Provided herein are regulatable expression systems and methods of using said regulatable expression systems to express proteins of interest. The regulatable expression systems comprise a unidirectional regulatable promoter operably linked to a single transcription unit encoding a protein of interest, a ribosome skip, and a transactivator protein.

The present disclosure is related to genetically engineered microbial strains and related bioprocesses for the production of products from acetyl-CoA. Specifically, the use of dynamically controlled synthetic metabolic valves to reduce the activity of certain enzymes, leads to increased product production in a two-stage process.

Recombinant adenovirus genomes that include a synthetic transcriptional circuit are described. Synthetic adenoviruses positively regulated using two-step transcriptional amplification (TSTA) are further described. Selection of the heterologous promoter is based on the desired replication characteristics of the synthetic virus. For example, the heterologous promoter can be a constitutive promoter, a tumor-specific promoter or a tissue-specific promoter.

The present disclosure provides expression constructs designed to provide for expression of therapeutic proteins from engineered cells. The engineered cells may be encapsulated into implantable elements that allow for the therapeutic protein to be released into from the capsule while protecting the cell from the immune system of a patient into which the capsule is implanted.

Recombinant expression vectors are disclosed that include a control sequence for recombinant expression of proteins of interest; the control sequence combines a mCMV enhancer sequence with a rat EF-1alpha intron sequence. Some of the vectors are useful for tetracycline-inducible expression. Some of the vectors contain a 5′ PiggyBac ITR and a 3′ PiggyBac ITR to promote genomic integration into a host cell chromosome. A method of selecting a stable production cell line for manufacturing a protein of interest is also disclosed. Also disclosed are mammalian host cells comprising the inventive recombinant expression vectors and a method of producing a protein of interest, in vitro, involving the mammalian host cell.

Microbial consortia exert great influence over the physiology of humans, animals, plants, and ecosystems. However, difficulty in controlling their composition and population dynamics have limited their application in medicine, agriculture, biotechnology, and the environment. The approach disclosed herein provides an effective method to dynamically control population compositions in microbial consortia, which we demonstrate in the context of co-culture fermentations for chemical production. Co-culture fermentations can improve chemical production from complex biosynthetic pathways over monocultures by distributing enzymes across multiple strains, thereby reducing metabolic burden, overcoming endogenous regulatory mechanisms, or exploiting natural traits of different microbial species. However, stabilizing and optimizing microbial sub-populations for maximal chemical production remains a major obstacle in the field. An optogenetic circuit, called OptoTA, is disclosed for regulating a toxin-antitoxin system, which enables tunability of, e.g., Escherichia coli growth using only blue light. With the disclosed system, one can control population ratios of co-cultures of, e.g., E. coli and Saccharomyces cerevisiae containing different metabolic modules of biosynthetic pathways. Results reveal that intermediate light duty cycles improve chemical production by establishing optimal co-culture populations.

The present disclosure provides a nucleic acid sequence for regulating the transcription of a nucleic acid fragment of interest, wherein the nucleic acid sequence comprises at least 2 copies of TetO-operator sequences capable of binding to a transactivator rtTA regulatable by tetracycline or a derivative thereof, and 1 copy of minimal promoter sequence containing a TATA box sequence, and at least 1 copy of a CuO-operator sequence capable of binding to a transcription repressor CymR regulatable by cumate. The present disclosure also provides a vector and a host cell containing the nucleic acid sequence, and a method for inducing the expression of a nucleic acid fragment of interest in a host cell.

An engineered genetic incompatibility (EGI) strain of a wild-type organism is designed to include a haplosufficient lethal allele and a haploinsufficient resistance allele. In another aspect, a biocontainment system generally includes a polynucleotide that encodes a coding region whose expression causes infertility or death, a transcription regulatory region operably linked upstream of the coding region and containing a silent mutation, and a polynucleotide that encodes a programmable transcription activator. The programmable transcription activator is engineered to bind to the transcription regulatory region in the absence of the silent mutation, thereby expressing the coding region in the absence of the silent mutation, but does not initiate expression of the coding region when the transcription regulatory region comprises the silent mutation.

The disclosure relates to compositions, methods, and processes for the preparation, use, and/or formulation of adeno-associated virus (AAV) particle comprising a viral gene and a capsid, wherein the viral genome comprises at least one Vectorized Editing of Nucleic acids to correct Overt Mutations (VENOM) element.

The present disclosure provides molecular tools, methods and kits for using cell division loci (CDLs) to control cell proliferation in animal cells. CDLs, as provided herein, are loci whose transcription product(s) are expressed during cell division. CDLs may be genetically modified, as described herein, to comprise a negative selectable marker and/or an inducible activator-based gene expression system, which allows a user to permit, ablate, and/or inhibit proliferation of the genetically modified cell(s) by adding or removing an appropriate inducer.