# Patent

## Title
Nucleic acid constructs containing orthogonal site selective recombinases (OSSRs)

## Abstract

The present invention provides for a recombinant nucleic acid comprising a nucleotide sequence comprising a plurality of constructs, wherein each construct independently comprises a nucleotide sequence of interest flanked by a pair of recombinase recognition sequences. Each pair of recombinase recognition sequences is recognized by a distinct recombinase. Optionally, each construct can, independently, further comprise one or more genes encoding a recombinase capable of recognizing the pair of recombinase recognition sequences of the construct. The recombinase can be an orthogonal (non-cross reacting), site-selective recombinase (OSSR).

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority as a continuation-in-part application of PCT International Patent Application No. PCT/US2009/060275, filed Oct. 9, 2009, which claims priority to U.S. Provisional Patent Application Ser. No. 61/104,239, filed on Oct. 9, 2008; which are hereby incorporated by reference.


STATEMENT OF GOVERNMENTAL SUPPORT
The invention described and claimed herein was made in part utilizing funds supplied by the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The government has certain rights in this invention.


FIELD OF THE INVENTION
This invention relates generally to the use of recombinases.
BACKGROUND OF THE INVENTION
Phage recombinases are splicing enzymes used by virions to insert or remove their genomic DNA from a host chromosome. The use of recombinases (or integrases) for genomic manipulation is well established. Site-specific recombinases are significant tools in a variety of applications in research, medicine, and biotechnology. Conditional gene targeting using site-specific recombinases has enabled the functional analysis of genes, which cannot be inactivated in the germline. Site-specific recombinases also allow the precise integration of open reading frames (ORFs) encoding proteins of interest into highly active gene loci in cell lines and transgenic animals. Recombinases are disclosed in the following references: Groth, Amy C.; Calos, Michele P. J. Mol. Biol (2004) 335, 667-678; Silver, Daniel P.; Livingston, David M. Molecular Cell (2001), 8, 233-243; Sauer, Brian; McDermott, Jeffrey. Nucleic Acids Research (2004) 32(20), 6086-6095; Yagil, Ezra; Dorgai, László; Weisberg, Robert A. J. Mol. Biol (1995) 252, 163-177; and, Dorgai, László; Yagil, Ezra; Weisberg, Robert A. J. Mol. Biol (1995) 252, 178-188.
SUMMARY OF THE INVENTION
The present invention provides for a recombinant nucleic acid comprising a nucleotide sequence comprising a plurality of constructs, wherein each construct independently comprises a nucleotide sequence of interest flanked by a pair of recombinase recognition sequences. Each pair of recombinase recognition sequences is recognized by a distinct recombinase. Optionally, each construct can, independently, further comprise one or more genes encoding a recombinase capable of recognizing the pair of recombinase recognition sequences of the construct.
The present invention provides for a recombinant nucleic acid comprising a first construct and a second construct; wherein the first construct comprises a nucleotide sequence encoding a first recognition sequence of a first recombinase, a second recognition sequence of the first recombinase, and a first nucleotide sequence of interest located between the first and second recognition sequence of the first recombinase; wherein the second construct comprises a nucleotide sequence encoding a first recognition sequence of a second recombinase, a second recognition sequence of the second recombinase, and a second nucleotide sequence of interest located between the first and second recognition sequence of the second recombinase; wherein the second construct is located downstream of the first construct; wherein the first recombinase and the second recombinase do not cross react with the recognition sequence of the other.
A recombinase that can be used in the present invention is an orthogonal (non-cross reacting), site-selective recombinase (OSSR). An OSSR is a recombinase that recognizes a specific recognition site or nucleotide sequence and does not cross-react with the recognition site or nucleotide sequence of another recombinase.
The present invention also provides for a recombinant vector comprising the recombinant nucleic acid. The present invention also provides for a vector or expression vector comprising a recombinant nucleic acid of the present invention.
The present invention further provides for a host cell comprising any of the recombinant nucleic acid or vector of the present invention. In some embodiments, the recombinant nucleic acid is integrated into a chromosome or replicon of the host cell. The host cell can be an eukaryotic or a prokaryotic cell.
The present invention further provides for a host organism comprising one or more host cells of the present invention. In some embodiments, all of the cells of the host organism comprise a recombinant nucleic acid of the present invention.
The present invention provides for a method of excising or deleting one or more nucleotide sequence of interest from a host cell, comprising: (a) providing a signal to a host cell to activate expression from a promoter in the host cell, wherein the host cell comprises a promoter upstream of a plurality of constructs, wherein each construct independently comprises a nucleotide sequence of interest flanked by a pair of recombinase recognition sequences; and (b) excising or deleting one or more nucleotide sequence of interest.
The present invention provides for a method of excising or deleting a first nucleotide sequence of interest from a host cell, comprising: (a) providing a signal to a host cell to activate expression from a promoter in the host cell, wherein the host cell comprises a promoter upstream of a first construct and a second construct; and (b) excising or deleting a first nucleotide sequence of interest; wherein the first construct comprises a nucleotide sequence encoding a first recognition sequence of a first recombinase, a second recognition sequence of the first recombinase, and the first nucleotide sequence of interest located between the first and second recognition sequence of the first recombinase; wherein the second construct comprises a nucleotide sequence encoding a first recognition sequence of a second recombinase, a second recognition sequence of the second recombinase, and a second nucleotide sequence of interest located between the first and second recognition sequence of the second recombinase; wherein the second construct is located downstream of the first construct; wherein the first recombinase and the second recombinase do not cross react with the recognition sequence of the other.
The present invention further provides for a system capable of noise canceling with non-coding interfering RNA suppression.
The present invention further provides for a system capable of noise canceling with dominant negative complexation.
The present invention further provides for a system comprising a switch that is controlled by the relative expression of two variable promoters



BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.
FIG. 1 shows an illustration of an exemplary recombinant nucleic acid of the present invention. Three representative constructs are shown. Each horizontal bar represents a separate ORF of interest. Each vertical bar represents a recombinase gene and the triangles with the same hatch marks indicate its corresponding recognition sites. In all figures, each “T” represents a transcription terminator element. In all figures, each L-shaped line with an arrow represents a promoter.
FIG. 2 shows an exemplary Potter Standard Plasmid. The name of such a plasmid is the form of “Vector-Part”. For example, a “Part” is defined as the sequence between BglII and BamHI sites. For example, a “Vector” is defined as the sequence between BamHI and BglII sites. As such, the “Vector” can be defined as a special part containing EcoRI and XhoI restriction sites. Any number of “Parts” and “Vectors” can be individually defined as basic parts or composite parts.
FIG. 3 shows an exemplary standard assembly. The nucleotide sequences from top to bottom are SEQ ID NOs:4-7, respectively.
FIG. 4 shows an exemplary Type I coding sequences, wherein the start and stop codons are placed directly adjacent to the BglII and BamHI sites, respectively. The start codons can be ATG, CTG, TTG, or GTG. The top nucleotide sequence is SEQ ID NO:8, and the bottom nucleotide sequence is SEQ ID NO:9.
FIG. 5 shows exemplary Type II-Type IV coding sequences (SEQ ID NOs:10-12, respectively). The coding sequences allow the construction of ORF fusions for chimeric and tagged proteins. GlySer scars separate junctions between fused peptides.
FIG. 6 shows an exemplary Ribosome Binding Site (RBS), and the spacing of a ribosome binding site relative to the start codon is fixed. Shown is a (likely) strong RBS. The nucleotide sequences from top to bottom are SEQ ID NOs:13-15, respectively.
FIG. 7 shows an exemplary promoter (SEQ ID NO:16). The transcriptional start site (+1) is located at the position directly 5′ to the BamHI site (whenever possible).
FIG. 8 shows an exemplary terminator. The transcriptional termination site is located at the position directly 5′ to the BamHI site (whenever possible). The top nucleotide sequence is SEQ ID NO:17, and the bottom nucleotide sequence is SEQ ID NO:18.
FIG. 9 shows an example of cross pairing (of three different recombinases) which should not result in excision.
FIG. 10 shows a screen based on excision events. In this example, reactive pairs are identified by replica plating from ampicillin (Amp)/chloramphenicol (Cm) plates to kanamycin (Kan)/Cm plates.
FIG. 11 shows a representative testing of cross pairs and simultaneous expression. A large number of constructs is required (ΣN for N recognition sites +2 per recombinase using a three plasmid system) and extensive cross testing.
FIG. 12 shows a representative fusebox expression cassette.
FIG. 13 shows a representative synthetic teleomere.
FIG. 14 shows a system capable of noise canceling with non-coding interfering RNA suppression.
FIG. 15 shows a system capable of noise canceling with dominant negative complexation.
FIG. 16 shows a system comprising a switch that is controlled by the relative expression of two variable promoters
FIG. 17 shows a sample gel showing a cross-test involving Cre, Dre, Cre-Dre and the various site combinations giving the expected results quite cleanly by colony PCR analysis
FIG. 18 shows an “or” gate of the present invention.
FIG. 19 shows the expected results for a circuit in an agar-plate based assay.
FIG. 20 shows the result of a cross testing method using an active site knockout of Dre (DreX-Y324F). Tyrosine Y324 is annotated as the active site tyrosine for this enzyme. However, after replacing this residue with phenylalanine, as determined in the sequencing result shown, activity of the enzyme is still observed. The nucleotide sequences depicted for “p15a-cm-ptet (RK) DreX” and “DreX Second Read” are SEQ ID NO:19. The nucleotide sequence depicted for “p15a-cm-ptet (RK) Dre” is SEQ ID NO:20.
FIG. 21 shows a flowchart for a plate based assay for searching for and using orthogonal site selective recombinases.



DETAILED DESCRIPTION
Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a sequence” includes a plurality of such sequences, and so forth.
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.
In some embodiments, the recombinant nucleic acid comprises two or more constructs, three or more constructs, four or more constructs, five or more constructs, or ten or more constructs. In some embodiments, the recombinant nucleic acid comprises up to ten constructs.
In some embodiments, the recombinant nucleic acid further comprises a third construct comprising a nucleotide sequence encoding a first recognition sequence of a third recombinase, a second recognition sequence of the third recombinase, and a third nucleotide sequence of interest located between the first and second recognition sequence of the third recombinase. In some embodiment, the recombinant nucleic acid further comprises a fourth, fifth, or/and etc. construct(s), each construct comprising a nucleotide sequence encoding a first recognition sequence of a unique recombinase, a second recognition sequence of the unique recombinase, and a nucleotide sequence of interest located between the first and second recognition sequence of the unique recombinase, wherein the unique recombinase is a recombinase is distinct of any of the other recombinase which recognizes a recognition sequence within the recombinant nucleic acid.
See FIG. 1 for one embodiment of the invention.
The use of recombinases for manipulation of genomic sequences is well known to those skilled in the art. Commonly, the recombinase is used as follows: a target DNA containing a selection marker is initially inserted into a chromosome at a desired location. Following selection, an OSSR is used to extract the selection marker. In this method, the toolkit of two recombinases and one selection marker can be used repeatedly to significantly modify the organism under study. The orthogonality of the recombinase is important to prevent destructive or unpredictable recombination events.
A recombinase that can be used in the present invention is an orthogonal (non-cross reacting), site-selective recombinase (OSSR). An OSSR is a recombinase that recognizes a specific recognition site or nucleotide sequence and does not cross-react with the recognition site or nucleotide sequence of another recombinase. In some embodiments, each recombinase recognizes a pair of identical DNA sequences is that is about 50 to 60 basepair in length. In some embodiments, the recombinase recognizes a pair of identical DNA sequences is that is about 52 to 58 basepair in length. In some embodiments, the recombinase recognizes a pair of identical DNA sequences is that is about 53, 54, 55, 56 or 57 basepair in length. If only two sites are present, and they are oriented in the same direction on a DNA strand, the sequence between the sites is excised into a loop, with one recognition site remaining on the annealed DNA and one site incorporated into the loop. If more than two sites are present, or the orientation between sites is different, multiple activities can occur, including flipping and mis-annealing of the genomic DNA.
To be useful for the present invention, the recombinase and its recognition sites must display the following properties: (1) Engineered recognition sites should be unique to the cell (that is, no native genomic sequence matches the recognition site). (2) Recombinases must not bind to the recognition sites associated with other recombinases. (3) Recombinases bound to a recognition site must not be able to bind to the “partner” recombinase associated with a different recognition site. (The functional unit of a recombinase is a dimer of dimers. For example, dimer AA normally binds to site a. This then interacts with another dimer AA bound to a different site a to cause a recombination event. If dimer AA recognizes site a, and dimer BB recognizes site b, if AA is capable of binding to BB in the presence of a and b, a cross reaction would occur.) (4) Recognition sites should be kept to a total of two per cell per recombinase in all cases where this is possible during the operation of the invention. FIG. 9 shows an example of cross pairing (of three different recombinases) which should not result in excision. FIG. 10 shows a screen based on excision events. FIG. 11 shows a representative testing of cross pairs and simultaneous expression.
Recombinases useful for this invention include, but are not limited to, to the recombinases listed in Table 1.







TABLE 1







Recombinases.












#
Name
Host
Organism
Gene
Accession















1
BSu_xerC
Bacillus subtilis
chromosome
codV
P39776


2
BSu_xerD
Bacillus subtilis
chromosome
ripX
P46352


3
BSu_ydcL
Bacillus subtilis
chromosome
ydcL
A69774


4
CBu_tnpA
Clostridium butyricum
chromosome
tnpA
S40097


5
Col1D
Escherichia coli
plasmid F
D
P06615


6
CP4-57
Escherichia coli
chromosome
Int
P32053


7
Cre
Escherichia coli
phage P1
Int
P06956


8
D29
Mycobacterium smegmatis
phage D29
Int
AAC18476


9
DLP12
Escherichia coli
phage DLP12
Int
P24218


10
DNo_int
Dichelobacter nodosus
chromosome
Orf
AAB00935


11
ECo_fimB
Escherichia coli
chromosome
fimB
P04742


12
ECo_fimE
Escherichia coli
chromosome
fimE
P04741


13
ECo_orf
Escherichia coli
chromosome
b2442
A65019


14
ECo_xerC
Escherichia coli
chromosome
xerC
C37841


15
ECo_xerD
Escherichia coli
chromosome
xerD
P21891


16
HIn_orf
Haemophilus influenzae
chromosome
orf1572
P46495


17
HIn_rci
Haemophilus influenzae
chromosome
rci
P45198


18
HIn_xerC
Haemophilus influenzae
chromosome
xerC
P44818


19
HIn_xerD
Haemophilus influenzae
chromosome
xerD
P44630


20
HK22
Escherichia coli
phage HK022
int
AAF30377


21
HP1
Haemophilus influenzae
phage HP1
int
P21442


22
L2
Acholeplasma sp.
phage L2
int
AAA87961


23
L5
Mycobacterium tuberculosis
phage L5
int
CAA79409


24
L54
Staphylococcus aureus
phage L54
int
P20709


25
Lambda
Escherichia coli
phage lambda
int
AAA96562


26
LLe_orf
Lactobacillus leichmannii
chromosome
orf
CAA55635


27
LLe_xerC
Lactobacillus leichmannii
chromosome
xerC
CAA59018


28
phi10MC
Oenococcus oeni
phage phi10MC
int
AAD00268


29
MJa_orf
Methanococcus jannaschi
chromosome
orf
Q57813


30
MLe_xerD
Mycobacterium leprae
chromosome
xerD
S72959


31
MPa_int
Mycobacterium paratuberculosis
chromosome
int
AAA88834


32
MTu_int
Mycobacterium tuberculosis
chromosome
int
B70965


33
MTu_xerC
Mycobacterium tuberculosis
chromosome
xerC
Q10815


34
MV4
Lactobacillus delbrueckii
phage MV4
int
AAC48859


35
MX8
Myxococcus xanthus
phage Mx8
int
AAC48895


36
pAE1
Alcaligenes eutrophus
plasmid pAE1
orf
AAA87238


37
pCL1
Chlorobium limicola
plasmid pCL1
fim
AAB36935


38
pDU1
Nostoc sp.
plasmid pDU1
orf
AAA17517





plasmid




39
pMEA
Amycolatopsis methanolica
pMEA300
orf
AAB00469





plasmid




40
RSp_EF
Rhizobium sp.
pNG234a
EF
P55429





plasmid




41
RSp_GC
Rhizobium sp.
pNG234a
GC
P55459





plasmid




42
RSp_QK
Rhizobium sp.
pNG234a
QK
P55632





plasmid




43
RSp_RA
Rhizobium sp.
pNG234a
RA
AAB92467





plasmid




44
RSp_RB
Rhizobium sp.
pNG234a
RB
P55635





plasmid




45
RSp_RC
Rhizobium sp.
pNG234a
RC
P55636





plasmid




46
RSp_RD
Rhizobium sp.
pNG234a
RD
P55637





plasmid




47
RSp_RE
Rhizobium sp.
pNG234a
RE
P55638





plasmid




48
RSp_RF
Rhizobium sp.
pNG234a
RF
P55639


49
pSAM2
Streptomyces ambofaciens
plasmid pSAM2
orf
P15435


50
pSDL2
Salmonella dublin
plasmid pSDL2
resV
A38114


51
pSE101
Saccharopolyspora erythraea
plasmid pSE101
orf
S41725


52
pSE211
Saccharopolyspora erythraea
plasmid pSE211
orf
P22877


53
pWS58
Lactobacillus delbrueckii
plasmid pWS58
orf
CAA90472


54
phi-11
Staphylococcus aureus
phage phi11
int
AAA32198


55
phi-13
Staphylococcus aureus
phage phi13
int
S52761


56
phi-80
Escherichia coli phage
phage phi80
int
CAA27683


57
phi-adh
Lactobacillus gasseri
phage phi-adh
int
JN0535


58
phi-CTX
Pseudomonas aeruginosa
phage phiCTX
int
CAA74224


59
phi-g1e
Lactobacillus sp.
phage phi-g1e
int
T13182


60
phi-LC3
Lactococcus lactis
phage phiLC3
int
A47085


61
phi-R73
Escherichia coli
phage phi-R73
int
A42465


62
P186
Escherichia coli
phage 186
int
AAC34175


63
P2
Escherichia coli
phage P2
int
AAD03297


64
P21
Escherichia coli
phage P21
int
AAC48886


65
P22
Salmonella typhimurium
phage P22
int
AAF75002


66
P4
Escherichia coli
phage P4
int
CAA29379


67
P434
Escherichia coli
phage 434
int
P27078


68
PAe_xerC
Pseudomonas aeruginosa
chromosome
sss
AAG08665


69
PMi_fimB
Proteus mirabilis
chromosome
fimB
CAB61438





plasmid IncI2




70
R721
Escherichia coli
(R721)
rcb
G45252





plasmid IncI1




71
Rci
Escherichia coli
(R64)
rci
P10487


72
SF6
Shigella flexneri
phage Sf6
int
P37317


73
SLP1
Streptomyces coelicolor
plasmid SLP1
orf
CAC08268


74
IntI3
Serratia marcescens
chromosome
orf
BAA08929


75
SsrA
Methanosarcina acetivorans
plasmid pC2A
ssrA
AAB39744


76
SSV1
Sulfolobus sp.
phage SSV1
int
CAA30211


77
T12
Streptococcus pyogenes
phage T12
int
AAC488867


78
IntI1
Escherichia coli
transposon Tn21
int
AAA82254





transposon




79
Tn4430
Bacillus thuringiensis
Tn4430
int
CAA30491





transposon




80
Tn5041
Pseudomonas sp.
Tn5041
orfI
CAA67462





transposon




81
Tn5252
Streptococcus pneumoniae
Tn5252
int
A55863





transposon




82
Tn5276
Lactobacillus lactis
Tn5276
int
C55205





transposon




83
Tn554a
Staphylococcus aureus
Tn554
tnpA
P06696





transposon




84
Tn554b
Staphylococcus aureus
Tn554
tnpB
P06697


85
IntI2
Escherichia coli
transposon Tn7
int
CAA05031





transposon




86
Tn916
Entercoccus faecalis
Tn916
int
P22886


87
Tuc
Lactobacillus lactis
phage Tuc2009
int
AAA32608


88
BZo_int
Bergeyella zoohelcum
chromosome
orf
AAA50502


89
ASp_xisA
Anabaena sp.
chromosome
xisA
P08862


90
ASp_xisC
Anabaena sp.
chromosome
xisC
Q44217


91
FLP
Saccharomyces cerevisiae
plasmid 2μ 
FLP
J01347


92
pKD1
Kluyveromyces lactis
plasmid pKD1
FLP
P13783


93
pSB2
Zygosaccharomyces bailii
plasmid pSB2
FLP
M18274


94
pSB3
Zygosaccharomyces bisporus
plasmid pSB3
FLP
P13784


95
pSM1
Zygosaccharomyces fermentati
plasmid pSM1
FLP
P13770


96
pSR1
Zygosaccharomyces rouxii
plasmid pSR1
FLP
P13785


97
HPy_xerC
Helicobacter pylori
chromosome
xerC
C64604


98
HPy_xerD
Helicobacter pylori
chromosome
xerD
C64644


99
Eco_Rac
Escherichia coli
chromosome
int
P76056


100
Eco_Qin
Escherichia coli
chromosome
int
P76168


101
CP4-6
Escherichia coli
chromosome
orf
P71928


102
E14
Escherichia coli
chromosome
int
P75969


103
MGo_orf
Mycobacterium gordonae
chromosome
orf
AAB54012


104
MLe_xerC
Mycobacterium leprae
chromosome
xerC
CAB10656


105
MTu_xerD
Mycobacterium tuberculosis
chromosome
xerD
CAB10958


106
pEAF
Escherichia coli
plasmid EAF
rsv
AAC44039


107
PF1_xerC
Pseudomonas fluorescens
chromosome
sss
T10461


108
PWi_orf
Protothera wickerhamii
mitochondria
ymf42
T11912


109
Sfi21
Streptococcus thermophilus
phage Sfi21
int
AAD44095


110
phi-r1t
Lactobacillus lactis
phage r1t
int
AAB18676


111
STy_xerC
Salmonella typhimurium
chromosome
xerC
P55888


112
STy_xerD
Salmonella typhimurium
chromosome
xerD
P55889


113
SSp_orf
Synechocystis sp.
chromosome
orf
BAA16682


114
DNo_orf
Dichelobacter nodosus
chromosome
orf
AAB00935


115
VCh_orf
Vibrio cholerae
chromosome
orf
AAC44230




Methanothermobacter





116
MMa_xerC
marburgensis
chromosome
xerC
D69219


117
ECo_orf2
Escherichia coli
chromosome
intB
P39347


118
SIn_orf
Salmonella infantis
chromosome
orf
J03391


119
BK-T
Lactococcus lactis
phage BK-T
int
T13262


120
phi-42
Staphylococcus aureus
phage phi42
int
AAA91615


121
FRAT1
Mycobacterium sp.
phage FRAT1
int
P25426


122
HZe_vlf1
Helicoverpa zea
chromosome
vlf1
AAA58702


123
pKW1
Kluveromyces waltii
plasmid pKW1
FLP
X56553


124 
CBu_tnpB
Clostridium butyricum
chromosome
tnpB
S40098


125 
S2 
Haemophilus influenzae
phage S2
int
CAA96221


126
NBU1
Bacteroides uniformis
plasmid NBU1
int
AAF74437





transposon




127
Tn1545
Streptococcus pneumoniae
Tn1545
int
P27451


128
T270
Streptococcus pyogenes
phage T270
int
AAA85500


129
PMi_xerC
Proteus mirabilis
chromosome
xerC
AAB87500


130
PMi_xerD
Proteus mirabilis
chromosome
xerD
AAB87499


131
phiV
Shigella flexneri
phage V
int
AAB72135


132
O1205
Streptococcus thermophilus
phage O1205
int
T13289





transposon




133
Tn4556
Streptomyces fradiae
Tn4556
int
P20184


134
MS6
Mycobacterium sp.
phage Ms6
int
AAD03774





plasmid




135
pFAJ
Rhodococcus erythropolis
pFAJ2600
pmrA
AAC45806


136
SMa_xerC
Serratia marcescens
chromosome
xerC
AAC46276





plasmid




137
pTiA6
Agrobacterium tumefaciens
pTiA6NC
int
AAB91569


138
AAe_orf
Aquifex aeolicus
chromosome
int
G70397





transposon




139
Tn557
Staphylococcus aureus
Tn557
int
AAC28969


140
EAe_int
Enterobacter aerogenes
chromosome
int
AAB95339


141
SF2
Shigella flexneri
phage Sf2
int
AAC39270


142
ECo_yfdB
Escherichia coli
chromosome
yfdB
P37326


143
RP3
Streptomyces rimosus
phage RP3
int
X80661


144
VWB
Streptomyces venezuelae
phage VWB
int
CAA03882


145
SEx_vlf1
Spodoptera exigua
chromosome
vlf1
AAF33611


146
STy_rci
Salmonella typhimurium
chromosome
rci
AAC38070


147
PPu_orf
Pseudomonas putida
chromosome
orf
CAA06238


148
A2
Lactobacillus casei
phage A2
int
CAA73344


149
pECE1
Aquifex aeolicus
plasmid ece1
int
AAC07955


150
MLo_int
Mesorhizobium loti
chromosome
intS
AAC24508


151
SRu_orf
Selenomonas ruminantium
chromosome
orf
BAA24921


152 
pQPRS
Coxiella burnetti
plasmid pQPRS
int
CAA75853


153 
PRe_orf
Panagrellus redivivus
chromosome
orf
CAA43185


154
CEl_orf
Caenorhabditis elegans
chromosome
orf
Z82079


155
IntI4
Vibrio cholerae
chromosome
intI4
AAF71178


156
SMu_orf
Streptococcus mutans NG8
chromosome
orfA
AAC17173


157
phiU
Rhizobium leguminosarum
phage phiU
int
BAA25885


158
PHo_xerC
Pyrococcus horikoshii
chromosome
xerC
B71194


159
RCa_orf1
Rhodobacter capsulatus
chromosome
orf1
T03499


160
RCa_orf2
Rhodobacter capsulatus
chromosome
orf2
T03567





transposon




161
Tn5382
Enterococcus faecium
Tn5382
int
AAC34799




Methanothermobacter





162
psiM2
marburgensis
phage PsiM2
int
T12745


163
STy_orf
Salmonella typhimurium
chromosome
orf
T03001


164
MTu_orf
Mycobacterium tuberculosis
chromosome
Rv2659c
G70966


165
TPa_xerC
Treponema pallidum
chromosome
codV
AAC65375


166
TPa_xerD
Treponema pallidum
chromosome
xprB
AAC65379


167
CTr_xerC
Chlamydia trachomatis
chromosome
xerC
AAC67942


168
CTr_xerD
Chlamydia trachomatis
chromosome
xerD
AAC68462


169
phiPVL
Staphylococcus aureus
phage phiPVL
int
BAA31902


170
pNL1
Sphingomonas aromaticivorans
plasmid pNL1
int
AAD03886


171
CP4-157
Escherichia coli O157:H7
chromosome
int
AAC31482


172
SAu_xerD
Staphylococcus aureus
chromosome
xerD
AAC64162


173
YPe_orf
Yersinia pestis
chromosome
orf
AAC69581


174
RPr_xerD
Rickettsia prowazekii
chromosome
xerD
B71693


175
RPr_xerC
Rickettsia prowazekii
chromosome
xerC
B71643


176
VCh_SXT
Vibrio cholerae
chromosome
orf
AAF93686


177
AAc_orf
Actinob. actinomycetemcomitans
chromosome
orf
AAC70901


178
MAV1
Mycoplasma arthritidis
chromosome
int
AAC33780


179
fOg44
Oenococcus oeni
phage fOg44
int
AAD10711


180
SFX
Shigella flexneri
phage SFX
int
AAD10295





transposon




181
Tn4371
Ralstonia eutropha
Tn4371
int
CAA71790


182
HPy_orf
Helicobacter pylori
chromosome
orf
A71869


183
CPn_xerC
Chlamydia pneumoniae
chromosome
xerD
BAA99231


184
CPn_xerD
Chlamydia pneumoniae
chromosome
xerC
BAA98236


185
K139
Vibrio cholerae
phage K139
int
AAD22068


186
PPu_orf2
Pseudomonas putida
chromosome
orf
BAA75916





plasmid




187
pPZG
Pantoea citrea
pPZG500
int
AAD21210


188
H19J
Escherichia coli
phage H19J
int
CAB38715


189
phi304L
Corynebacterium glutamicum
phage phi304L
int
CAB38562


190
SCo_orf
Streptomyces coelicolor
chromosome
orf
T36198


191
phi16
Corynebacterium glutamicum
phage phi16
int
CAA73074


192
BHa_xerC
Bacillus halodurans
chromosome
codV
BAB06184


193
XFa_xerC
Xylella fastidiosa
chromosome
xerC
AAF84292


194
BHa_xerD
Bacillus halodurans
chromosome
xerD
BAB05248


195
PAe_xerD
Pseudomonas aeruginosa
chromosome
xerD
AAG07125


196
VCh_xerC
Vibrio cholerae
chromosome
xerC
AAF93305


197
VCh_xerD
Vibrio cholerae
chromosome
xerD
AAF95562


198
NMa_xerC
Neisseria meningitidis ser. A 
chromosome
xerC
CAB83879


199
NMb_xerC
Neisseria meningitidis ser. B 
chromosome
xerC
AAF42202


200
XFa_xerD
Xylella fastidiosa
chromosome
xerD
AAF84234


201
CMu_xerC
Chlamydia muridarum
chromosome
xerC
AAF73578


202
SAu_xerC
Staphylococcus aureus
chromosome
xerC
AAF89877


203
NMa_xerD
Neisseria meningitidis ser. B 
chromosome
xerD
AAF41164


204
NMb_xerD
Neisseria meningitidis ser. A 
chromosome
xerD
CAB84234


205
CMu_xerD
Chlamydia muridarum
chromosome
xerD
AAF39124


206
PAb_xerD
Pyrococcus abysii
chromosome
xerD
A75153


207
pI3
Deinococcus radiodurans
plasmid pI3
ResU
AAF44051





plasmid




208
pTiSAK
Agrobacterium tumefaciens
TiSAKURA
orf36
BAA87661


209
HPj_xerC
Helicobacter pylori J
chromosome
xerC
B71910


210
TMa_xerC
Thermotoga maritima
chromosome
xerC
D72312


211
CJe_xerD
Campylobacter jejuni
chromosome
xerD
CAB73128


212
APe_xerD
Aeropyrum pernix
chromosome
xerD
G72672


213
PSy_orf
Pseudomonas syringae
chromosome
orfF
CAB96970


214
MM1
Streptococcus pneumoniae
phage MM1
int
CAB96616


215
XNi_vlf1
Xestia nigrum
chromosome
vlf1
AAF05239


216
PXy_vlf1
Plutella xylostella
chromosome
vlf1
AAG27387


217
pXO1-132
Bacillus anthracis
plasmid pXO1
132
D59107





transposon




218
Tn4555
Bacteroides fragilis
Tn4555 
int
AAB53787


219
DRa_xer
Deinococcus radiodurans
chromosome
xerD
G75636


220
BJa_int
Bradyrhizobium japonicum
chromosome
intA
AAF64651


221
BHa_orf4
Bacillus halodurans
chromosome
BH2349
BAB06068


222
pXO1-103
Bacillus anthracis
plasmid pXO1
103
G59103


223
PAe_orf2
Pseudomonas aeruginosa
chromosome
orf2
AAG04117





plasmid




224
pLGV440
Chlamydia trachomatis
pLGV440
orf8
P08788





transposon




225
Tn5520
Bacteroides fragilis
Tn5520
bipH
AAC80279


226
pNL1_tnpA
Sphingomonas aromaticivorans
plasmid pNL1
tnpA
AAD03922


227
CTr_orf
Chlamydia trachomatis
chromosome
orf1
S44160


228
BHa_orf1
Bacillus halodurans
chromosome
BH3551
BAB07270


229
phi-933W
Escherichia coli
phage 933W
int
AAD25406


230
CPs_orf1
Chlamydia psittaci
chromosome
orf
B39999


231
VCh_orf2
Vibrio cholerae
chromosome
VC1758
AAF94908


232
DRa_orf2
Deinococcus radiodurans
chromosome
orf2
F75611


233
pCPnE1
Chlamydophila pneumoniae
plasmid pCPnE1 
orf2
CAA57585


234
ECo_intB
Escherichia coli
chromosome
intB
AAD37509


235
UUr_xerC
Ureaplasma urealyticum
chromosome
xerC
AAF30630


236
HK97
Escherichia coli
phage HK97
int
AAF31094


237
TPW22
Lactococcus sp.
phage TPW22
int
AAF12706


238
APSE-1
Acyrthosiphon pisum
phage APSE-1
int
AAF03981





plasmid




239
pURB500
Methanococcus maripaludis
pURB500
int
AAC45247


240
SFl_int
Shigella flexneri
chromosome
int
AAD44730


241
UUr_xerD
Ureaplasma urealyticum
chromosome
ripX
AAF30551


242
Wphi
Escherichia coli
phage Wphi
int
CAB54522


243
BHa_orf2
Bacillus halodurans
chromosome
BH2364
BAB06083


244
SEn_int
Salmonella enterica
chromosome
intI5
AAG03003


245
pCP1
Deinococcus radiodurans
plasmid pCP1
xerD
AAF12667


246
SCo_int
Streptomyces coelicolor
chromosome
int
CAB71253


247
PRi1724
Agrobacterium rhizogenes
plasmid pRi1724
orf9
BAB16128


248
SCo_traS
Streptomyces coelicolor
chromosome
traS
T35465


249
HPy_orf1
Helicobacter pylori
chromosome
orf
A71870


250
XFa_orf1
Xylella fastidiosa
chromosome
XF2530
AAF85328


251
UUr_codV
Ureaplasma urealyticum
chromosome
codV
AAF30942


252
pXO1-18
Bacillus anthracis
plasmid pXO1
18
B59093


253
CPs_orf2
Chlamydia psittaci
chromosome
orf2
A39999


254
SPBc2
Bacillus subtilis
phage SPBc2
yopP
T12850


255
D3
Pseudomonas aeruginosa
phage D3
int
AAF04808


256
XFa_orf2
Xylella fastidiosa
chromosome
XF1642
AAF84451


257
XFa_orf3
Xylella fastidiosa
chromosome
XF0678
AAF83488





plasmid




258
pLGV440-2
Chlamydia trachomatis
pLGV440
N1
S01180


259
pB171
Escherichia coli
plasmid pB171
rsvB
BAA84906


260
DRa_orf3
Deinococcus radiodurans
chromosome
orf
C75509


261
CPZ-55
Escherichia coli
phage CPZ-55
int
P76542





transposon




262
ICESt1
Streptococcus thermophilus
ICESt1
int
CAB70622


263
pGP7-D
Chlamydia trachomatis
plasmid pGP7-D 
TCA01
AAF39715


264
XFa_orf4
Xylella fastidiosa
chromosome
XF1718
AAF84527


265
HIn_orf2
Haemophilus influenzae
chromosome
int
AAF27347


266 
DNo_orf2
Dichelobacter nodosus
chromosome
intC
CAB57348





transposon




267
NBU2
Bacteroides fragilis
NBU2
intN2
AAF74726





plasmid Col1B-




268
pCol1B
Shigella sonnei
P9
resA
BAA75108


269
PSy_orf4
Pseudomonas syringiae
chromosome
orf
CAC14205





transposon




270
Tn4652
Pseudomonas putida
Tn4652
orf5
AAD44277





plasmid




271
pLGV440-3
Chlamydia trachomatis
pLGV440
orf7
P10561


272
pF
Escherichia coli
plasmid F
int
BAA97902


273
BHa_orf3
Bacillus halodurans
chromosome
BH4039
BAB07758


274 
XFa_orf5
Xylella fastidiosa
chromosome
XF2132
AAF84931





plasmid




275
pNRC100_1
Halobacterium sp.
pNRC100
H0618
T08273


276
SDy_orf
Shigella dysenteriae
chromosome
int
AAF28112


277 
pQpRS_2
Coxiella burnetti
plasmid pQpRS
orf410
CAA75839


278
PMu_rci
Pasteurella multocida
chromosome
rci
AAF68420


279
SPBc2
Bacillus subtilis
phage SPBc2
yomM
AAC13009


280
PPa_int
Pseudomonas pavonaceae
chromosome
intP
CAB65361





plasmid




281
pKLC102
Pseudomonas aeruginosa
pKLC102
xerC
AAG02084


282
XFa_orf6
Xylella fastidiosa
chromosome
XF0631
AAF83441


283
SCo_orf3
Streptomyces coelicolor
chromosome
int
CAC14368


284 
LLa_orf
Lactococcus lactis
chromosome
orf3
AAF86683


285
MSp_orf
Mycobacterium sp.
chromosome
intM
CAB65286


286
pNL1_tnpB
Sphingomonas aromaticivorans
plasmid pNL1
tnpB
AAD03921


287
XFa_orf7
Xylella fastidiosa
chromosome
XF0968
AAF83778


288
ECo_orf5
Escherichia coli
chromosome
int
AAF06962


289
AGe_vlf1
Anticarsia gemmatalis
chromosome
vlf-1
AAD54607


290
pLH1
Lactobacillus helveticus
plasmid pLH1
orf195
CAA10964


291
SAu_orf2
Staphylococcus aureus
chromosome
orf
AAG29618


292
LDi_vlf1
Lymantria dispar
chromosome
vlf-1
AAC70272


293
OPs_v1f1
Orgyia pseudotsugata
chromosome
vlf-1
AAC59079


294
SCo_orf2
Streptomyces coelicolor
chromosome
int
CAC08306


295
BBu_orf
Borrelia burgdorferi
chromosome
orf6
AAC34963


296 
pNOB8
Sulfolobus sp.
plasmid pNOB8
orf101
T31031


297 
pMT1
Yersinia pestis
plasmid pMT1
T1101
T15016


298
ACa_vlf1
Autographica californica
chromosome
vlf-1
AAA66707


299
VCh_orf3
Vibrio cholerae
chromosome
VC0821
AAF96190


300
BMo_vlf1
Bombyx mori
chromosome
vlf-1
AAC63749


301 
phi-PV83
Staphylococcus aureus
phage PV83
int
BAA97808


302
PGi_orf
Porphyromonas gingivalis
chromosome
orf6
BAA35089


303
AFu_orf
Archaeoglobus fulgidus
chromosome
AF0082
B69260


304 
pCHL1
Chlamydia trachomatis
plasmid pCHL1
orf7
AAA91567


305 
pR27
Salmonella typhi
plasmid R27
orf
AAF70020


306 
APe_orf
Aeropyrum pernix
chromosome
APE0818
E72674


307
PSy_orf2
Pseudomonas syringiae
chromosome
orfA
CAB96965





plasmid




308
pNRC100_2
Halobacterium sp.
pNRC100
H0928
T08297


309
MJa_orf2
Methanococcus jannaschi
chromosome
MJ0770
Q58180


310 
phi16-3
Rhizobium sp.
phage 16-3
int
CAB54831


311 
pCP32-1
Borrelia burgdorferi
plasmid cp32-1 
BBP37
AAF07426


312 
SAl_orf
Streptomyces albus
chromosome
orf
AAD46512





plasmid




313
pNRC100_3
Halobacterium sp.
pNRC100
H1373
T08333


314
VCh_orf4
Vibrio cholerae
chromosome
VC0185
AAF93361


315
Tec2
Euplotes crassus
transposon Tec2
orf2B
AAA91341


316
Tec1
Euplotes crassus
transposon Tec1
orf2B
AAA91341


317
PPu_orf3
Pseudomonas putida
chromosome
orf101
CAB54061


318
pCP32
Borrelia hermsii
plasmid cp32
orf6
AAF28881


319
NMe_int
Neisseria meningitidis
chromosome
int
CAB84481


320 
pCP32-4
Borrelia burgdorferi
plasmid cp32-4 
BBR38
AAF07512


321 
pCP18
Borrelia burgdorferi
plasmid cp18
orf6
AAB63432


322 
pCP18-2
Borrelia burgdorferi
plasmid cp18-2 
orf27
AAF29799





transposon




323
Tn5401
Bacillus thuringensis
Tn5401
int
P27451


324 
SMi_xerD
Streptococcus mitis
chromosome
xerD
CAC19443


325
SPn_xerD
Streptococcus pneumoniae
chromosome
xerD
CAC19448


326
EFa_orf
Enterococcus faecium
chromosome
intD
AAG42074





phage VT1-




327
VT1
Escherichia coli O157:H7
Sakai
int
BAB19626


328
psiM100
Methanothermobacter wolfeii
phage psiM100
int
AAG39942


329
CP-933C
Escherichia coli O157:H7
phage 933C
Z1835
AAG55933


330
CP-933I
Escherichia coli O157:H7
phage 933I
Z0324
AAG54584


331
CP-933M
Escherichia coli O157:H7
phage 933M
Z1323
AAG55457


332
CP-933U
Escherichia coli O157:H7
phage 933U
intU
AAG57039


333
CP-933T
Escherichia coli O157:H7
phage 933T
intT
AAG56898


334
CP-933N
Escherichia coli O157:H7
phage 933N
intN
AAG55869


335
CP-9330
Escherichia coli O157:H7
phage 933O
intO
AAG56112


336 
bIL310
Lactococcus lactis
phage bIL310
orf1
AAK08405


337 
bIL311
Lactococcus lactis
phage bIL311
int
AAK08433


338 
SPy_orf5
Streptococcus pyogenes
chromosome
int4
AAK34767


339 
bIL309
Lactococcus lactis
phage bIL309
int
AAK08349


340 
bIL312
Lactococcus lactis
phage biL312
int
AAK08454


341 
SPy_orf2
Streptococcus pyogenes
chromosome
int3
AAK33851


342
SPy_orf4
Streptococcus pyogenes
chromosome
int2
AAK34288


343 
bIL286
Lactococcus lactis
phage bIL286
int
AAK08288


344 
LLa_xerD
Lactococcus lactis
chromosome
xerD
AAK04743


345
LLa_ymfD
Lactococcus lactis
chromosome
ymfD
AAK05330


346
SPy_orf3
Streptococcus pyogenes
chromosome
spy1196
AAK34058


347
SPy_orf1
Streptococcus pyogenes
chromosome
spy0365
AAK33410


348
LLa_orf2
Lactococcus lactis
chromosome
ynbA
AAK05376


349
ECo_orf7
Escherichia coli O157:H7 
chromosome
Z4313
AAG58098


350
ECo_orf6
Escherichia coli O157:H7 
chromosome
Z1120
AAG55265


351
pMLa
Mesorhizobium loti
plasmid pMLa
mll9356
BAB54967


352
pMLb
Mesorhizobium loti
plasmid pMLb
mlr9649
BAB54839


353
pRi_orf2
Rhizobium rhizogenes
plasmid pRi
ri136
BAB16255


354
MLo_orf1
Mezorhizobium loti
chromosome
mll8495
BAB54366


355
MLo_orf2
Mezorhizobium loti
chromosome
mll7973
BAB53631


356
MLo_orf3
Mezorhizobium loti
chromosome
mlr7741
BAB54140


357
MLo_orf4
Mezorhizobium loti
chromosome
mlr6952
BAB53138


358
SEn_orf2
Salmonella enterica
chromosome
int2
AF261825


359
MLo_orf5
Mezorhizobium loti
chromosome
mll5763
BAB52151


360
ECo_orf8
Escherichia coli
chromosome
ILG1
AAK49816


361
MLo_orf6
Mezorhizobium loti
chromosome
mlr0958
BAB48432


362
CCr_orf1
Caulobacter crescentus
chromosome
CC2681
AAK24647


363
MLo_orf7
Mezorhizobium loti
chromosome
mll4043
BAB50796


364
MLo_orf8
Mezorhizobium loti
chromosome
mll0487
BAB48065


365
MLo_orf9
Mezorhizobium loti
chromosome
mlr0475
BAB48054


366
phi-ETA
Staphylococcus aureus
phage phi-ETA 
orf1
BAA97587


367
CCr_xerD
Caulobacter crescentus
chromosome
CC3006
AAK24968


368
CCr_xerC
Caulobacter crescentus
chromosome
CC0344
AAK22331


369
pRVS1
Vibrio salmonicida
plasmid pRVS1 
int
CAC35342


370
phiSLT
Staphylococcus aureus
phage phi-SLT 
int
BAB21695


371
SSo_xer
Sulfolobus solfataricus
chromosome
xerCD
AAK40704





transposon




372
CW459
Clostridium perfringens
CW459
int459
AAK17958


373
MPu_xerC
Mycoplasma pulmonis
chromosome
MY5310
CAC13704


374
TVo_xerC
Thermoplasma volcanium
chromosome
xerC
BAB59407


375
TAc_xerC
Thermoplasma acidophilum
chromosome
Tal314
CAC12435


376
TVo_orf1
Thermoplasma volcanium
chromosome
orf1
BAB59869


377
SEn_orf2
Salmonella enterica
chromosome
S020
AAK02039


378
PMu_xerC
Pasteurella multocida
chromosome
xerC
AAK03785


379
PMu_xerD
Pasteurella multocida
chromosome
xerD
AAK02177


380
MLo_xerD
Mesorhizobium loti
chromosome
mlr3575
NP_104652


381
DRa_orf4
Deinococcus radiodurans
chromosome
xerD
AA-F12544


382
HSp_orf1
Halobacterium sp.
chromosome
ssrA
AAG19292


383
PMu_orf1
Pasteurella multocida
chromosome
slpA
AAK03853


384
PGi_xerC
Porphyromonas gingivalis
chromosome
PG1732



385
PGi_xerD
Porphyromonas gingivalis
chromosome
PG0386



386
RCa_orf3
Rhodobacter capsulatus
chromosome
orf
U57682


387
MLo_orf10 
Mesorhizobium loti
chromosome
mlr9321
NP_085850


388
MLo_orf11 
Mesorhizobium loti
chromosome
mlr9323
NP_085851


389
MLo_orf12
Mesorhizobium loti
chromosome
mlr9324
NP_085852


390
MLo_orf13
Mesorhizobium loti
chromosome
mll9328
NP_085856


391
MLo_orf14
Mesorhizobium loti
chromosome
mll9329
NP_085857


392
MLo_orf15 
Mesorhizobium loti
chromosome
mll9330
NP_085858


393
MLo_orf16
Mesorhizobium loti
chromosome
mll9331
NP_085859









In some embodiments, the suitable recombinases are the recombinases listed as numbers 7, 12, 93, 95, 97, and 98 in Table 1.
A method to identify an OSSR is by determining by identifying the catalytic residues. Identifying orthogonality is done by preparing a plasmid containing a gene of interest (such as Kanamycin resistance) that is flanked by putative recombinase recognition sites. Co-transformation of a cell with this plasmid and a plasmid containing the putative recombinase will either result in excision of the gene of interest or no reaction. If a reaction occurs, the cell will then be susceptible to treatment with Kanamycin. This is identifiable by replica plating of viable colonies onto an agar plate containing Kanamycin.
In some embodiments, an integration construct is constructed that can be integrated into a genome using lambda red (a promiscuous recombinase). The integration construct would comprise a promoter blocked by a terminator, with the terminator flanked by recognition sites of one of the six recombinases. By making competent cell stocks of each cell line, each recombinase gene is added as a plasmid and then the activity of the reporter gene is measured. This would determine if each protein was capable of working on multiple sequences.
The paper “DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages” (Sauer and McDermott, Nucleic Acids Research, 2004, Vol. 32, No. 20, 6086-6095; hereby incorporated by reference) performed a similar experiment using a multi-copy plasmid to check cross reactivity between the recombinase Cre and a homolog protein labeled Dre. A marker for Zeomycin resistance was placed between the recognition sites, and then cell lines were transformed with a separate plasmid containing the recombinase under study, either Cre or Dre. By growing these cells in the presence or absence of the antibiotic, a perfect record of viability was reported. When using the non-cross reacting gene, a perfect record of death was reported when using the appropriate gene to remove the resistance marker. This screen addresses point (2) listed above, that a recombinase will not bind to the recognition sites of another. However, this screen does not determine whether a mixed recognition site package (site cre, GENE, site dre, as well as the reverse) is capable of initiating recombination.
The recombinant nucleic acids of the invention can be constructed using methods well known to one skilled in the art. One such method includes the “West Coast BioBricks System” (BioBricks) for which separate constructs have been made for many recognition sites, promoters, resistance markers, and other biological device pieces. These pieces allow sequential assembly of complicated constructs. Exemplary components useful for the BioBricks system are shown in FIGS. 2-9. Coding Sequences (C) are complete open reading frames (type I), or sequences encoding polypeptides but lacking either a stop codon (type II), a start codon (type III), or both (type IV). Ribosome Binding Sites (RBS) are sequences encoding a ribosome binding site, fused 5′ to an ORF part. Terminators (TT) are sequence causing transcription termination.
One can use the BioBricks system to make constructs similar to those described in the referenced paper, with many different recognition sites flanking an antibiotic resistance marker. All relevant combinations will be made for each construct. For example, for recombinases A, B, and C that recognize sites a, b, and c, respectively, the following constructs can be made:

    
    
        a Resistance Marker1 a//Resistance Marker 2
        a Resistance Marker1 b//Resistance Marker 2
        a Resistance Marker1 c//Resistance Marker 2
        b Resistance Marker1 a//Resistance Marker 2
        b Resistance Marker1 b//Resistance Marker 2
        b Resistance Marker1 c//Resistance Marker 2
        c Resistance Marker1 a//Resistance Marker 2
        c Resistance Marker 1 b//Resistance Marker 2
        c Resistance Marker1 c.//Resistance Marker 2.
    
    


As described above, Marker 1 is removable but Marker 2 is not. Stocks of competent cells are made for each construct. These cells are then transformed with plasmids containing one or more recombinases each to cover all potential combinations. Those plasmids should also harbor resistance marker 3. These transformations are then be plated on agar plates containing antibiotics 2 and 3, and incubated to give rise to resultant colonies. Using the replica plating technique, colonies are then transferred to a plate containing antibiotic 1. Colonies are counted to assess viability.
In some embodiments, the recombinant nucleic acid further comprises a promoter that is upstream of the constructs and is capable of transcribing one or more of the constructs in a suitable host cell.
In some embodiments, the recombinant nucleic acid further comprises one or more target nucleotide sequences that are downstream of the constructs, wherein the one or more target sequences are transcribed when all of the constructs are deleted or excised. The target nucleotide sequences can encode an ORF, interference RNA, antisense RNA, or the like.
In some embodiments, the nucleotide sequence of interest comprises one or more ORF, interference RNA, antisense RNA, or the like, or one or more a terminator, or both thereof.
In some embodiments, the ORF encodes a polypeptide. The polypeptide can be a selective marker, an enzyme, a polypeptide that causes the death of the host cell in which the recombinant nucleic acid is located, or the like.
In some embodiments, each construct can further comprise a terminator located between the nucleotide sequence of interest and the second recognition sequence.
In some embodiments, the construct is located or inserted within two ORFs, that when the construct is excised from the recombinant nucleic acid, the two ORFs form a single ORF encoding a polypeptide of a certain biological function. In some embodiments, the certain biological function is one that causes the death of a host cell comprising the recombinant nucleic acid.
In some embodiment of the present invention, the recombinant nucleic acid comprises a synthetic telomere. The synthetic telomere is one application of multiple OSSRs that function as a counting mechanism for host cell, such as a bacteria, such as E. coli. The synthetic telomere makes use of cell cycle sensitive, low level expression of recombinases that, over time, cleaves inhibitory sequences from the genome, and concludes in the expression of a target gene. Such a device requires OSSRs that do not prevent miscounting, or worse, genomic scrambling. In some embodiments, the synthetic telomere is capable of a time dependence that based on the number of OSSRs present. The recombinases can be used to remove a section of the recombinant nucleic acid, such as DNA. Using the synthetic telomere, nucleotide sequence is removed as it is processed, preventing it from being read or interfering with the counting mechanism.
In some applications of the synthetic telomere, the synthetic telomere can function as a fuse that “burns down” until it reaches its target (i.e., the promoter becomes adjacent or operably linked to a gene or nucleotide sequence) and causes the expression of the gene or nucleotide sequence. The expression of the gene or nucleotide sequence can in turn directly or indirectly cause the activation or up regulation of the expression of one or more genes, and/or the repression or down regulation of the expression of one or more genes.
A synthetic teleomere functions as follows: (1) A suitable signal from the cell causes the transcription of the first recombinase. The second recombinase cannot be transcribed because of the presence of a terminator element. (2) Once the recombinase is translated and folded, the gene for the recombinase and the associated terminator element are excised by a recombination event. This DNA loop will be broken down by the host cell. (3) The next time the initiating signal fires, the second recombinase is transcribed. The third recombinase cannot be transcribed because of the presence of a terminator element. (4) The process repeats until all recombinase-terminator elements have been removed, and a reporter gene at the end of the sequence is expressed.
The synthetic teleomere acts as a time delay between the activation of transcription and the expression of a target gene. However, if the signal pulses are associated with a distinct phenomenon, such as cell cycles, night and day, or chemical washes, the invention now serves as a counting mechanism (albeit one that always counts down).
The present invention also provides for a recombinant vector comprising the recombinant nucleic acid. The recombinant nucleic acid can be a double-stranded or single-stranded DNA, or RNA. In some embodiments, the recombinant nucleic acid is integrated into a chromosome of a host cell. In some embodiments, the recombinant nucleic acid can further comprise sequences sufficient for having the recombinant nucleic acid stably replicate in a host cell. The recombinant nucleic acid can be replicon capable of stable maintenance in a host cell. In some embodiments, the replicon is a plasmid. The present invention also provides for a vector or expression vector comprising a recombinant nucleic acid of the present invention.
It will, be apparent to one of skill in the art that a variety of recombinant vectors can be utilized in the practice of aspects of the invention. As used herein, “vector” refers to polynucleotide elements that are used to introduce recombinant nucleic acid into cells for either expression or replication. Selection and use of such vehicles is routine in the art. An “expression vector” includes vectors capable of expressing DNAs that are operatively linked with regulatory sequences, such as promoter regions. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those that integrate into the host cell genome.
Selectable markers can also be included in the recombinant expression vectors. A variety of markers are known which are useful in selecting for transformed cell lines and generally comprise a gene whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium. Such markers include, for example, genes that confer antibiotic resistance or sensitivity.
Methods for introducing the recombinant vectors of the present invention into suitable hosts are known to those of skill in the art and typically include the use of CaCl2 or other agents, such as divalent cations, lipofection, DMSO, protoplast transformation, conjugation, and electroporation.
Activating expression from the promoter results in the expression of the first recombinase which in turn proceeds to recognize the two recognition sequence of the first recombinase and excise or delete the first nucleotide sequence of interest. Activating expression of each respective recombinase results in the recognition of the two recognition sequence of the respective recombinase and the excision or deletion of the corresponding nucleotide sequence of interest.
In some embodiments, the method further comprises: introducing a recombinant nucleic acid into a host cell prior to the providing step, wherein the recombinant nucleic acid comprises at least the first construct and the second construct.
The present invention further provides for a compositions and methods attenuating signal (non-coding RNA interference and Dominant-Negative complexation) by coupling to the activation of a recombinase, an enzyme which can toggle a DNA element between two states. A target DNA element can be designed to, the difference between the two states is essentially arbitrary and almost unlimited. It is not necessary that the same gene product be produced in both states. When used in traditional systems, these signal attenuation methods are limited by the fact that the intensity of the desired output is directly tied to the level of attenuation. When recombinases are used in traditional systems, they are typically limited by the noise generated in attempting to control the recombinase gene, which can cause the switch to enter the improper state. It has not been previously demonstrated that these various devices can be operated in concert to both: (1) have greater control over the properties of both devices, and (2) produce unique genetic devices that would not be possible without the marriage of these functions.
Non-coding RNA interference is a method of preventing protein expression from a given mRNA. by the introduction of a second, non-coding RNA (hereafter ncRNA) that renders the target mRNA unreadable by the protein synthesis machinery. A number of design styles have been used to demonstrate this, but the key component is that the non-coding RNA typically binds the coding RNA in a 1:1 ratio. Thus, an effect is produced in direct proportion with the number of readable copies, where readable copies=(mRNA−(non-coding RNA)), modulated by the binding constant. This system is itself subject to noise near the equivalence point, but is otherwise a very tuneable system.
Dominant-Negative complexation involves intentional production of a non-functional version of the active enzyme (usually a point mutation in the active site) that attenuates the active enzyme in two ways: (1) Competition for the binding site, and (2) Interference in the formation of functional multimers. In both cases, the level of attenuation is a function of concentration of the active enzyme relative to inactive enzyme. However, in systems where every subunit of a complex must be functional to produce an active enzyme (as with recombinases), the multimer effect allows even small amounts of inactive protein to nullify an otherwise-active complex. The intensity of this effect is increased as the number of monomers required in the active complex grows (recombinases are functional tetramers.) Of particular importance is that the recombinase reaction is fully reversible if it does not go to completion! Therefore, a “mixed” multimer of functional and non-functional monomers will not produce a side-product, but will instead continue to suppress function of active multimers by competing for the binding site.
Recombinases are unique among DNA manipulating enzymes for several reasons: (1) The output of their function is digital. When properly designed, the two states can be assigned any arbitrary function. The intensity or type of activation event has no mandatory influence on the two states in any way. It is even possible to toggle a switch between a “sensitive” and “insensitive” state, so that the output can be made dependent or independent to any other control mechanism. (2) The mechanism of action allows for large numbers of recombinases to be used without concern for exhausting available sites or causing cross reactions between multiple sites. Thus one well-designed circuit could easily be adapted for a different output, and multiple circuits could be used in the same cell. This is important in the construction of logic circuits or decision trees that require multiple events recognized over time before a final state is reached. (3) In addition to not interfering with each other, recombinases have a very limited set of required interactions to perform their function: they need to bind identical monomers and bind to DNA. By not involving any other cellular machinery in their function, the system is simplified and the risk of side reactions and cell overload is reduced.
Two different modes of use are possible, integrated/excised or “facing left”/“facing right”. To simplify, further circuits are described using the “facing left”/“facing right” convention although this system works with both designs. The main difference between the two modes is that integrated/excised is less reversible and thus less likely to be scrambled. However, it functions by removing a DNA element, so genetic information is lost permanently when the switch is activated. Alternatively, the “facing left/facing right” circuit is reusable, but can lead to scrambling of the switch if not properly controlled.
A system, for noise canceling in recombinase circuits, works as follows (FIG. 14):
1. A constitutive promoter is followed by a non-coding, interfering RNA, producing that RNA at a set level.
2. An inducible promoter is followed by a recombinase gene. This mRNA is interfered with by the product of promoter 1.
3. Leaky expression of the inducible promoter does not lead to recombinase expression, and the switch remains “unflipped”
4. Induction of promoter 2 produces recombinase mRNA in sufficient quantity that it cannot entirely be inhibited, and recombinase is produced.
5. The produced recombinase “flips” the switch.
Another system follows a similar plan, but uses a Dominant-Negative enzyme (FIG. 15):
1. A constitutive promoter is followed by an active-site knockout of a recombinase, producing RNA and protein at a set level.
2. An inducible promoter is followed by a functional recombinase.
3. Leaky expression of the inducible promoter does not lead to recombinase function, and the switch remains “unflipped”.
4. Induction of promoter 2 produces recombinase in sufficient quantity that (a) functional tetramers form and (b) can compete with non-functional tetramers for DNA binding sites.
5. The functional recombinase tetramers “flip” the switch.
Note that system requires lower levels of its inhibitory product to cause inhibition. This makes it stronger, but also harder to titrate exact levels. The above two systems allow for noise canceling. By replacing promoter 1 with an inducible promoter, the threshold of activation is no longer a static value, and the amount of induction required for flipping the switch changes based on the cellular environment. This is summarized for both types of systems in FIG. 16. This is an extremely useful and powerful device. The inducible promoters can be of any type, with particular applications that can be tied to the activation and deactivation of different metabolic pathways in the cell because of the sensitivity of measuring relative abundance. The flipping of switches can be tied to growth, or change in carbon or nitrogen source. The systems can be based on either attenuation method.
With careful system construction any two promoters can have an output tied to a change in their expression level ratio rather than in absolute abundance, if they dominant-negative system is used. This is because various ribosome binding sites can be used to alter the relationship between RNA level and protein level to a range where the system responds.
The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.
Example 1
A Fusebox Expression Cassette
An embodiment of the invention is a nucleic acid comprising a nucleotide sequence comprising: promoter—ribosome binding site—recognition site 1a—terminator—recognition site 1b—recognition site 2a—terminator—recognition site 2b—gene of interest, the gene of interest cannot be expressed until the sequence is removed of terminators by treatment with both recombinase 1 and recombinase 2. The expression cassettes for recombinase 1 and 2 do not need to be part of the named construct, nor do they need to be activated simultaneously (hence the name “fusebox”). Once each of the recombinases is activated a single time, it activates a permanent change in cellular state, bringing the cell closer to the expression of the gene of interest. Such a system would allow for multiple, non-simultaneous “checkpoints” to be identified before the gene of interest would be expressed. FIG. 12 shows a representative fusebox expression cassette. A system like this allows for the one-time recognition of multiple signals that result in a permanent change in the cell state. The two (or more) signals do not need to occur simultaneously. This system requires no feedback loop and is low load on the cell.
Example 2
A Synthetic Telomere
FIG. 13 shows a representative synthetic teleomere. This device employs recombinases to catalyze the excision of their own gene from a genomic insert. Only one recombinase can be expressed per promoter cycle, resulting in a change in cell state after a given number of events.
Example 3
Cross Testing of Recombinases and Circuit Design
This example demonstrates the efficacy of the cross testing method, initiation the development of our compatibility grid, and the design a circuit using the information thus gained.
Three of the most commonly used (in recombinant systems) recombinases are tested: cre, dre, and FlpSc (here the Sc annotation means from S. cerevisiae), each paired with one of their better characterized att sites:









lox, acted on by Cre:



(SEQ ID NO: 1)



ATAACTTCGTATAGCATACATTATACGAAGTTAT,



 



rox, acted on by Dre:



(SEQ ID NO: 2)



TAACTTTAAATAATGCCAATTATTTAAAGTTA,



and



 



frt:



(SEQ ID NO: 3)



TTTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGG



AACTT.






As multiple sequences for multiple att sites appear across publications, an internal reference code is prepared for each att site. For example, the above sites are referred to as CreA, DreA, and ScA, respectively. This allows keeping track of the various sequences independent of their references while still retaining the reference and sequence information for later publication. It should also be noted that each recombinase has more than a single att site. Most have either two or four, although for commonly used proteins like lox, more are known. These sites need to be cross-tested against each other as well as with att sites that putatively respond to a different recombinase. Such experiments produce independent, non-cross reactive effects across an entire genome with a single enzyme.
Two separate classes of constructs are prepared:
Recombinase constructs: p15A origin, Cm resistance, pTET promoter, and one or two recombinases in an operon.
Att excision constructs: ColE1 origin, Amp resistance, pLacUV5 promoter, attx—Red Fluorescent Protein—atty, where attx and atty are various att recognition sites described above.
These vectors are then co-transformed into E. coli strain DH10B, outgrown for one hour in SOC media, and plated on LB plates containing Ampicillin, Chloroamphenicol, IPTG (to induce RFP expression) and aTc (to induce recombinase expression). Although RFP is induced, the constructs are tested using colony PCR to determine if the corresponding part of the test construct is short, indicating an excision product, or long, indicating that RFP has not been excised. These tests can also be performed using more recombinases and att sites, as well as different copy number of the target plasmid and multiple excision targets, including Kan resistance and removal of a terminator that is blocking beta-galactosidase production (i.e. an “on” switch).
Below, these pairs are drawn out in a grid. The grid is filled in with the expected behavior of each crosstest. Table 2 shows the expected cutting patterns for various combinations of recombinases and att sites.














TABLE 2







CreA-
DreA-
ScA-
CreA-
DreA-
CreA-



CreA
DreA
ScA
ScA
ScA
DreA






















Cre
cut
no cut
no cut
no cut
no cut
no cut


Dre
no cut
cut
no cut
no cut
no cut
no cut


FlpSc
no cut
no cut
cut
no cut
no cut
no cut


Cre-Dre
cut
cut
no cut
no cut
no cut
no cut


Dre-FlpSc
no cut
cut
cut
no cut
no cut
no cut


Cre-FlpSc
cut
no cut
cut
no cut
no cut
no cut









As can be see in the right half of Table 2, the absence of cutting on “mis-matched” att sites is critical to the function of our recombinase circuits. While it would be difficult to test greater numbers of recombinases or sites simultaneously, we hope to map out possible conflicts by analyzing each pairwise combination. If future discrepancies arise in more complicated systems, using this information will allow us to more readily identify the cause of the incompatability.
FIG. 14 is a sample gel showing the results the above cross tests. The cross-tests involving Cre, Dre, Cre-Dre and the various site combinations give the expected results quite cleanly by colony PCR analysis. FIG. 14 combines three separate gels and is coded to show the excision behavior of the recombinases on the left when expressed in the presence of the att sites shown above, where those att sites are flanking an RFP production cassette. As standards, there are an unexcisable RFP cassette that can be amplified using the same PCR primers as in the experiment, as well as a “pseudoscar” PCR product that corresponds to the size of a CreA scar after a successful CreA-CreA excision. The PCR primers are universal to the test construct and bind outside of the att sites in order to perform this analysis.
In each set of experiments, the desired behavior is observed. Most importantly, when Cre and Dre are co-expressed, the mixture can correctly excise a CreA-CreA construct, a DreA-DreA construct, but shows no activity on a CreA-DreA construct. This was not necessarily true because the proteins are so similar that antibodies raised to Cre will bind to Dre in a western blot. The exact reason for this relationship is not known, but may have to do with the nature of the Holliday junction resolution (i.e., which nucleotides immediately flank the recombination site) rather than the protein quaternary structure.
A number of cross tests are also performed using FlpSc. Surprisingly, FlpSc is a “bad partner” in crosstests involving Cre. Since FlpSc and Cre are not even in the same sub-family, this behavior is not expected. This result may be related to copy number or att site choice or FlpSc is a less accessible enzyme for advanced circuit design.
The current data of the compatibility grid is in Table 3 below.














TABLE 3







CreA-
DreA-
ScA-
CreA-
DreA-
CreA-



CreA
DreA
ScA
ScA
ScA
DreA






















Cre
cut
no cut
some
no cut
no cut
no cut





cut


Dre
no cut
cut
no cut
no cut
no cut
no cut


FlpSc
some
no cut
cut
no cut
no cut
no cut



cut


Cre-Dre
cut
cut
no cut
no cut
no cut
no cut


Dre-FlpSc
no cut
cut
cut
N/D
N/D
N/D


Cre-FlpSc
cut
no cut
cut
N/D
N/D
N/D









It currently appears that both Cre and Dre show some cross activity on at least one att site that is normally associated with the other protein. Although it may be possible to work around this problem by using alternate aft sites, other behaviors of FlpSc suggest that screening other recombinases will prove more immediately fruitful. When FlpSc is co-expressed with another protein, we have been unable to obtain a PCR product when we analyze the colonies. FlpSc may be reacting between sites on multiple plasmids, a behavior reported in the literature for Cre and Dre, but this not yet conclusively verified. The inclusion of copy number variation experiments and changing the reporter to Kanamycin resistance and testing for cell survivability, as described previously, may resolve this.
Using the information obtained regarding the compatibilities of Cre and Dre recombinases, an “or” gate is designed as shown in FIG. 15. In this circuit, neither gene A nor gene B can be expressed from their promoters because of the presence of a strong, bi directional terminator in the middle. By differentially expressing either cre or dre, either the white att sites or the striped att sites will excise, respectively. This will allow one of the two target genes to express, but will prevent expression of the other gene by removing its promoter. As described herein, one can control cell fate by differential recombinase expression and can also build towards more complex circuits as additional compatible parings are determined. This circuit for the desired activity can be tested in an agar-plate based assay, where, in the image shown in FIG. 19. The agar-plate based assay comprises the following: Gene A—RFP, Gene B—GFP, promoters in top construct are lacUV5, cre promoter is pTET and dre promoter is pBAD. An agar plate is grown to a lawn, then small disks of filter paper that have been soaked in either aTC (to induce pTET) or arabinose (to induce pBAD) are added to either side of this dish. After diffusion of the inducer, one expects to see a plate as shown below, where no cells produce both fluorescent proteins. As a control, one directly expresses GFP from pBAD and RFP from pTET, in which case the entire plate should express both proteins.
Example 4
New Aft Sites that Make an Orthogonal Pair but Still Respond to an Existing Recombinase
As discussed above, the following describes a method for discovering new att sites that make an orthogonal pair but still respond to an existing recombinase. For example, there are many pairs of sites known that the Cre recombinase can act on, but that the individual sites themselves are not competent to resolve, and thus form exclusive pairs. The information gained from performing the crosstesting work, together with sequence information of the known competent pairs of att sites allows one to determine which residues of the crossover region of the att sites generate this site-match specificity that is independent of the recombinase recognition affinity. Increasing the number of att sites that respond to a single recombinase has great potential for introducing genome-wide modifications in a living organism simultaneously and without side-reactions in response to a single stimulus event. Such a tool would be useful both in optimization of fermentative production of small molecules and in the control of genetically engineered organisms in the field, for example plants that begin breaking down their own cellulose in response to a certain signal.
A primary concern in the use of recombinase based circuits is that since the input signal intensity is effectively decoupled from the output signal intensity (i.e. a different promoter can be used for the recombinase and the target gene, as shown in the “or” gate circuit), there may be problems with noise and fidelity. Unlike traditional promoter systems which respond to a variety of inducer concentrations, a recombinase circuit can be a “single fire” system: as soon as the target DNA sequence is excised (or inverted, in some designs), the new circuit becomes active and typically cannot be undone in a controlled fashion. As such controlling more complex circuits requires compensation for this sensitivity and unidirectional. Allowing the att sites and inverted DNA sections to remain in the cell is more likely to scramble the circuit than to allow for the generation of more intricate pathways. However, since the system is effectively completely irreversible, a mechanism is needed to prevent noisy signal from cutting our circuits. This can be done by expressing dominant negative recombinases along side the functional circuits, as described herein, that is using dominant-negative repression and non-coding RNA interference for tuning recombinase based genetic circuits to allow for decoupling of input and output signal strengths by setting a threshold effect (i.e. band-pass filter).
In a simple system, dominant negative recombinase monomers can be expressed with a constitutive promoter of known output. This drastically reduces the chance that 4 functional monomers can access the target sequence and fire until they are in a concentration in excess of the dominant negative monomers. One then measures the ratio of inactive to active monomers that allows activation. Over time, any circuit could fire regardless of the amount of inactive monomer present, so one can test these constructs in longitudinal experiments to assess their stability, which is especially relevant for field applications. The dominant negative recombinases are expected to be specific for their parent construct. Thus, expressing CreX (where the X signifies the inactive dominant-negative mutant) inhibits the activity of Cre but not Dre, while expressing DreX inhibits the activity of Dre and not Cre. By differentially expressing both CreX and DreX, one is able to set independent thresholds for the activity of each enzyme. This type of behavior is useful for therapeutic applications, where a “tumor-hunter” bacteria must carefully calibrate multiple signals in identifying a tumor before it is allowed to initiate production of a toxic chemical.
In another application, the dominant-negative mutant is put under control of a variable strength promoter, while the active recombinase is also under control of a variable strength promoter. As before, when thee construct is tuned so that the initial active:inactive ratio is below the activity threshold, it will not fire. In this case however, since both promoter strengths are changing, the difference in initial promoter strength can be lower (while protein copy number difference is maintained through differences in ribosome binding site strength). This allows the use of native promoter mechanisms to control a circuit in response to a change in cell physiology. This allows access to more “hands-off” circuits, where a cell responds to natural changes in its own environment rather than induced changes (i.e., adding IPTG or arabinose), and to do so in such a way where a strong response can still be generated by use of the active recombinase causing a much stronger promoter to join up to a target DNA.
This approach is also useful in that, in many circuit designs, one can think of a circuit as being made of “cheap” DNA and “expensive” proteins. In other words, it is energetically costly to maintain a protein gradient of activator and repressor pairs, but energetically cheaper to maintain the DNA that encodes those proteins and the DNA that encodes the target DNA elements they act upon. Since recombinases can induce a permanent change in the DNA of a cell without the constant presence of the protein, circuits can be designed that excise both the recombinase and the dominant negative mutant when they are no longer needed, lowering the protein burden on the cell, and only then expressing the next set of necessary proteins. Using this “sliding window” approach allows the circuit to progress towards completion without an exponential increase in protein production to maintain it.
A knockout of Dre, named DreX(Y324F) is prepared. Tyrosine Y324 is annotated as the active site tyrosine for this enzyme. However, after replacing this residue with phenylalanine, as determined in the sequencing result shown below, one still observes activity of the enzyme using our crosstesting method described herein. See FIG. 20. Activity identical to the native Dre even when DreX(Y324F) is expressed alone. This annotation as the active site residue is incorrect and have moved on to knocking out additional tyrosine residues in Dre, Cre, and FlpSc. This process continues until active site knockouts are identified. One then determines the activation threshold for each pair, determines if the dominant negative pairs are exclusive through crosstesting, and initiates longitudinal studies to determine the fidelity of the thresholds through time.
Example 5
Method of Searching for and Using Orthogonal Site Selective Recombinases
Seven recombinases (Cre, Dre, FlpSc, FlpZb, FlpZf, ΦC31, and Bxb1, where Sc Zb and Zf are different species of yeast) are analyzed for compatibility (ability to act specifically and independently upon their own attachment, att, sites when present in a single cell) in an experimental system (see Tables 4-7). Each is able to produce the desired recombination product using its cognate att sites in our system. Cre is compatible with Dre under some conditions, although some recombination is observed upon sequences flanked by mismatched CreA-DreA att sites (half-sites). This is the only half-site combination that resulted in an excision product. Analysis of the CreA and DreA sites suggest that a functional Holliday junction could be formed, although the identity of the cleavage product has not been determined. Dre and FlpSc are compatible under all conditions, while FlpSc and Cre demonstrated cross-reactivity. The remaining four recombinases show high levels of cross reactivity with non-cognate att sites.
The incompatibility between Cre and Dre is easily resolvable by changing the bases of the crossover region to make CreA and DreA an incompatible pair (one that cannot form a holiday junction that permits strand exchange.) The incompatibility between FlpSc and Cre may not be resolvable, but this pairing (and many additional enzymes and binding sites) is currently being testing using a plate based assay that significantly increases our analytical throughput. A flowchart of this process is shown in FIG. 21.














TABLE 4






CreA-
DreA-
ScA-
ZbA-
ZfA-
ΦC31B
Bxb1B-



CreA
DreA
ScA
ZbA
ZfA
ΦC31B
Bxb1B







Cre
12/12
 0/12
12/12
12/12
12/12
12/12
12/12


Dre
 0/12
12/12
 0/12
12/12
12/12
12/12
11/12


FlpSc
10/12
 0/11
12/12
8/8
8/8
12/12
10/11


FlpZb
12/12
12/12
12/12
12/12
12/12
12/12
12/12


FlpZf
12/12
12/12
11/12
12/12
12/12
12/12
12/12


ΦC31
12/12
12/12
12/12
12/12
11/12
12/12
11/12


Bxb1
10/12
 7/10
10/11
12/12
12/12
12/12
8/8





X/Y - fraction of colonies with observed excision


Bold = desired


Normal = undesired













TABLE 5







Cre and Dre appear to form a highly compatible pair, as do 


Dre and FlpSc. FlpSc and Cre cross react, and all other 


combinations show a high incidence of background activity. Only 


Cre, Dre, and FlpSc are further analyzed for cross-compatability.











X/Y - fraction of colonies with observed excision


Bold = desired


Normal = undesired













TABLE 6







Cross compatability tests are performed by expression 


one or two enzymes in the presence of either “full” targets 


(2 identical att sites) or “half” targets (2 non identical att sites).


The first 9 boxes are carried over from Table 5.











X/Y - fraction of colonies with observed excision


Bold = desired


Normal = undesired













TABLE 7







The blue shaded boxes show that while Dre does not change it's 


behavior under any new conditions, Cre alone is able to recombine 


one CreA site + one DreA site. Further analysis of these att sites 


suggests a change in the crossover region will resolve this problem. 


Cre and FlpSc continue to show cross-cutting behavior in the presence


of Dre. However, neither Cre nor FlpSc show activity on Cre cognate


sites when co-expressed (creA-creA column/Cre-FlpSc row).











X/Y - fraction of colonies with observed excision


Bold = desired


Normal = undesired






While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

## Claims

1. A recombinant nucleic acid comprising a nucleotide sequence comprising (a) a plurality of constructs comprising a first pair of recombinase recognition sequences which is recognized by a first recombinase, wherein the first pair of recombinase recognition sequences flank a first terminator, and a second pair of recombinase recognition sequences which is recognized by a second recombinase, wherein the second pair of recombinase recognition sequences flank a second terminator, (b) a promoter upstream of the plurality of constructs capable of transcribing the plurality of constructs, wherein each construct independently comprises a nucleotide sequence of interest flanked by a pair of recombinase recognition sequences, wherein each pair of recombinase recognition sequences is recognized by a recombinase that does not cross-react with the recombinase recognition sequences of another construct, and the two recombinase recognition sequences of each pair are oriented in the same direction to each other, and (c) a target gene downstream of the plurality of constructs; wherein the first recombinase and the second recombinase are (i) Escherichia coli Cre and Dre, or (ii) Dre and Saccharomyces cerevisiae FLP.

2. The recombinant nucleic acid of claim 1 wherein each construct independently further comprises one or more genes encoding a recombinase capable of recognizing the pair of recombinase recognition sequences of the construct.

3. A vector comprising the recombinant nucleic acid of claim 1.

4. An ex vivo host cell comprising the vector of claim 3.

5. The recombinant nucleic acid of claim 1, wherein the target gene encodes an open reading frame (ORF), interference RNA, or antisense RNA.

6. The recombinant nucleic acid of claim 5, wherein the target gene encodes the ORF and the ORF encodes a selective marker, an enzyme, or a polypeptide that cause the death of a host cell.

7. The recombinant nucleic acid of claim 1, wherein the nucleotide sequences of interest are terminators.

8. The recombinant nucleic acid of claim 1, wherein each nucleotide of interest comprises (i) an open reading frame (ORF) of a recombinase which recognizes the pair of recombinase recognition sequences flanking the nucleotide of interest, and (ii) a terminator downstream of the ORF.

9. The recombinant nucleic acid of claim 8, wherein the target gene encodes an open reading frame (ORF) and the ORF encodes a polypeptide that cause the death of a host cell.

10. The recombinant nucleic acid of claim 1, wherein the first pair of recombinase recognition sequences flank a first gene encoding the first recombinase wherein the first gene is upstream of the first terminator, and the second pair of recombinase recognition sequences flank a second gene encoding the second recombinase wherein the second gene is upstream of the second terminator.