iGEM 2024 — Team boku-vienna

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}A peek into our lab journals.{% endblock %}
{% block page\_content %}

* Expression
* Preliminary Tests
* Biosensor
* Immobilization

## Expression lab journal

## Preliminary tests lab journal

## Lab journal parts:

[Part 1](#note_biosensor1)
[Part 2](#note_biosensor2)
[Part 3](#note_biosensor3)
[Part 4](#note_biosensor4)
[Part 5](#note_biosensor5)
[Part 6](#note_biosensor6)
[Part 7](#note_biosensor7)
[Part 8](#note_biosensor8)

## Biosensor lab protokolls

### part 1

### part 2

### part 3

### part 4

### part 5

### part 6

### part 7

### part 8

## Immobilization with:

[Agar Agar](#note_imobilisation_agar)
[Biochar](#note_imobilisation_carbon)

## Immobilization with Agar Agar

## Immobilization with activated carbon

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %} Going beyond base metal alloys.{% endblock %}
{% block page\_content %}

* Expression
* Immobilization
* Biosensor

## Menu

[Overview](#exp_overview)
[Cycle A](#cycleA)
[Cycle B](#cycleB)
[Cycle C](#cycleC)
[Cycle D](#cycleD)
[Conclusion](#exp_conclusion)

## Overview

Our iGEM team sets out to engineer and express various laccase constructs originating from the organisms
*Coriolopsis trogii*, *Trametes hirsuta*, *Trametes pubescens*, and *Trametes
versicolor*. The goal was secretory and extracellular production of these enzymes using a signal
peptide in the yeast *Pichia pastoris*, purify these laccases, assess, and compare their enzymatic
activities and evaluate their potential for degrading NSAIDs (non-steroidal anti-inflammatory drugs). To
achieve our goal we utilized several methods, including Golden Gate Assembly, heat shock transformation,
electroporation, and different fermentation strategies.

## Cycle A - The cloning cycle

### Design

We selected six laccases from the organisms *Coriolopsis trogii*, *Trametes hirsuta*,
*Trametes pubescens*, and *Trametes versicolor* after research on their potential for
degrading NSAIDs. Alongside we did all cloning steps using GFP as a control. These sequences were codon
optimized for expression in *Pichia pastoris* and synthesized by Integrated DNA Technologies
(IDT). We included a pre-Ost1-pro-α secretion factor from *Saccharomyces cerevisiae*, a TEV site,
and
a His-tag, along with fusion sites for Golden Gate Assembly. It was decided that the His-tag and TEV
site should be added to the C-terminal end of our enzymes. A protein structure prediction using
AlphaFold showed that the N-terminus would be internalized, increasing the risk of misfolding. To
further mitigate that risk on the C-terminus, we designed primers to remove TEV site and His-tag.

![Fig 1](https://static.igem.wiki/teams/5329/pages/research-related/engin-expression-insert.webp)

**Fig 1)** *example of synthesized laccase gene*

### Build

After receiving the DNA, we performed PCR to remove the cleavable His-tag, to have two versions of each
laccase, with and without tags. We cloned them into our BB1\_FS\_23 vector, followed by heat shock
transformation of *Escherichia coli* for amplification. The subsequent selection on Kanamycin
plates was successful, and plasmid DNA extraction delivered a good yield. We then performed Golden Gate
Assembly with BB2\_L\_AB vector assembling the genes with a pGAP promoter and RPS25Att terminator, forming
a functional expression cassette. We then performed Golden Gate to assemble a *Pichia pastoris*
compatible expression vector using the 100\_BB3rN\_Fs1\_Fs4 plasmid, which was used in heat shock
transformation of *E. coli*. After amplification using the bacteria, plasmid DNA extract and
electroporation transformation of *Pichia pastoris*, we finally had cells that, in theory,
produce our laccases.

![fully assembled BB3](https://static.igem.wiki/teams/5329/pages/research-related/engin-expression-vector.webp)

**Fig 2)** *fully assembled BB3*

### Test

Colony PCR was performed after every transformation to check for successful take up of the plasmid.
Furthermore, extracted plasmid DNA was sent in for sequencing to double check for mutations. When
examining the sequencing results, we noticed that for two of our laccases, from the organism
*Trametes pubescens*, the sequence length was off. It turns out that during optimization for
synthesis through IDT, an additional BsaI restriction site was added, which during Golden Gate assembly
using BsaI, lead to problems.

### Learn

The main learning outcome from the cloning cycle, was to always double and triple check your sequences
before ordering. We decided to continue with our ten remaining laccase constructs.

## Cycle B - First expression cycle

### Design

We planned to produce our laccases in 15 mL YPD-NTC media using 100 mL shake flasks which would be
incubated for about 72 hours.

### Build

Media was prepared, shake flasks were filled and inoculated with a little dab of one colony . Shake
flasks were then incubated at 29°C at 200 RPM over the weekend. 1 mL of each flask was centrifuged to
only test activity in the supernatant, where the laccase should be due to the secretion factor.

### Test

Laccase activity of the supernatant was measured with a spectrophotometer using an ABTS activity assay
at pH 3, as these conditions were determined to work best by preliminary tests done on commercially
available laccase. No measurable laccase activity could be detected.

### Learn

Improvements were brainstormed, and we decided on three main differences to our first approach. We would
start a preculture containing NTC from which cells would be taken, washed, and used to inoculate a
production culture without NTC at a specific starting OD. Additional copper ions would be added as
laccases are multicopper oxidases and need copper to function. The starting OD600 of our
production culture would be much higher.

## Cycle C – Second Expression Cycle

### Design

A preculture would be started from a colony on a plate, grown for about 24 hours, subsequently the
OD600 measured, and it was calculated what volume would be needed to get a starting
OD600 of 8 for the production culture. The copper ion concentration we would add would be
50μM.

### Build

We again prepared the media and inoculated straight prom the plate. After one day we had an
OD600 of around forty in each flask. The correct volume of each flask was taken, washed with
fresh YPD media, and used to inoculate the production cultures without antibiotic but this time
containing additional copper.

### Test

We tried to determine Laccase activity like before, but after initial spectrophotometric tests showed no
activity, we decided to do a western blot of the supernatant and the pellet of cells. The western blot
did not show any laccase, which explained the lack of laccase activity.

### Learn

We will screen for other colonies, this time using primers which would bind to regions in the genome
flanking the sites where the plasmid should integrate. This was done to rule out transient expression of
cells only containing the plasmid, which would give them a temporary antibiotic resistance without
integrating into the yeast genome. We might also increase the chances of protein expression using
minimal medium such as Yeast Nitrogen Base (YNB).

## Cycle D - Third Expression Cycle

### Design

A new extensive round of colony PCR will be performed, screening eight colonies of each transformation
for the integration of the plasmid into their genome. Moreover, we will use buffered YNB media at a pH
of six and a copper concentration of 50µM for the production culture.

### Build

After the colony PCR screening process using the new primers, new YPD-NTC precultures were inoculated
with the promising candidates and left to incubate overnight. Meanwhile we prepared the YNB media which
would be buffered using a phosphate buffer and again added copper ions to reach the desired
concentration of 50μM. The next day, the precultures were harvested, washed, and again used to inoculate
the production culture to an OD600 of 8.

### Test

The same ABTS activity assay at pH 3 was performed and for the first time we had positive results
showing laccase activity in the supernatant of our media. This activity could only be detected after
extensive incubation of the assay which lead us to suspect that the concentration of laccase was very
low. We then used a SpeedVac vacuum concentrator to reduce the volume of liquid and increase
concentration of laccase. This was then used to run an SDS-PAGE which was then partly stained with
Coomassie blue and partly using silver staining. Unfortunately, we were not yet able to detect the
laccase proteins using SDS-PAGE. However, diclofenac concentration has been reduced by more than 40% in
our samples analyzed by means of HPLC.

![photo of ABTS assay, different concentrations of laccase and two positive samples](https://static.igem.wiki/teams/5329/pages/research-related/engin-expression-abtsassay.webp)

**Fig 3)** *photo of ABTS assay, different concentrations of laccase and two positive
samples*

### Learn

YNB media and the additional screening for colonies yielded positive results. To further improve the amount of expressed protein, switching to a promoter such as AOX1 might help, as an inducible expression system could be better suited to produce laccases. We had already started the cloning process, but due to time constraints we could not finish and test that theory. Also, improving detection of laccase, either through preceding purification using chromatography or using either laccase specific antibodies for western blotting or mass spectrometry would help to detect lower amounts of the enzyme.

## Conclusion and Future Outlook

By following the DBTL cycle, we were able to successfully express and secrete functional laccase enzymes, as evidenced by measurable activity in the culture’s supernatant using the ABTS assay. Future work has to be done to determine if the TEV site and His-tag have a negative influence on laccase activity as we were only able to detect activity in strains which had the tags removed. Purification with chromatography and mass spectrometry detection of our enzymes would help to determine if the protein was expressed and just did not show any activity or if there was no laccase enzyme in the supernatant. Continuing the work on the inducible AOX1 promoter-based expression system might also hold a lot of potential for future iGEM teams.

![graphical summary of our project and future possibilities](https://static.igem.wiki/teams/5329/pages/research-related/engin-expressionn-cylcessummary.webp)

**Fig 4)***graphical summary of our project and future possibilities*

## Menu

[Overview](#immobilisation_overview)
[Cycle A](#immobilization_cycleA)
[Cycle B](#immobilization_cycleB)
[Cycle C](#immobilization_cycleC)
[Cycle D](#immobilization_cycleD)

# Laccase Immobilization for Diclofenac/Ibuprofen Degradation

## Overview

Our team focused on addressing the challenge of degrading pharmaceutical pollutants, such as Diclofenac
and Ibuprofen, in wastewater using the fungal laccase  *Trametes versicolor*. Initial experiments
indicated that free laccase showed minimal activity at the neutral pH typical of wastewater, leading us
to explore enzyme immobilization as a strategy to enhance its stability and performance. We utilized
agar-agar as an immobilization matrix and intended to compare it with activated carbon; however, time
constraints limited this comparison. The project aimed to determine the most effective method for
enhancing laccase performance under wastewater conditions.

## Cycle A – Preliminary Experiments

### Design

The first step was to establish the optimal working conditions for  *Trametes
versicolor* laccase, particularly its pH and temperature stability. Our goal was to understand
how these factors influenced the enzyme’s activity, especially under neutral pH conditions that
resemble those in wastewater treatment plants.

### Build

We conducted a series of experiments in which laccase was incubated in different pH buffers and
temperatures. Enzymatic activity was assessed using ABTS as a substrate, with conversions monitored
photometrically.

### Test

The initial results suggested that laccase exhibited high activity at lower pH values but showed
almost no activity at pH 7, which raised concerns about the viability of using free laccase directly
in wastewater. However, further investigations revealed that the low activity at pH 7 was due to the
limitations of ABTS as a substrate at neutral pH. When tested with other substrates, such as
Diclofenac and Ibuprofen, laccase demonstrated activity even at pH 7, confirming that the enzyme can
function effectively under neutral conditions with appropriate substrates. This insight underscores
the importance of understanding the specific environmental conditions that influence enzymatic
activity for effective bioremediation strategies.

![Fig1: pH-optimum of T. versicolor ](https://static.igem.wiki/teams/5329/pages/research-related/engsuccessimob/engin-immobilization-fig1.webp)

**Fig 1)** *pH-optimum of T. versicolor*

![pH-stability of T. versicolor](https://static.igem.wiki/teams/5329/pages/research-related/engsuccessimob/engin-immobilization-fig2.webp)

**Fig 2)** *pH-stability of T. versicolor*

### Learn

Initially, we believed that *T. versicolor*laccase had minimal activity at pH 7, leading us to
focus on stabilizing the enzyme to make it functional in the neutral pH conditions of wastewater. This
prompted us to explore immobilization strategies to enhance its stability and activity. Later, we
discovered that free laccase could still function at pH 7 under specific conditions, so the pH issue
was no longer a critical barrier. However, even though the pH problem was resolved, the practical
challenges of using free laccase remained. Releasing free enzymes directly into wastewater would not
be feasible or environmentally sustainable, as the enzyme could degrade or disperse without providing
consistent treatment. This reinforced our decision to focus on immobilization as a necessary approach
to ensure controlled and effective degradation of pharmaceutical pollutants.

## Cycle B – Fe-Doped ZIF-8 Immobilization

### Design

To improve laccase stability and catalytic efficiency, we planned to co-immobilize *T.
versicolor*laccase and the mediator ABTS in Fe-doped ZIF-8. This matrix was chosen for its
ability to enhance enzyme stability, thermal resistance, and reusability, as well as its capacity to
minimize secondary pollution caused by free ABTS.

### Build

We aimed to co-immobilize laccase and ABTS using Fe-induced mineralization in a 1:1 ratio of Fe
2+to Zn2+, forming a stable ZIF-8 structure around the enzyme and mediator. This
process would enhance laccase performance in degradation.

### Test

We discovered that 2-Methylimidazole, a key component in ZIF-8 synthesis, had been listed by the ECHA
as a substance of very high concern due to its reproductive toxicity. This regulatory issue prevented
us from proceeding with the experiment.

### Learn

The inability to complete the ZIF-8 immobilization highlighted the importance of considering
regulatory restrictions in material selection. This experience emphasized that, beyond technical
feasibility, we must always take environmental impact and the well-being of our communities into
account when choosing materials and methods.

## Cycle C – Agar-Agar-Immobilization

### Design

Given the challenges faced in the preliminary experiments and ZIF-8 immobilization, we proceeded with
agar-agar as a more accessible and safer immobilization matrix for laccase. Our objective was to
enhance enzyme stability and reusability for pharmaceutical degradation, specifically targeting
Diclofenac and Ibuprofen.

### Build

We immobilized *T. versicolor*laccase within agar-agar cubes and planned to test its
degradation efficiency by exposing the immobilized enzyme to solutions containing Diclofenac and
Ibuprofen.

![Immobilization cubes in the making](https://static.igem.wiki/teams/5329/pages/research-related/engsuccessimob/engin-immobilization-fig3.webp)

**Fig 3)** *Immobilization cubes in the making*

### Test

Using HPLC, we monitored the degradation of Diclofenac and Ibuprofen over a 4-day period. Immobilized
laccase degraded 57% of Diclofenac at the higher enzyme concentration (0.60 mg/mL), outperforming the
48% achieved by free laccase. For Ibuprofen, degradation was similar between free and immobilized
laccase, with both around 32% degradation.

![Degradation of Diclofenac with Immobilized Laccase - Graphic](https://static.igem.wiki/teams/5329/pages/research-related/engsuccessimob/engin-immobilization-fig4-jpg.webp)

**Fig 4)** *Degradation of Diclofenac with Immobilized Laccase - Graphic*

We also compared the performance of reused and new immobilized laccase cubes. In this case, "reused
cubes" refers to cubes that were washed multiple times after their first immobilization cycle before
being used again in the next cycle. For Ibuprofen, both the reused and new cubes showed the same
degradation efficiency, indicating that the immobilized enzyme retained its activity after reuse. This
suggests that the immobilization method is suitable for long-term, cost-effective applications in
wastewater treatment. Interestingly, for Diclofenac, the reused cubes performed better than the new
ones, achieving 56% degradation within the first hour, likely due to variations in storage conditions
that may have enhanced enzyme stability. This highlights the robustness of the immobilized laccase,
particularly in its ability to be reused while maintaining or even improving degradation efficiency.

![Newly immobilized cubes vs. reused cubes – Ibuprofen degradation](https://static.igem.wiki/teams/5329/pages/research-related/engsuccessimob/engin-immobilization-fig5-jpg.webp)

**Fig 5)** *Newly immobilized cubes vs. reused cubes – Ibuprofen degradation*

![Newly immobilized cubes vs. reused cubes – Diclofenac degradation                      ](https://static.igem.wiki/teams/5329/pages/research-related/engsuccessimob/engin-immobilization-fig6-jpg.webp)

**Fig 6)** *Newly immobilized cubes vs. reused cubes – Diclofenac degradation*

To confirm that laccase remained contained within the agar-agar cubes, we conducted enzyme
inactivation experiments. After incubation, two aliquots were taken: one was directly analyzed by
HPLC, and the other was heat-treated to inactivate any free laccase that may have leaked out before
analysis. Both aliquots showed similar degradation results, confirming that no enzyme had leaked from
the cubes during the process. This makes our immobilization method a strong candidate for sustainable
and scalable wastewater treatment solutions.

### Learn

Through our experiments, we confirmed that agar-agar is an effective immobilization matrix for *T.
versicolor*laccase, allowing for stable and reusable enzyme activity in the degradation of
pharmaceutical pollutants. The enzyme retained its activity over multiple cycles, showing consistent
degradation efficiency for both Diclofenac and Ibuprofen. Additionally, the enzyme inactivation
experiments confirmed that the laccase remained securely contained within the agar-agar cubes, with no
leakage into the surrounding environment. This ensures that our immobilization method offers both
environmental safety and operational control.

We also learned that reused immobilized laccase cubes can sometimes outperform new ones, particularly
for Diclofenac, potentially due to changes in storage conditions that may improve enzyme stability.
This highlights the importance of monitoring storage parameters such as temperature and pH in future
experiments to maintain or improve enzyme activity. Additionally, further use of the reused cubes over
multiple cycles would be beneficial to fully assess their long-term efficiency and cost-effectiveness
in practical wastewater treatment applications. Based on these findings, it would be beneficial to
repeat the cycle with different laccase concentrations to further optimize degradation efficiency.

## Cycle D – Activated Carbon Immobilization

### Design

Following the success of agar-agar immobilization, we intended to explore activated carbon as another
immobilization method. Activated carbon's high surface area and strong adsorptive properties were
expected to support effective enzyme attachment and catalytic performance. We aimed to compare this
method with the agar-agar approach to identify the most efficient immobilization strategy.

### Build

We prepared activated carbon and attempted to immobilize laccase on its surface. However, during the
process, we encountered difficulties filtering the fine activated carbon particles after treatment
with HCl. Time constraints further prevented us from completing this method and making a direct
comparison with agar-agar immobilization.

### Test

Due to the challenges faced during preparation, we were unable to test the activated carbon
immobilization method in time. As a result, no direct comparison with the agar-agar method was made.

### Learn

Despite the setbacks, we gained valuable insights into the complexities of using activated carbon for
enzyme immobilization. Future experiments could focus on optimizing the carbon treatment process and
successfully completing the comparison with agar-agar to evaluate which immobilization technique is
more effective for large-scale applications in wastewater treatment.

## Conclusion

Through our engineering cycles, we confirmed that free laccase can function at neutral pH levels, such
as those found in wastewater. However, we recognized that immobilization is essential for enhancing its
stability and maintaining catalytic activity over time. The success of agar-agar immobilization
demonstrates a promising solution for degrading pharmaceutical pollutants, with the added benefit of
reusability.

Additionally, our enzyme inactivation experiments confirmed that laccase remained securely contained
within the agar-agar cubes, with no leakage into the surroundings, ensuring controlled and
environmentally safe application. Furthermore, reused immobilized laccase cubes retained or even
exceeded the efficiency of new cubes, particularly for Diclofenac, likely due to storage effects,
highlighting the potential for long-term, cost-effective wastewater treatment solutions.

## Menu

[Introduction and background](#bs_intro)
[Design](#bs_design)
[Engineering Success & Next Steps](#bs_success)

## Introduction and background

The biosensor project is inspired by previous publications, which showed that diclofenac may interfere
with certain cellular pathways in *S. cerevisiae*, eventually leading to upregulation of genes,
notably PDR5
(Pleiotropic Drug Resistance 5), through promoter activation [1]. PDR5 gene encodes an ABC
transporter. One key transcription factor that activates PDR5 expression in response to diclofenac
treatment is pdr1p/pdr3p heterodimer. **Schuller and colleagues**[1] have shown
previously that modifications of the PDR5 promoters, essentially increasing copy numbers of the
pdr1p/pdr3p binding consensus sequences increases sensitivity of the promoter response to diclofenac,
measured by fluorescent reporter gene assay. Further characterisations of additional promoters containing
pdr1p and pdr3p binding sites[2] encouraged us to include more promoter candidates to test
whether and to what extend do they respond to diclofenac.

As for ibuprofen, to our knowledge, current challenge has it that there is no known direct and native
pathway in *S. cerevisiae* that can be modified and utilised for our biosensor project. Previous
genome-wide
deletion screening against chemical and oxidative sensitivity has shown knockout of various candidate
genes confer susceptibility to ibuprofen[3], implying existing coping mechanism(s) that
underlie fitness of WT cells under ibuprofen treatment. Furthermore, He and colleagues[4] have
elucidated that ibuprofen induces degradation of high affinity aromatic amino acid transporter tat2p;
under ibuprofen treatment, cells are deprived of aromatic amino acids, particularly tryptophan. Thus, we
are driven to look for genes that are potentially upregulated under low tryptophan level to test our
hypothesis whether such promoters will also be upregulated with the presence of ibuprofen. We selected 4
promoters that could potentially respond to ibuprofen, namely pPYK1, pCYS3, pSHM1, and
pHIS4[5].

## Design

### Reporter Assay

To assess whether or to what extend these promoters respond to the NSAIDs, we designed reporter gene
essay, namely fusing various promoters with the downstream reporter gene (sfGFP). By comparing levels of
fluorescence emission among different cells transformed with various promoter-sfGFP constructs, we might
be able to observe and compare the dynamic ranges of these biosensors across various drug concentrations
(**Fig.1**).

![Conceptualisation of the reporter gene assay.](https://static.igem.wiki/teams/5329/pages/research-related/engin-bs1.webp)

### Gibson Assembly

We use Gibson assembly as a restriction enzyme-independent cloning strategy to make our constructs due to
the fact that some of our selected promoters contain BbsI and BsaI cutting sites. These might affect
Golden Gate Cloning efficiency. Our cloning strategy for Biosensor sub-project consists of promoter
isolation and addition of homology sequencing to DNA fragments by extended primer (overhang primer) PCR
(**Fig.2**).

![an illustration of how our Gibson Assembly would work](https://static.igem.wiki/teams/5329/pages/research-related/engin-bs2.webp)

## Engineering success and next steps

Despite that we only obtained one single colony of one construct which showed positive after colony PCR at
the point of Wiki-freeze (**Fig.3a**), we have gone through at least one design, build, test,
and learn cycle. The Sanger sequencing further validated our assembly (**Fig.3b**). We
firstly designed four-fragment Gibson assembly reactions to bring together our genetic and regulatory
elements to the goal plasmid; we executed our subsequent experiments to build and acquire our basic parts
for our Gibson assembly to work. In these processes, we went through multiple failures and additional
trouble-shooting steps, such as the promoter amplification inefficiency and determining best annealing
temperature for our promoter isolation PCR by thermogradient PCR. Without these steps, we would never have
learnt from a practical perspective that a four-part Gibson assembly reaction seemed to be very
inefficient in our current settings, given that **a)** We observed an increase in
transformation efficiency when we switched from heat-shock to electroporation; and **b)** We
only obtained one single positive colony. Through these small intermediate conclusions, we are inspired to
further change and test with different parameters. From current results, we believe that it would be
meaningful to **a)** Further increase the reaction concentration of Gibson assembly;
**b)** Test new Gibson compatible commercial kits; and **c)** Consulting experts
(such as Janoš) for advices and experiences. From a bigger picture, we believe it is worthy to try
additional promoters from previous study[5] that responded to Ibuprofen.

In the earlier phase of the Sub-project, we have mistakenly matched the wrong name to the gene number
denoted in the expression heat map (see **Liu et al. 2021**[5]). As a result, only
3 out of 6 selected promoters will potentially respond to ibuprofen and diclofenac treatment. Thus, we
will also include addition promoters which potentially respond to ibuprofen and our current knowledge
about them in our basic part contribution entry. It would be absolutely inspiring and proud to see future
generation iGEMers to continue our journey to design, build, and test their biosensor systems that detect
micropollutants, and learn from their own mistakes, experiences, and successes.

As for our next step in the biosensor project, we will continue with our trouble shooting and optimisation
for Gibson Assembly. Transformation in *S. cerevisiae* with various concentrations of diclofenac
with
negative control (transformation with backbone plasmid) will further elucidate whether at all will this
promoter indeed or to what extent will it respond to diclofenac. We are looking forward to sharing these
exciting results with judges at the Grand Jamboree 2024 in Paris.

![Fig.3) a) colony PCR with common primers and amplifying primers. A band from the pCWP2 colony is observed at just below the 3kb marker, very close to the expected electrophoretic mobility (2.5kb by common primers and about 2.2kb by amplification primers). b) Sequencing result aligned to designed plasmid. Our assembly success is validated by Sanger sequencing (Microsynth) with both common and amplifying primer pairs. Red arrows denotes sequence coverage.](https://static.igem.wiki/teams/5329/pages/research-related/engin-bs3.webp)

## References

---

1. Schuller, A., Rödel, G. & Ostermann, K. Tuning the Sensitivity of the PDR5 Promoter-Based Detection of
   Diclofenac in Yeast Biosensors. Sensors 17, 1506 (2017).
2. Buechel, E. R. & Pinkett, H. W. Activity of the pleiotropic drug resistance transcription factors
   Pdr1p and Pdr3p is modulated by binding site flanking sequences. FEBS Lett 598, 169–186 (2024).
3. Tucker, C. L. & Fields, S. Quantitative genome-wide analysis of yeast deletion strain sensitivities to
   oxidative and chemical stress. Comp Funct Genomics 5, 216–224 (2004).
4. He, C. et al. Enhanced Longevity by Ibuprofen, Conserved in Multiple Species, Occurs in Yeast through
   Inhibition of Tryptophan Import. PLoS Genet 10, e1004860 (2014).
5. Liu, H.-L., Wang, C. H.-T., Chiang, E.-P. I., Huang, C.-C. & Li, W.-H. Tryptophan plays an important
   role in yeast’s tolerance to isobutanol. Biotechnol Biofuels 14, 200 (2021).

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Tracing new paths.{% endblock %}
{% block page\_content %}

* Expression
* Preliminary Tests
* Immobilization
* Biosensor

## Expression Protocolls:

[Colony PCR](#exp_cpcr)
[PCR](#exp_pcr)
[Gelelectrophoresis](#exp_gelel)
[Golden Gate
Cloning](#exp_goldengate)
[Heatshock E.coli](#exp_heatshock)
[Mediarecipes](#exp_mediar)

## Colony PCR Laboratory Protocol

## PCR Laboratory Protocol

## Gelelectrophoresis Laboratory Protocol

## Golden Gate Cloning Laboratory Protocol

## E.coli Heatshock Protocol

## Mediarecipe Protocol

## Preliminary Tests lab protocoll

## Immobilization with:

[Agar Agar](#exp_agar)
[Activated Carbon](#exp_carbon)

## Laboratory Protocol with Agar Agar Immobilisation

## Laboratory Protocol with Active Carbon Immobilisation

## Biosensor lab protocoll

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Our Lab Results: Precision Meets Transparency.{% endblock %}
{% block page\_content %}

## Introduction

Our project was built up of three different parts: The expression – The immobilization – The Biosensor.

The expression part of our project aims to express different fungal laccases using the yeast *Pichia
pastoris*. We want to compare the ability of these laccases to degrade pharmaceuticals like
diclofenac and ibuprofen and explore if the laccases are suitable for wastewater treatment.

Furthermore, we wanted to investigate if we can add a cleavable His-tag to the C-terminus of the laccases. A His-tag
would prove beneficial for production because it would facilitate the purification. The structure prediction model AlphaFold shows
that the C-terminus of the laccase would be on the inside of the protein which might interfere with protein folding
and therefore with the activity. We planned to express each laccase with and without this tag to investigate this
issue.

Laccases often show decreased or no activity at a neutral pH, making them unsuitable for wastewater treatment at
that pH. We circumvent this problem by immobilizing the laccase on a matrix. Through immobilization, the laccases
show activity under neutral pH conditions at which they were not active when they were not immobilized. Furthermore,
through immobilization, we avoid releasing GMOs into the environment.

As a side project, we want to expand the promoter collection for yeasts by finding promoters that respond to
diclofenac and/or ibuprofen in the yeast's surroundings.

* Expression
* Preliminary Tests
* Immobilization
* Biosensor

## Menu

[Short Description](#exp_shortInfo)
[Detailed Results](#exp_detailInfo)

## Short Summary

### Goal:

* Expression of six different fungal laccases in *Pichia pastoris* and confirm enzyme
  activity

### Methods:

* Producing the expression vectors using Golden Gate cloning
* Optimization of the production by optimizing the media and growth conditions
* Determining laccase activity in the culture media using ABTS

### Results

* We managed to establish two yeast cultures that secrete active laccases into the media and have
  proven that they degrade diclofenac

## Detailed Results

One goal of our project is to compare different laccases. We have selected six laccases that looked
promising for our planned application (Table 1). The genes were optimized for expression by the yeast
*Pichia pastoris*. The genes were synthesized by the company IDT. Using PCR the TEV site and
His-tag were removed and the corresponding fusion site was added to the 3’ end of the laccase genes.
This left us with 12 different laccase genes.

| Registry Number | Laccase | Origin Species | GenBank Entry Number |
| --- | --- | --- | --- |
| BBa\_K5329009 | CVL3 | *T. versicolor* | D13372 |
| BBa\_K5329010 | LAC1 | *T. hirsuta* | EU492907 |
| BBa\_K5329011 | LCC1 | *C. trogii* | KU055621 |
| BBa\_K5329012 | LCC1 | *T. versicolor* | AY693776 |
| BBa\_K5329013 | LCC2 | *T. versicolor* | Y18012 |

Using golden gate assembly, we successfully cloned the laccases into different plasmid backbones (BB1).
The BB1 assemblies were inserted into *Escherichia coli* using heat-shock transformation. We
confirmed the insertion of the genes into BB1 by colony PCR and Sanger sequencing.

![Fig 1](https://static.igem.wiki/teams/5329/pages/research-related/results-expression-1.webp)

**Fig 1)***Colony PCR of *E.coli* colonies to check for the presence of the BB1
containing
plasmids with laccase genes. The expected size of the amplified fragments is 2 kb*

The expression cassettes consisting of the constitutive pGAP promoter, the different laccase genes, and
the rps25aTT terminator were assembled using Golden Gate cloning into the BB3 (100\_BB3rN). The assembled
BB3s were again transformed into *E.coli* (NEB10beta) using heat shock transformation. The presence
of the laccase genes in the plasmids was confirmed through restriction digest.

![Fig 2](https://static.igem.wiki/teams/5329/pages/research-related/results-expression-2.webp)

**Fig 2)***Gel Electrophoresis of the BB3 plasmids digested with the restriction enzymes
AvrII and AscI to confirm the presence of the laccase genes (Gel1)*

![Fig 3](https://static.igem.wiki/teams/5329/pages/research-related/results-expression-3.webp)

**Fig 3)***Gel Electrophoresis of the BB3 plasmids digested with the restriction enzymes
AvrII and AscI to confirm the presence of the laccase genes (Gel2)*

After confirming that the assembly was successful the BB3s were linearized and transformed into *Pichia
pastoris* using electroporation. The transformation was confirmed by colony PCR.

![Fig 4](https://static.igem.wiki/teams/5329/pages/research-related/results-expression-4.webp)

**Fig 4)***Results of the colony PCR to check for the insertion of our expression vector
into the yeast genome*

The first attempts to express the laccases were made by inoculating 10 mL of YPD and growing the cells at
30°C for two days. To check for expression an ABTS Assay of the supernatant was made. No laccase activity
could be detected in the media when performing an ABTS Assay. A Western Blot did show that no laccases
were secreted into the media.

![Fig 5a](https://static.igem.wiki/teams/5329/pages/research-related/results-expression-5a.webp)
![Fig 5b](https://static.igem.wiki/teams/5329/pages/research-related/results-expression-5b.webp)

**Fig 5)***Left: Western Blot (His-Blot; 1min exposure time) of the first expression
experiments. The laccases have an expected size of about 55kDa
Right: Ponceau staining of the blot
did not detect any laccases in the supernatant.*

To improve the expression of laccases we switched to YNB media. We made an overnight preculture and
inoculated the YNB Media at an OD600 8. After incubating for 48 hours laccase activity could be
detected using the ABTS assay after extended incubation in two cultures. The culture expressing the
*Trametes hirsuta* laccase and the culture expression the *Trametes versicolor* LCC2 laccase. Both of the expressed laccases do not contain the HIS-tag.

![Fig 6](https://static.igem.wiki/teams/5329/pages/research-related/engin-expression-abtsassay.webp)

**Fig 6)***photo of ABTS assay, different concentrations of laccase and two positive samples*

SDS-PAGE and staining (Coomassie and Silver) were performed on concentrated supernatants, to confirm that
the laccases were indeed present in the supernatant. We expected a signal at the size of about 55 kDa but
no band can be observed.

![Fig 7](https://static.igem.wiki/teams/5329/pages/research-related/results-expression-7.webp)

**Fig 7)***Silver-stained SDS-PAGE gel checking for laccase presence in the supernatant*

Because we could detect laccase activity with the ABTS Assay we decided to test if the culture supernatant can
degrade Diclofenac. For this experiment, we mixed 1mL of culture supernatant with 1mL of Diclofenac stock
solution (100µg/mL). The same reaction mix was also performed with the supernatant of a yeast culture
which did not express the laccases (empty control – EC).

After incubation of the mix over the weekend the concentration of the pharmaceuticals was analyzed using
HPLC. The results of the HPLC showed a 51,9 % reduction of diclofenac in the samples where laccases are
produced compared to the sample containing the supernatant of yeasts not producing laccases (EC).

Table 2: Results of the HPLC analysis

| Sample | concentration of diclofenac  [µg/mL] | Reduction relative  to empty control [%] |
| Empty control | 45,21 | - |
| LCC2 *T. versicolor* 10C | 27,36 | –39,5 |
| Laccase *T. hirsuta* 4D | 21,73 | –51,9 |

![Fig 8](https://static.igem.wiki/teams/5329/pages/research-related/results-expression-8.webp)

**Fig 8)***Diclofenac concentration after the incubation with the supernatant. Starting
concentration 50µg/mL. Sample description: EC = empty control, 10C = LCC2 (*t.v.*), 4D = laccase (*t.h.*)*

An attempt was made to further improve the protein expression by switching to an inducible AOX1 promoter.
The golden gate assembly was made, and the insertion of the expression cassette into the BB3 was confirmed
by restriction digest but the transformation into *P. pastoris* was unsuccessful. Due to time
constraints, no second attempt was made.

In summary, the team successfully performed cloning steps and managed, through optimization of cultivation
conditions to successfully produce two different laccases. Although we could not detect our laccases with
the SDS-PAGE and silver staining we were able to detect the enzyme activity and prove the ability of the
laccases to degrade diclofenac.

## Menu

[Short Summary](#pretest_shortInfo)
[Detailed Results](#pretest_detailedInfo)

* [Introduction](#pretest_intro)
* [Experimental Design & Setup](#pretest_experimentalDesign)
* [Data Collection & Analysis](#pretest_data)
* [Suitability](#pretest_suitability)
* [Visualization & Interpretation](#pretest_visualization)
* [Conclusion](#pretest_conclusion)

Determination of the optimal working conditions of the enzymes, including the identification of their pH
stability and temperature optimum.

## Short Summary

### Goal:

Determine the optimal working conditions for enzymes, focusing on identifying their pH and temperature
stability.

### Method:

Enzymes were incubated in different pH buffers to determine both their optimal pH and pH stability.
Similarly, the temperature optimum was assessed by incubating the enzymes at various temperatures. The
conversion of the substrate ABTS was used to measure enzymatic activity, which was monitored
spectrophotometrically at 420 nm.

### Hypothesis:

The pH optimum of the enzymes is expected to be around pH 4, with the temperature optimum expected to be
approximately 45°C.

### Conclusion:

Enzyme activity was higher at lower pH values, with the highest activity measured at the lowest pH
tested. At pH 7, there was almost no activity left. While increased temperatures led to slightly higher
activity, the effect of temperature on enzyme activity was minimal compared to the impact of pH.

## Detailed Results

### Introduction

In our preliminary experiments, we studied laccases from two different organisms: *Trametes versicolor*
and *Agaricus bisporus*. These enzymes were investigated to determine their optimal pH and
temperature, as well as their stability under varying pH and temperature conditions.

To measure enzymatic activity, we used 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS) as a substrate. In the presence of the enzyme, ABTS is oxidized to its radical cation (ABTS•+),
which has a characteristic green color. This reaction is monitored spectrophotometrically at 420 nm, as
the absorbance of ABTS•+ at this wavelength correlates with the enzyme’s catalytic activity. By
tracking the increase in absorbance, we can quantify the rate of substrate conversion, providing a
reliable measure of enzyme activity.

### Experimental Design and Setup

We conducted pH stability tests for both *Trametes versicolor* and *Agaricus bisporus* by
incubating the enzymes in different pH buffers ranging from 4-6. Enzymatic activity was assessed by
measuring the conversion of ABTS at 420 nm to determine the optimal pH for both enzymes.

During the pH tests, it became evident that Agaricus bisporus exhibited very low activity across all pH
levels compared to *T. versicolor*. Given the low performance of *A. bisporus* enzyme, we decided
not to proceed with further temperature tests for this enzyme. As a result, subsequent experiments
focused solely on *T. versicolor* to study its temperature and pH stability.

![Fig 1](https://static.igem.wiki/teams/5329/pages/research-related/results-pretest-1.webp)

**Fig 1)***pH stability of *T. versicolor* (Absorption maximum vs. incubation
time).*

### Data Collection and Analysis

The highest activity was observed at pH 5. At pH 7, no activity was detected, indicating that *T.
versicolor* is less stable at neutral pH. For the pH stability testings *T. versicolor* was
incubated at room temperature for 72 hours at different pH (pH 4, 5, 6, 7). The results are shown in the
figure above. Due to the fact that no laccase activity was left at pH 7, the values were not included
in the plot.

For *Trametes versicolor* temperature tests were conducted to determine its optimal operating
temperature.

### Suitability for Wastewater Treatment and Enzyme Immobilization

Since wastewater typically has a neutral pH, the low activity of *T. versicolor* at pH 7 presents
a challenge for its direct use in wastewater treatment. To overcome this, we explored enzyme
immobilization as a strategy to improve stability and activity at higher pH levels. Immobilizing the
enzyme may offer protection against pH fluctuations and enhance its long-term functionality in neutral
wastewater environments. Further investigations showed that the ABTS conversion does not work at neutral
pH when used as substrate for *T. versicolor*, which may have caused the low activity at this pH.

Despite knowing that the free laccase might be active at pH 7 and that ABTS could be the limiting
factor, we continued working at pH 7 with immobilization. This is because the reusability and increased
stability of the immobilized enzyme are crucial for practical applications in wastewater treatment.
Moreover, immobilization ensures that the laccase remains contained within the matrix, preventing its
release into the environment, which is important for maintaining ecological safety.

### Visualization and Interpretation

In the figure below (Figure 2) the activity data for T. versicolor across different pH levels
are shown, with the y-axis representing the degradation rate and the x-axis showing the pH. Similarly,
Figure 3 presents the activity data for different temperatures. These figures illustrate that *T.
versicolor* exhibits absorption maximum at pH 3 and 65 °C.

![Fig 2](https://static.igem.wiki/teams/5329/pages/research-related/results-pretest-2.webp)

**Fig 2)***pH-optimum of *T. versicolor**

![Fig 3](https://static.igem.wiki/teams/5329/pages/research-related/results-pretest-3.webp)

**Fig 3)***pH-optimum of *A. bisporus.**

![Fig 4](https://static.igem.wiki/teams/5329/pages/research-related/results-pretest-4.webp)

**Fig 4)***T-optimum of *T. versicolor**

### Conclusion

Our preliminary experiments indicate that *Trametes versicolor* laccase exhibits optimal activity
at pH 3 and a temperature of 65°C. Initially, we assumed the enzyme would have minimal activity at
neutral pH, which is typical of wastewater environments. However, further investigations suggest that
*T. versicolor* laccase may still be active at pH 7. Despite this, we are proceeding with enzyme
immobilization due to the numerous advantages it offers.

Immobilization is crucial for improving the enzyme’s reusability, stability, and long-term
functionality, especially in continuous wastewater treatment systems. It also prevents the release of
free laccase into the environment, ensuring better control over its application. These benefits make
immobilization an essential strategy for maximizing the potential of *T. versicolor* in practical
wastewater treatment.

## Menu

[Short Summary](#immobilization_shortInfo)
[Degradation of
Diclofenac](#immobilization_diclo)
[Degradation of Ibuprofen](#immobilization_ibu)
[Summary](#immobilization_summary)
[New vs. Used Cubes](#immobilization_cubes)

## Short Summary

### Goal:

Investigate the efficiency of laccase immobilized in agar-agar for the degradation of pharmaceutical
pollutants like Diclofenac and Ibuprofen in wastewater.

### Method:

Laccase is immobilized in agar-agar cubes, followed by exposure to solutions containing Diclofenac and
Ibuprofen. The degradation of these pollutants is monitored over time using HPLC to measure
concentration changes.

### Hypothesis:

It is expected that the immobilized laccase will exhibit a higher degradation rate for Diclofenac and
Ibuprofen compared to free laccase, due to increased enzyme stability and reusability in the agar-agar
matrix.

### Conclusion:

For Ibuprofen, the reused immobilized laccase cubes demonstrated the same degradation efficiency as the
new cubes. However, for Diclofenac, reused cubes showed improved efficiency, possibly due to variations
in storage conditions. With free laccase, we achieved a 48% degradation of Diclofenac, while the
immobilization process was slower but ultimately more effective, with 57% degradation using immobilized
laccase—representing a clear improvement. For Ibuprofen, the degradation was 31% with free laccase and
slightly higher, at 32%, with immobilized laccase.

## Degradation of Diclofenac with immobilized laccase

The HPLC results of our first immobilization approach reflect the degradation of Diclofenac and Ibuprofen
after being treated with agar-agar immobilized laccase. The initial concentrations of the drugs were set
at 100 µg/mL for Diclofenac and 500 µg/mL for Ibuprofen. The results show a varying degree of degradation
across different samples, indicating the enzymatic activity of laccase.

This diagram illustrates the degradation process of Diclofenac using two different concentrations of
immobilized laccase: **0.15 mg/mL** and **0.60 mg/mL**. The results indicate
that the degradation efficiency is markedly higher with **0.60 mg/mL** of laccase,
demonstrating that increased concentrations of immobilized laccase lead to a more effective degradation of
Diclofenac. Consequently, this higher concentration results in a faster and greater reduction in
Diclofenac levels compared to the 0.15 mg/mL concentration.

![Fig 1](https://static.igem.wiki/teams/5329/pages/research-related/results-immobilisation-1.webp)

**Fig 1)***Degradation of Diclofenac with Immobilized Laccase*

The degradation process is more effective with the higher laccase concentration, and after 2 hours, both
concentrations reach a plateau, indicating that no further degradation occurs beyond this point.
Additionally, since no further degradation was observed after 24 hours, we excluded day 4 from the plot.

![Fig 2](https://static.igem.wiki/teams/5329/pages/research-related/results-immobilisation-2.webp)

**Fig 2)***Degradation of Diclofenac with Immobilized Laccase after 2 hours*

Additionally, during the reaction, the solution changes from transparent to yellow after 2 hours, showing
that the immobilized laccase is active.

![Fig 3](https://static.igem.wiki/teams/5329/pages/research-related/results-immobilisation-3.webp)

**Fig 3)***Degradation of Diclofenac with Immobilized Laccase - Graphic*

## Degradation of Ibuprofen with Immobilized Laccase

The diagram below shows the degradation process of Ibuprofen using two different concentrations of
immobilized laccase: 0.15 mg/mL and 0.60 mg/mL. Like Diclofenac, the degradation efficiency is higher with
increasing concentration. However, the degradation of Ibuprofen was less effective than Diclofenac. Same
as for Diclofenac after 2 hours of incubation no further degradation occurs.

![Fig 4](https://static.igem.wiki/teams/5329/pages/research-related/results-immobilisation-4.webp)

**Fig 4)***Degradation of Ibuprofen with Immobilized Laccase*

## Summary

Overall, the laccase enzyme showed higher degradation for Diclofenac compared to Ibuprofen, with
degradation percentages for Diclofenac reaching as high as 57.3%, whereas the highest reduction for
Ibuprofen was 25.9%. The immobilization of laccase in agar-agar was effective in breaking down both
pharmaceuticals, but it appears to work more efficiently on Diclofenac. This experiment demonstrates the
potential of laccase to mitigate pharmaceutical pollution, with varying effectiveness depending on the
compound.

To further compare degradation results a new batch of agar-agar cubes was prepared. The subsequent
experiment, which examined the degradation of Ibuprofen and Diclofenac using reused immobilized laccase
and free laccase. Additionally, the experiment aimed to investigate whether the immobilized laccase
retains its activity after being heat inactivated at 95°C post-immobilization and how its performance
compares to free laccase. To assess this, we incubated the free laccase with the drug solution, taking two
aliquots at specific time points during the incubation. One aliquot was then heat inactivated, allowing us
to compare it with the untreated sample and determine if the laccase remained effectively immobilized
within the agar cubes and was not leaking into the solution.

### Degradation of Ibuprofen with Reused Immobilized Laccase

Table 1: Degradation of Ibuprofen with Reused Immobilized Laccase over time

| Time [h] | Ibuprofen [µg/mL] |
| 0 | 1000 |
| 1 | 683.72 |
| 2 | 674.26 |
| 24 | 682.70 |
| 96 | 686.71 |

Ibuprofen was tested at a concentration of **1000 µg/mL** using reused immobilized laccase
for degradation. The initial concentration of Ibuprofen was 1000 µg/mL, which showed a decrease to
**683.72 µg/mL** after 1 hour. However, the degradation did not progress significantly over
the next 96 hours, and the concentration remained constant after 31,6% degradation.

### Degradation of Diclofenac with Reused Immobilized Laccase

Table 2: Degradation of Diclofenac with Reused Immobilized Laccase over time

| Time [h] | Ibuprofen [µg/mL] |
| 0 | 200 |
| 1 | 88.49 |
| 2 | 87.49 |
| 24 | 87.72 |
| 96 | 85.45 |

The degradation of **200 µg/mL** Diclofenac using reused immobilized laccase showed a
substantial reduction to **88.49 µg/mL** (56% degradation) and did not further degrade
after
1 hour.

## New cubes vs. reused cubes

![Fig 5](https://static.igem.wiki/teams/5329/pages/research-related/results-immobilisation-5.webp)

**Fig 5)** *Comparison Ibuprofen degradation new versus reused cubes.*

![Fig 6](https://static.igem.wiki/teams/5329/pages/research-related/results-immobilisation-6.webp)

**Fig 6)***Comparisson Diclofenac degradation new versus reused cubes.*

There was no difference in Ibuprofen degradation between the reused and new immobilized laccase cubes, as
both demonstrated the same degradation efficiency. In contrast, for Diclofenac, the reused cubes showed
improved efficiency compared to the new ones, potentially due to differences in storage conditions.

### Degradation of Ibuprofen with Immobilized Laccase vs. post-incubation inactivated laccase

Table 3: Degradation of Ibuprofen with Immobilized Laccase vs. post-incubation inactivated
laccase

| Time [h] | Ibuprofen [µg/mL] | Ibuprofen [µg/mL]  heat treated |
| 0 | 500.00 | 500.00 |
| 1 | 336.68 | 338.82 |
| 2 | 335.76 | 338.37 |
| 24 | 337.05 | 337.06 |
| 96 | 336.49 | 336.54 |

The results show that both the immobilized laccase and the heat-inactivated laccase after incubation
achieved the same **32%** degradation of Ibuprofen. This confirms that the laccase remained
effectively immobilized within the cubes, as the degradation was not impacted by heat inactivation,
indicating no enzyme leakage from the cubes.

### Degradation of Diclofenac with Immobilized Laccase vs. post-incubation inactivated laccase

Table 4: Degradation of Diclofenac with Immobilized Laccase vs. post-incubation inactivated
laccase

| Time [h] | Diclofenac [µg/mL] | Diclofenac [µg/mL]  heat treated |
| 0 | 100.00 | 100.00 |
| 1 | 69.96 | 90.61 |
| 2 | 69.25 | 74.83 |
| 24 | 67.16 | 72.64 |
| 96 | 70.25 | 73.96 |

The results show that both the immobilized laccase and the heat-inactivated laccase after incubation
degraded Diclofenac, with the heat-treated sample achieving **27%** degradation and the
non-heat-treated sample achieving **32%**. This suggests that while some activity was lost
after heat treatment, the majority of the laccase remained immobilized within the cubes, as the
degradation difference was relatively small, indicating minimal enzyme leakage.

### Degradation of Ibuprofen and Diclofenac with free laccase

Table 5: Degradation of Ibuprofen and Diclofenac with free laccase

| Time [h] | Diclofenac [µg/mL] | Diclofenac [µg/mL]  heat treated |
| 0 | 1000.00 | 200.00 |
| 1 | 683.29 | 170.61 |
| 2 | 680.35 | 163.92 |
| 24 | 681.98 | 122.26 |

The degradation of Ibuprofen at **1000 µg/mL** using free laccase showed a promising
degradation of 31.6 % after just 1 hour. The concentration did not decrease further. In the experiment
with free laccase degrading 200 µg/mL Diclofenac, the initial concentration of 200
µg/mL
was **reduced by 15%** after 1 hour and was further degraded to **48%**.

#### Comparative Analysis of Laccase Performance

Across all experiments, both Ibuprofen and Diclofenac concentrations exhibited a similar trend of
reduction, confirming that laccase is effective at degrading these pharmaceutical pollutants. Notably,
the reused immobilized laccase performed as well as the new immobilized laccase for Ibuprofen,
demonstrating that it retains its effectiveness even after being washed and reused. This is
particularly
promising, as it suggests the potential for cost-effective applications in bioremediation without
compromising degradation efficiency. Interestingly, for Diclofenac, the reused cubes showed even
greater
efficiency than the new ones, possibly due to differences in storage conditions. The ability to reuse
the immobilized laccase enzyme not only maintains degradation performance but also adds value to its
application in environmental remediation efforts.

## Menu

[Promoter Isolation](#biosensor_promoter)
[Primer PCR](#biosensor_primerPCR)
[Gibson Assembly](#biosensor_gibson)
[References](#biosensor_references)

## Promoter isolation

We first designed primers to isolate promoters from *S. cerevisiae* genomic DNA (yeast strain
S288c). These primers include homology overhangs that allow annealing during the assembly step. The
melting temperatures we set to amplify our sequences from genomic DNA are initially calculated by NEB
website (Tm calculator). Since primers are designed with homologous overhangs, we used a two-step PCR
strategy to amplify the sequences; namely, the first 5 thermocycles are done with the primer melting
temperature without the homology part, and the rest (about 30 thermocycles) are done with 72 degrees
Celsius (maximal) annealing temperature. However, we have observed that our promoters are amplified with
various efficiencies (see **Fig.1a** and **b**). At first, we hypothesised that
this may be due to the bias that our target promoters might not be evenly distributed across the templates
we took each time for our PCR reactions. Next, we used the amplicons that we purified from gels as
templates to amplify more PCR products with maximal annealing temperature, that is, 72 degrees Celsius.
However, we were not able to identify any amplification at the expecting electromobility
(**Fig.1c** lane pHIS4 and opPDR5, **Fig.1d** lane pCWP2 and pSHM1).
Furthermore, we repeatedly observed seemingly consistently low amplification efficiency from certain
promoters (exemplified in Fig.1a, lane pHIS4, pPYK1; **1b**, opPDR5;
**1c**, pPYK1; **1d**, pPYK1, pHIS4, and opPRDR5). We thus postulated that
primers might not have bound to our gel-purified templates.

![Fig 1](https://static.igem.wiki/teams/5329/pages/research-related/results-biosensor-fig1.webp)
![Fig 2](https://static.igem.wiki/teams/5329/pages/research-related/results-biosensor-fig2.png)

We hypothesised that the estimated annealing temperature might not be accurate in this particular
instance, and we sought an optimal melting temperature for each promoter by setting up thermogradient PCR
reactions with melting temperatures ranging from 62 to 72 Celsius to amplify target sequences from gDNA.
We assumed annealing temperature “sweet spots” for each corresponding promoter if the intensity of that
band is the highest and that such band is surrounded by lower intensity bands, resembling a gaussian
distribution, even though fluctuations in band intensity across temperatures due to sampling bias (that
the amount of target is not always the same when the template is taken from the gDNA) is frequently
observed (**Fig.2**). Next, we used the annealing temperature at which the band intensities
are brightest for amplification from gDNA, and we were able to amplify a decent amount of product at
expected electrophoretic mobility (**Fig.3**).

![Fig 3](https://static.igem.wiki/teams/5329/pages/research-related/results-biosensor-fig3.webp)

## Extended primer PCR to amplify reporter gene, terminator and the backbone

We used similar approaches to amplify our fragments with overhang primer PCR. The primers have been
designed such that each fragment will have overlapping sequences with the upstream and downstream elements
in our goal plasmid (see [Engineering
Success - Biosensor – Introduction and Background, **Fig.2)**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%2B%20%27#engin_gibson'}}).
In general, we observed our target amplicon at the expected electrophoretic
mobility for both our reporter gene (**Fig.4a** and **b**), terminator
(**Fig.4c**), and BB3(2μ) backbone (**Fig.4d** and **e**). In
addition to the 2μ plasmid, which is a high copy number plasmid for *S. cerevisiae*, we also
amplified a low copy number plasmid with a centromere binding region (BB3\_ARS/CEN) which integrates to the
yeast genome and should contain the same sequence near multicloning site as in BB3\_2μ plasmid. However,
our initial attempt to use the same overhang primers to amplify BB3\_ARS/CEN was unsuccessful
(**Fig.4f**). We reasoned that the smear might be due to either one or more of at least three
following reasons: a), artefacts from unspecific primer binding, b), amplification artefacts due to
secondary structures of the plasmid and/or primers, and c), nuclease contamination. After using BamI
(which cleaves once between the designed primer binding sites) linearised and PCR cleaned up plasmid as
template, we still overserved an unspecific smear (**Fig.4g**). This excludes nuclease
contamination as PCR clean-up kit (NEB #T1030S) should have removed nucleases. We then sought an optimal
melting temperature for our primers to bind to the template, as at this point it appeared to us that
unspecific binding might be the main cause of the smear (i.e., there are multiple similar elements in the
template that could bind to our primers; thus, the amplicons will have various lengths). We observed a
specific band at 62.9 degrees Celsius when we did a thermogradient PCR (**Fig.4h**). However,
we were not able to reproduce this result when we attempted to amplify the target again
(**Fig.4i** and **j**). From this point, we went on to our assembly step with
BB3\_2μ plasmid. Though, copy number can have a modulating effect on the sensitivity of our biosensor, it
is more pressing to build a construct for the proof of concept.

![Fig 4](https://static.igem.wiki/teams/5329/pages/research-related/results-biosensor-fig4.jpg)

## Gibson Assembly and *E. coli* transformation

After we have amplified and purified all of our parts, we went on to assemble our target plasmids. We
first set out a 0.1 pmol reaction, i.e., that all of our parts are at concentration of 0.1 pmol. Our
initial transformation strategy was conventional heat shock transformation. However, we did not observe
any colonies after our first transformation (data not shown). We confirmed by positive control that the
assembly reaction indeed worked as cells transformed with assembled positive control DNA sample provided
by the manufacturer showed colonies that are larger in size and higher in density (**Fig.5b**
and **c**) than
cells transformed with the same DNA sample without assembly reaction (**Fig.5a**). We
hypothesized that this
is due to poor assembly efficiency since our transformation positive control, in which cells were
transformed by empty backbone BB3\_2μ (**Fig.5d** and **e**) and BB3\_ARS/CEN (not
shown), showed decent number of
colonies, implying that the transformation is not likely to be the cause. We thus increased the
concentration of fragments to 0.25 pmol. After heat shock transformation of the assembled products, we
still did not observe any colonies. We occasionally observed a few very tiny colonies, further colony PCR
analysis showed negative results. We questioned whether our fragments indeed contained homologous sites
and whether these sites are flanking the target sequence with designated order. To test this, we sent our
fragments for Sanger Sequencing (Microsynth). All our BB3 plasmids, the 5’ of reporter genes, and
3’ of terminator sequence have shown desired homology sequence (data not shown); forward sequencing
of pPYK1 and pCWP2 showed ambiguous results suggesting impurity (**Fig.5f** and
**h**). However, this is not seen
in the reverse sequencing data of the same primers (**Fig.5g** and **i**). We
believed that these impurities may
result from unspecific forward primer binding at the very 5’ sequence of these promoters as
promoters frequently contain multiple copies of very similar sequences. We nonetheless assumed that these
PCR products do contain desired promoter sequences, albeit only in a fraction. Taken together, these data
suggest that we have to a large extent overestimated the actual amount of target plasmid within the Gibson
Assembly mix. Since we do not have much time left before Wiki freeze, instead of using a higher
concentration of fragments for the assembly reaction, we decided to transform *E.coli* cells via
electroporation as electroporation has much higher transformation efficiency than heat shock1.

![Fig 5](https://static.igem.wiki/teams/5329/pages/research-related/results-biosensor-fig5.jpg)

## References

1. Lessard, J. C. Transformation of E. coli Via Electroporation. in 321–327 (2013).
   doi:10.1016/B978-0-12-418687-3.00027-6.

{% endblock %}