iGEM 2021 — Team Aboa

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# Team:Aboa/Notebook

![](https://static.igem.org/mediawiki/2021/6/67/T--Aboa--logo.svg)

[Home](https://2021.igem.org/Team%3AAboa)

About Us

[Team Members](https://2021.igem.org/Team%3AAboa/Team)
[Collaborations](https://2021.igem.org/Team%3AAboa/Collaborations)
[Partnership](https://2021.igem.org/Team%3AAboa/Partnership)
[Attributions](https://2021.igem.org/Team%3AAboa/Attributions)

Project

[Overview](https://2021.igem.org/Team%3AAboa/Overview)
[Pharmaceutical Waste](https://2021.igem.org/Team%3AAboa/Pharmaceutical_Waste)
[Description](https://2021.igem.org/Team%3AAboa/Description)
[Contribution](https://2021.igem.org/Team%3AAboa/Contribution)
[Engineering Success](https://2021.igem.org/Team%3AAboa/Engineering)
[Design](https://2021.igem.org/Team%3AAboa/Design)
[Proposed Implementation](https://2021.igem.org/Team%3AAboa/Implementation)

Lab

[Methods & Experiments](https://2021.igem.org/Team%3AAboa/Experiments)
[Results](https://2021.igem.org/Team%3AAboa/Results)
[Safety](https://2021.igem.org/Team%3AAboa/Safety)
[Notebook](https://2021.igem.org/Team%3AAboa/Notebook)

Human Practices

[Human Practices](https://2021.igem.org/Team%3AAboa/Human_Practices)
[Integrated Human Practices](https://2021.igem.org/Team%3AAboa/Human_Practices#Integrated)
[Education](https://2021.igem.org/Team%3AAboa/Education)
[Communication](https://2021.igem.org/Team%3AAboa/Communication)

Inclusivity

[In General](https://2021.igem.org/Team%3AAboa/Inclusivity)
[Plain Language](https://2021.igem.org/Team%3AAboa/Plain_Language)
[Supporting SL](https://2021.igem.org/Team%3AAboa/SupportingSL)

Achievements

[Judging Form](https://igem.org/2021_Judging_Form?id=3872)
[Medal Criteria](https://2021.igem.org/Team%3AAboa/Medal_criteria)

☰

Team Aboa 2021

# Notebooks

We have carefully documented every step we have taken throughout this iGEM year.
Below you can find both our general notebook including all meetings and Human Practices activities and our laboratory notebook with more detailed descriptions about the days in the laboratory.
We have added the names of attending team members to every activity, but we want to emphasize that the absence in some meetings doesn’t mean that the members wouldn’t have done anything.
Lots of the work regarding our project has been done independently - which is of course impossible to document in these notebooks.

If you want to get more information about the meeting topics in meetings with our partnership team UChicago, check this [Partnership page](https://2021.igem.org/Team%3AAboa/Partnership).

## Our general notebook

Also downloadable from [here](https://static.igem.org/mediawiki/2021/2/23/T--Aboa--notebook.pdf).

## Our laboratory notebook

Also downloadable from [here](https://static.igem.org/mediawiki/2021/5/57/T--Aboa--lab_notebook.pdf).

[## Home](https://2021.igem.org/Team%3AAboa)

## About us

[Team Members](https://2021.igem.org/Team%3AAboa/Team)
[Collaborations](https://2021.igem.org/Team%3AAboa/Collaborations)
[Partnership](https://2021.igem.org/Team%3AAboa/Partnership)
[Attributions](https://2021.igem.org/Team%3AAboa/Attributions)

## Project

[Overview](https://2021.igem.org/Team%3AAboa/Overview)
[Pharmaceutical Waste](https://2021.igem.org/Team%3AAboa/Pharmaceutical_Waste)
[Description](https://2021.igem.org/Team%3AAboa/Description)
[Contribution](https://2021.igem.org/Team%3AAboa/Contribution)
[Engineering Success](https://2021.igem.org/Team%3AAboa/Engineering)
[Design](https://2021.igem.org/Team%3AAboa/Design)
[Proposed Implementation](https://2021.igem.org/Team%3AAboa/Implementation)

## Lab

[Methods & Experiments](https://2021.igem.org/Team%3AAboa/Experiments)
[Results](https://2021.igem.org/Team%3AAboa/Results)
[Safety](https://2021.igem.org/Team%3AAboa/Safety)
[Notebook](https://2021.igem.org/Team%3AAboa/Notebook)

## Human Practices

[Human Practices](https://2021.igem.org/Team%3AAboa/Human_Practices)
[Integrated Human Practices](https://2021.igem.org/Team%3AAboa/Human_Practices#Integrated)
[Education](https://2021.igem.org/Team%3AAboa/Education)
[Communication](https://2021.igem.org/Team%3AAboa/Communication)

## Inclusivity

[Inclusivity](https://2021.igem.org/Team%3AAboa/Inclusivity)
[Plain Language](https://2021.igem.org/Team%3AAboa/Plain_Language)
[Supporting Sign Language](https://2021.igem.org/Team%3AAboa/SupportingSL)

## Achievements

[Judging Form](https://2021.igem.org/Team%3AAboa/)
[Medal Criteria](https://2021.igem.org/Team%3AAboa/Medal_criteria)

## Sponsors

![](https://static.igem.org/mediawiki/2021/0/0d/T--Aboa--UTU.png)
![](https://static.igem.org/mediawiki/2021/3/38/T--Aboa--IDT.png)
![](https://static.igem.org/mediawiki/2021/2/2a/T--Aboa--Benchling.png)

[![](https://static.igem.org/mediawiki/2021/1/1c/T--Aboa--instagram_icon.svg)](https://instagram.com/igem.aboa)
![](https://static.igem.org/mediawiki/2021/b/b3/T--Aboa--email_icon.svg)
[![](https://static.igem.org/mediawiki/2021/8/8b/T--Aboa--website_icon.svg)](https://aboaturku.fi)

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

# Team:Aboa/Engineering

![](https://static.igem.org/mediawiki/2021/6/67/T--Aboa--logo.svg)

[Home](https://2021.igem.org/Team%3AAboa)

About Us

[Team Members](https://2021.igem.org/Team%3AAboa/Team)
[Collaborations](https://2021.igem.org/Team%3AAboa/Collaborations)
[Partnership](https://2021.igem.org/Team%3AAboa/Partnership)
[Attributions](https://2021.igem.org/Team%3AAboa/Attributions)

Project

[Overview](https://2021.igem.org/Team%3AAboa/Overview)
[Pharmaceutical Waste](https://2021.igem.org/Team%3AAboa/Pharmaceutical_Waste)
[Description](https://2021.igem.org/Team%3AAboa/Description)
[Contribution](https://2021.igem.org/Team%3AAboa/Contribution)
[Engineering Success](https://2021.igem.org/Team%3AAboa/Engineering)
[Design](https://2021.igem.org/Team%3AAboa/Design)
[Proposed Implementation](https://2021.igem.org/Team%3AAboa/Implementation)

Lab

[Methods & Experiments](https://2021.igem.org/Team%3AAboa/Experiments)
[Results](https://2021.igem.org/Team%3AAboa/Results)
[Safety](https://2021.igem.org/Team%3AAboa/Safety)
[Notebook](https://2021.igem.org/Team%3AAboa/Notebook)

Human Practices

[Human Practices](https://2021.igem.org/Team%3AAboa/Human_Practices)
[Integrated Human Practices](https://2021.igem.org/Team%3AAboa/Human_Practices#Integrated)
[Education](https://2021.igem.org/Team%3AAboa/Education)
[Communication](https://2021.igem.org/Team%3AAboa/Communication)

Inclusivity

[In General](https://2021.igem.org/Team%3AAboa/Inclusivity)
[Plain Language](https://2021.igem.org/Team%3AAboa/Plain_Language)
[Supporting SL](https://2021.igem.org/Team%3AAboa/SupportingSL)

Achievements

[Judging Form](https://igem.org/2021_Judging_Form?id=3872)
[Medal Criteria](https://2021.igem.org/Team%3AAboa/Medal_criteria)

☰

Team Aboa 2021

# Engineering Success

![Four arrows forming a circle. Arrows have texts: design, build, test and learn.](https://static.igem.org/mediawiki/2021/f/f2/T--Aboa--Engineering-Cycle2.png)

**Figure 1. Engineering cycle.** In our project, the purpose was to fulfill all the phases in a successful engineering cycle; design, build, test and learn.

## Design

The project objective was to contribute to the development of a microbial wastewater treatment system for the detoxification of pharmaceutical waste, especially diclofenac (2-[2-(2,6-dichloroanilino)phenyl]acetic acid).
Diclofenac is a non-steroidal anti-inflammatory drug which causes major environmental stress in our local area, the Baltic Sea.
Additionally, the current removal efficiency of diclofenac is only 27% at our local wastewater treatment plant.
Therefore, a solution is urgently needed to tackle this problem.

The plan for this was to utilize laccases, specific enzymes that are capable of catalyzing the conversion of diclofenac into less harmful derivatives.
Only bacterial laccase options were considered to facilitate production of active enzymes in prokaryotic organisms. The laccases chosen for the work were CotA from *B. subtilis*, CueO from *E. coli* and Yak from *Y. enterocolitica subsp. palearctica* strain 7 (read more about these laccases from our [Design page](https://2021.igem.org/Team%3AAboa/Design)).
These were selected as they have shown high activity in near neutral pH conditions, suitability for diclofenac detoxification, or do not yet have wet lab data from previous iGEM teams by whose projects we were inspired (Team Darmstadt 2020: [https://2020.igem.org/Team:TU\_Darmstadt](https://2020.igem.org/Team%3ATU_Darmstadt), Team Kaiserslautern 2020: [https://2020.igem.org/Team:TU\_Kaiserslautern](https://2020.igem.org/Team%3ATU_Kaiserslautern) and Team Stuttgart 2020: [https://2020.igem.org/Team:Stuttgart](https://2020.igem.org/Team%3AStuttgart)).

The laccases were to be overexpressed in the *E. coli* BL21(DE3) strain utilizing the pET system, which is one of the most powerful recombinant protein production systems (Mierendorf et al., 1998).
The expression vector of choice was pET36b(+) as it allowed for the addition of a C-terminal His-tag to the laccases. A C-terminal His-tag was selected as it enables a simple protein purification technique while minimizing enzyme activity loss (Li et al., 2017).
The produced laccases would be extracted using Ni2+ affinity chromatography after cell lysis through sonification.

Next, the enzyme efficiencies of the three heterologous laccases would be compared at conditions simulating our local wastewater treatment plant through enzymatic assays.
The enzyme activities would first be determined by applying the ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay, a spectrophotometric technique based on the oxidation of ABTS by laccase (More et al., 2011).
Our plans also included measurements with LC-MS to obtain diclofenac-specific results about the produced laccases.
The most suitable laccase for us would be the laccase that is found to be most active especially in regards to diclofenac in wastewater conditions.

This laccase would then be overexpressed in the model cyanobacterium *Synechocystis* sp. PCC 6803, which is the chassis of our proposed implementation.
The expression vector for this would be the shuttle vector pDF-lac2, modified from the pDF-lac plasmid, and the expression construct transformed by natural transformation (Thiel et al., 2018).
Cyanobacteria were chosen for the proposed implementation as they are photoautotrophic, thus providing a sustainable alternative for heterotrophic production platforms.

Please find more information on the entire design process and details about our chosen elements on our [design page](https://2021.igem.org/Team%3AAboa/Design).

## Build

In the build phase of the project we assembled our expression constructs and generated our engineered *E. coli* BL21(DE3) strains for laccase comparison. The three laccase coding genes were ordered as synthetic DNA fragments from IDT.
The fragments were designed to be flanked by the NdeI and XhoI restriction sites. They arrived in pUCIDT-Kan plasmids from which they were subcloned into the pET36b(+) expression vector received from a biochemist at our university.
This resulted in three final expression constructs, pET36b-*cotA*, pET36b-*cueO* and pET36b-*yak*. These subcloning steps were performed in the *E. coli* DH5ɑ strain. Once the final expression constructs were assembled, they were transformed into the *E. coli* BL21(DE3) strain.
The generated strains were verified through restriction digestion with NdeI and XhoI. As the ordered genes were obtained as fully verified, sequenced fragments, there was no need to confirm the final constructs by DNA sequencing, as PCR amplification was not used in expression plasmid construction.

Generation of the expression constructs for our proposed implementation chassis, *Synechocystis* sp. PCC 6803 was done simultaneously with the pET36b constructs. For these, another set of the three laccase genes were ordered as synthetic fragments from IDT.
The difference between these and the genes that were used for the pET36b based expression constructs was only the initial restriction site, NsiI instead of NdeI, that flanked the gene.
The laccase genes received in the pUCIDT-Kan plasmids were first subcloned directly downstream of the S3 RBS sequence in the pNiv plasmid (NsiI-XhoI).
Another set of restriction enzymes, BspHI for pUCIDT-*cotA* and pUCIDT-*cueO*, and PvuII for pUCIDT-*yak*, were used to avoid digestion products similar in size.
A combined fragment consisting of the RBS and laccase gene was then digested from the pNiv carrier plasmid. These were to be subcloned into the pDF-lac2 expression plasmid (SpeI-SalI) to generate the final expression constructs.
Their assembly was, however, never completed as work with the generated *E. coli* BL21(DE3) strains for evaluation of laccase function was prioritized.

Please find more information on the build phase including gel pictures on our [results page](https://2021.igem.org/Team%3AAboa/Results).

## Test

Once the three *E. coli* BL21(DE3) laccase overexpression strains were generated, and the proteins of the correct size were obtained, the aim was first to see if any enzyme activity could be observed.
This was first attempted by using ABTS as substrate. However, during the initial analytical trials, we observed that the ABTS stock we had obtained was no longer useful, possibly due to oxidation and subsequent decomposition during earlier storage.
To continue the analysis we decided to use an alternative substrate syringaldazine which has been previously used for laccase characterization.

The kinetic enzyme activity assays unambiguously demonstrated the laccase CotA from *Bacillus subtilis* was catalytically active, and able to oxidize syringaldazine.
This demonstrated that one of the three enzyme targets selected for the project was successfully overexpressed in a heterologous host, purified, and confirmed functional, therefore completing one of the primary goals of the iGEM project.

Please find more information on the build phase on our [methods page](https://2021.igem.org/Team%3AAboa/Experiments) and [results page](https://2021.igem.org/Team%3AAboa/Results).

## Learn

The project allowed us to walk through the entire step-wise process from construct design, genetic assembly of multiple alternative expression plasmids, generation of corresponding *E. coli* overproduction strains, protein overexpression and purification, and finally, the characterization of the enzyme activities.
As part of the process, we learned that things in science do not always work as anticipated, and that many side-tracks can be avoided by careful planning, and preparation for alternative execution plans at every stage of the project.

As an example of the practical lessons of the work, one of the three enzyme targets in this work (Yak from *Yersinia enterocolitica*) was not expressed in soluble form, a second enzyme (CueO from *E. coli*) was not catalytically active although successfully purified, while the third enzyme (CotA from *Bacillus subtilis*) was expressed, purified and verified to be catalytically active *in vitro*.

Please find more information on the learn phase on our [results page](https://2021.igem.org/Team%3AAboa/Results).

## Design in the following engineering cycle

From the beginning, the work was designed to look beyond the expected achievements in the project, and plan for the next steps. In practise, these included specific plans for:

1. comprehensive kinetic characterization and optimization of the laccase reaction conditions,
2. structure-based site-directed mutagenesis to improve the catalytic performance of the successful target enzyme CotA,
3. completion of the corresponding cyanobacterial expression constructs and the generation of the *Synechocystis* sp. PCC 6803 expression strains,
4. introduction of signal peptides to allow the excretion of the expressed CotA to the extracellular medium,
5. further improvement of the cyanobacterial expression system by translational optimization, and
6. incorporation of a molecular kill-switch that would be required for the use or large-scale confined testing of the system outside the laboratory.

Please find more information on the things we would like to perform in the following engineering cycle on our [design page](https://2021.igem.org/Team%3AAboa/Design) and our [proposed implementation page](https://2021.igem.org/Team%3AAboa/Implementation).

## References

* Li, L., Xie, T., Liu, Z., Feng, H., & Wang, G. (2017). Activity enhancement of CotA laccase by hydrophilic engineering, histidine tag optimization and static culture. Protein Engineering, Design and Selection, 31(1), 1–5, <https://doi.org/10.1093/protein/gzx064>.
* More, S. S., P S, R., K, P., M, S., Malini, S., & S M, V. (2011). Isolation, Purification, and Characterization of Fungal Laccase from Pleurotus sp. Enzyme research, 2011, 248735, <https://doi.org/10.4061/2011/248735>.

[## Home](https://2021.igem.org/Team%3AAboa)

## About us

[Team Members](https://2021.igem.org/Team%3AAboa/Team)
[Collaborations](https://2021.igem.org/Team%3AAboa/Collaborations)
[Partnership](https://2021.igem.org/Team%3AAboa/Partnership)
[Attributions](https://2021.igem.org/Team%3AAboa/Attributions)

## Project

[Overview](https://2021.igem.org/Team%3AAboa/Overview)
[Pharmaceutical Waste](https://2021.igem.org/Team%3AAboa/Pharmaceutical_Waste)
[Description](https://2021.igem.org/Team%3AAboa/Description)
[Contribution](https://2021.igem.org/Team%3AAboa/Contribution)
[Engineering Success](https://2021.igem.org/Team%3AAboa/Engineering)
[Design](https://2021.igem.org/Team%3AAboa/Design)
[Proposed Implementation](https://2021.igem.org/Team%3AAboa/Implementation)

## Lab

[Methods & Experiments](https://2021.igem.org/Team%3AAboa/Experiments)
[Results](https://2021.igem.org/Team%3AAboa/Results)
[Safety](https://2021.igem.org/Team%3AAboa/Safety)
[Notebook](https://2021.igem.org/Team%3AAboa/Notebook)

## Human Practices

[Human Practices](https://2021.igem.org/Team%3AAboa/Human_Practices)
[Integrated Human Practices](https://2021.igem.org/Team%3AAboa/Human_Practices#Integrated)
[Education](https://2021.igem.org/Team%3AAboa/Education)
[Communication](https://2021.igem.org/Team%3AAboa/Communication)

## Inclusivity

[Inclusivity](https://2021.igem.org/Team%3AAboa/Inclusivity)
[Plain Language](https://2021.igem.org/Team%3AAboa/Plain_Language)
[Supporting Sign Language](https://2021.igem.org/Team%3AAboa/SupportingSL)

## Achievements

[Judging Form](https://2021.igem.org/Team%3AAboa/)
[Medal Criteria](https://2021.igem.org/Team%3AAboa/Medal_criteria)

## Sponsors

![](https://static.igem.org/mediawiki/2021/0/0d/T--Aboa--UTU.png)
![](https://static.igem.org/mediawiki/2021/3/38/T--Aboa--IDT.png)
![](https://static.igem.org/mediawiki/2021/2/2a/T--Aboa--Benchling.png)

[![](https://static.igem.org/mediawiki/2021/1/1c/T--Aboa--instagram_icon.svg)](https://instagram.com/igem.aboa)
![](https://static.igem.org/mediawiki/2021/b/b3/T--Aboa--email_icon.svg)
[![](https://static.igem.org/mediawiki/2021/8/8b/T--Aboa--website_icon.svg)](https://aboaturku.fi)

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

# Team:Aboa/Experiments

![](https://static.igem.org/mediawiki/2021/6/67/T--Aboa--logo.svg)

[Home](https://2021.igem.org/Team%3AAboa)

About Us

[Team Members](https://2021.igem.org/Team%3AAboa/Team)
[Collaborations](https://2021.igem.org/Team%3AAboa/Collaborations)
[Partnership](https://2021.igem.org/Team%3AAboa/Partnership)
[Attributions](https://2021.igem.org/Team%3AAboa/Attributions)

Project

[Overview](https://2021.igem.org/Team%3AAboa/Overview)
[Pharmaceutical Waste](https://2021.igem.org/Team%3AAboa/Pharmaceutical_Waste)
[Description](https://2021.igem.org/Team%3AAboa/Description)
[Contribution](https://2021.igem.org/Team%3AAboa/Contribution)
[Engineering Success](https://2021.igem.org/Team%3AAboa/Engineering)
[Design](https://2021.igem.org/Team%3AAboa/Design)
[Proposed Implementation](https://2021.igem.org/Team%3AAboa/Implementation)

Lab

[Methods & Experiments](https://2021.igem.org/Team%3AAboa/Experiments)
[Results](https://2021.igem.org/Team%3AAboa/Results)
[Safety](https://2021.igem.org/Team%3AAboa/Safety)
[Notebook](https://2021.igem.org/Team%3AAboa/Notebook)

Human Practices

[Human Practices](https://2021.igem.org/Team%3AAboa/Human_Practices)
[Integrated Human Practices](https://2021.igem.org/Team%3AAboa/Human_Practices#Integrated)
[Education](https://2021.igem.org/Team%3AAboa/Education)
[Communication](https://2021.igem.org/Team%3AAboa/Communication)

Inclusivity

[In General](https://2021.igem.org/Team%3AAboa/Inclusivity)
[Plain Language](https://2021.igem.org/Team%3AAboa/Plain_Language)
[Supporting SL](https://2021.igem.org/Team%3AAboa/SupportingSL)

Achievements

[Judging Form](https://igem.org/2021_Judging_Form?id=3872)
[Medal Criteria](https://2021.igem.org/Team%3AAboa/Medal_criteria)

☰

Team Aboa 2021

# Methods & Experiments

The aim of the project was to contribute to the development of a microbial wastewater treatment system for the detoxification of pharmaceutical waste, especially targeting the non-steroidal anti-inflammatory drug diclofenac (2-[2-(2,6-dichloroanilino)phenyl]acetic acid).
The approach was to overexpress and extract three heterologous laccase enzymes in engineered *E. coli*. Laccases are enzymes which are capable of catalyzing the conversion of diclofenac into less harmful derivatives.
The laccases compared in this study were CotA from *B. subtilis*, CueO from *E. coli* and Yak from *Y. enterocolitica subsp. palearctica* strain 7.

First, we will describe our generally done experiments and then our protein production and purification methods.
Finally, we will go through the measurements we performed to measure laccase activities. To get to know more about our project, read our [project description](https://2021.igem.org/Team%3AAboa/Description).
And if you want to know the results we obtained with these methods, please check our [results page](https://2021.igem.org/Team%3AAboa/Results)!

## Generally done experiments

Here we describe the general experiments we performed, such as making competent cells, transformation of cells and preparing of gel electrophoresis.

## Preparation of plates:

LBA plates were made from LB media which contained LB Broth (Sigma-Aldrich) and BactoAgar (Sigma-Aldrich).
Kanamycin and ampicillin were used as antibiotics since pUCIDT-Kan and pET36b plasmids carry the kanamycin resistance gene and pNiv and pDF-lac2 plasmids, in turn, carry the ampicillin resistance gene.
The plates were stored at +4&degC.

## Preparation of 1X BG11 medium for growth and maintenance of *Synechocystis* spp. PC 6803:

To be able to prepare 1X BG11 medium for cyanobacteria, multiple stock solutions need to be done. In Table 1, you can see the reagents and solutions that are needed for the preparation of BG11.
In the same table, there are also the reagents needed for 1X BG11. All the solutions are made in MQ water. In addition, the following stock solutions also need to be prepared:

* 175 mM K2HPO4 (100 ml)
* 189 mM Na2CO3 (100 ml)
* 6 mg/ml Fe-NH4-Citrate (50 ml)
* 1 M TES-KOH pH 8.0 (250 ml)

**Table 1. The reagents needed for 100X micronutrient, 1000X trace metal mix and 1X BG11.** Note that Fe-NH4-Citrate is added after autoclaving.

![100X BG11 (500 ml) solution contains 74.8 g NaNo3, 3.75 g MgSO4 x 7 H2O, 1.8 g CaCl2 x 2 H2O, 0.325 g (0.355 g) citric acid (monohydrate) and 0.05 g EDTA-Na2. 1000X trace metal mix (1000 ml) contains 2.86 g H3BO3, 1.81 g MnCl2 x 4 H2O, 222 mg ZnSO4 x 7 H2O, 391 mg Na2MoO4 x H2O, 79 g CuSO4 x 5 H2O and 49.47 mg Co(No3)2 x 6 H2O. 1M Hepes-NaOH buffer (pH 7.5, 500 ml) contains 119.15 g Hepes-NaOH and NaOH as much as needed to adjust the pH of the solution to 7.5. Finally, 1X BG11 (1000 ml) contains 10 ml 100X BG11, 20 ml 1M Hepes-NaOH buffer, 1 ml 1000X trace metal mix, 1 ml 175 mM K2HPO4 and 1 ml 189 nM Na2CO3.](https://static.igem.org/mediawiki/2021/8/82/T--Aboa--BG11new.jpg)

## Making competent cells:

Competent *E. coli* cells were made according to the following protocol.

1. Grow the cells in 5 ml LB until the absorbance at OD600 is between 0.4-0.5. (It will take approximately 1-2 hours, check every 30 minutes).
2. Keep on ice for 5 minutes.
3. Centrifuge at 5 000 rpm at +4°C for 30 seconds (or 2 min if swing-out). Collect the pellet. Discard the supernatant.
4. Keep the tubes on ice from now on.
5. Resuspend the pellet in 1 ml of 0.1 M MgCl2 (sterile) gently with a pipette and transfer to 1.5 ml microcentrifuge tubes.
6. Centrifuge at Max speed for 30 seconds with a tabletop centrifuge. Collect the pellet. Discard the supernatant.
7. Repeat the MgCl2 washing steps (5 and 6) for two times.
8. Resuspend the pellet in 100 µl of 0.1 M CaCl2 (sterile) gently with a pipette.
9. Keep on ice for 15 minutes and use for transformation.

## Transformation of the cells:

To transform our *E. coli* cells with laccase-containing plasmids, the following protocol was used.
After the transformation, plasmids were isolated and DNA digested for further measurements.

### Heat-shock transformation protocol for *E. coli* cells

1. Thaw the competent cells on ice for 15 min.
2. Add 3 µl DNA to the cells.
3. Incubate on ice for 30 min.
4. Place the cell + DNA mixture on a water bath (or in a heat block), keep 45 s at 42 °C.
5. Keep 2 min on ice.
6. Add 500 µl of LB (+ copper) in the laminar.
7. Incubate at +37°C for 1 h with 200 rpm shaking.
8. Plate 100 μl on a LA plate with appropriate antibiotics. (plate 1)
9. Plate the remaining cell culture on another LA plate with appropriate antibiotics. (plate 2)
10. Incubate plates at +37°C for 16 h.
11. Count the colonies from the plates and take a picture.

### Plasmid isolation

To isolate plasmids from cell cultures, QIAGEN QIAprep® Spin Miniprep Kit was used according to the manufacturer’s instructions (Qiagen, 2015).

### Digestion

Digestion reaction mixtures are prepared into PCR tubes by calculating and adding following reagents to it.

| **Reagent** | **Volume** |
| --- | --- |
| DNA | Calculate to equal 1 µg or 2 µg |
| rCutSmart Buffer (NEB) | 5 µl |
| Restriction enzyme | Equal to DNA (1 µl if 1 µg, 2 µl if 2 µg) |
| MQ |  |
| **Reaction volume** | **50 µl** |

After preparing reaction mixtures, incubate mixtures at 37°C for 4 h to allow enzymes to work efficiently.
Then, inactivate the enzymes by incubating at 80°C for 20 min. Store the products at 4°C.

The restriction enzymes we used over this project were NdeI, XhoI, NsiI, BspHI, SalI, SpeI and PvuII.
Read more about the specific phases in which they were used from our [laboratory notebook](https://2021.igem.orf/Team%3AAboa/Notebook).

## Agarose gel electrophoresis

To separate and analyze digestion samples, gel electrophoresis was performed.

### Agarose gel & Agarose gel electrophoresis

1. Weigh the correct mass of agarose into an erlenmeyer flask (0.78 g for 1.2 % gel and 0.98 g for 1.5 % gel).
2. Add 65 ml of 1xTAE buffer into the agarose.
3. Boil in a microwave until all agarose has melted and slightly simmered.
4. Add 6.5 µl SyBR Safe DNA Gel Stain 10 000x to the gel mixture and pour it onto the tray.
5. When the gel has solidified, put the tray in the gel electrophoresis apparatus. Add 10 µl of NEB purple loading dye 6x to samples and pipet the samples into wells. Start running the gel, for example, at 50 V for 60-100 min (read more specific times we used from our [laboratory notebook](https://2021.igem.org/Team%3AAboa/Notebook).

### Gel extraction

After running agarose gel electrophoresis, the desired fragments are cut from a gel and the DNA is extracted using the QIAGEN QIAquick® Gel Extraction Kit according to the manufacturer's instructions (Qiagen, 2018).

### Ligation

After the gel extraction, the ligation reaction was performed. It was done with a 3:1 ratio, i.e. the amount of DNA was three times bigger than the amount of a plasmid.
The reagents needed for the reaction are as follows:

| **Reagent** | **Volume** |
| --- | --- |
| DNA | Calculate to equal 150 ng |
| plasmid | Calculate to equal 50 ng |
| T4 Ligase Reaction Buffer | 2 µl |
| T4 DNA Ligase | 1 µl |
| MQ |  |
| **Reaction volume** | **20 µl** |

After preparing ligation reaction mixtures, incubate the samples at 19°C for 16 h to enable the enzymes to work efficiently.
After that, incubate them at 65°C for 10 min to inactivate the enzymes. Store the ligation products at 4°C.

## Protein production and purification

The laccase production of *E. coli* BL21(DE3) cells was induced by the IPTG system and the His-tagged laccases were extracted with Ni2+ affinity chromatography.

## Production

1. Incubate transformed cells in LB media at 37°C at 220 rpm for 16 h.
2. Add 400 µl kanamycin and 5 ml of cell culture in 400 ml LB media. Incubate at 37°C at 220 rpm for an hour. Measure OD600 values every 30 min until they are over 0.6A.
3. Add 400 µl of 1M IPTG to cells and incubate with the same settings for 3 h.
4. Measure OD600 values.
5. Centrifuge at 4°C at 20 000 rpm for 10 min. Discard the supernatant and add 15 ml of TrisA buffer to the pellets.
6. Sonicate in 4 x 30 s cycles to break the cell membranes. Amplitude should be between 8 and 12.
7. Centrifuge at 4°C at 20 000 rpm for 30 min.
8. Measure the volume of the supernatant and discard the pellet.

## Purification

Two buffers, Tris A buffer and B buffer, needed in the affinity chromatography were prepared according the following amounts of reagents:

**Tris A buffer, pH 7.5:**

* 20 mM Tris-Cl
* 150 mM NaCl
* 20 mM imidazole

**B buffer:**

* 20 mM Tris-Cl, pH 7.5
* 150 mM NaCl
* 300 mM imidazole

### Affinity chromatography:

1. Add 1 ml Ni2+ -affinity resin and a filter to the bottom of the column.
2. Add 3 CV (column volume) of MQ to remove the ethanol and wash the hartz.
3. Balance column by adding 3 CV of Tris A buffer.
4. Add produced proteins to the column.
5. Wash the column with 3 CV of Tris A buffer.
6. Add 7 CV of B buffer to the column.
7. Collect the run-through.
8. Wash column with 10 ml MQ.

## Measurements

## SDS-PAGE

SDS-PAGE was performed to verify the sizes of purified laccase proteins.
Bio-Rad Ready Gel® Precast Polyacrylamide Gel was used and the gel was run using Bio-Rad Mini Protean 3. The SDS buffer was prepared as follows:

**10x SDS stock buffer, pH 8,3**

* 0.25 M Tris
* 1.92 M glycine
* 1 % SDS

### Making samples:

Samples for SDS-PAGE were made from both raw extracts (1-2 mg/ml) and purified proteins (0.1-0.5 mg/ml).
The total sample size for both was 40 µl and they were made as follows:

1. Add 10µl of 4 x SDS buffer and 30µl of protein
2. Boil samples with SDS for 5 minutes.
3. Centrifuge at 12 000 x g for 1 minute.
4. Add 10 µl of samples to gel.

### Visualization of samples:

To visualize the proteins separated in the SDS-PAGE run, the gel was dyed with Coomassie blue stain. It was done as follows:

1. Mix Coomassie Brilliant Blue G-250 stain (70 mg) with 950 ml of MQ for 2-3 hours.
2. Add 3 ml of HCl and adjust the volume to 1 liter with MQ.

## ABTS assay

An ABTS assay was conducted to measure the laccase activities.
ABTS was dissolved in water according to the manufacturer’s recommendations.
To find out if the laccases were active at all, the following filter paper test was performed. A colour change would indicate the laccase activity.

Filter paper test

1. First, soak the filter paper in ABTS solution (2 mM ABTS, 100 mM phosphate-citrate buffer, pH 4).
2. Dry the paper at 60°C and add 10 µl of enzymes glycerol preps.
3. Incubate at 30°C for 10 min.

In addition, a 96-well plate assay was used to test the activity of the laccases.
The experiments were done in two different pHs, pH 5 and pH 7, of phosphate-citrate buffer (100 mM). Absorbances were measured at A420 in one minute intervals with the Tecan M200 plate reader.

## Syringaldazine assay

A syringaldazine assay (modified from Enzymatic Assay of Laccase (EC 1.10.3.2) | Sigma-Aldrich) was performed to measure laccase activities.
In the assay, the substrate was syringaldazine and a commercial SAE0050-laccase from *Aspergillus SP*. was used as a control.
Syringaldazine was dissolved in methanol and the Potassium-phosphate buffer (0.1 mM, pH 6.5) was used.
The absorbances were measured at A530 in one minute intervals at 30°C with the Tecan M200 plate reader.

## References

* Qiagen. (2015). Quick-Start Protocol for QIAprep® Spin Miniprep Kit.
* Qiagen. (2018). Quick-Start Protocol for QIAquick® Gel Extraction Kit.
* Sigmaaldrich.com. *Enzymatic Assay of Laccase (EC 1.10.3.2)*. [online] Available at:
  https://www.sigmaaldrich.com/FI/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-laccase [Accessed 2 September 2021].

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Team Aboa 2021

# Results

The objective of the project was to contribute to the development of a microbial wastewater treatment system for the detoxification of pharmaceutical waste, especially targeting the non-steroidal anti-inflammatory drug diclofenac (2-[2-(2,6-dichloroanilino)phenyl]acetic acid).
The approach was to overexpress and extract three heterologous laccase enzymes in engineered *E. coli*. Laccases are enzymes which are capable of catalyzing the conversion of diclofenac into less harmful derivatives.
The laccases compared in this study were CotA from *B. subtilis*, CueO from *E. coli* and Yak from *Y. enterocolitica subsp. palearctica* strain 7.
We were able to successfully produce and purify CotA and CueO, of which CotA was shown to have catalytic activity *in vitro*.

On this page we will go more in depth on these steps forming the build and test phase of our project (see [engineering success)](https://2021.igem.org/Team%3AAboa/Engineering).
Information about the design phase can be found [here](https://2021.igem.org/Team%3AAboa/Design).

## Construct assembly and generation of the *E. coli* strains for evaluating laccase function

The genes coding for the three laccases compared in the study, CotA, CueO and Yak, were synthesized by IDT and received in pUCIDT-Kan plasmids.
To enable the expression in *E. coli* BL21(DE3), the laccase genes were subcloned into the pET36b(+) expression vector (NdeI-XhoI) (Fig. 1A).
The *cotA* gene is 1542 bp, *cueO* 1551 bp, and *yak* 1602 bp, whereas the pUCIDT-Kan backbone is around 2500 bp, and the empty pET36b(+) plasmid 5248 bp and 669 bp after digestion.
Fragments of these lengths were obtained, which verified successful gene flanking design (Fig. 1A).
The pET36b plasmid seemed to however only have been partially digested as bands corresponding to the entire plasmid size were also obtained (Fig. 1A).
Selection was done through antibiotic selection using kanamycin as both pUCIDT-Kan and pET36b plasmids carry the kanamycin resistance gene.

The *E. coli* expression strains for evaluation of laccase function were then generated by transforming the three pET36b based expression constructs, pET36b-*cotA*, pET36b-*cueO* and pET36b-*yak*, into *E. coli* BL21(DE3).
Three colonies from each strain, selected by antibiotic resistance, were analyzed by restriction digestion with NdeI and XhoI. This should result in fragments the size of the genes and the pET36b backbone.
All of the screened *E. coli* BL21(DE3)/pET36b-*cotA* were true transformants, whereas only two of the *E. coli* BL21(DE3)/pET36b-*cueO* and one of the *E. coli* BL21(DE3)/pET36b-*yak* showed bands of expected size (Fig. 1B).
For each of the laccase producing strains a successfully transformed colony was selected for the continuation of the work.

![Two black figures next to each other named A and B. They both have names of enzymes written on the top and varying numbers written on both sides under the term ladder. There are white lines as bands between them.](https://static.igem.org/mediawiki/2021/4/48/T--Aboa--fig1.jpg)

**Figure 1. Agarose gel electrophoresis.** A) The digestion products of the pUCIDT-Kan plasmids carrying cotA, cueO and yak, and pET36b(+) expression vector. B) The verification digestions of the pET36b-cotA, pET36b-cueO and pET36b-yak expression constructs. The expression constructs were digested with NdeI and XhoI restriction enzymes. The 1 kb DNA ladder (NEB) was used as a molecular weight standard, the DNA was colored with SyBR Safe DNA Gel Stain (ThermoFisher), and the gel was visualized under UV light. The agarose gel used was 1.2 %.

The laccase genes for the *Synechocystis* sp. PCC 6803 expression constructs were subcloned from the pUCIDT-Kan plasmids directly downstream of the S3 RBS sequence in the pNiv plasmid (NsiI-XhoI).
Since the pUCIDT-Kan plasmid also contained additional NsiI restriction sites resulting in bands of similar size to the laccase genes, BspHI was added to the *cotA* and *cueO* digestions and PvuII to the *yak* digestion.
These digestions were successful (Fig. 2A, Fig. 2B).

The combined RBS and laccase gene fragments were then digested from the pNiv carrier plasmid as well as the pDF-lac2 expression plasmid (SpeI-SalI) (Fig. 2C).
Subcloning was, however, not completed as work with the generated *E. coli* BL21(DE3) strains for laccase overexpression was prioritized.

![Three black figures named A, B and C of which A and B are as smaller picctures on top of the figure C. They all have names of enzymes written on the top and varying numbers written on either the left or both sides under the term ladder. There are white lines as bands evenly distributed in each figure.](https://static.igem.org/mediawiki/2021/9/9c/T--Aboa--fig2.jpg)

**Figure 2. Agarose gel electrophoresis.** A) The digestion products of the pUCIDT-Kan plasmids carrying *cotA*, *cueO* and *yak* (NsiI-XhoI). B) The digestion products of the carrier plasmid pNiv(S3) (SpeI-SalI). C. Agarose gel electrophoresis of digestion products of the pNiv(S3)-*cotA*, pNiv(S3)-*cueO*, pNiv(S3)-*yak* and pDF-lac2 plasmids.

## Overexpression and laccase purification

The generated *E. coli* BL21(DE3) strains were induced by IPTG, the cells lysed by sonication, and His-tagged laccases extracted with Ni2+ affinity chromatography.
The purified proteins were analyzed by SDS-PAGE to verify their sizes (Fig. 3). Raw extracts consisted of proteins of all sizes as expected (Fig. 3).
SDS-PAGE analysis of purified CotA and CueO enzyme fractions showed clear bands around 60 kDa and 65 kDa, expectedly corresponding to CueO (calculated size 53.75 kDa (Roberts et al., 2002)) and CotA (calculated size 59.23 kDa (Enguita et al., 2003)), respectively, in addition to several minor impurity bands (Fig. 3).
As the elution volumes and the sample loading volumes were equal, the intensity of the target bands suggested that the concentration of CueO was approximately two-fold in comparison to CotA. A band corresponding to Yak was not obtained, and hence work with the enzyme terminated.
It is possible that Yak was not expressed in soluble form in *E. coli* and aggregated after expression, or was misfolded and proteolytically degraded.

![A white picture with three sections. On the left, there’s written Raw extract on the top. Under it there’s three blue lines from the top to bottom. They’re the width of a finger. In the middle section there’s written purified on top. Under it there’s written ladder and the names of the three used enzymes next to it. Under the ladder there are numbers from 200 to 10 starting from the top. Next to each numbers, there’s a blue line. Under the names of the enzymes, there are some blue lines. The right section is similar to the middle section. There are just even weaker and less blue lines next to the ladder.](https://static.igem.org/mediawiki/2021/f/fb/T--Aboa--fig3.jpg)

**Figure 3.** SDS-PAGE of raw extracts and purified enzyme fractions. Bio-Rad Ready Gel® Precast Polyacrylamide Gel was used and the ladder was the PageRuler Unstained Protein Ladder (Thermo Fisher).

## Qualitative colorimetric laccase assay with ABTS

First we tried to observe enzyme activity qualitatively using filter paper that was first soaked in ABTS and then dried before aliquoting laccase on it.
The addition of laccase should result in a blue-greenish color.
This would have indicated enzyme activity that could then have been studied in more detail quantitatively.
The paper was, however, already tinted green after ABTS treatment, and the laccases were found to either decolorize the paper or have no further effect on it, implying that the ABTS was already oxidized before enzyme addition, and thus unsuitable for determination of laccase activity.
Instead of wasting time to repeat the unsuccessful qualitative trial, we continued directly to the quantitative kinetic assay.

## Quantitative kinetic laccase assay with ABTS as substrate

The ABTS *in vitro* assay was based on laccase-catalyzed ABTS oxidation, which can be monitored spectrophotometrically as absorbance signal increase at 420 nm.
The purified laccase fractions were analysed using three different enzyme volumes (1 µl, 5 µl and 10 µl) and five ABTS concentrations (2 mM, 4 mM, 6 mM, 8 mM, 10 mM) in addition to zero controls, at two different buffer pHs (pH 5 and pH 7).
The resulting reaction absorbance profiles were not as anticipated (Fig. 4), and displayed a slight initial signal increase over the first minutes, followed by signal decrease.
The profiles clearly indicated that the response was not enzyme concentration -dependent for either CotA or CueO, suggesting that either the enzymes or the substrate were defective.
The progress curve of the zero enzyme control, and the control with only buffer, were similar to the actual reaction profiles (Fig. 4B), confirming that there was something wrong with the actual reaction setup.
In addition, the ABTS solution was not completely colourless at the beginning but slightly yellow, and did not show proper color change under any condition, indicating that the stock had already been oxidized and possibly further degraded.

![Two figures named A and B. Both of them have many graphs shaped of round parabels opening downwards in the beginning and then the curves go horizontally. On the right figure the multiple graphs distribute more apart from each other.](https://static.igem.org/mediawiki/2021/1/11/T--Aboa--fig4.jpg)

**Figure 4.** Representative kinetic activity trials with the purified laccase fractions and ABTS measured at 420 nm at pH 5. A) Reactions with 0.16 mM ABTS and 0 µl (blue) 1 µl (red line), 5 µl (green line) and 10 µl (purple line) CotA purification fraction. B) Reactions with 5 µl CotA and varying concentrations of the substrate: 0.01 mM (red line), 0.02 mM (black line), 0.04 mM (green line), 0.08 mM (purple line), 0.16 mM (cyan line). In addition, a control with no substrate (blue line) and a negative control with neither substrate nor enzyme (orange line).

## Kinetic laccase assay using syringaldazine as substrate

Next we decided to test the reaction with an alternative commercial substrate syringaldazine. The reaction is based on substrate oxidation which can be spectrophotometrically monitored as signal increase at 530 nm.
The reaction was performed on a 96-well plate using three different enzyme volumes (1 µl, 5 µl, 10 µl), three substrate concentrations (5 mM, 10 mM, 20 mM) in addition to zero controls.
The activity of the purified target enzymes CotA and CueO were measured in parallel to a commercial control laccase SAE0050 from *Aspergillus SP.*.

The CueO elution fraction was not active towards syringaldazine, whereas CotA clearly catalyzed substrate oxidation as seen in increasing progress curves (Fig.5).
While the signal in the zero enzyme control remained near the baseline (Fig. 5A-B blue line), the progress curve slopes of the enzyme containing reactions seemed to correlate with the enzyme concentrations:
Linear signal increase was observed with the different enzyme dilutions and substrate concentrations (Fig. 5A-B). The 20 mM reaction profiles using 5 µl of the CotA fraction were identical between the graphs (Fig. 5A-B green lines), showing that the result was reproducible.
The reaction slope of the 10 µl reaction in Fig.5 (purple line) was not as steep as anticipated, suggesting that there may have been difficulties in the sample preparation.
The catalytic activity of the positive control reaction with the commercial SAE0050-laccase was unexpectedly high, catalyzing complete substrate oxidation in a matter of seconds, even at the lowest enzyme dilutions.
These control reactions did not need to be repeated with lower enzyme concentrations because the activity of the CotA reaction under investigation was confirmed even in the absence of a positive control reference curve.

The reaction series confirmed that the laccase CotA was catalytically active and capable of oxidizing syringaldazine.
Therefore, the design of the His-tagged expression construct, over-expression in *E. coli*, and nickel affinity purification had all been successful.

![Two figures named A and B. The left one has four lines with different slopes, the right one has three lines with different slopes.](https://static.igem.org/mediawiki/2021/c/c7/T--Aboa--fig5.jpg)

**Figure 5**. Representative kinetic activity assays with the purified CotA laccase fractions and syringaldazine measured at 530 nm A) Reactions with 20 mM syringaldazine and 0 µl (green line), 1 µl (red line), 5 µl (blue line) and 10 µl (purple line) CotA purification fraction. B) Reactions with 5 µl CotA and varying concentrations of the syringaldazine: 0 mM (green line), 5 mM (red line), 20 mM (blue line).

## References

* Enguita, F. J., Martins, L. O., Henriques, A. O., Carrondo, M. A. Crystal structure of a bacterial endospore coat component: A laccase with enhanced thermostability properties. The Journal of Biological Chemistry, 278(21), 19416-25, <https://doi.org/10.1074/jbc.M301251200>.
* Roberts, S. A., Weichsel, A., Grass, G., Thakali, K., Hazzard, J. T., Tollin, G., Rensing, C., Montfort, W. R. Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 99(5), 2766-71, <https://doi.org/10.1073/pnas.052710499>.

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## Inclusivity

[Inclusivity](https://2021.igem.org/Team%3AAboa/Inclusivity)
[Plain Language](https://2021.igem.org/Team%3AAboa/Plain_Language)
[Supporting Sign Language](https://2021.igem.org/Team%3AAboa/SupportingSL)

## Achievements

[Judging Form](https://2021.igem.org/Team%3AAboa/)
[Medal Criteria](https://2021.igem.org/Team%3AAboa/Medal_criteria)

## Sponsors

![](https://static.igem.org/mediawiki/2021/0/0d/T--Aboa--UTU.png)
![](https://static.igem.org/mediawiki/2021/3/38/T--Aboa--IDT.png)
![](https://static.igem.org/mediawiki/2021/2/2a/T--Aboa--Benchling.png)

[![](https://static.igem.org/mediawiki/2021/1/1c/T--Aboa--instagram_icon.svg)](https://instagram.com/igem.aboa)
![](https://static.igem.org/mediawiki/2021/b/b3/T--Aboa--email_icon.svg)
[![](https://static.igem.org/mediawiki/2021/8/8b/T--Aboa--website_icon.svg)](https://aboaturku.fi)