We hereby disclose and claim a mathematically based system for design and implementation synthetic biology genetic circuitry architecture which uses genetic expression circuits which are linked together to achieve a therapeutic or diagnostic effect. The genetic architecture that enables the reversible and tunable desired gene product output is achieved by genetic control elements upstream and downstream of a promoter region. Reversible and tunable control is achieved through the computational design and pairing of transcriptional repression elements with their cognate transcriptional promoters to enable a broad range of therapeutic and diagnostic applications.

The present invention provides compositions and methods for programming mammalian cells to perform desired functions. In particular, the present invention provides compositions and methods for programming stem cells to differentiate into a desired cell type. A quorum sensing systems that regulates the expression of cell fate regulators is introduced into mammalian host cells, such as stem cells. The quorum sensing systems generally comprises vectors that express the components of a bacterial quorum sensing pathway, including proteins which catalyze the synthesis of an autoinducer and a gene encoding a regulatory partner of the autoinducer, and vectors in which genes encoding cell fate regulators are operably linked to a promoter induced by the autoinducer/regulatory partner complex. The system can also comprise vectors in which genes encoding additional cell fate regulators are operably linked to a promoter that is induced by a factor synthesized in response to a first stage of differentiation, so that a second stage of differentiation is triggered.

Provided is a modular construction of synthetic gene circuits in mammalian cells using TALE transcriptional repressors. Provided is a method for achieving regulated expression of two proteins: an expression cassette A comprises a feedback element coding sequence, a promoter A, a protein A and TALER protein A encoding gene linked by means of a self-cleaving polypeptide, and a target sequence A (comprising an shRNA1 target sequence); an expression cassette B comprises a feedback element coding sequence, a promoter B, a protein B and TALER protein B encoding gene linked by means of a self-cleaving polypeptide, and a target sequence B (comprising an shRNA2 target sequence); an expression cassette C comprises a constitutive promoter and an activating element-coding sequence; a recombinant vector A having the expression cassette A, a recombinant vector B having the expression cassette B, and a recombinant vector C having the expression cassette C are introduced into host cells to regulate the expression of protein A and protein B by adding shRNA1 or shRNA2.

Provided herein are CRISPR-based synthetic repression systems as well as methods and compositions using the synthetic repression systems to treat septicemia, an adverse immune response in a subject and Waldenstrm macroglobulinemia.

Provided herein are systems that provide a genetic program to control bacterial life cycle and function execution, thereby conferring programmable microbial transition between planktonic and biofilm states and facilitating the development of cellular functions across physiological domains.

The invention features compositions and methods for identifying orthogonal transcriptional switches derived from Tet repressor homologs for Saccharomyces cerevishiae regulated by 2,4-diacetylphloroglucinol (DAPG) and other ligands.

Provided herein are CRISPR-based synthetic repression systems as well as methods and compositions using the synthetic repression systems to treat septicemia, an adverse immune response in a subject and Waldenstr?m macroglobulinemia.

A nucleic acid molecule is uniquely designed and encodes an entire CRISPRi or CRISPRa system, while being sized for packaging within a single adeno-associated virus (AAV) vector. Examples of the nucleic acid molecule include about 4600 to 4700 base pairs. Examples of the nucleic acid molecule can include a nucleotide encoding a Cas polypeptide; a nucleotide encoding a repressor or an activator domain attached to the nucleotide encoding the Cas polypeptide via a linker; a first promoter operably connected to the nucleotide encoding the repressor or activator domain or the nucleotide encoding the Cas polypeptide; a nucleotide encoding an alpha-helical connecting the nucleotide encoding the Cas polypeptide to a nuclear localization signal (NLS); and a second promoter operably connected to a guide RNA (gRNA).

The invention relates to the production and use of Cas-encoding sequences and vectors comprising these. Aspects of the invention provide products, vectors, delivery vehicles, uses and methods for producing Cas-encoding sequences in bacterial or archaeal cells.

The invention relates to a seamless cloning method, comprising a single assembly step of two or more polynucleotides, e.g., a plasmid vector and a gene insert, conducted at 58 to 100? C., the preferred temperature(s) being the same or greater than a particular one of the following temperatures: 58? C., 59? C., 60? C., 61? C., 62? C., 63? C., 64? C., 65? C., 66? C., 67? C., 68? C., 69? C., 70? C., 71? C., 72? C., 73? C., 74? C., 75? C., 76? C., 77? C., 78? C., 79? C., 80? C., 84? C., 88? C., 92? C., 96? C. and 100? C., and a transformation into chemically competent cells for covalent linking, preferably including a static recovery period.