iGEM 2021 — Team UNESP_Brazil

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:UNESP Brazil/Notebook

Notebook

![](https://static.igem.org/mediawiki/2021/a/a9/T--UNESP_Brazil--logo.png)

# Notebook

* [HOME](https://2021.igem.org/Team%3AUNESP_Brazil)
* [PROJECT](https://2021.igem.org/Team%3AUNESP_Brazil/Description)
* [MODEL](https://2021.igem.org/Team%3AUNESP_Brazil/Model)
* [PARTS](https://2021.igem.org/Team%3AUNESP_Brazil/Parts)
* [HARDWARE](https://2021.igem.org/Team%3AUNESP_Brazil/Hardware)
* [HUMAN PRACTICES](https://2021.igem.org/Team%3AUNESP_Brazil/Human_Practices)
* [TEAM](https://2021.igem.org/Team%3AUNESP_Brazil/Team)
* [COLLABORATIONS](https://2021.igem.org/Team%3AUNESP_Brazil/Collaborations)
* [SAFETY](https://2021.igem.org/Team%3AUNESP_Brazil/Safety)
* [FOR JUDGES](Judging%20Form)

![](https://static.igem.org/mediawiki/2021/9/9a/T--UNESP_Brazil--menu.jpg)

* ### Test-tube culture for PCR (tetR and SpdCas9)

  Test-tube 405 (Pveg-cre-tetR) - Chloramphenicol

  Test-tube 472 - Ampicillin

  ### Plasmid Miniprep (405 and 472)

  Pveg-cre-tetR: 100,1 ng/uL

  SpdCas9: 37,3 ng/uL

  #### February 2021
* ### PCR (SpdCas9 and tetR)

  SpdCas9: 4327 bp - Tm: 65°C - Extension time: 2:30 min

  tetR: 796 bp - Tm: 60 °C - Extension time: 1 min

  Template - 50ng

  ### Digestion (SpdCas9,TetR, pSB1A3 - 37°C - 30 min)

  pSB1A3: 100 ng - EcoRI + PstI + FastAP

  tetR: 1000 ng - EcoRI + BcuI

  SpdCas9: 1000 ng - XbaI + PstI

  ### Ligation (22°C - 1h)

  Ligation calculator - 1:3

  pSB1A3(25 ng) + tetR(27,7 ng) + SpdCas9(150,5 ng)

  ### Transformation

  50 uL E. coli + 10-50 ng DNA (~10 uL)

  Heat-shock: ice (30 min) + 42°C (30 sec) + ice (2 min)

  Incubation with SOC: 37°C - 1 hour

  ### Test-tube culture (six colonies)

  **Digestion (EcoRI + PstI and EcoRI + SpeI and EcoRI + XhoI)**

  #### March 2021
* ### gBlocks PCR

  BBprefix\_fw + BBsuffix\_rv + Taq DNA pol + 50 ng DNA template

  ### E. coli Top10 chemically competent

  Follow protocol - dropbox

  ### Digestion (pSB1A3, pSB1K3, 9.1, 9.2)

  pSB1A3: 500 ng - XbaI + PstI + FastAP

  pSB1K3: 500 ng - XbaI + PstI + FastAP

  9.1: 500 ng - XbaI + PstI

  9.2: 500 ng - XbaI + PstI

  ### Ligation (22°C - 1h)

  Ligation calculator - 1:3

  pSB1K3(25 ng) + 9.1(9,75 ng)

  pSB1K3(25 ng) + 9.2(12,5 ng)

  ### Transformation pSB1K3 (iGEM Kit)

  **Miniprep pSB1K3 + Criotube**

  ### Digestion (10.1, 10.2)

  10.1: 500 ng - XbaI + PstI

  10.2: 500 ng - XbaI + PstI

  pSB1A3: 1000 ng - XbaI + PstI + FastAP

  Ligation + Transformation

  ### Digestion pSB1K3

  Overnight - too many red colonies

  ### Transformation 10.1/10.2 + pSB1K3

  ### Test-tube 10.1, 10.2

  **Digestion to confirm cloning**

  **AsiA PCR to correct reading frame**

  2-3 ng DNA template

  **Digestion pSB1A3\_tetR\_SpdCas9**

  **Digestion AsiA**

  AsiA: 500 ng - BcuI + PstI - overnight

  #### August 2021
* ### Ligation pSB1K3 + 9.1/9.2/10.1/10.2

  ### Criotube 9.1/9.2

  ### Digestion to confirm cloning 9.1/9.2/AsiA

  XbaI + PstI

  ### Sequencing AsiA - primer 618 - Tm 60°C

  **Digestions 9.1, 9.2, 10.1, 10,2 and ligation with 11**

  ### Test-tube pSB1K3\_(9.1/9.2/10.1/10.2)\_11 - SUCCESS WITH 9.1 AND 9.2

  **Golden Gate 8.1+8.2**

  **PCR after Golden Gate** - Tm 58°C - primers 123+124 (BBprefix and BBsuffix)

  #### September 2021
* ### Repeat AsiA PCR

  Ta 55°C - Time-Hifi - primers 124+635 - **SUCCESS**

  **PCR target\_broccoli**

  Ta 55°C - Time-Hifi - primers 123+124

  ### Digestion pSB1A3\_tetR\_SpdCas9, AsiA, target-broccoli, 11

  pSB1A3\_tetR\_SpdCas9: 500 ng - BcuI + PstI + FastAP - 3 hours - 37°C

  AsiA: 500 ng - PstI + XbaI - 3 hours - 37°C

  target-brococli: 500 ng - EcoRI + BcuI - 1 hour - 37°C

  11: 500 ng - EcoRI + BcuI - 1 hour - 37°C

  ### Ligation

  9.1\_11/9.2\_11 + target-broccoli

  10.1/10.2 + 11 - **SUCCESS - DIGESTION ELECTROPHORESIS**

  pSB1A3\_tetR\_SpdCas9 + AsiA

  Negative control

  ### Repeat AsiA ligation

  1:3, 1:6, 1:10, 1:50

  AsiA: 5 ng

  plasmid: 25 ng

  ### Transformation AsiA + Incubation (overnight, 37°C)

  ### Repeat PCR target-broccoli

  Criotube pSB1A3\_tetR\_SpdCas9\_AsiA

  Digestion to confirm Asia: colonies 1, 2 and 9 - PCR

  PCR to confirm: primers 635/124 - Tm 60°C

  Colony (1) - OK

  ### Transformation 9.1/9.2 + target-broccoli

  Digestion to confirm target-broccoli: E + P - **SUCCESS**

  ### Transformation BL21

  1. pSB1A3\_tetR\_SpdCas9\_AsiA + 9.1\_8/9.2\_8/10.1\_11/10.2\_11 (4 plates Kanamycin+Chloramphenicol+Ampicilin)

  2. pSB1A3\_tetR\_SpdCas9\_AsiA (1 plate Chloramphenicol+Ampicilin)

  3. 9.1\_8/9.2\_8/10.1\_11/10.2\_11 (4 plates Kanamycin+Chloramphenicol)

  #### October 2021

![](https://static.igem.org/mediawiki/2021/e/e7/T--UNESP_Brazil--agoraVAIfooter.png)

CONTACT

# Get in touch!

# Sponsors

![](https://static.igem.org/mediawiki/2021/c/c6/T--UNESP_Brazil--sponsorsOUTUBRO.png)

# Contact

São Paulo State University

Araraquara/SP - CEP 14800-903

igemunespbrazil@gmail.com

+55 11 99768-2689

Copyright iGEM UNESP Brazil

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:UNESP Brazil/Engineering

![](https://static.igem.org/mediawiki/2021/a/a9/T--UNESP_Brazil--logo.png)

* [HOME](https://2021.igem.org/Team%3AUNESP_Brazil)
* [PROJECT](https://2021.igem.org/Team%3AUNESP_Brazil/Description)
* [MODEL](https://2021.igem.org/Team%3AUNESP_Brazil/Model)
* [PARTS](https://2021.igem.org/Team%3AUNESP_Brazil/Parts)
* [HARDWARE](https://2021.igem.org/Team%3AUNESP_Brazil/Hardware)
* [HUMAN PRACTICES](https://2021.igem.org/Team%3AUNESP_Brazil/Human_Practices)
* [TEAM](https://2021.igem.org/Team%3AUNESP_Brazil/Team)
* [COLLABORATIONS](https://2021.igem.org/Team%3AUNESP_Brazil/Collaborations)
* [SAFETY](https://2021.igem.org/Team%3AUNESP_Brazil/Safety)
* [FOR JUDGES](https://igem.org/2021_Judging_Form?id=3749)

![](https://static.igem.org/mediawiki/2021/9/9a/T--UNESP_Brazil--menu.jpg)

Project

# Engineering Success

DESIGN

     We made the design to optimize the project's innovation pillars, the autonomous and temporal regulation of the system. Thus, we divided the design into three modules: control, message, and response. Each module is responsible for regulating the circuit, carrying out the progression and continuity of the code, and issuing the signals, respectively.

     The control module consists of 3 elements: the TetR repressor protein, controlled by the J23100 promoter (constitutive and strong of the Anderson family for *E. coli*); SpdCas9 fused to bacteriophage T4 anti-sigma 70 protein, AsiA, controlled by the Tet promoter (regulated by TetR); and gRNA-S, controlled by the T7-lacO promoter (related to T7 RNA polymerase and with a regulatory site for the LacI protein). To better understand and visualize the control module, you can join the previous information with the image below.

![](https://static.igem.org/mediawiki/2021/d/d0/T--UNESP_Brazil--Project1.png)

Image 1. Graphical representation of the control module. (a) Constitutive PJ23117 promoter controlling the synthesis of the TetR repressor protein. (b) TetR protein negatively regulating the Ptet promoter, which regulates the synthesis of SpdCas9 protein fused to the AsiA activator protein. In the presence of anhydrotetracycline (aTc), the promoter repression does not occur due to the formation of TetR-aTc complex. (c) LacI protein negatively controlling the PT7-lacO promoter, through interaction with the lac operator site, which controls the transcription of the gRNA-S.

     After the start of gRNA-S transcription, Message Module will start. The Message Module is composed of seven blocks, composed of the following parts: a target site (Sa and 3a to 7a) of activation for the SpdCas9\_AsiA\_gRNA complex, the PJ23117 promoter (weak promoter, constitutive of the Anderson family for *E. coli*), the ribozyme RiboJ, gRNAs 0 and 1 and gRNAs 3 to 7. The system will work through the transcription with time spacing of each of the blocks. The 0 and 1 gRNAs are responsible for guiding the activation complex to the response module and thus transcribing the fluorescent RNAs. On the other hand, gRNAs 3 to 7 have the function of interspersing the synthesis of gRNAs 0 and 1 because they guide the activation complex through parts of the Message Module (3 to 7), producing a temporal variation in the transcription of gRNAs 0 and 1, hence a variation in fluorescent RNAs. In addition, the gRNAs in this module contain RiboJ, a self-cleaving ribozyme that will separate the two gRNAs after transcription, in addition to increasing their stability.

![](https://static.igem.org/mediawiki/2021/4/47/T--UNESP_Brazil--Project2.png)

     The last division of our system corresponds to the Response Module. This circuit module contains the parts necessary to create the binary code message through fluorescence. For this, there are two parts composed of the RNAs Broccoli and Corn genes, which will be the numbers 0 and 1 of the code, controlled by the PJ23117 promoter. In addition, there are four target sites: 0i, 0a, 1i, and 1a. The "a" sites are for activating gene transcription, and the "i" sites are for repressing the gene. Thus, when gRNA-0 reaches a minimum concentration in the system, the transcription of RNA Broccoli and repression of RNA Corn will occur. When the system transcribes the gRNA-1, the opposite happens. With this, it will be possible to differentiate the message through the time needed to change the concentration of gRNAs 0 and 1.

![](https://static.igem.org/mediawiki/2021/7/7a/T--UNESP_Brazil--Project3.png)
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BUILD

     The Benchling platform was used to design all project parts. The table below show all the parts synthesized in gBlocks, by IDT.

![](https://static.igem.org/mediawiki/2021/9/9c/T--UNESP_Brazil--Project-gBlocks.png)

The parts have restriction sites following the BioBrick and Golden Gate cloning patterns, to be later joined in plasmids pSB1A3 (Control Module) and pSB1K3 (Message and Answer Module). The dcas9 and tetR genes were amplified by PCR from a laboratory plasmid. The other sequences were synthesized by IDT in gBlocks. The gBlocks containing the gRNA-S and all other gRNAs (3 to 8) contain the BsaI enzyme site to be joined by Golden Gate. The gBlock of the target-0a\_PJ23117\_target-1i\_broccoli part has been split into two parts and has been joined by Golden Gate. The union of non-equal parts was also performed:

PT7-lacO\_gRNA-0+target-1a\_PJ23117\_target-0i\_corn

PT7-lacO\_RiboJ\_gRNA-0+target-1a\_PJ23117\_target-0i\_corn

PT7-lacO\_gRNA-1+target-1a\_PJ23117\_target-0i\_corn

PT7-lacO\_RiboJ\_gRNA-1+target-1a\_PJ23117\_target-0i\_corn

     These parts will work as a proof of concept for the system, as they do not have the complete message, only a Corn activation gRNA (1) and a repression gRNA (0).

     Initially, parts 1-4 (pSB1K3) and tetR-dcas9 (pSB1A3) were cloned in *E. coli* Top10, as this strain has greater replication capacity. These clonings were confirmed by digestion and agarose gel electrophoresis, in addition to Sanger sequencing. To prove the functionality of the genetic circuit the parts will be cloned into BL21 since this bacterium has T7 RNA polymerase.

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TEST

     Tests were performed in silico with iBioSim 3.0 software. [Click Here](https://2021.igem.org/Team%3AUNESP_Brazil/Model) for more data.

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---

LEARN

     As engineering success we were able to ligate gBlocks 8.1 and 8.2 through GoldenGate with the *Bsa*I enzyme. The image below shows the PCR extension of the target-0a\_J23117\_target-1i\_broccoli sequence, confirming the success of GoldenGate.

![](https://static.igem.org/mediawiki/2021/9/9b/T--UNESP_Brazil--BroccoliElectrophoresisGel.png)

Image 4. Electrophoresis gel of target0a\_PJ23117\_target1i\_broccoli after Golden Gate reaction.

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---

* [Design](#item-1)
* [Build](#item-2)
* [Test](#item-3)
* [Learn](#item-4)

---

* [Project](https://2021.igem.org/Team%3AUNESP_Brazil/Description)
* [Implementation](https://2021.igem.org/Team%3AUNESP_Brazil/Implementation)
* [Notebook](https://2021.igem.org/Team%3AUNESP_Brazil/Notebook)

![](https://static.igem.org/mediawiki/2021/e/e7/T--UNESP_Brazil--agoraVAIfooter.png)

CONTACT

# Get in touch!

# Sponsors

![](https://static.igem.org/mediawiki/2021/c/c6/T--UNESP_Brazil--sponsorsOUTUBRO.png)

# Contact

São Paulo State University

Araraquara/SP - CEP 14800-903

igemunespbrazil@gmail.com

+55 11 99768-2689

Copyright iGEM UNESP Brazil

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:UNESP Brazil/Experiments

![](https://static.igem.org/mediawiki/2021/a/a9/T--UNESP_Brazil--logo.png)

* [HOME](https://2021.igem.org/Team%3AUNESP_Brazil)
* [PROJECT](https://2021.igem.org/Team%3AUNESP_Brazil/Description)
* [MODEL](https://2021.igem.org/Team%3AUNESP_Brazil/Model)
* [PARTS](https://2021.igem.org/Team%3AUNESP_Brazil/Parts)
* [HARDWARE](https://2021.igem.org/Team%3AUNESP_Brazil/Hardware)
* [HUMAN PRACTICES](https://2021.igem.org/Team%3AUNESP_Brazil/Human_Practices)
* [TEAM](https://2021.igem.org/Team%3AUNESP_Brazil/Team)
* [COLLABORATIONS](https://2021.igem.org/Team%3AUNESP_Brazil/Collaborations)
* [SAFETY](https://2021.igem.org/Team%3AUNESP_Brazil/Safety)
* [FOR JUDGES](https://igem.org/2021_Judging_Form?id=3749)

![](https://static.igem.org/mediawiki/2021/9/9a/T--UNESP_Brazil--menu.jpg)

# 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.

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).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [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)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:UNESP Brazil/Results

![](https://static.igem.org/mediawiki/2021/a/a9/T--UNESP_Brazil--logo.png)

* [HOME](https://2021.igem.org/Team%3AUNESP_Brazil)
* [PROJECT](https://2021.igem.org/Team%3AUNESP_Brazil/Description)
* [MODEL](https://2021.igem.org/Team%3AUNESP_Brazil/Model)
* [PARTS](https://2021.igem.org/Team%3AUNESP_Brazil/Parts)
* [HARDWARE](https://2021.igem.org/Team%3AUNESP_Brazil/Hardware)
* [HUMAN PRACTICES](https://2021.igem.org/Team%3AUNESP_Brazil/Human_Practices)
* [TEAM](https://2021.igem.org/Team%3AUNESP_Brazil/Team)
* [COLLABORATIONS](https://2021.igem.org/Team%3AUNESP_Brazil/Collaborations)
* [SAFETY](https://2021.igem.org/Team%3AUNESP_Brazil/Safety)
* [FOR JUDGES](https://igem.org/2021_Judging_Form?id=3749)

![](https://static.igem.org/mediawiki/2021/9/9a/T--UNESP_Brazil--menu.jpg)

# Results

You can describe the results of your project and your future plans here.

### 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](http://parts.igem.org/Main_Page).** 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.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)