Biology subjects

Velazquez, E.

Publications and source records attributed to Velazquez, E..

3 recordsLinked to original sources

bioRxiv2021

Genome-wide protein-DNA interaction site mapping using a double strand DNA-specific cytosine deaminase

DNA-protein interactions (DPIs) are central to such fundamental cellular processes as transcription and chromosome maintenance and organization. The spatiotemporal dynamics of these interactions dictate their functional consequences; therefore, there is great interest in facile methods for defining the sites of DPI within cells. Here, we present a general method for mapping DPI sites in vivo using the double stranded DNA-specific cytosine deaminase toxin DddA. Our approach, which we term DddA-sequencing (3D-seq), entails generating a translational fusion of DddA to a DNA binding protein of interest, inactivating uracil DNA glycosylase, modulating DddA activity via its natural inhibitor protein, and DNA sequencing for genome-wide DPI detection. We successfully applied this method to three Pseudomonas aeruginosa transcription factors that represent divergent protein families and bind variable numbers of chromosomal locations. 3D-seq offers several advantages over existing technologies including ease of implementation and the possibility to measure DPIs at single-cell resolution.

genetics↗

bioRxiv2021

Targetron-assisted delivery of exogenous DNA sequences into Pseudomonas putida through CRISPR-aided counterselection

Genome editing methods based on Group II introns (known as Targetron technology) have been long used as a gene knock-out strategy in a wide range of organisms in a fashion independent of homologous recombination. Yet, their utility as delivery systems has been typically suboptimal because of their reduced efficiency of insertion when they carry exogenous sequences. We show that this limitation can be tackled and Targetron adapted as a general tool in Gram-negative bacteria. To this end, a set of broad host range standardized vectors were designed for conditional expression of the Ll.LtrB intron. After testing the correct functionality of these plasmids in Escherichia coli and Pseudomonas putida, we created a library of Ll.LtrB variants carrying cargo DNA sequences of different lengths to benchmark the capacity of intron-mediated delivery in these bacteria. Next, we combined CRISPR/Cas9-facilitated counterselection to increase the chances of finding genomic sites inserted with the thereby engineered introns. By following this pipeline, we were able to insert exogenous sequences of up to 600 bp at specific genomic locations in wild-type P. putida KT2440 and its {Delta}recA derivative. Finally, we were able to apply this technology to successfully tag this strain with an orthogonal short sequence (barcode) that acts as a unique identifier for tracking this microorganism in biotechnological settings. The results with P. putida exemplified the value of the Targetron approach for unrestricted delivery of small DNA fragments to the genomes of Gram-negative bacteria for a suite of genome editing endeavours.

synthetic biology↗

bioRxiv2021

Versioning Biological Cells for Trustworthy Cell Engineering

"Full-stack" biotechnology platforms for cell line (re)programming are on the horizon, due mostly to (a) advances in gene synthesis and editing techniques as well as (b) the growing integration with informatics, the internet of things and automation. These emerging platforms will accelerate the production and consumption of biological products. Hence, transparency, traceability and -ultimately-trustworthiness is required -from cradle to grave- for engineered cell lines and their engineering processes. We report here the first version control system for cell engineering that integrates a new cloud-based version control software for cell lines digital footprint with molecular barcoding of living samples. We argue that version control for cell engineering marks a significant step towards more open, reproducible, easier to trace and share, and more trustworthy engineering biology. One Sentence SummaryWe demonstrate a transparent and open way of engineering and sharing cell lines.

synthetic biology↗