Team:Lethbridge HS/Parts

Parts

Part Design

Commercial RNA is often produced through solid-phase synthesis, but this method can be costly. Other methods include in vitro transcription using RNA polymerase from bacteriophage T7. While versatile, in vitro transcription has issues in transcript inhomogeneity and low yield.

Karolinska Institute’s Petzold lab in Stockholm, Sweden, developed a simple yet cost-effective strategy for the enzymatic production of short, non-modified RNAs in high yield and purity. Their 2020 paper (DOI: 10.3390/molecules25051142) describes a “one-pot” method for producing siRNA from a plasmid encoding tandem repeats of the target RNA sequence followed by cleavage with RNase H guided by a chimeric cleavage DNA splint. This method could produce single repeat units of precise length, simplifying downstream purification and improving yields.

We adapted this method to produce the siRNA needed to combat knapweed using a DNA template consisting of a T7 promoter (BBa_I719005) which includes the optimal initiation sequence (5’ - GGG AGA - 3’), and a number of tandem repeats of the target RNA sequence - in our case the unique ClpP or ADT2 gene fragments. We synthesized the DNA template as a G-block and it can be cloned into a pSB1C3 plasmid as it is BioBrick compatible and contains the appropriate prefix and suffix sequences. This approach for siRNA production has not been attempted by any iGEM teams to date and may be a new way for teams to easily produce siRNA for their projects.

Basic Parts

Our basic parts were designed to include 26 repeats of a 19 base pair siRNA template sequence. The siRNA DNA sequences were designed using the IDT Custom Dicer-Substrate siRNA Generator.

Composite Parts

The composite parts were designed for easy cloning into pSB1C3 and include the iGEM prefix and suffix sequences. All composite parts also contain the T7 RNA Polymerase promoter BBa_I719005 including the preferred initiation sequence. No terminator was included in our design as these constructs can be used as DNA template for in vitro transcription by either linearizing the plasmid or PCR amplifying the sequence.

Part Table

<groupparts>iGEM21 Lethbridge_HS</groupparts>