iGEM 2018 — Team USP-Brazil

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# Team:USP-Brazil/Notebook

Wiki - iGEM Brazil

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  * Project

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    [Improvement](https://2018.igem.org/Team%3AUSP-Brazil/Improve)

    * Wetlab

      [Results](https://2018.igem.org/Team%3AUSP-Brazil/Results)
      [Notebook](https://2018.igem.org/Team%3AUSP-Brazil/Notebook)
      [Demonstrate](https://2018.igem.org/Team%3AUSP-Brazil/Demonstrate)

      * Modelling

        [Overview](https://2018.igem.org/Team%3AUSP-Brazil/Modelling)
        [Single System Model](https://2018.igem.org/Team%3AUSP-Brazil/Model)
        [Simulations](https://2018.igem.org/Team%3AUSP-Brazil/Simulations)
        [Multi-Systems Model](https://2018.igem.org/Team%3AUSP-Brazil/MultiModel)
        [Statistical Analysis](https://2018.igem.org/Team%3AUSP-Brazil/Statistics)

        * Human

          [Overview](https://2018.igem.org/Team%3AUSP-Brazil/Overview_Human)
          [Public Engagement](https://2018.igem.org/Team%3AUSP-Brazil/Public_Engagement)
          [Integrated](https://2018.igem.org/Team%3AUSP-Brazil/Human_Practices)
          [Collaborations](https://2018.igem.org/Team%3AUSP-Brazil/Collaborations)

          * Resources

            [Safety](https://2018.igem.org/Team%3AUSP-Brazil/Safety)
            [Interlab](https://2018.igem.org/Team%3AUSP-Brazil/InterLab)
            [Achievements](https://2018.igem.org/Team%3AUSP-Brazil/Achievements)
            [Collection of Parts](https://2018.igem.org/Team%3AUSP-Brazil/Part_Collection)

![](https://static.igem.org/mediawiki/2018/d/d3/T--USP-Brazil--logo-nm.png "Hello there! Come to our poster and know us!")

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#### Wetlab

* [Results](#results)
* [Notebook](#note)
* [Demonstrate](#demons)

## Notebook

### Circuit construction

To build up all the 12 plasmids, we mostly used biobricks from the 2017 distribution kit. The flowchart below shows all the cloning procedures we have planned.

![](https://static.igem.org/mediawiki/2018/d/d3/T--USP-Brazil--clone.png)

### Protocols

The circuit construction was done following these protocols:

### Heat shock transformation

After thawing the cells on ice for 10 minutes, add 1-5uL of plasmid to 50uL of competent cells and mix gently. Incubate the cells on ice for 20-13 minutes and spread 20uL of antibiotics for each plate. Afterwards, waterbath the cells at 42°C for 1 minute. Put the tube back on ice for 2 minutes. Add 950uL of LB to the tube and incubate at 37°C for 30-60 minutes. Centrifuge and ressuspend the cells with 100uL LB. Plate and incubate the cells ovvernight at 37°C.

### PCR Protocol

| DNA | Forward primer 10mM | Reverse primer 10mM | dNTPs 10mM | 10x Buffer | MgCl2 50mM | Taq | H2O |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1uL | 0,5uL | 0,5uL | 0,5uL | 2,5uL | 0,75uL | 0,1uL | to 25uL |

We have ordered from Exxtend the following primers for PCR of BioBricks. They are complementary to the BioBricks prefix and suffix, and have a 5' overhang for the ocasion of wanting to amplify a DNA fragment for cloning, as the proximity of the EcoRI and PstI restriction sites to the end of the fragment would hinder the enzymes activity:

Primer Forward: 5’-TGATTACTGAATTCGCGGCCGCTTCTAG-3’

Primer Reverse: 5’-TGATTACTCTGCAGCGGCCGCTACTAGTA-3’

Cycles for the pcr were as following:

| Step 1 | Step 2 | Step 3 | Step 4 | Step 5 | Step 6 | Step 7 |
| --- | --- | --- | --- | --- | --- | --- |
| 95ºC 5min 95ºC 30s 60ºC 30s 72ºC 1min/Kb Return to Step 2 30x 72ºC 5min 4ºC | | | | | | |

### Digestion Protocol

We used EcoRI, SpeI, NotI, XbaI or PstI for digestion of biobricks. Depending on the use we were going to make of the parts, we used different quantities of DNA for digestion (to obtain a linearized vector for 3A assembly, for example, we used less DNA to minimize the amount of background, empty vector transformations)

| Plasmid | 10x 2.1 Buffer | Enzymes | H2O |
| --- | --- | --- | --- |
| 15uL | 2uL | 1uL each | To 20uL |

Incubate the restriction digest at 37°C for 30 minutes and then 80°C for 20 minutes, for heat-inactivation in case of a subsequent 3A assembly. If the fragments were to be used in conventional ligation, we purified it from an agarose gel, loading all of the digestion.

### Ligation Protocol

|  | Vector | Insert | 10x Ligase Buffer | T4 Ligase | H2O |
| --- | --- | --- | --- | --- | --- |
| Stdt. Ligation | 1,5uL | 10uL | 2uL | 1uL | To 20uL |
| 3A assembly | 2,3uL | 2,3uL each | 1uL | 1uL |

Ligate 16°C for 60 minutes and heat-inactivate at 80°C for 20 minutes

## PDF NOTEBOOK

[Notebook.pdf](https://static.igem.org/mediawiki/2018/f/f2/T--USP-Brazil--Notebook.pdf)

iGEM USP-Brazil

* [Home](https://2018.igem.org/Team%3AUSP-Brazil)
* [Team](https://2018.igem.org/Team%3AUSP-Brazil/Team)
  + [People](https://2018.igem.org/Team%3AUSP-Brazil/Team)
  + [Attributions](https://2018.igem.org/Team%3AUSP-Brazil/Attributions)
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* [Project](https://2018.igem.org/Team%3AUSP-Brazil/Overview)
  + [Overview](https://2018.igem.org/Team%3AUSP-Brazil/Overview)
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  + [Project Design](https://2018.igem.org/Team%3AUSP-Brazil/Design)
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  + [Measurement](https://2018.igem.org/Team%3AUSP-Brazil/Measurement)
  + [Part Improvement](https://2018.igem.org/Team%3AUSP-Brazil/Improve)
* [WetLab](https://2018.igem.org/Team%3AUSP-Brazil/Results)
  + [Results](https://2018.igem.org/Team%3AUSP-Brazil/Results)
  + [Notebook](https://2018.igem.org/Team%3AUSP-Brazil/Notebook)
  + [Demonstrate](https://2018.igem.org/Team%3AUSP-Brazil/Demonstrate)
* [Modelling](https://2018.igem.org/Team%3AUSP-Brazil/Model)
  + [Overview](https://2018.igem.org/Team%3AUSP-Brazil/Modelling)
  + [Single System Model](https://2018.igem.org/Team%3AUSP-Brazil/Model)
  + [Simulations](https://2018.igem.org/Team%3AUSP-Brazil/Simulations)
  + [Multi-Systems Model](https://2018.igem.org/Team%3AUSP-Brazil/MultiModel)
  + [Statistical Analysis](https://2018.igem.org/Team%3AUSP-Brazil/Statistics)
* [Human](https://2018.igem.org/Team%3AUSP-Brazil/Overview_Human)
  + [Overview](https://2018.igem.org/Team%3AUSP-Brazil/Overview_Human)
  + [Public Engagement](https://2018.igem.org/Team%3AUSP-Brazil/Public_Engagement)
  + [Integrated](https://2018.igem.org/Team%3AUSP-Brazil/Human_Practices)
  + [Collaborations](https://2018.igem.org/Team%3AUSP-Brazil/Collaborations)
* [Resources](https://2018.igem.org/Team%3AUSP-Brazil/Safety)
  + [Safety](https://2018.igem.org/Team%3AUSP-Brazil/Safety)
  + [Interlab](https://2018.igem.org/Team%3AUSP-Brazil/InterLab)
  + [Achieviements](https://2018.igem.org/Team%3AUSP-Brazil/Achievements)
  + [Colection of Parts](https://2018.igem.org/Team%3AUSP-Brazil/Part_Collection)


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# Team:USP-Brazil/Experiments

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[Parts Overview](https://2018.igem.org/Team%3AUSP-Brazil/Parts)
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[SAFETY](https://2018.igem.org/Team%3AUSP-Brazil/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AUSP-Brazil/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AUSP-Brazil/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AUSP-Brazil/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AUSP-Brazil/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AUSP-Brazil/Hardware)
[Measurement](https://2018.igem.org/Team%3AUSP-Brazil/Measurement)
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[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=USP-Brazil)

# 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.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:USP-Brazil/Results

Wiki - iGEM Brazil

* [Home](https://2018.igem.org/Team%3AUSP-Brazil)
* Team

  [People](https://2018.igem.org/Team%3AUSP-Brazil/Team)
  [Attributions](https://2018.igem.org/Team%3AUSP-Brazil/Attributions)
  [Sponsors](https://2018.igem.org/Team%3AUSP-Brazil/Sponsors)

  * Project

    [Overview](https://2018.igem.org/Team%3AUSP-Brazil/Overview)
    [Applications](https://2018.igem.org/Team%3AUSP-Brazil/Applications)
    [Project design](https://2018.igem.org/Team%3AUSP-Brazil/Design)
    [Parts](https://2018.igem.org/Team%3AUSP-Brazil/Parts)
    [Measurement](https://2018.igem.org/Team%3AUSP-Brazil/Measurement)
    [Improvement](https://2018.igem.org/Team%3AUSP-Brazil/Improve)

    * Wetlab

      [Results](https://2018.igem.org/Team%3AUSP-Brazil/Results)
      [Notebook](https://2018.igem.org/Team%3AUSP-Brazil/Notebook)
      [Demonstrate](https://2018.igem.org/Team%3AUSP-Brazil/Demonstrate)

      * Modelling

        [Overview](https://2018.igem.org/Team%3AUSP-Brazil/Modelling)
        [Single System Model](https://2018.igem.org/Team%3AUSP-Brazil/Model)
        [Simulations](https://2018.igem.org/Team%3AUSP-Brazil/Simulations)
        [Multi-Systems Model](https://2018.igem.org/Team%3AUSP-Brazil/MultiModel)
        [Statistical Analysis](https://2018.igem.org/Team%3AUSP-Brazil/Statistics)

        * Human

          [Overview](https://2018.igem.org/Team%3AUSP-Brazil/Overview_Human)
          [Public Engagement](https://2018.igem.org/Team%3AUSP-Brazil/Public_Engagement)
          [Integrated](https://2018.igem.org/Team%3AUSP-Brazil/Human_Practices)
          [Collaborations](https://2018.igem.org/Team%3AUSP-Brazil/Collaborations)

          * Resources

            [Safety](https://2018.igem.org/Team%3AUSP-Brazil/Safety)
            [Interlab](https://2018.igem.org/Team%3AUSP-Brazil/InterLab)
            [Achievements](https://2018.igem.org/Team%3AUSP-Brazil/Achievements)
            [Collection of Parts](https://2018.igem.org/Team%3AUSP-Brazil/Part_Collection)

![](https://static.igem.org/mediawiki/2018/d/d3/T--USP-Brazil--logo-nm.png "Hello there! Come to our poster and know us!")

![](https://static.igem.org/mediawiki/2018/9/98/T--USP-Brazil--banner_wetlab.png)

#### Wetlab

* [Results](#results)
* [Notebook](#note)
* [Demonstrate](#demons)

## Results

After some trouble with gene synthesis and building our constructs, we got to doing experiments with the first three candidates for Sender systems (Lux, Las, Rhl) and all of the six Receiver systems. In this page, we will go through each one of the Sender systems and report how our reporters responded to the signal.

Our experiments were normally done in 15mL falcon tubes or 24-well plates (with admitted differences in aeration) and in 2 mL LB or M9 (for kinetic experiments) medium, at 37ºC and shaking at 180~200RPM. As the auto-induced quorum sensing system should be kickstarted by arabinose (see [Design](https://2018.igem.org/Team%3AUSP-Brazil/Design)), after growing an overnight culture diluted 1:10 for one hour, we induced our cells with 2.5mM L-arabinose and started measurements.

### Lux Sender

The Lux Sender was our first system to yeld positive results, as it had the most straightforward cloning steps from the Distribution Kit parts. To test if the system would respond, we first plated our co-transformations in solid medium, slathered with 20uL of 1M arabinose. Also, to check if it was really our quorum sensing signal inducing the fluorescence, we tested inducing with arabinose in a single point of the petri dish.

![](https://static.igem.org/mediawiki/2018/3/38/T--USP-Brazil--Lux_Lux_petri_dish.png)

After seeing that our system worked, we proceeded to the liquid medium assays. They were done as reported above, then fitted for a dose-response curve, assuming an increasing amount of HSL signal over time. The promoters that showed the most significant response were pLux and pLas, with the latter having a lower sensitivity (~3.3x pLux EC50) but plateauing at a higher expression (~1.7x pLux expression). The other promoters only showed significant expression at later stages of our experiment, and at a much lower strength, but it at least indicated that they were working. We credit these differences in timing and expression strenght, comparing our data and our references, to the amount of proteins being expressed. We have a synthase, a receptor, EYFP, ECFP, the araC gene for pBad and two antibiotic resistances, so low signals get nearly unnoticeable.

![](https://static.igem.org/mediawiki/2018/5/5c/T--USP-Brazil--Good_assay.png)

![](https://static.igem.org/mediawiki/2018/3/3f/T--USP-Brazil--Bad_assay_Lux.png)

Team [Düsseldorf](https://2018.igem.org/Team%3ADuesseldorf) helped us by providing some 3OC6 HSL, which we could use in an assay to discern the amount of HSL in our auto-inducer assays, this time in M9 medium, so protein production and population growth were overall slower. We were able to see, as expected, that directly inducing our system with HSL makes for a faster response than inducing with arabinose (EC50 was 2.7x lower), and also reaching a smaller plateau (7x smaller). This leads us to the conclusions that there is a delay to the start of the producion of reporter protein, due to the need of producing signal synthase; and that our auto-inducer system leads to an HSL concentration that allows for a much higher equilibrium, resulting in increased promoter strength. The HSL concentration we used for this assay was 10-4mM, as literature showed that this concentration already showed the maximum transcription rate by the Lux promoter. We expect the HSL concentration reached using the synthase, then, to be higher than this quantity.

![](https://static.igem.org/mediawiki/2018/3/3e/T--USP-Brazil--HSL_assay.png)

While testing the Las promoter in M9, however, we got an interesting result: activity was only as high as in LB if induced with synthetic HSL. When induced with arabinose, the system presented activity much closer to controls, what leads us to conclude that in M9 medium the LuxI synthase has trouble reaching HSL concentrations of 10-4mM.

![](https://static.igem.org/mediawiki/2018/6/61/T--USP-Brazil--Lux_Las_1_17_10.png)

![](https://static.igem.org/mediawiki/2018/9/96/T--USP-Brazil--Lux_Las_2_17_10.png)

### Las Sender

After finishing building this construct, we tried to do the same experiments as with the Lux Sender, but we didn't get a response for either with arabinose or directly with HSLs (hoping that the synthase would be the problem and the receptor would be able to have chemical crosstalk with 3OC6 HSL), despite the construct having correct DNA sizes in analytical digestions along the construction. We still intend to sequence this part to check for any mutations, but unfortunately the result should only be available after the Jamboree.

### Rhl Sender

When testing with the Rhl system's Sender plasmid, the only promoter that showed significant expression in relation to previously tested controls was pLas (showing that the sender plasmid really works). As with the Lux Sender test, the other promoters could start showing activity at later stages, but we couldn't proceed with the assay at that point. It can be seen, also, that this first promoter showed less activity and sensibility than in the first assay, so if we consider the two synthases doing HSL at the same rate, we should be able to get values for RhlR crosstalk strength and sensibility for the positive promoter. One relevant piece of data here was the lack of activity of pRhl, Rhl system's native promoter. As with the other promoters in the first assay, we credit this to metabolic burden. This promoter already showed little expression from the part's experience page and, at the levels of HSL concentration that could be deduced from the Lux Sender assay using synthetic HSL, some times higher than 10-4 mM, this promoter should not show a lot of expression, specially in this conditions and timeframe.

![](https://static.igem.org/mediawiki/2018/a/a8/T--USP-Brazil--Rhl_assay.png)

We later tested the activity of this Sender with pLux and pLas in M9 medium, induced with both arabinose (2.5mM) and synthetic HSL (10-4mM). Again, the only promoter showing activity was pLas, although only when induced by arabinose. This suggests that the Rhl synthase can generate concentrations of HSL higher than 10-4mM even in M9 medium and that the lack of activity of the other promoters is really due to a lack of crosstalk.

![](https://static.igem.org/mediawiki/2018/1/19/T--USP-Brazil--Rhl_17_10.png)

### References

* Grant, Paul K et al. “Orthogonal Intercellular Signaling for Programmed Spatial Behavior.” Molecular Systems Biology 12.1 (2016): 849. PMC. Web. 16 Oct. 2018.
* Spencer R. Scott and Jeff Hasty. “Quorum Sensing Communication Modules for Microbial Consortia” ACS Synthetic Biology 5.9 (2016), 969-977 doi: 10.1021/acssynbio.5b00286
* N.Kylilis, Z.A. Tuza, G. Stan, K.M. Polizzi. “Tools for engineering coordinated system behaviour in synthetic microbial consortia” Nature Communications, volume 9 (2018), Article number: 2677.
* R.J.Case, M.Labbate, S.Kjelleberg. “AHL-driven quorum-sensing circuits: their frequency and function among the Proteobacteria” The ISME Journal, volume 2, pages 345–349 (2008)

iGEM USP-Brazil

* [Home](https://2018.igem.org/Team%3AUSP-Brazil)
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  + [Attributions](https://2018.igem.org/Team%3AUSP-Brazil/Attributions)
  + [Sponsors](https://2018.igem.org/Team%3AUSP-Brazil/Sponsors)
* [Project](https://2018.igem.org/Team%3AUSP-Brazil/Overview)
  + [Overview](https://2018.igem.org/Team%3AUSP-Brazil/Overview)
  + [Applications](https://2018.igem.org/Team%3AUSP-Brazil/Applications)
  + [Project Design](https://2018.igem.org/Team%3AUSP-Brazil/Design)
  + [Parts](https://2018.igem.org/Team%3AUSP-Brazil/Parts)
  + [Measurement](https://2018.igem.org/Team%3AUSP-Brazil/Measurement)
  + [Part Improvement](https://2018.igem.org/Team%3AUSP-Brazil/Improve)
* [WetLab](https://2018.igem.org/Team%3AUSP-Brazil/Results)
  + [Results](https://2018.igem.org/Team%3AUSP-Brazil/Results)
  + [Notebook](https://2018.igem.org/Team%3AUSP-Brazil/Notebook)
  + [Demonstrate](https://2018.igem.org/Team%3AUSP-Brazil/Demonstrate)
* [Modelling](https://2018.igem.org/Team%3AUSP-Brazil/Model)
  + [Overview](https://2018.igem.org/Team%3AUSP-Brazil/Modelling)
  + [Single System Model](https://2018.igem.org/Team%3AUSP-Brazil/Model)
  + [Simulations](https://2018.igem.org/Team%3AUSP-Brazil/Simulations)
  + [Multi-Systems Model](https://2018.igem.org/Team%3AUSP-Brazil/MultiModel)
  + [Statistical Analysis](https://2018.igem.org/Team%3AUSP-Brazil/Statistics)
* [Human](https://2018.igem.org/Team%3AUSP-Brazil/Overview_Human)
  + [Overview](https://2018.igem.org/Team%3AUSP-Brazil/Overview_Human)
  + [Public Engagement](https://2018.igem.org/Team%3AUSP-Brazil/Public_Engagement)
  + [Integrated](https://2018.igem.org/Team%3AUSP-Brazil/Human_Practices)
  + [Collaborations](https://2018.igem.org/Team%3AUSP-Brazil/Collaborations)
* [Resources](https://2018.igem.org/Team%3AUSP-Brazil/Safety)
  + [Safety](https://2018.igem.org/Team%3AUSP-Brazil/Safety)
  + [Interlab](https://2018.igem.org/Team%3AUSP-Brazil/InterLab)
  + [Achieviements](https://2018.igem.org/Team%3AUSP-Brazil/Achievements)
  + [Colection of Parts](https://2018.igem.org/Team%3AUSP-Brazil/Part_Collection)