iGEM 2019 — Team Amazonas-Brazil

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# Team:Amazonas-Brazil/Notebook

* Project

  [Description and Inspiration](https://2019.igem.org/Team%3AAmazonas-Brazil/Description)
  [Design](https://2019.igem.org/Team%3AAmazonas-Brazil/Design)
  [Safety](https://2019.igem.org/Team%3AAmazonas-Brazil/Safety)
  [Results](https://2019.igem.org/Team%3AAmazonas-Brazil/Results)
  [Hardware](https://2019.igem.org/Team%3AAmazonas-Brazil/Hardware)
  [Notebook](https://2019.igem.org/Team%3AAmazonas-Brazil/Notebook)
* Biobricks

  [Basic Part](https://2019.igem.org/Team%3AAmazonas-Brazil/BasicPart)
  [Composite Part](https://2019.igem.org/Team%3AAmazonas-Brazil/CompositePart)
* [Model](https://2019.igem.org/Team%3AAmazonas-Brazil/Model)
* Human Practices

  [Overview](https://2019.igem.org/Team%3AAmazonas-Brazil/Human_Practices)
  [Education and Public Engagement](https://2019.igem.org/Team%3AAmazonas-Brazil/Public_Engagement)
  [Integrated Human Practices](https://2019.igem.org/Team%3AAmazonas-Brazil/Integrated)
* People

  [Attributions](https://2019.igem.org/Team%3AAmazonas-Brazil/Attributions)
  [Collaborations](https://2019.igem.org/Team%3AAmazonas-Brazil/Collaborations)
  [Team](https://2019.igem.org/Team%3AAmazonas-Brazil/Team)
* For judges

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

Team:Amazonas Brazil - 2019.igem.org

Notebook

MAY

**(05.31)** -Late Registration is done.

JULY

**(07.03)** -Our iGEM box was detained in Brazilian Customs

AUGUST

**Week 0** (08.05-08.12)
-Our Twist’s sequence was detained in Brazilian Customs

-Plasmidial extraction of EcN

-Stock preparation of EcN

-Trial of Confirmation of EcN strain

**Week 1** (08.19-08.26)
-Our IDT sequences arrived safely!

-Arrived parts were cloned in E.coli DH5alpha and streaked

-Biobricks Inoculation

-Miniprep of BioBricks

**Week 2** (08.26-09.02)
-Analytical Digestion of Secretion System Biobricks

-Purification, quantification and Ligation of Secretion System Biobricks

-Transformation and Inoculum of Secretion System Biobricks

-Digestion of Biobricks

-Plasmid extraction for confirmation of cloning.

-Blue White screening of Secretion System

SEPTEMBER

**Week 3** (09.02-09.09)
After dealing with customs, our Twist Bioscience sequences arrived safely!

-Blue White screening

-Analytical digestion of Secretion System and plasmidial extraction

-Inoculation and Eletrocompetence of JM110

-Plate streaking of EcN and DH5alpha

-Digestion of Twist parts

-Digestion of EcN Cryptid plasmids (pMUT1 and pMUT2)

-MiniPrep of parts

-Quantification of parts by QuBit

**Week 4** (09.09-09.16)
-Precipitation

-Plasmidial extraction

-PCR and colony PCR

-Digestion of part from USP and from secretion system

-Inocule of EcN and DH5alpha

-Purification, quantification and ligation of parts from secretion system

**Week 5** (09.16-09.23)
-Quantification of secretion system by NanoDrop

-Ligation of Secretion system biobricks

-Precipitation of secretion system

-Digestion of Secretion system

-Gblock Purification

-Transformation of system on DH5alpha

-Inocule of JM110 with secretion system

-Plating of DH5alpha

**Week 6** (09.23-09.30)
-Transformation of secretion system

-Digestion of secretion system

-Quantification of gBlocks

-Ligation of gBlocks to pUC19 vector

OCTOBER

**Week 7** (09.30-10.07)
-Precipitation of ligation of gBlock7 to TolC\_B0015\_pSB1C3

-Transformation of the ligation in DH5alpha

-Cloning of Secretion and AND gate modules in EcN and DH5alpha

-InPreparation of backbones and acquiring of already made backbones

-Trial of validation of AND gate

**Week 8** (10.07-10.14)
-Padronization of Growth curves

-Trial of Validation of AND gate and secretion module

-Cloning of Secretion and AND gate modules in EcN and DH5alpha

-Inocules of EcN

-Preparation of DH5alpha electrocompetent and Chemocompetent

-Preparation of EcN electrocompetent and chemocompetent

**Week 9** (10.14-10.21)
-Preparation of EcN electrocompetent cells

-Transformation of RFP\_pSB1C3, GFP\_pSB1C3, gblock3\_pUC19, gfpmut3\_pUC19, ANDgate\_pUC19 in E coli Nissle

-Cell lyses, protein dosage and SDS-Page of E coli Nissle, gBlock3\_pUC19 in EcN, ANDgate\_pUC19 in EcN and gfpmut3\_pUC19 in EcN

-Growth curve of E coli Nissle with the plasmids GFP\_pSB1C3, RFP\_pSB1C3, GFPmut3\_pUC19, gBlock3\_pUC19, pUC19 and ANDgate\_pUC19.

-Characterization of ANDgate\_pUC19 with hypoxia and lactate

![]()
![](https://static.igem.org/mediawiki/2019/9/96/T--Amazonas-Brazil--patroc%C3%ADnio.png)


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# Team:Amazonas-Brazil/Experiments

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[HOME](https://2019.igem.org/Team%3AAmazonas-Brazil)

TEAM

[Team Members](https://2019.igem.org/Team%3AAmazonas-Brazil/Team)
[Collaborations](https://2019.igem.org/Team%3AAmazonas-Brazil/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3AAmazonas-Brazil/Description)
[Design](https://2019.igem.org/Team%3AAmazonas-Brazil/Design)
[Experiments](https://2019.igem.org/Team%3AAmazonas-Brazil/Experiments)
[Notebook](https://2019.igem.org/Team%3AAmazonas-Brazil/Notebook)
[Contribution](https://2019.igem.org/Team%3AAmazonas-Brazil/Contribution)
[Results](https://2019.igem.org/Team%3AAmazonas-Brazil/Results)
[Demonstrate](https://2019.igem.org/Team%3AAmazonas-Brazil/Demonstrate)
[Improve](https://2019.igem.org/Team%3AAmazonas-Brazil/Improve)
[Attributions](https://2019.igem.org/Team%3AAmazonas-Brazil/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AAmazonas-Brazil/Parts)
[Basic Parts](https://2019.igem.org/Team%3AAmazonas-Brazil/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AAmazonas-Brazil/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AAmazonas-Brazil/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3AAmazonas-Brazil/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AAmazonas-Brazil/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AAmazonas-Brazil/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AAmazonas-Brazil/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AAmazonas-Brazil/Hardware)
[Measurement](https://2019.igem.org/Team%3AAmazonas-Brazil/Measurement)
[Model](https://2019.igem.org/Team%3AAmazonas-Brazil/Model)
[Plant](https://2019.igem.org/Team%3AAmazonas-Brazil/Plant)
[Software](https://2019.igem.org/Team%3AAmazonas-Brazil/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Amazonas-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)

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

# Team:Amazonas-Brazil/Results

* Project

  [Description and Inspiration](https://2019.igem.org/Team%3AAmazonas-Brazil/Description)
  [Design](https://2019.igem.org/Team%3AAmazonas-Brazil/Design)
  [Safety](https://2019.igem.org/Team%3AAmazonas-Brazil/Safety)
  [Results](https://2019.igem.org/Team%3AAmazonas-Brazil/Results)
  [Hardware](https://2019.igem.org/Team%3AAmazonas-Brazil/Hardware)
  [Notebook](https://2019.igem.org/Team%3AAmazonas-Brazil/Notebook)
* Biobricks

  [Basic Part](https://2019.igem.org/Team%3AAmazonas-Brazil/BasicPart)
  [Composite Part](https://2019.igem.org/Team%3AAmazonas-Brazil/CompositePart)
* [Model](https://2019.igem.org/Team%3AAmazonas-Brazil/Model)
* Human Practices

  [Overview](https://2019.igem.org/Team%3AAmazonas-Brazil/Human_Practices)
  [Education and Public Engagement](https://2019.igem.org/Team%3AAmazonas-Brazil/Public_Engagement)
  [Integrated Human Practices](https://2019.igem.org/Team%3AAmazonas-Brazil/Integrated)
* People

  [Attributions](https://2019.igem.org/Team%3AAmazonas-Brazil/Attributions)
  [Collaborations](https://2019.igem.org/Team%3AAmazonas-Brazil/Collaborations)
  [Team](https://2019.igem.org/Team%3AAmazonas-Brazil/Team)
* For judges

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

Team:Amazonas Brazil - 2019.igem.org

Results

Characterization of Escherichia coli Nissle 1917

**Escherichia coli Nissle 1917 (EcN)** is a powerful chassis for synthetic biology and have been widely used as probiotic since its discovery in the second half of the 19th century by Alfred Nissle1. We aimed to engineer and standardize Escherichia coli Nissle 1917 for iGEM, providing tools for boolean logic operations executed through its robust machinery. In that direction, we successfully transformed Nissle by electroporation and characterized its EcN growth curve, as well as quantified the expression of GFP and RFP using standardized BioBrick parts cloned in the pSB1C3 backbone.

E. coli Nissle 1917 strains were grown overnight in Luria-Bertani (LB) containing ampicilin (100 µg/mL) at 37°C and 200 rpm. Cultures were diluted in fresh LB until achieve 0,1 OD with the corresponding antibiotic and transferred to a 96-well plate (50 µL/well). Samples were always made in triplicates and a blank of LB. During 8h the absorbance at OD600 and fluorescence (excitation 488 nm and emission 530 nm) were measured with intervals of 1 hour.

![](https://static.igem.org/mediawiki/2019/f/fb/T--Amazonas-Brazil--expressao-rfp.png)

Fig. 1. - RFP sinalization system in pSB1C3 running on E. coli Nissle 1917.

Characterization of AND gate

We quantified the dynamic range of our genetic circuit (BBa\_K3320006) in response to different concentrations of hypoxia and lactate. To simulate different concentrations of oxygen for the hypoxia-inducible promoter (BBa\_K387003) we used syringes, as you can see in Figure 2, Inspired by the protocol established by the iGEM Team McMaster II for the same promoter in the composite biobrick BBa\_K2493001. The construction was transformed in EcN and inoculated in LB broth in different conditions of concentration of lactate and oxygen and then incubated at 37° C in shaker for 16 hours at 220 rpm. After the incubation, the GFP fluorescence was measured by Hidex Chameleon Microplate Reader.

![](https://static.igem.org/mediawiki/2019/6/68/T--Amazonas-Brazil--Seringas.jpeg)

Fig. 2 - Simulation of different concentrations of oxygen

In this experiment, we expected to understand the input-output relation with the goal to better design a real-world model with potential for environment recognition and drug delivery. The data is shown below.

![](https://static.igem.org/mediawiki/2019/8/89/T--Amazonas-Brazil--hyplac.png)

Figure: GFP expression under control of our AND logic gate in different hypoxic and lactate conditions.

The first condition indicates the state of hypoxia, the second hypoxia and 0,1 mM of lactate and the third hypoxia and 1 mM. It is possible to conclude that there is an increase of GFP expression related to lactate increase, as indicates the fluorescence difference between samples with 0,1 and 1 mM. However, in the condition of hypoxia and absence of lactate, the system is still ON, which indicates a leaky regulation of the hypoxia-dependent promoter. We hope to continue our study to reach an AND gate behavior.

Secretion System

As a result of postal service problems, we were not able to clone all the secretion system biobricks. We hope to continue our experiments and clone this unit since this is an essential part to perform the drug delivery

Future Perspectives

a) Characterization of the current genetic construction using advanced and accurate methods such as RNAseq and flow cytometry

b) Individual characterization of each promoter from the synthetic genetic circuit

c) Test of the logic of the circuit using well-characterized promoters

d) Replace the inputs for other characteristics from the tumor microenvironment

e) Adoption of other protocols for simulation of hypoxia

f) Identification of others hypoxia-inducible promoters by genome mining or;

g) Fine-tuning of the promoter fdhF by mutagenesis or change of ribosome-biding site

h) Complete the cloning of the secretion system

i) Replace the GFP for therapeutic compounds with immunostimulant or anticancer activity

![]()
![](https://static.igem.org/mediawiki/2019/9/96/T--Amazonas-Brazil--patroc%C3%ADnio.png)