Recombinant clone selection system

A method for cloning and isolating recombinant nucleic acid comprising the steps of providing a first nucleic acid comprising:

• a surface-expressed-moiety gene;
• a first promoter sequence for promoting the expression of the surface-expressed-moiety gene;
• and
• an operator, said operator being functionally linked to the expression of the surface-expressed-moiety gene such that when a complementary repressor is bound to the operator expression of the surface-expressed-moiety gene is repressed;
• and
• a second nucleic acid comprising:
• a repressor gene;
• a second promoter sequence for promoting the expression of the repressor gene;
• and
• an insertion site located within the repressor gene such that when a foreign nucleic acid is inserted at the insertion site expression of the repressor gene is insertionally inactivated;
• cleaving the second nucleic acid at the insertion site forming a cleaved second nucleic acid;
• contacting the cleaved second nucleic acid with a foreign nucleic acid under conditions sufficient to incorporate the foreign nucleic acid into the cleaved second nucleic acid, thereby forming a recombinant nucleic acid;
• inserting the recombinant nucleic acid into a host organism containing the first nucleic acid, wherein the host organism is a cultured cell;
• growing the host organism for a time sufficient to express the surface-expressed-moiety gene;
• and
• contacting the host organism with a solid support having attached thereto a moiety that specifically binds to the surface-expressed moiety.

A cloning system for the isolation of recombinant nucleic acid comprising:

• a tag moiety sequence, said sequence being adapted for linking to a foreign nucleic acid thereby forming an insertion sequence;
• and a nucleic acid comprising:
• a surface-protein gene, said gene coding for a protein which is expressed on the surface of a host organism, wherein the host organism is a cultured cell;
• a promoter sequence for promoting the expression of the surface-protein gene;
• and
• an insertion site located within the surface-protein gene such that when the insertion sequence is inserted at the insertion site the tag moiety sequence is expressed at the surface of the host organism.

A method for cloning and isolating recombinant nucleic acid comprising the steps of:

• providing a tag moiety sequence, said sequence being adapted for linking to a foreign nucleic acid thereby forming an insertion sequence;
• and a nucleic acid comprising:
• a surface-protein gene, said gene coding for a protein which is expressed on the surface of a host organism, wherein the host organism is a cultured cell;
• a promoter sequence for promoting the expression of the surface-protein gene;
• and
• an insertion site located within the surface-protein gene such that when the insertion sequence is inserted at the insertion site the tag moiety sequence is expressed at the surface of the host organism;
• cleaving the nucleic acid at the insertion site thereby forming a cleaved nucleic acid;
• linking the tag moiety sequence with a foreign nucleic acid, thereby forming the insertion sequence;
• contacting the cleaved nucleic acid with the insertion sequence under conditions sufficient to incorporate the insertion sequence into the cleaved nucleic acid, thereby forming a recombinant nucleic acid;
• inserting the recombinant nucleic acid into a host organism;
• growing the host organism for a time sufficient to express the tag moiety sequence;
• and
• contacting the host organism with a solid support having attached thereto a moiety that specifically binds to the tag moiety.

A cloning system, including a nucleic acid that is expressed in a host organism that is a cultured cell, for the isolation of recombinant nucleic acid comprising:

• a surface-expressed-moiety gene;
• a first promoter sequence for promoting the expression of the surface-expressed-moiety gene;
• a lethal gene, the expression product of the lethal gene being lethal to the host organism;
• a second promoter sequence for promoting the expression of the lethal gene;
• and an insertion site located within the lethal gene such that when foreign nucleic acid is inserted at the insertion site, the lethal gene is insertionally inactivated.

A method for cloning and isolating recombinant nucleic acid comprising the steps of providing a nucleic acid comprising:

• a surface-expressed-moiety gene;
• a first promoter sequence for promoting the expression of the surface-expressed-moiety gene;
• a lethal gene, the expression product of the lethal gene being lethal to the host organism;
• second promoter sequence for promoting the expression of the lethal gene;
• and
• an insertion site located within the lethal gene such that when foreign nucleic acid is inserted at the insertion site, the lethal gene is insertionally inactivated;
• cleaving the nucleic acid at the insertion site thereby forming a cleaved nucleic acid contacting the cleaved nucleic acid with a foreign nucleic acid under conditions sufficient to incorporate the foreign nucleic acid into the cleaved nucleic acid, thereby forming a recombinant nucleic acid;
• inserting the recombinant nucleic acid into a host organism that is a cultured cell;
• growing the host organism for a time sufficient to express the lethal gene and the surface-expressed-moiety gene;
• and
• contacting the host organism with a solid support having attached thereto a moiety that specifically binds to the surface-expressed moiety.

A method for cloning and isolating recombinant nucleic acid comprising the steps of:

• providing a tag moiety sequence, said sequence being adapted for linking to a foreign nucleic acid thereby forming an insertion sequence;
• and
• a nucleic acid comprising:
• a surface-protein gene, said gene coding for a protein which is expressed on the surface of a host organism, wherein the host organism is cultured cell;
• a promoter sequence for promoting the expression of the surface-protein gene;
• and
• an insertion site located within the surface-protein gene such that when the insertion sequence is inserted at the insertion site the tag moiety sequence is expressed at the surface of the host organism;
• cleaving the nucleic acid at the insertion site thereby forming a cleaved nucleic acid;
• linking the tag moiety sequence with a foreign nucleic acid, thereby forming the insertion sequence;
• contacting the cleaved nucleic acid with the insertion sequence under conditions sufficient to incorporate the insertion sequence into the cleaved nucleic acid, thereby forming a recombinant nucleic acid;
• inserting the recombinant nucleic acid into a host organism;
• growing the host organism for a time sufficient to express the tag moiety sequence;
• and
• contacting the host organism with a solid support having attached thereto a moiety that specifically binds to the tag moiety.

A cloning system, including nucleic acid that expresses in a host organism that is a cultured cell, useful for the isolation of recombinant nucleic acid comprising:

• a surface-expressed-moiety gene;
• a first promoter sequence for promoting the expression of the surface-expressed-moiety gene;
• a lethal gene, the expression product of the lethal gene being lethal to the host organism;
• a second promoter sequence for promoting the expression of the lethal gene;
• and
• an insertion site located within the lethal gene such that when foreign nucleic acid is inserted at the insertion site, the lethal gene is insertionally inactivated.