iGEM 2019 — Team USP-Brazil

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# Team:USP-Brazil/Notebook

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* [Medal Criteria](https://2019.igem.org/Team%3AUSP-Brazil/MedalCriteria)

# Lab experiments

Our daily experiments involved the manipulation of E. coli, thus needing its cultivation. These bacteria was mainly grown in the lysogy broth (LB) following the stablished protocol for such medium. The LB agar plates were made adding 1,5% of bacto agar. Such medium were all incubated at 37°C. All the antibiotic concentration for strains harboring plasmids was followed as stated by the CLSI standards.

PCRs were performed utilizing either Taq DNA Polymerase (Sinapse Inc - P1011) for routine amplification or Q5® High-Fidelity 2X Master Mix (NEB - M0492S) for high-fidelity amplification. The reactions were performed as recommended by its protocol. All electrophoresis were performed in TBE 1x with 0,8% agarose gel at 90 volts.

Digestion and ligation reactions were all performed as stated by its protocols. We utilized mainly NEB enzymes for such experiments, following the molar ratio and concentration per enzymes units stated.

Bacterial transformation was performed either with electrocompetent cells for higher rate of transformants or chemically competent cells for routine transformations. Electrocompetent cells were accomplished through several washes with Milli-Q water and glycerol 10%, the transformation protocol were made at 1800V in an electroporation cuvette (1mm gap width). Chemically competent cells were prepared washing cells with divalent cations solutions (such as Mg2+ and Ca2+), the transformation protocol were made heat shocking the cells at 42°C.

Cell density were measured with a spectrophotometer and cell fluorescence were measured with SpectraMax Paradigm in a 96-well black plate to avoid neighbour well interference.

Two important experiments that need to be featured are:

## Golden gate assay

Both approach A and approach B circuit were too big to be ordered in a single block. Because of that both IDT and Twist synthesis were fragmented and ordered in several blocks, which were linked through the NEB® Golden Gate Assembly Kit (BsaI-HF® v2), according to the Instruction Manual bellow.

## Comparison efficiency test of pBAD and pLac promoters

These experiments was used in order to analysis the chemical promoters, pLac and pBAD, and the light promoter. During the experiment, all the promoters mentioned in the project were tested. Each one was grown in two types of media: 0.2% glicerol mineral media and 0.2% glucose mineral media. For pBAD different concentrations of arabinose were used and for pLac different concentrations of IPTG were used. All the experiments occurred overnight.

[Notebook 1](https://static.igem.org/mediawiki/2019/e/e5/T--USP-Brazil--note1.pdf)

[Notebook 2](https://static.igem.org/mediawiki/2019/0/06/T--USP-Brazil--note2.pdf)

[Notebook 3](https://static.igem.org/mediawiki/2019/5/57/T--USP-Brazil--neb.pdf)

[Notebook 4](https://static.igem.org/mediawiki/2019/d/df/T--USP-Brazil--note4.pdf)

## USP-Brazil

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# Team:USP-Brazil/Experiments

Giu's Stellar Project

![](https://uploads-ssl.webflow.com/5da2956e848a163ca2d7c583/5da41c804981196cdb777d65_TEAMUSP-LOGO-VARIASAPLICACOES.png)PROJECThumanNOTEBOOKsafetyTEAM

# Rediscovering bioproduction

genSwitch is a genetic circuit able to make E.coli recognize blue led light and switch between two different states of genetic activation

![](https://uploads-ssl.webflow.com/5da2956e848a163ca2d7c583/5da414065ac6b0b940261dd4_videoigem.gif)

## What is bioproduction?

![](https://uploads-ssl.webflow.com/5da2956e848a163ca2d7c583/5da413225ac6b02857261b5f_layout7-05.gif)

Bioproduction is a process used in industries that generates useful products through microorganisms. The most common inductor used in bioproduction are chemical compounds (like IPTG and arabinose) that are, not only expensive, but also lacks fine control of induction. Our goal is to built a genetic circuit that can switch between two different states of activation with just a single input of blue led light. The circuit was built in E.coli and works based on inhibition handles and recombinases that can invert the promoter region according to the presence and absence of the same type of blue light. In addition, fluorescent proteins such as GFP, RFP and YFP function as outps for our circuit. We also compared the efficiency between chemical induced promotors and light-induced ones by evaluating strength and lack. Finally, with our circuit, bioproduction can become more practical, controlled and economical.

## Why use light?

![](https://uploads-ssl.webflow.com/5da2956e848a163ca2d7c583/5da41731ae7ebd1d5ddd8159_image52.png)

Light is a fine, precise inductor, as the specificity of the signal is in the amissor, the wavelength and intensity, and in the receptor, in our case, a protein, that recognize a specific wavelength ortogonali. Besides that, light can be activated instantaneously, simply by turning on it’s source. To stop the induction, it’s not necessary to change the culture media or wash the culture, a process that can make some bioproduction impossible. Using light opens new possibilities for this industry.

## Why make a switch?

![](https://uploads-ssl.webflow.com/5da2956e848a163ca2d7c583/5da417fa20ae1b223c3b0ea9_eeee.png)

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# 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

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the part's Main Page on the Registry.** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)