iGEM 2019 — Team Tec-Monterrey

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# Team:Tec-Monterrey/Notebook

[![](https://static.igem.org/mediawiki/2019/thumb/4/4c/T--Tec-Monterrey--white-logo-breathe-in.png/240px-T--Tec-Monterrey--white-logo-breathe-in.png)
Breathe-In](https://2019.igem.org/Team%3ATec-Monterrey)

* TEAM

  [Members](/Team%3ATec-Monterrey/Team)
  [Attributions](/Team%3ATec-Monterrey/Attributions)
  [Collaborations](/Team%3ATec-Monterrey/Collaborations)[Special Thanks](/Team%3ATec-Monterrey/Thanks)
* PROJECT

  [Description](/Team%3ATec-Monterrey/Description)
  [Design](/Team%3ATec-Monterrey/Design)
  [Notebook & Protocols](/Team%3ATec-Monterrey/Notebook)
  [Experiments](/Team%3ATec-Monterrey/Experiments)
  [Demonstrate](/Team%3ATec-Monterrey/Demonstrate)
  [Model](/Team%3ATec-Monterrey/Model)
  [Hardware](/Team%3ATec-Monterrey/Hardware)
  [Safety](/Team%3ATec-Monterrey/Safety)
* PARTS

  [Parts Overview](/Team%3ATec-Monterrey/Parts)[Basic Parts](/Team%3ATec-Monterrey/Parts#basic-parts)
  [Improved Parts](/Team%3ATec-Monterrey/Parts#improved-parts)
  [Composite Parts](/Team%3ATec-Monterrey/Parts#composite-parts)
* HUMAN

  [Human Practices](/Team%3ATec-Monterrey/Human_Practices)
  [Integrated Human](/Team%3ATec-Monterrey/Human_Practices)[Education and Public Engagement](/Team%3ATec-Monterrey/Public_Engagement)
* [CONTACT](/Team%3ATec-Monterrey/Contact)

# NOTEBOOK

In order to accomplish the objectives previously established on our working plan
[(see the “Design” page)](/Team%3ATec-Monterrey/Design), we
divided the team into different modules, so we could work simultaneously in the laboratory as you can see
in
the next sections.

Microfluidic device

The hardware module started working in the AutoCAD designs in June in order to have time to send the
mask
for fabrication. As soon as the mask arrived in August, hardware started working in the microfluidic
device.

![](https://static.igem.org/mediawiki/2019/a/a0/T--Tec-Monterrey--notebook-ricardo.jpg)
![](https://static.igem.org/mediawiki/2019/c/c6/T--Tec-Monterrey--notebook-ricardo2.jpg)
![](https://static.igem.org/mediawiki/2019/5/5e/T--Tec-Monterrey--notebook-ricardo3.jpg)
![](https://static.igem.org/mediawiki/2019/6/61/T--Tec-Monterrey--notebook-ricardo4.jpg)

3A Assembly

3A Assembly module started the lab work on August, since the IDT and Twist parts we needed arrived at
the
end of July.
You can see the schedule on the following images:

![](https://static.igem.org/mediawiki/2019/1/1d/T--Tec-Monterrey--notebook-3a1.jpg)
![](https://static.igem.org/mediawiki/2019/0/02/T--Tec-Monterrey--notebook-3a2.jpg)
![](https://static.igem.org/mediawiki/2019/6/6f/T--Tec-Monterrey--notebook-3a3.jpg)

Golden Gate

Golden Gate module started its work the third week of august because we had to obtain the X-gal
necessary
to perform blue-white screening in order to confirm the success of the Golden Gate procedure.

![](https://static.igem.org/mediawiki/2019/4/45/T--Tec-Monterrey--notebook-golden1.jpg)
![](https://static.igem.org/mediawiki/2019/9/93/T--Tec-Monterrey--notebook-golden2.jpg)
![](https://static.igem.org/mediawiki/2019/0/0e/T--Tec-Monterrey--notebook-golden3.jpg)

Heavy Metal 3A

As part of our experiments, we wanted to run a proof of concept for the amplification cascade using
toxic
metal inducible promoters. We worked for three weeks on this module and only missed the last
transformation of our final product ligations.

![](https://static.igem.org/mediawiki/2019/7/7f/T--Tec-Monterrey--notebook-metales.jpg)
![](https://static.igem.org/mediawiki/2019/f/fa/T--Tec-Monterrey--notebook-metales2.jpg)

Mucolytic

The team in charge of this module started its work on the first week of August, with the purpose of
characterizing an easy method for protein recovery for further assays.

![](https://static.igem.org/mediawiki/2019/1/1d/T--Tec-Monterrey--notebook-mucolytic1.png)
![](https://static.igem.org/mediawiki/2019/b/b9/T--Tec-Monterrey--notebook-mucolytic2.jpg)
![](https://static.igem.org/mediawiki/2019/a/ad/T--Tec-Monterrey--notebook-mucolytic4.jpg)

![](https://static.igem.org/mediawiki/2019/thumb/1/1f/T--Tec-Monterrey--sponsors-acceso-lab.png/320px-T--Tec-Monterrey--sponsors-acceso-lab.png)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Tec-Monterrey/Experiments

[![](https://static.igem.org/mediawiki/2019/thumb/4/4c/T--Tec-Monterrey--white-logo-breathe-in.png/240px-T--Tec-Monterrey--white-logo-breathe-in.png)
Breathe-In](https://2019.igem.org/Team%3ATec-Monterrey)

* TEAM

  [Members](/Team%3ATec-Monterrey/Team)
  [Attributions](/Team%3ATec-Monterrey/Attributions)
  [Collaborations](/Team%3ATec-Monterrey/Collaborations)[Special Thanks](/Team%3ATec-Monterrey/Thanks)
* PROJECT

  [Description](/Team%3ATec-Monterrey/Description)
  [Design](/Team%3ATec-Monterrey/Design)
  [Notebook & Protocols](/Team%3ATec-Monterrey/Notebook)
  [Experiments](/Team%3ATec-Monterrey/Experiments)
  [Demonstrate](/Team%3ATec-Monterrey/Demonstrate)
  [Model](/Team%3ATec-Monterrey/Model)
  [Hardware](/Team%3ATec-Monterrey/Hardware)
  [Safety](/Team%3ATec-Monterrey/Safety)
* PARTS

  [Parts Overview](/Team%3ATec-Monterrey/Parts)[Basic Parts](/Team%3ATec-Monterrey/Parts#basic-parts)
  [Improved Parts](/Team%3ATec-Monterrey/Parts#improved-parts)
  [Composite Parts](/Team%3ATec-Monterrey/Parts#composite-parts)
* HUMAN

  [Human Practices](/Team%3ATec-Monterrey/Human_Practices)
  [Integrated Human](/Team%3ATec-Monterrey/Human_Practices)[Education and Public Engagement](/Team%3ATec-Monterrey/Public_Engagement)
* [CONTACT](/Team%3ATec-Monterrey/Contact)

# EXPERIMENTS

The experiments and protocols followed an organized sequence of
steps that allowed us to validate the results obtained from our
laboratory work. We focus on having several lines in our experiments
that were carried out separately but still converge in the same
goal:

* 3A Assembly of biosensor promoters
* 3A Assembly of Heavy metals
* Signal amplification cascade
* Mucolytic production in E. Coli

In order to develop our project, we looked for the appropriate
protocols to success, however, it was necessary to modify some of
them in order to adapt them for our needs and goals. The following
list shows all the protocols used, please read the PDF document for
the complete steps. We hope they could be easily repeated by future
iGEM teams to ensure the repeatability of each experience.

* PCR amplification
* PCR clean-up
* Digestion
* Agarose gel electrophoresis
* DNA gel extraction
* Ligation
* 3A assembly
* Golden
* Competent Cells (E. Coli)
* Electro (stbl4)
* Calcium (dh5alpha and BL21)
* Transformation protocol (E. Coli)
* Heatshock
* Electroporation
* Competence and transformation (Bacillus subtilis)
* Glycerol stocks
* Plasmidic DNA extraction (Miniprep)
* Protein induction assay
* Protein purification assay
* Fluorescence kinetic curve
* Bradford

[Experiments file](https://static.igem.org/mediawiki/2019/6/6d/T--Tec-Monterrey--experi-2.1.pdf)

![](https://static.igem.org/mediawiki/2019/thumb/1/1f/T--Tec-Monterrey--sponsors-acceso-lab.png/320px-T--Tec-Monterrey--sponsors-acceso-lab.png)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Tec-Monterrey/Results

[![](https://static.igem.org/mediawiki/2019/thumb/4/4c/T--Tec-Monterrey--white-logo-breathe-in.png/240px-T--Tec-Monterrey--white-logo-breathe-in.png)
Breathe-In](https://2019.igem.org/Team%3ATec-Monterrey)

* TEAM

  [Members](/Team%3ATec-Monterrey/Team)
  [Attributions](/Team%3ATec-Monterrey/Attributions)
  [Collaborations](/Team%3ATec-Monterrey/Collaborations)[Special Thanks](/Team%3ATec-Monterrey/Thanks)
* PROJECT

  [Description](/Team%3ATec-Monterrey/Description)
  [Design](/Team%3ATec-Monterrey/Design)
  [Notebook & Protocols](/Team%3ATec-Monterrey/Notebook)
  [Experiments](/Team%3ATec-Monterrey/Experiments)
  [Demonstrate](/Team%3ATec-Monterrey/Demonstrate)
  [Model](/Team%3ATec-Monterrey/Model)
  [Hardware](/Team%3ATec-Monterrey/Hardware)
  [Safety](/Team%3ATec-Monterrey/Safety)
* PARTS

  [Parts Overview](/Team%3ATec-Monterrey/Parts)[Basic Parts](/Team%3ATec-Monterrey/Parts#basic-parts)
  [Improved Parts](/Team%3ATec-Monterrey/Parts#improved-parts)
  [Composite Parts](/Team%3ATec-Monterrey/Parts#composite-parts)
* HUMAN

  [Human Practices](/Team%3ATec-Monterrey/Human_Practices)
  [Integrated Human](/Team%3ATec-Monterrey/Human_Practices)[Education and Public Engagement](/Team%3ATec-Monterrey/Public_Engagement)
* [CONTACT](/Team%3ATec-Monterrey/Contact)

# RESULTS

![](https://static.igem.org/mediawiki/2019/a/a1/T--Tec-Monterrey--results-portada.jpg)

## Goals

At the beginning of Breathe-in, we decided to set modular objectives in order to achieve our main goal. The
modules were not divided into the typical iGem areas but according to the intentions of the different areas
that compose the design section. We also included the mathematical model to have a proper complementarity
between the theoretical and experimental work.

### Whole cell biosensors

* To assemble five different constructs that will allow us to detect the presence of molecules generating
  different types of cellular damage.
* To assemble constructs that allow us to amplify the signal and reduce the limit of detection of the
  different biosensors.
* To characterize both the amplified and non amplified biosensors.

### Microfluidic platform

* To create a microfluidic device that could multiflex the amount of reactions for the detection of
  antibiotics.
* To produce Bacillus subtilis culture droplets.
* To simulate the conditions of biosensors activation in droplets with the use of antibiotics.

### Mucolytic

* To characterize an easy method for protein recovery for further assays.
* To measure the effect of sialidase employment over the viscosity of a CF lung simulated environment.
* To increment the effect of antibiotics over P. aeruginosa by the
  addition of sialidases to the media.

## Microfluidic platform

* Designed and successfully manufactured microfluidic devices for droplet generation using soft
  photolithography techniques.
* Modelled and simulated microfluidic geometries to validate the proposed flow rates in a T-junction using
  COMSOL Multiphysics.
* Generated microdroplets in the microfluidic d evice with the validated conditions from the simulations.

[
](https://static.igem.org/mediawiki/2019/3/35/T--Tec-Monterrey--hw-droplets-generation.mp4)

## Whole cells biosensors

### 3A assembly

* The plasmid for B. subtilis genome integration (PBS1C) was successfully transformed and digested for the
  3A assembly strategy, using EcoR1 and Pst1 restriction enzymes.
* After amplification, the synthetized gblocks corresponding for each of the promoters were successfully
  ligated to an RGT part, using the pSB1C3 plasmid as a vector, digested and confirmed by running an
  electrophoresis gel.

![](https://static.igem.org/mediawiki/2019/d/da/T--Tec-Monterrey--results-3aypua.jpg)

### Amplification cascade

The 5 promoters were successfully ligated to the cascade through the Golden Gate assembly method.

## Mucolytic

* E. Coli containing the corresponding constructs of this module were
  successfully transformed.
* Both the bacteria transformed with the sfGFP and that with the chimeric protein showed to be highly
  expressed according to their phenotypes.
* The expression of the chimeric protein was IPT induced.
* The expressed chimeric protein was correctly purificated using the HisTag.

![](https://static.igem.org/mediawiki/2019/5/52/T--Tec-Monterrey--results-gelnufos.jpg)

![](https://static.igem.org/mediawiki/2019/thumb/1/1f/T--Tec-Monterrey--sponsors-acceso-lab.png/320px-T--Tec-Monterrey--sponsors-acceso-lab.png)