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Summary DNA assembly systems based on the Golden Gate method are popular in synthetic biology but have several limitations: small insert size, incompatibility with other cloning platforms, DNA domestication requirement, generation of fusion scars, and lack of post‐assembly modification. To address these obstacles, we present the DASH assembly toolset, which combines features of Golden Gate‐based cloning, recombineering, and site‐specific recombinase systems. We developed (1) a set of donor vectors based on the GoldenBraid platform, (2) an acceptor vector derived from the plant transformation‐competent artificial chromosome (TAC) vector, pYLTAC17, and (3) a re‐engineered recombineering‐readyE. colistrain, CZ105, based on SW105. The initial assembly steps are carried out using the donor vectors following standard GoldenBraid assembly procedures. Importantly, existing parts and transcriptional units created using compatible Golden Gate‐based systems can be transferred to the DASH donor vectors using standard single‐tube restriction/ligation reactions. The cargo DNA from a DASH donor vector is then efficiently transferredin vivoinE. coliinto the acceptor vector by the sequential action of a rhamnose‐inducible phage‐derived PhiC31 integrase and arabinose‐inducible yeast‐derived Flippase (FLP) recombinase using CZ105. Furthermore, recombineering‐based post‐assembly modification, including the removal of undesirable scars, is greatly simplified. To demonstrate the utility of the DASH system, a 116 kilobase (kb) DNA construct harbouring a 97 kb cargo consisting of 35 transcriptional units was generated. One of the coding DNA sequences (CDSs) in the final assembly was replaced through recombineering, and thein plantafunctionality of the entire construct was tested in both transient and stable transformants. 

SUMMARY Genome editing technologies like CRISPR/Cas have greatly accelerated the pace of both fundamental research and translational applications in agriculture. However, many plant biologists are functionally limited to creating small, targeted DNA changes or large, random DNA insertions. The ability to efficiently generate large, yet precise, DNA changes will massively accelerate crop breeding cycles, enabling researchers to more efficiently engineer crops amidst a rapidly changing agricultural landscape. This review provides an overview of existing technologies that allow plant biologists to integrate large DNA sequences within a plant host and some associated technical bottlenecks. Additionally, this review explores a selection of emerging techniques in other host systems to inspire tool development in plants. 

RationaleMass spectrometry imaging of young seedlings is an invaluable tool in understanding how mutations affect metabolite accumulation in plant development. However, due to numerous biological considerations, established methods for the relative quantification of analytes using infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) mass spectrometry imaging are not viable options. In this study, we report a method for the quantification of auxin‐related compounds using stable‐isotope‐labelled (SIL) indole‐3‐acetic acid (IAA) doped into agarose substrate. MethodsWild‐typeArabidopsis thalianaseedlings,sur2andwei8 tar2loss‐of‐function mutants, andYUC1gain‐of‐function line were grown for 3 days in the dark in standard growth medium. SIL‐IAA was doped into a 1% low‐melting‐point agarose gel and seedlings were gently laid on top for IR‐MALDESI imaging with Orbitrap mass spectrometry analysis. Relative quantification was performed post‐acquisition by normalization of auxin‐related compounds to SIL‐IAA in the agarose. Amounts of auxin‐related compounds were compared between genotypes to distinguish the effects of the mutations on the accumulation of indolic metabolites of interest. ResultsIAA added to agarose was found to remain stable, with repeatability and abundance features of IAA comparable with those of other compounds used in other methods for relative quantification in IR‐MALDESI analyses. Indole‐3‐acetaldoxime was increased insur2mutants compared with wild‐type and other mutants. Other auxin‐related metabolites were either below the limits of quantification or successfully quantified but showing little difference among mutants. ConclusionsAgarose was shown to be an appropriate sampling surface for IR‐MALDESI mass spectrometry imaging ofArabidopsisseedlings. SIL‐IAA doping of agarose was demonstrated as a viable technique for relative quantification of metabolites in live seedlings or tissues with similar biological considerations.