Toggle Cloning - proposal for a new standard:
Researchers are in the comfortable position to be able to choose from a diversity of cloning and expression systems for building complex multi-gene assemblies. Yet, proprietary designs often run contrary to efforts towards standardizing assembly strategies. Some plasmid lay-outs accomodate compatibility, but truly universal systems are hard to come by.ATG:biosynthetics GmbH has developed a vector assembly system with standardized modular elements that adhere to a uniform design logic. This flexTEC system allows easy modification, exchange, addition, removal and regeneration of components or higher-order gene assemblies. This system has been de-signed with the idea of an industrial standard in mind and has successfully been implemented for multi-gene assemblies in metabolic engineering of bacteria.
AbsI(XhoI) and SgrDI(SalI) are compatible sites which can be used to combine several multi-gene constructs assembled by AscI(BssHII) and MauBI(BssHII). In addition theses sites are flanking theAbsI(XhoI)-Rc-x- AscI(BssHII) and MauBI(BssHII)-Rc-y- SgrDI(SalI) recombination sites for (toggle) recombination assembly.
Figure1.
FlexTEC - Vector systems for all genetic systems are all pre-designed according to the same basic principle as modular sequence elements which always fit into the sequence context.
A-BoX – expression elements are equipped with species specific regulatory functions - into the desired BoX-segment. Transcriptional promoters and terminators or polyA-sites
and translational control elements (Shine-Dalgarno, Kozak elements) need to be sequence adapted. Proper initiation function on the RNA-level can be calculated by ATG:
EvoMAG ™
SgrDI-S-BoX-AsiSI – selector elements are available for various resistance genes. AsiSI-T-BoX–PacI – transfer elements (like: shuttle host oris, oriV, oriT or recombination sites) etc. can be toggle assembled to contain multiple functional transfer elements. The PacI-M-BoX-AbsI(XhoI) vector mainatenance box (ColEI*high copy) can be equipped with alternative oris like p15a, R6Kgamma, RK2 etc.
SgrDI-S-BoX-AsiSI – selector elements are available for various resistance genes. AsiSI-T-BoX–PacI – transfer elements (like: shuttle host oris, oriV, oriT or recombination sites) etc. can be toggle assembled to contain multiple functional transfer elements. The PacI-M-BoX-AbsI(XhoI) vector mainatenance box (ColEI*high copy) can be equipped with alternative oris like p15a, R6Kgamma, RK2 etc.
At present available pre-designed:
- FlexTEC - Vector system
- FlexColi (Escherichia coli)
- FlexBACTurbo (Baculovirus)
- FlexMAM (Mammalian cells)
The Toggle System:
TOGGLE Assembly Systems adhere to a standardized unified constructive principle. Toggle plasmid vectors generally consist of four modular elements: applications (A-Box), selectors (S-Box), as well as target (T-Box) and maintenance (M-Box) functions (see fig. 1).
The system is designed as an open standard for the synthetic biology research community but also to researchers in other fields, academic as well as industrial. The standard encourages more intense exchange of parts and modules between researchers.
TOGGLE Assembly Systems adhere to a standardized unified constructive principle. Toggle plasmid vectors generally consist of four modular elements: applications (A-Box), selectors (S-Box), as well as target (T-Box) and maintenance (M-Box) functions (see fig. 1).
The system is designed as an open standard for the synthetic biology research community but also to researchers in other fields, academic as well as industrial. The standard encourages more intense exchange of parts and modules between researchers.
Toggle Basic Systems
Currently, we have two ready-to-use versions of the Toggle system (the so called flexTEC line of expression systems). One for E.coli and one for insect cells.
In principle, Toggle can be custom-adapted to any organism that can be genetically modified.
Currently, we have two ready-to-use versions of the Toggle system (the so called flexTEC line of expression systems). One for E.coli and one for insect cells.
In principle, Toggle can be custom-adapted to any organism that can be genetically modified.