Browsing by Author "Shis, David L."

Now showing 1 - 6 of 6
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    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, Liskin
    Protein 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.
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    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.
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    Stable Maintenance of Multiple Plasmids in E. coli Using a Single Selective Marker
    (American Chemical Society, 2012) Schmidt, Calvin M.; Shis, David L.; Nguyen-Huu, Truong D.; Bennett, Matthew R.; Institute of Biosciences and Bioengineering
    Plasmid-based genetic systems in Escherichia coli are a staple of synthetic biology. However, the use of plasmids imposes limitations on the size of synthetic gene circuits and the ease with which they can be placed into bacterial hosts. For instance, unique selective markers must be used for each plasmid to ensure their maintenance in the host. These selective markers are most often genes encoding resistance to antibiotics such as ampicillin or kanamycin. However, the simultaneous use of multiple antibiotics to retain different plasmids can place undue stress on the host and increase the cost of growth media. To address this problem, we have developed a method for stably transforming three different plasmids in E. coli using a single antibiotic selective marker. To do this, we first examined two different systems with which two plasmids may be maintained. These systems make use of either T7 RNA polymerase-specific regulation of the resistance gene or split antibiotic resistance enzymes encoded on separate plasmids. Finally, we combined the two methods to create a system with which three plasmids can be transformed and stably maintained using a single selective marker. This work shows that large-scale plasmid-based synthetic gene circuits need not be limited by the use of multiple antibiotic resistance genes.
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    Synthetic biology: the many facets of T7 RNA polymerase
    (Wiley, 2014) Shis, David L.; Bennett, Matthew R.
    Split T7 RNA polymerase provides new avenues for creating synthetic gene circuits that are decoupled from host regulatory processesﾗbut how many times can this enzyme be split, yet retain function? New research by Voigt and colleagues (Segall-Shapiro et al, 2014) indicates that it may be more than you think.
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    Tuning the dynamic range of bacterial promoters regulated by ligand-inducible transcription factors
    (Springer Nature, 2018) Chen, Ye; Ho, Joanne M.L.; Shis, David L.; Gupta, Chinmaya; Long, James; Wagner, Daniel S.; Ott, William; Josić, Krešimir; Bennett, Matthew R.
    One challenge for synthetic biologists is the predictable tuning of genetic circuit regulatory components to elicit desired outputs. Gene expression driven by ligand-inducible transcription factor systems must exhibit the correct ON and OFF characteristics: appropriate activation and leakiness in the presence and absence of inducer, respectively. However, the dynamic range of a promoter (i.e., absolute difference between ON and OFF states) is difficult to control. We report a method that tunes the dynamic range of ligand-inducible promoters to achieve desired ON and OFF characteristics. We build combinatorial sets of AraC-and LasR-regulated promoters containing -10 and -35 sites from synthetic and Escherichia coli promoters. Four sequence combinations with diverse dynamic ranges were chosen to build multi-input transcriptional logic gates regulated by two and three ligand-inducible transcription factors (LacI, TetR, AraC, XylS, RhlR, LasR, and LuxR). This work enables predictable control over the dynamic range of regulatory components.
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    Tuning the dynamic range of bacterial promoters regulated by ligand-inducible transcription factors
    (Springer Nature, 2018) Chen, Ye; Ho, Joanne M.L.; Shis, David L.; Gupta, Chinmaya; Long, James; Wagner, Daniel S.; Ott, William; Josić, Krešimir; Bennett, Matthew R.
    One challenge for synthetic biologists is the predictable tuning of genetic circuit regulatory components to elicit desired outputs. Gene expression driven by ligand-inducible transcription factor systems must exhibit the correct ON and OFF characteristics: appropriate activation and leakiness in the presence and absence of inducer, respectively. However, the dynamic range of a promoter (i.e., absolute difference between ON and OFF states) is difficult to control. We report a method that tunes the dynamic range of ligand-inducible promoters to achieve desired ON and OFF characteristics. We build combinatorial sets of AraC-and LasR-regulated promoters containing -10 and -35 sites from synthetic and Escherichia coli promoters. Four sequence combinations with diverse dynamic ranges were chosen to build multi-input transcriptional logic gates regulated by two and three ligand-inducible transcription factors (LacI, TetR, AraC, XylS, RhlR, LasR, and LuxR). This work enables predictable control over the dynamic range of regulatory components.