iGEM 2021 — Team Ecuador

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# Team:Ecuador/Notebook

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Project IGem Ecuador

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* [HOME](https://2021.igem.org/Team%3AEcuador)
* [TEAM](https://2021.igem.org/Team%3AEcuador/Team)
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* [PARTS](https://2021.igem.org/Team%3AEcuador/Parts)
* [SAFETY](https://2021.igem.org/Team%3AEcuador/Safety)
* [HUMAN PRACTICES](https://2021.igem.org/Team%3AEcuador/Human_Practices)
* [AWARDS](https://2021.igem.org/Team%3AEcuador/awards)

igemecuador@gmail.com

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#### Description

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#### Engineering

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#### Model

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#### Hardware

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#### Experiments

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#### Implementation

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#### Safety

## Notebook

Dry-Lab

Wet-Lab

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# Team:Ecuador/Engineering

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Project IGem Ecuador

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* [HOME](https://2021.igem.org/Team%3AEcuador)
* [TEAM](https://2021.igem.org/Team%3AEcuador/Team)
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* [RESULTS](https://2021.igem.org/Team%3AEcuador/Results)
* [PARTS](https://2021.igem.org/Team%3AEcuador/Parts)
* [HUMAN PRACTICES](https://2021.igem.org/Team%3AEcuador/Human_Practices)
* [MEDALS](https://2021.igem.org/Team%3AEcuador/Medals)

igemecuador@gmail.com

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#### Description

[![Snow](https://static.igem.org/mediawiki/2021/0/06/T--Ecuador--Proyect_Model.png)](https://2021.igem.org/Team%3AEcuador/Model)

#### Model

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#### Experiments

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#### Engineering

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#### Safety

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#### Implementation

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#### Hardware

[○ Design](#design)

[○ Build](#build)

[○ Test](#test)

[○ Learn](#learn)

## Engineering: The DBTL cycle

We describe a core set of activities that enable each step of the Design-Build-Test-Learn (DBTL) cycle of synthetic biology, focusing particularly on the development of Agrobactory's Module 2-Delivery and Realease (composite part [BBa\_K3893030](http://parts.igem.org/Part%3ABBa_K3893030)).

![](https://static.igem.org/mediawiki/2021/9/96/T--Ecuador--Eng_SBCycle.png)

## Design (Re-design)

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

Module 2 was designed for delivering and realeasing dsRNA molecules of the *velvet gene* produced by Module 1. However, we replaced Module 1 by a fluorescent reporter to test the behaviour of the genetic device and the effectiveness of cell lysis. Thus, LuxR-AHL transcription factor activates simoultanously expression of GFP and the lysis protein PhiX174E, when sufficient amounts of cells in the population produce AHL.

![](https://static.igem.org/mediawiki/2021/1/18/T--Ecuador--Eng_DBcircuit.png)

Before building our device in the lab, we made a [mathematical ODE model](https://2021.igem.org/Team%3AEcuador/Model?ref-tab=tab2#m2model) to check this design was able to produce the desired oscilations. Although the ODE model only considers N=20 cells, we see qualitatively confirm that our device is able to develop the desired behaviour, i.e. the population oscilations based on quorum sensing.

**ODE Model**

As the ODE model of Module 2-Delivery and Release, this new model has 7 states. Particularly N is the number cells growing in the culture, and GFP is the system output. Ae is the number of molecules in the culture medium, and the remaining species are intracellular ones.

![](https://static.igem.org/mediawiki/2021/e/e8/T--Ecuador--EngModel.png)

**Computational simulation**

From the computational simulation performed with our model (see the detailes in our [modeling page](https://2021.igem.org/Team%3AEcuador/Model?ref-tab=tab2#m2model)), as expected, we can confirm that the designed gene circuit shows an oscillatory behaviour since cells die only when the lysis protein is activated. Therefore, this ODE model used as a proxy of Module 2-Delivery and Release fulfills the requirements for delivering and releasing dsRNA molecules due to cell lysis.

![](https://static.igem.org/mediawiki/2021/d/da/T--Ecuador--EngOscilatorGFP.png)

## Build

![](https://static.igem.org/mediawiki/2021/0/00/T--Ecuador--build_icon.png)

In the lab, we needed to build our device. For this we planned a golden gate assembly with 3 levels using the [Golden Braid assembly method](https://2018.igem.org/Team%3AValencia_UPV/Design#GB) from Valencia\_UPV. First, we combined together two composite parts that were already in the Part Registry ([BBa\_K2656122](http://parts.igem.org/Part%3ABBa_K2656122)  a Level 1 transcriptional unit expresing GFP under the control of the pux promoter and [BBa\_K2656114](http://parts.igem.org/Part%3ABBa_K2656114)  a Level 1 transcriptional unit constitutively expressing luxR gene) to create part [BBa\_K3893028](http://parts.igem.org/Part%3ABBa_K3893028) .

Then we created 2 more Level 1 Transcriptional units using basic parts from the [Valencia\_UPV Part Collection](https://2018.igem.org/Team%3AValencia_UPV/Parts): parts [BBa\_K3893026](http://parts.igem.org/Part%3ABBa_K3893026)  (a Level 1 transcriptional unit constitutively expressing luxI gene) and [BBa\_K3893027](http://parts.igem.org/Part%3ABBa_K3893027)  (a Level 1 transcriptional unit expresing phiX174 lysis protein under the control of the pLux promoter). This way, we combined these two parts and we created a new composite part [BBa\_K3893029](http://parts.igem.org/Part%3ABBa_K3893029). Finally, combining these two Level 2 parts we obtained a contruction with four transcriptional units implementing our initial design [BBa\_K3893030](http://parts.igem.org/Part%3ABBa_K3893030).

### Level 2 parts

#### Part K3893028: AHL induced expression of GFP and luxR constitutive expression

[![](https://static.igem.org/mediawiki/parts/d/d7/T--Ecuador--K3893028_sbolv.png)](http://parts.igem.org/Part%3ABBa_K3893028)

#### Part K3893029: AHL induced expression of phiX174 lysis protein and luxI constitutive expression

[![](https://static.igem.org/mediawiki/parts/8/8d/T--Ecuador--K3893029_sbolv.png)](http://parts.igem.org/Part%3ABBa_K3893029)

### Level 3 part

#### Part K3893030: QS-based lysis protein/population oscilator

[![](https://static.igem.org/mediawiki/parts/c/c1/T--Ecuador--K3893030_sbolv.png)](http://parts.igem.org/Part%3ABBa_K3893030)

## Test

![](https://static.igem.org/mediawiki/2021/2/28/T--Ecuador--test_icon.png)

We designed a temporal experiment to assess and quantify (if possible) cell lysate. We collected absorbance and fluorescence data from the gene circuit in vivo. One of the assays is shown in the Figure below.
**Figures A and B** depict how cells death when the lysis protein is activated by the lux promoter K2656003. The number of cells was calibrated using standardized particle units from Engineering Committee (Measurement Committee) and iGEM Interlab study 2018-2019.

The total GFP fluorescence expressed by the population and a single-cell is shown in **Figures C and D**, respectively. At the beginning of the experiment, the number of cells producing GFP is very low (OD 600=3.05e-6). But after lysis, GFP molecules are released to the medium and some cells are still producing GFP. We used MEFL/Particle (molecules of equivalent fluorescein per particle) as a standardized unit to quantify GFP expression per cell.

![](https://static.igem.org/mediawiki/2021/thumb/1/1f/T--Ecuador--EngOscilator2.png/609px-T--Ecuador--EngOscilator2.png)

**Materials and Methods**

1. All experimental measurements were taken with Biotek CytationTM 3, using a 96-well plate. The experimental conditions used in this work are in the Table below
2. The cells were E. cloni® cells (Lucigen) transformed with the corresponding plasmid.
3. For all experiments, we worked with an isolated colony that contains the corresponding plasmid.
4. Cultures of 3mL falcon tubes with sLB medium with the corresponding antibiotic were prepared and incubated overnight (18 hours) at 37°C, 200 rpm.
5. The overnight cultures were refreshed in 3 mL of sBL with the corresponding antibiotic and incubated at 37°C, 200 rpm for 4 hours.
6. Culture tubes at OD600=0.05 were sit in cold water for 30 mins.
7. We measured absorbance and fluorescence of four replicas of the sample starting with OD600=3.05e-6 during 14 hours.
8. PROTOCOL: Time between measures 5 min, Temperature 37°C, Shaking Double orbital (Continuously), Absorbance wavelength 600 nm, Excitation wavelength 485 nm, Emission wavelength 528 nm, 96-well volume (individual) 200 μl.

## Learn

![](https://static.igem.org/mediawiki/2021/e/e3/T--Ecuador--learn_icon.png)

In parallel to this DBTL cycle to develop our Module 2, we performed a smaller inned DBTL cycle including design, assembly (build), measurements (test), and model and characterization of the part (learn) using part [BBa\_K3893028](http://parts.igem.org/Part%3ABBa_K3893028) and [contributing to characterize the pLux promoter](https://2021.igem.org/Team%3AEcuador/Contribution) (Part [BBa\_K2656003](http://parts.igem.org/Part%3ABBa_K2656003) which is the Golden Braid compatible, together with its Biobrick starndard sister part [BBa\_R0062](http://parts.igem.org/Part%3ABBa_R0062)) in the context of the transcriptional unit built in part [BBa\_K2656122](http://parts.igem.org/Part%3ABBa_K2656122).

![](https://static.igem.org/mediawiki/parts/thumb/d/dc/T--Ecuador_UPV--Model_pLux_Charact.png/320px-T--Ecuador_UPV--Model_pLux_Charact.png)

Using the information we obtained from this smaller DBTL, we could characterize the pLux promoter (in the context of the transcriptional units data was taken, which is the same as the context of our oscilator).
From this characterization of the pLux promoter in the mentioned context, we extracted the following parameters and used them to learn:

1. Effective dissociation constant: Kd = 15.34 nM
2. Hill coefficient: n = 0.958
3. Basal expression: 𝛽 = 0.1049

With these parameters from the experimental data and model of pLux promoter, we went back to our model and learned, by including the new values and performing new computational simulations.

![](https://static.igem.org/mediawiki/2021/2/29/T--Ecuador--M2simulation_with_param.png)

Here, we show the results of the new computational simulations. And we can see that the information we learnt and pluged back into the initial model makes our model better. By comparing the **in silico vs. in vivo results**, we see that our model captures the temporal dynamics of the oscilator, and also recapitulates with the Total dsRNA being similar to the Total GFP in the experimental data.

An interesting point is that we did not fit the oscillator model with data from the experiment from part [BBa\_K3893030](http://parts.igem.org/Part%3ABBa_K3893030), but we incorporated the characterization made of one of the components of the system, and still, we got good agreement between the model of Module 2 and its corresponding experimental data. This, speaks itself about the power of modeling and the DBTL cycle in Synthetic Biology.
Finally, our newly adjusted ODE model can be used to redesign our device, by predicting outcomes for different conditions of components, and also it can be used in an optimization process to improve other aspects of our devices we need to improve. Now we have a starting point for optimization that can be done in silico, instead of building a new set of circuits to see which one performs better than the original.

![](https://static.igem.org/mediawiki/2021/d/d9/T--Ecuador--IgemECSponsors.png)

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# Team:Ecuador/Experiments

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Project IGem Ecuador

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* [TEAM](https://2021.igem.org/Team%3AEcuador/Team)
* [PROJECT](https://2021.igem.org/Team%3AEcuador/Description)
* [RESULTS](https://2021.igem.org/Team%3AEcuador/Results)
* [PARTS](https://2021.igem.org/Team%3AEcuador/Parts)
* [HUMAN PRACTICES](https://2021.igem.org/Team%3AEcuador/Human_Practices)
* [MEDALS](https://2021.igem.org/Team%3AEcuador/Medals)

igemecuador@gmail.com

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#### Description

[![Snow](https://static.igem.org/mediawiki/2021/0/06/T--Ecuador--Proyect_Model.png)](https://2021.igem.org/Team%3AEcuador/Model)

#### Model

[![Snow](https://static.igem.org/mediawiki/2021/3/3a/T--Ecuador--Proyect_Experiments.png)](https://2021.igem.org/Team%3AEcuador/Experiments)

#### Experiments

[![Snow](https://static.igem.org/mediawiki/2021/7/77/T--Ecuador--Proyect_Engineering.png)](https://2021.igem.org/Team%3AEcuador/Engineering)

#### Engineering

[![Snow](https://static.igem.org/mediawiki/2021/9/93/T--Ecuador--Proyect_Safety.png)](https://2021.igem.org/Team%3AEcuador/Safety)

#### Safety

[![Snow](https://static.igem.org/mediawiki/2021/c/cd/T--Ecuador--Proyect_Implementation.png)](https://2021.igem.org/Team%3AEcuador/Implementation)

#### Implementation

[![Snow](https://static.igem.org/mediawiki/2021/b/b7/T--Ecuador--Proyect_Hardware.png)](https://2021.igem.org/Team%3AEcuador/Hardware)

#### Hardware

[○ Design of target-gene dsRNAs](#mref1)

[○ Target-gene fragment production](#mref2)

[○ Bacterial production of dsRNA using pL4440](#mref3)

[○ pL4440 improvement](#mref4)

[VIEW NOTEBOOK](https://2021.igem.org/Team%3AEcuador/Notebook)

[○ QS-based lysis protein/population oscillator](#mref1)

[VIEW NOTEBOOK](https://2021.igem.org/Team%3AEcuador/Notebook)

[○ In vitro inhibition test](#mref1)

[VIEW NOTEBOOK](https://2021.igem.org/Team%3AEcuador/Notebook)

Module 01

Module 02

Module 03

## Production of dsRNA

### 1. Design of target-gene dsRNAs

Agrobactory 593 is a bacterial modular platform in which it is possible to change the target pathogen by changing the gene of interest and therefore the design of the interfering Ribonucleic acid (RNAi). To apply the RNAi approach in *Fusarium oxysporum*  f. sp.  *cubense*  tropical race 4 (Foc TR4), we began a review of the literature to find genes involved in the pathogenicity of the fungus, we select only those that have been previously used in RNAi assays. Next, because to date Foc TR4 has not been reported in Ecuador, we use the *Fusarium oxysporum* f. sp. *cubense* race 1 (Foc R1) strain (less aggressive) available in our laboratory, therefore we look for the candidate genes to have homology between Foc TR4 and Foc R1. The genes SGE1, ERG11, Velvet, and SIX1 were chosen for their role in the development of diseases (banana Fusarium Wilt) such as conidia, virulence, synthesis of secondary metabolites (fumonisins and fusarins), and wilt [1]. Subsequently, the design of double stranded ribonucleic acid (dsRNA) was carried out using the E-RNAi [2]. For more details check  [Part collection](https://2021.igem.org/Team%3AEcuador/Part_Collection%20) .

![](https://static.igem.org/mediawiki/2021/9/96/T--Ecuador--Experiments_1.jpg)

:   **a. Desing of the dsRNA**

    Several critical candidate genes were investigated in Foc R4T to choose the genes for the dsRNA design. The silencing of these genes could limit the fungus' survival or dispersal capacity. In addition, because the entry of Foc R4T for investigation is forbidden and also it is not reported in the country, Foc R1 was considered for in vitro testing and a confirmation of the homology of these genes was performed. The candidate genes SGE1, ERG11, Velvet, and SIX1 were discovered based on these considerations. SGE1 promotes the expression of SIX, which is required for the formation of conidia. ERG11 plays a role in the synthesis of ergosterol as well as conidia formation. Velvet is involved in the development of hyphae, the generation and germination of conidia, and the formation of virulence-inducing secondary metabolites. SIX1 plays the role of a virulence factor. Furthermore, it was discovered that ERG11 and SIX1 were used to silence Foc R4T [3], [4]. In Foc R1, SGE1 and Velvet have also been used [5], [6].

    For the design of the dsRNA, we used the [E-RNAi](https://www.dkfz.de/signaling/e-rnai3/) program and we validated its physicochemical characteristics. As criteria, we selected 21 nucleotide target sequences that begin with AA and are located within a region of the coding sequence, 50-100 nucleotides of the AUG start codon, and within 50-100 nucleotides of the stop codon. The presence of AA at the beginning of the sequence allows the use of dTdT at the 3 'end of the antisense sequence. We considered that the GC content should be less than 50% and we avoided sequences with repeats of three or more G or C, since its presence initiates intramolecular secondary structures that prevent silencing hybridization. Furthermore, we confirmed that RFC10-compatible restriction sites are not found in the sequences [6].

     **b. Primers design for gene identification**

    | Primer | Sequence 5 '- 3' | Length (bp) | PCR product (bp) |
    | --- | --- | --- | --- |
    | ERG11\_F  ERG11\_R | TTCCACTGGTTCCCTTTCGT  TTGGCATTAGGGCAATCGTA | 20  20 | 340 |
    | SGE\_F  SGE\_R | ATGTCTGGAACCATGCCACTC  CTACCACCACGCCTGACCATA | 21  21 | 951 |
    | Sixpb\_F  Sixpb\_R | ACCCGAGGACCGAGACTTTA  CTGCGTGTCCGTATCATCCA | 20  20 | 180 |
    | Six1\_F  Six1\_R | CCGGATTTTGAGCTTTCGAC  GTAGTGTTTGGAGGGCTTTAG | 21  22 | 720 |
    | Velvet\_F  Velvet\_R | ACTTTTGACTACAACGCCA  CCCGCTTCCTCATACGAAC | 19  19 | 407 |

     **c. DNA extraction**

    From the culture of the Foc R1 fungus in potato dextrose broth (PDB) medium with two weeks of growth, we proceeded with the extraction of deoxyribonucleic acid (DNA) [(Benchling protocol)](https://benchling.com/s/prt-4aJtGgWc7n40GCiqN0bv) .

     **d. PCR**

    Using polymerase chain reaction (PCR), the genes of interest Velvet, Six1, Erg11, SGE were amplified  [(Benchling protocol)](https://benchling.com/s/prt-9oNqw3iLpPQp1zKen8kW).

### 2. Target-gene fragment production

In order to design interference RNAs that silence the virulence and viability genes of Foc R1 and TR4, we generate fragments for the production of specific dsRNAs for each gene of interest (SGE, ERG11, Velvet, and SIX1), which will be assembled (using restriction enzymes) with the widely used plasmid [7] (L4440 and L4440 enhanced).

![](https://static.igem.org/mediawiki/2021/f/f6/T--Ecuador--Experiments_2.png)

:   **a. Primers design with overhangs**

    To add restriction sites (RS) to the fragments for the dsRNA production, primers overhangs with RSs were designed for PstI and XbaI, compatible with the RFC10 assembly, described below. The fragments obtained will be assembled in the conventional plasmid L4440.

    | Primer | Sequence 5 '- 3' | Length (bp) | PCR product (bp) |
    | --- | --- | --- | --- |
    | dsRNA\_ERG11\_F  dsRNA\_ERG11\_R | attctgcagCTAAGGACAACGATGACAC  acttctagaGTCGGGATGGTGTACTTG | 28  27 | 403 |
    | dsRNA\_Velvet\_R  dsRNA\_Velvet\_F | ttactgcagGGCGGACAGAAAGGT  cagtctagaTGTGGAAGAGGGCAAG | 24  25 | 215 |
    | dsRNA\_SGE1\_R  dsRNA\_SGE1\_F | cgttctagaGGTACTGTTGTTGTGGCT  actctgcagTGCCCAGTGATGATGA | 27  25 | 498 |
    | dsRNA\_Six\_F  dsRNA\_Six\_R | cgttctagaCGCCAGGAAGTCTACCAGA  attctgcagTCCACCGTCGGCACCTT | 28  26 | 548 |

    Due to the suggestion of our advisors, we decided to change the design of our chassis, so we changed the design and assembly of our parts, being necessary to add RS for the BglII (forward end) and KpnI (reverse end) enzymes, as described below. The obtained fragments will be assembled into the improved plasmid L4440.

    | Primer | Sequence 5 '- 3' | Length (bp) | PCR product (bp) |
    | --- | --- | --- | --- |
    | dsRNA\_ERG11\_F\_2  dsRNA\_ERG11\_R\_2 | aaaaagatctGACACCATTAAGGAGCGAC  aaaaggtaccAAGTAGGCCGAGTCAGTAGC | 29  30 | 481 |
    | dsRNA\_SGE1\_F\_2  dsRNA\_SGE1\_R\_2 | aaaagatctCCCTTTCCAAACAGGCC  aaaggtaccCCGTGTTGAGCTTCCTC | 26  26 | 324 |
    | dsRNA\_SIX1\_F\_2  dsRNA\_SIX1\_R\_2 | aaaagatctAGACACACATCTAGGCGA  aaaggtaccACCTTGAGCAATTCGACC | 27  27 | 639 |
    | dsRNA\_Velvet\_F\_2  dsRNA\_Velvet\_R\_2 | aaaagatctGCAGCCTCTGGTATCAGA  aaaggtaccACTCTCCATCAATCCAGGA | 27  28 | 548 |

     **b. RNA extraction**

    The process began with the cultivation of Foc R1 in a static PDB medium, two growth stages were evaluated at 7 days and 14 days. Subsequently, RNA extraction was carried out, using a Kit that includes DNA degradation using DNase, ensuring the obtaining of good quality RNA  [(Benchling protocol)](https://benchling.com/s/prt-hhOxzDXFPfY49lt3TOHB%20) .

     **c. Reverse transcription**

    Reverse transcription, a process in which strands of RNA are converted into complementary DNA (cDNA) using enzymes called reverse transcriptases. We use RNA (extracted the same day) and random primers and Olido dT that hybridize with the template RNA and generate a starting point for synthesis  [(Benchling protocol)](https://benchling.com/s/prt-m0ED40Gni5ZS82GAqYhy).

     **d. Overhang PCR**

    Amplification of fragments for the production of dsRNA by overhang PCR
    In order to add restriction enzymes sequences to our PCR product, we performed an overhang PCR. This technique uses the fidelity of the 3’ end of primers for a specific target to allow the addition of more sequence to the 5’ end  [(Benchling protocol)](https://benchling.com/s/prt-nzD4vkib4usLLqesWeHy).

### 3. Bacterial production of dsRNA using pL4440

Conventional dsRNA production is performed in  *Escherichia coli*  HT115 because it is a strain deficient in RNase III, an enzyme that could degrade dsRNA [1]. We decided to transform the L4440 plasmid linked to the target-gene fragment into  *E. coli*  DH5α, a strain with which the transformation protocol had previously been standardized in our laboratory. Once this step was completed, the aim was to extract the ligated plasmid from  *E. coli*  DH5α and bring it to a concentration suitable for transformation to  *E. coli*  HT115. Conventional dsRNA production is performed by induction with isopropyl-β-D-1-thiogalactopyranoside (IPTG) to activate the T7 promoter and initiate transcription [3].

![](https://static.igem.org/mediawiki/2021/0/0d/T--Ecuador--Experiments_3.png)

:   **a. Plasmid extraction (Miniprep)**

    The extraction of the L4440 plasmid was performed using the lysis-alkaline protocol, which is based on the fact that the chromosomal and plasmid DNA denature at different conditions, which can be used to separate them  [(Benchling protocol)](https://benchling.com/s/prt-OPgZSM1TDy232kMlKBgz%20) .

     **b. Digestion and ligation**

    Target-gene fragments and L4440 plasmid were digested using enzymes Xba I and Pst I following instructions of the insert supplied by Promega corporation. Ligation was carried out using T4 DNA ligase of the same brand  [(Benchling protocol)](https://benchling.com/s/prt-fLukou9FLJaQwBAhjiRR%20) .

     **c. Competent cells preparation**

    Competent cells were obtained from an  *E. coli*  culture with an OD=0.45. CCMB80 buffer widely used in iGEM was used to prepare chemically competent cells  [(Benchling protocol)](https://benchling.com/s/prt-nxiZxvFheoJ2Cif6qXBb%20) .

     **d. Bacterial transformation**

    - DH5α:  *E. coli*  DH5α strain was the first host considered due to the high efficiency of transformation reported in the literature. The bacterial transformation was developed by thermic shock following a variation of Hannah protocol.

    - HT115 (DE3):  *E. coli*  HT115 strain was transformed by thermic shock as mentioned above  [(Benchling protocol)](https://benchling.com/s/prt-JxXcDGKZF7Z1MI7vYfp6%20) .

 **e. dsRNA production in HT115 strain**

Ligation to plasmid L4440 was verified by Colony PCR using the following primers:

| Primer | Sequence 5 '- 3' | Length (bp) | PCR product (bp) |
| --- | --- | --- | --- |
| pL4440C\_F  pL4440C\_R | gttttcccagtcacgacgtt  tcagtgagcgaggaagcaac | 20  20 | 304 |

Subsequently, the transformed strain HT115 was cultivated until an OD = 0.4, at which time the inducer of IPTG would be added to start the production of dsRNA  [(Benchling protocol)](https://benchling.com/s/prt-hAg1hnrJudDbVqTDewL2%20) .

 **f. dsRNA extraction and purification**

dsRNA was obtained for conventional purification as reported by Seung-Joon Ahn and collaborators [1]  [(Benchling protocol)](https://benchling.com/s/prt-hAg1hnrJudDbVqTDewL2%20) .

### 4. pL4440 improvement

In order to improve the production of dsRNA in  *E. coli*  DH5α and HT115 strains, we enhance the conventional plasmid L4440, bidirectional sequences of the T7 promoter, and two terminators were introduced into the plasmid backbone using 3A assembly and simple assembly. Also, ribosome binding sites and Shine-Dalgarno were added to provide stability to the dsRNA. For more details check  [Part collection](https://2021.igem.org/Team%3AEcuador/Part_Collection%20) .

![](https://static.igem.org/mediawiki/2021/5/55/T--Ecuador--Experiments_4.png)

:   **a. Primer design**

    Primers were designed to add RSs for the EcoRI, PstI, and SpeI enzymes to the conventional plasmid L4440 (subsequent simple and 3A Assembly). In addition, primers were designed for the verification of 3A Assembly.

    | Primer | Sequence 5 '- 3' | Length (bp) | PCR product (bp) |
    | --- | --- | --- | --- |
    | pL4440Modf\_F  pL4440Modf\_R | aaaGAATTCGCGGCCGCTTCTAGccttcctcgctcactga  aaaTACTAGTAGCGGCCGCTGCAGtcgttttacaacgtcgtg | 40  42 | 2548 |
    | BBa\_G00100  BBa\_G00101 | tgccacctgacgtctaagaa  attaccgcctttgagtgagc | 20  20 | 314 |

    **b. Obtention of Backbones**

    Plasmids (pSB1C3 and pL4440 ) were extracted from bacteria grown in Luria Bertani medium
     [(Benchling protocol)](https://benchling.com/s/prt-PAlASiKuvnqytvowqThc) .

    **c. L4440 plasmid modification by PCR**

    Amplification and modification of plasmid L4440 was performed using a high fidelity polymerase
     [(Benchling protocol)](https://benchling.com/s/prt-9oNqw3iLpPQp1zKen8kW) .

    **d. Digestion and ligation**

    The gBlocks being designed in two parts (UP and DOWN), and it should be noted that both parts have the green fluorescence protein (GFP) indicator protein for easy visualization of a correct binding. Both necessary parts were digested with the enzymes EcoRI and PstI. The same enzymes were used for the digestion of the Backbones (pSB1C3 and pL4440)
     [(Benchling protocol)](https://benchling.com/s/prt-fLukou9FLJaQwBAhjiRR) .

    **e. Production of the assembled plasmid**

    After obtaining competent  *E. coli*  DH5α cells, the bacterial transformation was carried out. This was done in order to obtain a greater amount of gBlocks, for both processes the previously mentioned protocols were used.

     **f. Digestion for 3A Assembly**

    After obtaining the assembled plasmids with the UP and DOWN parts, we proceeded with their digestion and ligation. The UP parts were digested with EcoRI and SpeI and the DOWN parts with PstI and XbaI. In both cases, gel purification was performed and the band corresponding to ~ 185 bp was cut. A similar process was carried out with the Backbones, using the EcoRI and PstI enzymes (gel and plasmid purification ~ 2 Kb)
     [(Benchling protocol)](https://benchling.com/s/prt-SxZE0if249NJwhbIGIzO) .

     **g. Assemblies**

    Two types of assemblies were tried

    **- 3A Assembly:**  For the 3A assembly, the molar ratio used was vector: part UP: part DOWN of 1:25:25
     [(Benchling protocol)](https://benchling.com/s/prt-36qip0dOLZL3W8HKjLwW) .

    **- Simple Assembly:**
    From the assembled plasmids with the UP and DOWN parts, we proceeded to perform a simple assembly, in which the plasmid with the UP part was digested with PstI and SpeI enzymes (used as Backbone ~ 2 Kp) and the plasmid with the DOWN part was digested with PstI and XbaI enzymes (used as Insert ~ 185 bp). Expecting a final plasmid of ~ 2400 bp which has the UP and DOWN parts separated by the BglII and KpnI enzyme restriction sites.
    Subsequently, the transformation was carried out in *E. coli* DH5α.

     **h. Colony PCR**

    Ligation of the insert into the plasmid was verified by colony PCR using primers BBa\_G00100 and BBa\_G00101. The expected fragment is 646 bp
     [(Benchling protocol)](https://benchling.com/s/prt-9oNqw3iLpPQp1zKen8kW) .

     **i. Addition of the fragments for dsRNA production**

    The resulting plasmid from simple assembly (~ 648 pb) and the fragments for dsRNA production were digested with KpnI and BglII enzymes
     [(Benchling protocol)](https://benchling.com/s/prt-fLukou9FLJaQwBAhjiRR) .
    Both the final plasmids and the amplicons for each gene were purified. Then, they were ligated (expected size of 1.1 Kb) and transformed into E. coli HT115 (DE3) strain.

     **j. dsRNA production in HT115 strain**

    The production of dsRNA was carried out by induction with IPTG. Finally. DsRNA extraction and purification was performed
     [(Benchling protocol)](https://benchling.com/s/prt-QD0McGgLLmA9CH0v3BzF) .

## References

[1] V. Gurdaswani, S. B. Ghag, y T. R. Ganapathi, “FocSge1 in Fusarium oxysporum f. sp. cubense race 1 is essential for full virulence”, BMC Microbiology, vol. 20, núm. 1, p. 255, ago. 2020, doi: 10.1186/s12866-020-01936-y

[2] H. Thomas, and M. Boutros. “E-RNAi: a web application for the multi-species design of RNAi reagents--2010 update.” Nucleic acids research vol. 38,Web Server issue (2010): W332-9. doi:10.1093/nar/gkq317

[3] T. Dou et al., “Host‐induced gene silencing of Foc TR4 ERG6/11 genes exhibits superior resistance to Fusarium wilt of banana,” Plant Biotechnol J, vol. 18, no. 1, pp. 11–13, Jan. 2020, doi: 10.1111/pbi.13204.

[4] S. Widinugraheni et al., “A SIX1 homolog in Fusarium oxysporum f.sp. cubense tropical race 4 contributes to virulence towards Cavendish banana,” PLOS ONE, vol. 13, no. 10, p. e0205896, Oct. 2018, doi: 10.1371/journal.pone.0205896.

[5] V. Gurdaswani, S. B. Ghag, and T. R. Ganapathi, “FocSge1 in Fusarium oxysporum f. sp. cubense race 1 is essential for full virulence,” BMC Microbiology, vol. 20, no. 1, p. 255, Aug. 2020, doi: 10.1186/s12866-020-01936-y.

[6] S. B. Ghag, U. K. S. Shekhawat, and T. R. Ganapathi, “Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana,” Plant Biotechnol J, vol. 12, no. 5, pp. 541–553, Jun. 2014, doi: 10.1111/pbi.12158.

[7] S. Ahn, K. Donahue, Y. Koh, et al., “Microbial-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management” International Journal of Insect Science, vol. 11, pp. 1-8, 2019. doi; 10.1177/1179543319840323

## Delivery and Release

### QS-based lysis protein/population oscillator

This test was performed to characterize the influence of the Quorum Sensing (QS) system, based on the Lux operon, on the expression of a gene of interest To accomplish so, the plasmid including the LuxI, LuxR, and GFP components regulated by the pLUx promoter, as well as the iGEM parts of LOR and the chloramphenicol resistance gene, was assembled. A 4kpb plasmid was obtained after assembly and converted into E. cloni® cells (Lucigen). Fluorescence and absorbance measurements were taken with an OD600 greater than 0,05. Due to the increase in the bacterial population, QS signals begin to increase, due to the secretion of acyl-homoserine lactone (AHL) molecules, which promotes the expression of the Lux operon and leads to the production of GFP. As a result, enhanced GFP expression is a good indicator of how well the QS-based expression system is working.

![](https://static.igem.org/mediawiki/2021/1/1a/T--Ecuador--Experiments_6.png)

 **a. Procedure**

- All experimental measurements were taken with Biotek CytationTM 3, using a 96-well plate.

- The cells were E. cloni® cells (Lucigen) transformed with the corresponding plasmid.

- For all experiments, we worked with an isolated colony that contains the corresponding plasmid.

- Cultures of 3mL falcon tubes with sLB medium with the corresponding antibiotic were prepared and incubated overnight (18 hours) at 37°C, 200 rpm.

- The overnight cultures were refreshed in 3 mL of sBL with the corresponding antibiotic and incubated at 37°C, 200 rpm for 4 hours.

- Culture tubes at OD600=0.05 were sit in cold water for 30 mins.

- We measured absorbance and fluorescence of four replicas of the sample starting with OD600=3.05e-6 during 14 hours.

 **b. Protocol**

Time between measures 5 min, Temperature 37°C, Shaking Double orbital (Continuously), Absorbance wavelength 600 nm, Excitation wavelength 485 nm, Emission wavelength 528 nm, 96-well volume (individual) 200 μl.

## RNA silencing

### *In vitro* inhibition test

One approach to validate a candidate gene is to introduce an RNAi elicitor molecule into pest cells and assess its effect on the silencing phenotype [1]. These investigations were conducted to assess the effects of synthetic dsRNA molecules homologous to selected target genes on Foc R1. The assessment was performed using *in vitro* assays to measure the effects of the dsRNAs on spore germination and subsequent colony establishment.

:   **a. Conidial suspension**

    This protocol aims to obtain conidia from Foc R1, this will be used in the inhibition assays  [(Benchling protocol)](https://benchling.com/s/prt-bKj3nb3408JViyNAb4m8) .

    ![](https://static.igem.org/mediawiki/2021/c/cb/T--Ecuador--Experiments_5.png)

     **b. Inhibition test**

    The antifungal activities of the dsRNA molecules were tested by a reduction in colony number with the following administration by imbibition into spores of Foc R1, as described by Bailey and Niblett [2]. We conducted experiments to determine the optimal test concentration of dsRNAs to use in the spore germination inhibition bioassays  [(Benchling protocol)](https://benchling.com/s/prt-OB66bjs1j2aZ93NP6NY5) .

![](https://static.igem.org/mediawiki/2021/7/74/T--Ecuador--Experiments_5.2.png)

## References

[1] F. M. Mumbanza, A. Kiggundu, G. Tusiime, W. K. Tushemereirwe, C. Niblett, and A. Bailey, “*In vitro* antifungal activity of synthetic dsRNA molecules against two pathogens of banana, *Fusarium oxysporum* f. sp. *cubense* and *Mycosphaerella fijiensis*,” Pest Manag. Sci., vol. 69, no. 10, pp. 1155–1162, Oct. 2013, doi: 10.1002/ps.348

[2] A. Bailey and C. Niblett, “Bioassay for gene silencing constructs,” US20100257634A1, 2010 [Online]. Available: https://patents.google.com/patent/US20100257634A1/en

![](https://static.igem.org/mediawiki/2021/d/d9/T--Ecuador--IgemECSponsors.png)

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

# Team:Ecuador/Results

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Project IGem Ecuador

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#### Results

[![](https://static.igem.org/mediawiki/2021/a/a0/T--Ecuador--Results_ProfConceptIcon.png)](https://2021.igem.org/Team%3AEcuador/Proof_Of_Concept)

#### Proof Of Concept

[![](https://static.igem.org/mediawiki/2021/1/1e/T--Ecuador--Results_ContributionIcon.png)](https://2021.igem.org/Team%3AEcuador/Contribution)

#### Contribution

Module 01

Module 02

Module 03

## Production of dsRNA

### 1. Design of target-gene dsRNAs

We worked out a PCR for recognizing and amplifying the selected genes.
These results allowed us to continue with Velvet (450 pb), Erg11 (400 pb), Sge( 1089 pb) and Six1 (246 pb).

![](https://static.igem.org/mediawiki/2021/2/21/T--Ecuador--gelcarito.png)

In the gel, it is observed that the Six1a gene was not successfully identified,
so it was omitted for subsequent experiments. Foc TR4 harbors three SIX homologs:
SIX1a, b and c that may be present in other races [1].
However, the sequence from which the primers were designed was not scored.
It is therefore concluded that the sequence chosen was not present in Foc R1.

The dsRNAs designed for each deleted gene are described below:

| Gene | dsRNA Sequence | Secondary structures (R-RNAi) |
| --- | --- | --- |
| ERG11 Gene ID: 28942710 | ACGCGCTAAGGACAACGATGACACCGAGCACGAT  ATGATGAAGCATCTCATGAACTCTACTTACAAGAA  CGGCACCCCTGTCCCTGATCATGAAGTCGCCCACA  TGATGATTGCTCTCCTCATGGCTGGCCAGCACTCT  TCTTCTTCTACCAGCTCTTGGATCATGCTCCGTCTC  GCTCAGTACCCTCACATCATGGAGGAGCTGTACC  AAGAGCAGGTCAGAGAACTCGGTGCTGATTTGCC  TCCCCTGACTTACGACAACCTTGCCAAGCTGCCCC  TCAACCAGGCCATTATCAAGGAGACTCTCCGCCTT  CACGCCCCTATCCACTCTATCATGCGCGCCGTCAA  GTCTCCCATGCCTGTCCCTGGAACCAAGTACACCA TCCCGACCTCGCAC | ![](https://static.igem.org/mediawiki/2021/6/64/T--Ecuador--ERG11.jpg) |
| Velvet    GenBank: KF800754.1 | GCAACCTGAGCGTGCCCGAGCCTGCGGTTCTGGT  TCAAAAGCCAACAGTGACCGCCGACCTGTTGATCC  TCCCCCAGTTGTCGAGCTTCGGATTATCGAGGGAC  CTAGTGTGGAAGAGGGCAAGGACATCACTTTTGA  CTACAACGCCAACTTCTTTCTCTACGCCAGTCTCGA  GCATGCCCGTCCACTTGCACGCGGTCGTGTTAATA  CTCCAGCTGCTGGCAACCCACCCATTCTGACTGGT  GTTCCCGCTTCGGGCATGGCCTATCTCGACCGTCC  CACCGAAGCTGGTTATTTCATCTTTCCCGACCTTTC  TGTCCGCCACGAGGGCCTCTACATACTGACCTTCA  GTTTGTTTGAGACTACCAAGGAGGAAAGAGACTA  TGATCTGGAACCTGCCGATGGCGATCTTCCACCCG  GAGTCGACTATAGAATGGAAATCAAGACTGAACC  TTTCAGCGTCTACAGCGCT | ![](https://static.igem.org/mediawiki/2021/f/f6/T--Ecuador--Velvet.jpg) |
| SGE1    GenBank: KU048919.1 | CTGTGCCCAGTGATGATGAGCGCCATCTGGTCGG  TTCACTCGTCGACTCGTACGACTTCAAGGAACAAG  GACTGGTCAAGAAGACTATCAGCATCACATACCAA  GGCGTGCCTCACCACCTCGTCAGTTACTACCATGT  GGAAGACGTCAAGGCTGGACTTTTGCCTAGTCCT  GTGGATGATCCCAGACTCCGCGGCGTCGTCCCTCG  AACCGAGCTCCTGACAGGTCAGAACTTCCGCGCCC  CGATCGAGGAAGCTCAACACGGTACCTACTTGAG  CGGCGCCTACATGCCAAACATGGATCACCAGTATG  CTCCTATTGGCTTTCCCACACACACACAGCAACCG  GCACTCCAACAGCAGCCACAACAGCAGCCACAGC  CACAGCACCAGCCACAACTACAGTATCAGCCACAG  CCGCACCAGCACCAACCACAGCTGCAGTACCAGCC ACAGCAGCAGCACCAGCCACAACAACAGTACC | ![](https://static.igem.org/mediawiki/2021/3/3a/T--Ecuador--SGE1.jpg) |
| Six1 | CGCCAGGAAGTCTACCAGAGCAGCGGATCGCGTC  GAGCTCTCGTACGACGGCTTCGCGAAGCCCTCATC  AAGACCACCATCCTCGTGGGCGTGTGCAAGCCCCT  CGAAGCCATCCTGTCAATCATGAAGGTAGAGAAA  CCCGAGGACCGAGACTTTAGCCAAACGCGCGACG  GATGGCAGGCTGATCAAGCAAACCACGACCGCGG  AATCGATTGGTTCAAGAAAGTGTATACTCGAAACG  CTGGGGATACGATGGGATTGTTTGACGCGCACAA  GGATTTCGCATGGGTCTCGGCGGAGATTACATAT  GGATTGTTTCTATCGGATCGGCAAGTCTTGGATGA  TACGGACACGCAGCTTGTGGTGCTGCCGGCGATC  ATGAGCCAGAATCTTCCGCTGGAGACGAATTGGC  ATATTCGGGGAACGCGGCGAATTGGGGTGTCGAA  GGAGGATGTTCAAGTCATCTGGGATAGCGTCAAG GAAGTG | ![](https://static.igem.org/mediawiki/2021/3/39/T--Ecuador--SIX1.jpg) |

### 2. Target-gene fragment production

Fragments for dsRNA production were successfully obtained with the PCR overhang.
Reporting fragments of Six1 (548 bp) and Velvet (215 bp) and Erg11 (403 bp).
The size obtained confirmed the addition of restriction sites for the PstI and XbaI enzymes.
However, there was no amplification for the Sge1 gene.
Because we noticed an error in the primer design for this gene,
it was omitted for subsequent tests in this module.
These fragments were used in the production of dsRNA using the conventional plasmid L4440.

![](https://static.igem.org/mediawiki/2021/2/2a/T--Ecuador--Results2_1eros_genes.png)

Similarly, the addition of the rectriction rates for the BglII and KpnI
enzymes was successfully achieved in all selected genes.
Obtaining the expected fragments of Six1 (639 bp) and Velvet (548 bp) and Erg11 (418 bp)
and Sge (324 bp). These fragments were used in the production of dsRNa with the improved
plasmid L4440.

![](https://static.igem.org/mediawiki/2021/5/51/T--Ecuador--Results2_SIX_ERG11_amplification.png)

![](https://static.igem.org/mediawiki/2021/3/35/T--Ecuador--Results2_Velvet_sge_amplification.png)

### 3. Bacterial production of dsRNA using pL4440

L4440 Plasmids were extracted at concentrations ranging from 100 ng/ul to 150 ng/ul. The quality ranges were acceptable, approaching values of 1.8 for the 260/280 parameter and 2 in the case of 260/230. Plasmids were run on 1% agarose gel electrophoresis.

![](https://static.igem.org/mediawiki/2021/f/ff/T--Ecuador--Results3_L4440.1.png)

Agarose gel electrophoresis of L4440 plasmid digest (M) DNA size marker, 1 Kb (ABM). (1-7) Undigested plasmid (3-7) XbaI and PstI digested plasmid.

Enzymatic digestion with XbaI and PstI enzymes was checked by 1% agarose gel electrophoresis. In the figure below, specifically in line 1, the different conformations of the plasmid in line 1 can be seen. After enzymatic digestion, only the linearised form was observed.

![](https://static.igem.org/mediawiki/2021/3/3c/T--Ecuador--Results3_L4440dig.png)

Agarose gel electrophoresis of L4440 plasmid digestion. (1) Undigested plasmid (3-7) Xba I and PST I digested plasmid. (M) DNA size marker, 1 kb (ABM).

To test the ligation, the target gene fragment Velvet was chosen, which was digested in the same way as the L4440 plasmid, i.e. with Xba I and PST I. After transformation, Colony PCR was performed to evaluate the presence of the ligated plasmid. However, in all the attempts performed so far, only the amplicon corresponding to the recirculated vector (304 bp) was found.

![](https://static.igem.org/mediawiki/2021/d/d8/T--Ecuador--Results3_L4440lig.png)

The literature reports some reasons for the presence of too much plasmid background among them: Restriction enzyme(s) didn't cleave plasmid completely. To test this possibility, we will perform sequential cleavages with each enzyme separately. Another possible reason is deficient dephosphorylation. Recently the enzyme alkaline phosphatase arrived in our laboratory due to the sponsorship of Promega Corporation. We are planning to implement a dephosphorylation process in the workflow.

### 4. L4440 plasmid improvement and dsRNA production

In Phase I we successfully obtained bacterial colonies with the gBlock Up and DOWN assembled inside the backbones. This result was verified by colony PCR obtaining the expected 1250 bp fragment.

![](https://static.igem.org/mediawiki/2021/a/a8/T--Ecuador--Results4_Fase1.png)

After verifying the success of Phase I, the bacteria cultured in LB medium showed fluorescence characteristic of the GFP indicator, corroborating its correct binding.

![](https://static.igem.org/mediawiki/2021/6/66/T--Ecuador--Results4_fluorescent_culture.png)

In Phase II, after digestion and 3A assembly, bacterial colonies were obtained and colony PCR was performed expecting fragments of size 511 bp and 566 bp, which would indicate a correct assembly. However, as amplification results, fragments of approximately 1.1Kb were obtained in the pSB1C3 backbone (primers pSB1C3\_F, pSB1C3\_R) and of approximately 300 bp for p4440 (primers BBa\_G00100, BBa\_G00101), reflecting negative results of 3A assembly.

![](https://static.igem.org/mediawiki/2021/4/4b/T--Ecuador--Results4_PCR_COLONY_FALLIDA_cds_gfp.png)

![](https://static.igem.org/mediawiki/2021/e/eb/T--Ecuador--Results4_PCR_COLONY_FALLIDA_L4440.png)

Considering the previous negative results, 3A assembly was re-designed to a simple assembly. The UP fragments were digested using the restriction enzymes PstI and SpeI, while the DOWN sequence was digested with PstI and XbaI. This ligation was tested with primers BBa\_G00100 and BBa\_G00101, obtaining in one of the colonies the expected fragment of ~648 bp (image bellow 627 pb), indicating a successful assembly of the UP and DOWN parts.

![](https://static.igem.org/mediawiki/2021/c/c3/T--Ecuador--Results4_3AAssembly.png)

![](https://static.igem.org/mediawiki/2021/e/e3/T--Ecuador--Results4_3Acolonias.jpeg)

As part of Phase III, we continued with the addition of the fragments for dsRNA production on the improved plasmid L4440 in the  *E. coli*  HT115 strain. We started with the ERG11 gene, since it is a virulence gene, was chosen to initiate the assemblies. The dsRNA production plasmid was generated from these constructs. Identification of this correct assembly was performed with primers BBa\_G00100 and BBa\_G00101 obtaining a 1.1Kb fragment.

![](https://static.igem.org/mediawiki/2021/f/f4/T--Ecuador--Gel_inserto_final.png)

We were pleased to present these results at the the Giant Jamboree  [Meet up BRAZIL](https://jamboree.igem.org/meetups/sao-carlos). We will continue in dsRNA production and forward inhibition testing using Foc R1.

## References 1. Widinugraheni, S., Niño-Sánchez, J., van der Does, H. C., van Dam, P., García-Bastidas, F. A., Subandiyah, S., Meijer, H., Kistler, H. C., Kema, G., & Rep, M. (2018). A SIX1 homolog in Fusarium oxysporum f.sp. cubense tropical race 4 contributes to virulence towards Cavendish banana. PloS one, 13(10), e0205896. https://doi.org/10.1371/journal.pone.0205896 2. Deng, G. , Yang, Q. , He, W. , Li, C. , Yang, J. , Zuo, C. , Gao, J. et al . ( 2015 ) Análisis proteómico de la germinación de conidios en Fusarium oxysporum f. sp. cubense tropical race 4 revela nuevos objetivos en la ruta de biosíntesis de ergosterol para controlar la marchitez por Fusarium del banano . Apl. Microbiol. Biot. 99 , 7189 - 7207

## Delivery and Release

### QS-based lysis protein/population oscillator

The results of the oscillator experiment QS-based lysis protein population oscillator are described in  [Engineering: The DBTL cycle](https://2021.igem.org/Team%3AEcuador/Engineering%20) .

## RNA silencing

### *In vitro* inhibition test

We planned to test the antifungal activity of gene-specific dsRNAs, produced in  *E. coli*  and previously purified, by a reduction in colony number following administration by imbibition into spores of FOC TR1. For this purpose, we first developed a protocol for obtaining a conidial suspension with the appropriate yield to obtain a minimum concentration of 1 x 105 conidia/mL.

However, due to the delays associated with the COVID-19 pandemic, we are unable to complete all the inhibition assays in the laboratory. We continue the lab work and hope to present these and more results at the Giant Jamboree  [Meet up BRAZIL](https://jamboree.igem.org/meetups/sao-carlos) .

![](https://static.igem.org/mediawiki/2021/8/8a/T--Ecuador--microconidia.png)

*Microconidia of Foc R1 at 1000 X.*

![](https://static.igem.org/mediawiki/2021/d/d9/T--Ecuador--IgemECSponsors.png)