Agrobactory 593 was designed as a modular platform to produce double-stranded RNA (dsRNA) of one or several genes from the pathogen of interest genome. Once inside the pathogen, this dsRNA is further processed into RNA interference that can regulate the expression of any gene in the genome. Therefore, these RNA molecules can become a biopesticide to treat plant diseases.
Our collection has two groups of parts, the specific dsRNA to prevent Fusarium wilt by Foc TR4 (as an example of what can be done) and the parts that are necessary to build the modular platform. This second group is a library of constructs with the T7 promoter and combinations of RBSs and Shine-Dalgarno sequences. Once assembled, they become a destination vector (promoter, rbs, recognition sites, and terminator in both forward and reverse directions) that can receive dsRNA sequences in the desired location when digested with the appropriate enzymes.
Gere, we describe the procedure to create a dsRNA collection for your pathogen of interes with the example of FOC TR4.
Design of dsRNA for target gene
dsRNA design workflow
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 , . In Foc R1, SGE1 and Velvet have also been used , .
For the design of the dsRNA, we used the E-RNAi 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 . Subsequently, we analyzed the homology with other species, to ensure the specificity of the designed dsRNAs and thus maintain biosafety and biosecurity of environmental organisms .
|BBa_K3893001||dsRNA designed for the target gene Velvet|
|BBa_K3893002||dsRNA designed for the target gene ERG11|
|BBa_K3893003||dsRNA designed for the target gene SGE1|
|BBa_K3893004||dsRNA designed for the target gene SIX1|
Modular Platform Assembly System
To use, test, and produce the dsRNA we design before, we created a plasmid backbone with an insert that can receive different sequences of dsRNA with a BglII and KpnI digestion.
When designing this insert, we has several requirements and also found several problems.
The original design for producing dsRNA (plasmid L4440) has the T7 promoter but no transcriptional terminator. This originates the problem of T7 polymerase producing lengthy and nonspecific RNA fragments during transcription . To solve this problem we incorporated high-efficiency transcriptional terminators on both sides of our design. Then, we also incorporated an RBS and Shine Dalgarno sequences, not to start transcription, but to boost the stability of the dsRNA generated, while incorporating an inverted T7 polymerase promoter sequences for the creation of double-stranded RNA.
With this in mind, we face some shortcomings: first, it is a short fragment, with many sequence repeats, which makes it difficult to synthesis. Second, we want this insert to behave as Biobrick compatible once it has the desired dsRNA sequence cloned inside.
This is the design that solves all the problems.
The basic parts to make our insert are:
Basic Parts Table
|BBa_K3893000||Shine-Dalgarno for E coli|
|BBa_K3893006||rpoC Transcriptional terminator|
|BBa_K3893007||Reverse of BBa_M36010 / rnpB_T1|
|BBa_K3893019||Reverse of BBa_B0034 / RBS2 o RBS|
|BBa_K3893020||Reverse of BBa_I712074 / Promoter T7|
|BBa_K3893021||Reverse of BBa_K3893000 / Shine-Dalgarno sequence for E. coli|
|BBa_K3893022||Reverse of BBa_B0032 / RBS3|
GFP was expressed as a reporter in a translational unit constructed to characterize the up and down parts that will make up the dsRNA production cassettes. This characterization requires that the transcriptional unit be designed in a forward and reverse direction.
The GFP transcriptional unit within the cassette functions as a dropout in each up and down the part because the gene is left out when the vector formation with the entire cassette is effective
Estructure of GFP Translational unit
|BBa_K3893017||BBa_B0030||BBa_E0040||BBa_K2598024||GFP translational unit|
Reverse of GFP Transcriptional unit
|BBa_K3893018||Reverse transcriptional unit for GFP|
To prevent repetitive sequences in the synthesis process, the cassette was divided into two parts. The terminator is present in all designed cassettes, with the following variations:
○ RBS weak and medium
○ presence or absence of the Shine Dalgarno sequence
Before constructing the modified plasmids and using them, we took into account the following:
○ Have a Biobrick-compatible backbone, in case you don't have it you could modify it with PCR overhang and add RFC10 prefix and suffix.
○ The following restriction enzymes are used: EcoRI, PstI, SpeI, XbaI, BglII, and KpnI. Therefore the dsRNA sequence should not contain these restriction sites.
○ Previously carry out PCR overhang to the dsRNA sequence to add the restriction sites: BglII and KpnI.
Once the above considerations have been met, the following assemblies are performed:
1) We synthesized the up (UP) and down (DP) parts and cloned them into Biobrick-compatible backbones with EcoRI and PstI.
2) Assembly 3A adapted: We digested the UP plasmid with EcoRI - SpeI, the DP plasmid with XbaI - PstI, and a Biobrick compatible backbone with EcoRI - PstI. Then we ligated and obtained as a product the modified plasmid (dsRNA receptor).
3) We inserted the dsRNA into the plasmid by digesting and ligating both sequences with BglII and KpnI.
Table of uppers parts (UP)
|Composite part||Terminator||Promoter||RBS||SD||TU GFP||Description|
Table of lowers parts (DP)
|Composite part||TU GFP||SD||RBS||Promoter||Terminator||Description|
The L4440 backbone is the standard for producing dsRNA, but it is incompatible with biobricks, thus it was changed with a PCR overhang that included the RFC10 prefix and suffix in order to convert it to our modular platform afterwards.
|BBa_K3893016||BioBricks compatible L4440 plasmid|
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