination band disappeared.

![PCR contamination troubleshooting in a 2% agarose gel. New polymerase and polymerase buffer (lane 1), new dNTPs (lane 2), new Forward (lane 3) and Reverse primers (lane 4) aliquots were tested.](https://static.igem.org/mediawiki/2021/7/72/T--Stockholm--img--contamination-troubleshooting.png)

Figure 7: PCR contamination troubleshooting in a 2% agarose gel. New polymerase and polymerase buffer (lane 1), new dNTPs (lane 2), new Forward (lane 3) and Reverse primers (lane 4) aliquots were tested.

In the example shown in Figure 7, bands can be seen in lane 1, 3 and 4, although very faint in the latters. The band disappears when a fresh dNTP aliquot was used, therefore we concluded that it was the component contaminated with template.

## Phase V - Elution

Proceeding with our SELEX protocol we faced other difficulties. Elution and desalting of the selected aptamers from the beads were the last steps of the protocol. Unfortunately, we were not able to detect DNA in the flow-through of our desalting column. At first, we thought the desalting was the striking point, however, after checking DNA concentration in our sample pre and post elution, as well as post desalting, we saw that no DNA was detectable already after elution. Thus, we concluded that the critical point was the elution of the aptamers from the beads.

To fix this problem we tested different elution strategies with different elution buffers and different degrees of heat. Still, the elution was unsuccessful. By discussing with instructors we realised that we were assuming the wrong DNA concentration in our samples. The expected DNA concentrations after PCR amplification were based on the Nanodrop measurement of the PCR products. However, due to primer and other components in the PCR product, the measurement is highly inaccurate.

A better approach to determine the DNA amount is by analysing a gel image using the ladder as reference for DNA amount. We concluded that we had too little DNA input to measure anything with the Nanodrop after elution and desalting. We solved this problem by increasing the PCR product used for elution.

## Phase VI - Desalting

During the desalting step, a major issue was the elution volume. In our first attempts, the volume of the buffer used to elute the DNA from the NAP-5 column was too high, resulting in the dilution of our library. If the library is too diluted, few aptamer sequences will be incubated with the targets in the next SELEX rounds.

We are currently working on optimising this last step.

# Learning and Improving

As mentioned before, learning and improvement are not only final steps at the end of a project. They continuously occur during the building and testing of the project. In our aptamer selection journey we have learned a lot about structured troubleshooting. We faced multiple problems in one step of our protocol, the PCR. It was of greatest importance to discuss with teammates, instructors, advisors and sometimes senior researchers to understand the continuous erroneous results. In the end, we succeeded with our PCR. However, we learned from our project that success in the learning and improvement step is only the beginning of another engineering cycle, and that many rounds will eventually lead to a finished project.

Although we did not have enough time to develop high-affinity aptamers, which was our desired end goal for this part of the project, we optimised our methodology. We gathered our gained knowledge during this journey in the SELEX troubleshooting guides (seen below), where detailed protocols, tips to keep in mind and tricks for accurate troubleshooting can be found. Future iGEM teams working with aptamers will greatly benefit from them!

* [In vitro SELEX Troubleshooting Guide](https://static.igem.org/mediawiki/2021/9/93/T--Stockholm--Education-SELEX-Troubleshoot.pdf)
* [Cell SELEX Troubleshooting Guide](https://static.igem.org/mediawiki/2021/a/ab/T--Stockholm--CellSELEX-Troubleshoot.pdf)

Additionally to our gained knowledge in the design, build and testing phase of our project, we helped our entrepreneurial subteam to refine the design of our end product. From an at-home use rapid test for detecting acne and dermatitis, to a customisable high throughput test. Learning and improvement is not the last part of the cycle but is conducted after every small step!

# PCDA-Based Detection Method

## Research

When researching a suitable detection method for our biosensor, we focused on the desired features of our end product. We wanted the test to be easily performed and interpreted without the need for special equipment. Hence, we searched for colorimetric methods, which can be observed by the naked eye, but at the same time allow for quantification by measuring absorbance, which is necessary for an accurate laboratory characterisation.

During the research step, we came across 10,12-Pentacosadiynoic acid (PCDA) (Figure 9). PCDA has special spectrophotometric properties upon polymerisation. Sonication is used to assemble the PCDA monomers into vesicles. Vesicles turn from white to dark blue upon UV irradiation and then red if an external factor such as chemicals, heat or radiation stresses the vesicle surface (5). This colour change is visible with the nake eye and we took advantage of this property to develop the detection method for our biosensor.

![Chemical structure and blue to red colour transition of polydiacetylene in polymerised vesicles.](https://static.igem.org/mediawiki/2021/5/56/T--Stockholm--img--Experiments-PCDA-vesicle.jpg)

Figure 8: Chemical structure and blue to red colour transition of polydiacetylene in polymerised vesicles.

## Design

We aim to conjugate our aptamers with blue PCDA vesicles. The premise of this idea is that the aptamer undergoes conformational changes upon target binding; inducing stress to the PCDA vesicles surface, thus changing colour from blue to red.

The colour transition would happen regardless of the target being lipoteichoic acid or a *C. acnes* cell and would be proportional to the amount of target bound to the aptamers. Moreover, the colour change will not only be visible by the naked eye, but will also be quantifiable by absorbance measurement in a spectrophotometer. The ratio between *C. acnes*/LTA absorbance makes our biosensor a semi-quantitative test for dysbiosis detection.

In addition, this detection method could be used for high throughput screening tests, if we decide to scale up our prototype and increase the number of targets.

## Building and Testing

As in the aptamer selection part of the project, the Build & Test steps of the engineering cycle are strongly linked to the learning and improvement steps of the cycle. Again we will guide you through different phases of the detection method building and testing process, which all led to learning and improvement. Hence, they all display little engineering cycles in themselves.

## Phase I - PCDA Polymerisation and Vesicle Synthesis

We started the Build & Test phase with the synthesis of our PCDA vesicles, which are the base of every further step of the project.

The synthesis itself was challenging at first, as none of us had any experience with the procedures.

After following the protocol for the first time, we successfully produced a white substance with little blue and red sparks of PCDA vesicles. We were unsure about the concentration we produced, therefore, we re-calculated the amount of PCDA in our solution. Additionally, we realised that the nitrogen flow used to dry the chloroform when dissolving the PCDA needs to be very gentle to avoid spilling. Furthermore, we should only use a small amount of chloroform, as it easily dissolves.

We learned from these adjustments to improve the protocol. As a result, we were able to produce PCDA vesicles with the desired concentration and a pure-white colour.

## Phase II - Inducing Colour Change

After successfully achieving both the PCDA vesicles polymerisation and the colour change from white to blue by sonication, we moved on to induce the blue to red transition.

First, we tried UV radiation as an external stressor, but O/N irradiation only induced a slight colour change. We still wondered if our vesicles were able to turn red, so we tried heat as a source of external stress by using a Bunsen burner for a few seconds. Heat prompted a fast colour change and the blue solution turned into a bright orange within seconds (Figure 9). Thus, we concluded that our PCDA vesicles are susceptible to change colour upon mechanical stress.

![PCDA solutions subjected to different stress conditions (heat, UV radiation, normal), and subsequently diluted for spectrophotometric measurements](https://static.igem.org/mediawiki/2021/d/d5/T--Stockholm--img--Results1.jpg)

Figure 9: PCDA solutions subjected to different stress conditions (heat, UV radiation, normal), and subsequently diluted for spectrophotometric measurements

## Phase III - Testing the detection method

Next, we proceeded to the aptamer-PCDA conjugation and test of the detection method.

Even though we ordered an already published aptamer against protein A, we were not able to see any colour change in the PCDA-aptamer conjugates upon incubation with the aptamer target in 24-well plates.

This was a major drawback in our project. We analysed the potential failures of our biosensor and came up with the following potential explanations for the lack of colour change:

* The PCDA and the aptamer do not conjugate effectively
* The bought protein A aptamer is not binding its target effectively
* Protein A was damaged due to incorrect storage conditions

The latter could be ruled out easily. Even though protein A was stored at room temperature instead of -20 degrees Celsius for several days, according to the advice of senior researchers of the department, protein A is very stable and should still be functional.

To evaluate if the conjugation of aptamer and PCDA vesicles was successful we needed access to advanced imaging methods that were not available for our iGEM team. This is why we reached out to MEPhI (Moscow Engineering Physics Institute) iGEM team. We started a collaboration and sent samples to the Russian team, who tried helping us out. Even though they were not able to condusct the planned tests before the wiki freeze, we were really happy to discuss those techniques and learned a lot about scientific collaborations.

Next, we discussed strategies for testing the affinity of the protein A aptamer with our advisors. We designed a series of experiments aiming to characterise the aptamer affinity.

## Phase IV - Low-affinity aptamer

After successfully dissecting possible explanations for the unsuccessful biosensor function, we conducted several experiments to test our aptamer's affinity. Using chromatography, bead based assays and binding assays based on fluorescent detection, we concluded that our aptamer has low affinity for its target.

This taught us not only to think about experimental errors but also analyse published data in a critical way - already published data does not always work perfectly in your set-up.

## Learning and Improving

Again, learning and improvement occurred during every phase of the development, building and testing of the PCDA-based detection method.

Learning was done by reaching out for help to people with more experience or access to special equipment needed for the experiment.

We also realised the weight of creativity for the experimental design and how important it is to break down big experiments into smaller ones in order to rule out the different hypotheses during troubleshooting.

# References

1. Keefe AD, Pai S, Ellington A. Aptamers as therapeutics. Nature reviews Drug discovery. 2010 Jul ; 9 (7) : 537 - 50.
2. Sun H, Zhu X, Lu PY, Rosato RR, Tan W, Zu Y. Oligonucleotide aptamers: new tools for targeted cancer therapy. Molecular Therapy-Nucleic Acids. 2014 Jan 1 ; 3 : e182.
3. Grice, E. A., & Segre, J. A. (2011). The skin microbiome. Nature reviews. Microbiology, 9 (4) , 244 â€“ 253. https://doi.org/10.1038/nrmicro2537
4. Dreno B, Dagnelie MA, Khammari A, Corvec S. The skin microbiome: a new actor in inflammatory acne. American Journal of Clinical Dermatology. 2020 Sep 10 : 1 - 7 .
5. Scoville SP, Shirley WM. Investigations of chromatic transformations of polydiacetylene with aromatic compounds. Journal of applied polymer science. 2011 Jun 5 ; 120 (5) : 2809 - 20.
6. Kim, Young-Rok & Jung, Sungho & Ryu, Hyunil & Yoo, Yeong-Eun & Kim, Sun Min & Jeon, Tae-Joon. (2012). Synthetic Biomimetic Membranes and Their Sensor Applications. Sensors (Basel, Switzerland). 12. 9530 - 50. 10.3390 / s120709530.
7. De Grazia A, Mikhael M, Stojanovska N, Reedy B, Shimmon R, Tahtouh M. Diacetylene copolymers for fingermark development. Forensic science international. 2012 Mar 10 ; 216 ( 1 - 3 ) : 189 - 97.

[![](https://static.igem.org/mediawiki/2021/b/bb/T--Stockholm--img--ki-whiteback-modified.png)](https://ki.se)[![](https://static.igem.org/mediawiki/2021/e/e2/T--Stockholm--img--kthwhite-modified.png)](https://kth.se)[![](https://static.igem.org/mediawiki/2021/d/da/T--Stockholm--img--stockholmuni-modified.png)](https://su.se)

Sample template built using the iGEM Wiki Starter Pack by BITS Goa.

Code released under the MIT license.

Based on [Bootstrap](https://getbootstrap.com) and themes [Flatly](https://bootswatch.com/flatly/) and [Darkly](https://bootswatch.com/darkly/) from [Bootswatch](https://bootswatch.com/).

Some content from the [iGEM Example Wiki](https://2020.igem.org/Team%3AExample). Images from [Unsplash](https://unsplash.com). Web fonts from [Google](https://fonts.google.com).

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Stockholm/Experiments

Experiments | iGEM Stockholm

* Team

  [Team](https://2021.igem.org/Team%3AStockholm/Team)[Attributions](https://2021.igem.org/Team%3AStockholm/Attributions)[Collaborations](https://2021.igem.org/Team%3AStockholm/Collaborations)[Partnership](https://2021.igem.org/Team%3AStockholm/Partnership)[Inclusivity](https://2021.igem.org/Team%3AStockholm/Inclusivity)[Sponsors](https://2021.igem.org/Team%3AStockholm/Sponsors)
* Project

  [Description](https://2021.igem.org/Team%3AStockholm/Description)[Contribution](https://2021.igem.org/Team%3AStockholm/Contribution)[Engineering](https://2021.igem.org/Team%3AStockholm/Engineering)[Experiments](https://2021.igem.org/Team%3AStockholm/Experiments)[Notebook](https://2021.igem.org/Team%3AStockholm/Notebook)[Proof Of Concept](https://2021.igem.org/Team%3AStockholm/Proof_Of_Concept)[Results](https://2021.igem.org/Team%3AStockholm/Results)
* [Model](https://2021.igem.org/Team%3AStockholm/Model)
* Human Practices

  [Human Practices](https://2021.igem.org/Team%3AStockholm/Human_Practices)[Implementation](https://2021.igem.org/Team%3AStockholm/Implementation)[Entrepreneurship](https://2021.igem.org/Team%3AStockholm/Entrepreneurship)[Communication](https://2021.igem.org/Team%3AStockholm/Communication)[Education](https://2021.igem.org/Team%3AStockholm/Education)
* [Safety](https://2021.igem.org/Team%3AStockholm/Safety)

[ ]

# Experiments

An overview of how we explored the skin

---

##### Contents

# Overview

The goal of this year's iGEM Stockholm team was to create a detection method based on aptamers bound to PCDA vesicles. When the aptamers bind to their target due to mechanical stress the PCDA will change colour indicating that the target is present. Quantification of the intensity of the colour change would be proportional to the concentration of the bacteria present in the skin.

# PCDA Polymerisation

Polydiacetylenes (PCDA) are a family of polymers created by the polymerisation of diacetylene creating vesicles or tube structures. (1). Due to their unique chromatic properties, they have frequently been used as a detection method. 10,12-Pentacosadiynoic acid (PCDA) belongs in the family of polydiacetylenes and yields a blue colour in its primary form. Under heat exposure (thermochromism), mechanical stress (mechanochromism) or solvent (solvatochromism), PCDA changes colour from blue to red (2). Our biosensor is based on mechanical stress induction: as mentioned before, it is caused by target binding of the aptamer, which results in a colour change.

## Formation of PCDA vesicles

In order to create the PCDA vesicles, we first dissolved the PCDA in chloroform, which we then we evaporated using gentle nitrogen flow. Following this, we dissolved the PCDA in water using sonication. The end product was an emulsion with a white colour. The next step was exposure to UV light to induce polymerisation which in turn results in a colour change from white to blue. This protocol has been used before for the creation of PCDA vesicles (3, 4). The solution was stored at 4 degrees Celcius until further use.

![Schematic representation of PCDA-vesicle formation and color change induction.](https://static.igem.org/mediawiki/2021/5/56/T--Stockholm--img--Experiments-PCDA-vesicle.jpg)

## Conjugation of the PCDA vesicles to the aptamers

The conjugation of the PCDA vesicles to the aptamers is based on the Carbodiimide method. This method is used for the creation of amide bonds between carboxylates and amines by adding dicyclohexylcarbodiimide (DCC). DCC is a crosslinker that creates a reactive intermediate which reacts with nucleophiles like amines resulting in an amide bond (5, 6). In our experiments EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) was used. EDC is the most popular carbodiimide for conjugating carboxylates with amines and it is used together with NHS (N-hydroxysulfosuccinimide) in order to stabilise the reactive intermediate.

We chose this method since PCDA has carboxyl groups and the aptamer is a small DNA sequence with amine groups. The protocol we followed was based on a paper by Wu et al. (4).

![Schematic illustration of the mode of action of our biosensor. PCDA-vesicles conjugated to an aptamer change colour upon target binding.](https://static.igem.org/mediawiki/2021/a/aa/T--Stockholm--img--Experiments-biosensor-mode-of-action.jpg)

Figure 2: Schematic illustration of the mode of action of our biosensor. PCDA-vesicles conjugated to an aptamer change colour upon target binding.

# Bacteria Cultivation

Another important part of the project was the cloning of a biobrick, which was made by assembling two existing parts from the registry: part BBa\_K103003 coding for staphylococcal protein A, and part BBa\_K1073024 amilGFP. Firstly, the two biobricks had to be expressed and harvested through PCR and gel extraction. After that, the two parts were ligated and assembled into a vector. In order to have a functioning protein, the stop codon that was left between the biobricks had to be removed using site-directed mutagenesis. Finally, the new biobrick was cloned into Top10 competent E. coli cells and expression could be confirmed by observing the colonies. The plasmid was sent for sequencing to verify the plasmid sequence and to validate the part.

## SELEX

SELEX or Systematic Evolution of Ligands by Exponential Enrichment is a screening technique based on selecting specific targets from a large pool of random oligonucleotides which could be a mixture of RNA, DNA, single or double-stranded, by an iterative process of separation and amplification. The products of SELEX are DNA or RNA aptamers that mimic the properties of antibodies. They can further bind to a big variety of molecules like proteins, peptides, drugs, organic molecules or even metal ions (7).

The reason why we used aptamers as our detection method this year, is that the development of aptamers is cheaper and more flexible compared to antibodies. Moreover, aptamers are easier to manipulate biochemically. For example, it is simple to add functional groups to the aptamers; in our case we conjugated them to PCDA.

The main principle of making aptamers remains the same no matter the target of interest. The random pool of DNA is incubated with the target. After the incubation, the binding complex is separated from the unbound sequences and then eluted in order to have the DNA sequences only. Sequential positive and negative selection steps allow retrieving the aptamer sequences with the highest sensitivity and selectivity for the target of interest.

Then, an error-prone PCR amplifies the selected aptamers and introduces point mutations in some of the sequences, resulting in a library of variants. Ultimately, we select the sense single DNA strands from the PCR product to build a new library, which will be used in the next SELEX round.

This concludes the first round of SELEX. In order to obtain aptamers with high affinity for the target, multiple rounds of SELEX need to be performed (8).

# Cell-SELEX

The first part of the experiment was to create an aptamer that can detect *Cutibacterium acnes*, a bacteria linked to acne but at the same time belongs in the natural flora of the skin (9). In order to do that, we used two types of *C. acnes*. More specifically, CCUG 1794 T *Cutibacterium acnes* (Type I) and CCUG 6369 *Cutibacterium acnes* (Type II) were used for negative and positive control, respectively. At first, we incubated a random pool of DNA with our CCUG 6369 *C. acnes* cells after measuring the right amounts of cells by OD600 measurement.

Next, we centrifuged the cell-aptamer mixture and removed the supernatant and, as a result, we had only the cell-aptamer complexes. Then, we eluted the aptamers by heat denaturation and separated them from the cells by centrifugation at 14 000 rpm. Next, we amplified the aptamers with PCR. The end product was a double stranded DNA with the antisense sequence having a biotinylated primer.

This primer was used so we can separate the sense from the antisense sequence using streptavidin beads and NaOH elution. At the end of the first round, we had only a pool of aptamers. In the second round, we used the pool of aptamers from the previous round. We incubated it with the positive and negative bacterial clones; we then followed the same steps as in the first round. The protocol we used was optimised from the protocol from Sefah et al (8) and Simaeys et al (10).

![Representation of a bacterial cell membrane with multiple different surface targets, in the form of proteins, lipids or similar.](https://static.igem.org/mediawiki/2021/3/3e/T--Stockholm--img--Experiments-bacteria-surface.jpg)

Figure 4: Representation of a bacterial cell membrane with multiple different surface targets, in the form of proteins, lipids or similar.

# Bacteria Cultivation

*Cutibacterium acnes*, formerly known as *Proprionibacterium acnes*, is a rod-shaped gram-positive commensal skin bacterial species (11).

*C. acnes* sub-types I and II, used in the cell SELEX experiments, were cultured using Brain Heart Infusion media or Blood Agar.

All *C. acnes* subtypes, including the aforementioned, are known to be facultatively anaerobic (11). We had to adapt the standard bacteria culture to this metabolic demand. In order to reduce the oxygen in contact with the liquid culture, we used long glass tubes filled with media almost to the top and closed them with sealing lids.

![C. acnes cultured anaerobically in Brain Heart Infusion media.](https://static.igem.org/mediawiki/2021/f/f6/T--Stockholm--img--cacnes-liquid-culture.jpeg)

Figure 5: C. acnes cultured anaerobically in Brain Heart Infusion media.

When *C. acnes* was cultured in plates, we came up with the following strategy: the Blood Agar plates were piled inside of an anaerobic jar. Then, we placed a lit candle on top of the pile of plates and closed the jar with a sealing lid. The candle would light until the oxygen was consumed. This way, we ensured the anaerobic conditions required by the bacteria were maintained inside the jar.

![C. acnes culture on a Blood agar plate.](https://static.igem.org/mediawiki/2021/a/a0/T--Stockholm--img--cacnes-blood-agar.png)

Figure 6: C. acnes culture on a Blood agar plate.

[Your browser does not support the video tag.](https://static.igem.org/mediawiki/2021/9/94/T--Stockholm--anaerobic-jar.mp4)

This footage shows how we achieved anaerobic conditions by consuming the oxygen inside the sealed jar.

# LTA-SELEX

The second step of our experiment was to create an aptamer that is specific for Lipoteichoic acid (LTA), which is a molecule that exists in the membrane of all gram-positive bacteria (12). Using the carbodiimide method, we conjugated LTA from *Staphylococcus aureus* to carboxyl-activated sepharose beads. The beads were used as a means of LTA immobilisation and to facilitate the target-aptamer complex separation during the SELEX cycle.

In order to obtain our LTA aptamer, we used the method described earlier, but here we replaced the cells with our LTA-beads as positive control. For the negative control, we used bovine serum albumin (BSA).

![Illustration of the cell wall of gram positive bacteria.](https://static.igem.org/mediawiki/2021/e/ef/T--Stockholm--img--Experiments-gram-positive-bacteria.jpg)

Figure 7: Illustration of the cell wall of gram positive bacteria.

# Protein-A Aptamer

For our proof of concept, we set out to test a published protein A aptamer. Protein A is a protein that exists in the membrane of *S. aureus* and an aptamer targeting this specific protein already exists (13). However, when we incubated this aptamer conjugated to PCDA with protein A, we could not observe any colour change. For this reason, we aimed to characterise protein A aptamer binding affinity to our target. To this end, we used HPLC with a protein A column and protein A-bound beads. Unfortunately, both of these methods showed poor target affinity of the aptamer so we tried another approach. We coated wells with protein A and we incubated it with protein A aptamer attached to a fluorophore. From this binding affinity assay, we concluded that our protein A aptamer is not capable of binding protein A efficiently.

# BioBrick Engineering

Another important part of the project was building a new BioBrick: [BBa\_K4071000](http://parts.igem.org/Part%3ABBa_K4071000). The idea was to create a fusion protein consisting of protein A labelled with a reporter protein in order to be visible with the naked eye. Thus, we decided to assemble two pre-existing parts from the registry: part [BBa\_K1073024](http://parts.igem.org/Part%3ABBa_K1073024), coding for the yellow chromoprotein amilGFP and part [BBa\_K103003](http://parts.igem.org/Part%3ABBa_K103003), coding for the Staphylococcal protein A.

![Vector map of pSB1C3 containing the amilGFP BioBrick (BBa_K1073024). Retrieved from the iGEM Registry of Standard Biological Parts.](https://static.igem.org/mediawiki/2021/a/a2/T--Stockholm--img--pSB1C3-BBa_K1073024-wiki.png)

Figure 8: Vector map of pSB1C3 containing the amilGFP BioBrick (BBa\_K1073024). Retrieved from the iGEM Registry of Standard Biological Parts.

![Vector map containing the functional parts of the amilGFP BioBrick (BBa_K1073024).](https://static.igem.org/mediawiki/2021/c/c7/T--Stockholm--img--amilGFP-BBa_K1073024-Map.png)

Figure 9: Vector map containing the functional parts of the amilGFP BioBrick (BBa\_K1073024).

![Vector map of pSB1C3 containing the protein A BioBrick (BBa_K103003). Retrieved from the iGEM Registry of Standard Biological Parts.](https://static.igem.org/mediawiki/2021/d/df/T--Stockholm--img--pSB1C3-BBa_K103003wiki.png)

Figure 10: Vector map of pSB1C3 containing the protein A BioBrick (BBa\_K103003). Retrieved from the iGEM Registry of Standard Biological Parts.

![Vector map containing the functional parts of the protein A BioBrick (BBa_K103003).](https://static.igem.org/mediawiki/2021/6/67/T--Stockholm--img--protein-A-BBa_K103003-Map.png)

Figure 11: Vector map containing the functional parts of the protein A BioBrick (BBa\_K103003).

Our cloning strategy was designed based on the BioBrick RFC10 assembly standard:

We decided our BioBrick will first have the amilGFP part, since it contains a promoter and an RBS sequence, and then the protein A construct.

Based on this, we planned to:

* Isolate the protein A CDS
* Linearise the pSB1C3 vector containing amilGFP
* Ligate protein A part into the linearised vector containing amilGFP

![Schematic representation of the cloning strategy used to build the amilGFP-proteinA BioBrick (BBa_K4071000). Retrieved from SnapGene.](https://static.igem.org/mediawiki/2021/a/a2/T--Stockholm--img--cloning-wiki.png)

Figure 12: Schematic representation of the cloning strategy used to build the amilGFP-proteinA BioBrick (BBa\_K4071000). Retrieved from SnapGene.

With these aims, we decided to digest the BioBricks in the following way:

* Protein A BioBrick was digested with XbaI and PstI restriction enzymes:
  + This creates sticky ends compatible with SpeI and PstI, respectively.
  + Since this part has to go in second position, the BioBrick prefix was removed while the BioBrick suffix was kept.
* AmilGFP plasmid was linearised by digesting with SpeI and PstI restriction enzymes:
  + This creates sticky ends compatible with XbaI and PstI, respectively.
  + Since this construct has to go in first position, the BioBrick prefix was kept while the BioBrick suffix was removed.

The sticky ends generated in both parts allow directional cloning and regeneration of the BioBrick suffix. Thus, the new BioBrick preserves the standard prefix and suffix needed for RFC10 cloning.

Ultimately, to have a fusion protein, two adjacent STOP codons at the end of the amilGFP CDS had to be removed by using site-directed mutagenesis.

![TAA STOP codons (red) at the end of the amilGFP CDS (BBa_K1073024). Retrieved from SnapGene.](https://static.igem.org/mediawiki/2021/c/cf/T--Stockholm--img--Stop-codon-wiki.png)

Figure 13: TAA STOP codons (red) at the end of the amilGFP CDS (BBa\_K1073024). Retrieved from SnapGene.

Quikchange, a PCR site-directed mutagenesis method, allows the introduction of point mutations in specific locations of interest (14).

The first step we performed was designing two primers that hybridise with the target sequence and, at the same time, contain the modifications we want to incorporate in the template. These primers were then used in a PCR reaction together with a high copy fidelity polymerase, Phusion polymerase in our case. Since the primers have mismatches, the optimal annealing temperature is hard to determine, therefore three different annealing temperatures were used: 55 degrees Celsius, 58 degrees Celsius and 60 degrees Celsius.

Our primers were designed by Kozane.app, a primer design tool for site-directed mutagenesis.

The As in second position in both TAA STOP codons were substituted by Cs in order to create TCA Serine codons. The small size and lack of charge of Serine make the amino acid perfect for this linker region, which should be flexible in order to allow proper folding of both amilGFP and protein A.

![Quikchange Forward and Reverse primers are complementary to the target sequence at the end of the amilGFP CDS (BBa_K1073024), but include the desired point mutations (mismatches) that would be incorporated by PCR. Retrieved from SnapGene.](https://static.igem.org/mediawiki/2021/8/88/T--Stockholm--img--Quikchange-primers-wiki.png)

Figure 14: Quikchange Forward and Reverse primers are complementary to the target sequence at the end of the amilGFP CDS (BBa\_K1073024), but include the desired point mutations (mismatches) that would be incorporated by PCR. Retrieved from SnapGene.

After the PCR, the amplified product consisted of two different populations of DNA molecules: DNA strands which had incorporated the mutation and parental not-mutated strands.

The next step was digesting with DpnI, an endonuclease that recognises methylated and hemimethylated DNA. Since most E. coli strains express the DNA adenine methyltransferase enzyme (15), the parental DNA would be methylated. Therefore, the parental DNA (original template) was susceptible to being digested by DnpI. The PCR product, however, would be unmethylated and would not be digested.

![Overview of the Quikchange site-directed mutagenesis method. Image retrieved from Agilent (14).](https://static.igem.org/mediawiki/2021/b/bd/T--Stockholm--img--Quikchange-method-wiki.png)

Figure 15: Overview of the Quikchange site-directed mutagenesis method. Image retrieved from Agilent (14).

The last step was transforming TOP10 E. coli competent cells with the mutated DNA molecules, so the bacteria can perform nick repair.

Plasmids extracted from these bacteria would be the newly engineered BioBrick [(BBa\_K4071000)](http://parts.igem.org/Part%3ABBa_K4071000), coding for protein A labelled with the reporter chromoprotein amilGFP.

![Vector map of pSB1C3 containing the final version of the amilGFP-protein A BioBrick (BBa_K4071000).](https://static.igem.org/mediawiki/2021/d/d7/T--Stockholm--img--amilGFP-protA-BioBrick-Map.png)

Figure 16: Vector map of pSB1C3 containing the final version of the amilGFP-protein A BioBrick (BBa\_K4071000).

# References

1. Reppy M, Pindzola B. Biosensing with polydiacetylene materials: structures, optical properties and applications. Chemical Communications. 2007 ; (42) : 4317.
2. Lebegue E, Farre C, Jose C, Saulnier J, Lagarde F, Chevalier Y et al. Responsive Polydiacetylene Vesicles for Biosensing Microorganisms. Sensors. 2018 ; 18 (2) : 599.
3. Wen JT, Bohorquez K, Tsutsui H. Polydiacetylene-coated polyvinylidene fluoride strip aptasensor for colorimetric detection of zinc (II). Sensors and Actuators B: Chemical. 2016 Sep 1 ; 232 : 313 - 7.
4. Wu W, Zhang J, Zheng M, Zhong Y, Yang J, Zhao Y, Wu W, Ye W, Wen J, Wang Q, Lu J. An aptamer-based biosensor for colorimetric detection of Escherichia coli O157: H7. PloS one. 2012 Nov 7 ; 7 (11) : e48999.
5. Shah TR, Misra A. Proteomics Challenges in Delivery of Therapeutic Genomics and Proteomics.
6. Hermanson GT. Bioconjugate techniques. Academic press; 2013 Jul 25.
7. Chai C, Xie Z, Grotewold E. SELEX (Systematic Evolution of Ligands by EXponential Enrichment), as a powerful tool for deciphering the proteinâ€“DNA interaction space. InPlant Transcription Factors 2011 (pp. 249 - 258). Humana Press.
8. Sefah K, Shangguan D, Xiong X, O'donoghue MB, Tan W. Development of DNA aptamers using Cell-SELEX. Nature protocols. 2010 Jun ; 5 (6) : 1169 - 85.
9. Spittaels KJ, Ongena R, Zouboulis CC, Crabbe A, Coenye T. Cutibacterium acnes phylotype I and II strains interact differently with human skin cells. Frontiers in Cellular and Infection Microbiology. 2020 Nov 16 ; 10 : 712.
10. Van Simaeys D, Lopez-Colon D, Sefah K, Sutphen R, Jimenez E, Tan W. Study of the molecular recognition of aptamers selected through ovarian cancer cell-SELEX. PloS one. 2010 Nov 1 ; 5 (11) : e13770.
11. Elston MJ, Dupaix JP, Opanova MI, Atkinson RE. Cutibacterium acnes (formerly Proprionibacterium acnes) and Shoulder Surgery. Hawaii J Health Soc Welf. 2019 ; 78 (11 Suppl 2) : 3 - 5.
12. Poxton IR. Teichoic acids, lipoteichoic acids and other secondary cell wall and membrane polysaccharides of gram-positive bacteria. InMolecular Medical Microbiology 2015 Jan 1 (pp. 91 - 103). Academic Press.
13. Stoltenburg R, Krafcikova P, Viglasky V, Strehlitz B. G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA. Scientific reports. 2016 Sep 21 ; 6 (1) : 1 - 2.
14. Agilent. QuikChange Site-Directed Mutagenesis Kit Instruction Manual. Available from: https://www.agilent.com/cs/library/usermanuals/public/200518.pdf [Accessed 14 Oct 2021].
15. New England Biolabs. Dam and Dcm Methylases of E. coli. Available from https://international.neb.com/tools-and-resources/usage-guidelines/dam-and-dcm-methylases-of-e-coli [Accessed 14 Oct 2021].

[![](https://static.igem.org/mediawiki/2021/b/bb/T--Stockholm--img--ki-whiteback-modified.png)](https://ki.se)[![](https://static.igem.org/mediawiki/2021/e/e2/T--Stockholm--img--kthwhite-modified.png)](https://kth.se)[![](https://static.igem.org/mediawiki/2021/d/da/T--Stockholm--img--stockholmuni-modified.png)](https://su.se)

Sample template built using the iGEM Wiki Starter Pack by BITS Goa.

Code released under the MIT license.

Based on [Bootstrap](https://getbootstrap.com) and themes [Flatly](https://bootswatch.com/flatly/) and [Darkly](https://bootswatch.com/darkly/) from [Bootswatch](https://bootswatch.com/).

Some content from the [iGEM Example Wiki](https://2020.igem.org/Team%3AExample). Images from [Unsplash](https://unsplash.com). Web fonts from [Google](https://fonts.google.com).

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Stockholm/Results

Results | iGEM Stockholm

* Team

  [Team](https://2021.igem.org/Team%3AStockholm/Team)[Attributions](https://2021.igem.org/Team%3AStockholm/Attributions)[Collaborations](https://2021.igem.org/Team%3AStockholm/Collaborations)[Partnership](https://2021.igem.org/Team%3AStockholm/Partnership)[Inclusivity](https://2021.igem.org/Team%3AStockholm/Inclusivity)[Sponsors](https://2021.igem.org/Team%3AStockholm/Sponsors)
* Project

  [Description](https://2021.igem.org/Team%3AStockholm/Description)[Contribution](https://2021.igem.org/Team%3AStockholm/Contribution)[Engineering](https://2021.igem.org/Team%3AStockholm/Engineering)[Experiments](https://2021.igem.org/Team%3AStockholm/Experiments)[Notebook](https://2021.igem.org/Team%3AStockholm/Notebook)[Proof Of Concept](https://2021.igem.org/Team%3AStockholm/Proof_Of_Concept)[Results](https://2021.igem.org/Team%3AStockholm/Results)
* [Model](https://2021.igem.org/Team%3AStockholm/Model)
* Human Practices

  [Human Practices](https://2021.igem.org/Team%3AStockholm/Human_Practices)[Implementation](https://2021.igem.org/Team%3AStockholm/Implementation)[Entrepreneurship](https://2021.igem.org/Team%3AStockholm/Entrepreneurship)[Communication](https://2021.igem.org/Team%3AStockholm/Communication)[Education](https://2021.igem.org/Team%3AStockholm/Education)
* [Safety](https://2021.igem.org/Team%3AStockholm/Safety)

[ ]

# Results

What did we find?

---

##### Contents

# PCDA Synthesis and Polymerisation

Our detection method is based on the 10,12-Pentacosadiynoic acid (PCDA) polymer vesicles which can change colour when subjected to various kinds of stress, such as chemical, mechanical, heat and radiation.

The first step was to synthesise PCDA polymers from crystalline PCDA monomers. In their polymer form, PCDAs adopt vesicle formations in a water solution. This solution can then be stored at 4 degrees Celsius for extended periods of time.

We tested the PCDA solution for reactivity to heat and UV-radiation. The results of this test are shwon in Figure 1. For heat, a few mL of the solution was poured into a glass test tube and subjected to a burner flame. After a few seconds, the solution turned to a bright orange, indicating a structural change. When it comes to UV-radiation, the colour change is a lot more subtle. By leaving it overnight with UV-radiation, the colour changed to a deep purple. This is probably due to the UV lamps' supplied energy being relatively low, especially compared to the burner's energy flux into the solution.

![PCDA solutions subjected to different stress conditions (heat, UV radiation, normal), and subsequently diluted for spectrophotometric measurements. ](https://static.igem.org/mediawiki/2021/d/d5/T--Stockholm--img--Results1.jpg)

Spectrophotometric measurements of the colour change were performed with the solutions from Figure 1, and the results are shown in Figure 2. The absorbance maxima changes from around 640 nm to around 500 nm. Both peaks visible on the spectrum are shifted to lower wavelengths when the PCDA solution is heated.

![Absorbance spectrums, shown with different coloured curves of A: Blue PCDA at 1 mM (Black), 0.1 mM (Red) and 0.01 mm (Blue); B: Red PCDA at 1 mM (Blue), 0.01 mM (Purple), 0.01 mM (Red) and 0.001 mM (Black).](https://static.igem.org/mediawiki/2021/0/0a/T--Stockholm--img--Results_2.png)

Figure 2: Absorbance spectrums, shown with different coloured curves of A: Blue PCDA at 1 mM (Black), 0.1 mM (Red) and 0.01 mm (Blue); B: Red PCDA at 1 mM (Blue), 0.01 mM (Purple), 0.01 mM (Red) and 0.001 mM (Black).

# PCDA - Protein A Aptamer Conjugation

The next step was to conjugate our PCDA with the aptamer and then test it using protein A. After incubation of the protein A with the PCDA-aptamer conjugate we did not detect any colour change so we needed to test if the protein A aptamer is actually binding protein A. We investigated the protein A aptamer binding affinity with three different experiments: first, using protein A-coated beads, then with chromatography, and finally by performing a binding assay.

## Protein A coated beads

Early on, after noticing that the protein A aptamer might not bind to its target, we used protein A coated beads for testing the binding qualitatively. After following the protocol provided with the beads, we ran the fraction from elution in an agarose gel, which is shown in Figure 3. After elution, the one sample of the fraction was desalted, in order to have a less harsh environment for the aptamers. However, as can be seen on the gel, the wells appear empty, which means that the aptamers most likely did not bind to protein A. It is nonetheless possible for the binding to be too weak, resulting in very little aptamers being eluted and thus not showing up on the gel.

![Protein A products were run in 2% agarose gel shared with samples from a PCR troubleshooting. Wells 1-5 have PCR products with different annealing temperatures. Wells 6 and 7 show the product after elution of beads, before and after desalting.](https://static.igem.org/mediawiki/2021/7/7f/T--Stockholm--img--proofofconcept2.png)

Figure 3: Protein A products were run in 2% agarose gel shared with samples from a PCR troubleshooting. Wells 1-5 have PCR products with different annealing temperatures. Wells 6 and 7 show the product after elution of beads, before and after desalting.

## Chromatography using a protein A coated column

Since the test using the magnetic beads could have a too low concentration in the elution for it to be detectable on a gel, we set up a chromatography experiment with a protein using the HiTrap Mabselect SuRe protein A affinity column. The resulting chromatogram is shown in Figure 4. Fractions from the sample application run-through and from the elution were analysed using an agarose gel, which is also shown in Figure 4. The wells containing the sample application run-through had a lot of contents, while the fractions from the elution were empty. This again shows that the protein A aptamer was not binding to protein A.

![Chromatography results performed using the protein A affinity column. Top part shows the chromatogram, where the y-axis displays UV-absorbance and conductivity and the x-axis displays volume in mL. Lower part shows chromatography fractions, run in 2% agarose gel in TBE electrophoresis. Lanes 1-4 are from fractions during sample application and lanes 15-24 indicates fractions from elution.](https://static.igem.org/mediawiki/2021/a/a7/T--Stockholm--img--results4.png)

Figure 4: Chromatography results performed using the protein A affinity column. Top part shows the chromatogram, where the y-axis displays UV-absorbance and conductivity and the x-axis displays volume in mL. Lower part shows chromatography fractions, run in 2% agarose gel in TBE electrophoresis. Lanes 1-4 are from fractions during sample application and lanes 15-24 indicates fractions from elution.

## Binding assay using a fluorescent protein A aptamer

The previous results suggest that our protein A aptamer is not binding protein A as expected. In order to finally figure out the aptamer affinity issues we designed the following experiment using protein A aptamer labelled with the fluorophore fluorescein (5' 6-FAM aptamer).

We incubated 100pmol, 200pmol and 300pmol of fluorescent aptamer in triplicates in a 96-well plate coated with protein A for 45 minutes. Then, washed the wells with PBS to remove unbound aptamer molecules and measured the fluorescence intensity in a plate reader.

To infer the pmol of aptamer that had successfully bound to protein A, we created the following standard curve using known pmol quantities of 5' 6-FAM aptamer.

![5' 6-FAM aptamer standard curve showing the relationship between RFUs (Relative fluorescence units) and aptamer amount (pmol). A linear regression model was used to determine the line, regression equation and R^2 coefficient.](https://static.igem.org/mediawiki/2021/c/cf/T--Stockholm--img--results_5.JPG)

Figure 5: 5' 6-FAM aptamer standard curve showing the relationship between RFUs (Relative fluorescence units) and aptamer amount (pmol). A linear regression model was used to determine the line, regression equation and R^2 coefficient.

Using the regression equation, we inferred the pmol of aptamer that bound to the protein A.

The total percentage of bound aptamer was then calculated for each sample by dividing the pmol of aptamer bound by total pmol aptamer incubated.

*Table 1. Amount of aptamer bound (pmol) and percentage of bound aptamer determined by the binding affinity assay.*

| Amount [pmol] | RFU | Inferred amount bound aptamer [pmol] | Percentage bound aptamer |
| --- | --- | --- | --- |
| 100 | 62 | 0.527 | 0.527 |
| 62 | 0.527 | 0.527 |
| 51 | 0.434 | 0.434 |
| 200 | 92 | 0.782 | 0.391 |
| 171 | 1.45 | 0.727 |
| 93 | 0.791 | 0.395 |
| 300 | 137 | 1.16 | 0.388 |
| 114 | 0.969 | 0.323 |
| 63 | 0.536 | 0.179 |

Based on these results, we concluded that the protein A aptamer we chose for our proof of concept does not have sufficient binding affinity to its target.

This conclusion is also supported by protein A - aptamer docking [model](https://2021.igem.org/Team%3AStockholm/Model), where no evidence of obvious strong interactions was found.

# SELEX

Another crucial part of our project was to create the two aptamers, one for lipoteichoic acid and one for *C. acnes*, using SELEX. We faced a lot of problems during this process. The main obstacle was the optimization of the PCR. From wrong concentrations of PCR reagents to contaminations and wrong primers we spent a lot of time troubleshooting in order to make the PCR work since it is the most important part of the SELEX process. One example is shown in Figure 6, which displays an agarose gel with PCR troubleshooting products, where two different polymerases, temperatures and primer concentrations were tried. Everything is written in detail on the [Engineering page](https://2021.igem.org/Team%3AStockholm/Engineering) and in our troubleshooting guide found on the [Contributions page](https://2021.igem.org/Team%3AStockholm/Contributions).

![Troubleshooting of the contaminated PCR products in a 2% agarose gel. Two different polymerases: Takara Titanium Taq DNA Polymerase (lanes 1,2,5,7,8,11) and DreamTaq DNA Polymerase (lanes 3,4,6,9,10,12), two different primer concentrations: 0.5 mM (lane 1,3,7,9) and 1mM (lanes 2,4,8,10); and two annealing temperatures: 55Â°C (lanes 1-6) and 60Â°C (lanes 7-12) were tested. Additionally, a non template control (NTC) for each polymerase and annealing temperature was run.](https://static.igem.org/mediawiki/2021/e/e1/T--Stockholm--img--Results_6.png)

Figure 6: Troubleshooting of the contaminated PCR products in a 2% agarose gel. Two different polymerases: Takara Titanium Taq DNA Polymerase (lanes 1,2,5,7,8,11) and DreamTaq DNA Polymerase (lanes 3,4,6,9,10,12), two different primer concentrations: 0.5 mM (lane 1,3,7,9) and 1mM (lanes 2,4,8,10); and two annealing temperatures: 55Â°C (lanes 1-6) and 60Â°C (lanes 7-12) were tested. Additionally, a non template control (NTC) for each polymerase and annealing temperature was run.

After all the hard work over the summer, we were delighted to get positive SELEX results during our last few days in the lab. We performed a first successful library amplification and determined the right amount of PCR amplification cycles needed for the second amplification based on Figure 7. Then the remainder of both libraries was amplified with 45 cycles of PCR in total, since the pre-amplification accounted for 15 cycles and the optimal number of cycles after pre-amplification was chosen to be 30.

![2% agarose gel showing library amplification PCR products, with pre-amplified library (lane 1), LTA SELEX (lanes 2-7), cell SELEX (lane 8-13) and non template control, NTC (lane 15). Samples indicate increasing PCR amplification cycles for LTA and Cell SELEX, from left to right, respectively. Highlighted bands display selected conditions for library amplification.](https://static.igem.org/mediawiki/2021/f/f4/T--Stockholm--img--Results_7.png)

Figure 7: 2% agarose gel showing library amplification PCR products, with pre-amplified library (lane 1), LTA SELEX (lanes 2-7), cell SELEX (lane 8-13) and non template control, NTC (lane 15). Samples indicate increasing PCR amplification cycles for LTA and Cell SELEX, from left to right, respectively. Highlighted bands display selected conditions for library amplification.

The PCR products from the second amplification can be seen in Figure 8. Then, the PCR products were pooled for each SELEX type, after which the sense and antisense strands were separated. The sense strands were eluted and desalted, which yielded very little product as can be seen in Figure 9, and we are unsure whether it is the right product that was obtained.

![2% agarose gel of the amplification products for (A): LTA SELEX, and (B): Cell SELEX; after 45 cycles. For each gel, lanes 1-10 display separate PCR products. Lane 11 has non-amplified libraries as control.](https://static.igem.org/mediawiki/2021/1/1a/T--Stockholm--img--Results_8png.png)

Figure 8: 2% agarose gel of the amplification products for (A): LTA SELEX, and (B): Cell SELEX; after 45 cycles. For each gel, lanes 1-10 display separate PCR products. Lane 11 has non-amplified libraries as control.

![2% agarose gel of fractions from desalting after the elution of the sense DNA strands. Run-through of the sample in lane 1, sample from the eluted fraction in lane 2, and sample from the left-over volume, after elution in lane 3.](https://static.igem.org/mediawiki/2021/e/eb/T--Stockholm--img--Results_9.png)

Figure 9: 2% agarose gel of fractions from desalting after the elution of the sense DNA strands. Run-through of the sample in lane 1, sample from the eluted fraction in lane 2, and sample from the left-over volume, after elution in lane 3.

As SELEX is a repetitive process, we were able to prove that each part of our protocol works. Given more time, we would have worked towards achieving multiple successful rounds of SELEX.

# BioBrick Engineering

We set out to create a fusion protein A labelled with the yellow chromoprotein amilGFP as a reporter. In order to do this, we selected pre-existing parts from the registry: the amilGFP BioBrick [(BBa\_K1073024)](https://parts.igem.org/Part%3ABBa_K1073024), as well as staphylococcal protein A BioBrick [(BBa\_K103003)](http://parts.igem.org/Part%3ABBa_K103003).

Following our cloning strategy, as seen on our [Experiments page](https://2021.igem.org/Team%3AStockholm/Experiments), we first successfully linearised the plasmid containing the amilGFP BioBrick by digesting with the SpeI and PstI restriction enzymes.

![Digested and undigested product in 1% agarose gel. Lane C shows the undigested plasmid. Two different bands can be observed, corresponding to two different conformations of the DNA plasmid: open coiled (OC) and Covalently Closed Circle (CCC). Lane D shows the digested plasmid product.](https://static.igem.org/mediawiki/2021/8/89/T--Stockholm--img--amilGFP-vector-digestion.png)

Figure 10: Digested and undigested product in 1% agarose gel. Lane C shows the undigested plasmid. Two different bands can be observed, corresponding to two different conformations of the DNA plasmid: open coiled (OC) and Covalently Closed Circle (CCC). Lane D shows the digested plasmid product.

Once we confirmed the plasmid had been linearised, we isolated it by performing gel extraction.

![1% agarose gel with linearised amilGFP plasmid, before (A) and after (B) the gel extraction. (A) AmilGFP plasmid was digested with SpeI and PstI.  (B) Using the UV table, the fragment of interest was isolated, leaving out the smear.](https://static.igem.org/mediawiki/2021/a/ac/T--Stockholm--img--amilGFP-gel-extraction.png)

Figure 11: 1% agarose gel with linearised amilGFP plasmid, before (A) and after (B) the gel extraction. (A) AmilGFP plasmid was digested with SpeI and PstI. (B) Using the UV table, the fragment of interest was isolated, leaving out the smear.

Then, we moved on to amplify the protein A BioBrick by PCR, using VF2 and VR standard primers. The PCR product had the expected size: ~ 500 bp.

![PCR amplification products on a 2% agarose gel. Lanes 1 to 3 contain the same PCR product. Lane 4 is PCR negative control (non-template control).](https://static.igem.org/mediawiki/2021/8/81/T--Stockholm--img--PCR-amplification-protein-A.png)

Figure 12: PCR amplification products on a 2% agarose gel. Lanes 1 to 3 contain the same PCR product. Lane 4 is PCR negative control (non-template control).

After performing a PCR clean up, the amplified protein A BioBrick was digested using XbaI and PstI restriction enzymes. The expected fragments were:

* Protein A BioBrick -> ~ 235 bp.
* VF2 - XbaI -> 133 bp
* PstI - VR -> 154 bp

We could not use PCR clean up to get rid of the digestion leftovers, since it removes oligos up until 40 bp. Therefore, the protein A BioBrick (digestion product) was isolated by gel extraction.

![1% agarose gel with digested protein A BioBrick, before (A) and after (B) the gel extraction. (A) The electrophoresis was run until the protein A BioBrick (~235 bp) was separated from other digestion products (~150 bp).  (B) Using the UV table, the fragment of interest was isolated successfully.](https://static.igem.org/mediawiki/2021/e/e6/T--Stockholm--img--Protein-A-gel-digestion.png)

Figure 13: 1% agarose gel with digested protein A BioBrick, before (A) and after (B) the gel extraction. (A) The electrophoresis was run until the protein A BioBrick (~235 bp) was separated from other digestion products (~150 bp). (B) Using the UV table, the fragment of interest was isolated successfully.

The next step was to ligate the protein A BioBrick and the vector containing amilGFP plasmid to create the fusion protein. Compatibility between XbaI - SpeI and PstI - PstI sticky ends allowed for directional cloning of the protein A BioBrick into the amilGFP plasmid. Ligation was performed by incubation of both parts with T4 ligase. The ligation product was then used to transform TOP10 E. coli competent cells.

Several yellow colonies, indicating amilGFP expression, were selected to set O/N cultures, from which glycerol stocks and minipreps were prepared. Sanger sequencing confirmed our plasmid had the desired sequence.

![Yellow E. coli colonies due to amilGFP expression on LB-agar plate.](https://static.igem.org/mediawiki/2021/5/5b/T--Stockholm--img--Yellow-colonies.png)

Figure 14: Yellow E. coli colonies due to amilGFP expression on LB-agar plate.

Ultimately, in order to create a functional fusion protein, we had to mutate two sequential stop codons at the end of the amilGFP coding sequence by using Quikchange site-directed mutagenesis. During the PCR step, TAA STOP codons were substituted by TCA Serine codons thanks to the primers, which had these modifications incorporated (Figure 15). Three different annealing temperatures were used in the PCR: 55 degrees Celsius, 58 degrees Celsius and 60 degrees Celsius.

Then, DpnI endonuclease digested the non-mutated parental DNA strands, and the final product was transformed into TOP10 E. coli competent cells.

![Quikchange Forward and Reverse primers are complementary to the target sequence at the end of  the amilGFP CDS (BBa_K1073024), but include the desired point mutations (mismatches) that were incorporated by PCR. Retrieved from SnapGene.](https://static.igem.org/mediawiki/2021/8/88/T--Stockholm--img--Quikchange-primers-wiki.png)

Figure 15: Quikchange Forward and Reverse primers are complementary to the target sequence at the end of the amilGFP CDS (BBa\_K1073024), but include the desired point mutations (mismatches) that were incorporated by PCR. Retrieved from SnapGene.

Plates transformed with products from 55 degrees Celsius and 58 degrees Celsius PCR annealing temperatures had colonies. Expression of functional amilGFP could be confirmed with the presence of bright yellow colonies in LB-agar plates and yellow cell pellets in liquid culture.

Finally, the sequence of the newly engineered amilGFP-proteinA BioBrick [(BBa\_K4071000)](http://parts.igem.org/Part%3ABBa_K4071000) was verified by Sanger sequencing and the part was validated.

![Vector map of pSB1C3 containing the final version of the amilGFP-protein A BioBrick (BBa_K4071000).](https://static.igem.org/mediawiki/2021/d/d7/T--Stockholm--img--amilGFP-protA-BioBrick-Map.png)

Figure 16: Vector map of pSB1C3 containing the final version of the amilGFP-protein A BioBrick (BBa\_K4071000).

[![](https://static.igem.org/mediawiki/2021/b/bb/T--Stockholm--img--ki-whiteback-modified.png)](https://ki.se)[![](https://static.igem.org/mediawiki/2021/e/e2/T--Stockholm--img--kthwhite-modified.png)](https://kth.se)[![](https://static.igem.org/mediawiki/2021/d/da/T--Stockholm--img--stockholmuni-modified.png)](https://su.se)

Sample template built using the iGEM Wiki Starter Pack by BITS Goa.

Code released under the MIT license.

Based on [Bootstrap](https://getbootstrap.com) and themes [Flatly](https://bootswatch.com/flatly/) and [Darkly](https://bootswatch.com/darkly/) from [Bootswatch](https://bootswatch.com/).

Some content from the [iGEM Example Wiki](https://2020.igem.org/Team%3AExample). Images from [Unsplash](https://unsplash.com). Web fonts from [Google](https://fonts.google.com).