# Patent

## Title
Polypeptides that include conformation-constraining groups which flank a

## Abstract

This is a Continuation In Part of application Ser. No. 08/511,486, filed Aug. 7, 1995.

## Background
This invention relates to the identification and selection of genetically transformed cells. More specifically, this invention describes a vector-host cloning system in which genetic transformation with foreign nucleic acid is accompanied by a measurable change in surface binding properties of a host organism.

The ability to selectively recombine genetic material from different organisms in vitro and to cause the resulting "recombinant" material to be replicated and/or used to direct the expression of proteins in a host organism, has transformed the study of biology and greatly enhanced its practical utility, e.g., Cohen et al., U.S. Pat. No. 4,237,224. Applications of this "cloning" or "recombinant nucleic acid" technology include the expression of medically useful human proteins in bacterial cell culture, the creation of immortalized genetic "libraries" containing the entire genome of a particular organism, and the segregation and replication of foreign nucleic acid in preparation for nucleic acid sequencing.

Generally, the cloning process includes the following steps: (i) foreign nucleic acid fragments are prepared having the appropriate size and cohesive end regions, the foreign nucleic acid being either a digest of genomic nucleic acid or cDNA; (ii) a vector nucleic acid is cleaved with one or more restriction enzymes, preferably at a unique site, to form a vector nucleic acid having cohesive end regions suitable for binding to the cohesive end regions of the foreign nucleic acid fragments, where the vector nucleic acid includes (a) elements which allow the inserted foreign nucleic acid fragments to survive and autonomously replicate in a host organism and (b) a selectable marker in order to facilitate the recognition and isolation of cells carrying the foreign nucleic acid; (iii) the foreign nucleic acid fragments are contacted with the cleaved vector nucleic acid under conditions favoring combination of the foreign nucleic acid fragments and the vector nucleic acid, and the vector and foreign nucleic acid are ligated together, thereby forming a recombinant vector-foreign nucleic acid; (iv) the recombinant vector-foreign nucleic acid is introduced into the host organism, thereby genetically transforming the host organism; (v) the host organisms containing the recombinant vector-foreign nucleic acid are separated from untransformed cells and cells containing only parent vector nucleic acid, and optionally, (vi) the separated cells are subjected to a secondary screen for specific nucleic acid inserts, e.g., by contacting with nucleic acid probes or by contacting their expressed proteins with antibody probes.

A key step in the above cloning process is the identification and isolation of host organisms which have been successfully transformed with recombinant vector-foreign nucleic acid, i.e., step (v) above. A number of products can result from the reaction of the cleaved vector nucleic acid with the foreign nucleic acid fragments. The products of the recombination reaction are a heterogeneous mixture of recombinant vector-foreign nucleic acid molecules together with religated parental vector molecules. In fact, for many systems, the vast majority of cells are transformed with parental vector nucleic acid only, e.g., less than 0.1% of the host organisms are transformed with vector-foreign nucleic acid. This low frequency of vector-foreign nucleic acid transformation creates a large background of cells containing no foreign nucleic acid, thereby dramatically increasing the effort required to perform any secondary screening. The automation of step (v) is a particularly important issue for large-scale cloning projects where millions of potential clones must be screened, e.g., the construction of libraries for genomic sequencing projects.

There are four methods that are commonly used to identify host organisms that contain recombinant vector-foreign nucleic acid, including (i) restriction analysis of small-scale preparations of plasmid nucleic acid, (ii) .alpha.-complementation, (iii) insertional inactivation, and, (iv) screening by hybridization (see Molecular Cloning 2nd Ed., Sambrook et al, Chapter 1, Cold Spring Harbor Laboratory Press (1989)).

In the restriction analysis method, a number of independently transformed host organisms are picked from a parent culture and grown up in small-scale cultures. Plasmid nucleic acids are isolated from each culture and then analyzed by digestion with restriction enzymes followed by gel electrophoresis.

In the .alpha.-complementation method, the cloning vectors carry a short segment of Escherichia coli nucleic acid that contains the regulatory sequences and the coding information for the first 146 amino acids of the .beta.-galactosidase gene (lacZ). Embedded in this coding region is a polycloning site. These vectors are used in combination with host organisms that code for the carboxy-terminal portion of .beta.-galactosidase. While neither the host-encoded nor the vector-encoded fragments are themselves active, they can associate to form an enzymatically active protein. This type of complementation is called .alpha.-complementation. The Lac+ bacteria that result from .alpha.-complementation are easily recognized because they form blue colonies in the presence of the chromogenic substrate X-gal. However, insertion of a fragment of foreign nucleic acid into the polycloning site of the vector results in the production of an amino-terminal fragment that is not capable of .alpha.-complementation. Host organisms carrying recombinant vectors therefore form white colonies. The recombinant vectors can then be visually identified and manually "picked" from the culture.

The insertional inactivation method is used with vectors that carry two or more antibiotic resistance genes and an appropriate distribution of restriction enzyme cleavage sites. The foreign nucleic acid and the vector nucleic acid are digested with restriction enzymes that recognize sites located in only a first antibiotic resistance gene. After ligating the two nucleic acids, the ligation mixture is used to transform E. coli. Transformants are selected that are resistant to the second antibiotic. Some of the colonies that grow in the presence of the second antibiotic will contain recombinant vectors; others will contain parental vector nucleic acid that has religated during ligation without insertion of foreign nucleic acid. To discriminate between the two types of transformants, separate plates containing the first or second antibiotic are inoculated with a number of colonies in patches in identical locations. The colonies that grow in the presence of the first antibiotic contain plasmids with active resistance genes, and it is unlikely that such plasmids contain insertions of foreign nucleic acid. It is likely that the colonies that do not grow in the presence of the first antibiotic but do grow in the presence of the second antibiotic carry the foreign nucleic acid sequences.

In hybridization screening techniques, a replica of the host organism colonies is transferred to a filter support, the cells are lysed releasing their nucleic acid, and the nucleic acid is then probed with labeled sequence-specific nucleic acid probes. The location of probe-positive colonies is then used to identify the location of recombinant cells on the original culture plate.

The above methods for identifying cells which have been transformed with recombinant vectors each require a number of cumbersome steps resulting in significant barriers to intelligent automation. Even the most streamlined .alpha.-complementation method requires the analyst to (i) grow-up the putatively transformed cells in a culture plate, (ii) visually identify cells having a specified color, (iii) pick cells having a specified color, and (iv) transfer the picked cells to a secondary location. While automated systems for picking individual colonies have been demonstrated, they require complicated robotic and image processing apparatus, making them unsuitable for cost effective, routine application, e.g., Uber et al., Biotechniques 11: 642-648 (1991); Jones et al., Nucleic Acids Research 20: 4599-4606 (1992). Moreover, because the .alpha.-complementation approach requires cells to be plated out, it is not amenable to the selection of cells which can not be grown on culture plates, e.g., certain eucaryotic tissue cells.

The present invention is directed toward our discovery of a cloning system which facilitates the isolation of recombinant nucleic acid. Our system links the recombination event to the expression of a moiety at the surface of a host organism such that when recombination occurs, the moiety is expressed on the surface of the host, and in the absence of recombination, the moiety is not expressed on the surface of the host. Moreover, this differentially-expressed moiety is a member of a binding pair, the other member of which is attached to a solid support. Thus, when the moiety is expressed on the surface of the host organism, the host organism can be captured by binding to the solid support. The invention includes methods, apparatus, and compositions for using the cloning system.

An object of our invention is to provide a cloning system which is well suited for practical, low-cost automation of the recombinant selection process.

A further object of our invention is to provide a cloning system which does not traditionally require growing host organisms on a culture plate to identify and isolate cells which have been transformed with recombinant nucleic acid.

An additional object of our invention is to provide a cloning system which does not require visual identification of host organisms which have been transformed with recombinant nucleic acid.

Another object of our invention is to provide a cloning system which does not require mechanically picking selected cells from a culture plate.

Yet another object of our invention is to provide a cloning system which results in the placement of recombinant-transformed organisms at discrete, pre-defined locations on a solid support, each location containing only a single recombinant organism.

In a first aspect, the foregoing and other objects of our invention are achieved by a cloning system referred to herein as the "trans cloning system", such system including a vector nucleic acid for expression in a host cell. The vector nucleic acid of the trans cloning system includes a repressor gene coding for a repressor and a first promoter sequence for promoting the expression of the repressor gene. The repressor gene contains an insertion site located such that when a foreign nucleic acid is inserted at the insertion site, expression of the repressor gene is insertionally inactivated. The vector further includes a surface-expressed moiety gene for directing the expression of a surface expressed moiety. An operator is functionally linked to the expression of the surface-expressed-moiety gene such that when the repressor is bound to the operator, expression of the surface-expressed-moiety gene is repressed.

One embodiment of the first aspect of the present invention includes a method for cloning and isolating recombinant nucleic acids using the above-described trans cloning system. In the method, the vector is cleaved at the insertion site forming a cleaved vector nucleic acid, and the cleaved vector is contacted with a foreign nucleic acid under conditions sufficient to incorporate the foreign nucleic acid into the cleaved vector nucleic acid, thereby forming a recombinant nucleic acid. The recombinant nucleic acid is inserted into a host organism and the host organism is grown up for a time sufficient to express the surface-expressed-moiety gene. The host organism is then contacted with a solid support having attached thereto a binding moiety capable of specifically binding to the surface-expressed moiety.

In a second aspect referred to herein as the "cis cloning system", the present invention provides a nucleic acid capable of expression in a host organism useful for the isolation of recombinant nucleic acid which includes a surface-expressed-moiety gene along with a promoter sequence for promoting the expression of the surface-expressed-moiety gene. The nucleic acid of the invention further includes a first insertion site located downstream from the promoter sequence and upstream from the surface-expressed-moiety gene and a second insertion site located between the first insertion site and the surface-expressed-moiety gene. Finally, the nucleic acid of the invention includes an expression blocking sequence, located between the first insertion site and the second insertion site, which serves to block the expression of nucleic acid sequence located downstream from the expression blocking sequence, e.g., the SEM gene.

In one preferred embodiment, the present invention provides a method for cloning and isolating recombinant nucleic acid using the above described nucleic acid. In the method, the nucleic acid is cleaved at the first insertion site and at the second insertion site thereby forming a cleaved nucleic acid. Next, the cleaved nucleic acid is mixed 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. The recombinant nucleic acid is then inserted into a host organism and the host organism is grown up for a time sufficient to express the surface expressed moiety gene. Finally, the host organism is contacted with a solid support having attached thereto a moiety capable of specifically binding to the surface expressed moiety.

In yet another aspect referred to herein as the "tag cloning system", the present invention provides a cloning system useful for the isolation of recombinant nucleic acid which includes a tag moiety sequence which is adapted for linking to a foreign nucleic acid thereby forming an insertion sequence. In addition to the tag moiety sequence, the system includes a nucleic acid having a surface-protein gene coding for a protein which is expressed on the surface of a host organism. The nucleic acid further includes 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.

One embodiment of the present invention includes a method for cloning and isolating recombinant nucleic acid using the above-described tag cloning system. In this method, the nucleic acid of the invention is cleaved at the insertion site thereby forming a cleaved nucleic acid. The tag moiety sequence is then linked to a foreign nucleic acid forming an insertion sequence. Next, the insertion sequence is contacted with the cleaved nucleic acid under conditions sufficient to incorporate the insertion sequence into the cleaved nucleic acid, thereby forming a recombinant nucleic acid. The recombinant nucleic acid is then inserted into a host organism and the host organism is grown up for a time sufficient to express the tag moiety sequence. Finally, the host organism is contacted with a solid support having attached thereto a moiety capable of specifically binding to the tag moiety.

In yet another aspect, referred to herein as the "lethal gene inactivation system", the present invention provides a nucleic acid capable of expression in a host organism useful for the isolation of recombinant nucleic acid. The nucleic acid of the invention includes a surface-expressed-moiety gene along with a first promoter sequence for promoting the expression of the surface-expressed-moiety gene. The nucleic acid of the invention further includes a lethal gene whose expressed protein is lethal to the host organism and a second promoter sequence for promoting the expression of the lethal gene. Finally, the nucleic acid includes an insertion site located within the lethal gene such that when foreign nucleic is inserted at the insertion site the lethal gene is insertionally inactivated.

In one preferred embodiment, the invention includes a method for cloning and isolating recombinant nucleic acid using the lethal gene inactivation system. In this method, the nucleic acid of the invention is cleaved at the insertion site. The cleaved nucleic acid is then contacted 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. The recombinant nucleic acid is then inserted into a host organism and the host organism is grown up for a time sufficient to express the lethal gene and the SEM gene. Finally, the host organism is contacted with a solid support having attached thereto a moiety capable of specifically binding to the SEM.

In yet another aspect, the present invention provides a host organism capture system. The system includes a solid support having a plurality of binding moieties attached thereto. The binding moieties are members of binding pairs, the complementary member of the binding pairs being differentially expressed on the surface of a host organism using any one of the above-described cloning systems. Preferably the binding moieties are located in a plurality of discrete anchor sites, each anchor site having dimensions such that only a single host organism can bind to a single anchor site.

These and other objects, features, and advantages of the present invention will become better understood with reference to the following description, drawings, and appended claims.

FIG. 1 shows a schematic diagram of the trans cloning system of the present invention.

FIG. 2 shows a schematic diagram of the of cis cloning system of the present invention.

FIG. 3 shows a schematic diagram of the tag cloning system of the present invention.

FIG. 4 shows a schematic diagram of the lethal gene inactivation system of the present invention.

FIG. 5 shows a proposed E. coli K-12 host strain useful in the trans cloning system of the invention.

FIG. 6 shows a proposed plasmid for the trans cloning system of the invention.

Generally, the present invention is directed toward our discovery of cloning systems useful for the isolation of recombinant nucleic acid. These systems link the recombination of vector nucleic acid and foreign nucleic acid to the expression of a moiety which is located at the surface of a host organism, the moiety being a first member of a binding pair. When recombination occurs between vector nucleic acid and foreign nucleic acid, the moiety is expressed on the surface of the host organism. Alternatively, when no recombination occurs, the moiety is not expressed on the surface of the host organism. The isolation of the recombinant nucleic acid is then performed by attaching a second member of the binding pair to a solid support and contacting the host organism with the support. When the first member of the binding pair is expressed on the surface of the host organism, the host organism binds to the second member of the binding pair attached to the solid support, thereby selectively removing those organisms from the host-organism suspension. In this way host organisms containing recombinant nucleic acid can be physically isolated from host organisms which lack such nucleic acid.

In a first aspect of the present invention, hereinafter referred to as the "trans cloning system", the recombination of vector nucleic acid and foreign nucleic acid is linked to the expression of a surface-expressed moiety through the disruption of a repressor gene. When recombination has occurred, the repressor gene is insertionally inactivated, thereby allowing the expression of a SEM gene which is functionally linked to an operator which binds the repressor. Therefore, in the absence of recombination, a functional repressor is expressed, leading to the repression of the SEM gene. As used herein, "repressed" means that the level of expression is reduced by at least a factor of three relative to the level of expression observed in the absence of repression.

In the preferred embodiment shown in FIG. 1, the vector nucleic acid includes (i) a repressor gene; (ii) an insertion site located within the repressor gene or its associated promoter such that when a foreign nucleic acid is inserted at the insertion site, the repressor gene is insertionally inactivated; (iii) a promoter functionally linked to the repressor gene for promoting expression of the repressor gene; (iv) a SEM gene for directing the expression of a surface-expressed moiety; and (v) an operator functionally linked to the SEM gene such that when a repressor is bound to the operator, expression of the SEM gene is repressed.

As used herein, the term "insertion site" refers to a location on a double or single stranded nucleic acid at which the nucleic acid may be cleaved in a sequence-specific manner, using a restriction endonuclease or any other like sequence-specific cleaving means, such that properly treated foreign nucleic acid may be ligated therein, e.g., a restriction site. As used herein, the term "ligate" refers to the joining of two strands of nucleic acid by either enzymatic means, e.g., using T4 nucleic acid ligase, E. coli nucleic acid ligase, or other like nucleic acid ligases, e.g., Current Protocols in Molecular Biology, Ausubel et al. eds., Current Protocols (1993), or using chemical means, e.g., Herrlein et al., Nucleic Acids Research, 22: 5076-78 (1994), and Shabarova et al., Nucleic Acids Research, 19: 4247-51 (1991).

In the present invention, the purpose of the vector nucleic acid is to (i) provide a means for the transport of foreign nucleic acid into a host organism, (ii) provide for the maintenance and/or replication of the foreign nucleic acid in the host organism, and (iii) provide a linkage between genetic recombination and the expression of the SEM molecule at the surface of the host organism. Preferred vectors may be based on plasmids, cosmids, viruses, or retroviruses, e.g., M13mp, bacteriophage .lambda., cos 202, cos 203, bovine papilloma virus, Epstein-Barr virus, or any other like vectors, e.g., Molecular Cloning Second Edition, Sambrook et al., eds., Cold Spring Harbor Laboratory Press (1989). The vector nucleic acid may be either linear or circular in form. However, preferably the vector nucleic acid is a circular plasmid.

As used herein, the term "repressor" refers to molecules which act to inhibit the expression of a target structural gene by binding to a specific operator functionally linked to the target structural gene, and the term "repressor gene" refers to a gene which codes for the repressor molecule. The term "operator" as used herein refers to a specific site on a nucleic acid at which a repressor specifically binds, such specific binding blocking the expression of an associated structural gene. Typically, repressors act by preventing the transcription of a structural gene into mRNA by blocking the attachment of RNA polymerase to the nucleic acid. Therefore, when repressor molecules are not present, RNA polymerase can bind to nucleic acid and initiate transcription, and, when repressor molecules are present, the RNA polymerase is prevented from binding to the nucleic acid, and transcription, and therefore expression, is effectively blocked.

The repressor gene may be either constitutively expressed or inducibly expressed. Preferably, the repressor gene is constitutively expressed. More preferably, the repressor gene is overexpressed, where, as used herein, the term "overexpressed" refers to a level of expression which is greater than normal physiological levels. Such constitutive overexpression provides tight control of the expression of the structural gene with which the repressor molecule is associated.

Preferred repressor-operator systems include the lac operon, the gal operon, the alpha2-ste6 system, the cI repressor of the .lambda..sub.PL promoter, or any other like repressor-operator system, e.g., Maximizing Gene Expression, Reznikoff et al., Butterworths (1986).

In an important feature of the present invention, an insertion site is located within the repressor gene, where, as used herein, the term "insertion site" refers to a location on a nucleic acid at which the nucleic acid can be cleaved at a sequence-specific location using a sequence-specific cleaving agent, e.g., a restriction enzyme, or chemical cleaving agents, e.g., Dervan, Nature 359: 87-88, and foreign nucleic acid can be inserted. The location of the insertion site within the repressor gene is such that when a foreign nucleic acid is inserted at the site, the expression of the repressor gene is effectively inactivated, i.e., the repressor gene is insertionally inactivated. Typically, an insertion site is a nucleic acid sequence at which one or more restriction enzymes specifically cut the nucleic acid, the ends of the cuts being such that properly treated foreign nucleic acid can be ligated into the insertion site. Preferably, the insertion site may be cut by a plurality of different restriction enzymes or other sequence-specific cutting agents, thereby providing greater flexibility as to the types of foreign nucleic acids which can be accommodated. Numerous such restriction enzymes are available from commercial sources, each enzyme recognizing a different nucleic acid sequence, e.g., Stratagene Cloning Systems, La Jolla Calif., 1994 Catalog. Preferably, the restriction site defining the insertion site occurs only once in the vector nucleic acid so that the insertion site is uniquely defined in the vector, e.g., cleaving agents which recognize sequences of at least six bases in length.

In one preferred embodiment of the present invention, the vector nucleic acid includes a promoter for promoting the expression or overexpression of the repressor gene. The promoter may be either constitutive, e.g., tet promoter, bla promoter, or any other like constitutive promoter, or regulatory, e.g., lac, .lambda..sub.PL, Tac, or any other like regulatory promoter. The promoter is located so as to be functionally linked to the expression of the repressor gene. Preferably, the promoter is regulatory.

Preferably, the vector nucleic acid of the present invention further includes a forward selectable marker for selecting between transformed and untransformed host organisms. Preferred forward selectable markers include genes conferring antibiotic resistance. Thus, when a population of host organisms is challenged with a selection agent, e.g., an antibiotic, only those organisms which have been transformed with the vector nucleic acid will survive. Exemplary forward selectable markers include the amp.sup.R gene.

The vector nucleic acid of the trans cloning system further includes a SEM gene which codes for a moiety that is expressed in the host, then transported to the surface of the host such that it is presented to the exterior surface of the host organism. Moreover, the SEM is a member of a binding pair, where, as used herein, the term "binding pair" refers to a pair of molecular entities, e.g., binding moieties, which specifically and strongly bind to one another under suitable conditions, one member of the binding pair being differentially expressed on the surface of a host organism, and the other member of the binding pair being attached to a solid support. Exemplary binding pairs include ligand/receptor pairs, antigen/antibody pairs, the biotin/avidin pair, and the like. A more thorough discussion of the properties of preferred SEM molecules and preferred binding pairs will be presented in a following section.

The vector also includes an operator associated with the SEM gene which serves to regulate the expression of the SEM gene. Moreover, the operator is functionally linked to the expression of the SEM gene such that when the repressor is bound to the operator, expression of the SEM gene is repressed. Preferably the operator is located upstream from the SEM gene. As used herein the term "upstream" refers to a direction towards the 5' end of a sense strand of a double-stranded nucleic acid, and the term "downstream" refers to a direction towards the 3' end of a sense strand of a double-stranded nucleic acid. Preferred operator/repressor pairs have been discussed above.

The host organism may be eucaryotic, viral, bacterial, or plant. The host organism must be (i) capable of being transformed with the vector nucleic acid and (ii) provide a suitable environment for the maintenance and/or replication of the vector nucleic acid and the host nucleic acid. Exemplary host organisms include COS cells, CHO cells, E. coli, or any other suitable host organism. Preferably, the host organism is a bacteria, e.g., E. coli.

One particularly preferred host/vector trans cloning system is based on a modified host E. coli K-12 strain. In this strain, the regulatory fimE and fimB genes, and the fimH gene are deleted causing the resulting host strain to be non-fimbriated and "locked on" for type 1 fimbriae when complemented in trans with a wild-type fimH gene (FIG. 13). The preferred minimal elements of a cloning plasmid in this preferred system include an antibiotic resistance marker, a pUC replicon for high-copy number, an M13 origin of replication, the fimH gene cloned behind the lambda left promoter, and the cI repressor gene of lambda FIG. 14). When the above E. coli host strain is transformed with this plasmid, the resulting tranformant will still be non-fimbriated because the cI protein will repress the fimH gene, resulting in lack of complementation. When foreign DNA is cloned into the cI gene, the cI gene will be insertionally inactivated, relieving the repression of the fimH gene and consequently complementing the fimH defect in the chromosome. The resulting host will become fimbriated and hence capable of capture on an R-NAse B-functionalized substrate. A plasmid cloning system based upon insertional inactivation of the lambda cI857 repressor gene has been previously described, e.g., P. J. Solenberg and S. G. Burgett, J. Bacteriol. 171: 4807-4813 (1989).

Methods utilizing the trans cloning system utilize cloning techniques well known in the art of molecular biology. First, the vector nucleic acid is cleaved at the insertion site using an appropriate restriction endonuclease under conditions sufficient to render the restriction enzyme active. The cleaved vector nucleic acid is contacted with a foreign nucleic acid under conditions sufficient to incorporate the foreign nucleic acid into the vector nucleic acid at the insertion site, e.g., by enzymatic ligation, thereby forming a recombinant vector nucleic acid. The recombinant vector nucleic acid is then inserted into the host organism by either transfection, transformation, or conjugation, the particular mode of insertion being dependent on the nature of the recombinant vector nucleic acid and the host organism. The host organism is then grown up to provide an opportunity for the expression of the surface-expressed moiety. Finally, the host organism is contacted with a solid support which has been functionalized with a moiety capable of specifically binding to the SEM (see below). If a particular host organism contains a recombinant vector nucleic acid, the host organism will express the SEM at its outer surface and will therefore bind to the functionalized solid support.

In a second aspect of the present invention, hereinafter referred to as the "cis cloning system", the recombination of vector nucleic acid and foreign nucleic acid is linked to the expression of a SEM by linking the recombination event with the removal of a segment of vector nucleic acid which blocks the expression of the SEM gene.

As shown in FIG. 2, the preferred vector nucleic acid of the cis cloning system includes five elements: (i) a promoter sequence, (ii) a first insertion site, (iii) a second insertion site, (iv) an expression blocking sequence, and (v) a SEM gene. Preferably, the vector is a circular nucleic acid.

The purpose of the promoter sequence is to promote the expression of downstream nucleic acid sequence, particularly the SEM gene. Preferred promoter sequences have been previously described herein.

Preferably, the first and second insertion sites are chosen such that different restriction enzymes cleave each site, and that each restriction site occurs only once in the vector nucleic acid. By making each of the two restriction sites different from the other, the opportunity for religation of the parent vector after restriction without incorporation of the foreign nucleic acid is reduced, and, CDNA fragments, for example, may be directionally inserted.

In a significant feature of the present invention, the nucleic acid sequence located between the first and second insertion sites forms an expression blocking sequence (EBS), where as used herein, the term "expression blocking sequence" refers to a nucleic acid sequence which serves to block the expression of downstream nucleic acid, particularly the SEM gene. The EBS is located between the first and second insertion sites forming a removable "EBS cassette".

One preferred embodiment of the EBS acts by blocking the translation of mRNA transcribed from the EBS and associated downstream transcript by ribosomes. Such an EBS is referred to herein as a "translation blocking sequence". A preferred translation blocking sequence includes a sequence which, when transcribed into mRNA, forms a hairpin or other like self-associated structure in the mRNA molecule, e.g., tetra loops, stem loops, pseudo knots, and the like. By forming such a self-associated structure, the efficiency of translation of downstream messenger nucleic acid sequence is greatly reduced.

A second preferred embodiment of the EBS acts by reducing the efficiency of transcription of nucleic acid sequence into mRNA, such a sequence being referred to herein as a "transcription blocking sequence." Rather than disrupting the ribosome-mRNA interaction, the transcription blocking sequence interferes with the RNA-DNA-polymerase interaction. Many such transcription blocking sequences are well known, e.g., stem loops. Preferably, the EBS contains multiple transcription blocking sequences arranged serially.

A third preferred embodiment of the EBS utilizes both a translation blocking sequence and a transcription blocking sequence to block the expression of downstream nucleic acid.

The SEM gene in the cis cloning system is located downstream from the EBS and is physically linked to the EBS such that when the EBS is present, expression of the SEM gene is blocked. Otherwise, the SEM gene performs essentially the same function and has the same properties as the SEM gene described above with reference to the trans cloning system.

The host organism in the cis cloning system serves to provide an environment suitable for maintenance and/or replication of the vector nucleic acid.

Methods employing the cis cloning system utilize cloning techniques well known in the art of molecular biology. First, the vector nucleic acid is cleaved at the first and second insertion sites using an appropriate cleaving agent or combination of cleaving agents under reaction conditions sufficient to render all the cleaving agents active. In the event that the reaction conditions of the two agents are not compatible, two separate reactions may be performed serially. This double cleavage reaction serves to remove the EBS cassette.

The cleaved vector nucleic acid is then contacted with a foreign nucleic acid under conditions sufficient to incorporate the foreign nucleic acid into the vector nucleic acid between the first and second insertion sites, e.g., by enzymatic ligation, thereby forming a recombinant vector nucleic acid. The recombinant vector nucleic acid is then inserted into the host organism by either transfection, transformation, or conjugation, the particular mode of insertion being dependent on the nature of the recombinant vector nucleic acid and the host organism. The host organism is then grown up to provide an opportunity for the expression of the surface-expressed moiety. Finally, the host organism is contacted with a solid support which has been functionalized with a moiety capable of specifically binding to the SEM (see below). If a particular host organism contains a recombinant vector nucleic acid, the host organism will express a SEM at its outer surface and will therefore bind to the functionalized solid support.

In yet a third aspect of the present invention, hereinafter referred to as the "tag insertion cloning system", the recombination of vector nucleic acid and foreign nucleic acid is linked to the expression of a tag moiety (TM) on the surface of the host organism by linking the recombination event with the insertion of a tag moiety sequence into a gene for a protein which is normally expressed on the surface of the host organism. By inserting the TM sequence into the surface-protein gene, the product of the tag moiety sequence is presented to the outside surface of the host organism.

As shown in FIG. 3, the tag cloning system includes (i) a vector nucleic acid, the vector nucleic acid including (a) a surface-protein gene, (b) an insertion site located within the surface-protein gene, and (c) a promoter functionally linked to the expression of the surface-protein gene; and (ii) an insertion sequence, the insertion sequence including a tag moiety sequence physically linked to a foreign nucleic acid.

The insertion sequence of the present invention is formed by physically linking a foreign nucleic acid sequence with a tag moiety sequence, e.g., by ligation. The foreign nucleic acid and the tag moiety may be linked by any well known ligation means, including chemical ligation or enzymatic ligation.

The surface-protein gene of the present invention codes for a protein that is expressed on the surface of the host organism, i.e., some portion of the surface protein is presented to the environment outside the host organism. Exemplary preferred surface proteins include epidermal growth factor, epidermal growth factor receptor, mating factor, and mating factor receptor. When E. coli is the host organism, preferred surface proteins include maltoporin (Lam B), K88ac and K88ad pilin proteins, TraT lipoprotein, PhoE and OmpA outer membrane proteins, and the OmpA-lipoprotein fusion protein (Lpp-OpmA), e.g., Georgiou et al., Trends Biotechnol., 11: 6-10 (1993).

The tag moiety sequence may be any sequence which codes for a moiety which is a member of a binding pair, e.g., an antigen, an antibody, a receptor ligand and the like, and which can be inserted into the surface-protein gene without disrupting the expression or localization of the surface protein. Alternatively, the tag moiety sequence may be a sequence which, when inserted into the surface protein gene, causes a change in the expression product of the surface protein gene which results in a change in the binding properties of the host organism, e.g., by altering the reading frame of the surface protein gene.

In an important feature of the present invention, the surface-protein gene includes an internal insertion site for inserting the insertion sequence. Preferably, the insertion site is unique within the vector nucleic acid. When the insertion sequence is inserted at the insertion site, the tag-moiety is expressed at the surface of the host organism. Preferably, the insertion site is located in a part of the surface-protein gene which is expected to be located at the outer surface of the host organism when expressed.

Preferably, the vector nucleic acid further includes a promoter sequence functionally linked to the surface-protein gene for promoting the expression of the surface-protein gene.

Methods utilizing the tag cloning system utilize traditional cloning techniques. First, the vector nucleic acid is cleaved at the insertion site using an appropriate sequence-specific cleaving agent, e.g., restriction endonucleases, under conditions sufficient to render the cleaving agent active. Next, the foreign nucleic acid is ligated to the tag moiety sequence, thereby forming an insertion sequence. The cleaved vector nucleic acid is then contacted with the insertion sequence under conditions sufficient to incorporate the insertion sequence into the vector nucleic acid at the insertion site, e.g., by enzymatic or chemical ligation, thereby forming a recombinant vector nucleic acid. The recombinant vector nucleic acid is then inserted into the host organism by either transfection, transformation, or conjugation, the particular mode of insertion being dependent on the nature of the recombinant vector nucleic acid and the host organism. The host organism is then grown up to provide an opportunity for the expression of the surface-protein gene/tag sequence. Finally, the host organism is contacted with a solid support which has been functionalized with a moiety capable of specifically binding to the tag moiety. If a particular host organism contains a recombinant vector nucleic acid, the host organism will express the tag moiety at its outer surface and will therefore specifically bind to the functionalized solid support.

In a fourth aspect of the present invention, hereinafter referred to as the "lethal gene inactivation system", the recombination of vector nucleic acid and foreign nucleic acid is linked to the insertional inactivation of a lethal gene in the vector. Note that in this system, recombination is indirectly linked to a SEM or tag molecule through the survival of the host organism. Thus, if the recombination event occurs, the host organism survives, the SEM is expressed, and the surviving host organisms can be isolated using a properly functionalized support as described elsewhere.

As shown in FIG. 4, the lethal gene inactivation system includes a nucleic acid which includes a lethal gene, the expression product of the lethal gene being lethal to a host organism. The nucleic acid of the present system further includes a promoter sequence functionally linked to the lethal gene and an insertion site located within the lethal gene. The insertion site is located such that when foreign nucleic acid is inserted at the insertion site, the lethal gene is insertionally inactivated. Also included in the vector nucleic acid is a surface-expressed-moiety gene, the purpose of which is as described above. In an alternative preferred embodiment, the SEM gene is located on a host nucleic acid.

Preferred lethal genes for E. coli include the active cytotoxic ccdB gene under the control of the lacP promoter, e.g., Bernard et al., Gene, 148: 71-74 (1994).

Methods employing the lethal gene inactivation cloning system utilize cloning techniques well known in the art of molecular biology. First, the nucleic acid of the invention is cleaved at the insertion site using an appropriate cleaving agent or combination of cleaving agents under reaction conditions sufficient to render all the cleaving agents active. The cleaved nucleic acid is then contacted with a foreign nucleic acid under conditions sufficient to incorporate the foreign nucleic acid into the nucleic acid at the insertion site, e.g., by enzymatic ligation, thereby forming a recombinant nucleic acid. The recombinant nucleic acid is then inserted into the host organism by either transfection, transformation, or conjugation, the particular mode of insertion being dependent on the nature of the recombinant vector nucleic acid and the host organism. The host organism is then grown up to provide an opportunity for the expression of the lethal gene and the surface-expressed moiety. Finally, the host organism is contacted with a solid support which has been functionalized with a moiety capable of specifically binding to the SEM (see below). If a particular host organism contains a recombinant nucleic acid, the host organism will survive to express a SEM at its outer surface and will therefore bind to the functionalized solid support.

The host capture system of the present invention provides a means for isolating a host organism which has been transformed with recombinant nucleic acid using any of the cloning systems described above. Generally, the host capture includes a plurality of binding moieties which are attached to a solid support. The binding moieties are members of a binding pair the complementary member of which is differentially expressed on the surface of a host organism, e.g., a SEM, or tag moiety.

Preferably, the binding pair should have an equilibrium dissociation constant (K.sub.d) which is in the micromolar or submicromolar range, where, as used herein, the term equilibrium dissociation constant refers to the ratio of unbound to bound binding moieties at equilibrium expressed in concentration units. A dissociation constant in this range facilitates the efficient capture of the recombinant host organisms and provides the requisite selectivity between specifically bound and non-specifically bound host organisms. More preferably, at least one member of the binding pair includes a reactive functional group enabling it to be covalently immobilized to a solid substrate, or to a linker attached to a solid substrate.

Preferably, the binding moiety that is differentially expressed at the surface of the host is chosen such that overexpression of that moiety in the host organism is possible without compromising the viability of the host organism. Such overexpression provides a multiplicity of possible binding sites for binding to the functionalized solid support.

The choice of a particular binding pair is a strong function of the host organism being used. Such binding pairs may be chosen generally among ligand/receptor pairs, antigen/antibody pairs, biotin/avidin pairs, metal/chelator pairs, and any other like binding pairs.

One preferred binding pair useful in cases where E. coli is used as the host organism is based on the outer membrane receptor protein FhuA. The FhuA protein binds specifically to a ferrichrome class of siderophores, where as used herein, the term "siderophore" refers to a low-molecular weight iron(III) transport agent produced and utilized by many aerobic microorganisms, e.g., H. Nikaido, Trends Microbiol., 1: 5-7 (1993), and V. Braun, Trends Biochem. Sci., 10: 75-78 (1985). A preferred ferrichrome class siderophore is ferricrocin, e.g., M. Llinas and J. B. Neilands, Bioinorg. Chem., 2: 159-165 (1972).

A particularly preferred binding pair useful in E. coli-based systems relies on the interaction of type 1 fimbrae of E. coli and bovine ribonuclease B (RNAse B). Most E. coli K-12 strains contain ca. 100-300 type 1 fimbriae at the cell surface, each fimbria having a width of ca. 7 nm and a length of 0.2 to 2 .mu.m, arranged peritrichously around the cell. These fimbriae are adhesive organelles important for successful bacterial recognition and colonization of specific host tissues. A single fimbria consists of ca. 1,000 repeating subunits of mostly a single polypeptide (FimA) having a molecular mass of ca. 17 kilodaltons (kDa). The minor subunit (FimH) mediates specific binding to D-mannosyl residues e.g., Klemm, P., and Krogfelt, K. A. in P. Klemm (ed.), Fimbriae, Adhesion, Genetics, Biogenesis and Vaccines, p 9-26, CRC Press, Boca Raton, Fla. (1994). The fact that type 1 fimbriae exhibit high affinity for D-mannosyl residues suggested the use of bovine ribonuclease B as the complementary member of the binding pair. Bovine ribonclease B is a glycoprotein with a molecular mass of 15.5 kDa which consists of 124 amino acids with a unique glycosylation site including five glycoforms with oligosaccharides of five to nine mannose residues, e.g., D. Fu, L. Chen, and R. A. O'Neill Carbohydr. Res. 261: 173-186 (1994). The well known ability of RNAse B to "stick" to hydrophobic substrates such as glass and polystyrene suggested that suitably patterned substrates with adsorbed RNAse B could be used to capture single E. coil cells (1.times.3 .mu.m).

Additional preferred binding pairs useful in systems using E. coli as the host organism include the cell surface determinants flagella/antiflagellin protein, e.g., Macnab, Annu. Rev. Genet., 26: 131-158 (1992) and Silverman et al., Nature, 249: 73-74 (1974); fimbriae/antifimbriae protein or cognate receptors, e.g., Ofek et al., Curr. Top. Microbiol. Immunol., 151: 91-113 (1990), and Gbarah et al., Infect. Immun., 59: 4524-4530 (1991), and Klemm et al., Mol. Microbiol., 4: 553-559 (1990), and Nilsson et al., Bio/Technology, 12: 1376-78 (1994); and lipopolysaccharide (LPS)/anti-LPS protein.

Yet another preferred binding pair system useful in systems using E. coli as the host organism is the outer membrane receptor protein BtuB which selectively binds the vitamin B12 class of molecules, e.g., Holroyd et al., in Leive et al. (eds.), Microbiology-1994, p. 21-23, American Society for Microbiology, Washington, D.C. (1994).

For gram positive host organisms, preferred binding pairs include the membrane receptor protein FhuD of Bacillus subtilis which binds members of the ferrichrome family of siderophores including ferricrocin, e.g., Schneider et al., Mol. Microbiol. 8: 111-121 (1993).

For systems using yeast as a host organism, a preferred binding pair is the membrane .alpha.-factor pheromone receptor (Ste2) of the yeast Saccharomyces cerevisiae which binds to the mating .alpha.factor, e.g., Kurjan, Annu. Rev. Biochem. 61: 1097-1129 (1992).

For systems utilizing mammalian host organisms, preferred binding pairs include the epidermal growth factor/epidermal growth factor receptor pair and the bombesin/bombesin receptor pair, e.g., fantl et al., Annu. Rev. Biochem., 62: 453-481 (1993) and Spindel et al., Recent Prog. Horm. Res. 48: 365-391 (1993).

The solid supports used to immobilize one of the binding moieties are preferably flat, chemically well defined, i.e., chemically homogenous, not water soluble, capable of chemical modification, and wettable by an aqueous solvent. Preferred materials include glass, quartz, silicon, plastic, metal, and the like.

In a preferred embodiment of the present invention, the immobilized binding moiety is attached to the solid support at a plurality of discrete anchor sites, where, as used herein, the term "anchor site" refers to a location on the solid substrate having one or more binding moieties attached thereto such that there is an essentially binding-moiety-free region separating each anchor site. Such attachment may be based on covalent attachment or physical adsorption, e.g., hydrophobic adsorption or charged-based adsorption. Preferably, each anchor site has dimensions such that only a single host organism can bind to a single anchor site. When E. coli is the host organism, each anchor site should have an area of at least approximately 1 square micrometer.

In a more preferred embodiment, the anchor sites are arranged in a high density regular array. By arranging the anchor sites in such a way, the location of each bound host organism can be uniquely determined.

One preferred method of forming an ordered array of covalently immobilized moieties is by using linking groups, where, as used herein, the term "linking group" refers to a chemical species which serves to link a binding moiety to a solid support. Preferred linker molecules have both aqueous and membrane solubility. Particularly preferred linking groups include homopolymers of .omega.-aminopolyethyleneglycol (PEG) carboxylic acids or .omega.-aminoalkanoic acids such as .epsilon.-aminocaproic acid.

A particularly preferred method of forming a high-density array uses long-chain alkanethiols as linking groups, i.e., molecules having the formula HS(CH.sub.2).sub.n X, where n is preferably greater than 10, and X is an attachment group which facilitates covalent attachment of the binding moiety to the linking group. Long-chain alkanethiols rapidly form ordered, oriented, self-assembled monolayers on evaporated gold films deposited on glass substrates with the attachment group X facing away from the solid support. High resolution patterns of self-assembled monolayers (SAM) of alkanethiolates on gold substrates can be readily fabricated with dimensions as small as 1 .mu.m, e.g., Kumar et al., J Amer. Chem. Soc. 114:9188-9189 (1992). The dimension of the SAM at each array location or anchor site should not be larger than the projected area of a host organism to ensure that only a single host organism is captured at each location.

An alternative method for forming a high density array utilizes photolithography techniques. In this method, the surface of the solid substrate is treated with an agent that facilitates attachment of a binding moiety, e.g., an organosilane. The substrate is then treated with a photoresist agent to form a layer of photoresist material and baked to cure the photoresist. Next, the surface is exposed with ultraviolet light through a mask having a desired pattern, the mask defining the pattern of the array. The exposed substrate is then developed with developing agent removing either the exposed regions of photoresist, i.e., a positive photoresist, or the masked regions of photoresist, i.e., a negative photoresist. The steps of exposing and developing create regions of masked and unmasked surface. The substrate is then contacted with a solution of binding moiety, the binding moiety attaching to the substrate only at locations not coated with the photoresist, i.e, unmasked surface. Finally, the remaining photoresist is removed, resulting in a surface patterned with zones of binding moiety.

The invention will be further clarified by a consideration of the following examples, which are intended to be purely exemplary of the invention and not to limit its scope. adsorbed with RNAse B

Slides were functionalized with RNAse B by adding 10-20 .mu.l of RNAse B (0.5-500 .mu.g/ml) in 0.1 M Tris, pH 8.3 buffer, and placing slides at 4.degree. C. for 16 h in a water saturated chamber. Slides were then rinsed first with PBS, 0.1% Tween 20, 0.1 mM EDTA, followed with sterile water before incubation with E. coli. Strains were grown in LB medium at 37.degree. C. until log phase, washed with PBS, 1% BSA, 0.1 mM EDTA, and resuspended in one-half of the original volume of the culture in the same buffer.

RNAse B-adsorbed substrates were incubated with bacterial suspension for 40 min at room temp, then washed gently several times with cold PBS, 0.1% Tween 20, 0.1 mM EDTA. Substrates were viewed by light microscopy. Escherichia coli AAEC356 (M. S. McClain, I. C. Blomfield, K. J. Eberhardt, and B. I. Eisenstein J. Bacteriol. 17:4335-4344 (1993)), expressing type 1 fimbriae, bound confluently to areas of glass microscope slides, previously adsorbed with RNAse B. Conversely, no binding of E. coli AAEC072 (I. C. Blomfield, M. S. McClain, and B. I. Eisenstein, Mol. Microbiol. 5:1439-1445 (1991)), an isogenic non-fimbriated strain, was observed with RNAse B-adsorbed glass sides. Similar results were observed with RNAse B adsorbed on polystyrene microscope slides (data not shown).

These results demonstrate that E. coli, expressing type 1 fimbriae, can be specifically captured on substrates previously adsorbed with RNAse B. Non-fimbriated E. coli are not captured.

Patterning of Silanized Quartz Substrates with RNAse B

In order to capture single E. coli cells expressing type 1 fimbriae, it is necessary to adsorb RNAse B to discrete areas of a substrate. This adsorption was accomplished by microlithography as follows.

Four-inch diameter quartz wafers were silanized with octadecyltrichlorosilane to increase the hydrophobicity of the surface. This treatment increased the ability of the substrate to adsorb RNAse B. The silanization procedure was as follows. Wafers were "puddled" with 10% octadecyltrichlorosilane in toluene and then spun to fully coat the wafers. This procedure was repeated three times. Wafers were then "puddled" with toluene and spun to remove unreacted silane. This procedure was repeated several times. Wafers were then "puddled" with isopropanol and spun, "puddled" with 90% isopropanol-water and spun, and finally "puddled" with water and spun several times. Wafers were cured at 110.degree. C. for 15 min.

The silanized quartz wafers were then puddled with Shipley microposit S1813 photoresist (Marlborough, Mass.) and then spun. The resulting wafers were soft baked at 90.degree. C. for 30 min.

A mask (designated mask 1) containing arrays of 50 .mu.m, 25 .mu.m, 10 .mu.m, 5 .mu.m, 2.5 .mu.m, 1.0 .mu.m, 0.75 .mu.m, and 0.5 .mu.m squares was constructed. Within a given size array, squares were separated by 100 .mu.m, i.e., the array had a pitch of 100 .mu.m. A second mask (designated mask 2) consisting of lettering was also used. Following exposure of ultraviolet light through the masks for 8 sec, the wafers were developed with Shipley microposit MF-319 for 30 sec in the dark.

Each photoresist-patterned wafer was treated with 40 ml of 100 .mu.g/ml RNAse B in 0.1 M Tris, pH 8.3 buffer, at 4.degree. C. for 16 h. Wafers were washed first with PBS, 0.1% Tween 20, 0.1 mM EDTA, then with sterile water. These wafers were then allowed to dry at ambient temperature.

Photoresist from the above wafers was removed by stripping with acetone for 20 seconds at room temp, followed by rinsing with fresh acetone. The resulting wafers contained RNAse B adsorbed only within the squares from the mask. B-Adsorbed Silanized Quartz

Fimbriated strain AAEC356 was grown and washed as described above in Example 1. RNAse B-adsorbed silanized quartz wafers prepared as in Example 2 were incubated with bacterial suspension as described above. Wafers were viewed by light microscopy.

For wafers generated with mask 2, lettering formed with confluent fimbriated E. coli bacteria resulted. See FIG. 7. For wafers generated with mask 1 consisting of arrays of different-sized squares, confluent cells were observed within the 50, 25, and 10 .mu.m squares. Two or three cells were observed within the 5 .mu.m squares. A single cell was captured within virtually every 2.5 .mu.m square, i.e., 24 of 25 No cells were captured within the other smaller squares (data not shown).

Although only a few embodiments have been described in detail above, those having ordinary skill in the molecular biology art will clearly understand that many modifications are possible in the preferred embodiment without departing from the teachings thereof. All such modifications are intended to be encompassed within the following claims.

## Claims

1. A method of isolating individual cell of interest said method comprising the steps of contacting a cell population with a host organism capture system comprising: moieties being members of binding pairs, the complementary member of the binding pairs being expressed on the surface of a host organism, wherein the binding moieties are located in a plurality of discrete anchor sites, each anchor site having dimensions such that only a single host organism can bind to a single anchor site.

2. The method according to claim 1, wherein the binding moieties are located in a plurality of discrete anchor sites, each anchor site having dimensions such that only a single host organism can bind to a single anchor site.

3. The method according to claim 1, wherein the binding moiety is RNAse B.

4. A method according to claim 1, wherein the host organism capture system is fabricated by a method comprising: moiety; coating the surface with a photoresist agent; regions of unmasked surface; binding moiety attaches only at regions of unmasked surface; and

5. The method of claim 4 wherein the solid support is quartz.

6. The method of claim 4 wherein the step of treating the surface of the solid support to facilitate attachment of a binding moiety includes treatment with n organosilane reagent.