iGEM 2020 — Team UPF_Barcelona

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# Team:UPF Barcelona/Notebook

# Notebook

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

### What should this page have?

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

### Inspiration

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

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

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# Team:UPF Barcelona/Engineering

UPF\_Barcelona

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* Project

  [Description](https://2020.igem.org/Team%3AUPF_Barcelona/Description)
  [Design](https://2020.igem.org/Team%3AUPF_Barcelona/Design)
  [Engineering](https://2020.igem.org/Team%3AUPF_Barcelona/Engineering)[Implementation](https://2020.igem.org/Team%3AUPF_Barcelona/Implementation)
  [Entrepreneurship](https://2020.igem.org/Team%3AUPF_Barcelona/Entrepreneurship)
  [Safety](https://2020.igem.org/Team%3AUPF_Barcelona/Safety)[Poster](https://2020.igem.org/Team%3AUPF_Barcelona/Poster)
* Labwork

  [T3 biosensor](https://2020.igem.org/Team%3AUPF_Barcelona/Intein)
  [Proof of concept](https://2020.igem.org/Team%3AUPF_Barcelona/Proof_Of_Concept)[Experiments](https://2020.igem.org/Team%3AUPF_Barcelona/Experiments)
* DryLab

  [Hardware](https://2020.igem.org/Team%3AUPF_Barcelona/Hardware)
  [Software](https://2020.igem.org/Team%3AUPF_Barcelona/Software)
  [Contribution](https://2020.igem.org/Team%3AUPF_Barcelona/Contribution)[Model](https://2020.igem.org/Team%3AUPF_Barcelona/Model)
* Results

  [Results](https://2020.igem.org/Team%3AUPF_Barcelona/Results)
  [Improve](https://2020.igem.org/Team%3AUPF_Barcelona/Improve)
* [Parts](https://2020.igem.org/Team%3AUPF_Barcelona/Parts)
* Human Practices

  [Human Practices](https://2020.igem.org/Team%3AUPF_Barcelona/Human_Practices)
  [Public Engagement](https://2020.igem.org/Team%3AUPF_Barcelona/Education)
* Team

  [Team](https://2020.igem.org/Team%3AUPF_Barcelona/Team)
  [Attributions](https://2020.igem.org/Team%3AUPF_Barcelona/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AUPF_Barcelona/Collaborations)
* Awards

  [Medals](https://2020.igem.org/Team%3AUPF_Barcelona/Medals)
  [Awards](https://2020.igem.org/Team%3AUPF_Barcelona/Awards)

In this page we will introduce the Engineering Design Cycle that we followed in order to create our Intein Mediated T3
(IMT3) Biosensor. We made our first version IMT3 sensor, then we made another design round that led us to the IMT3 sensor
that we present with Hormonic. However, a third engineering process to enhance it to a better version was already being
planned. Here you can see the journey we went through to generate the **BBa\_K3484000**.

Engineering success

* [The enginnering cycle](#cycle)* [T3 biosensor development](#Improvement)
  * [References](#ref)

# The enginnering cycle

![](https://static.igem.org/mediawiki/2020/9/91/T--UPF_Barcelona--cycle.png)

**Synthetic biology** is a science discipline that differs a lot from the others. Usually, you would use the scientific method to obtain
results in the majority of life sciences, centering your work on making observations and performing experiments. However, synthetic biology goes
beyond that, it is all about constructing something new from parts (biological parts if you may). That is why, sometimes the
**engineering design process** fits better with these projects, since a product has to be built, a machine that performs a task,
where it is more important what it does rather than how, yet still has to be tested. Funnily enough, that's why iGEM stands for
genetically engineered machines.

# T3 biosensor development

![](https://static.igem.org/mediawiki/2020/2/26/T--UPF_Barcelona--researchIcon.png)

## **Research:** *How can we sense T3?*

We went through the literature trying to find any previous T3 sensor in bacteria. We found the sensor used by researchers in
the University of Princeton, where they used the ΔI-SMTR mini intein coupled to the TS enzyme creating an OD sensor [1]. To see
more of our biosensor background go to our [T3 biosensor design page](https://2020.igem.org/Team%3AUPF_Barcelona/Intein).

![](https://static.igem.org/mediawiki/2020/0/0c/T--UPF_Barcelona--arrow.png)

![](https://static.igem.org/mediawiki/2020/a/a3/T--UPF_Barcelona--imagine.png)

## **Imagine:** *How can our bacteria communicate with a computer?*

Since the main characteristic of inteins is their ability to reconstruct virtually any split protein, we thought that,
if split correctly, we could use a Fluorescent Protein. Using light as a reporter we can quantitatively and continuously
know the amount of hormone. That's why we thought of splitting eGFP, since we saw in papers that the ΔI-SMTR mini intein
without the THR-β could put together the two parts of the eGFP [2].

![](https://static.igem.org/mediawiki/2020/0/0c/T--UPF_Barcelona--arrow.png)

![](https://static.igem.org/mediawiki/2020/8/88/T--UPF_Barcelona--designIcon.png)

## **Design:** *How can our bacteria express our protein the best way?*

We want to couple our ΔI-SMTR mini intein, which consist of the THR-β flanked by the two intein splicing domains. To this we added
the eGFP split between residues 70 and 71 to keep the necessary cysteine after the second splicing domain. To see more in detail
the construction of this sensing protein, we recommend you visit our [T3 biosensor design page](https://2020.igem.org/Team%3AUPF_Barcelona/Intein).

We had our idea for chimeric protein, however, in the same study where they showed the reconstruction of the eGFP, they also
mentioned the propensity of forming inclusion bodies. This normally happens when bacteria are transformed to express at a high
rate big proteins, the machinery of protein folding probably falls short. So in our design we had to include a solubility tag,
and we found 4 possible candidates [3], based on size (Tab.1).

|  |  |
| --- | --- |
| Solubility Tag | Length (aa) |
| FH8 | 69 |
| SUMO | 100 |
| GB1 | 56 |
| MBP | 396 |

Table 1. List of candidates for our solubility tag.

We wanted a small tag since we did not want to enlarge our protein, so even though Maltose Binding Protein (MBP) showed
good solubility, we considered it too big. From the other 3 options, GB1 was the smallest, however, neither GB1 nor SUMO
solubility ability was good compared to Fh8. The MBP and Fh8 are ranked among the best solubility enhancer tags [4]. So we placed the latter in the N-terminal of the first half of eGFP.

Another tag that we wanted to add was the Flagx3 tag in the C-Terminal of the second half of eGFP, for immunodetection
purposes. Although any tag would have worked for these detection, experts working within the Transational Synthetic Biology Lab, suggested us to work with this one due to its great performance in past experiments.

For the non-coding sequences, we tried to use the most standard parts already used in iGEM. We used the [BBa\_K880005](http://parts.igem.org/Part%3ABBa_K880005)
which contains a strong constitutive promoter ([BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100)) and a strong RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034))
as well, perfect for recombinant protein expression. For terminators, we used the common
double terminator [BBa\_B0014](https://parts.igem.org/Part%3ABBa_B0014). These parts were recommended to us by Dr. Carlos Toscano,
who was an advisor for our University team in 2018 and used these parts.

![](https://static.igem.org/mediawiki/2020/0/0c/T--UPF_Barcelona--arrow.png)

![](https://static.igem.org/mediawiki/2020/7/7a/T--UPF_Barcelona--build.png)

## **Build:** *How can we build it?*

After the design phase of our Biosensor and taking into account the difficult access to the lab for confinement, we made the
decision of synthesizing it directly as a clonal plasmids. We ordered to IDT the *IMT3\_eGFP\_pUC-Kan* sequence with a high copy number vector.

![](https://static.igem.org/mediawiki/2020/0/0c/T--UPF_Barcelona--arrow.png)

![](https://static.igem.org/mediawiki/2020/9/9c/T--UPF_Barcelona--testIcon.png)

## **Test:** *Do we get the results we expected?*

We transformed our plasmid to *E. Coli* BL21 cells and performed tests to check how the sensor was working.
We performed a Western blot to check the expression of our protein, and we saw that splicing was occurring when induced with T3 and that the solubility tag avoided major protein aggregates. We then tested it quantitatively by placing our sensing cells
with different T3 concentrations ranging from 10uM to 80 uM, and unfortunately we obtained some random behaviour.
To see the final results visit our [Results page](https://2020.igem.org/Team%3AUPF_Barcelona/Results).

![](https://static.igem.org/mediawiki/2020/0/0c/T--UPF_Barcelona--arrow.png)

![](https://static.igem.org/mediawiki/2020/7/70/T--UPF_Barcelona--learnIcon.png)

## **Learn:** *What we did not know from our design?*

From the results of the initial plate readers, we saw that a lot of times higher values of T3 in the media did not correspond to
higher fluorescence. Moreover, different plate reader results showed different behaviours between the ranges 20 uM to 80 uM.
This made us think that maybe we were exploring the wrong range of concentrations of our sensor to report correctly. If the sensor needed more concentration to report correctly, it meant that it needed concentrations higher than 80uM. However, we do not think this was the case since T3 is insoluble at concentrations higher than 80uM. So, having in mind the low levels of T3 the Thyroid hormone receptors normally
sense in the body (around 1 pM), the sensor was probably saturated, raising that random behaviour. We had to explore concentrations lower than 20uM.

![](https://static.igem.org/mediawiki/2020/0/0c/T--UPF_Barcelona--arrow.png)

![](https://static.igem.org/mediawiki/2020/0/03/T--UPF_Barcelona--improveIcon.png)

## **Improve:** *Is our reporter the best one?*

We also realised that maybe eGFP wasn't the best GFP we could use. Fluorescent levels were very low, and having in
mind that we had to detect them from our own fluorescent sensor, we needed more signal. Looking for other reporters,
we found a version of the GFP called superfolder GFP (sfGFP). This variation had two main characteristics: it was faster and brighter.

![](https://static.igem.org/mediawiki/2020/0/0c/T--UPF_Barcelona--arrow.png)

![](https://static.igem.org/mediawiki/2020/8/88/T--UPF_Barcelona--designIcon.png)

## **Design:** *How can we introduce the new reporter?*

Once we decided to use sfGFP as our new reporter, we could also vary the split point. We looked up in the literature splitting
points and created two versions. The first one was keeping the same split region as the eGFP because the residue sequence is very
similar. The 70/71 split point from the eGFP corresponded to the residues 69/70 for the sfGFP, in which residue 70, just after the
C-terminal of the intein, had to be changed to a Cysteine.

The second version came from the [2014 Heidelberg iGEM team](https://2014.igem.org/Team%3AHeidelberg), where they used trans splicing inteins (two different proteins)
to join two halves of sfGFP. The split point was between the residues 64 and 65, and once again, residue 65 had to be changed to a cysteine.

![](https://static.igem.org/mediawiki/2020/0/0c/T--UPF_Barcelona--arrow.png)

![](https://static.igem.org/mediawiki/2020/7/7a/T--UPF_Barcelona--build.png)

## **Build:** *How can we build it?*

Once again, we decided to synthesize it again the same way as the IMT3\_eGFP\_pUC-Kan, same promoter, rbs, tags and terminator.
Hence we ordered the two second versions of our sensor: *IMT3\_sfGFP70/71\_pUC-Amp* and *IMT3\_sfGFP64/65\_pUC-Amp*,
changing the antibiotic resistance to avoid any possible cross contamination, since we would work with both strains at the same time a lot.

However, IDT warned us that the sequence for *IMT3\_sfGFP64/65\_pUC-Amp* “included a partial or full coding region from Brucella melitensis”
and could not be sent to us from the United States to Spain. So at the end, the *IMT3\_sfGFP70/71\_pUC-Amp* became the only *IMT3\_sfGFP\_pUC-Amp* sequence.

![](https://static.igem.org/mediawiki/2020/0/0c/T--UPF_Barcelona--arrow.png)

![](https://static.igem.org/mediawiki/2020/9/9c/T--UPF_Barcelona--testIcon.png)

## **Test:** *Does it perform better?*

This time, knowing that we were probably saturating our sensor, we chose to test our construct with T3 concentrations ranging from 1pM to 100uM.
We tested both constructs: *IMT3\_eGFP\_pUC-Kan* and *IMT3\_sfGFP\_pUC-Amp* and results showed a better performance of the sfGFP version,
with less randomness, brighter and more precise. To see the final sfGFP sensor results visit our  [results](https://2020.igem.org/Team%3AUPF_Barcelona/Results) page.

![](https://static.igem.org/mediawiki/2020/0/0c/T--UPF_Barcelona--arrow.png)

![](https://static.igem.org/mediawiki/2020/7/70/T--UPF_Barcelona--learnIcon.png)

## **Learn:** *Why do these improvements work?*

As we expected, since sfGFP has more brightness, the fluorescence values related to each T3 concentration escalated,
so the difference between values also escalated. Also, thinking that the IMT3 sensor was saturating at values between
20uM and 80uM, increasing the testing range to a logarithmic scale from 1pM to 100uM (10-12 to 10-4 M),
allowed us to appreciate the sensing capacities of our sensor and allowed us to fit it better to the model.

![](https://static.igem.org/mediawiki/2020/0/0c/T--UPF_Barcelona--arrow.png)

![](https://static.igem.org/mediawiki/2020/0/03/T--UPF_Barcelona--improveIcon.png)

## **Improve:** *In what other ways can we modify our sensor?*

So far we have achieved a sensor able to detect T3 concentrations down to 10-7 M and that has a dynamic range that goes as far as 10-4 M. However,
the IMT3 biosensor can be further improved. Decreasing even more the detection limit is something that should be done if
we would like to directly detect T3 concentrations similar to the ones found in the blood and interstitial liquid (around 1 pM).
Another improvement would be to widen the dynamic range, making the sensor more sensible, or even changing the reporter for other applications.

![](https://static.igem.org/mediawiki/2020/0/0c/T--UPF_Barcelona--arrow.png)

![](https://static.igem.org/mediawiki/2020/8/88/T--UPF_Barcelona--designIcon.png)

## **Design:** *How can we make new versions?*

#### **Low Copy Vectors**

One approach that can be done to reduce the random noise from the sensor, thus making it more precise, is to place our IMT3 sensor in
a low copy vector.

At the moment IMT3\_sfGFP\_pUC-Amp is in a high copy pUC vector, meaning that for each cell there are between 500 and 700
plasmids per cell. These 200 plasmid variations can cause noise. We think that cloning our IMT3\_sfGFP sensor to a low copy number vector,
such as the pSC101 [5], with a compy number of ~5 could reduce the noise, thus decreasing the lower detection limit. However, we might lose
some intensity of the signal. We did this assembly via Golden Gate assembly using BsaI Type IIS restriction enzyme. but we couldn't
characterise its performance due to time limitations. See Golden Gate Assembly in the [Protocol page](https://2020.igem.org/Team%3AUPF_Barcelona/Experiments).

#### **Double LBD**

Talking with our instructors, they told us that, since our sensor had a ligand and receptor dynamic, increasing the number of receptors could modify the sensibility or the ability of the sensor to detect lower T3 concentrations [6]. In our case, the receptors are the Ligand Binding Domains of the
THR-β. We thought that by placing one next to the other (Fig.1) in between the Intein Splicing domains we could tune the minimum detectable concentration.

![](https://static.igem.org/mediawiki/2020/1/16/T--UPF_Barcelona--figureEng.png)

Figure 1. Proposed design with two receptors (THR-β).

To implement that we thought of doing it via Golden Gate assembly. We could use PCR amplification to get the LBD of the THR-β
and insert it next to the other one, however, sine this fragment is around 780 kb, having such long equal sequences can easily
trigger homologous recombination mechanisms, making it very difficult to have this sequence repeated. That is why we ordered to Twist
the LBD THR-β codon optimized to differ from the sequence we had in the sensor yet produce the same amino acids. This fragment will
be placed next to the other receptor without creating homologous recombination.

#### **Directed evolution**

We realised that based on literature our modification options were limited. Not much has been published about mutant variants
of the THR-β besides the ones related with diseased, which often are inhibitory mutations. We know which residues directly
interact with T3 when binding at a molecular level [7], but we don't know what changes to do in order to improve its performance.
Actually we don't have to know what changes make to the protein, natural selection (in our case our selection) can lead to better sensor variants.

Here we are stepping in the field of Directed Evolution, a fairly recent academic field yet has been present since the origins of
humankind. It is based on creating diversity and applying a proper selective pressure to navigate towards a better sequence.
This has been already done in the past selecting the trees that produced more fruits or the cows that produced more milk. But now,
with better technologies, we can select, for example, brighter bacteria. Using the Mutagenesis plasmid 6 (MP6) [8] which randomly inserts mutations, we designed an experimental pipeline to be able to generate variability in our IMT3\_sfGFP biosensor, select them by
fluorescence via cell sorting and repeat it to make different evolution cycles (Fig.2).

[Directed evolution pipeline](https://static.igem.org/mediawiki/2020/c/ca/T--UPF_Barcelona--directed_evolution_new.pdf)

![](https://static.igem.org/mediawiki/2020/8/87/T--UPF_Barcelona--evolution.png)

Figure 2. T3 biosensor directed evolution approach.

## References

[1] Gierach, I., Li, J., Wu, W. Y., Grover, G. J., & Wood, D. W. (2012). **Bacterial biosensors for screening isoform-selective ligands for human thyroid receptors α-1 and β-1.** *FEBS open bio*, 2, 247–253. https://doi.org/10.1016/j.fob.2012.08.002

[2] Gangopadhyay, J. P., Jiang, S., & Paulus, H. (2003). **An in Vitro Screening System for Protein Splicing Inhibitors Based on Green Fluorescent Protein as an Indicator.** *Analytical Chemistry*, 75(10), 2456–2462. DOI:10.1021/ac020756b

[3] Costa, S., Almeida, A., Castro, A., Domingues, L. (2014). **Fusion tags for protein solubility, purification, and immunogenicity in Escherichia coli: the novel Fh8 system.** *Front. Microbiol.*, 5, 63, https://doi.org/10.3389/fmicb.2014.00063

[4] Costa, S. J., Almeida, A., Castro, A., Domingues, L., Besir, H. (2013). **The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology.** *Appl Microbiol Biotechnol*, 97, 6779–6791. DOI 10.1007/s00253-012-4559-1

[5] **Plasmids 101: Origin of Replication**. Retrieved July 25, 2020, from [https://blog.addgene.org/plasmid-101-origin-of-replication]

[6] Gonzalez-Flo, E., Alaball, M. E., Macia, J. (2020). **Two-Component Biosensors: Unveiling the Mechanisms of Predictable Tunability.** *ACS Synthetic Biology*, 9(6), 1328-1335. DOI: 10.1021/acssynbio.0c00010

[7] **UniProtKB - P10828 (THB\_HUMAN)**. Retrieved October 22, 2020, from [https://www.uniprot.org/uniprot/P10828]

[8] Badran, A., Liu, D. (2015). **Development of potent in vivo mutagenesis plasmids with broad mutational spectra.** *Nat Commun*, 6, 8425. https://doi.org/10.1038/ncomms9425

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# CONTACT US

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# Team:UPF Barcelona/Experiments

UPF\_Barcelona

[![](https://static.igem.org/mediawiki/2020/4/46/T--UPF_Barcelona--logowhite.png)](https://2020.igem.org/Team%3AUPF_Barcelona)

Menu

* Project

  [Description](https://2020.igem.org/Team%3AUPF_Barcelona/Description)
  [Design](https://2020.igem.org/Team%3AUPF_Barcelona/Design)
  [Engineering](https://2020.igem.org/Team%3AUPF_Barcelona/Engineering)[Implementation](https://2020.igem.org/Team%3AUPF_Barcelona/Implementation)
  [Entrepreneurship](https://2020.igem.org/Team%3AUPF_Barcelona/Entrepreneurship)
  [Safety](https://2020.igem.org/Team%3AUPF_Barcelona/Safety)[Poster](https://2020.igem.org/Team%3AUPF_Barcelona/Poster)
* Labwork

  [T3 biosensor](https://2020.igem.org/Team%3AUPF_Barcelona/Intein)
  [Proof of concept](https://2020.igem.org/Team%3AUPF_Barcelona/Proof_Of_Concept)[Experiments](https://2020.igem.org/Team%3AUPF_Barcelona/Experiments)
* DryLab

  [Hardware](https://2020.igem.org/Team%3AUPF_Barcelona/Hardware)
  [Software](https://2020.igem.org/Team%3AUPF_Barcelona/Software)
  [Contribution](https://2020.igem.org/Team%3AUPF_Barcelona/Contribution)[Model](https://2020.igem.org/Team%3AUPF_Barcelona/Model)
* Results

  [Results](https://2020.igem.org/Team%3AUPF_Barcelona/Results)
  [Improve](https://2020.igem.org/Team%3AUPF_Barcelona/Improve)
* [Parts](https://2020.igem.org/Team%3AUPF_Barcelona/Parts)
* Human Practices

  [Human Practices](https://2020.igem.org/Team%3AUPF_Barcelona/Human_Practices)
  [Public Engagement](https://2020.igem.org/Team%3AUPF_Barcelona/Education)
* Team

  [Team](https://2020.igem.org/Team%3AUPF_Barcelona/Team)
  [Attributions](https://2020.igem.org/Team%3AUPF_Barcelona/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AUPF_Barcelona/Collaborations)
* Awards

  [Medals](https://2020.igem.org/Team%3AUPF_Barcelona/Medals)
  [Awards](https://2020.igem.org/Team%3AUPF_Barcelona/Awards)

This section consists in a recopilation of the protocols that we have used during the development of Hormonic. It also contains the notebook of both wetlab parts of the project: the intein-mediated T3 biosensor and the proof of concept.

EXPERIMENTS

* [Protocols](#Protocols)* [T3-biosensor Notebook](#T3_biosensor_notebooks)
  * [Proof of concept Notebook](#Proof_notebook)

# Protocols

Experimentation is one of the fundamental parts related to our biosensor. The design of the experiments and also the adaptation of the protocols to each experiment allowed us to achieve the main objectives of our project. In this page you can find all the steps that we followed, because we want all the experiments to be repeatable and reproducible.

## Cell Lysis by Sonication

#### Description

This protocol uses the Branson Digital Sonifier 250 & Sonifier Sound Enclosure to lysate Bacterial *E. coli* Cells.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/a/ad/T--UPF_Barcelona--CellLysisbySonification.pdf)

## Digestion

#### Description

Protocol for the digestion of DNA fragments for Biobrick assembly. It is necessary to prepare one Eppendorf for each DNA fragment that needs to be digested.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/1/15/T--UPF_Barcelona--digestionProtocol.pdf)

## Electro-competent cells

#### Description

Protocol for the preparation of electro-competent cells for the use of electroporation, a widely used method for transformation of DNA extracts into cells based on electric pulses.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/9/93/T--UPF_Barcelona--electrocompetentcellpreparation.pdf)

## Electroporation transformation

#### Description

Electroporation is a widely used method for transformation of DNA extracts into cells. It is based on sending electrical pulses to the cell so its polarized membrane forms reversible transient pores that let the DNA flow into the cell. This protocol explains how to transform electro-competent cells via electroporation. This protocol is adapted from Woodall's work (Woodall, C. A. (n. d.). *Electroporation of E. coli. E. coli Plasmid Vectors, 55–60. doi:10.1385/1-59259-409-3:55)*.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/6/63/T--UPF_Barcelona--electroporationProtocol.pdf)

## Kappa PCR

#### Description

Protocol for PCR using Kappa polymerase. The Kappa polymerase is a high-fidelity polymerase so it is a good option for DNA that needs to be transformed. It is necessary to prepare one tube for each PCR reaction. This protocol is for a 25 μL reaction.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/1/18/T--UPF_Barcelona--kappaPCRprotocol.pdf)

## Ligation

#### Description

Protocol for the ligation of DNA fragments for Biobrick assembly. It is necessary to prepare in Eppendorfs a mix for every combination of DNA fragments that needs to be ligated.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/f/fe/T--UPF_Barcelona--ligationProtocol.pdf)

## Phusion PCR

#### Description

Protocol for PCR using Phusion polymerase. The Phusion polymerase is a high-fidelity polymerase so it is a good option for DNA that needs to be transformed. It is necessary to prepare one tube for each PCR reaction. This protocol is for a 20 μL reaction.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/5/50/T--UPF_Barcelona--phusionPCRprotocol.pdf)

## Probative Digestion

#### Description

Protocol for the digestion of DNA fragments for running a gel to check if an insert is present. It is necessary to prepare in Eppendorfs a mix for every combination of DNA fragments that we want to digest.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/0/00/T--UPF_Barcelona--probativeDigestion.pdf)

## Taq Colony PCR

#### Description

Protocol for a colony PCR using Taq polymerase. The Taq polymerase is not a high-fidelity polymerase so it is a good option for checking if a colony has an insert. It is necessary to prepare one tube for each PCR reaction. This protocol is for a 25 μL reaction.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/b/b9/T--UPF_Barcelona--taqcolonyPCRprotocol.pdf)

## Transformation

#### Description

Protocol for the transformation of DNA via heat shock using the DH5α strain of *E. coli*. It is necessary to prepare one Eppendorf for each DNA fragment that needs to be transformed.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/7/74/T--UPF_Barcelona--transformationProtocol.pdf)

## Western blot

#### Description

Protocol for Western blot procedure and the sample preparation. With this technique we can detect the presence of the desired protein. In order to quantify it, we have to apply the SDS-PAGE protocol.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/9/90/T--UPF_Barcelona--WesternBlotFinal.pdf)

# T3 biosensor notebooks

Experimentation is key in Synthetic Biology. Even though the design can be based on literature and theory, biology never reacts exactly as you planned, and that’s a fact. So, the only way to prove the design of our intein-mediated T3 (IMT3) biosensor was to actually bring it to reality. Here, we present the Benchling notebooks we used to track the different experiments we did to make our sensor come true.

***E. coli* DH5α Transformation:** Here we explain how we used *E. coli* DH5α cells as DNA storage.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/3/30/T--UPF_Barcelona--InteinNotebook.pdf)

***E. coli* BL21 Transformation:** Here we explain how we prepared *E. coli* BL21 (DE3) to be electro competent and how we transformed them to express both of our IMT3 biosensors as our main chassis for its good recombinant protein expression.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/b/bf/T--UPF_Barcelona--BL21transformation.pdf)

**Western Blots:** Here we have described the different Western Blots we did to check the correct expression and splicing of our protein.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/b/b3/T--UPF_Barcelona--WesternBlotInteinNotebook.pdf)

**Plate-readers:** This notebook describes the preparation of our strains and wells content to sense in a Plate-reader over the course of long time periods and see its reaction with different T3 concentrations. As expected, we had some biology related problems, but technology was not in our side all the time either.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/e/ed/T--UPF_Barcelona--PlatereaderInteinNotebook.pdf)

# Proof of concept notebook

As a Proof of Concept for our biosensor we decided to build a lactone biosensor. This notebook contains the different cloning strategies performed to build the genetic construct and transform it into our sensor cells. Once the sensor was complete, a full characterization of its performance was done, using both, fixed concentrations of lactone and our lactone producer cells to determine the dynamic range of our lactone sensor cells. This notebook also contains all the wetlab experiments performed to do these characterizations.

[DOWNLOAD](https://static.igem.org/mediawiki/2020/9/91/T--UPF_Barcelona--lactoneNotebook.pdf)

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# Team:UPF Barcelona/Results

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As a proof of concept for our device, which regulates a correct dosing depending on a measured hormonal level,
we designed a biological circuit capable of producing and sensing lactone in a controlled way. This circuit is formed
by two kinds of cells, ones responsible for producing lactone, and ones responsible for sensing the presence of lactone
and producing super-folder GFP (sfGFP) in response. With this circuit, we can simulate and understand how the thyroid axis
works and how our device would work.

Results

* [Results](#biosensor)

+ [T3 biosensor](#temperature)
+ [Proof of concept](#od)

* [Conclusions](#concl)
* [References](#ref)

# Results

* [T3 biosensor](#T3)
* [Proof of concept](#lactone)

#### Introduction

The design of the biosensor was made only with the bibliographic information available. Not being able to access the laboratory
made us fine tune the “in silico” sequence design as much as possible to cover all of our bases and expect as little experimental
surprises as possible. This would allow us to minimize the troubleshooting time as much as possible to seize the restricted
laboratory access time. This is the reason why we already added to the initial construct the **Fh8 solubility tag**, the **Flag tag**,
a strong pair of **Promoter-RBS** and the **BioBrick assembly** prefix/suffix, among others. However, since our biosensor had never
been done before in the literature, we surely had to test our silico design in vitro. All the protocols we used are available
as downloadable files at the [Experiments page](https://2020.igem.org/Team%3AUPF_Barcelona/Experiments).

#### Sequence Checking

The two final constructs were synthesized and provided by IDT. Despite how much we trust iGEM’s sponsors, we had to check the
sequences of both Intein Mediated T3 (IMT3) biosensors. To do this, we separately transformed *E. Coli* DH5α with our two plasmids:
*IMT3\_eGFP\_pUC-Kan* (enhanced GFP as a reporter version) and *IMT3\_sfGFP\_pUC-Amp* (super folder GFP as a reporter version).

**Colony PCR followed by sequencing**, both with the commonly used VF2 and VR primers, confirmed the sequence. The sequence-confirmed
plasmids were extracted and transformed into *E. Coli* BL21 (DE3) via electroporation, and Colony PCR was performed once again.

![](https://static.igem.org/mediawiki/2020/f/f6/T--UPF_Barcelona--inteinResults1.png)

Fig 1. Colony PCR of *IMT3\_eGFP\_pUC-Kan* confirmed a weight around 3,5 Kb. First well was a positive control, the next 7 were different colonies.

![](https://static.igem.org/mediawiki/2020/a/a1/T--UPF_Barcelona--inteinResults2.png)

Fig 2. Colony PCR of *IMT3\_sfGFP\_pUC-Amp* confirmed a weight around 3,4 Kb. Wells 1, 2 and 3 correspond to colonies 1, 2 and 3 respectively.

#### Biosensor Expression

To evaluate whether our IMT3 sensing protein was being expressed as well as its splicing abilities in a qualitative way, we performed
**Western blots** to detect our target proteins.

How?

#### Ensuring a proper sensing protein expression

×

BL21 (DE3) containing *IMT3\_sfGFP\_pUC-Amp* and *IMT3\_eGFP\_pUC-Kan* were grown overnight in LB with and without T3 present in the media.
A negative control of BL21 (DE3) without insert was prepared in the same way.

The day after, cells were lysed by sonication followed by a Western Blot. Samples were prepared with and without Urea treatment
before gel loading. This way we could see if the Fh8 solubility tag was working or major inclusion bodies were present.

Close

![](https://static.igem.org/mediawiki/2020/7/72/T--UPF_Barcelona--inteinResults3.png)

Fig 3. A) Western blot from BL21 with *IMT3\_eGFP\_pUC-Kan* where the whole construct (85 kDa) and after splicing joint eGFP (37kDa)
can be seen. B.1) Positive control: mammalian lysiate from a colleague with the same FLAGx3 tag. B.2) Negative control, BL21
without plasmid incubated overnight with 100uM of T3. C) Western blots from the whole construct. C.1 is a positive control.
C.2 and C.3 samples were treated with the boiling method. C.4 and C.5 were treated with Urea incubation before loading the gel.

The Wester Blot showed that the **full protein was being produced** (Fig.3A), corresponding to the 85kDa signal.
**Splicing activity** can be seen from the presence of the 37kDa protein, corresponding to the two halves of the GFP and both tags,
increasingly shown in the samples of induced cells with T3, suggesting that the **splicing** is indeed **coupled with presence with T3**.

We could also observe that there was no significant difference between the samples treated with Urea and without,
suggesting that the Fh8 solubility tag was avoiding major aggregate formation (Fig.3C).

#### Biosensor Performance

In order to characterize the functionality of both Intein Mediated T3 (IMT3) biosensors, in parallel to its expression study, **Long
plate reader Experiments** were set up to sense the amount of fluorescence produced by our biosensor with different T3 concentrations.

BL21 (DE3) containing *IMT3\_eGFP\_pUC-Kan*, *IMT3\_sfGFP\_pUC-Amp* and no plasmid were grown overnight in LB. The next day one part cells were inoculated in 30 fresh LB media and grown for 3 to 4 hours. They were induced with different
concentrations of T3 ranging from 1pM to 100uM plus controls (Tab.2). Fluorescence intensity and OD600 were analysed
every 30 minutes over 24 hours with a plate reader (Tab.1).

Click to see Plate-Reader parameters

#### Plate-reader parameters

×

| Experimental parameters and conditions | Description |
| --- | --- |
| Plate-Reader model | Synergy HTX |
| Plate type | Thermo Fisher 96-well microplates  black-walled clear bottom |
| Cell medium | LB |
| Time | 24 hours |
| Shake | Liner: Continuous  Frequency: 567 rpm (3 mm) |
| Temperature | 37 ºC |
| Gain | 50 |
| Optical Density (OD) measurement  (absorbance) | 660nm |
| GFP excitation wavelength | 485nm |
| GFP emission wavelength | 528nm |

Table 1. Experimental conditions and parameters used in the Plate-Reader analysis.

Close

![](https://static.igem.org/mediawiki/2020/e/e5/T--UPF_Barcelona--inteinResults5.png)

Table 2. Combinatios of T3 concentrations and sensor types. *IMT3\_eGFP\_pUC-Kan* (Orange), *IMT3\_sfGFP\_pUC-Amp* (green), Bl21 no plasmid (yellow) and Blank, LB+Amp (white).

Sensor fluorescence was obtained by subtracting the blank LB fluorescence and normalizing to the OD (Fig.4-5).
All the values were obtained from the mean of the 3 wells with the same conditions. Then it was analysed at steady state (time to infinity)
to generate the transfer functions (Fig.6-7).

![](https://static.igem.org/mediawiki/2020/d/d1/T--UPF_Barcelona--inteinResults6.png)

Equation 1. Normalization equations used to generate the results.

![](https://static.igem.org/mediawiki/2020/2/2f/T--UPF_Barcelona--inteinResults7.png)

Fig 4. Temporal behaviour of fluorescence under different T3 concentration with *IMT3\_eGFP\_pUC-Kan* over 24 hours. The values are the mean of the 3 same concentration wells.

![](https://static.igem.org/mediawiki/2020/2/20/T--UPF_Barcelona--inteinResults8.png)

Fig 5. Temporal behaviour of fluorescence under different T3 concentration with *IMT3\_sfGFP\_pUC-Amp* over 24 hours. The values are the mean of the 3 same concentration wells.

![](https://static.igem.org/mediawiki/2020/4/4c/T--UPF_Barcelona--inteinResults9_new.png)

Fig 6. Transfer function of *IMT3\_eGFP\_pUC-Kan* correlating the steady fluorescence with the amount of T3. Experimental data (red dots) and fitted model (blue line).

![](https://static.igem.org/mediawiki/2020/c/cc/T--UPF_Barcelona--inteinResults10_new.png)

Fig 7. Transfer function of *IMT3\_sfGFP\_pUC-Amp* correlating the steady fluorescence with the amount of T3. Experimental data (red dots) and fitted model (blue line.

*IMT3\_sfGFP\_pUC-Amp* fluorescence over the time showed an increase proportional to the concentration of T3 in the media (Fig.5).
*IMT3\_sfGFP\_pUC-Amp* also had higher fluorescent values (between 700 and 1800 RFU) (fig.5) compared with *IMT3\_eGFP\_pUC-Kan* (between 400 and 900 RFU) (fig.4).

Overall, the *IMT3\_eGFP\_pUC-Kan* experimental results were not concluding enough to see its sensing range.

However, with the *IMT3\_sfGFP\_pUC-Amp*, we have **successfully developed a biosensor able to detect changes in T3 concentration** and
report them with a fluorescent signal proportional to the ligand concentration. We could observe that the sensor response had a
**dynamic range of: 10-7 to 10-4 M of T3**, as can be seen above (Fig.7).

#### Introduction

We have already seen the characteristics of the lactone circuit and its analogy to the thyroid axis in the [Proof of Concept page](https://2020.igem.org/Team%3AUPF_Barcelona/Proof_Of_Concept)
. Here you can find the results obtained from the different tests and characterizations that were performed using this circuit.

#### Characterization of the lactone biosensor

To characterize the functioning of the lactone biosensor cell, different concentrations of
lactone going from 100 pM to 100 µM were used. The response to these concentrations over time was monitored using
a Plate-Reader analysis on GFP fluorescence emission (Fig.8). From this data we could fit the resulting sfGFP concentration
to our lactone biosensor model, showing a great correlation between the experimental and the theoretical results.

![](https://static.igem.org/mediawiki/2020/c/c9/T--UPF_Barcelona--resultsFig1.png)

Figure 8. GFP fluorescence emission with different AHL concentrations with respect to time.

In the graph below (Fig.9) we can see these fitting results. The graph represents fluorescence,
in Relative Fluorescence Units (RFU) normalized with respect to the optical density, relative to the different lactone
concentrations. The shape of this function follows a Hill function, showing a dynamic range that goes from 10-8 to 10-5 M.
This means that our biosensor would follow a sinusoidal dynamics.

![](https://static.igem.org/mediawiki/2020/9/96/T--UPF_Barcelona--resultsFig2.png)

Figure 9. Final GFP fluorescence emission with respect to different AHL concentrations.

#### Characterization of the lactone system: Combining the producer and the sensor cells

As for the genetic circuit, we characterized its behaviour under different concentrations of both arabinose and glucose.
In order to model these dynamics two transfer functions can be derived: one representing the producer cell and the other
representing the sensor cell. These models can be found in our [Modeling page](https://2020.igem.org/Team%3AUPF_Barcelona/Model).
The experimental results (Fig.10) showed a great similarity with the theoretical results previously obtained.

![](https://static.igem.org/mediawiki/2020/7/77/T--UPF_Barcelona--resultsFig3.png)

Figure 10. Experimental and modelling results of the genetic circuit.

As expected, the presence of sfGFP is dramatically reduced as the concentration of the inhibitor (glucose) increases
or as the concentration of the activator (arabinose) decreases. The graphs in Fig.10 can be simplified into the table below (Fig.11).

![](https://static.igem.org/mediawiki/2020/8/80/T--UPF_Barcelona--resultsTab1.png)

Figure 11. Truth table for the behaviour of genetic circuit with respect to glucose and arabinose.

# Conclusions

#### Future work: improve our biosensor

Alongside the good results, it is important to keep in mind that this is not the final state of our IMT3 sensor. From the first
version of the sensor IMT3\_eGFP, where the results were not as promising, we managed to improve it to the second version
(IMT3\_sfGFP), which had more stable results improving the accuracy and the precision from our previous version. We already
started the **third engineering cycle**, planning on how to **improve our sensor** to its next version to further enhance its characteristics.

We would like to study the **effect of having two Ligand Binding Domains in tandem** instead of only one.
Increasing the number of receptors could modify the sensibility or the ability of the sensor to detect lower
T3 concentrations [1]. A **Low copy number** vector was also considered, to reduce the randomness of the high copy
plasmid, where sometimes from cell to cell the number of plasmids can vary up to 200 copies. Changing the GFP split
point or **introducing mutations** to the intein or the residues that interact with the ligand are key targets that are meant to be explored.

Specially this last point, **mutagenesis**, can be extrapolated to **Directed Evolution** principles, which opens a realm of protein optimization,
in which our IMT3 sensor could excel. All these proposed modifications are further explained in our [Engineering Success](https://2020.igem.org/Team%3AUPF_Barcelona/Engineering) Page.

#### Extending our biosensor to other applications

In Hormonic, the IMT3 biosensor was built with the objective of finding a good way to continually report to a computational unit the levels of T3 to potentially
treat patients with **hypothyroidism**, but along the way, we envisioned plenty of other possible applications.

With **cell free system** technologies being cheaper every year, IMT3 biosensors could be introduced to the diagnosis field, by the
creation of quick tests, where the reporter protein, instead of being a fluorescent protein, could be a chromoprotein or any other
depending on the application. Specially in these times (2020), the adaptability of intein mediated biosensors could help develop
**new quick diagnosis systems for COVID-19**, considering the interaction of Human hACE2 protein with the Spike protein of the virus, finding the
binding domains and studying the pairing with the intein domains.

The use of Inteins as sensors has been studied for over two decades. However, this is the **first time** it has been shown that the two
intein domains can be coupled in the same chimeric protein with a ligand binding domain to report fluorescence. Only in the
human body there are several proteins that bind to other molecules or proteins that have been studied. This opens the potential
for the creation of other **Intein Mediated Biosensors**, that, not only will detect different molecules, but also could be reported
as fluorescence and coupled to computational devices.

Moreover, with the **proven capability of our PID to restore the feedback**, these types of sensors adapted to sense other molecules
allow the capacity of **restoring other hormonal disorders**, such as diabetes, menopause, imbalances in sexual hormones, or even osteoporosis.

All in all, **achieving a functional IMT3 biosensor** means much more than reporting T3 concentration. It means that the post-translational
protein splicing phenomena is a very strong and versatile tool.

We would also like to acknowledge that this IMT3 biosensor will be used as **educational material** as an example of a protein biosensor,
which will be explained at a high school summer camp organized by “La Fundacio La Pedrera” in Barcelona. For more information visit
our [Science communication page](https://2020.igem.org/Team%3AUPF_Barcelona/Education).

## References

[1] Gonzalez-Flo, E., Alaball, M. E., Macía, J. **Two-Component Biosensors: Unveiling the Mechanisms of Predictable Tunability.** *ACS Synthetic Biology*, 2020 9 (6), 1328-1335 DOI: 10.1021/acssynbio.0c00010

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