Browsing by Author "Swint-Kruse, Liskin"
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Item AlloRep: A Repository of Sequence, Structural and Mutagenesis Data for the LacI/GalR Transcription Regulators(Elsevier, 2016) Sousa, Filipa L.; Parente, Daniel J.; Shis, David L.; Hessman, Jacob A.; Chazelle, Allen; Bennett, Matthew R.; Teichmann, Sarah A.; Swint-Kruse, LiskinProtein families evolve functional variation by accumulating point mutations at functionally important amino acid positions. Homologs in the LacI/GalR family of transcription regulators have evolved to bind diverse DNA sequences and allosteric regulatory molecules. In addition to playing key roles in bacterial metabolism, these proteins have been widely used as a model family for benchmarking structural and functional prediction algorithms. We have collected manually curated sequence alignments for >ᅠ3000 sequences, in vivo phenotypic and biochemical data for >ᅠ5750 LacI/GalR mutational variants, and noncovalent residue contact networks for 65 LacI/GalR homolog structures. Using this rich data resource, we compared the noncovalent residue contact networks of the LacI/GalR subfamilies to design and experimentally validate an allosteric mutant of a synthetic LacI/GalR repressor for use in biotechnology. The AlloRep database (freely available at www.AlloRep.org) is a key resource for future evolutionary studies of LacI/GalR homologs and for benchmarking computational predictions of functional change.Item Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins(American Society for Biochemistry and Molecular Biology, 2015) Bondos, Sarah E.; Swint-Kruse, Liskin; Matthews, Kathleen S.To modulate transcription, a variety of input signals must be sensed by genetic regulatory proteins. In these proteins, flexibility and disorder are emerging as common themes. Prokaryotic regulators generally have short, flexible segments, whereas eukaryotic regulators have extended regions that lack predicted secondary structure (intrinsic disorder). Two examples illustrate the impact of flexibility and disorder on gene regulation: the prokaryotic LacI/GalR family, with detailed information from studies on LacI, and the eukaryotic family of Hox proteins, with specific insights from investigations of Ultrabithorax (Ubx). The widespread importance of structural disorder in gene regulatory proteins may derive from the need for flexibility in signal response and, particularly in eukaryotes, in protein partner selection.Item Modular, Multi-Input Transcriptional Logic Gating with Orthogonal LacI/GalR Family Chimeras(American Chemical Society, 2014) Shis, David L.; Hussain, Faiza; Meinhardt, Sarah; Swint-Kruse, Liskin; Bennett, Matthew R.In prokaryotes, the construction of synthetic, multi-input promoters is constrained by the number of transcription factors that can simultaneously regulate a single promoter. This fundamental engineering constraint is an obstacle to synthetic biologists because it limits the computational capacity of engineered gene circuits. Here, we demonstrate that complex multi-input transcriptional logic gating can be achieved through the use of ligand-inducible chimeric transcription factors assembled from the LacI/GalR family. These modular chimeras each contain a ligand-binding domain and a DNA-binding domain, both of which are chosen from a library of possibilities. When two or more chimeras have the same DNA-binding domain, they independently and simultaneously regulate any promoter containing the appropriate operator site. In this manner, simple transcriptional AND gating is possible through the combination of two chimeras, and multiple-input AND gating is possible with the simultaneous use of three or even four chimeras. Furthermore, we demonstrate that orthogonal DNA-binding domains and their cognate operators allow the coexpression of multiple, orthogonal AND gates. Altogether, this work provides synthetic biologists with novel, ligand-inducible logic gates and greatly expands the possibilities for engineering complex synthetic gene circuits.