Abstract: The disclosure provides methods of making a tetracycline inducible expression system in a cell. The methods include providing the cell with a first nucleic acid sequence comprising a first promoter operably linked to a tetracycline repressor gene coding sequence, providing the cell with a second nucleic acid sequence comprising a second promoter operably linked to a coding sequence of a gene of interest wherein the second promoter is modified to include one or more tetracycline repressor protein binding sites, and determining the expression of the gene of interest in the presence or absence of tetracycline. The disclosure further provides nucleic acid sequences, vectors and cells including the tetracycline inducible modified promoter.

Abstract: The disclosure provides methods for making a polynucleotide wherein the addition of nucleotides can be physically, chemically and/or enzymatically controlled. The methods include combining a selected nucleotide, cations, an error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3? terminal nucleotide, wherein the reaction reagents can be modulated and under conditions that allow covalent addition of one or more of a selected nucleotide to the 3? terminal nucleotide such that the selected nucleotide becomes a 3? terminal nucleotide, and repeating the addition step until the polynucleotide is formed.

Abstract: The disclosure provides methods for making a polynucleotide wherein the addition of nucleotides can be physically, chemically and/or enzymatically controlled. The methods include combining a selected nucleotide, cations, an error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3? terminal nucleotide, wherein the reaction reagents can be modulated and under conditions that allow covalent addition of one or more of a selected nucleotide to the 3? terminal nucleotide such that the selected nucleotide becomes a 3? terminal nucleotide, and repeating the addition step until the polynucleotide is formed.

Abstract: A method of processing a collection of nucleic acid sequences is provided including connecting an adaptor to one or more or each nucleic acid sequence in the collection to create a processed nucleic acid template library, wherein the adaptor includes a first DNA sequence encoding a PAM sequence and at least a tracr mate.

Abstract: The present disclosure provides methods of activating an enzyme, such as error prone or template independent polymerase, using electricity to alter pH of a reaction zone and reaction site from an inactivating pH at which the enzyme is inactive to an activating pH at which the enzyme is active to add a nucleotide to an initiator or growing polymer chain. The activating pH can then be changed back to an inactivating pH and the process repeated as many times as desired to produce a target nucleic acid sequence.

Abstract: Methods for making a polynucleotide is provided. The methods include (a) providing a first single stranded oligonucleotide, (b) providing a second single stranded oligonucleotide under conditions wherein the first single stranded oligonucleotide anneals to the second single stranded oligonucleotide thereby forming a double stranded oligonucleotide template having an extendible end comprising the 3? terminal nucleotide of the first single stranded oligonucleotide, (c) providing a reaction mixture to the double stranded initiator wherein the reaction mixture comprises an enzyme, a selected nucleotide triphosphate, and divalent cations, and wherein the enzyme extends the extendible end, (d) regenerating an extendible end of the extended template, and repeating steps (c) to (d) until a polynucleotide of a desired sequence or information content is formed, with the proviso that step (d) is not required to be performed after the polynucleotide is formed.

Abstract: The disclosure provides methods of making a tetracycline inducible expression system in a cell. The methods include providing the cell with a first nucleic acid sequence comprising a first promoter operably linked to a tetracycline repressor gene coding sequence, providing the cell with a second nucleic acid sequence comprising a second promoter operably linked to a coding sequence of a gene of interest wherein the second promoter is modified to include one or more tetracycline repressor protein binding sites, and determining the expression of the gene of interest in the presence or absence of tetracycline. The disclosure further provides nucleic acid sequences, vectors and cells including the tetracycline inducible modified promoter.

Abstract: Methods and compositions are provided for modulating expression of a target nucleic acid sequence within a non-E. coli cell. The method includes providing the cell with a guide RNA comprising a portion that is complementary to all or a portion of the target nucleic acid sequence, and providing the cell a Cas protein, wherein the guide RNA and the Cas protein co-localize at the target nucleic acid sequence and wherein the Cas protein modulate the expression of the target nucleic acid sequence.

Abstract: A method of processing a collection of nucleic acid sequences is provided including connecting an adaptor to one or more or each nucleic acid sequence in the collection to create a processed nucleic acid template library, wherein the adaptor includes a first DNA sequence encoding a PAM sequence and at least a tracr mate.

Abstract: The present disclosure provides methods of activating an enzyme, such as error prone or template independent polymerase, using electricity to alter pH of a reaction zone and reaction site from an inactivating pH at which the enzyme is inactive to an activating pH at which the enzyme is active to add a nucleotide to an initiator or growing polymer chain. The activating pH can then be changed back to an inactivating pH and the process repeated as many times as desired to produce a target nucleic acid sequence.

Abstract: The disclosure provides methods for making a polynucleotide wherein the addition of nucleotides can be physically, chemically and/or enzymatically controlled. The methods include combining a selected nucleotide, cations, an error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3? terminal nucleotide, wherein the reaction reagents can be modulated and under conditions that allow covalent addition of one or more of a selected nucleotide to the 3? terminal nucleotide such that the selected nucleotide becomes a 3? terminal nucleotide, and repeating the addition step until the polynucleotide is formed.