iGEM 2021 — Team TecCEM

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# Team:TecCEM/Notebook

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Untitled Document

Protocols

Laboratory protocols:

This document is a compilation of the protocols we followed all along our experimentation and work inside the laboratory. It contains the detailed procedure of 27 molecular biology, microbiology, and DLS protocols, as well as the recipes for 10 buffers and solutions. It also contains an index so that the desired protocol could be found in a quick and easy manner.

Through our lab notebook you will find which protocols we carried out (they are highlighted in color blue), but the procedure that we followed is not detailed. We decided to create this document, so that you know exactly how each and every one of our protocols were carried out. The purpose of this document was also for us to follow a standardized procedure for not having variations in our results due to differences in the way we carried out those 27 protocols.

We are also aiming to share this document with the Bioengineering Department of our school, for future use of iGEM teams or students in general. Having all molecular biology and microbiology protocols in one document makes the learning process so much easier.

Week 1

Week overview:

* Cleansing and organization of our drawers.
* Sterilization of water, flasks, microtubes, conic tubes and micropipette tips.
* Preparation of antibiotic stock solutions.
* Preparation and sterilization of LB+agar, LB+agar+CAM, LB+CAM and LB culture media (the media with antibiotic were sterilized before adding the antibiotic).
* Preparation and sterilization of cell preservation glycerol solutions.
* Propagation of E. coli strains in LB+agar culture medium.
* Chemocompetent cells preparation for Sure and C43 strains.
* Transformation of BL21 chemocompetent cells with laccase BBa\_K729006.
* Electrocompetent cells preparation for SURE and C43.
* Cell preservation in glycerol of Sure and C43 strains.

Week 2

Week overview:

* First Antibiogram-like assay with glycerol gradients 0-25% and DH5α strains.
* Inoculation and propagation of E. coli strains with their respective antibiotics.
* Electrotransformation of Sure and C43 with laccase, at 2000 and 2500 V
* Transformation of BL21 DE3 by heat shock with laccase plasmid.
* First glycerol gradient assay in LB culture medium.
* Induction with 1 M IPTG of electrotransformed C43 cells.
* Sure and C43 competent cells preservation in glycerol.
* Glycerol gradient assay was repeated.

Week 3

Week overview:

* Electrocompetent preparation of E. coli strains.
* BL21-laccase induction with IPTG and SDS-PAGE of induced samples and Semi-dry membrane transference.
* Measurement of optical density (OD600) of glycerol gradient assay cultures.
* Optical density measurement for competent cell cultures.

Week 4

Week overview:

* Optical density (OD600) for glycerol gradients were measured approximately every 40 minutes for almost 9 hours and graphed.
* Optical density (OD600) for glycerol gradients were also measured with Lector Híbrido Multi-Modal de Microplacas Synergy H4™️ on the CINVESTAV facilities every 20 minutes for 12 hours.
* Transformation with laccase biobricks 15 A, 13 I, 15 E, 13 M and PSB1C3.
* DH5α conservation in LB culture medium and glycerol.
* BBa\_K863010 recovery.
* Pictures of glycerol gradient assay results in LB+agar were taken.

Week 5

Week overview:

* Inoculation of DH5α in petri dishes with glycerol gradients.
* Inoculation of transformed bacteria with plasmids 13I, 15A, 13M and 15E in petri dishes with pizza format (1 UFC/”slice”).
* Inoculation of transformed bacteria with plasmids 13I, 15A, 13M and 15E in 10 mL of liquid LB+CAM culture media. 4 tubes per plasmid, 1 UFC/tube.
* Mini Prep pSB1C3 plasmid extraction.
* Agarose gel electrophoresis to verify integrity of our extracted plasmid plasmids 13I, 13M, 15E, 15A and psB1C3.
* Transformation by thermal shock of BL21 DE3 with plasmids 13 I, 15A, 13M and 15E.
* Laccase induction assay with IPTG.
* psB1C3 enzyme digestion with EcoR1 and Pst1. Agarose gel electrophoresis for digestion products.

Week 6

Week overview:

* Re-inoculation of BL21 strain with each laccase plasmid (15E, 15A, 13M and 13 I) in LB+agar+CAM and LB+CAM culture media and induction with IPTG.
* Laccase induction assay with colorants, acetone and guayacol. SDS-PAGE.
* psB1C3 enzyme digestion with EcoR1 and Pst1 was repeated. Agarose gel electrophoresis for digestion products.

Week 7

Week overview:

* Propagation of competent cells and strains with laccase 15 A, 15 E, 13 M and 13 I.
* Laccase precipitation assay. 2 SDS-PAGE to verify laccase concentration.
* Microplastic determination assay with DLS (Dynamic Light Scattering). The assay was not successful because the equipment didn’t work properly.
* psB1C3 enzyme digestion with EcoR1 and Pst1. Agarose gel electrophoresis for digestion products.
* We received the sequences that we sent to IDT for the enterprise to synthesize them for us (GBlocks ESR1 and EmGFP), along with the primers (M13 Fwd and M13 Rev) for us to make several copies of them. Amplification of received sequences.

Week 8

Week overview:

* Laccase induction assay with colorants.
* PCR for ESR1 and emRFP: Agarose gel electrophoresis for amplicons and DNA extraction from agarose gel with GenElute™ Gel Extraction Kit.
* Biobrick BBa\_B0010 extraction.
* Enzyme digestion for pSB1C3 with EcoR1 and Pst1.
* Chemocompetent BL21 preparation protocol.
* Laccase extraction assay with Nickel and repetition.
* Inoculation of J3100-17D in LB+agar+AMP culture media. No growth observed.
* Transformation by thermal shock of DH5α with psIJ8.
* BBa\_180005 propagation (promoter and RBS) in chemocompetent DH5α with LB + CAM culture medium.
* Transformation by thermal shock of DH5a with pSIJ8 and BBa\_K081005.
* KatE and PUL-ori-MCS ligation. They were cut with NcoI and HindIII with buffer 2.1.
* Ligation of GFP, ESR1 and pSB1C3.
* Ligation of KatE, PuDHT, AldoO and mRFP in backbone.
* pSB1C3 plasmid extraction.
* pSB1C digestion with EcoRI and PstI and agarose gel electrophoresis.

Week 9

Week overview:

* Inoculation of E. coli 10B cells with plasmid pSIJ8.
* Inoculation of E. coli DH5a cells with BBa\_K081005.
* Plasmid extraction from 10B-pSIJ8 and DH5a-BBa\_K081005 with PureLink® Quick Plasmid Mini Prep Kit and agarose gel electrophoresis.
* Laccase induction assay with colorants.
* Agarose gel electrophoresis to verify ligations from Week7 and DNA elution from agarose gel of each well, except marker, J23100 and CFP, with Gen Elute™ Gel Extraction Kit.
* Transformation by thermal shock of SoluBL21 with the laccases C- 15A and D - 13I. IPTG induction of BL21-15E culture.
* 4 SDS-PAGE from Week 8 laccase induction assays samples.
* Ligation of pUCori and pSB1C3 with different inserts (ESR1, PuDHT, AldO, mRFP and KatE).
* Transformation by thermal shock with ligation product. Competent DH5a and pSBIC3 + GFP (10 mL).
* pSIJ8 digestion with NcoI and HindIII.
* BBa\_K863010 (15A) induction with IPTG.

Week 10

Week overview:

* Two SDS-PAGE for pellet and media samples of laccase 15A in E. coli SoluBL21, obtained through laccase induction with IPTG in Week 9.
* E. coli 10β was transformed by thermal shock with the ligation products from Week 9.
* Nickel interaction assay with the soluble fraction and the media of our induced cultures with laccase 15 A
* Digestions for BBa\_K081005, J23100, KatE and PucOri.
* Laccase induction asay with colorants.
* Ligation of genes in pSB1C3.
* Transformation by thermal shock of DH5a with ligation products.
* Colony PCR of emGFP.
* J23100 enzyme digestion with PstI and SpeI and agarose gel electrophoresis.
* Laccase interaction assay with Nickel and SDS-PAGE.
* Electrotransformation of C43 with laccase 15A.

Week 11

Week overview:

* Re-inoculation of E.coli with pSB1C3+ESR1.
* Repetition of Electrotransformation of C43 with laccase 15A.
* Plasmid extraction (pSB1C3-ESR1) using Pure Link® Quick Plasmid Miniprep Kit.
* PCR of ESR1.
* DH5a Transformation by thermal shock of with the following ligation products
  + pSB1CE + KatE
  + pSB1C3 + aldo
  + pUCori + KatE
  + pSB1C3 + PuDAT
  + pSB1C3 + mRFP
* Preparation of chemocompetent E. coli C43.
* BBa\_K081005 (laccase 15 E) extraction
* Digestion of J23100, BBa\_K081005 and pSB1C3-ESR1, agarose gel electrophoresis of digestion products and elution of fragments J23100 S+N+P and ESR1 X+P.
* Plasmid extraction of DH5a pSIJ8, PucOri+KatE, pSB1C3+PucOri, pSB1C3+ESR1 and SoluBL21 laccase 15E (BBa\_K081005) using Pure Link® Quick Plasmid Miniprep Kit.
* Transformation by thermal shock of competent DH5a with BBa\_K081005.
* Cloning of AldO, PuDHT, mRFP, pUCori, KatE and ESR1 ¿ in cloning plasmid TA: TOPO-Ligation.
* Agarose gel electrophoresis of PCR and digestion products of ERS1, J23100 and pSB1C3+ESR1 and elution of J23100 S+N+P from electrophoresis using Gen Elute™ Gel Extraction Kit.
* Transformation by thermal shock of:
  + BB for UPIBI: CAM BBa\_K325909.
  + J251 - CAM.
  + J2CFF - CAM.
  + PCR products: ALDO, PUDHT, mRFP, pUC.ori, KatE, ESR1.

Week 12

Week overview:

* **BBa\_K081005 Plasmid extraction** using Pure Link® Quick Plasmid Miniprep Kit.
* **Transformation by thermal shock** of Biobricks and GBlocks.
* **BBa\_K081005 Plasmid extraction** of E. coli C43 with 15A laccase.
* IPTG induction of transformed products and BL21 DE3 + 15A.
* BBa\_K081005 in pSB1C3

  digestion

  with SpeI and PstI.
* **Protein precipitation assay** on SoluBL21 with 15A laccase culture.
* **Transformation by thermal shock** of DH5a with BBa\_K325409, for UPIBI.
* **Digestion** of ESR1 with XbaI, PstI and NcoI.
* **Digestion** of emRFP with XbaI and PstI.
* **Digestion** of BBa\_K081005 with SpeI and PstI.
* Agarose gel electrophoresis for digestion products and **elution** of fragments from previous gel using Gen Elute™ Gel Extraction Kit.
* **SDS-PAGE** for laccase induction assay samples (before and after induction, culture media and soluble fraction).
* **Transformation by thermal shock** of DH5a with BBa\_K325909.
* Propagation of pSB1C3-emGFP-DH5a in LB+CAM culture medium.
* Propagation of pSB1C3-ESR1-DH5a in LB+CAM culture medium.
* Propagation of BBa\_K081005-DH5a in LB+CAM culture medium.

Week 13

Week overview:

* Zetasizer protocol was carried out to quantify endocrine disruptive compounds on different water brands at different conditions.
* ESR1 plasmid extraction using Pure Link® Quick Plasmid Miniprep Kit, agarose gel electrophoresis and elution of DNA.
* BBa\_K081005 and GFP plasmid extraction using Pure Link® Quick Plasmid Miniprep Kit.
* IPTG induction of transformed products and BL21 DE3 + 15A.
* **Ligation** of ESR1 and GFP in BBa\_K081005, using New England Biolabs® kit.
* **PCR PolyA** of emRFP.
* **PCR** of aldO and PuDHT, agarose gel electrophoresis and elution of DNA.
* **Topo ligation** of mRFP for transformation of DH5a and agarose gel electrophoresis.
* **Laccase induction assay** with Nickel to purify laccase and measurement of absorbance at 280 nm of purification fractions.
* Genetic edition assay to DH5a (knockout).
* **Transformation by thermal shock** of DH5a ligation with ESR1 and GFP.

Week 14

Week overview:

* **SDS-PAGE** for laccase induction and purification assay carried out last week.
* **Colony PCR** of colonies with ERS1 and GFP to verify inserts on BBa\_K081005 and agarose gel electrophoresis for PCR products.
* Plasmid extraction of GFP+K081005 and ESR1+BBa\_K08005 ligation candidates, and agarose gel electrophoresis.
* Samples from laccase induction assay with colorants were analyzed with spectrophotometer: typical curves of each colorant were obtained as well as absorbance vs time curves.
* **Protein quantification with BCA assay.**
* Colony PCR of 30 different DH5a-pSB1C3+ESR1 colonies and agarose gel electrophoresis to verify ESR1 insert of Colony PCR products.
* AldO and puDHT PCR products precipitation with glycogen and ethanol, for genetic edition assay, agarose gel electrophoresis, and elution from gel.
* Plasmid extraction of DH5a pSB1C3-ESR1 colonies numbered as 8, 13 and 18, and agarose gel electrophoresis.
* Transformation of BL21 with pSB1C3-ESR1 candidates 8, 13 and 18.
* Transformation of DH5a with pSIJ8 and Knockout with pUCOri+KatE.
* Transformation of BL21 with BBa\_K081005-ESR1.
* Plasmid extraction of DH5a pSB1C3-ESR1 colonies numbered as 21, 23 and 26.
* Transformation of BL21 with pSB1C3-ESR1 candidates 21, 23 and 26.

Week 15

Week overview:

* 6 SDS-PAGE gels for ESR1 colonies 8, 13, 18, 21, 23 and 26 samples taken on Saturday 16 at 1.5, 3 , 4 and 5 hours, as well as a sample taken on monday (at 48 hours). Stained, faded and documented for Laccase protein view . It was found in Medium 48 hrs samples from ESRI colony 23 and weighted 70 +- kD.
* BPA degradation by Laccase from Trametes versicolor assay. To know if the commercial laccase from Trametes versicolor degrades EDCs like BPA. Through its spectrophotometric measurement in order to measure absorbance and identify if the BPA decreased in the samples. This analysis by means of graphs further in excel.
* UV- Spectrophotometer scanning of Microplastics solutions obtained from our Zeta sizer experiment of sanding the bottles exposed to different conditions such as solar radiation, agitation and without exposure, showing that there was greater in those exposed to UV radiation. being the lowest values ​​in which they were not exposed.

Dry lab

Week overview:

* The activities in our engineering notebook are mainly the following: research of concepts and circuits of quartz crystal microbalances, implementation of electronic devices: trial and failure, software development in Python and Arduino, CAD Fusion 360 design and testing the final QCM.

* Index
* Protocols
* Weeks 1-3
* Weeks 4-6
* Weeks 7-9
* Weeks 10-12
* Weeks 13-15
* Dry lab

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**iGEM Tec CEM 2021** ©
[Enrique Ramírez](https://www.linkedin.com/in/jairoenriqueramirezsanchez/), [Marianela Contreras](https://www.linkedin.com/in/marianela-contreras-dominguez/), [José del Angel &](https://www.linkedin.com/in/jos%C3%A9-%C3%A1ngel-del-%C3%A1ngel-6a9293175/) [Carlos Espejel](https://www.linkedin.com/in/carlos-espejel-salazar-4a8655192)

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# Team:TecCEM/Engineering

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Engineering Success | My Site

# Engineering success

## Quartz crystal evolution

Quartz crystal microbalance (QCM) sensors are frequently used as micro-weighing devices, thanks to their high sensitivity. This sensitivity is due to the frequency shifts due to mass increments that remain stuck on the sensing film surface of the sensor.

[![](https://static.igem.org/mediawiki/2021/2/25/T--TecCEM--biosensorcycle.jpeg)](https://static.igem.org/mediawiki/2021/2/25/T--TecCEM--biosensorcycle.jpeg)

![](https://static.igem.org/mediawiki/2021/9/90/T--TecCEM--EngineeringS1.png)

Figure 1. EWN 4Hz quartz crystal

![](https://static.igem.org/mediawiki/2021/5/59/T--TecCEM--EngineeringS2.png)

Figure 2. Inital electrical circuit photo

The sensitivity of the quartz crystal (QC) is directly proportional to the resonance frequency. As this QC didn't count with the expected resolution while doing our tests, we decided and learnt that it was necessary to look for another QC.

​

This QC did not work on our test because, the 2 electrodes needed to be connected at the edges of the crystal and the electrode pattern is a full pad, both sides type. So, it was not possible to functionalize it.

We learnt that the QC is for processes with high heat loads and for optical coatings with dielectric materials and semiconductor processes.

​

Once again with this outcomes, we looked for a different QC.

​

Finally, with all the testing previously done, we found the OpenQCM quartz crystal, that counted with a resonance of 10MHZ making it perfect for our project.

![](https://static.igem.org/mediawiki/2021/1/17/T--TecCEM--EngineeringS3.png)

![](https://static.igem.org/mediawiki/2021/b/ba/T--TecCEM--EngineeringS4.png)

Figures 3 and 4. First quartz 5Hz crystal used and final 10Hz crystal used for our design

## Electric circuit

For our first prototipe we built a circuit, where we could manipulate the type of signal we wanted to analyze and change the frequency that we will be using.

​

This very first prtototipe was used to incorporate the push buttons, and the Liquid Crystal Display to our Software.

​

With this prototipe we learnt that, the Arduino can only measure up to 5MHz of frequency, meaning that we had to redesign our circuit by changing our microcontroller from an Arduino to a Teensy 4.1.

![](https://static.igem.org/mediawiki/2021/6/6c/T--TecCEM--EngineeringS5.png)

Figure 5. Final circuit schematic.

## 3D Printing

## We use Z -ABS filament because we learnt that it is perfect for parts requiring testing, functional prototypes and concept models. To make some parts of the casing, such as the walls, a type of snap-fits was used as the first model, as the ones shown in the image on the right. However, when assembled and disassembled on different occasions, they did not hold and broke. This forced us to make changes in the design. We came up with a new idea: to use neodymium magnets of 8 mm diameter x 2 mm thickness.

![](https://static.igem.org/mediawiki/2021/7/7a/T--TecCEM--EngineeringS6.png)

Figure 6. "Snap-fit" parts sample.

![](https://static.igem.org/mediawiki/2021/7/7a/T--TecCEM--EngineeringS7.png)

Figure 7. Magnet based design.

The new designs had the corresponding spaces to accommodate the magnets in different areas. A 5% tolerance was used, so the diameter of the gaps was 8.4 mm and a height of 2.1 mm.

​

This seemed to work, but when printed, the magnets were forced in, so post-processing was necessary, which slowed down the working time.

​

So it was redesigned, with new tolerances of 7.5%, excellent results were obtained. It was learned that depending on the region, the 3D printer could have more or less accuracy, which can affect the assembly

## A limitation to our parts

**The problem:**

For Parts BBa\_K3809010 and BBa\_K3809011 we didn't add a promoter, so the part couldn't be expressed.

![](https://static.igem.org/mediawiki/2021/2/21/T--TecCEM--EngineeringS9.png)

**Results**

​

We managed to express the protein with the construct we built. It can be seen in the image at the right how the band after the 48 h growth. We can clearly see a band correspoding to the molecular weight of the protein.

![](https://static.igem.org/mediawiki/2021/4/43/T--TecCEM--EngineeringS8.png)

Figure 8. Digestion of fragments from BBa\_K081005, BBa\_K3809010 and BBa\_K3809011 to build BBa\_K3809012 and BBa\_K3809013.

**How did we solve it?**

​

We had to find a solution to this, so we used part BBa\_K081005 which has a strong constitutive promoter and an RBS and assembled it with the parts with the missing promoter. This led to the creation of parts BBa\_K3809012 and BBa\_K3809013.

![](https://static.igem.org/mediawiki/2021/d/d4/T--TecCEM--EngineeringS10.png)

Figure 9. Digestion of fragments from BBa\_K081005, BBa\_K3809010 and BBa\_K3809011 to build BBa\_K3809012 and BBa\_K3809013.

Figure 10. Protein development

* Index
* Quartz crystal evolution
* Electric circuit
* 3D printing
* A limitation to our parts

![TecCEM logo](https://static.igem.org/mediawiki/2021/1/1c/T--TecCEM--logo.png)

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# Team:TecCEM/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)

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# Team:TecCEM/Results

Untitled Document
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Home

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Team

* [Members](https://2021.igem.org/Team%3ATecCEM/Team)
* [Collaborations](https://2021.igem.org/Team%3ATecCEM/Collaborations)
* [Attributions](https://2021.igem.org/Team%3ATecCEM/Attributions)
* [Contribution](https://2021.igem.org/Team%3ATecCEM/Contribution)

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Project

* [Description](https://2021.igem.org/Team%3ATecCEM/Description)
* [Notebook](https://2021.igem.org/Team%3ATecCEM/Notebook)
* [Implementation](https://2021.igem.org/Team%3ATecCEM/Implementation)
* [Results](https://2021.igem.org/Team%3ATecCEM/Results)
* [Particle detection](https://2021.igem.org/Team%3ATecCEM/Results_Zetasizer)
* [Engineering success](https://2021.igem.org/Team%3ATecCEM/Engineering)

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Awards

* [Awards](https://2021.igem.org/Team%3ATecCEM/Awards)
* [Model](https://2021.igem.org/Team%3ATecCEM/Model)
* [Hardware](https://2021.igem.org/Team%3ATecCEM/Hardware)
* [Software](https://2021.igem.org/Team%3ATecCEM/Software)
* [Sustainable Development Goals](https://2021.igem.org/Team%3ATecCEM/Sustainable)
* [Safety](https://2021.igem.org/Team%3ATecCEM/Safety)

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Human Practices

* [Human Practices](https://2021.igem.org/Team%3ATecCEM/Human_Practices)
* [Education and Engagement](https://2021.igem.org/Team%3ATecCEM/Communication)
* [Survey Results](https://2021.igem.org/Team%3ATecCEM/Results_Survey)

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Parts

* [Overview](https://2021.igem.org/Team%3ATecCEM/Parts)
* [Biosensor Parts](https://2021.igem.org/Team%3ATecCEM/Biosensorparts)
* [Selection Marker Parts](https://2021.igem.org/Team%3ATecCEM/SelectionMarkerParts)

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Judging form

Results | My Site

# The results you’ve been waiting for.

## About the Biosensor.

Our first results: in silico analysis

Due to the pandemic, we weren’t able to perform any Lab Work in the first months. However, we found a way to begin to test our project through analysis in silico. From the protein we designed in part [BBa\_K3809010](http://parts.igem.org/Part%3ABBa_K3809010), we wanted to test if there would be a correct folding of the receptor, even after all the modifications and features we added. Furthermore, we wanted to verify that the His Tag we added and the Linker sequence would be exposed enough for us to perform an efficient purification and an adequate immobilization. We tested this through the prediction of a protein model made using the Software [Robetta](https://robetta.bakerlab.org/) [1]. We took the best model and verified the exposure of the features we added. To test if the folding was being done correctly, the protein was structurally aligned with the crystallized structure of the Human Estrogen Receptor Alpha used as reference, with a PDB code 2IOG ([Protein Data Bank](https://www.rcsb.org/)) [2]. What we found is that there was a correct folding of the modified protein, sustained by the fact that the structure was quite similar to that of protein 2IOG.

## Figure 1.

Protein designed by iGEM Teces. It includes the whole receptor hER⍺ (red), a linker sequence (green), a His Tag (magenta) and a Signal Peptide Sequence (blue). It can be seen that both the linker sequence and the His Tag are exposed. Protein visualized with Chimera [3].

![T--TecCEM--ResultsA1.png](https://static.igem.org/mediawiki/2021/e/ed/T--TecCEM--ResultsA1.png)

![T--TecCEM--ResultsA2.png](https://static.igem.org/mediawiki/2021/b/be/T--TecCEM--ResultsA2.png)

## Figure 2.

Structural alignment between the protein designed by iGEM TecCEM (red, magenta, blue and green) and the crystallized structure of the Human Estrogen Receptor Alpha 2IOG (white). The alignment shows a preserved structure in the Ligand Binding Domain of the modified ESR1. Protein visualized with Chimera [3].

The same was done for part [BBa\_K3809011](http://parts.igem.org/Part%3ABBa_K3809011), which was a reporter we designed to test experimentally in the lab whether the protein was folding correctly and if it could be immobilized in the QCM. The results we got from the alignment of the modified mRFP of [BBa\_K3809011](http://parts.igem.org/Part%3ABBa_K3809011) and mRFP were the same as for ESR1 of part [BBa\_K3809010;](http://parts.igem.org/Part%3ABBa_K3809010) both retained a similar structure to their reference structures.

To further prove the functionality of our protein, we conducted a Docking Analysis in which we tested the ability of the receptor to bind and capture a group of Endocrine Disruptive Chemicals which includes Estradiol (the natural ligand), Benzene, Bisphenol A, Dimethyl Phthalate, Ethylene Glycol, Phenol, Chloroprene and Perfluorooctanoic Acid. To prepare the model for the analysis, we added hydrogens to it, improving the quality of the assay. To this end, we used the tool [MolProbity](http://molprobity.biochem.duke.edu/index.php) [4]. The ligands were obtained from [PubChem [5]](https://pubchem.ncbi.nlm.nih.gov/) and the Docking was conducted using the Software tool [SwissDock](http://www.swissdock.ch/docking) [6]. The results we obtained were then visualized in Chimera in search of the type of interaction the ligands had with the receptor. We found that in most cases, the ligands bound to the protein formed a canal instead of a pocket. Besides, from the best result of the docking, we obtained its Free Gibbs Energy and used it to calculate the Association Constant. Furthermore, we evaluated the formation of hydrogen bonds between the receptor and the small molecule. The results are seen below.

![T--TecCEM--ResultsA3.png](https://static.igem.org/mediawiki/2021/6/61/T--TecCEM--ResultsA3.png)

## Figure 3.

Chloroprene-ESR1 Complex. The amino acids Thr71, Cys103 and Lys118 act as a spatial reference and are the ones closest to the molecule.

## Figure 4.

Chloroprene-ESR1 Complex. The surface of the protein is shown. It was the only ligand that had a pocket interaction.

![T--TecCEM--ResultsA4.png](https://static.igem.org/mediawiki/2021/2/25/T--TecCEM--ResultsA4.png)

![T--TecCEM--ResultsA5.png](https://static.igem.org/mediawiki/2021/1/17/T--TecCEM--ResultsA5.png)

## Figure 5.

Benzene-ESR1 Complex. The amino acids that interact with the molecule are mostly Arg142 and Glu157. The surface of the protein is shown and the type of interaction is the formation of a canal.

## Figure 6.

Estradiol-ESR1 Complex. The interaction is given by aminoacids Glu65, Thr76 and Leu77. The surface of the protein is shown and the type of interaction is the formation of a canal.

![T--TecCEM--ResultsA6.png](https://static.igem.org/mediawiki/2021/8/87/T--TecCEM--ResultsA6.png)

![T--TecCEM--tab1.jpeg](https://static.igem.org/mediawiki/2021/c/ca/T--TecCEM--tab1.jpeg)

## Table I.

Results obtained from the Docking Analysis. Estradiol (highlighted) is the natural ligand of the receptor.

The conclusion we got from these results was that our protein retained its functionality while keeping the characteristics we added and without compromising the affinity towards the ligands. As shown in Table I, we observed that EDCs had a similar affinity towards the receptor as Estradiol (natural ligand); and although the site of the interaction varied, the binding of the ligands with ESR1 was acceptable. What we concluded from this part of the experiments we performed is that we had reasons to believe that ESR1 would be able to work as the capturing molecule of EDCs.

## What did we accomplish in the Lab?

After we did the first analysis of the protein, we knew that we had what’s necessary to begin working in the Laboratory. We had gathered enough information and had the material in the Lab just to begin our experiments. So, after we got our permission to enter our Campus, we did just that.

We planned our experiments as follows:

* Obtain our Biobricks (either from the Synthesis of gBlocks through IDT or retrieving them from past iGEM plates).
* Insert our gBlocks in pSB1C3 vectors.
* Transform our DNA in a Cloning System to get more copies.
* Extract the plasmids out of the Cloning System.
* Make digestions of the plasmids containing the Biobricks.
* Ligate our Biobricks with the ones we obtained from iGEM plates.
* Transform our constructs in a Cloning System to get more copies.
* Extract the constructs out of the Cloning System.
* Transform our constructs in an Expression System.
* Retrieve our protein
* Immobilize the protein onto the Quartz Crystal Microbalance.

The parts we synthesized from IDT were BBa\_K3809010 and BBa\_K3809011 while the part we retrieved from iGEM Plates was BBa\_K081005. We assembled parts BBa\_K3809012 (ESR1)  and BBa\_K3809013 (mRFP) by merging  BBa\_K3809010 with BBa\_K081005 and BBa\_K3809011 with BBa\_K081005 respectively. For more information about our Parts, read [here.](https://2021.igem.org/Team%3ATecCEM/Parts)

However, due to the pandemic we were unable to achieve the goal of the protein purification and the subsequent immobilization onto the QCM. However, we did plan the immobilization protocol. It consists of the immobilization using the cysteine residue we added to the protein and a functionalized QCM. The functionalization would be done using L-cysteine, genipine and chitosan and the formation of a crosslinked structure that would give the protein stability.

![T--TecCEM--ResultsA7.png](https://static.igem.org/mediawiki/2021/3/3a/T--TecCEM--ResultsA7.png)

## pSB1C3, mRFP and ESR1

We started out by observing the plasmids pSB1C3 we had on an agarose gel electrophoresis to determine with which one was going to work.

​

We chose plasmid pSB1C3 labelled as B (lane 8), as it presented the clearest band between 2 and 3 Kb on the gel.

Next, we transformed 3 DH5a cultures with chosen pSB1C3 plasmid, made an extraction and ran an agarose gel electrophoresis to verify integrity and desired size.

​

We noted clear bands of approximately 2 kb. pSB1C3 desired plasmid is 2070 kb, so we were able to verify its size.

![T--TecCEM--ResultsA8.png](https://static.igem.org/mediawiki/2021/5/5c/T--TecCEM--ResultsA8.png)

Next, a digestion with restriction enzymes EcoRI and PstI was carried out to make sure those enzymes cut the plasmid at the desired sites, producing expected fragment sizes.

​

All fragments were the expected size, as well as the uncut plasmid in lane 8.

![T--TecCEM--ResultsA9.png](https://static.igem.org/mediawiki/2021/6/6d/T--TecCEM--ResultsA9.png)

![T--TecCEM--ResultsA10.png](https://static.igem.org/mediawiki/2021/d/df/T--TecCEM--ResultsA10.png)

At the same time, we amplified by PCR our mRFP and ESR1 genes in order to obtain multiple copies and be able to clone each one with pSB1C3.

​

Both amplified products were the desired size. mRFP amplification was expected to be 997 bases and ESR1 amplification was expected to be 1971 bases. Both amplifications are observed in the agarose gel near the desired sizes.

In parallel, DH5a chemocompetent cells were transformed with BBa\_K081005 and inoculated in a selective culture medium.

​

​Transformation was successful because the LB+agar+CAM culture medium presented growth, which means the bacteria acquired the CAM resistance gene inside BBa\_K081005.

![T--TecCEM--ResultsA11.png](https://static.igem.org/mediawiki/2021/1/18/T--TecCEM--ResultsA11.png)

## Next, we performed the cloning protocol for pSB1C3 plasmid with mRFP.

## One band of approximately 2.5-3 kb can be observed clearly in lanes 8-11. As pSB1C3 is 2070 kb and mRFP is 861 kb, the product of both genes is 2931 kb. With this agarose gel we could confirm that our cloning protocol to build pSB1C3+mRFP was successful.

![T--TecCEM--ResultsA12.png](https://static.igem.org/mediawiki/2021/9/97/T--TecCEM--ResultsA12.png)

![T--TecCEM--ResultsA13.png](https://static.igem.org/mediawiki/2021/b/b8/T--TecCEM--ResultsA13.png)

## After that, we performed the cloning protocol for pSB1C3 plasmid with ESR1. We proceeded to DH5a with pSB1C3+mRFP and pSB1C3+ESR1 constructions. Both plasmids were extracted. pSB1C3+mRFP was amplified by PCR and ran in agarose gel electrophoresis and pSB1C3+ESR1 was directly ran in agarose gel electrophoresis. We observed the desired amplification product size on lanes 2-5, since we expected a 997 base amplicon. We expected a bigger extraction product on lanes 6 and 7, so we think those extracted products were supercoiled, leading to smaller fragments.

## Next, a digestion with restriction enzymes SpeI and PstI was carried out to make sure those enzymes cut the plasmid at the desired sites, producing expected fragment sizes. All fragments were the expected size.

![T--TecCEM--ResultsA14.png](https://static.igem.org/mediawiki/2021/d/d3/T--TecCEM--ResultsA14.png)

![T--TecCEM--ResultsA15.png](https://static.igem.org/mediawiki/2021/2/2a/T--TecCEM--ResultsA15.png)

![T--TecCEM--ResultsA16.png](https://static.igem.org/mediawiki/2021/3/3a/T--TecCEM--ResultsA16.png)

## After that, DH5a transformed cells with both constructions (pSB1C3+mRFP and pSB1C3+ESR1) were propagated in culture media (solid and liquid). 30 colonies were chosen from the pSB1C3+ESR1 cultures and a colony PCR was performed to them in order to verify the presence of ESR1. The colonies that present DNA fragments of approximately 2 Kb, since ESR1 is 1971 bases. A few colonies of the 30 that were chosen presented the expected size (colonies 8, 13, 18, 21, 23 and 26, as illustrated above), so those DNA fragments were extracted and BL21 cells were transformed with them.

## Protein Expression: Our Final Result

## We were able to express the protein only for cells from colonies 18 and 23. The results were verified through an SDS-PAGE. However, the best band was seen for colony 23. The results are shown below.  We retrieved samples during the incubation period so that we could see the differential expression of ESR1. In conclusion: The results shown are promising and demonstrate that we can produce this protein in order to immobilize it in our QCM. Due to the pandemic, we weren’t able to do that just yet; but, we want to continue with this project and develop our biosensor so that we can fulfill the [implementation](https://2021.igem.org/Team%3ATecCEM/Implementation) we proposed.

![T--TecCEM--ResultsB1.png](https://static.igem.org/mediawiki/2021/0/0b/T--TecCEM--ResultsB1.png)

## This figure shows the band corresponding to the protein ESR1 (69.6 kDa).

## The other side of our project: Degradation through a Laccase.

As we stated in our Project Description, one of our goals was to find a Laccase that could degrade EDCs in water. Here we describe how our screening process was done and how we characterized the Laccase and establish a purification protocol.

​

In silico analysis

For this section of our project, we wanted to compare the Laccase we were using (BBa\_K863010) with a Commercial Laccase (if you want to see how we selected this Laccase, please read the information below). We did some experiments in which we predicted the structure of the enzyme to test the affinity of the Laccase towards any given substrate through Molecular Docking. We uploaded the amino acids sequence of the enzyme to I-TASSER, a server which runs a platform that predicts the structure of a protein and its function through a model [7] and compared the model we got with a Laccase from Trametes versicolor obtained from PDB under the code 1KYA [8] using a structural alignment (seen in figure 9).

## Figure 9.

Structural alignment of the model of the Laccase from [BBa\_K863010](http://parts.igem.org/Part%3ABBa_K863010) predicted using [I-TASSER](https://zhanggroup.org/I-TASSER/) (Light Brown) and the Laccase 1KYA from Trametes versicolor obtained from PDB (Light Blue). Image visualized using Chimera [3].

The results of these experiments showed a similar folding to the Laccase 1KYA from Trametes versicolor, which makes us think that the affinity for their substrates may be also similar. To test whether this last statement was true, we performed a Docking using SwissDock [6] and obtained the Free Gibbs Energy of the interaction, as well as the Affinity Constant for a group of EDCs (the same group we used for the Docking of ESR1). The Docking was done for the Modeled Protein and the Laccase 1KYA.

![T--TecCEM--ResultsA17.png](https://static.igem.org/mediawiki/2021/d/da/T--TecCEM--ResultsA17.png)

## Table II

Results obtained from the Docking Analysis for Laccase from BBa\_K863010

![T--TecCEM--Table2.jpeg](https://static.igem.org/mediawiki/2021/c/c9/T--TecCEM--Table2.jpeg)

![T--TecCEM--Table2.jpeg](https://static.igem.org/mediawiki/2021/2/2e/T--TecCEM--tab3.jpeg)

## Table III

Results obtained from the Docking Analysis for Laccase from Trametes versicolor.

​

​

In this case, the results were similar between both Laccases, with a similar affinity towards the same substrates.

## Color Assay:

To demonstrate activity of the lacasse purified, we conducted a dye degradation assay. Owing to their broad substrate specificity, laccases have attracted considerable interest in terms of applications to many fields, such as environmental detoxification. Some fungal laccases have been reported to perform dye decolorization of a variety of dyes, such as azo, anthraquinone, and aromatic methane. Synthetic dyes are widely used in several industries, including textiles, food processing, paper printing, cosmetics, and pharmaceuticals. Three dyes were selected according to previous reports of laccase degradation, malachite green, methylene blue and rose Bengal (Cheriaa J. & Bakhrouf A., 2009 [10]; Forootanfar, H., 2012; Pramanik, S., & Chaudhuri, S. 2018 [11]). Based on the chemical structure of chromogenic groups, dyes are classified as azo, heterocyclic/polymeric or triphenylmethanes. About 60% of produced dyes belong to the azo group which are categorized as monoazo, diazo, and triazo dyes. Malachite green is a triphenylmethane dye belonging to a basic dyes class, used extensively for dyeing silk, wool and cotton. Rose Bengal, an Azo dye is used in apoptosis assays, biological staining photography, recording industry, etc., this dye is genotoxic and microbial toxic. Methylene blue is a heterocyclic aromatic compound that has a wide use in biological and medicine applications in addition to textile-processing industries.

​

​

Selecting our Laccase:

The following table describes the four laccases with which we started our experiments. At first we carried out the assays described next to characterize each laccase and deduce which one works best.

​

​

We used three colorants (methylene blue, malachite green and bengal rose) at the beginning of our experimentation to verify laccase functionality. We observed the color changes in each colorant when interacting with the pellet (soluble fraction) and with the supernatant (culture medium) of the BL21 LB+CAM culture with which we expressed the laccases.

​

![T--TecCEM--tabla2.jpeg](https://static.igem.org/mediawiki/2021/8/80/T--TecCEM--tabla2.jpeg)

![T--TecCEM--ResultsFig1.png](https://static.igem.org/mediawiki/2021/0/09/T--TecCEM--ResultsFig1.png)

## Citrate Buffer With Colorants - August 18

No apparent difference between the RIPA buffer control and laccase B pellet laccase is found. The rest of the pellet tubes and all the supernatant tubes do present color differences compared to their controls. This suggests that the laccases A, C and D present functionality when they are and they are not secreted by the cells, and laccase B presents functionality only when it is not secreted by the cells.

![T--TecCEM--ResultsFig3.png](https://static.igem.org/mediawiki/2021/0/04/T--TecCEM--ResultsFig3.png)

All tubes do present color differences compared to their controls. This suggests that the four laccases present functionality when they are and they are not secreted by the cells.

​

Laccase B was discarded and we decided to keep working with laccase C (15 A, BBa\_K863010).

![T--TecCEM--ResultsFig4.png](https://static.igem.org/mediawiki/2021/4/4d/T--TecCEM--ResultsFig4.png)

## Citrate Buffer With Colorants - August 18

Once laccase functionality was verified and one laccase was chosen, the experiments that followed were carried out to quantify enzymatic activity.

​

The same three colorants were used to verify laccase enzymatic activity. We observed the color changes in each colorant when interacting with the pellet (soluble fraction) and with the supernatant (culture medium) of the BL21 DE3 LB+CAM culture with which we expressed laccase 15A. We confirmed our observations by measuring the absorbances (at the respective wavelength of each colorant) of our control, soluble fractions and culture medium samples.

![T--TecCEM--ResultsFig5.png](https://static.igem.org/mediawiki/2021/b/b3/T--TecCEM--ResultsFig5.png)

A constant behaviour of the interaction between laccase 15 A and the three colorants is that the absorbances of soluble fraction samples are smaller than their control (citrate buffer). In contrast, absorbances of culture medium samples are greater than their control (nicker affinity buffer). This demonstrates that protein in the soluble fraction presents greater enzymatic activity than protein in the culture medium.

![T--TecCEM--ResultsFig6.png](https://static.igem.org/mediawiki/2021/7/7f/T--TecCEM--ResultsFig6.png)

To rule out that the loss of color was due to a change in pH in the medium, pH strips were used to determine the pH of the tests carried out.

​

The samples for the 3 dyes and the two protein concentrates analyzed (soluble fraction or culture medium) were observed values between pH 8 and 7. pH to which no color change is attributed. This verifies / validates the activity observed in soluble fraction.

![T--TecCEM--ResultsFig7.png](https://static.igem.org/mediawiki/2021/b/b0/T--TecCEM--ResultsFig7.png)

Citrate Buffer With Colorants - October 02

Then again, the same three colorants were used to verify laccase enzymatic activity with the same procedure of the assay described above.

​

Interaction with methylene blue exhibits a different behaviour, compared to the interaction with the other two colorants. Soluble fraction and culture medium samples present smaller absorbance values than their respective colorants. So, in terms of absorbance, in methylene blue it is not very clear if the activity is greater in the soluble fraction or culture medium. However, we can tell from pure observation that it is more evident in the soluble fraction samples.

​

![T--TecCEM--ResultsFig8.png](https://static.igem.org/mediawiki/2021/1/18/T--TecCEM--ResultsFig8.png)

![T--TecCEM--ResultsFig9.png](https://static.igem.org/mediawiki/2021/2/20/T--TecCEM--ResultsFig9.png)

​

For malachite green assays, the absorbances of soluble fraction samples are smaller than their control (citrate buffer). In contrast, absorbances of culture medium samples are greater than their control (nicker affinity buffer). This demonstrates that protein in the soluble fraction presents greater enzymatic activity than protein in the culture medium.

​

For bengal rose assays, the absorbances of neither the soluble fraction samples nor the culture medium samples are smaller than their respective control (citrate buffer). This shows no enzymatic activity on any samples.

![T--TecCEM--ResultsFig10.png](https://static.igem.org/mediawiki/2021/1/1b/T--TecCEM--ResultsFig10.png)

![T--TecCEM--ResultsFig11.png](https://static.igem.org/mediawiki/2021/3/3c/T--TecCEM--ResultsFig11.png)

Again, to rule out that the loss of color was due to a change in pH in the medium, pH strips were used to determine the pH of the tests carried out.

​

The samples for the 3 dyes and the two protein concentrates analyzed (soluble fraction or culture medium) were observed values between pH 8 and 7. pH to which no color change is attributed. This verifies / validates the activity observed in soluble fraction.

​

​

​

![T--TecCEM--ResultsFig12.png](https://static.igem.org/mediawiki/2021/b/bf/T--TecCEM--ResultsFig12.png)

The typical curves were made for each type of dye. Thus, we obtained a percentage of degradation of the dye by the laccases obtained in soluble fraction or culture medium. The dye was prepared as indicated in the lab notebook and that color was analyzed at 0% degradation. The corresponding dilutions were made to dilute in 80%, 55% and 25% and only buffer without dye as 0%. Samples and readings were made in triplicate. The obtained curves and the corresponding average absorbance value are presented. The correlation factor R was> 0.98 for the three cases. The samples of culture medium concentrated by precipitation with ammonium sulfate always showed an intense brown color. So the absorbance was read directly from the sample for each of the wavelengths at which each dye absorbed.

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In this case, it was observed that the background noise due to the color of the sample was significant and contributed to the reading in the spectrophotometer. The background value of the samples and the typical curve obtained by diluting each dye with a citrate buffer were considered.

![T--TecCEM--ResultsFig13.png](https://static.igem.org/mediawiki/2021/d/d6/T--TecCEM--ResultsFig13.png)

Citrate Buffer With Colorants - October 16

For our next experiment, we now had standardized conditions and took absorbance measurements at the respective wavelength of each colorant (Bengal Rose: 562 nm, Malachite Green: 617 nm and Methylene Blue: 664 nm) to document absorbance through time. The first measurement was taken after 16 hours of incubation at 37°C, and then every 24 hours for 3 days. We had 3 repetitions of 5 different samples of each colorant: culture medium, soluble fraction, purified culture medium, purified soluble fraction and Trametes versicolor commercial laccase. Curves of absorbance vs time were built, as well as typical curves for each colorant. As we focused on the activity measurement as a percentage of degradation through time, the 3 controls from blue, green and rose were considered as the 100%.

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We didn’t have the exact concentration of colorants in our samples, so we measured the activity as a percentage of degradation of each colorant, where a 100% of substrate was considered as the absorbance of the control. Thus, the enzymatic degradation would be expressed as the loss of color. The samples we worked with were laccase in culture medium,  culture soluble fraction, purified culture medium, purified soluble fraction and commercial laccase from T. versicolor.

The results are presented below as graphs for each colorant.

![T--TecCEM--ResultsA18.png](https://static.igem.org/mediawiki/2021/a/a4/T--TecCEM--ResultsA18.png)

The samples from the soluble fraction were the ones that presented the greater colorant degradation at the end of the assay, followed by culture medium and T. versicolor samples. Purified soluble fraction samples were the ones that presented the least degradation.

![T--TecCEM--ResultsA19.png](https://static.igem.org/mediawiki/2021/e/e0/T--TecCEM--ResultsA19.png)

The samples from T. versicolor were the ones that presented the greater colorant degradation at the end of the assay, followed by purified culture medium samples. Purified soluble fraction samples were the ones that presented the least degradation.

![T--TecCEM--ResultsA20.png](https://static.igem.org/mediawiki/2021/9/95/T--TecCEM--ResultsA20.png)

The samples from T. versicolor were the ones that presented the greater colorant degradation, followed by soluble fraction samples. Purified soluble fraction samples were the ones that presented the least degradation.

From the previous graphs, we noted a similar behaviour in Methylene Blue and Bengal Rose, where soluble fraction and T. versicolor samples presented the greatest degradation. However, in Malachite Green, the Laccase from Trametes versicolor had a higher degradation rate.

The percentage of degradation of each sample for each colorant at the end of the assay are presented below:

![T--TecCEM--ResultsA21.png](https://static.igem.org/mediawiki/2021/b/bf/T--TecCEM--ResultsA21.png)

Overall, the best results were obtained by the T. versicolor commercial laccase, followed by the soluble fraction and the purified soluble fraction. However, it is important to remember that T. versicolor has a higher concentration since it was prepared at 1 mg/mL (purified laccases had concentrations of 0.334 µg/mL in the culture medium and 0.1298 µg/mL in the soluble fraction).

These concentrations were determined through BCA quantification protocol, by creating a BCA curve, which is shown below.

![T--TecCEM--ResultsB2.png](https://static.igem.org/mediawiki/2021/7/7f/T--TecCEM--ResultsB2.png)

## Expression Laccase Solubility Analysis - September 18

## We made polyacrylamide gels to verify if there is a difference in laccase expression when induced with IPTG in soluble fractions, insoluble fractions and culture medium samples. The first gel was not run with standardized conditions, so the second gel was made.

![T--TecCEM--ResultsFig20.png](https://static.igem.org/mediawiki/2021/8/8c/T--TecCEM--ResultsFig20.png)

![T--TecCEM--ResultsFig21.png](https://static.igem.org/mediawiki/2021/6/67/T--TecCEM--ResultsFig21.png)

## The appearance of a differentially expressed band observed in the total protein after induction with IPTG, with standardized electrophoretic profiles (same total protein load) proves that the expression of a higher quantity of laccase C (15 A) is potentially enhanced with IPTG.

Laccase Solubility Analysis - October 02

We repeated the SDS-PAGE assay

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The electrophoretic profiles before and after induction are not standardized, but present almost the same approximate total protein load. The appearance of a differentially expressed band is observed in lane 3, of approximately 48 kDa. Most of the protein is solubilized either in the cytoplasm or secreted into the culture medium. The band of the protein secreted to the culture medium is practically purified.

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These conclusions helped us determine if induction with IPTG was effective for our laccase. They also helped us know the approximate size of the protein and how purified the protein is both in soluble fraction and culture medium samples.

![T--TecCEM--ResultsFig22.png](https://static.igem.org/mediawiki/2021/7/7a/T--TecCEM--ResultsFig22.png)

Purification

As laccase 15A has an His-tag, we performed a purification assay with a Nickel column, and then carried out SDS-PAGE to observe if there is a difference between the samples immobilized to Nickel resin, and the not-immobilized ones. Also, we compared the amount of purified protein with each Nickel resin wash.

![T--TecCEM--ResultsFig23.png](https://static.igem.org/mediawiki/2021/c/cd/T--TecCEM--ResultsFig23.png)

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Laccase 15A shows specific interaction with the Nickel resin, as two bands are observed with higher intensity in the immobilized to Nickel resin sample (lane 2), in contrast with the total soluble fraction protein after induction sample (lane 1) or the flow through sample (lane 3). These two bands are gradually diminishing their intensity as protein is being washed from the Nickel resin.

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In order to elucidate a purification protocol that would give us the greatest possible amount of enzyme, absorbance of 21 cytoplasmic soluble fractions samples and 20 culture medium samples at 280 nm. Purification conditions were established with a gradient of the elution buffer, as shown in the following chromatograms. The first one is for cytoplasmic soluble fraction, and it shows a maximum absorbance at an elution volume between 15 to 20 mL and 30-50%  elution buffer; which corresponds to the fractions containing the Laccase.

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The second chromatogram is for culture medium, and it shows a maximum absorbance at an elution volume between 12 to 20 mL and 16-50% elution buffer; which corresponds to the fractions containing the Laccase.

![T--TecCEM--ResultsA22.png](https://static.igem.org/mediawiki/2021/3/3a/T--TecCEM--ResultsA22.png)

![T--TecCEM--ResultsA23.png](https://static.igem.org/mediawiki/2021/f/f1/T--TecCEM--ResultsA23.png)

Resuming and linking to the next part of degrading EDCs with Laccase.
The results shown above demonstrate that we were able to synthesize a functional laccase that can be directly purified and that is useful for endocrine disruptive compounds’ (EDCs) degradation.

BPA degradation by commercial Laccase from Trametes versicolor assay - October 20.

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Assay objective:
This experiment will allow us to know if the commercial laccase from Trametes versicolor really degrades EDCs like BPA. Through its spectrophotometric measurement in order to measure absorbance and identify if the BPA decreased in the samples. This analysis by means of graphs.

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BPA degradation and detection

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Bisphenol A (BPA), an alkylphenolic compound, is a main monomer in the production of polycarbonates, epoxy resins, and other plastics. Detection and removal of bisphenol A from industrial waste/soil/drinking water are critical to minimize human consumption.

Elimination of hazardous phenolic compounds using laccases has gained attention due to its efficiency. Purified laccase from Paraconiothyrium variabile (PvL) has been reported with high removal percentage of BPA [9].  Also, it was found that BPA degradation was along with the occurrence of laccase production in solid state fermentation with Trametes versicolor [10]. In vitro, the optimum pH range of BPA degradation with laccase was in the acidic region with the optimal performance around pH 5.0 [1]. As we have observed great enzymatic activity at pH of 5.7, we decided to perform our BPA degradation assay with this condition.

The main method for BPA determination is High-performance liquid chromatography (HPLC). However, it is not a very accessible method, and it is very expensive. Thus, we make a review in literature to know alternatives to monitor BPA degradation. Determination of BPA was reported also by spectrophotometer by Ali H. in 2019 [11] , where a molecularly imprinted nanocomposite composed of a covalently connected 3D network of reduced graphene oxide, b-cyclodextrin and polyacrylate was used for selective electrochemical detection of bisphenol A. The nanocomposite selectively captures bisphenol A, it was demonstrated by the removal of bisphenol A (figure 10) It was examined by UV-absorbance study along with HPLC as shown in figure 10, it was observed that the absorbance peak of bisphenol A at 276 nm was diminished, confirming the successful capture of bisphenol A by the nanocomposite.

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Figure 10. BPA absorbance spectra reported by different authors, the value of λmax is indicated by the pink arrow. A) Absorbance spectra showing the complete removal of bisphenol A (BPA) measured by spectrophotometer (a) and HPLC (b) [11]. B) Bisphenol A UV–VIS absorbance spectrum in water [12]. C) UV-VIS spectra of BPA in a reaction system with CC450 [13].

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![T--TecCEM--fig10.jpeg](https://static.igem.org/mediawiki/2021/1/1a/T--TecCEM--fig10.jpeg)

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To determine the concentration of BPA that can be detected by spectrophotometer, we found two reports where BPA was measured by this method, the first one reports a solution of 0.1 mM (25ppm) [12 and the second at 8.3 mM [11]. As BPA degradation by a laccase was reported to be initiated at 4 mM [1], we obtained the UV spectrum of this concentration in aqueous solution in the range of 200-600 nm. The results showed significant UV absorption with λmax at 290 nm (figure 11). Although we observe a very different wavelength from other references (274.6, 265 and 276 nm) as is shown in the figure 10, differences could be explained by the combination with other substances in those reports [11][12][13].

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Figure 11. UV spectrum of 4 mM BPA in aqueous solution in the range of 200-600 nm. Spectrophotometer Thermo Scientific Genesys 10S UV-VIS

![T--TecCEM--fig11.jpeg](https://static.igem.org/mediawiki/2021/8/8d/T--TecCEM--fig11.jpeg)

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Then we developed a standard curve with BPA (0.5-4 mM) and observed a linear behavior between 0.5-2.5 mM (figure 12).

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Image description. The BPA degradation assay calibration curve. Following the concentrations of the TABLE IV.

The standard calibration curve was obtained from the following samples of different concentrations, starting from 0.5 mM to 4 mM, following the amounts in microliters of the following image. These further UV-spectrophotometer each sample at a wavelength of 290 nm. And obtaining the results shown in Table IV. for the graphing of the standard curve.

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​Table V.  Preparation of each sample used in the obtention of the standard curve with the absorbance given by the UV- spectrophotometer. Using the values of H2O and citrate column for the BPA - absorbance graph in.

![1600px-T--TecCEM--fig12.png](https://static.igem.org/mediawiki/2021/thumb/3/3a/T--TecCEM--fig12.png/1600px-T--TecCEM--fig12.png)

![T--TecCEM--TableV.png](https://static.igem.org/mediawiki/2021/a/a0/T--TecCEM--TableV.png)

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BPA degradation assay by laccase

The laccase-catalyzed degradation of BPA was conducted. BPA stock solution was prepared at 25 mg/ml dissolved in methanol (according to the supplier's instructions, Aldrich 239658), then BPA working solution at 4mM was prepared in water. The reaction mixture (final volume of 1.1 mL) was prepared as follow: 0.5 ml of 4 mM BPA was added to 0.5 mL citrate buffer (100 mM Sodium citrate and 100 mM citric acid, pH 5.7), followed by introducing of the purified laccase from Trametes versicolor at 5 U/mL to the reaction mixture and incubation at 35°C and 250 rpm for different times. The negative control was designed by inserting a citrate buffer to the reaction mixture instead of laccase. To determine BPA degradation, the absorbance (at 290 nm) was measured.

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An average of the absorbances obtained was collected at the next image in order to graph further.

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![T--TecCEM--datatable.png](https://static.igem.org/mediawiki/2021/9/95/T--TecCEM--datatable.png)

![T--TecCEM--FIG12DATA.png](https://static.igem.org/mediawiki/2021/d/d7/T--TecCEM--FIG12DATA.png)

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Table VI. Results of averaging results obtained from graphing the absorbance averages measured at an initial time and after 16 hours have passed.

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Conclusion.

In this interpretation, It is noted that under the conditions carried out, the concentration of BPA decreases from time zero to the second dose 16 hours later. Since the control remains more or less constant with an indifferent variable. This being a support for mentioned literature where in the presence of laccase, BPA is degraded.
Further testing with different conditions, concentrations, temperatures, and different laccases is necessary to obtain more accurate information, as an assay with our purified laccase would be the next step in this assay. As well as performing this experiment with other EDCs other than BPA.

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## References:

[1] Y. Song et al., “High-resolution comparative modeling with RosettaCM”, Structure, vol 21, no 10, bll 1735–1742, Sep 2013.

[2] K. D. Dykstra et al., “Estrogen receptor ligands. Part 16: 2-Aryl indoles as highly subtype selective ligands for ERα”, Bioorganic & Medicinal Chemistry Letters, vol 17, no 8, bll 2322–2328, 2007.

[3] E. F. Pettersen et al., “UCSF Chimera--a visualization system for exploratory research and analysis”, J Comput Chem, vol 25, no 13, bll 1605–1612, Oct 2004.

[4]  C. J. Williams et al., “MolProbity: More and better reference data for improved all-atom structure validation”, Protein Sci, vol 27, no 1, bll 293–315, Nov 2017.

[5] S. Kim et al., “PubChem in 2021: new data content and improved web interfaces”, Nucleic Acids Res, vol 49, no D1, bll D1388–D1395, Jan 2021.

[6] A. Grosdidier, V. Zoete, en O. Michielin, “SwissDock, a protein-small molecule docking web service based on EADock DSS”, Nucleic Acids Res, vol 39, no Web Server issue, bll W270-7, May 2011.

[7]  A. Roy, A. Kucukural, en Y. Zhang, “I-TASSER: a unified platform for automated protein structure and function prediction”, Nature Protocols, vol 5, no 4, bll 725–738, Apr 2010.

[8] T. Bertrand et al., “Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics”, Biochemistry, vol 41, no 23, bll 7325–7333, Jun 2002.

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[9] Z. Asadgol, H. Forootanfar, S. Rezaei, A. H. Mahvi y M. A. Faramarzi, "Removal of phenol and bisphenol-A catalyzed by laccase in aqueous solution", Journal of Environmental Health Science and Engineering, vol. 12, n.º 1, junio de 2014. Accedido el 21 de octubre de 2021. [Online]. Available: [https://doi.org/10.1186/2052-336x-12-93](https://doi.org/10.1186/2052-336x-12-93%C2%A0)

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[10] S. Zeng, J. Zhao y L. Xia, "Simultaneous production of laccase and degradation of bisphenol A with Trametes versicolor cultivated on agricultural wastes", Bioprocess and Biosystems Engineering, vol. 40, n.º 8, pp. 1237–1245, mayo de 2017. Accedido el 21 de octubre de 2021. [Online]. Available: [https://doi.org/10.1007/s00449-017-1783-1](https://doi.org/10.1007/s00449-017-1783-1%C2%A0)

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[11] H. Ali, S. Mukhopadhyay y N. R. Jana. New Journal of Chemistry. [Online]. Available: [https://pubs.rsc.org/en/content/articlelanding/2019/nj/c8nj05883k](https://pubs.rsc.org/en/content/articlelanding/2019/nj/c8nj05883k%C2%A0)

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[12] R. Abo, N.-A. Kummer y B. J. Merkel, "Optimized photodegradation of Bisphenol A in water using ZnO, TiO<sub>2</sub> and SnO<sub>2</sub> photocatalysts under UV radiation as a decontamination procedure", Drinking Water Engineering and Science, vol. 9, n.º 2, pp. 27–35, september 19 of 2016. [Online]. Available: <https://doi.org/10.5194/dwes-9-27-2016>

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[13] Z. Jiao et al., "Degradation of Bisphenol A by CeCu Oxide Catalyst in Catalytic Wet Peroxide Oxidation: Efficiency, Stability, and Mechanism", International Journal of Environmental Research and Public Health, vol. 16, n.º 23, p. 4675, noviembre de 2019. Accedido el 21 de octubre de 2021. [Online]. Available: [https://doi.org/10.3390/ijerph16234675](https://doi.org/10.3390/ijerph16234675%C2%A0)

* Index
* About biosensor
* We accomplish in the lab
* ESR1 Expression
* Degradation throught laccase
* Laccase Expression
* BPA degradation
* Conclusion

![TecCEM logo](https://static.igem.org/mediawiki/2021/1/1c/T--TecCEM--logo.png)