iGEM 2021 — Team Patras

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# Team:Patras/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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

# Team:Patras/Engineering

iGEM Patras 2021

[![Project PGasus](https://static.igem.org/mediawiki/2021/d/d7/T--Patras--PGasus_logo.png)](https://2021.igem.org/Team%3APatras)

* [Home](https://2021.igem.org/Team%3APatras)
* Team
  + [Meet our Team](https://2021.igem.org/Team%3APatras/Team)
  + [Our road to Giant Jamboree](https://2021.igem.org/Team%3APatras/Road)
  + [Attributions](https://2021.igem.org/Team%3APatras/Attributions)
  + [Sponsors](https://2021.igem.org/Team%3APatras/Sponsors)
* Project
  + [Description](https://2021.igem.org/Team%3APatras/Description)
  + [Contribution](https://2021.igem.org/Team%3APatras/Contribution)
  + [Sustainable](https://2021.igem.org/Team%3APatras/Sustainable)
  + [Parts](https://2021.igem.org/Team%3APatras/Parts)
* Wet Lab
  + [Engineering](https://2021.igem.org/Team%3APatras/Engineering)
  + [Protocols](https://2021.igem.org/Team%3APatras/Protocols)
  + [Results](https://2021.igem.org/Team%3APatras/Results)
  + [Notebook](https://2021.igem.org/Team%3APatras/Wet_Lab_Notebook)
* Dry Lab
  + [Docking](https://2021.igem.org/Team%3APatras/Docking)
  + [Modeling](https://2021.igem.org/Team%3APatras/Model)
  + [Notebook](https://2021.igem.org/Team%3APatras/Dry_Lab_Notebook)
* Human Centric Design
  + [(Integrated) Human Practices](https://2021.igem.org/Team%3APatras/Human_Practices)
  + [Science Communication](https://2021.igem.org/Team%3APatras/Communication)
  + [Collaborations](https://2021.igem.org/Team%3APatras/Collaborations)
  + [Proposed Implementation](https://2021.igem.org/Team%3APatras/Implementation)
  + [Diversity & Inclusion](https://2021.igem.org/Team%3APatras/Inclusivity)
  + [Partnership](https://2021.igem.org/Team%3APatras/Partnership)
* [Safety](https://2021.igem.org/Team%3APatras/Safety)
* [Judging](https://igem.org/2021_Judging_Form?id=3839)

## Engineering

You've got to experiment, to figure out what works!

#### **NGS**

#### **Wet Lab**

## Nanopore and NGS analysis

Our novel CYP2D6 and CYP2C19 variants were obtained from 103 samples after performing targeted resequencing using MinION from Oxford Nanopore Technologies, all part of a previous research project within the Laboratory of Pharmacogenomics and Individualized Therapy.

* [**Nanopore**](#tab1)
* [**NGS Pipeline**](#tab2)
* [**Variants**](#tab3)

Oxford Nanopore MinION sequencer is an exciting third-generation sequencing paradigm. Third-generation sequencing is progressing rapidly, moving from a technology once only capable of providing data for small genome analysis or performing targeted screening to one that promises high quality de novo assembly and structural variation (insertions, deletions, duplications) detection for human-sized genomes. MinION is a portable, real-time, long-read, low-cost device capable of sequencing many gigabases in a single sequencing experiment, producing several GB of data per experiment.

MinION is also using the innovative technology of nanopores. Specifically, It contains an array of tiny holes called nanopores, embedded in a membrane, across which an electrical current is passed, and single-stranded DNA or RNA can be streamed through the pores and sequenced in real-time. This makes nanopore sequencing unique; it is the only sequencing technology that enables direct, real-time analysis of short to ultra-long fragments of DNA or RNA in fully scalable formats. MinION comes with a flow cell containing the nanopores and the sensing chemistry used in nanopore sequencing. It also contains the circuitry required to convert the current flow through the pore into an electrical signal that is used to sequence DNA. The flow cell sensor array has 2048 wells and typically contains over 1,000 nanopores. These wells are connected to electrical channels and can only record up to 512 channels at any one time.

![](https://static.igem.org/mediawiki/2021/7/7e/T--Patras--nanoporemech.gif)

Before sequencing, a software must be installed and a library must be prepared according to the given instructions from the MinION . MinKNOW is a software for testing hardware, checking flow cells, data acquisition, real-time analysis and feedback, basecalling, data streaming, and device control. MinKNOW takes the raw data and converts it into reads by recognizing the distinctive change in current that occurs when a DNA strand enters and leaves the pore. MinKNOW then basecalls the reads and writes out the data into .fast5 or FASTQ files.

The pipeline followed is presented below:

![Responsive image](https://static.igem.org/mediawiki/2021/5/56/T--Patras--ngs-pipeline.jpg)

Guppy was used to run basecalling and generate the FASTQ files. Proceeding to alignment, GRCh37/hg19 was used as a reference genome resulting in SAM/BAM files. Finally, the Nanopolish tool was used for variant calling, and final VCF files were obtained. At this analysis step, it was noticed that among the already reported variants according to the genomic databases, some single nucleotide variants are not included in these databases and are supported by acceptable quality scores. To determine the potential pathogenicity of the assessed novel variants, in silico scores were used to predict whether an amino acid substitution leads to a protein damaging effect. Specifically, SIFT, Clinpred, and DANN scores were used to indicate a potentially damaging effect of variants, resulting in the novel ones presented in the variants section.

| CYP2C19 Variants | CYP2D6 Variants |
| --- | --- |
| p.L15P | p.L61S |
| p.R26\* | p.F112S |
| p.E274G | p.S476I |
| p.N286D |  |
| p.D293G |  |
| p.T304A |  |
| p.I327V |  |
| p.L380P |  |
| p.L413M |  |
| p.F487S |  |
|

From the presented variants, the p.R26\* on CYP2C19, did not proceed to functional characterization because the mutation resulted in a stop codon an in a non-functional oligopeptide has not any function.

## Wet Lab

* All
* Learn from the existing
* Cloning of the pENTR/D-TOPO vector
* Site-directed mutagenesis
* TA Cloning
* Transfection

#### **Learn from the existing**

For the evaluation of the enzyme activity with in vitro assays, is usually used a heterologous expression system that provides the advantage of high reproducible conditions without patients undergoing invasive procedures and eliminating the risk of developing adverse drug reactions. According to literature, specifically for the functional characterization of the differential effects of CYP variants due to single nucleotide variants, there are various expression systems such as E. coli, bacteria, yeast, baculoviruses, and mammalian cell lines that are used in research.

Exogenous DNA delivery into animal cells is a widely used process for the expression of functional recombinant proteins. However, when applied in mammalian cells, we face the problem of low protein expression and consequently the increasing costs. Thus, for achieving a successful transfection in mammalian cell lines, Lipofectamine 3000 was used.

Additionally, 293FT cells were selected because they allow very high levels of protein to be expressed from vectors containing the SV40 origin, compared to other cell lines, COS-7, HepG2, and 293T cells.

pcDNA3.4 TOPO vector was used as it contains a full-length CMV promoter and other expression elements that allow for higher expression than other pcDNA-based expression constructs, making it suitable to achieve higher levels of CYP expression in 293FT cells.

In the exhibition of CYP catalytic activity, the redox partner NADPH-cytochrome P450 oxidoreductase (CPR) plays a vital role. CPR provides the essential first electron, whereas the second electron may come directly from CPR or via cytochrome b5 (CYB). According to several studies, CYP activities were increased when using a co-expression system of CPR and CYP compared to the expression of CYP alone. However, the mechanism underlying the benefits of CYP and CPR plasmid co-expression in mammalian cells is not yet found.

In addition to CPR, electron transfer via CYB also plays a role in improving the enzymatic activities of several CYP isoforms.

Therefore, the co-expression of CYP, CPR, and CYB is supported for achieving optimal conditions for the evaluation of enzymatic activity in vitro and, due to its advantages, was selected for our experiments.

#### **Design: cDNAs Amplification with TOPO sites**

In our experiments, we used a pENTR/D-TOPO system. The vector is linearized with two DNA topoisomerases I on each 5’ and 3’ ends. The enzyme DNA topoisomerase I acts as both a restriction enzyme and a ligase; thus, no ligase or any restriction enzyme is needed to be added for this cloning procedure. In this system, PCR products are directionally cloned by adding four bases to the forward primer (CACC). The overhang in the cloning vector (GTGG) invades the 5′ end of the PCR product, anneals to the added bases, and stabilizes the PCR product in the correct orientation. Inserts can be cloned in the correct orientation with efficiencies equal to or greater than 90%. The Forward primers of each gene of interest are designed with a TOPO site (CACC) at the 5’ end of their sequence for directional cloning of the blunt-end PCR product into a pENTR/D-TOPO vector as presented in the table below.

![](https://static.igem.org/mediawiki/2021/8/8e/T--Patras--topo.jpg)

| # | Name | Type | Description | Length |
| --- | --- | --- | --- | --- |
| 1 | [BBa\_K3839028](http://parts.igem.org/Part%3ABBa_K3839028) | Primer | CYP2D6 TOPO site Forward Primer | 20 |
| 2 | [BBa\_K3839029](http://parts.igem.org/Part%3ABBa_K3839029) | Primer | CYP2D6 TOPO site Reverse Primer | 22 |
| 3 | [BBa\_K3839030](http://parts.igem.org/Part%3ABBa_K3839030) | Primer | CYP2C19\_201 TOPO site Forward Primer | 26 |
| 4 | [BBa\_K3839031](http://parts.igem.org/Part%3ABBa_K3839031) | Primer | CYP2C19\_201 TOPO site Reverse Primer | 30 |
| 5 | [BBa\_K3839032](http://parts.igem.org/Part%3ABBa_K3839032) | Primer | POR-218 TOPO site Forward Primer | 23 |
| 6 | [BBa\_K3839033](http://parts.igem.org/Part%3ABBa_K3839033) | Primer | POR-218 TOPO site Reverse Primer | 20 |
| 7 | [BBa\_K3839034](http://parts.igem.org/Part%3ABBa_K3839034) | Primer | CYB5A\_202 TOPO site Forward Primer | 19 |
||  |  |  |  |  |
| --- | --- | --- | --- | --- |
| 8 | [BBa\_K3839036](http://parts.igem.org/Part%3ABBa_K3839036) | Primer | CYB5A\_202 TOPO site Reverse Primer | 23 |
 9 | [BBa\_K3839035](http://parts.igem.org/Part%3ABBa_K3839035) | Coding | pENTR/D-TOPO | 2580 |

The initial wild-type templates for CYP2D6, CYP2C19, CPR, and cytochrome b5 cDNAs were purchased from IDT as gBlocksTM Gene Fragments. We amplified these cDNAs using a high-fidelity Q5 polymerase.

In our first experiments, we chose the Standard PCR for the amplification of the cDNA fragments. However, following this protocol resulted in a not satisfactory amount of PCR products. Consequently, we performed a Touch-down PCR to achieve high specificity and efficiency of the amplification.

The optimized conditions of the Touch-down PCR are shown in the table below.

| Component | 50μl Reaction | Final Concentration |
| --- | --- | --- |
| 5x Q5 |  |  |
| Reaction Buffer | 10 μl | 1x |
| 10 mM dNTPs | 1 μl | 200 μM |
| 10 µM Forward Primer | 2.5 µl | 0.5 μM |
| 10 µM Reverse Primer | 2.5 µl | 0.5 μM |
| Template DNA | variable | < 1,000 ng |
| Q5 High-Fidelity DNA Polymerase | 0.5 µl | 0.02 U/µl |
||  |  |  |
| --- | --- | --- |
| 5x Q5 High GC Enhancer (optional) | (10 µl) | (1x) |
 Nuclease-Free Water | to 50 µl |  |

| Step | Temp | Time |
| --- | --- | --- |
| Initial Denaturation | 95°C | 10 minutes |
| Denaturation | 94°C | 30 seconds |
| Touchdown | 62-54°C\* | 30 seconds |
|  | 72°C | 30 seconds |
| Final Extension | 72°C | 10 minutes |
| Hold | 4 | infinity |

| Temp | Cycles |
| --- | --- |
| 62°C | 5 cycles |
| 60°C | 5 cycles |
| 58°C | 5 cycles |
| 56°C | 5 cycles |
| 54°C | 5 cycles |

#### **Build: Cloning of the pENTR/D-TOPO vector**

All the pENTR/D-TOPO vectors cloned with the wild-type CYP2D6, CYP2C19, CPR, and cytochrome b5 cDNAs ([BBa\_K3839038](http://parts.igem.org/Part%3ABBa_K3839038), [BBa\_K3839039](http://parts.igem.org/Part%3ABBa_K3839039), [BBa\_K3839040](http://parts.igem.org/Part%3ABBa_K3839040), [BBa\_K3839041](http://parts.igem.org/Part%3ABBa_K3839041)), were transferred into NEB 5-Alpha High-Efficiency Competent E. coli cells according to the transformation protocol. For the clone selection, the antibiotic Kanamycin was used in the plates. After the incubation, single colonies were selected and cultured in liquid SOB Medium overnight. Finally, the plasmid DNAs were extracted and purified with Monarch® Plasmid Miniprep Kit.

You can preview the vector map on our [Parts](https://2021.igem.org/Team%3APatras/Parts) page.

![Responsive image](https://static.igem.org/mediawiki/2021/8/8c/T--Patras--liquid-cultures.jpg)

![Responsive image](https://static.igem.org/mediawiki/2021/8/8c/T--Patras--single-colony-selection.jpg)

#### **Design: Site-directed Mutagenesis**

The plasmids with the wild-type CYP2D6 cDNA and the plasmids containing the wild-type CYP2C19 cDNA were used each as a template to generate the novel enzymatic allelic variant constructs, using a Q5® Site-Directed Mutagenesis Kit. The primers for the mutagenesis of the variants CYP2D6 and CYP2C19 cDNAs are shown in the table below.

All the studied variants are substitutions, and for that reason, the mutagenic sites were placed onto the forward primer as a miss-match. The substitution was created by incorporating the desired nucleotide change in the center of the forward primer, including at least ten (10) complementary nucleotides on the 3´side of the mutation(s). The reverse primer was designed so that the 5´ ends of the two primers anneal back-to-back.

![](https://static.igem.org/mediawiki/2021/a/a8/T--Patras--topoprocedure.jpg)

Subsequently, were designed 12 pairs of primers.

| # | Name | Type | Description | Length |
| --- | --- | --- | --- | --- |
| 1 | [BBa\_K3839000](http://parts.igem.org/Part%3ABBa_K3839000) | Primer | Forward mutagenic primer CYP2D6 L61S | 21 |
| 2 | [BBa\_K3839001](http://parts.igem.org/Part%3ABBa_K3839001) | Primer | Reverse primer CYP2D6 L61S | 27 |
| 3 | [BBa\_K3839002](http://parts.igem.org/Part%3ABBa_K3839002) | Primer | Forward mutagenic primer CYP2D6 F112S | 21 |
| 4 | [BBa\_K3839003](http://parts.igem.org/Part%3ABBa_K3839003) | Primer | Reverse primer F112S | 19 |
| 5 | [BBa\_K3839004](http://parts.igem.org/Part%3ABBa_K3839004) | Primer | Forward mutagenic primer S476I | 21 |
| 6 | [BBa\_K3839005](http://parts.igem.org/Part%3ABBa_K3839005) | Primer | Reverse primer CYP2D6 S476I | 19 |
| 7 | [BBa\_K3839006](http://parts.igem.org/Part%3ABBa_K3839006) | Primer | Forward mutagenic primer L15P | 23 |
| 8 | [BBa\_K3839007](http://parts.igem.org/Part%3ABBa_K3839007) | Primer | Reverse primer CYP2C19 L15P | 24 |
| 9 | [BBa\_K3839008](http://parts.igem.org/Part%3ABBa_K3839008) | Primer | Forward mutagenic primer CYP2C19 E274G | 21 |
| 10 | [BBa\_K3839009](http://parts.igem.org/Part%3ABBa_K3839009) | Primer | Reverse primer CYP2C19 E274G | 18 |
| 11 | [BBa\_K3839010](http://parts.igem.org/Part%3ABBa_K3839010) | Primer | Forward mutagenic primer CYP2C19 N286D | 24 |
| 12 | [BBa\_K3839011](http://parts.igem.org/Part%3ABBa_K3839011) | Primer | Reverse primer CYP2C19 N286D | 20 |
| 13 | [BBa\_K3839012](http://parts.igem.org/Part%3ABBa_K3839012) | Primer | Forward mutagenic primer CYP2C19 D293G | 21 |
| 14 | [BBa\_K3839013](http://parts.igem.org/Part%3ABBa_K3839013) | Primer | Reverse primer CYP2C19 D293G | 27 |
| 15 | [BBa\_K3839014](http://parts.igem.org/Part%3ABBa_K3839014) | Primer | Forward mutagenic primer CYP2C19 T304A | 21 |
| 16 | [BBa\_K3839015](http://parts.igem.org/Part%3ABBa_K3839015) | Primer | Reverse primer CYP2C19 T304A | 18 |
| 17 | [BBa\_K3839016](http://parts.igem.org/Part%3ABBa_K3839016) | Primer | Forward mutagenic primer CYP2C19 I327V | 21 |
| 18 | [BBa\_K3839017](http://parts.igem.org/Part%3ABBa_K3839017) | Primer | Reverse primer CYP2C19 I327V | 19 |
| 19 | [BBa\_K3839018](http://parts.igem.org/Part%3ABBa_K3839018) | Primer | Forward mutagenic primer CYP2C19 L380P | 21 |
| 20 | [BBa\_K3839019](http://parts.igem.org/Part%3ABBa_K3839019) | Primer | Reverse primer CYP2C19 L380P | 21 |
| 21 | [BBa\_K3839020](http://parts.igem.org/Part%3ABBa_K3839020) | Primer | Forward mutagenic primer CYP2C19 L413M | 21 |
| 22 | [BBa\_K3839021](http://parts.igem.org/Part%3ABBa_K3839021) | Primer | Reverse primer CYP2C19 L413M | 21 |
| 23 | [BBa\_K3839022](http://parts.igem.org/Part%3ABBa_K3839022) | Primer | Forward mutagenic primer CYP2C19 F487S | 21 |
| 24 | [BBa\_K3839023](http://parts.igem.org/Part%3ABBa_K3839023) | Primer | Reverse primer CYP2C19 F487S | 21 |

#### **Build: Cloning of the obtained variant vectors**

The following procedures of transformation and plasmid extraction were performed for each designed vector separately.

After receiving the vectors containing the genes’ variants, High-Efficiency E.coli cells (50 μL of NEB 5-Alpha competent cells) were transformed with 1 μg of designed plasmids. The mixture was allowed to stand on ice for 30 minutes, followed by heat shock at 42 oC for 30sec, placed on ice for 5 minutes, added 950 μL of S.O.C. Medium to the resultant mix, and cultured with shaking vigorously (250 rpm) at 37 oC for 30 min. After that, 100 mL of the culture solution was spread on a kanamycin-containing SOB agar plate and cultured at 37 oC for 16 h. Single colonies were selected and transplanted into 5.0 mL of kanamycin-containing liquid medium (SOB) and cultured at 37 oC while shaking at 250 rpm for 18 h.

After that, plasmids from twelve colonies were subsequently extracted and purified using the Monarch® Plasmid Miniprep Kit. 5mL of bacterial cultures was centrifuged at 13,000 RPM, and the pellets were resuspended in 200μl of resuspension buffer. After lysis and two stages of washing buffers, the extracted plasmids were diluted in 30μl of elution buffer.

![](https://static.igem.org/mediawiki/2021/0/0e/T--Patras--kanamycin-plates.jpg)

#### **Digestion of the pENTR/D-TOPO vectors + mutated inserts**

In order to remove the desired sequence containing the gene variants, digestion with two restriction enzymes, NotI and AscI, was performed. 5-6 ng of the pENTR/D-TOPO vector subcloned with the CYP2D6 and CYP2C19 variants were digested with 1μl of each enzyme (10,000 units/ml) at a final volume of 20μl, at 37oC for 30 minutes, following the digestion protocol and conditions of each enzyme.

#### **Test: Electrophoresis**

All the digestion mixture was run at a low-melting agarose gel of 0,8%, and the DNA band of interest was removed from the gel with cutting. The low-melting gel provides us with greater sieving properties and higher clarity than standard melting temperature agarose.

![](https://static.igem.org/mediawiki/2021/1/16/T--Patras--digestion2cyp.png)

Purification of the extracted DNA band was performed using a Gel extraction kit.

![](https://static.igem.org/mediawiki/2021/5/51/T--Patras--gelextr.png)

#### **Building the mammalian expression vector**

The next step was to subclone the DNA fragments with the designed gene variants into a pcDNA3.4 vector with TOPO TA cloning sites. The pcDNA 3.4 TOPO plasmid is a constitutive mammalian expression vector designed to deliver high levels of transgene expression.

DNA Pol I, Large (Klenow) fragment without the 3’- 5’ exonuclease activity, was used to generate blunt-ends with A and T overhangs on the DNA fragments, setting the conditions for achieving the directional TOPO cloning with the vector.

![](https://static.igem.org/mediawiki/2021/4/4f/T--Patras--tacloning.jpg)

Wild-type and variant CYP2C19 cDNAs subcloned into the pcDNA3.4 vector. Plasmids carrying CPR or cytochrome b5 cDNAs were also subcloned into the pcDNA3.4 vector.

Competent E. coli cells were transformed with the final vectors subcloned with the DNA fragments, according to the given protocol. After incubation for 24 hours and the colony selection, the E.coli cells were cultured for 16 hours in SOB medium containing Ampicillin. Finally, the plasmid DNA was extracted and purified at a final volume of 30μL.

![Responsive image](https://static.igem.org/mediawiki/2021/0/0c/T--Patras--plasmid2.png)

![Responsive image](https://static.igem.org/mediawiki/2021/6/6d/T--Patras--plasmid1.png)

#### **Test: Electrophoresis**

To verify the integrity of the produced subcloned vectors, electrophoresis on agarose gel 1% was run.

![](https://static.igem.org/mediawiki/2021/6/60/T--Patras--gelelectrophoresis.jpg)

#### **Prepartion**

Before the transfection of the eukaryotic cells, 5ng of the mammalian expression vectors were prepared in a solution of H2O, CH3COONa, and absolute ethanol to purify and precipitate the DNA. This solution was stored at -20°C overnight.

#### **Transfection**

293FT cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum at 37°C under 5% CO2.

24 h before transfection, the cells were plated at a density of 2\*106
cells/100-mm dish. Subsequently, Opti-MEM medium (Invitrogen) was added to the culture medium.

After 24 hours of incubation, cells were transfected with the designed plasmid encoding the wild type and variant CYP2D6 and CYP2C19 cDNAs, CPR cDNA, and Cytochrome B5 cDNA using Lipofectamine 3000, at concentrations of 7,5μl/1x106 cells. 3-5 ng of the designed vectors were transfected into the cells.

For selecting the transfected cells, Neomycin (G418) was utilized at a concentration of 800μg/mL of full medium.

After 6 h of incubation at 37°C, the culture medium was replaced with 10% FBS-DMEM. The incubation continued for 16 hours at 37°C, followed by dispension of medium and washing with PBS, the 293FT cells were scraped off.

The cells were centrifuged at 1500g for 5 minutes and resuspended in a homogenization buffer [10 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 10% glycerol].
Microsomal fractions were prepared by differential centrifugation at 9000g for 20 minutes so as the components of the cell were separated according to their density, followed by centrifugation of the resulting supernatant at 105,000g for 60 minutes.

The microsomal pellet was resuspended in 50 mM Tris-HCl (pH 7.4) containing 1 mM EDTA, 20% glycerol, 150 mM KCl, and Protease Inhibitor Cocktail for Use with Mammalian Cell and Tissue Extract and stored at –80°C.

* References

  1. Kono, N, Arakawa, K. Nanopore sequencing: Review of potential applications in functional genomics. Develop Growth Differ. 2019; 61: 316– 326.

  ---

  2. Alberto Magi, Roberto Semeraro, Alessandra Mingrino, Betti Giusti, Romina D’Aurizio, Nanopore sequencing data analysis: state of the art, applications and challenges, Briefings in Bioinformatics, Volume 19, Issue 6, November 2018, Pages 1256–1272

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  3. Jain, M., Olsen, H. E., Paten, B., & Akeson, M. (2016). The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biology, 17(1)

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  4. Ammar R, Paton TA, Torti D et al. Long read nanopore sequencing for detection of HLA and CYP2D6 variants and haplotypes [version 2; peer review: 2 approved]. F1000Research 2015, 4:17

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  5. Zanger, U. M. & Schwab, M. Cytochrome P450 enzymes in drug metabolism: Regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol. Ter. 138, 103–141 (2013).

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  6. Zhou, S. F., Liu, J. P. & Chowbay, B. Polymorphism of human cytochrome P450 enzymes and its clinical impact. Drug Metab. Rev. 41, 89–295 (2009).

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  7. Schenkman, J. B. & Jansson, I. Te many roles of cytochrome b5. Pharmacol. Ter. 97, 139–152 (2003).

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  8. Henderson, C. J., McLaughlin, L. A. & Wolf, C. R. Evidence that cytochrome b(5) and cytochrome b(5) reductase can act as sole electron donors to the hepatic cytochrome P450 systems. Mol. Pharmacol. 83, 1209–1217 (2013).

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  9. Ibeanu GC, Goldstein JA, Meyer U, Benhamou S, Bouchardy C, Dayer P et al. Identification of new human CYP2C19 alleles (CYP2C19\*6 and CYP2C19\*2B) in a Caucasian poor metabolizer of mephenytoin. J Pharmacol Exp Ther 1998; 286: 1490–1495.

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  10. Saito, T. et al. Functional characterization of 50 CYP2D6 allelic variants by assessing primaquine 5-hydroxylation. Drug Metab. Pharmacokinet. 33, 250–257 (2018).

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  11. Ibeanu GC, Goldstein JA, Meyer U, Benhamou S, Bouchardy C, Dayer P et al. Identification of new human CYP2C19 alleles (CYP2C19\*6 and CYP2C19\*2B) in a Caucasian poor metabolizer of mephenytoin. J Pharmacol Exp Ther 1998; 286: 1490–1495.

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  12. Dai, D. P. et al. 293FT is a highly suitable mammalian cell line for the in vitro enzymatic activity analysis of typical P450 proteins. Pharmazie 70, 33–37 (2015

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  13. Iwata, H. et al. High catalytic activity of human cytochrome P450 co-expressed with human NADPH-cytochrome P450 reductase in Escherichia coli. Biochem. Pharmacol. 55, 1315–1325 (1998).

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  14. Pham, P. L., Kamen, A. & Durocher, Y. Large-scale transfection of mammalian cells for the fast production of recombinant protein. Mol. Biotechnol. 34, 225–237 (2006).

  ---

![]()![iGEM Patras 2021 logo](https://static.igem.org/mediawiki/2021/f/f9/T--Patras--patras-footer.png)

University of Patras, Campus, Rio

**Email:** igempatras@gmail.com

#### About Us

iGEM Patras is proposing a comprehensive workflow for the identification & functional characterization of PGx variants.

#### Sponsors

We are thankfull to all our sponsors. Without them this project would not be possible.

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# Team:Patras/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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

# Team:Patras/Results

iGEM Patras 2021

[![Project PGasus](https://static.igem.org/mediawiki/2021/d/d7/T--Patras--PGasus_logo.png)](https://2021.igem.org/Team%3APatras)

* [Home](https://2021.igem.org/Team%3APatras)
* Team
  + [Meet our Team](https://2021.igem.org/Team%3APatras/Team)
  + [Our road to Giant Jamboree](https://2021.igem.org/Team%3APatras/Road)
  + [Attributions](https://2021.igem.org/Team%3APatras/Attributions)
  + [Sponsors](https://2021.igem.org/Team%3APatras/Sponsors)
* Project
  + [Description](https://2021.igem.org/Team%3APatras/Description)
  + [Contribution](https://2021.igem.org/Team%3APatras/Contribution)
  + [Sustainable](https://2021.igem.org/Team%3APatras/Sustainable)
  + [Parts](https://2021.igem.org/Team%3APatras/Parts)
* Wet Lab
  + [Engineering](https://2021.igem.org/Team%3APatras/Engineering)
  + [Protocols](https://2021.igem.org/Team%3APatras/Protocols)
  + [Results](https://2021.igem.org/Team%3APatras/Results)
  + [Notebook](https://2021.igem.org/Team%3APatras/Wet_Lab_Notebook)
* Dry Lab
  + [Docking](https://2021.igem.org/Team%3APatras/Docking)
  + [Modeling](https://2021.igem.org/Team%3APatras/Model)
  + [Notebook](https://2021.igem.org/Team%3APatras/Dry_Lab_Notebook)
* Human Centric Design
  + [(Integrated) Human Practices](https://2021.igem.org/Team%3APatras/Human_Practices)
  + [Science Communication](https://2021.igem.org/Team%3APatras/Communication)
  + [Collaborations](https://2021.igem.org/Team%3APatras/Collaborations)
  + [Proposed Implementation](https://2021.igem.org/Team%3APatras/Implementation)
  + [Diversity & Inclusion](https://2021.igem.org/Team%3APatras/Inclusivity)
  + [Partnership](https://2021.igem.org/Team%3APatras/Partnership)
* [Safety](https://2021.igem.org/Team%3APatras/Safety)
* [Judging](https://igem.org/2021_Judging_Form?id=3839)

## Results

Once you see results, it becomes an addiction!

[![](https://static.igem.org/mediawiki/2021/d/df/T--Patras--western.png)](#tabs1-5b)

#### **Western Blotting**

[![](https://static.igem.org/mediawiki/2021/8/83/T--Patras--enzyme-activity.png)](#tabs1-5b1)

#### **Enzymatic Properties**

## Western Blotting

Western blotting was performed to determine the protein expression levels of CYP2D6 and CYP2C19 wild type and variant proteins. The analysis was performed according to the standard protocol with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis; for performing Western using a software to evaluate and visualize the protein expression levels.

* [**CYP2D6**](#tab1)
* [**CYP2C19**](#tab2)

For CYP2D6, each well was loaded with 30 μg/mL microsomes, and CYP2D6 protein was detected using a polyclonal anti-human CYP2D6 antibody (diluted 1:250). Secondary antibody detection was carried out with horseradish peroxidase-conjugated goat anti-rabbit IgGcat. Additionally, a total protein assay was performed to normalize each signal using 30 μg/mL microsomes.

For CYP2C19, each well was loaded with 100 μg/mL microsomes, and CYP2C19 protein was detected using a polyclonal anti-human CYP2C19 antibody (diluted 1:100). Secondary antibody detection was carried out with horseradish peroxidase-conjugated goat anti-rabbit IgGcat. Additionally, a total protein assay was performed to normalize each signal using 100 μg/mL microsomes.

## Enzymatic Properties

* [**Introduction**](#tab3)
* [**CYP2D6**](#tab4)
* [**CYP2C19**](#tab5)
* [**Summary**](#tab6)

The enzymatic properties of wild-type and CYP2D6 and CYP2C19 variants were measured through pharmacokinetic parameters from in vitro drug metabolism data. Enzyme kinetics deals with a quantitative description of the rates of enzyme-catalyzed chemical reactions, mainly how experimental variables affect reaction rates. Enzyme kinetics combined with related approaches can show how the functional properties of a variant or engineered enzyme compare to those of its wild-type.

To display information about enzyme kinetics, Michaelis-Menten graphs (graphing reaction rate as a function of substrate concentration) are used to provide information, such as kinetic parameters: Km, Vmax, and CLint

![](https://static.igem.org/mediawiki/2021/4/4d/T--Patras--enzymatic-kinetics.png)

According to the graph, at a low substrate concentration, there is an abrupt increase in the rate of reaction (V0) as the substrate's concentration increases, then level off to a flat plateau at high substrate concentrations. This plateau occurs because the enzyme is saturated, and any additional substrate molecules will not have an effect due to the saturation and will wait until another enzyme becomes available and so the rate of reaction and product formation is limited and is only dependable on the enzyme’s activity. This maximum rate of reaction is called maximum velocity Vmax.

Km is the substrate concentration that gives the rate that is halfway to Vmax. It measures how quickly reaction rate (V0) increases with substrate concentration, how easily the enzyme can be saturated by the substrate, and also a measure of the affinity of the enzyme for its substrate. Lower Km represents a lower affinity for the substrate.

CLint (CLint = Vmax/Km) is a measure of enzyme activity measured by measuring the increase in the metabolite.

To display information about enzyme kinetics, Michaelis-Menten graphs (graphing reaction rate as a function of substrate concentration) are used to provide information, such as kinetic parameters: Km, Vmax, and CLint

For CYP2D6, the microsomal fraction obtained from 293FT cells were incubated in a mixture containing reagents and as substrate dextromethorphan in variable concentrations (1, 2, 4, 8, 10, 20, 40, 80 μM).

The determination of dextromethorphan O-demethylation in 30 μg of the microsomal protein fractions (containing wild-type CYP2D6 or CYP2D6 variants) showed that dextromethorphan formation was linear for incubations of up to 30 min. Following incubation and protein removal by centrifugation at 14,000 g for 5 min, 50 μL of the resultant supernatant was injected into an ultra-performance liquid chromatography (UPLC)-fluorescence (FLR) detector system.

![](https://static.igem.org/mediawiki/2021/5/53/T--Patras--cyp2d6-curves.jpg)

Michaelis-Menten curves for wild type and variant CYP2D6. Determination of kinetic parameters of dextromethorphan O-demethylation.

The Michaelis-Menten kinetic parameters were measured for the dextromethorphan O-demethylation for the wild type and the 3 CYP2D6 variant proteins. Below are represented the means ± standard deviation (SD) of as derived from the three independently performed catalytic assays compared with wild-type CYP2D6.
Differences with \*p < 0.05 were considered significant.

| # | CYP2D6 Variants | Km (μM) | V max (nmol/min/mg protein) | CLint (mL/min/mg protein) (% of Wild-type) |
| --- | --- | --- | --- | --- |
| 1 | Wild-type | 2.01 ± 0.24 | 25.31 ± 0.51 | 12.72 ± 1.35 (100.00) |
| 2 | p.L61S | 1.53 ± 0.45 | 2.26 ± 0.15\* | 1.54 ± 0.31\* (12.11) |
| 3 | p.F112S | 3.09 ± 0.84 | 15.60 ± 1.86 | 5.19 ± 0.76\* (40.80) |
| 4 | p.S476I | N.D. | N.D. | N.D. |
|

Among the three types of CYP2D6 variants tested, the kinetic parameters of 1 (p.S476I) could not be determined because the amount of the metabolite produced was at or below the detection limit.

![](https://static.igem.org/mediawiki/2021/5/53/T--Patras--chart1-cyp2d6.png)

Representation of kinetic parameters of dextromethorphan O-demethylation by microsomes from 293FT cells expressing wild-type and variant CYP2D6 proteins.

The CLint of the wild type CYP2D6 represents 100% of the enzymatic activity, resulting in a diagram representing the enzymatic activity of variant CYP2D6 as a % of the wild type.

![](https://static.igem.org/mediawiki/2021/f/fc/T--Patras--chart2-cyp2d6.png)

Dextromethorphan O-demethylation activity by wild-type and variant CYP2D6 proteins.

Compared to wild type, p.L61S variant CYP2D6 exhibited significantly decreased activity with 12.1 % of the activity of wild type. This hint indicates that maybe in clinical studies, such variant could lead to a Poor Metabolizer.

Additionally, the p.F112S variant showed a decreased activity, 40.7% of CYP2D6. This substitution may be located far from the site that interacts with the substrate and thus does not affect its metabolism as on p.L61S.

* More details about UPLC for CYP2D6

  On CYP2D6, dextromethorphan O-demethylation activity was measured as described below.

  1) An incubation mixture prepared containing:
  + • microsomal fraction (30 μg) obtained from 293FT cells
  + • dextromethorphan in concentrations: 1, 2, 4, 8, 10, 20, 40, 80 μM
  + • 3.3 mM MgCl2
  + • and 100 mM potassium phosphate buffer (pH 7.4) in a final volume of 150 μL

  2) Incubation at 37°C for 3 min

  3) Initiation of reactions by adding 15 μL of a mixture containing:
  + • 1.3 mM NADP+
  + • 3.3 mM glucose-6-phosphate
  + • and 0.4 U/mL glucose-6-phosphate dehydrogenase

  4) Incubation at 37°C for 30 min

  5) Termination of reactions by adding 150 μL methanol containing 200 nM levallorphan, as an internal standard

  6) Incubation 30 min

  7) Protein removed with centrifugation at 14,000 g for 5 min

  8) 50 μL of the resultant supernatant was injected into an ultra-performance liquid chromatography (UPLC)-fluorescence (FLR) detector system

  ##### Conditions of LC-MS/MS performance

  LC-MS/MS system was used to measure the (S)-Mephenytoin 4'-hydroxylation activity on CYP2C19.

  4'-Hydroxy mephenytoin was measured using the LC-MS/MS system in the positive ion detection mode at the electrospray ionization interface. Separation by LC was conducted using NANOSPACE SI-2. Chromatographic separation was performed using a Luna C18 100A column (2×150 mm, 5.0-μm particle size) maintained at 40°C. The flow rate was set at 300 μL/min, and the mobile phases were formed using water containing 0.1% formic acid as eluent A and acetonitrile containing 0.1% formic acid as eluent B.

  The gradient program was as follows: initial elution with 100% A, followed by a linear gradient to 50% B from 3.0 to 4.0 min to 90% B from 4.0 to 6.0 min, and to 100% B from 6.0 to 8.0 min, held at 100% B for 12.0 min, and then immediately returned to initial conditions and maintained for 3.0 min until the end of the run.

  LC effluent was introduced into the mass spectrometer between 3.0 and 12.0 min after injection. Quantification analyses were performed in the selected reaction monitoring mode, in which transitions from the precursor into the product ion were monitored: m/z 219.1→134.2 for (S)-mephenytoin (collision energy, 10 V), m/z 235.1→150.2 for 4'-hydroxy mephenytoin (collision energy, 17 V), and m/z 238.1→150.2 for 4'-hydroxy mephenytoin-d3 (collision energy, 19 V).

  The optimized parameters for MS were as follows: spray voltage, 3.0 kV; sheath gas pressure, 50 psi; vaporizer temperature, 450°C; capillary temperature, 300°C; and collision pressure, 1.5 mTorr. The sheath gas was nitrogen, and the collision gas was argon. The LC-MS/MS system was controlled by Xcalibur software, which was also used to analyze the attained data. Standard curves for 4'-hydroxy mephenytoin were constructed in the 0.25-5.0 μM range using authentic metabolite standards.

For CYP2C19, the microsomal fraction obtained from 293FT cells was incubated in a mixture containing reagents and as substrate (S)-mephenytoin in variable concentrations (5, 10, 25, 50, 100, 250, 500, 1000 μM).

The determination of (S)-mephenytoin 4'-hydroxylation in 50 μg of the microsomal protein fractions (containing wild-type CYP2C19 or CYP2C19 variants) showed that 4'-hydroxy mephenytoin formation was linear for incubations of up to 30 min. After protein removal by centrifugation at 14,000 g for 5 min, 10 μL of the supernatant was injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system.

The kinetic parameters (Michaelis constant (Km), maximum velocity (Vmax), and intrinsic clearance (CLint = Vmax/Km) were determined using the Enzyme Kinetics Module of SigmaPlot 12.5.

![](https://static.igem.org/mediawiki/2021/a/af/T--Patras--mephenytoin-curves.png)

Michaelis-Menten curves for wild type and variant CYP2C19. Determination of kinetic parameters of (S)-mephenytoin 4'-hydroxylation

The Michaelis-Menten kinetic parameters were measured for the mephenytoin 4'-hydroxylation for the wild type and the 9 CYP2C19 variant proteins, excepted the variant p.R26\* because it results in a stop codon and no protein can be synthesized. Below are the means ± standard deviation (SD) of as derived from the three independently performed catalytic assays compared with wild-type CYP2C19. Differences with \*p < 0.05 and \*\*\*p <0.005 were considered significant

| # | CYP2C19 Variants | Km (μM) | V max (nmol/min/mg protein) | CLint (mL/min/mg protein) (% of Wild-type) |
| --- | --- | --- | --- | --- |
| 1 | Wild-type | 0.118 ± 0.013 | 0.278 ± 0.005 | 2.368 ± 0.230 (100.00) |
| 2 | p.L15P | 0.119 ± 0.002 | 0.152 ± 0.001\*\*\* | 1.274 ± 0.029 (53.80) |
| 3 | p.E274G | 0.136 ± 0.010 | 0.041 ± 0.001\*\*\* | 0.306 ± 0.023\* (12.92) |
| 4 | p.N286D | 0.177 ± 0.013 | 0.027 ± 0.001\*\*\* | 0.152 ± 0.009\* (6.42) |
| 5 | p.D293G | N.D. | N.D. | N.D. |
| 6 | p.T304A | 0.219 ± 0.007\* | 0.114 ± 0.002\*\*\* | 0.521 ± 0.011\* (22.00) |
| 7 | p.I327V | 0.132 ± 0.005 | 0.073 ± 0.001\*\*\* | 0.550 ± 0.018\* (23.22) |
| 8 | p.L380P | N.D. | N.D. | N.D. |
| 9 | p.L413M | 0.133 ± 0.013 | 0.080 ± 0.003\*\*\* | 0.605 ± 0.047\* (25.55) |
| 10 | p.F487S | N.D. | N.D. | N.D. |

Representation of kinetic parameters of mephenytoin 4'-hydroxylation by microsomes from 293FT cells expressing wild-type and variant CYP2C19 proteins.

From the measurement of 9 types of CYPC19 variants, the kinetic parameters of p.D293G, p.L380P, and p.F487S could not be determined because the amount of the metabolite produced was at or below the detection limit. Proceeding to the analysis of the enzymatic activity of each variant compared against the wild type CYP2C19, the following charts are obtained.

![Responsive image](https://static.igem.org/mediawiki/2021/f/fd/T--Patras--chart1-cypc19.png)

![Responsive image](https://static.igem.org/mediawiki/2021/b/b3/T--Patras--chart2-cyp2c19.jpg)

Mephenytoin 4'-hydroxylation activity by wild-type and variant CYP2C19 proteins

It is observed that all the variant CYP2C19 exhibited a decreased enzyme activity, with most of them appearing a 20% activity of the wild type CYP2C19. Precisely, for p.T304A, p.I327V, and p.L413M the enzymatic activity of variant CYP2C19 was 22.02%, 23.25%, and 25.84%, respectively.

Among the variant, two showed significantly decreased activity, the p.E274G with 12.9 % of the activity of wild type and p.N286D with even more significant reduction of the activity, presenting a 6.42% of the activity of wild type.

The variant, p.L15P appears a 53.80% of the activity of wild type, indicating that the substitution may occur at a site that does not affect the interaction with the substrate as on the other variants.

* More details about LC/MS-MS for CYP2C19

  On CYP2C19, dextromethorphan O-demethylation activity was measured as described below.

  1) An incubation mixture prepared containing:
  + • microsomal fraction (50 μg) obtained from 293FT cells
  + • (S)-mephenytoin in concentrations 5, 10, 25, 50, 100, 250, 500, 1000 μ
  + • 3.3 mM MgCl2
  + • and 100 mM potassium phosphate buffer (pH 7.4) in a final volume of 150 μL

  2) Incubation at 37°C for 3 min

  3) Initiation of reactions by adding 15 μL of a mixture containing:
  + • 1.3 mM NADP+
  + • 3.3 mM glucose-6-phosphate
  + • and 0.4 U/mL glucose-6-phosphate dehydrogenase

  4) Incubation at 37°C for 30 min

  5) Termination of reactions by adding 150 μL acetonitrile containing 10 μM 4'-hydroxy mephenytoin-d3, as an internal standard

  6) Incubation 30 min

  7) Protein removed with centrifugation at 14,000 g for 5 min

  8) 10 μL of the resultant supernatant was injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system

  ##### Conditions of UPLC performance

  UPLC was performed to measure the dextromethorphan O-demethylation activity on CYP2D6.

  The UPLC-FLR system was comprised of an ACQUITY UPLC H-Class, connected to an ACQUITY UPLC FLR Detector. Separation was performed on an analytical column (ACQUITY UPLC CSH Fluoro-Phenyl, 2.1⋅75 mm, 1.7-μm particle size; Waters Corporation) maintained at 40°C. The mobile phase was composed of 75% buffer (20 mM potassium phosphate and 20 mM hexane sulfonic acid, pH 4.0) and 25% acetonitrile. The flow rate was set at 0.5 mL/min with fluorescence detection at excitation and emission wavelengths of 280 nm and 310 nm, respectively. Standard curves for dextrorphan were constructed in the 0.5-100 μM range using authentic metabolite standards.

From the in-vitro assays, we have evidence about how every variant, alongside its consequent substitution on the amino acid sequence, affects the enzyme’s activity and determines the variant CYP2D6/CYP2C19 enzyme activity compared against the wild type’s activity. However, it would be difficult to assess the clinical outcome in subjects who express those CYP2D6 and CYP2C19 variants without in vivo data. To better understand our findings, it would be great to examine the clinical relationships among CYP2D6 and CYP2C19 variants and the plasma concentrations of the given drug-substrate and its metabolite. Thus, further studies will be needed to define the importance of the above novel variants in clinical settings.

\*The results presented in this section are a part of the collaboration between our Laboratory of Pharmacogenomics and Individualized Therapy, School of Pharmacy, University of Patras with the Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences at Tohoku University, Japan.

![]()![iGEM Patras 2021 logo](https://static.igem.org/mediawiki/2021/f/f9/T--Patras--patras-footer.png)

University of Patras, Campus, Rio

**Email:** igempatras@gmail.com

#### About Us

iGEM Patras is proposing a comprehensive workflow for the identification & functional characterization of PGx variants.

#### Sponsors

We are thankfull to all our sponsors. Without them this project would not be possible.

#### Follow Us

© Copyright **iGEM Patras 2021**. All Rights Reserved