import os
from pathlib import Path
import libsbml
from combine_notebooks.helper_functions.utils import *
from combine_notebooks.validation.validation_sbml import validate_sbml
def create_repressilator(sbml_path: Path) -> libsbml.SBMLDocument:
"""Create repressilator."""
print("Create SBML model")
# create a new document
doc: libsbml.SBMLDocument = libsbml.SBMLDocument(3, 2)
model: libsbml.Model = doc.createModel()
# --- compartments ---
# add compartment and set annotation
c1 = add_compartment(model, "cell", 1.0, "meta_cell", "SBO:0000290")
c1_cv = create_annotation(
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS,
"http://identifiers.org/GO:0005623",
)
c1.addCVTerm(c1_cv)
# --- species ---
# add species for the 3 mRNAs and 3 Proteins and set annotations
s1 = add_species(model, "PX", "PX", "cell", "LacI protein", "SBO:0000252", 0, True)
s1_cv = create_annotation(
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS,
"http://identifiers.org/uniprot/P03023",
)
s1.addCVTerm(s1_cv)
s2 = add_species(model, "PY", "PY", "cell", "TetR protein", "SBO:0000252", 0, True)
s2_cv = create_annotation(
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS,
"http://identifiers.org/uniprot/P04483",
)
s2.addCVTerm(s2_cv)
s3 = add_species(model, "PZ", "PZ", "cell", "cI protein", "SBO:0000252", 0, True)
s3_cv = create_annotation(
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS,
"http://identifiers.org/uniprot/P03034",
)
# example setting a model qualifier
# s3_cv.setQualifierType(libsbml.MODEL_QUALIFIER)
# s3_cv.setBiologicalQualifierType(libsbml.BQM_IS)
s3.addCVTerm(s3_cv)
s4 = add_species(model, "X", "meta_X", "cell", "LacI mRNA", "SBO:0000250", 0, True)
s4_cv = create_annotation(
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/CHEBI:33699",
)
s4_cv = add_annotation_resource(
s4_cv, libsbml.BQB_IS_VERSION_OF, "http://identifiers.org/kegg.compound/C00046"
)
s4.addCVTerm(s4_cv)
s4_cv = add_annotation_resource(
s4_cv, libsbml.BQB_ENCODES, "http://identifiers.org/uniprot/P03023"
)
s4.addCVTerm(s4_cv)
s5 = add_species(model, "Y", "meta_Y", "cell", "TetR mRNA", "SBO:0000250", 20, True)
s5_cv = create_annotation(
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/CHEBI:33699",
)
s5_cv = add_annotation_resource(
s5_cv, libsbml.BQB_IS_VERSION_OF, "http://identifiers.org/kegg.compound/C00046"
)
s5.addCVTerm(s5_cv)
s5_cv = add_annotation_resource(
s5_cv, libsbml.BQB_ENCODES, "http://identifiers.org/uniprot/P04483"
)
s5.addCVTerm(s5_cv)
s6 = add_species(model, "Z", "meta_Z", "cell", "cl mRNA", "SBO:0000250", 0, True)
s6_cv = create_annotation(
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/CHEBI:33699",
)
s6_cv = add_annotation_resource(
s6_cv, libsbml.BQB_IS_VERSION_OF, "http://identifiers.org/kegg.compound/C00046"
)
s6.addCVTerm(s6_cv)
s6_cv = add_annotation_resource(
s6_cv, libsbml.BQB_ENCODES, "http://identifiers.org/uniprot/P03034"
)
s6.addCVTerm(s6_cv)
# --- parameters ---
# <parameter id="tau_mRNA" name="mRNA half life" value="2" constant="true"/>
add_parameters(model, "tau_mRNA", "mRNA half life", True, value=2.0)
# <parameter id="kd_mRNA" constant="false" name="kd_mRNA" sboTerm="SBO:0000356">
add_parameters(model, "kd_mRNA", "kd_mRNA", False, sbo_term="SBO:0000356")
# <parameter metaid="metaid_0000233" sboTerm="SBO:0000016" id="k_tl" name="k_tl" constant="false">
add_parameters(model, "k_tl", "k_tl", False, sbo_term="SBO:0000016")
# <parameter metaid="metaid_0000025" id="eff" name="translation efficiency" value="20">
add_parameters(model, "eff", "translation efficiency", True, value=20)
# <parameter metaid="metaid_0000032" sboTerm="SBO:0000348" id="t_ave" name="average mRNA life time" constant="false"/>
add_parameters(
model, "t_ave", "average mRNA life time", False, sbo_term="SBO:0000348"
)
# <parameter metaid="metaid_0000133" sboTerm="SBO:0000356" id="kd_prot" name="kd_prot" constant="false">
add_parameters(model, "kd_prot", "kd_prot", False, sbo_term="SBO:0000356")
# <parameter metaid="metaid_0000128" sboTerm="SBO:0000332" id="tau_prot" name="protein half life" value="10"/>
add_parameters(model, "tau_prot", "protien half life", True, value=10)
# <parameter metaid="metaid_0000234" sboTerm="SBO:0000485" id="a0_tr" name="a0_tr" constant="false">
add_parameters(model, "a0_tr", "a0_tr", False, sbo_term="SBO:0000485")
# <parameter metaid="metaid_0500235" sboTerm="SBO:0000485" id="ps_0" name="tps_repr" value="0.0005">
add_parameters(model, "ps_0", "tps_repr", True, value=0.0005)
# <parameter metaid="metaid_0900235" sboTerm="SBO:0000186" id="a_tr" name="a_tr" constant="false">
add_parameters(model, "a_tr", "a_tr", False, sbo_term="SBO:0000186")
# <parameter metaid="metaid_0800235" sboTerm="SBO:0000186" id="ps_a" name="tps_active" value="0.5">
add_parameters(model, "ps_a", "tps_active", True, value=0.5)
# <parameter metaid="metaid_0000027" sboTerm="SBO:0000288" id="KM" name="KM" value="40">
add_parameters(model, "KM", "KM", True, value=40)
# <parameter metaid="metaid_0000026" sboTerm="SBO:0000190" id="n" name="n" value="2">
add_parameters(model, "n", "n", True, value=2.0)
# TODO: a0_tr, a_tr, n, KM
# --- rules ---
add_assignment_rule(model, "kd_mRNA", "ln(2) / tau_mRNA")
add_assignment_rule(model, "t_ave", "tau_mRNA / ln(2)")
add_assignment_rule(model, "k_tl", "eff / t_ave")
add_assignment_rule(model, "kd_prot", "ln(2) / tau_prot")
add_assignment_rule(model, "a0_tr", "ps_0 * 60")
add_assignment_rule(model, "a_tr", "(ps_a - ps_0) * 60")
# --- reaction ---
# X -> None
r1 = add_reactions(
model,
"Reaction1",
"meta_Reaction1",
"SBO:0000179",
"degradation of LacI transcripts",
False,
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/GO:0006402",
"kd_mRNA * X",
"SBO:0000049",
)
add_reactant_to_reaction(r1, "X")
r2 = add_reactions(
model,
"Reaction2",
"meta_Reaction2",
"SBO:0000179",
"degradation of TetR transcripts",
False,
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/GO:0006402",
"kd_mRNA * Y",
"SBO:0000049",
)
add_reactant_to_reaction(r2, "Y")
r3 = add_reactions(
model,
"Reaction3",
"meta_Reaction3",
"SBO:0000179",
"degradation of CI transcripts",
False,
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/GO:0006402",
"kd_mRNA * Z",
"SBO:0000049",
)
add_reactant_to_reaction(r3, "Z")
r4 = add_reactions(
model,
"Reaction4",
"meta_Reaction4",
"SBO:0000184",
"translation of LacI",
False,
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/GO:0006412",
"k_tl * X",
"SBO:0000049",
)
add_product_to_reaction(r4, "PX")
add_modifier_to_reaction(r4, "X", "SBO:0000461")
r5 = add_reactions(
model,
"Reaction5",
"meta_Reaction5",
"SBO:0000184",
"translation of TetR",
False,
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/GO:0006412",
"k_tl * Y",
"SBO:0000049",
)
add_product_to_reaction(r5, "PY")
add_modifier_to_reaction(r5, "Y", "SBO:0000461")
r6 = add_reactions(
model,
"Reaction6",
"meta_Reaction6",
"SBO:0000184",
"translation of CI",
False,
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/GO:0006412",
"k_tl * Z",
"SBO:0000049",
)
add_product_to_reaction(r6, "PZ")
add_modifier_to_reaction(r6, "Z", "SBO:0000461")
r7 = add_reactions(
model,
"Reaction7",
"meta_Reaction7",
"SBO:0000179",
"degradation of LacI",
False,
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/GO:0030163",
"kd_prot * PX",
"SBO:0000049",
)
add_reactant_to_reaction(r7, "PX")
r8 = add_reactions(
model,
"Reaction8",
"meta_Reaction8",
"SBO:0000179",
"degradation of TetR",
False,
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/GO:0030163",
"kd_prot * PY",
"SBO:0000049",
)
add_reactant_to_reaction(r8, "PY")
r9 = add_reactions(
model,
"Reaction9",
"meta_Reaction9",
"SBO:0000179",
"degradation of CI",
False,
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/GO:0030163",
"kd_prot * PZ",
"SBO:0000049",
)
add_reactant_to_reaction(r9, "PZ")
r10 = add_reactions(
model,
"Reaction10",
"meta_Reaction10",
"SBO:0000183",
"transcription of LacI",
False,
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/GO:0006351",
"a0_tr + (a_tr * power(KM, n)) / (power(KM, n) + power(PZ, n))",
None,
)
add_product_to_reaction(r10, "X")
add_modifier_to_reaction(r10, "PZ", "SBO:0000536")
r11 = add_reactions(
model,
"Reaction11",
"meta_Reaction11",
"SBO:0000183",
"transcription of TetR",
False,
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/GO:0006351",
"a0_tr + (a_tr * power(KM, n)) / (power(KM, n) + power(PX, n))",
None,
)
add_product_to_reaction(r11, "Y")
add_modifier_to_reaction(r11, "PX", "SBO:0000536")
r12 = add_reactions(
model,
"Reaction12",
"meta_Reaction12",
"SBO:0000183",
"transcription of CI",
False,
libsbml.BIOLOGICAL_QUALIFIER,
libsbml.BQB_IS_VERSION_OF,
"http://identifiers.org/GO:0006351",
"a0_tr + (a_tr * power(KM, n)) / (power(KM, n) + power(PY, n))",
None,
)
add_product_to_reaction(r12, "Z")
add_modifier_to_reaction(r12, "PY", "SBO:0000536")
print("-" * 80)
print(libsbml.writeSBMLToString(doc))
print("-" * 80)
# write to file
os.makedirs(os.path.dirname(str(sbml_path)), exist_ok=True)
libsbml.writeSBMLToFile(doc, str(sbml_path))
# validate file
validate_sbml(doc, units_consistency=False)
return doc
if __name__ == "__main__":
from combine_notebooks import RESULTS_DIR
# RESOURCES_DIR: Path = Path(__file__).parent / "resources"
# RESULTS_DIR: Path = RESOURCES_DIR / "results"
doc: libsbml.SBMLDocument = create_repressilator(
sbml_path=RESULTS_DIR / "repressilator_sbml_libsbml.xml"
)
Create SBML model
--------------------------------------------------------------------------------
<?xml version="1.0" encoding="UTF-8"?>
<sbml xmlns="http://www.sbml.org/sbml/level3/version2/core" level="3" version="2">
<model>
<listOfCompartments>
<compartment metaid="meta_cell" sboTerm="SBO:0000290" id="cell" size="1">
<annotation>
<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:vCard="http://www.w3.org/2001/vcard-rdf/3.0#" xmlns:vCard4="http://www.w3.org/2006/vcard/ns#" xmlns:bqbiol="http://biomodels.net/biology-qualifiers/" xmlns:bqmodel="http://biomodels.net/model-qualifiers/">
<rdf:Description rdf:about="#meta_cell">
<bqbiol:is>
<rdf:Bag>
<rdf:li rdf:resource="http://identifiers.org/GO:0005623"/>
</rdf:Bag>
</bqbiol:is>
</rdf:Description>
</rdf:RDF>
</annotation>
</compartment>
</listOfCompartments>
<listOfSpecies>
<species metaid="PX" sboTerm="SBO:0000252" id="PX" name="LacI protein" compartment="cell" initialAmount="0" hasOnlySubstanceUnits="true">
<annotation>
<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:vCard="http://www.w3.org/2001/vcard-rdf/3.0#" xmlns:vCard4="http://www.w3.org/2006/vcard/ns#" xmlns:bqbiol="http://biomodels.net/biology-qualifiers/" xmlns:bqmodel="http://biomodels.net/model-qualifiers/">
<rdf:Description rdf:about="#PX">
<bqbiol:is>
<rdf:Bag>
<rdf:li rdf:resource="http://identifiers.org/uniprot/P03023"/>
</rdf:Bag>
</bqbiol:is>
</rdf:Description>
</rdf:RDF>
</annotation>
</species>
<species metaid="PY" sboTerm="SBO:0000252" id="PY" name="TetR protein" compartment="cell" initialAmount="0" hasOnlySubstanceUnits="true">
<annotation>
<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:vCard="http://www.w3.org/2001/vcard-rdf/3.0#" xmlns:vCard4="http://www.w3.org/2006/vcard/ns#" xmlns:bqbiol="http://biomodels.net/biology-qualifiers/" xmlns:bqmodel="http://biomodels.net/model-qualifiers/">
<rdf:Description rdf:about="#PY">
<bqbiol:is>
<rdf:Bag>
<rdf:li rdf:resource="http://identifiers.org/uniprot/P04483"/>
</rdf:Bag>
</bqbiol:is>
</rdf:Description>
</rdf:RDF>
</annotation>
</species>
<species metaid="PZ" sboTerm="SBO:0000252" id="PZ" name="cI protein" compartment="cell" initialAmount="0" hasOnlySubstanceUnits="true">
<annotation>
<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:vCard="http://www.w3.org/2001/vcard-rdf/3.0#" xmlns:vCard4="http://www.w3.org/2006/vcard/ns#" xmlns:bqbiol="http://biomodels.net/biology-qualifiers/" xmlns:bqmodel="http://biomodels.net/model-qualifiers/">
<rdf:Description rdf:about="#PZ">
<bqbiol:is>
<rdf:Bag>
<rdf:li rdf:resource="http://identifiers.org/uniprot/P03034"/>
</rdf:Bag>
</bqbiol:is>
</rdf:Description>
</rdf:RDF>
</annotation>
</species>
<species metaid="meta_X" sboTerm="SBO:0000250" id="X" name="LacI mRNA" compartment="cell" initialAmount="0" hasOnlySubstanceUnits="true">
<annotation>
<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:vCard="http://www.w3.org/2001/vcard-rdf/3.0#" xmlns:vCard4="http://www.w3.org/2006/vcard/ns#" xmlns:bqbiol="http://biomodels.net/biology-qualifiers/" xmlns:bqmodel="http://biomodels.net/model-qualifiers/">
<rdf:Description rdf:about="#meta_X">
<bqbiol:isVersionOf>
<rdf:Bag>
<rdf:li rdf:resource="http://identifiers.org/CHEBI:33699"/>
<rdf:li rdf:resource="http://identifiers.org/kegg.compound/C00046"/>
</rdf:Bag>
</bqbiol:isVersionOf>
<bqbiol:encodes>
<rdf:Bag>
<rdf:li rdf:resource="http://identifiers.org/uniprot/P03023"/>
</rdf:Bag>
</bqbiol:encodes>
</rdf:Description>
</rdf:RDF>
</annotation>
</species>
<species metaid="meta_Y" sboTerm="SBO:0000250" id="Y" name="TetR mRNA" compartment="cell" initialAmount="20" hasOnlySubstanceUnits="true">
<annotation>
<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:vCard="http://www.w3.org/2001/vcard-rdf/3.0#" xmlns:vCard4="http://www.w3.org/2006/vcard/ns#" xmlns:bqbiol="http://biomodels.net/biology-qualifiers/" xmlns:bqmodel="http://biomodels.net/model-qualifiers/">
<rdf:Description rdf:about="#meta_Y">
<bqbiol:isVersionOf>
<rdf:Bag>
<rdf:li rdf:resource="http://identifiers.org/CHEBI:33699"/>
<rdf:li rdf:resource="http://identifiers.org/kegg.compound/C00046"/>
</rdf:Bag>
</bqbiol:isVersionOf>
<bqbiol:encodes>
<rdf:Bag>
<rdf:li rdf:resource="http://identifiers.org/uniprot/P04483"/>
</rdf:Bag>
</bqbiol:encodes>
</rdf:Description>
</rdf:RDF>
</annotation>
</species>
<species metaid="meta_Z" sboTerm="SBO:0000250" id="Z" name="cl mRNA" compartment="cell" initialAmount="0" hasOnlySubstanceUnits="true">
<annotation>
<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:vCard="http://www.w3.org/2001/vcard-rdf/3.0#" xmlns:vCard4="http://www.w3.org/2006/vcard/ns#" xmlns:bqbiol="http://biomodels.net/biology-qualifiers/" xmlns:bqmodel="http://biomodels.net/model-qualifiers/">
<rdf:Description rdf:about="#meta_Z">
<bqbiol:isVersionOf>
<rdf:Bag>
<rdf:li rdf:resource="http://identifiers.org/CHEBI:33699"/>
<rdf:li rdf:resource="http://identifiers.org/kegg.compound/C00046"/>
</rdf:Bag>
</bqbiol:isVersionOf>
<bqbiol:encodes>
<rdf:Bag>
<rdf:li rdf:resource="http://identifiers.org/uniprot/P03034"/>
</rdf:Bag>
</bqbiol:encodes>
</rdf:Description>
</rdf:RDF>
</annotation>
</species>
</listOfSpecies>
<listOfParameters>
<parameter id="tau_mRNA" name="mRNA half life" value="2" constant="true"/>
<parameter sboTerm="SBO:0000356" id="kd_mRNA" name="kd_mRNA" constant="false"/>
<parameter sboTerm="SBO:0000016" id="k_tl" name="k_tl" constant="false"/>
<parameter id="eff" name="translation efficiency" value="20" constant="true"/>
<parameter sboTerm="SBO:0000348" id="t_ave" name="average mRNA life time" constant="false"/>
<parameter sboTerm="SBO:0000356" id="kd_prot" name="kd_prot" constant="false"/>
<parameter id="tau_prot" name="protien half life" value="10" constant="true"/>
<parameter sboTerm="SBO:0000485" id="a0_tr" name="a0_tr" constant="false"/>
<parameter id="ps_0" name="tps_repr" value="0.0005" constant="true"/>
<parameter sboTerm="SBO:0000186" id="a_tr" name="a_tr" constant="false"/>
<parameter id="ps_a" name="tps_active" value="0.5" constant="true"/>
<parameter id="KM" name="KM" value="40" constant="true"/>
<parameter id="n" name="n" value="2" constant="true"/>
</listOfParameters>
<listOfRules>
<assignmentRule variable="kd_mRNA">
<math xmlns="http://www.w3.org/1998/Math/MathML">
<apply>
<divide/>
<apply>
<ln/>
<cn type="integer"> 2 </cn>
</apply>
<ci> tau_mRNA </ci>
</apply>
</math>
</assignmentRule>
<assignmentRule variable="t_ave">
<math xmlns="http://www.w3.org/1998/Math/MathML">
<apply>
<divide/>
<ci> tau_mRNA </ci>
<apply>
<ln/>
<cn type="integer"> 2 </cn>
</apply>
</apply>
</math>
</assignmentRule>
<assignmentRule variable="k_tl">
<math xmlns="http://www.w3.org/1998/Math/MathML">
<apply>
<divide/>
<ci> eff </ci>
<ci> t_ave </ci>
</apply>
</math>
</assignmentRule>
<assignmentRule variable="kd_prot">
<math xmlns="http://www.w3.org/1998/Math/MathML">
<apply>
<divide/>
<apply>
<ln/>
<cn type="integer"> 2 </cn>
</apply>
<ci> tau_prot </ci>
</apply>
</math>
</assignmentRule>
<assignmentRule variable="a0_tr">
<math xmlns="http://www.w3.org/1998/Math/MathML">
<apply>
<times/>
<ci> ps_0 </ci>
<cn type="integer"> 60 </cn>
</apply>
</math>
</assignmentRule>
<assignmentRule variable="a_tr">
<math xmlns="http://www.w3.org/1998/Math/MathML">
<apply>
<times/>
<apply>
<minus/>
<ci> ps_a </ci>
<ci> ps_0 </ci>
</apply>
<cn type="integer"> 60 </cn>
</apply>
</math>
</assignmentRule>
</listOfRules>
<listOfReactions>
<reaction metaid="meta_Reaction1" sboTerm="SBO:0000179" id="Reaction1" name="degradation of LacI transcripts" reversible="false">
<annotation>
<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:vCard="http://www.w3.org/2001/vcard-rdf/3.0#" xmlns:vCard4="http://www.w3.org/2006/vcard/ns#" xmlns:bqbiol="http://biomodels.net/biology-qualifiers/" xmlns:bqmodel="http://biomodels.net/model-qualifiers/">
<rdf:Description rdf:about="#meta_Reaction1">
<bqbiol:isVersionOf>
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</sbml>
--------------------------------------------------------------------------------
--------------------------------------------------------------------------------
validation error(s) : 0
validation warning(s) : 0
--------------------------------------------------------------------------------