Team:Ecuador/Results

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igemecuador@gmail.com

Results

Proof Of Concept

Contribution

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).

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

Velvet

 

GenBank: KF800754.1

GCAACCTGAGCGTGCCCGAGCCTGCGGTTCTGGT

TCAAAAGCCAACAGTGACCGCCGACCTGTTGATCC

TCCCCCAGTTGTCGAGCTTCGGATTATCGAGGGAC

CTAGTGTGGAAGAGGGCAAGGACATCACTTTTGA

CTACAACGCCAACTTCTTTCTCTACGCCAGTCTCGA

GCATGCCCGTCCACTTGCACGCGGTCGTGTTAATA

CTCCAGCTGCTGGCAACCCACCCATTCTGACTGGT

GTTCCCGCTTCGGGCATGGCCTATCTCGACCGTCC

CACCGAAGCTGGTTATTTCATCTTTCCCGACCTTTC

TGTCCGCCACGAGGGCCTCTACATACTGACCTTCA

GTTTGTTTGAGACTACCAAGGAGGAAAGAGACTA

TGATCTGGAACCTGCCGATGGCGATCTTCCACCCG

GAGTCGACTATAGAATGGAAATCAAGACTGAACC

TTTCAGCGTCTACAGCGCT

SGE1

 

GenBank: KU048919.1

CTGTGCCCAGTGATGATGAGCGCCATCTGGTCGG

TTCACTCGTCGACTCGTACGACTTCAAGGAACAAG

GACTGGTCAAGAAGACTATCAGCATCACATACCAA

GGCGTGCCTCACCACCTCGTCAGTTACTACCATGT

GGAAGACGTCAAGGCTGGACTTTTGCCTAGTCCT

GTGGATGATCCCAGACTCCGCGGCGTCGTCCCTCG

AACCGAGCTCCTGACAGGTCAGAACTTCCGCGCCC

CGATCGAGGAAGCTCAACACGGTACCTACTTGAG

CGGCGCCTACATGCCAAACATGGATCACCAGTATG

CTCCTATTGGCTTTCCCACACACACACAGCAACCG

GCACTCCAACAGCAGCCACAACAGCAGCCACAGC

CACAGCACCAGCCACAACTACAGTATCAGCCACAG

CCGCACCAGCACCAACCACAGCTGCAGTACCAGCC

ACAGCAGCAGCACCAGCCACAACAACAGTACC
Six1

CGCCAGGAAGTCTACCAGAGCAGCGGATCGCGTC

GAGCTCTCGTACGACGGCTTCGCGAAGCCCTCATC

AAGACCACCATCCTCGTGGGCGTGTGCAAGCCCCT

CGAAGCCATCCTGTCAATCATGAAGGTAGAGAAA

CCCGAGGACCGAGACTTTAGCCAAACGCGCGACG

GATGGCAGGCTGATCAAGCAAACCACGACCGCGG

AATCGATTGGTTCAAGAAAGTGTATACTCGAAACG

CTGGGGATACGATGGGATTGTTTGACGCGCACAA

GGATTTCGCATGGGTCTCGGCGGAGATTACATAT

GGATTGTTTCTATCGGATCGGCAAGTCTTGGATGA

TACGGACACGCAGCTTGTGGTGCTGCCGGCGATC

ATGAGCCAGAATCTTCCGCTGGAGACGAATTGGC

ATATTCGGGGAACGCGGCGAATTGGGGTGTCGAA

GGAGGATGTTCAAGTCATCTGGGATAGCGTCAAG

GAAGTG

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

We were pleased to present these results at the the Giant Jamboree Meet up BRAZIL. 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 .

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 .


Microconidia of Foc R1 at 1000 X.