iGEM 2023 — Team tecmonterreygdl

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## FRET-EryK Benchling Notebook

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## AtPCS Benchling Notebook

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## ECFP cloning and device calibration Benchling Notebook

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## Hardware Timeline

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### 📍 February

**February 28, 2023**

* First meeting with the 'iGem-NeoTech' group and consolidation of the Hardware team.

### 📍 March

**March 10, 2023**

* First internal meeting of the 'hardware team', background research and brainstorming for the generation of a device.

**March 21, 2023**

* Introduction to the laboratory, the team learns in more detail about the objectives and progress of the laboratory work, and makes a first approach to a concept for the development of a device.

**March 25, 2023**

* First concept generation in CAD (including geometry, manufacturing and material selection) using SolidWorks.

### 📍 April

**April 5, 2023**

* 3D printing of the first model using PLA.

**April 6, 2023**

* First test of the assembled model, improvements and corrections are proposed.

**April 7, 2023**

* The hardware team collaborates and it is introduced as a section on the Wiki.

**April 10, 2023**

* Weekly 'hardware team' meeting; areas are created to distribute assignments (CAD design, circuits-instrumentation, wiki and finance).

**April 15, 2023**

* Modifications and improvements in the CAD design for the first prototype (LED-LDR) and re-printing.

**April 28, 2023**

* Instrumentation of the model using different excitation sources (LEDs).

### 📍 May

**May 17, 2023**

* Tests in the biotech lab with the fluorescent protein 'mCherry' on the first prototype.

**May 23, 2023**

* Weekly team meeting, model reassessment (introduction of TCS320 RGB color sensor module).

### 📍 June

**June 8, 2023**

* CAD model redesign, validation and approval of model dimensions.

**June 15, 2023**

* Printing and device assembly tests.

**June 26, 2023**

* Final print of the second prototype, assembly and electrical instrumentation.

### 📍 July

**July 10, 2023**

* 3D model assembly in the laboratory. Tests were performed with various materials to test and characterize the system. Finally we tested with 'mCherry' protein in different concentrations, the reading of the samples was performed in order to evaluate fluorescence.

**July 23, 2023**

* Initiates development of software for intensity scaling.
* Transilluminator tests for evaluation of mCherry excitation.
* New dilutions of mCherry and mCherry induced in E.Coli were tested.
* An adjustment is made to the model, and two UV light LEDs are introduced to the design.

**July 24, 2023**

* The model is adjusted in the CAD software, and re-printed, generating the third prototype.

**July 25, 2023**

* Implementation of UV excitation source (UV led 390-400 nm) in the device and tests with pure mCherry.
* Prototype redesign, the geometry of the model is restructured and the sensor is repositioned. The sensor model is changed and a sketch of the new model is generated. More UV LEDs are added to the model.
* New CAD model is generated and 3D printed (fourth prototype). Assembly tests are performed and the model is electronically instrumented.
* Implementation of a new color sensor TCS34725 (Color Light-to-Digital Converter with IR Filter TCS34725, TAOS™).
* Laboratory tests with the new color sensor and mCherry.

**July 26, 2023**

* Dataset development started.
* Tests and measurements with EGFP, RFP and YFP (donated by Mexico's City Monterrey Institute of Technology and Higher Education).

### 📍 August

**August 2, 2023**

* Compilation of photos from results and advances.
* Started Wiki formatting.

**August 9, 2023**

* Laboratory tests and collecting data from four different proteins (EGFP, RFP, YFP, and ECFP).

**August 11, 2023**

* Laboratory tests and collecting data from four different proteins (EGFP, RFP, YFP, and ECFP).

**August 23, 2023**

* Continue to describe more in depth prototypes revisions in Wiki.

### 📍 September

**September 3, 2023**

* Welding of electronic components.

**September 5, 2023**

* Started development of an interface.
* Implementation of a bluetooth module.
* New CAD model is designed.

**September 15, 2023**

* Due to time constraints, bluetooth and interface development were suspended.
* The 3D model is printed.
* Device assembly and testing.
* Device completed.

**September 22, 2023**

* Laboratory tests and collecting data from EGFP, RFP, YFP, and ECFP.

### 📍 October

**October 5, 2023**

* Laboratory tests and collecting data from EGFP, RFP, YFP, and ECFP.

**October 6, 2023**

* Laboratory tests and collecting data from EGFP, RFP, YFP, and ECFP.

**October 9, 2023**

* Laboratory tests and collecting data from EGFP, RFP, YFP, and ECFP.

**October 10, 2023**

* Cleaned and shaped the database.
* Development of machine learning algorithm K-Nearest Neighbors.
* Training the algorithm.
* Testing, tuning and evaluation of the machine learning model.
* Wiki corrections and concluding.

## Human Timeline

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### 📍 January

**January 07, 2023**

* First meeting with the human practices team.

### 📍 February

**February 14, 2023**

* Project planning session with the advisor.

**February 19, 2023**

* Conference and feedback day with Dr. Pablo Nikel.

**February 23, 2023**

* Meeting with the communication leader to plan work resources via Zoom.

**February 28, 2023**

* First meeting with the 'iGem-NeoTech' group.

### 📍 March

**March 1, 2023**

* Meeting with Ernesto, contact to do workshops for blind children.

**March 6, 2023**

* Meeting with the team to create the questions for the interviews and the surveys in the town "El Salto".

**March 16, 2023**

* Meeting with the group "Women for the future" to organize a collaboration for Women in STEM.

**March 19, 2023**

* Second meeting with the group "Women for the future" to organize the event.

**March 23, 2023**

* Sensing day to the community "El Salto" with the entire team.

**March 29, 2023**

* First meeting with the "wiki" team.

### 📍 April

**April 17, 2023**

* Scientific lottery design

**April 24, 2023**

* Organization of the "Women in STEM" round table and conference.

### 📍 May

**May 17, 2023**

* We sent emails to experts requesting interviews via Zoom for feedback on the project.
* The round table “Women in STEM” was carried out.

**May 17, 2023**

* The round table “Women in STEM” was carried out.

**May 20, 2023**

* The association SINGREM was contacted via email.

**May 23, 2023**

* Weekly team meeting.

**May 25, 2023**

* The coordinator of SINGREM OCCIDENTE replied to the email.

**May 30, 2023**

* The coordinator of SINGREM OCCIDENTE was contacted through a cell phone call so that the project could be proposed.

**May 31, 2023**

* Interview with expert Dr. Luis Figueroa for feedback via zoom.
* The coordinator of SINGREM OCCIDENTE sent the archives for the collaboration.

### 📍 June

**June 8, 2023**

* Interview with expert Dr. Jorge Rodriguez for feedback via zoom.

**June 12, 2023**

* Meeting with entrepreneurship specialist.

**June 15, 2023**

* We had the first meeting to start with the entrepreneurship part of the project.

**June 21, 2023**

* The academic advisor, Carlos Domenzain, helped us to send the archives of SINGREM to all the areas of the campus that were involved in the installation of the containers.

### 📍 July

**July 10, 2023**

* Weekly team meeting.

**July 29, 2023**

* The project of the installation of the containers was proposed to “Planta física” and approved by them.

### 📍 August

**August 2, 2023**

* Compilation of photos from results and advances.
* Started Wiki formatting.

**August 7, 2023**

* First check up entrepreneurship information for Wiki.

**August 9, 2023**

* Weekly team meeting.

**August 16, 2023**

* The legal leader Western Region . GDA Campus, Guillermo Solorio, was contacted via email.

**August 22, 2023**

* The Urban Forest Agency of the Guadalajara Metropolitan Area was contacted via whatsapp.

**August 23, 2023**

* Revisions in Wiki.

**August 24, 2023**

* Reunion with The Urban Forest Agency of the Guadalajara Metropolitan Area to propose the idea of the workshops.

### 📍 September

**September 9, 2023**

* The document where the project is presented in detail is sent to The Urban Forest Agency of the Guadalajara Metropolitan Area through email.

**September 6, 2023**

* The legal leader Western Region validates with different areas of the campus, the installment of the containers.

**September 8, 2023**

* The Urban Forest Agency of the Guadalajara Metropolitan Area through email approved the project of imparting workshops in the Bosque Urbano Tlaquepaque.

**September 14, 2023**

* The legal leader asked for more details about the containers and the legal documentation of SINGREM i.
* The itinerary for the workshops with the collaboration of The Urban Forest Agency of the Guadalajara Metropolitan Area was approved i.

**September 20, 2023**

* Via email the details and the legal documentation of SINGREM were asked for.

**September 21, 2023**

* The coordinator of SINGREM OCCIDENTE sent the documents that were asked for.

**September 23, 2023**

* Workshops in “Bosque urbano Tlaquepaque”.

**September 25, 2023**

* The School for Blind Children A.C. was contacted.

**September 27, 2023**

* The legal documentation of SINGREM was sent to the legal leader as well as a document with the specifications he asked for.
* The nursing home Residencia Guadalupe was contacted via email.

**September 28, 2023**

* The coordinator of Residencia Guadalupe responded and established a date for the visit.

**September 29, 2023**

* The School for Blind Children A.C deliberated if the collaboration could be made.

### 📍 October

**October 2, 2023**

* The collaboration with the School for Blind Children A.C was approved.

**October 4, 2023**

* Visit to the School for Blind Children A.C.

**October 5, 2023**

* Visit to the nursing home “Casa Guadalupe”.

**October 6, 2023**

* Meeting with the team for wiki corrections.
* Specialists entrepreneurship meeting.
* The green light is given to the signing of the agreement between our university and the association SINGREM to proceed with the containers installation.

**October 7, 2023**

* Team member, Sofia Méndez, at the Latin American Congress of Engineers in Medicine and Biology.

**October 9, 2023**

* Meeting with the team for wiki corrections.

**October 10, 2023**

* Wiki corrections and concluding.
* Meeting with the team for wiki corrections.

**October 11, 2023**

* Entrepreneurship Wiki corrections and concluding.

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## EryK-FRET Overview

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Emerging contaminants in water pose a significant risk to long-term health (Pereira *et al*., 2015; Stuart *et al*., 2012). Current detection methods require specialized laboratory equipment and trained personnel, thus we aimed to develop a quick and easy fluorescence-based detection method for erythromycin. To generate a prototype, we went through iterations of the engineering design process to test and improve upon different aspects of the project such as transformation efficiency and protein overexpression yield.

For donor fluorescence to occur, the chromophores in the system must be close enough in space for FRET to be possible, allowing acceptor fluorescence to occur. With our ECFP-EryK-mVENUS fusion construct, if erythromycin is present, EryK will ideally undergo a conformational change that should alter the distance between the donor, ECFP, and the acceptor, mVENUS **(Figure 1)**.

![](https://static.igem.wiki/teams/4763/wiki/engineering/eryk/fig1.png)
**Figure 1.** Schematic diagram of a single molecule FRET system in the presence of erythromycin. The expected conformational change upon erythromycin binding is denoted, as well as the change in fluorescence signal.

To achieve this, we started by first transforming *Escherichia coli* TOP10 as a maintenance strain and then *E. coli* BL21 with a plasmid encoding the 6xHis tagged ECFP-EryK-mVENUS fusion construct (Chan *et al*., 2013; Jeong *et al*., 2015). After expression, our construct will be purified which will produce a detectable fluorescent signal change when bound to erythromycin **(Figure 2)**.

![](https://static.igem.wiki/teams/4763/wiki/engineering/eryk/fig2-n.png)
**Figure 2.** The ECFP-eryK-mVENUS fusion construct will be obtained through transformation of *E. coli* BL21 with a plasmid encoding our construct, inducing overexpression, and subsequent purification from the cell lysate.

### 📐 Design

To assess the feasibility of our approach, protein structure predictions and molecular dynamics simulations were carried out to ensure the fluorophores of the fusion construct were within a functional FRET distance.

#### ECFP-EryK-mVENUS model

Using the *ab initio* structure prediction workflow and ColabFold (Mirdita *et al*., 2022), a predicted structure of the ECFP-EryK-mVENUS fusion construct was obtained **(Figure 3)**. ColabFold produces 5 predictions, however only the one with the best predicted aligned error (PAE) will be considered, as we are mainly interested in the 3D arrangement of the three constituent proteins.

![](https://static.igem.wiki/teams/4763/wiki/engineering/eryk/fig3.png)
**Figure 3.** Predicted structure with the best PAE obtained from ColabFold showing ECFP (blue), EryK (gray), and mVENUS (yellow).

While the 3D fold of each constituent protein matches previously characterized structures (Montemiglio *et al*., 2013; Pletnev *et al* 2012; Park *et al*., 2016), the connector regions and geometrical relationship between each protein is predicted with low confidence. This is indicated by the PAE values **(Figure 4)**.

![](https://static.igem.wiki/teams/4763/wiki/engineering/eryk/fig4.png)
**Figure 4.** PAE (top) and predicted local difference test (bottom) values obtained from the 5 ColabFold predicted structures.

It should be noted that ColabFold uses multiple sequence alignments and neural networks to predict a 3D structure, however it doesn't consider real-world biophysics and interactions with solvent, spontaneous changes between open and closed conformations, and other possible variables.

#### Molecular dynamics

To increase our confidence in the predicted structure of our construct, molecular dynamics (MD) simulations of the ColabFold model were performed to assess if the predicted structure obtained was maintained after biophysical interactions were introduced to the system.

To achieve this, a preliminary simulation was run with GROMACS using model 5 from Colabfold to assess whether the structure eventually converges over the course of the 10 ns simulation when including biophysical potentials. First, the topology was generated from the ColabFold model with the AMBER99SB-ILDN force field (Lindorff-Larsen *et al*., 2010) and a TIP3P (Jorgensen *et al*., 1983) water model. The model was put in a simulation box with a distance to the edge of 1.2 nm. The system was solvated in water and the charges were neutralized with Na+ ions. After this, the system was energy minimized to avoid problems that arise from disagreement between ColabFold's predicted structure and the energy minimum of our system according to the AMBER99SB-ILDN forcefield. To equilibrate the system, first a NVT simulation was run for 200 ps followed by a NPT simulation for 1 ns, both with 2 fs timesteps. The production MD was run for a total of 10 ns with 2 fs timesteps under NPT conditions **(Figure 5)**. Simulations were performed according to previous work (Lemkul, J. A., 2019) and available documentation (Hess *et al*., 2008).

![](https://static.igem.wiki/teams/4763/wiki/engineering/eryk/fig5.gif)

**Figure 5.** Trajectory (100 ps frame-1) of ECFP-EryK-mVENUS compared to predicted ColabFold (gray) structure.

To ensure the reliability of the simulation, thermodynamic variables such as the temperature, pressure, density, and total energy of the system were monitored for convergence. It should also be noted that after 4 ns the root-mean-square deviation of the simulation stopped diverging from the energy-minimized ColabFold prediction. Interestingly, the most dynamic regions of our system **(Figure 5)** correspond to the regions that display the greatest conformational change when comparing X-ray structures of the open and closed conformations of EryK **(Figure 6)**. A potential limitation of our *in silico* system is that the ionic strength may not match *in vitro* conditions thus altering the strength of electrostatic interactions (Zhou & Pang, 2018). Nevertheless, the simulation was promising and yields greater confidence in the predicted structure.

![](https://static.igem.wiki/teams/4763/wiki/engineering/eryk/fig6.gif)

**Figure 6.** Morph of EryK in an open conformation (PDB ID: 3ZKP) to a closed conformation (PDB ID: 2JJN).

### 🏗️ Build

The original plasmid was made up of a pUC57 backbone and the insert for ECFP-EryK-mVENUS. As an initial step we digested and ligated the insert into a pBAD/HisB and pBAD/HisC backbone with XhoI and NcoI (New England Biolabs). Both the vector and the insert were digested with the enzymes **(Figure 7A)** and, after gel purification and ligation, transformed into *E. coli* TOP10 as a maintenance strain **(Figure 7B)**.

![](https://static.igem.wiki/teams/4763/wiki/engineering/eryk/fig7.png)
**Figure 7.** **A)** Electrophoresis gel results on (1% agarose) from digestion of pBAD/hisB, pBAD/hisC, and ECFP-EryK-mVENUS with XhoI and NcoI and **B)** Transformation into TOP10.

Colony polymerase chain reaction (PCR) was performed to confirm successful transformation **(Figure 8)**. Following this, miniprep was performed on 5 mL liquid cultures from the transformation palate, these were grown in LB and carbenicillin and incubated overnight at 37 ºC with agitation at 200 rpm. For miniprep the Wizard Plus SV Minipreps DNA Purification Systems (Promega) was used.

![](https://static.igem.wiki/teams/4763/wiki/engineering/eryk/fig8-not8.png)
**Figure 8.** Electrophoresis gel results (1 %) of colony PCR from TOP10 transformation

The subsequent PCR step was performed several times varying annealing temperatures, number of cycles, and elongation step duration. The backbone **(Figure 9 A)** and the insert **(Figure 9 B)** were successfully amplified. After this the fragments were ligated together using T4 DNA Ligase Reaction Buffer and T4 DNA Ligase (New England Biolabs).

![](https://static.igem.wiki/teams/4763/wiki/engineering/eryk/fig8.png)
**Figure 9.** PCR reaction results for **A)** pET28b backbone and **B)** insert.

The next step was to transform pET28b(+)/ECFP-EryK-mVENUS into *E. coli* BL21, an expression strain. This proved difficult as the process was repeated several times varying amounts of plasmid added and incubation times with no success. Some of the mistakes made include not using enough plasmid during the transformation, not letting the transformation reaction recover for enough time, or the temperature for heat shock being too high. Once optimized, the transformation was successful **(Figure 10)**.

![](https://static.igem.wiki/teams/4763/wiki/engineering/eryk/fig9.png)
**Figure 10.** Transformation of BL21 with **A)** ECPF-EryK-mVENUS and **B)** pET28b(+) empty vector. Both plates were exposed to UV light to confirm fluorescence.

### 📊 Test and Optimization

Once the plasmid with the whole construct was successfully transformed into *E. coli* BL21, the cultures were induced with 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) to stimulate protein overexpression (Studier, F. W., 2014). This was repeated several times with different temperatures and for different periods of time, the last attempt at 16 ºC for 12 hours yielded a faint band at the approximate expected molecular weight of our construct (~83 kDa) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) **(Figure 11)**. Despite not seeing a clear band from the expression, we proceeded with the purification.

![](https://static.igem.wiki/teams/4763/wiki/engineering/eryk/fig10.png)
**Figure 11.** SDS-PAGE gel (8 %) ran to confirm overexpression of ECFP-EryK-mVENUS after induction with IPTG.

Purification was done through a Ni-NTA resin (Sigma-Aldrich) and all elutions were run on an SDS-PAGE gel to confirm the presence of our protein. Unfortunately, no protein was purified **(Figure 12)**.

![](https://static.igem.wiki/teams/4763/wiki/engineering/eryk/fig11.png)
**Figure 12.** SDS-PAGE gel (8 % and 4 %) ran to confirm purification of ECFP-EryK-mVENUS.

As we couldn't purify the complex, different tests were performed to assess viability of transformed *E. coli* in the presence of erythromycin. LB plates were made with different concentrations of erythromycin 0.2 μg mL-1, 2.5 μg mL-1, and 10 μg mL-1. Liquid cultures were made of transformed *E. coli* BL21 and plated onto these petri dishes. This was done to determine if *E. coli* remained viable after exposure to the antibiotic. Once colony growth and fluorescence were observed **(Figure 13)**, it was determined that there is a detectable level of fluorescence when exposed to erythromycin, however, it was not established whether fluorescence was directly related to erythromycin or was basal.

![](https://static.igem.wiki/teams/4763/wiki/engineering/eryk/fig12.png)
**Figure 13.** Transformed colonies on erythromycin plates at **A)** 0 μg mL-1, **B)** 0.02 μg mL-1, **C)** 2.5 μg mL-1, and **D)** 10 μg mL-1.

### 🦾 Future steps

Optimizing overexpression for subsequent protein purification is the next step. This will be done in the hopes of adding the purified protein directly to the sample with erythromycin to ensure proper detection and remove the risk of unwanted mutations and genetic variability caused by evolutionary pressure. Additionally, this will help calibrate the baseline of fluorescence with different concentrations of antibiotic to improve read accuracy and effectiveness.

In the future, and provided that ligand binding induces conformational changes in the sensor, EryK could be switched out for a different protein. This would allow detection of different emerging contaminants such as other antibiotics, per- and polyfluorinated substances, and heavy metals.

## AtPCS as a new biopart overview

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### 🤔 What's the deal with AtPCS?

Current FRET system design incorporating biopart **BBa\_K4447001** would allow us to determine the presence of erythromycin in water bodies, which represents a first step towards the detection of antibiotics, emerging contaminants of significant importance. An advantage of our FRET system is the potential to replace biopart **BBa\_K4447001** with other genes that are compatible with different compounds such as other antibiotics or heavy metals.

* Pollution of water bodies with heavy metals is very threatening as they are persistent, non-biodegradable and toxic.This applies to any metal with a density greater than 5 g cm-3 which is harmful even at low concentrations. Arsenic, cadmium, cupper, chromium, mercury, lead, nickel, and zinc are classified as heavy metals. These often originate from various sources such as volcanic activities, soil erosion, fertilizers, pesticides, industrial waste, and others. For each of the aforementioned metals, there are maximum tolerable levels that, when exceeded, can cause poisoning, brain damage, and even initiate cancer in humans (Jyoti *et al*., 2022).

* Cadmium is classified as one of the five priority heavy metals for public health due to its high degree of toxicity. Various studies prove carcinogenic properties linked to cadmium and prolonged exposure to it both in humans and animals (Tchounwou *et al*., 2012). Additionally, cadmium occupies the second position of the metals with the highest toxicity in cells, according to Lin *et al*. (2016), surpassed only by mercury.

The aforementioned reasons led us to search for an enzyme that catalyzes a reaction involving this metal, with the aim of generating a new biopart and be able to incorporate it into the biosensor, as a step towards detecting heavy metals in water bodies. The enzyme chosen for the generation of the biopart was phytochelatin synthase (PCS) (EC 2.3.2.15), which catalyzes the synthesis of glutathione (GSH) polymers called phytochelatins (PCs), with cadmium being the main inducer in plants and other organisms (García-García *et al*., 2014; 2020).

### ⚡ Molecular dynamics

As with the structure for ECFP-EryK-mVENUS, a ColabFold model was obtained using the provided sequence for AtPCS (UniProt: Q9S7Z3) **(Figure 14)**.

![](https://static.igem.wiki/teams/4763/wiki/engineering/atpcs/fig14.png)
**Figure 14.** Predicted structure with the best PAE obtained from ColabFold showing the modeled AtPCS sequence.

The sequence corresponds to a monomer and this was properly modeled by ColabFold with a high amount of confidence according to the PAE and PLDDT values **(Figure 15)**.

![](https://static.igem.wiki/teams/4763/wiki/engineering/atpcs/fig15.png)
**Figure 15.** PAE (top) and predicted local difference test (bottom) values obtained from the 5 ColabFold predicted structures of AtPCS.

MD simulations were performed to assess if the predicted structure obtained of the system was conserved after including biophysical potentials. A preliminary simulation was run with GROMACS using model 5 from ColabFold. The topology was generated with the same force field (AMBER99SB-ILDN) and water model (TIP3P) that was used for the simulation of the other construct (Lindorff-Larsen *et al*., 2010; Jorgensen *et al*., 1983) . The simulation was run in the same way and with the same parameters as the one for ECFP-EryK-mVENUS **(Figure 16)**.

![](https://static.igem.wiki/teams/4763/wiki/engineering/atpcs/fig16.gif)
**Figure 16.** Trajectory (100 ps frame-1) of AtPCS compared to predicted ColabFold (gray) structure.

Contrary to the ECFP-EryK-mVENUS model, there are no published experimental structures of AtPCS1. The simulation shows that the protein structure does not significantly diverge from the ColabFold model and, while it doesn't encompass our entire construct, it is still informative as the fluorophores will not induce conformational changes. On the other hand, it would be necessary to run a simulation with the presence of the ion to determine whether the enzyme would undergo a conformational change when catalyzing the reaction.

### 🧪 Experimental design and future perspectives

#### Clone AtPCS into expression vector pET-28b(+)

The goal of this was to construct the vector that would allow the expression of AtPCS in *E. coli*. In order to achieve this, the following workflow was necessary:

* Amplification of AtPCS by PCR.
* Confirmation of the correct amplification and integrity of the DNA by agarose gel electrophoresis and subsequent purification.
* Digestion reaction of AtPCS and pET-28b(+) with EcoRI and NdeI (New England Biolabs) to create complementary sticky ends.
* Ligation reaction with T4 DNA ligase (New England Biolabs) to generate the complete expression vector.
* Transformation of competent *E. coli* TOP10 cells by heat shock.
* Selection of transformants by cultivating with kanamycin.
* Confirmation of plasmid length by colony PCR and enzymatic digestion.

This goal was successfully achieved as shown in **Figure 17**, where a band corresponding to the length of the AtPCS gene was identified in sample D1 from colony 1 after a digestion assay with enzymes NdeI and EcoRI HF.

![](https://static.igem.wiki/teams/4763/wiki/engineering/atpcs/fig-16-n.png)

**Figure 17.** Digestion of pET28b(+) plasmid with NdeI and EcoRI HF restriction enzymes. The ladder used was 1kb Plus ladder from Invitrogen. A positive result is observed in colony 1.

#### Induce protein overexpression to confirm the production of the enzyme in *E. coli*

The goal of this step was to confirm that *E. coli* was able to synthesize the AtPCS enzyme. To achieve this, the following workflow was carried out:

* Transformation of competent *E. coli* BL21 cells with AtPCS-pET-28b(+).
* Induction with 0.4 mM IPTG.
* Cell lysis by sonication.
* SDS-PAGE to confirm the presence of the expected enzyme.

This goal was partially achieved, as several attempts were made to induce expression of AtPCS. Induction was attempted with different temperatures and for different periods of time, the last attempt at 16 ºC overnight produced a very faint band (like a stain) on the SDS-PAGE gel, as shown in **Figure 18**.

![](https://static.igem.wiki/teams/4763/wiki/engineering/atpcs/fig-17-n.png)
**Figure 18.** SDS-PAGE gel showing protein overexpression results for induced colonies. In wells corresponding to colonies 4-7 supernatants, a very faint blurred band near to 55 kDa (AtPCS molecular weight) is visible, contrary to negative control.

Multiple attempts were carried out to overexpress the protein, varying the induction conditions (incubation time and temperature) in an attempt to optimize them; however, no band was observed on the gel after performing the purification process using the colony 1 culture. After setting a 5 mL pre-culture from colony 4, we proceeded with inoculation, cell harvesting and sonication. This was done to continue with a final attempt of purification through a Ni-NTA resin (ThermoFisher). An SDS-PAGE was run to confirm the presence of our protein. Unfortunately, no protein was purified **(Figure 19)**.

![](https://static.igem.wiki/teams/4763/wiki/engineering/atpcs/fig-18-n.png)
**Figure 19.** SDS-PAGE gel (8 % and 4 %) ran to confirm purification of AtPCS.

Optimization of induction conditions are needed for confirmation of AtPCS enzyme production. To complete the incorporation of the enzyme into the FRET system, the plans for future activities are as follows:

#### 1. Purify AtPCS and characterize its activity.

This is meant to indicate whether AtPCS produced by *E. coli* is able to interact with cadmium, which is necessary for its incorporation into the FRET system, as it would allow the detection of this heavy metal. Furthermore it would help determine whether a conformational change occurs or not.

#### 2. Clone AtPCS into an expression vector with ECFP and mVENUS to generate the full FRET system by Gibson assembly, a method that has advantages including its speed, versatility, and scarless vector assembly (Thomas *et al*., 2015).

Linking AtPCS with ECFP and mVENUS will allow the generation of a fluorescent signal once the enzyme interacts with cadmium. This step is meant to assemble the components of the FRET system so it can be produced by *E. coli*.

#### Overexpress the FRET system, purify and characterize it.

Once the protein construct is overexpressed it will need to be tested. To do so, the construct will be purified and then tested with different amounts of cadmium. We expect to be able to detect a change in FRET signal with an increasing intensity as the concentration of cadmium becomes higher.

Experiments undertaken and future endeavors are summarized and displayed in **Figure 20**.

![](https://static.igem.wiki/teams/4763/wiki/engineering/atpcs/fig-19.png)
**Figure 20.** AtPCS as a new biopart: Experimental design and future perspectives.

---

### 📖 References

1. Chan, W. T., Verma, C. S., Lane, D. P., & Gan, S. K. (2013). A comparison and optimization of methods and factors affecting the transformation of Escherichia coli. *Bioscience reports*, 33(6), e00086. <https://doi.org/10.1042/BSR20130098>
2. García-García, J. D., Sánchez-Thomas, R., Saavedra, E., Fernández-Velasco, D. A., Romero-Romero, S., Casanova-Figueroa, K. I., … Moreno-Sánchez, R. (2020). Mapping the metal-catalytic site of a zinc-activated phytochelatin synthase. *Algal Research*, 101890. doi:10.1016/j.algal.2020.101890
3. García-García, J. D., Girard, L., Hernández, G., Saavedra, E., Pardo, J. P., Rodríguez-Zavala, J. S., Encalada, R., Reyes-Prieto, A., Mendoza-Cózatl, D. G., & Moreno-Sánchez, R. (2014). Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metal-hyperaccumulator Euglena gracilis. *Metallomics : integrated biometal science*, 6(3), 604-616. <https://doi.org/10.1039/c3mt00313b>
4. Hess, B., Kutzner, C., van der Spoel, D., & Lindahl, E. (2008). GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. *Journal of chemical theory and computation*, 4(3), 435-447. <https://doi.org/10.1021/ct700301q>
5. Jeong, H., Kim, H. J., & Lee, S. J. (2015). Complete Genome Sequence of Escherichia coli Strain BL21. *Genome announcements*, 3(2), e00134-15. <https://doi.org/10.1128/genomeA.00134-15>
6. Jyoti, D., Sinha, R., & Faggio, C. (2022). Advances in biological methods for the sequestration of heavy metals from water bodies: A review. *Environmental toxicology and pharmacology*, 94, 103927. <https://doi.org/10.1016/j.etap.2022.103927>
7. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R., & Klein, M. L. (1983). Comparison of simple potential functions for simulating liquid water. *Journal of Chemical Physics*, 79(2), 926-935. <https://doi.org/10.1063/1.445869>
8. Lemkul, J.A. (2019). From Proteins to Perturbed Hamiltonians: A Suite of Tutorials for the GROMACS-2018 Molecular Simulation Package [Article v1.0]. *Living Journal of Computational Molecular Science*. <https://doi.org/10.33011/livecoms.1.1.5068>
9. Lin, X., Gu, Y., Zhou, Q., Mao, G., Zou, B., & Zhao, J. (2016). Combined toxicity of heavy metal mixtures in liver cells. *Journal of Applied Toxicology*, 36(9), 1163-1172. doi:10.1002/jat.3283
10. Lindorff-Larsen, K., Piana, S., Palmo, K., Maragakis, P., Klepeis, J. L., Dror, R. O., & Shaw, D. E. (2010). Improved side-chain torsion potentials for the Amber ff99SB protein force field. *Proteins*, 78(8), 1950-1958. <https://doi.org/10.1002/prot.22711>
11. Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S., & Steinegger, M. (2022). ColabFold: making protein folding accessible to all. *Nature methods*, 19(6), 679-682. <https://doi.org/10.1038/s41592-022-01488-1>
12. Montemiglio, L. C., Macone, A., Ardiccioni, C., Avella, G., Vallone, B., & Savino, C. (2013). Redirecting P450 ERYK specificity by Rational Site-Directed Mutagenesis. *Biochemistry*, 52(21), 3678-3687. <https://doi.org/10.1021/bi400223j>
13. Park, S. W., Kang, S. K., & Yoon, T. S. (2016). Crystal structure of the cyan fluorescent protein Cerulean-S175G. *Acta Crystallographica Section F: Structural Biology Communications*, 72(7), 516-522. <https://doi.org/10.1107/s2053230x16008311>
14. Pereira, L. C., de Souza, A. O., Bernardes, M. F. F., Pazin, M., Tasso, M. J., Pereira, P. H., & Dorta, D. J. (2015). A perspective on the potential risks of emerging contaminants to human and environmental health. *Environmental Science and Pollution Research*, 22, 13800-13823.
15. Pletnev, S., Subach, F. V., Dauter, Z., Wlodawer, A., & Verkhusha, V. V. (2012). A Structural Basis for Reversible Photoswitching of Absorbance Spectra in Red Fluorescent Protein rsTagRFP. *Journal of Molecular Biology*, 417(3), 144-151. <https://doi.org/10.1016/j.jmb.2012.01.044>
16. Stuart, M., Lapworth, D., Crane, E., & Hart, A. (2012). Review of risk from potential emerging contaminants in UK groundwater. *Science of the Total Environment*, 416, 1-21.
17. Studier F. W. (2014). Stable expression clones and auto-induction for protein production in E. coli. *Methods in molecular biology (Clifton, N.J.)*, 1091, 17-32. <https://doi.org/10.1007/978-1-62703-691-7_2>
18. Tchounwou, P. B., Yedjou, C. G., Patlolla, A. K., & Sutton, D. J. (2012). Heavy metal toxicity and the environment. *Experientia supplementum (2012)*, 101, 133-164. <https://doi.org/10.1007/978-3-7643-8340-4_6>
19. Thomas, S., Maynard, N. D., & Gill, J. (2015). DNA library construction using Gibson Assembly®. Nature Methods, 12(11), i-ii. doi:10.1038/nmeth.f.384
20. Zhou, H. X., & Pang, X. (2018). Electrostatic Interactions in Protein Structure, Folding, Binding, and Condensation. *Chemical reviews*, 118(4), 1691-1741. <https://doi.org/10.1021/acs.chemrev.7b00305>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Want to replicate our project?{% endblock %}
{% block page\_content %}

## EryK-FRET Protocols

---

### Transformation of BL21 by heat shock

**Transformation of BL21 by heat shock**

1. Add 100 - 200 ng of DNA plasmid to a 100 μL aliquot of competent cells
2. Incubate on ice for 45 minutes
3. Heat shock at 42 ºC for 45 seconds
4. Transfer to ice for 10 minutes
5. Add 1 mL LB
6. Incubate at 37 ºC for 1 hour with shaking (200 rpm)

### Sonication

**Sonication protocol**

1. To each sample add 5 mL of 50 mM tris
2. Sonicate at 70 % repeating 3 cycles of 15 seconds on, 60 seconds off
3. Centrifuge at 9,838 g during 10 min at 4 ºC

### SDS-PAGE gel (12% and 10%)

**12% resolving SDS-PAGE gel**

1. To a falcon tube add the components shown in the table in order
2. Using a micropipette add the prepared solution until below the top of the gel mold
3. Add ethanol until fully covered

| Reagent | Quantity [μL] |
| --- | --- |
| Acrylamide 30% | 3, 250 |
| Tris pH 8.8 | 1, 005 |
| Distilled water | 3, 370 |
| SDS 10% | 78 |
| APS | 45 |
| TEMED | 4 |

**10% stacking SDS-PAGE gel**

1. To a falcon tube add the components shown in the table below
2. If resolving gel is solidified, remove ethanol by tipping
3. Using a micropipette add the prepared solution until the top of the gel mold
4. Add the comb ensuring a good fit

| Reagent | Quantity [μL] |
| --- | --- |
| Acrylamide 30% | 500 |
| Tris pH 6.8 | 200 |
| Distilled water | 224 |
| SDS 10% | 30 |
| APS | 11.5 |
| TEMED | 2.25 |

**Notes**

* Add Ammonium persulfate (APS) and Tetramethylethylenediamine (TEMED) right before adding gel to the mold as it will begin polymerizing
* Use the remaining mixture in the tube to determine when gel has solidified

### Miniprep with QIAprep Spin Miniprep Kit

**Miniprep with QIAprep Spin Miniprep Kit**

1. Pellet overnight cultures by centrifugation (13,000 rpm, 2 min) adding 1.5 mL at a time
2. Decant supernatant
3. Add 250 μL of Cell Lysis Solution and invert until clear
4. Add 10 μL of Alkaline Protease Solution and invert to mix
5. Incubate at room temperature for 5 min
6. Add 350 μL of Neutralization Solution and invert until homogenized
7. Centrifuge at maximum speed (11.4 g) for 10 min
8. Assemble spin column and add lysate
9. Centrifuge at maximum speed for 1 min
10. Discard flow through
11. Add 750 μL of Wash Solution
12. Centrifuge at 11.4g for 1 min
13. Add 250 μL of Wash Solution
14. Centrifuge at maximum speed for 2 min
15. Transfer spin column to a new and sterile 1.5 mL Eppendorf tubes
16. Add 80 μL of nuclease free water
17. Centrifuge at maximum speed for 1 min
18. Discard column
19. Measure yields on Nanodrop
20. Store at -20 ºC until further usev

**Note**

* When loading the blank on the Nanodrop for calibration use the same liquid (buffer or water) used to elute

### Miniprep - Promega

**Cleared lysate**

1. Harvest cells by centrifugation at 4,000 rpm, 10 min, 4 ºC
2. Decant supernatant
3. Resuspend in 250 μL of Cell Lysis Solution
4. Invert 4 times to mix
5. Add 10 μL of Alkaline Solution
6. Invert 4 times to mix
7. Incubate at room temperature for 5 min
8. Add 350 μL of Neutralization Solution
9. Invert 4 times to mix
10. Centrifuge at 14.1 g (top speed) for 10 min

**DNA Plasmid binding**

1. Assemble spin column and collection tube
2. Add supernatant into the column
3. Centrifuge at top speed for 1 min
4. Discard flow through

**Washing**

1. Add 750 μL of Wash Solution
2. Centrifuge at top speed for 1 min
3. Discard flow through
4. Add 250 μL of Wash Solution
5. Centrifuge at top speed for 2 min
6. Discard flowthrough
7. Centrifuge at top speed for 2 min
8. Discard flowthrough

**Elution**

1. Transfer spin column to a sterile 1.5 mL eppendorf
2. Add 100 μL room-temperature nuclease free water
3. Centrifuge at top speed for 1 min
4. Discard column
5. Measure yields on Nanodrop

### Miniprep - Jena Bioscience

**Miniprep – Fast-n-easy plasmid miniprep kit PP-2045**

1. Harvest cells by centrifugation (4000 rpm, 10 min, 4 ºC)
2. Add 300 μL of Lysis Buffer and resuspend with micropipette
3. Add 300 μL of Neutralization Buffer
4. Invert 5 times to mix
5. Centrifuge (10,000g, 5 min)
6. Assemble binding column
7. Add 100 μL of Activation Buffer into the column
8. Centrifuge column (10,000 g, 30 sec)
9. Micropipette supernatant to activated column
10. Centrifuge (10,000 g, 30 sec)
11. Discard flowthrough
12. Add 500 μL of Washing Buffer
13. Centrifuge (10,000g, 30 sec)
14. Discard flowthrough
15. Centrifuge (10,000 g, 30 sec)
16. Discard flowthrough
17. Place column into clean 1.5 mL tube
18. Add 100 μL of Nuclease Free H2O at 60 ºC
19. Incubate 5 min at 60 ºC
20. Centrifuge (11, 000 rpm, 1 min)
21. Quantify yields on Nanodrop

**Notes**

* Some deviations from suppliers instructions due to advice from PI

+ Secondary wash with 700 μL of Washing Buffer is not done
+ During elution step: instead of 30 - 50 μL of Elution Buffer, 100 μL of warm nuclease-free H2O were added
+ During elution step: prior to final centrifugation, a 5 minute incubation at 60 ºC was added

* When loading the blank on the Nanodrop for calibration use the same liquid (buffer or water) used to elute

### Ligation

**Ligation**

1. Add the reagents of the table in order

| Reagent | Quantity [μL] |
| --- | --- |
| Insert | Dependent on ratio |
| T4 DNA ligase buffer | 2 |
| Vector | Dependent on ratio |
| T4 DNA ligase | 1 |
| H2O | To 20 μL |

2. Incubate at room temperature for 1 hour
3. Proceed to transformation

**Notes**

* Any remaining ligation that wasn't used for the transformation was left overnight at room temperature then stored at 4 ºC
* NEB calculator was used to determine possible ratios

### LB + carbenicillin plates

**Preparing LB + carbenicillin plates**

1. Add 0.5 g of agarose to 25 mL of distilled water (20 g L-1)
2. Homogenize by shaking
3. Sterilize at 121 °C
4. Once cool to touch add 25 μL of carbenicillin (1 μL mL-1)
5. Pour into the plate and let harden

**Note**

* Multiply agarose, distilled water, and carbenicillin based on number of plates to prepare

### Gel purification - QIAGEN

**QIAprep Spin Miniprep Kit**

1. Cut target band as accurately as possible
2. Add QG buffer at a 1:3 ratio
3. Melt the gel completely in a 50 ºC water bath

1. Heat up nuclease free water simultaneously
2. Invert every 2 minutes

4. Add 1 gel volume of isopropanol
5. Assemble column
6. Add 500 μL of sample to the column
7. Centrifuge for 1 min at 13, 500 rpm
8. Discardo flowthrough
9. Repeat steps 6 - 8 until all sample is run through the column
10. Add 750 μL of PE buffer
11. Centrifuge for 1 min at 13, 500 rpm
12. Discard flowthrough
13. Incubate at room temperature for 2 min
14. Centrifuge for 1 min at 13, 500 rpm
15. Transfer column to clean 1.5 mL eppendorf
16. Add 50 μL EB buffer
17. Centrifuge for 1 min at 13, 500 rpm
18. Measure yield on Nanodrop

**Note**

* When loading the blank on the Nanodrop for calibration use the same liquid (buffer or water) used to elute

### Coomassie staining SDS-PAGE

**Coomassie staining SDS-PAGE**

1. Carefully remove gel from glass molds and place gel in plastic container
2. Add coomassie blue stain to gel until fully submerged
3. Let stain for at least 1 hour at gentle agitation (40 rpm)
4. Remove coomassie blue and submerge in distilled water with gentle agitation overnight

**Note**

* When adding coomassie stain avoid pouring directly on the gel to prevent uneven staining

### Colony PCR

**Colony PCR**

1. Add 50 μL of LB to a microtube
2. In a separate microtube add the reagents listed in the following table

| Reagent | Amount [μL] |
| --- | --- |
| Q5 master mix | 25 |
| DNA | < 1 000 ng |
| FW primer | 5 |
| RV primer | 5 |
| nuclease free H2O | to 50 |

3. Pick a colony with a sterile micropipette tip
4. Leave the tip in the PCR tube for 15 seconds
5. Transfer the tip to the LB tube and leave for 30 seconds
6. Put PCR tube in thermocycler with the following conditions (35 cycles)

| Temperature [ºC] | Duration |
| --- | --- |
| 98 | 00:00:30 |
| 98 | 00:00:10 |
| 61 | 00:00:30 |
| 72 | 00:01:15 |
| 72 | 00:02:00 |
| 4 | ∞ |

### Calcium-competent cells

**Calcium-competent cells**

1. Incubate 50 mL of E. coli on ice for 1 hr
2. Harvest cells by centrifuging (4,000 rpm, 10 min, 4 ºC)
3. Decant supernatant and resuspend in 10 mL of cold CaCl2
4. Centrifuge 4,000 rpm, 10 min, 4 ºC
5. Decant supernatant and resuspend in 1 mL cold 0.1 M CaCl2 + 15 % glycerol
6. Make 50 μL aliquots and store at -80 ºC

### Acetone precipitation for SDS-PAGE

**Acetone precipitation for SDS-PAGE (sample preparation)**

1. To 100 μL of sonication supernatant add 700 μL of cold acetone
2. Incubate at -20 ºC for at least 2 hours
3. Briefly spin down samples
4. Remove excess acetone with syringe and discard
5. Evaporate remaining acetone at 30 ºC for 20 minutes
6. Resuspend in 100 μL tris buffer
7. To each sample 50 μL of Laemmli and homogenize by pipetting
8. Add samples to 96 ºC water bath for 5 min
9. Transfer to ice for 5 min
10. Load samples onto gel

### SDS-PAGE gel (8% and 4%)

**8% resolving SDS-PAGE gel**

1. To a falcon tube add the components shown in the table in order
2. Using a micropipette add the prepared solution until below the top of the gel mold
3. Add ethanol until fully covered

| Reagent | Quantity [μL] |
| --- | --- |
| Acrylamide 30% | 1, 200 |
| Tris pH 8.8 | 1, 950 |
| Distilled water | 2, 700 |
| SDS 10% | 150 |
| APS | 15 |
| TEMED | 6 |

**4% stacking SDS-PAGE gel**

1. To a falcon tube add the components shown in the table below
2. If resolving gel is solidified, remove ethanol by tipping
3. Using a micropipette add the prepared solution until the top of the gel mold
4. Add the comb ensuring a good fit

| Reagent | Quantity [μL] |
| --- | --- |
| Acrylamide 30% | 375 |
| Tris pH 6.8 | 375 |
| Distilled water | 2, 925 |
| SDS 10% | 7.5 |
| APS | 12 |
| TEMED | 6 |

**Notes**

* Add APS and TEMED right before adding gel to the mold as it will begin polymerizing
* Both layers of the gel will take longer to solidify compared to a 12% and 10% gel

## AtPCS as a new biopart

---

### Transformation by heat shock

**Transformation by heat shock**

1. Use 100-200 ng of plasmid DNA
2. Place it into a tube with 60 μL (approximately) of competent cells
3. Incubate on ice for 45 minutes
4. Transfer to a heat bath or Thermoblock at 42 ºC for 45 seconds
5. Return to ice and incubate for 10 minutes
6. Add 1000 μL of liquid LB medium (without antibiotic)
7. Incubate for 1 hour at 37 ºC, 200 rpm
8. Take 200-500 μL and plate it onto Petri dishes with LB agar medium (with antibiotic)
9. Incubate at 37 ºC overnight

### SDS-PAGE: Gel and sample preparation

**8% resolving SDS-PAGE gel**

1. After assembling the gel mold, add the following reagents to a 15 mL Falcon tube

| Reagent | Quantity [μL] |
| --- | --- |
| Acrylamide 30% | 1, 200 |
| Tris pH 8.8 | 1, 950 |
| Distilled water | 1, 179 |
| SDS 10% | 150 |
| APS | 15 |
| TEMED | 6 |

2. Invert several times to homogenize and then add it to the mold using a 1000 μL micropipette (up to the first line that forms)
3. Add ethanol up to the edge of the glass

**4% stacking SDS-PAGE gel**

1. Add the following reagents to a 15 mL Falcon tube

| Reagent | Quantity [μL] |
| --- | --- |
| Acrylamide 30% | 375 |
| Tris pH 6.8 | 375 |
| Distilled water | 1, 844 |
| SDS 10% | 150 |
| APS | 24 |
| TEMED | 12 |

2. After confirming that the resolving gel has polymerized (approximately 30 minutes), remove the ethanol by inverting the mold
3. Add it using a 1000 μL micropipette into the mold until it reaches the edge of the glass
4. Place the comb inside the mold and wait for the gel to polymerize (approximately 30 minutes)
5. When the gel has polymerized, place it inside the chamber, add running buffer, and remove the comb

**Notes**

* Add APS and TEMED right before adding gel to the mold as it will begin polymerizing
* Both layers of the gel will take longer to solidify compared to a 12% and 10% gel
* On several occasions, the gel was prepared with an acrylamide concentration of 5.97% for the resolving gel and 2.98% for the stacking gel. To achieve this, the following quantities were added to the Falcon tubes

| Reagent | Quantity [μL] |
| --- | --- |
| Acrylamide 30% | 1, 200 |
| Tris pH 6.8 | 1, 950 |
| Distilled water | 2, 700 |
| SDS 10% | 150 |
| APS | 15 |
| TEMED | 6 |

| Reagent | Quantity [μL] |
| --- | --- |
| Acrylamide 30% | 275 |
| Tris pH 8.8 | 375 |
| Distilled water | 2, 925 |
| SDS 10% | 75 |
| APS | 12 |
| TEMED | 6 |

**Sample preparation**

1. To 100 μL of sonication supernatant or samples from protein purification, add 700 μL of cold acetone
2. Incubate at -20 ºC for at least 2 hours
3. Briefly spin down samples until reaching maximum speed in the microcentrifuge (13,500 rpm)
4. Remove excess acetone with a syringe and discard
5. Evaporate remaining acetone at 30 ºC for 20 minutes
6. Resuspend in 100 μL TRIS buffer 10 mM
7. To each sample, add 50 μL of Laemmli and homogenize by pipetting
8. Incubate for 5 min at room temperature
9. Add samples to 96 ºC water bath for 5 min
10. Load samples onto gel

### Protein purification

**Resin preparation**

1. Add 10 mL of buffer A (100 mM TRIS, 300 mM NaCl, 2 mM Imidazole) and 0.5 mL resin to a 15 mL Falcon tube
2. Shake for 5 minutes
3. Centrifuge for 5 minutes at 500 rpm, 4°C
4. Discard the supernatant and retain the resin

**AtPCS purification**

1. Add the supernatant recovered from sonication to the tube with the resin
2. Resuspend the precipitates with 4 mL of distilled water, take a sample (500 μL), and transfer it to a microcentrifuge tube (precipitate)
3. Shake for 30 minutes at 80 rpm
4. Centrifuge for 5 minutes at 500 rpm, 4°C
5. Take a sample (500 μL) of the supernatant and transfer it to a microcentrifuge tube (flow through)
6. Discard the remaining supernatant using a suction device
7. Add 10 mL of buffer B (100 mM TRIS, 300 mM NaCl, 10 mM Imidazole)
8. Invert for 5 minutes to homogenize
9. Centrifuge for 5 minutes at 500 rpm, 4°C
10. Take a sample (500 μL) of the supernatant and transfer it to a microcentrifuge tube (first wash)
11. Discard the rest of the supernatant using a suction device
12. Add 10 mL of buffer B (100 mM TRIS, 300 mM NaCl, 10 mM Imidazole)
13. Invert for 5 minutes to homogenize
14. Transfer the tube contents to a 10 mL syringe with a filter paper inside
15. Take a sample (500 μL) of the filtrate and transfer it to a microcentrifuge tube (second wash)
16. Add 1 mL of buffer E (100 mM TRIS, 300 mM NaCl, 200 mM Imidazole) and recover it in a microcentrifuge tube (eluted protein)
17. Recover the resin from the syringe by adding distilled water and transfer it to the 15 mL tube
18. Store samples and resin at 4°C

### Preparation of calcium competent E. coli cells

**Protocol:**

1. *Overnight culture and pre-culture*

1. Pour 3 ml of LB medium into a sterile 15 ml conical tube and inoculate with 1 Escherichia coli colony or 50 μL of liquid inoculum. Incubate at 37°C and 200 rpm overnight
2. Add 0.5 ml of the overnight culture into 49.5 of sterile LB medium, in a sterile Erlenmeyer flask
3. Incubate at 37°C with 200 rpm during 2~4h, until the optical density of the culture reading at 600 nm (OD600) reaches 0.3~0.4. Once the OD600 reaches 0.15, it is recommended to take measurements each 30 min; and if it is over 0.21, each 15 min

2. *CaCl2 washes*

1. Keep all reagents at 4°C, on ice
2. Transfer the E. coli culture into a sterile 50 ml conical tube. Incubate on ice for 1 h
3. Centrifuge at 4°C with 4,000 rpm for 10 min, discard the supernatant
4. Resuspend the cell pellet in 5 ml of cold 0.1 M CaCl2 and incubate on ice for 45 min
5. Centrifuge at 4°C with 4,000 rpm for 10 min, discard the supernatant
6. Resuspend the cell pellet in 600 μL of 0.1 M CaCl2 with 15% glycerol\*
*\*To prepare 0.6 ml of 0.1 M CaCl2 + 15% glycerol, mix 112.5 μL of 80% glycerol with 487.5 μL of 0.1 M CaCl2*7. Prepare ten 60 μL aliquots of competent cells in sterile 1.5 ml or 1.0 ml microtubes and refrigerate at -80°C until use

### Polymerase Chain Reaction with Q5 Master Mix

**Protocol**

1. Place all reaction components in a 0.2 ml microtube and quickly transfer the reaction to the thermocycler, according to the table below (for smaller volumes, adjust the volume of the Master Mix and primers proportionately). All components should be mixed prior to use

| Component | Amount [μL] |
| --- | --- |
| Q5® High-Fidelity 2X Master Mix | 25 |
| Forward primer | 2.5 |
| Reverse primer | 2.5 |
| Template DNA | Variable (20-100 ng) |
| Nuclease-free H2O | To 50 μL |
| Total volume | 50 |

2. onfigure the thermocycler according to the length of the fragment to amplify and to the annealing temperature of the primers [(https://tmcalculator.neb.com/#!/main)](https://tmcalculator.neb.com/#!/main). The usual configuration for the purpose of amplifying the AtPCS gene is shown as follows

| Stage |  | Temperature | Time |
| --- | --- | --- | --- |
| Initial denaturation |  | 98°C | 30 s |
| **Denaturation** | **[35 x]** | 98°C | 10 s |
| **Annealing** | 61°C\* | 30 s |
| **Extension** | 72°C | 1.5 min\*\* |
| Final Extension |  | 72°C | 2 min |
| Hold |  | 4°C | ∞ |

*\* According to annealing temperature of the primers in use*
*\*\* 30 s/kb, then multiplied by 2*3. After the reaction is done, perform agarose gel electrophoresis to verify the integrity and identity of the amplicon. Further purification can be performed from the gel or directly from the reaction mixture

### Miniprep - Promega

**Current protocol**

1. Harvest cells from overnight culture (1-10 mL) by centrifugation at 4,000 rpm, 10 min, 4 ºC
2. Discard supernatant
3. Resuspend in 250 μL of Cell Resuspension Solution
4. Place the resuspended cells in a microcentrifuge tube
5. Add 250 μL of Cell Lysis Solution and invert 4 times to mix
6. Add 10 μL of Alkaline Protease Solution and invert 4 times to mix
7. Incubate at room temperature for 5 min
8. Add 350 μL of Neutralization Solution and invert 4 times to mix
9. Centrifuge at top speed (13 500 rpm) for 10 min
10. Assemble spin column and collection tube
11. Add 700 - 800 μL of cleared lysate into the spin column
12. Centrifuge at top speed (13 500 rpm) for 1 min and discard flowthrough
13. Add 750 μL of Wash Solution with EtOH added
14. Centrifuge at top speed (13 500 rpm) for 1 min and discard flowthrough
15. Repeat steps 13 and 14 with 250 μL of Wash Solution with EtOH added
16. Centrifuge at top speed (13 500 rpm) for 2 min and discard flowthrough
17. Transfer spin column to a sterile 1.5 mL microtube
18. Add 80 μL of nuclease free water at 60 °C to the spin column
19. Centrifuge at top speed (13 500 rpm) for 1 min
20. Discard column
21. Measure yields on Nanodrop

### Miniprep - Jena Bioscience

**Current protocol**

1. Harvest cells from overnight culture (1-10 mL) by centrifugation at 4,000 rpm, 10 min, 4 ºC
2. Discard supernatant
3. Resuspend pellet in 300 μL of Lysis Buffer by vortex (around 1 min)
4. Transfer the sample to a new microcentrifuge tube
5. Add 300 μL of Neutralization Buffer and invert 4-6 times
6. Centrifuge for 5 min at 10,000 g (the color of the sample should change to yellow)
7. Place a column into a 2 mL collection tube
8. Add 100 μL of Activation Buffer to the column
9. Centrifuge for 30 sec at 10,000 g
10. Place the supernatant from step number 6 into the previously activated column
11. Centrifuge for 30 sec at 10,000 g and discard the supernatant
12. Add 500 μL of Wash Buffer (with EtOH) to the column
13. Centrifuge for 30 sec at 10,000 g and discard the supernatant
14. Centrifuge for 2 min at 10,000 g to remove residual buffer
15. Place the column inside a microcentrifuge tube
16. Add 100 μL of nucleases-free water to the center of the column
17. Incubate for 5 min at 60 ºC
18. Centrifuge for 1 min at 11,000 g
19. Quantify concentration using Nanodrop

### Gel purification QIAGEN

**Name of the experiment**
Gel extraction protocol: QIAquick Gel Extraction Kit

**Brief description of the procedure**
The QIAquick Gel Extraction Kit procedure involves running an agarose gel, cutting out the desired DNA fragment, dissolving the gel piece, binding DNA to a QIAquick silica membrane, washing, and eluting the DNA; resulting in a purified DNA sample

**Current protocol**

1. Cut the band(s) and place it/them into a microcentrifuge tube
2. Weigh the tube with the band and verify that the weight is within the range of 100-200 mg approximately after subtracting the weight of the empty tube (approximately 1 g)
3. Add 700 μL of buffer QG to the tube with the band
4. Incubate at 50°C for 10 minutes. It is recommended to vortex the tube every 3 minutes during incubation and at the end of the incubation
5. After ensuring that the sample is homogeneous, transfer it to a kit column. (It may be necessary to divide the volume of the tube to transfer it entirely to the column, so proceed with step 6 and repeat the process with the rest of the sample)
6. Centrifuge for 1 minute at maximum speed (13500 rpm)
7. Discard the supernatant and repeat with the remaining samples
8. Add 750 μL of buffer PE containing ethanol to each column
9. Centrifuge for 1 minute at maximum speed
10. Discard the supernatant
11. Centrifuge for 2 minutes at maximum speed
12. Transfer the column to a new microcentrifuge tube
13. Elute with 40-50 μL of nuclease-free warm water. It is important to make sure to add the water directly into the center of the column
14. Incubate for 5 minutes at 60°C
15. Centrifuge for 1 minute at maximum speed
16. Quantify using Nanodrop

**Notes**

* The protocol provided by QIAGEN for using the kit indicates that for each gel volume (exemplified as 100 mg), 3 volumes of QG buffer are added. As previously stated in the protocol, the final amount was standardized, and 700 LL of buffer are used for samples with an approximate weight of 100-200 mg
* The instruction to vortex during the first incubation is to ensure that no gelified remains of the sample are left before transferring it to the column
* It is essential to mention that after adding the QG buffer and starting the first incubation, preferably no more than 20 minutes should pass before centrifuging on the column for subsequent washing. This should be considered when working with multiple samples
* In the current protocol, the use of isopropanol after the first incubation was omitted, as similar results have been obtained with or without it. This step is optional
* The current protocol includes an additional centrifugation step (Step 11) after adding the PE buffer. This is done to minimize any remaining traces of PE buffer in the column
* Finally, a 5-minute incubation at 60°C was added after elution, which has resulted in improved DNA quantification results

### DNA Restriction Digestion

**Protocol**

1. For a 50 μL reaction, add the following components into a 0.2 ml microtube:

| Component | Amount [μL] |
| --- | --- |
| DNA | Variable (~1 µg) |
| 10X NEBuffer | 5.0 |
| Enzyme 1 | 1.0 |
| Enzyme 2 | 1.0 |
| Nuclease-free H2O | To 50 μL |
| Total volume | 50 |

2. Incubate overnight at 37°C
3. Inactivate the enzymes at 65°C for 20 min
4. Perform agarose gel electrophoresis of the sample to identify the relevant DNA fragment and purify it

### DNA Ligation with T4 Ligase

**Protocol**

1. Define the amount of vector to use and the vector-insert proportion
2. Place the reagents into a 0.2 ml microtube according to the following table:

| Component | Amount [μL] |
| --- | --- |
| Vector | Variable (Ideally 100 ng) |
| Insert | Variable |
| T4 DNA ligase 10X buffer | 2.0 |
| T4 DNA ligase | 1.0 |
| Nuclease-free H2O | To 20 μL |
| Total volume | 20 |

3. Leave the reaction at room temperature overnight
4. After 1 hour, take 5-10 μL of the reaction and transform competent cells or store at -20°C
5. The next day, take another 5-10 μL sample and transform competent cells or store at -20°C

### Colony PCR

**Colony PCR**

1. At sterile conditions, add 20 µL of LB to a PCR microtube (repeat for the total number of selected colonies for amplification)
2. Prepare a master mix depending on the samples (considering a final reaction volume of 15 µL):

| Reagent | Amount per sample [μL] |
| --- | --- |
| Q5 master mix | 7.7 |
| FW primer | 0.75 |
| RV primer | 0.75 |
| nuclease free H2O | to 15 |

3. Distribute the master mix into PCR microtubes (one for each colony plus a positive control)
4. Using a sterile toothpick or micropipette tip, pick up the first colony and place it into the first microtube with the master mix
5. Rotate the toothpick or tip inside the tube about twenty times and then transfer it to the microtube with LB medium
6. Repeat the last two steps with the rest of the selected colonies
7. For the positive control, take 1 µL of the sample and place it in a microtube with the master mix
8. Remove the toothpicks or tips from the microtubes with LB medium and store them at 4°C
9. Place the PCR tube in the thermocycler with the appropriate conditions depending on the length of the gene to be amplified and the Tm of the primers (35 cycles):

| Temperature [ºC] | Duration |
| --- | --- |
| 98 | 00:00:30 |
| 98 | 00:00:05 - 00:00:10 |
| 50-72 | 00:00:10 - 00:00:30 |
| 72 | 00:00:20 - 00:00:30 |
| 72 | 00:02:00 |
| 4 | ∞ |

### Cell induction, harvesting and lysis by sonication

***Preculture***

1. At sterile conditions, add 5 mL of LB medium and 5 μL of kanamycin antibiotic to a 50 mL Falcon tube
2. Select a colony of *Escherichia coli* BL21 (containing the plasmid with the gene) and pick it up with a metal loop
3. Submerge the tip of the loop into the tube with medium and antibiotic, and shake until no traces of the colony are visible
4. Repeat the steps above with the remaining chosen colonies
5. Place the tubes in an incubator at 37°C, 200 rpm, overnight

***Inoculation***

1. At sterile conditions, place 100 mL of LB medium in a sterile flask (if purification will be done after lysis) or 20 mL in a 50 mL Falcon tube (if no purification is planned)
2. Inoculate with 1 mL or 200 μL of the preculture
3. Incubate at 37°C, 200 rpm until the optical density reaches 0.5-0.6
4. Store at 4°C until the induction moment

***Induction with IPTG***

1. At sterile conditions, add 40 μL or 8 μL to achieve a concentration of 0.4 mM IPTG
2. Incubate tubes at 25°C (sometimes also done at 16°C), 200 rpm, overnight

***Cell harvesting***

1. Centrifuge the tubes at 4000 rpm, 10 minutes, 4°C
2. Discard the supernatant
3. Resuspend the pellets in 5 mL of buffer A (100 mM TRIS, 300 mM NaCl, 2 mM Imidazole)

***Cell lysis by sonication***

1. Sonicate at 75%, repeating 3 cycles of 15 seconds on, 60 seconds off. In some cases, 5 cycles of 30 seconds were also used
2. Centrifuge at 4000 rpm, 10 minutes, 4°C
3. Store the supernatants at 4°C and the pellets at -20°C

### 1% Agarose Gel Electrophoresis

**Protocols**

1. Weigh 5 g of agarose on a weighing tray
2. Measure 50 ml of 1X TAE buffer on a graduated cylinder
3. Pour the buffer into an Erlenmeyer flask and add the agarose. Microwave in short intervals (i.e. less than 1 min) until the agarose dissolves. The solution should be clear, with no agarose granules left
4. Quickly, pour the liquid into a gel mold tray with a comb. Wait 25-30 min for the gel to set
5. Remove the comb carefully and transfer the gel into the electrophoresis tank. The comb side should be next to the negative electrode
6. Fill the tank with 1X TAE buffer up to the maximum volume line. The gel is now ready for the samples to be loaded
7. To load the sample, mix the desired amount of sample with the corresponding amount of 6X loading dye. Place the samples carefully into the wells. It is important to note that SYBR has been added to the dye to achieve fluorescence of DNA present in the samples; thus, from the moment the dye is used until the gel is done running, the gel must be protected from light
8. Close the tank, making sure that the black electrode is on the side of the samples and the red electrode is on the opposite side. Verify that the electrodes are plugged in correctly into the power supply
9. Program the power supply as needed. In our lab, we use 60 V for 15 min and then 80 V for 55 min. Press start
10. Once the time is over, turn off the power supply, remove the lid of the tank and transfer the gel onto an UV transilluminator
11. Turn the transilluminator on to visualize the DNA samples on the gel
12. Recover the relevant samples or discard the gel. The gel can be reused once or twice for purposes such as visualizing PCR products or digestion reactions, but not if further purification is needed

### Fluorescent protein washes

**Protocol**

1. In sterile conditions, harvest 2-4 mL of *Escherichia coli* BL21 cells with the fluorescent protein of interest (YFP, GFP, RFP, or ECFP) in a microcentrifuge tube
2. Centrifuge at 10,000 rpm for 1 minute between every milliliter harvested
3. Discard the supernatant after each centrifugation
4. After obtaining the desired pellet, add 1 mL of M63 medium supplemented with 1 mM MgSO4, 0.1 μM thiamine, and the corresponding antibiotic (Carbenicillin for YFP and GFP, Chloramphenicol for RFP, and Kanamycin for ECFP) to the microcentrifuge tube
5. Centrifuge at 10,000 rpm for 1 minute.
6. Discard the supernatant
7. Resuspend the pellet in 1 mL of supplemented M63 medium
8. Take 75 μL from each sample and place them in cells for optical density measurement, each with an additional 1425 μL of distilled water
9. Measure the optical density of each sample using only distilled water as the blank
10. After obtaining the optical density, multiply the value by 20 to determine the optical density of the samples obtained in step number 7
11. Use the formula C1V1 = C2V2 to determine how much volume to add from the samples in step 7 to obtain dilutions at different optical density values with M63 medium
12. Prepare the dilutions in new microcentrifuge tubes
13. Analyze the samples in the device for constructing the calibration curve

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Achievements and future plans{% endblock %}
{% block page\_content %}

## FRET-EryK Results

---

### 🧬 Ligation

Our initial goal was to ligate the gene for ECFP-eryK-mVENUS from a pUC57 to a pET28b backbone. After several cycles of digestion, miniprep and ligation, the goal was finally achieved.

**Figure 1** shows a colony PCR gel run with primers for the entire construct (ECFP-eryK-mVENUS) and primers for only the mVENUS protein. Controls were run for both of these to ensure the band we saw corresponded to the protein. The observed bands were at the expected weights for both samples and matched the controls loaded. This indicates that the pET28b plasmid contains the entire protein sequence and can be used for transformation.

![](https://static.igem.wiki/teams/4763/wiki/results/eryk/fig1-colonypcr.png)

**Figure 1.** Colony PCR results confirming the presence of the ECFP-EryK-mVENUS protein construct within the plasmid.

### 💫 Transformation

Once the colony PCR showed our insert correctly ligated to the plasmid backbone, we transformed into BL21 E. coli and plated. The control was BL21 *E. coli* transformed with an empty pET28b vector. Colonies were observed which was a good sign, however the real test was exposing both plates to UV.

![](https://static.igem.wiki/teams/4763/wiki/results/eryk/fig2-n.png)

**Figure 2.** Transformation of BL21 with **A)** ECPF-EryK-mVENUS and **B)** pET28b(+) empty vector. Both plates were exposed to UV light to confirm fluorescence.

Once both plates were placed on the UV transilluminator it was clear that the colonies transformed with the whole FRET system emitted a higher fluorescence signal than those with only the pET28b backbone **(Figure 2)**. This indicates that the transformation was successful and our gene sequence is being translated.

### ✨ Overexpression

Having transformed *E. coli* with our FRET system, we could find the optimum conditions for overexpression. After testing several conditions, we induced at 16 ºC overnight, lyzed through sonication and measured intracellular protein presence and concentration through a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel showed a very faint band present at the appropriate weight for the ECFP-eryK-mVENUS.

![](https://static.igem.wiki/teams/4763/wiki/results/eryk/fig3-overexpression-sds-page.png)

**Figure 3.** SDS-PAGE gel (8 %) showing protein overexpression results for induced colonies.

## AtPCS Results

---

### 🧬 Cloning of AtPCS into pET28b

Our initial goal was to clone the AtPCS gene from a pUC57 to a pET28b backbone. After several cycles of optimizing digestion, miniprep and ligations, the goal was finally achieved. **Figure 4** and **5** show LB agar and Kanamycin, where 300 μL of the transformation reaction of *E. coli* TOP10 cells with the pET28b(+)\_AtPCS plasmids were added, which confirmed that the cells acquired antibiotic resistance through the plasmid.

![](https://static.igem.wiki/teams/4763/wiki/results/atpcs/fig-1.png)

**Figure 4.** Transformation of pET28b(+)\_AtPCS plasmid into *E. coli* TOP10 cells (1 hour ligation).

![](https://static.igem.wiki/teams/4763/wiki/results/atpcs/fig-2.png)

**Figure 5.** Transformation of pET20b(+)\_AtPCS plasmid into *E. coli* TOP10 cells (O/N ligation).

Additionally, **Figure 6** shows a colony PCR performed with five colonies from the previous plates to verify the presence of AtPCS gene in the pET28b(+) plasmids. Band was observed near the 1.5kbp mark, which matches the length of the gene (1461 bp).

![](https://static.igem.wiki/teams/4763/wiki/results/atpcs/fig-6.png)

**Figure 6.** Amplification of AtPCS gene from random colonies selected from the transformation of 1 hour ligation into *E. coli* TOP10 cells. The ladder used was the 1kb Plus ladder (Invitrogen) .

To identify, through an additional methodology, the clones that were correctly transformed with the gene, restriction assays were performed on plasmids obtained from colonies 1-3 using the NdeI and EcoRI-HF enzymes (New England Biolabs). **Figure 7** shows the agarose gel (1 %) with the results of the enzymatic digestions, where a band corresponding to the length of the AtPCS gene was identified in sample from colony 1.

![](https://static.igem.wiki/teams/4763/wiki/results/atpcs/fig-7.png)

**Figure 7.** Digestion of pET28b(+) plasmid with NdeI and EcoRI-HF restriction enzymes. The ladder used was the 1kb Plus ladder (Invitrogen). A positive result is observed in the lane corresponding to plasmid from colony 1.

### ✨ Protein overexpression

Having transformed *E. coli* BL21 with pET28b(+)\_AtPCS plasmid, we tested different conditions for overexpression. Finally, induction was carried out at 16 ºC overnight. Cell lysis was performed through sonication and intracellular protein presence and concentration was measured through a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as shown in **Figure 8**. The gel showed a very faint band present at the appropriate weight for the AtPCS gene (1461 bp).

![](https://static.igem.wiki/teams/4763/wiki/results/atpcs/fig-8.png)

**Figure 8.** SDS-PAGE gel (8 %) showing protein overexpression results for induced colonies. In wells corresponding to colonies 4-7 supernatants, a very faint blurred band near 55 kDa (AtPCS molecular weight) is thought to be seen.

{% endblock %}