Methods for dynamic vector assembly of dna cloning vector plasmids

Abstract

A method for using cloning vector plasmids to produce DNA molecules, such as transgenes, in a single cloning step. The transgenes can be used for the purpose of gene expression or analysis of gene expression. The plasmid cloning vectors are engineered to minimize the amount of manipulation of DNA fragment components by the end user of the vectors and the methods for their use. Transgenes produced using the invention may be used in a single organism, or in a variety of organisms including bacteria, yeast, mice, and other eukaryotes with little or no further modification.

1 . A method for constructing a transgene, comprising the steps of:
 a. providing a cloning vector plasmid with a backbone able to accept a sequential arrangement of inserts;   b. providing at least a first insert and a second insert to be included in the transgene; and   c. transferring both the first insert and the second insert into the backbone in a single reaction.   
     
     
         2 . A method for making a transgene, comprising the steps of:
 a. providing a cloning vector plasmid comprising first and second docking points;   b. introducing first nucleotide sequences to be included in the transgene into a first shuttle vector;   c. introducing second nucleotide sequences to be included in the transgene into a second shuttle vector; and   d. transferring simultaneously the first nucleotide sequences and the second nucleotide sequences from the shuttle vectors to the cloning vector plasmid, between the first and second docking points.   
     
     
         3 . A method for making a transgene, comprising the steps of:
 a. providing a cloning vector plasmid comprising first and second docking points;   b. introducing Promoter nucleotide sequences to be included in the transgene into a Promoter shuttle vector;   c. introducing Expression nucleotide sequences to be included in the transgene into an Expression shuttle vector;   d. introducing Regulatory nucleotide sequences to be included in the transgene into a Regulatory shuttle vector; and   e. transferring simultaneously the Promoter, Expression and Regulatory nucleotide sequences from the Promoter, Expression and Regulatory shuttle vectors to the cloning vector plasmid, between the first and second docking points.   
     
     
         4 . A method for simultaneously synthesizing an array of transgenes, comprising the steps of:
 a. providing a primary cloning vector plasmid comprising a first and a second docking point;   b. introducing at least one Promoter nucleotide sequence to be included in the transgene into a corresponding Promoter shuttle vector;   c. introducing at least one Expression nucleotide sequence to be included in the transgene into a corresponding Expression shuttle vector;   d. introducing at least one Regulatory nucleotide sequence to be included in the transgene into a corresponding Regulatory shuttle vector; and   e. transferring simultaneously the Promoter, Expression and Regulatory nucleotide sequences from the Promoter, Expression and Regulatory shuttle vectors to the cloning vector plasmid, between the first and second docking points;   f. wherein at least two combinations of one Promoter module, one Expression module, and one Regulatory module are transferred into two distinct primary cloning vector molecules.   
     
     
         5 . A method for making a modular cloning vector plasmid for the synthesis of a transgene or other complicated DNA construct, the method comprising the steps of:
 a. providing the cloning vector plasmid comprising a backbone, the backbone comprising first and second docking points, each docking point being fixed within the backbone and comprising at least one non-variable rare endonuclease site for an endonuclease enzyme;   b. cleaving the first docking point with a first endonuclease enzyme corresponding to the at least one non-variable rare restriction site of the first docking point, leaving the cleaved first docking point with a 3′ end;   c. cleaving the second docking point with a second nuclease enzyme corresponding to the at least one non-variable rare endonuclease site of the second docking point, leaving the cleaved second docking point with a 5′ end;   d. providing at least a first and a second insert, each insert comprising a 5′ end, a nucleotide sequence of interest and a 3′ end, wherein the 5′ end of the first insert is compatible to the 3′ end of the cleaved first docking point, the 3′ end of the second insert is compatible to the 5′ end of the cleaved second docking point, the 3′ end of the first insert being compatible to the 5′ end of the second insert to form a third non-variable rare endonuclease site for a third endonuclease enzyme; and   e. placing the inserts and the cleaved cloning vector plasmid into an appropriate reaction mixture to cause simultaneous ligation and self-orientation of the first and second inserts between the first and second docking points within the backbone, re-forming the first and second docking points, and forming the modular cloning vector plasmid.   
     
     
         6 . The method of  claim 5  that provides for modifying the modular cloning vector plasm further comprising the steps of:
 f. subsequently removing the first insert by cleaving the modular cloning vector plasmid at the first docking point and the third non-variable rare endonuclease site with the first and the third endonuclease enzymes, leaving a 3′ end at the cleaved first docking point and a 5′ end at the cleaved third endonuclease site;   g. providing a third insert comprising a 5′ end, a nucleotide sequence of interest and a 3′ end, wherein the 5′ end of the third insert is compatible to the 3′ end of the cleaved first docking point′, and the 3′ end of the third insert is compatible to the 5′ end of the cleaved third endonuclease site; and   h. placing the third insert and the cleaved modular cloning vector plasmid into an appropriate reaction mixture to cause simultaneous ligation and self-orientation of the third insert between the first docking point and the third endonuclease site, and re-forming the first docking point and the third endonuclease site.   
     
     
         7 . The method of  claim 5  that provides for modifying the modular cloning vector plasm further comprising the steps of:
 i. subsequently removing the second insert by cleaving the modular cloning vector plasmid at the first docking point and the third non-variable rare endonuclease site with the second and the third endonuclease enzymes, leaving a 3′ end at the cleaved third endonuclease site and a 5′ end at the cleaved second docking point;   j. providing a fourth insert comprising a 5′ end, a nucleotide sequence of interest and a 3′ end, wherein the 5′ end of the fourth insert is compatible to the 3′ end of the cleaved third endonuclease site, and the 3′ end of the fourth insert is compatible to the 5′ end of the cleaved second docking point; and   k. placing the fourth insert and the cleaved modular cloning vector plasmid into an appropriate reaction mixture to cause simultaneous ligation and self-orientation of the fourth insert between the third endonuclease site and the second docking point, and re-forming the third endonuclease site and the second docking point.   
     
     
         8 . The method of  claim 5 , wherein the inserts are created by a method selected from the group consisting of de novo synthesis, recombineering, and PCR terminator over-hang cloning. 
     
     
         9 . A method for synthesizing a transgene or other complicated DNA construct, comprising the steps of:
 a. providing a primary cloning vector plasmid comprising a backbone, the backbone comprising at least a first docking point and a second docking point, each docking point being fixed within the backbone and comprising at least one rare restriction site for a non-variable rare restriction enzyme;   b. cleaving the first docking point with a first non-variable rare restriction enzyme corresponding to one of the rare restriction sites of the first docking point, leaving the cleaved backbone with a 3′ end;   c. cleaving the second docking point with a second non-variable rare restriction enzyme corresponding to one of the restriction sites of the second docking point, leaving the cleaved backbone with a 5′ end;   d. providing a Promoter insert into which a Promoter sequence of interest, a 5′ end that is compatible to the 3′ end of the first docking point, and a 3′ end;   e. providing an Expression insert comprising an Expression sequence of interest, a 5′ end that is compatible to the 3′ end of the Promoter insert to form a rare restriction site for a third non-variable rare restriction enzyme, and a 3′ end;   f. providing a Regulatory insert comprising a Regulatory sequence of interest, a 5′ end that is compatible to the 3′ end of the Expression insert to form a rare restriction site for a fourth non-variable rare restriction enzyme, and a 3′ end that is compatible to the 5′ end of the cleaved second docking point which was cleaved in step ‘c’; and   g. placing the Promoter, Expression and Regulatory inserts and the cleaved cloning vector plasmid into an appropriate reaction mixture to cause simultaneous ligation, self-orientation and sequential placement of the Promoter, Expression and Regulatory inserts between the first and second docking points, reforming the first and second docking points, and forming a modular primary cloning vector plasmid.   
     
     
         10 . The method of  claim 9  that provides for modifying the modular primary cloning vector plasmid, further comprising the steps of:
 h. cleaving the modular primary cloning vector plasmid with a pair of rare restriction enzymes at a corresponding pair of rare restriction sites of at least one of the Promoter, Expression and Regulatory inserts, leaving the cleaved vector plasmid with a 3′ end and a 5′ end;   i. providing at least one fifth insert selected from a Promoter insert, an Expression insert, and a Regulatory insert, the fifth insert having a 5′ end that is compatible to the 3′ end of the cleaved vector plasmid, and a 3′ end that is compatible to the 5′ end of the cleaved vector plasmid; and   j. placing the fifth insert and the cleaved vector plasmid into an appropriate reaction mixture to cause simultaneous ligation and self-orientation of the fifth insert into the modular primary cloning vector plasmid, in the same sequence, and re-forming the pair of rare restriction sites.   
     
     
         11 . The method of  claim 10 , further comprising the step of repeating steps h, i, and j for one or more additional inserts selected from a Promoter insert, an Expression insert, and a Regulatory insert. 
     
     
         12 . A method for simultaneously synthesizing an array of transgenes or other complicated DNA constructs, comprising the steps of:
 a. providing at least one primary cloning vector plasmid comprising a backbone into which inserts having a 5′ end, a nucleotide sequence of interest and a 3′ end can be inserted, the backbone operable to accept a sequential arrangement of Promoter, Expression, and Regulatory inserts and comprising at least a first and a second docking point, each docking point being fixed within the backbone and comprising at least one restriction site for a non-variable rare restriction enzyme;   b. cleaving the first docking point with a first non-variable rare restriction enzyme corresponding to one of the restriction sites of the first docking point;   c. cleaving the second docking point with a second non-variable rare restriction enzyme corresponding to one of the restriction sites of the second docking point;   d. providing at least one Promoter insert into which a Promoter nucleotide sequence has been inserted, the 5′ end of the at least one Promoter insert compatible to the 3′ end of the first docking point which was cleaved in step ‘b’;   e. providing at least one Expression insert into which an Expression nucleotide sequence has been inserted, the 5′ end of the at least one Expression insert being compatible to the 3′ end of the at least one Promoter insert to form a restriction site for a third non-variable rare restriction enzyme;   f. providing at least one Regulatory insert into which a Regulatory nucleotide sequence has been inserted, the 5′ end of the at least one Regulatory insert being compatible to the 3′ end of the at least one Expression insert to form a restriction site for a fourth non-variable rare restriction enzyme, the 3′ end of the at least one Regulatory insert compatible to the 5′ end of the of the second docking point which was cleaved in step ‘c’; and   g. thereafter placing at least two different types of at least one of the Promoter, Expression and Regulatory inserts, at least one of each of the remaining inserts, and the cleaved cloning vector plasmid into an appropriate reaction mixture to cause simultaneous ligation, self-orientation and sequential placement of one each of the Promoter, Expression and Regulatory inserts between the first and second docking points within the backbone, thereby creating an array of plasmids having different combinations of Promoter, Expression and Regulatory inserts within their backbone.   
     
     
         13 . The method of  claim 12 , wherein step ‘g’ comprises placing at least two different types of at least two of the Promoter, Expression and Regulatory inserts, at least one of each of the remaining inserts, and the cleaved cloning vector plasmid into an appropriate reaction mixture to cause simultaneous ligation, self-orientation and sequential placement of one each of the Promoter, Expression, and Regulatory inserts between the first and second docking points within the backbone, thereby creating an array of plasmids having different combinations of Promoter, Expression and Regulatory inserts within their backbone. 
     
     
         14 . The method of  claim 12 , wherein step ‘g’ comprises placing at least two different types of each of the Promoter, Expression and Regulatory inserts and the cleaved cloning vector plasmid into an appropriate reaction mixture to cause simultaneous ligation, self-orientation and sequential placement of one each of the Promoter, Expression, and Regulatory inserts between the first and second docking points within the backbone, thereby creating an array of plasmids having different combinations of Promoter, Expression and Regulatory inserts within their backbone. 
     
     
         15 . The method of  claims 12 ,  13 , or  14  that provides for modifying the modular primary cloning vector plasmid, further comprising the step of:
 h. assaying the products of step ‘g’ using high throughput screening;   i. isolating a specific product of step ‘g’ which is a primary cloning vector plasmid;   j. cleaving the backbone of primary cloning vector plasmid from step ‘i’ at the 5′ and 3′ ends of one of the Promoter, Expression and Regulatory inserts with the pair of rare restriction enzymes corresponding thereto;   k. providing a sixth insert, the 5′ end of the sixth insert compatible to the 3′ end of the backbone which was cleaved in step ‘j’, the 3′ end of the sixth insert compatible to the 5′ end of the backbone which was cleaved in step ‘j’; and   l. thereafter placing the sixth insert and the backbone which was cleaved in step ‘j’ into an appropriate reaction mixture to cause simultaneous ligation and self-orientation of the sixth insert into the backbone.   
     
     
         16 . The method of  claim 13 , further comprising the step of repeating steps h, i, j, k and I for any number of inserts in order to sequentially replace one insert for another insert into the backbone. 
     
     
         17 . The method of  claim 12 , wherein the inserts are created by a method selected from the group consisting of de novo synthesis, recombineering, PCR, terminator over-hang cloning technology, and restriction endonuclease mapping. 
     
     
         18 . The method of any of  claims 5 ,  9  or  12 , wherein the backbone further comprises a unique HE site in a forward orientation located upstream from the 5′ end of the first docking point and a unique HE site in a reverse orientation located downstream from the 3′ end of the second docking point, the method further comprising the steps of:
 m. cleaving the backbone at each of the unique HE sites with a unique HE restriction enzyme;   n. purifying the cleaved portion containing the inserts; and   o. inserting the cleaved portion into a genome host of interest.