Development of novel vectors for synthetic biology industrial applications which require capture of large, difficult to clone repetitive DNA fragments

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The key requirement in area of synthetic biology is development of vectors for cloning, sequencing and**expression of large DNA fragments for industrial applications. Such designer vectors are currently lacking, but**are particularly needed for the capturing of DNA sequences that are difficult to clone due to their size and/or**repetitive nature. To optimize, test and improve the vector development we need well annotated genes that are**large and/or have repetitive nature. Our team from Western pioneered genome sequencing of spider mites, the**Two Spotted Spider Mite (TSSM) - Tetranychus urticae and Tetranychus lintearius that have small and**compact genomes (90 MBp) and represent one of best annotated arthropod genomes. These mites spin silk that**is made of fibers consisting of high molecular weight proteins (fibroins) with highly repetitive sequences. Due**to their length and the extreme repetitive nature of their sequences, fibroin genes are extremely challenging to**sequence and clone even with the application of the most advanced sequencing technologies currently available**(e.g. Oxford Nanopore). In this application we propose to establish new collaboration between our lab and**company Designermicrobes (DMI) and to perform following experiments aimed at development of novel**vectors for synthetic biology:**1) We will isolate and annotate fibroin genes in spider mite genomes defining repetitive genes to be cloned**into DMI vectors**2) We will optimize and improve DMI vectors and clone and sequence spider mite fibroin genes cloned in**DMI vector**Fibroin genes from Tetranychid mites are excellent DNA fragments for the improvement of the DMI vectors**and platform for optimizing the DMI technology. The ability to capture, clone and sequence long and repetitive**DNA fragments will be an important next step in improving and expanding the DMI capabilities and allow the**expansion of products in the area of synthetic biology.**