iGEM 2022 — Team tecmonterrey-gdl

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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# Introducing Notebook

# WET LAB

* 31 Jan 2022 - 10 Feb 2022: Bibliography research of relevant emerging contaminants in our country, focusing on water bodies in the Jalisco region.
* 11 Feb 2022 - 28/03/2022: Bioinformatic research of enzymes that can degrade selected contaminants into something we can measure.
* 06 May 2022 - 01 Oct 2022: Cloning EryK (BBa\_K4447001) and FRET system (BBa\_K4447004) into pBAD/Myc-HisB vector for protein overexpression in Escherichia coli BL21.
* 02 Oct 2022 - 10 Oct 2022: Analysis of protein expression through SDS-PAGE and EryK (BBa\_K4447001) enzymatic activity analysis.
* 10 Oct 2022: Bacterial expression system (BBa\_K4447005) synthesized with Twist arrived.

[Mixtec](https://static.igem.wiki/teams/4447/wiki/mixteco-explorando-las-ra-ces-de-nuestro-planeta-docx.pdf)

# DRY LAB

* 18 – 24 February 2022: Bibliography research of FRET systems similar to the project, including enhanced cyan fluorescent protein (ECFP) used as the donor, and Venus, used as a yellow fluorescent protein (YFP).
* 25 – 28 February 2022: Selection of two systems between the literature to use as a comparison.
* 01 – 07 August 2022: Transfer of data from the selected papers into Matlab and search of models that best fit the data.
* 08 – 14 August 2022: Interpretation of results and comparison of parameters.
* 15 – 21 August 2022: Written explication of results.

# PROTOCOLS

[Preparation of calcium competent cells](https://static.igem.wiki/teams/4447/wiki/preparation-of-calcium-competent-cells.pdf)
[Plasmid extraction with qiaprep spin miniprep kit](https://static.igem.wiki/teams/4447/wiki/plasmid-extraction-with-qiaprep-spin-miniprep-kit.pdf%20%20%20%20%20%20%20%20)
[DNA digestion with two restriction enzymes](https://static.igem.wiki/teams/4447/wiki/dna-digestion-with-two-restriction-enzymes.pdf)
[Competent cell transformation](https://static.igem.wiki/teams/4447/wiki/competent-cell-transformation.pdf)
[Cell lysis](https://static.igem.wiki/teams/4447/wiki/cell-lysis.pdf)
[Agarose gel electrophoresis](https://static.igem.wiki/teams/4447/wiki/agarose-gel-electrophoresis.pdf)

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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#### Silver Medal Criterion #1

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.

If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page) for your team to be eligible for this criteria.

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Please see the [2022 Medals Page](https://competition.igem.org/judging/medals) for more information.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

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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).

## Inspirations

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* [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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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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# Results

# a) For EryK (BBa\_K4447001)

The first induction was performed following our induction protocol, with a temperature of 37 °C for 4 hours after reaching OD greater than 0.4 and adding arabinose.

![](https://static.igem.wiki/teams/4447/wiki/geles-1.png)

Figure 1: Each sample corresponds to a colony, where the sample number corresponds to the colony number, plus a negative control. Each lane was loaded with 20 uL, 10 uL of supernatant and 10 uL loading buffer. The ladder used was: PageRuler Plus Prestained Protein Ladder. The red rectangle shows the location of our EryK protein with a weight of 43.5 kDa.

In these gels, ten samples and a control were run, where each sample corresponds to a different colony. To avoid the use of a possible cheater in addition to evaluate and determine the best colony, a densitometric analysis was performed to determine the concentration of the protein by comparing it with the intensity of a band of the ladder that has an already defined concentration.

![](    https://static.igem.wiki/teams/4447/wiki/tabla2-ultima-1.png
      )

Table 1: Estimated concentration of each sample. this was processed thanks to ImageJ (NIH, USA)

As we can see in the results of the densitometric analysis, it resulted in a null expression of our protein both in the control and in colony number 10, telling us that it is possibly cheater and that our induction protocol is successful, because of the way they were expressed in the other colonies and in the negative control the expected results were obtained.

# b) For FRET System (BBa\_K4447004)

The second experiment was performed following our induction protocol, with a temperature of 37 °C until it reach OD greater than 0.4, and after adding arabinose, we let bacterias growth overnight at 25 °C.

Figure 2: Each sample corresponds to a colony, where the sample number corresponds to the colony number. Each lane was loaded with 20 uL, 10 uL of supernatant and 10 uL loading buffer. M: PageRuler Plus Prestained Protein Ladder. C1 and C2: controls not induced with L-arabinose.

![](https://static.igem.wiki/teams/4447/wiki/sds-page-ecfp-eryk-mvenus.jpeg)

In our gel we did not see conclusive results, we did not see evidence of our enzyme in the supernatant, so we can analyze different possibilities, that the expression at 25 °C was not adequate and as the enzyme is quite large a more accelerated cellular machinery is necessary, another option is the formation of inclusion bodies, protein aggregates, which are formed by a recombinant protein, being not native to the species and being of a fairly large size tends to precipitate. The other option is that we find cheaters or self-ligating colonies.

# COLLABORATION

Tec-Chihuahua and our team organized a collaboration to validate the enzyme activity of our BioBrick BBa\_K4447001. To carry out these experiments, we cloned this part into pBAD/Myc-HisB and sent it to Tec-Chihuahua. Afterward, we requested them to induce protein overexpression at 16 °C and 0.2% arabinose, assuming slower cellular metabolism would reduce protein misfolding. After induction, Tec-Chihuahua ran an SDS-PAGE.

![](https://static.igem.wiki/teams/4447/wiki/sds-page-chihuahua-from-eryk.jpeg)

Figure 3. SDS-PAGE results of the EryK protein. M: Molecular mass marker (Thermo Scientific, Waltham, MA, USA)

As shown in

Figure 3

, no sign of our protein was seen, so we can conclude that these conditions were not favorable for producing our protein. For instance, we opted to change our protein overexpression conditions to 25 °C but with the same arabinose concentration.

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