Team:HiZJU-China/Experiments
petDuet:
The pETDuet vector can combined both monoamine oxidase and hydroxylamine Oxidase coding gene. The petDuet vector use T7 promoter and lac promoter to express the protain also contains AmpR gene for selection.
pGADT7:
The pGADT7 vector expresses proteins fused to amino acids of the GAL4 activation domain (AD). In yeast, fusion proteins are expressed at high levels from the constitutive ADH1; transcription is terminated by the ADH1 transcription termination signals. The pGADT7 vector also contains T7 promoter, SV40 NLS and Kozak sequence. The vector carries the Amp+ for selection in yeast. Yeast strains containing pGADT7 exhibit a higher transformation efficiency than strains carrying other AD protein vectors. The pGADT7 vector is always used in yeast two-hybrid technology for detection.
pGBKT7:
It is a plasmid which can express a fusion protein of BD and ERa-421F. Coding sequence of target gene was inserted into an expression vectors with ERa-421F and Plasmid(BBa_K289008) to obtain BBa_K3978004. We transformed the constructed plasmid into yeast Y2H to construct yeast two-hybrid technology system.
amoA
ATGGTGAGTATATTTAGAACGGAAGAAATCCTGAAAGCGGCCAAGATGCCGCCGGAAGCGGTTCATATGTCACGTCTGATTGATGCAGTTTATTTTCCAATTCTGATTATTTTGCTGGTGGGTACCTACCACATGCACTTTATGCTGCTGGCAGGTGACTGGGATTTCTGGATGGACTGGAAAGATCGTCAATGGTGGCCGGTTGTAACGCCAATCGTGGGGATCACCTACTGTTCGGCAATCATGTATTACTTGTGGGTCAACTACCGCCAACCGTTTGGTGCAACGTTGTGTGTGGTGTGTCTGCTGATTGGTGAGTGGCTGACACGTTACTGGGGATTCTACTGGTGGTCACACTACCCCATCAACTTCGTAACACCGGGCATTATGCTTCCGGGTGCGCTGATGCTGGACTTCACGCTGTATCTGACACGCAACTGGCTGGTGACGGCTCTGGTTGGAGGTGGATTCTTCGGTCTGCTGTTCTATCCGGGTAACTGGCCGATTTTTGGACCAACCCATTTGCCAATCGTTGTAGAAGGCACATTGCTGTCGATGGCTGATTACATGGGACATCTGTATGTTCGTACAGGTACACCCGAGTATGTTCGTCATATTGAGCAAGGTTCACTGCGTACCTTTGGTGGTCATACCACAGTTATTGCAGCATTCTTCTCTGCGTTCGTATCAATGTTGATGTTCACCGTATGGTGGTATCTTGGAAAAGTTTACTGTACAGCCTTTTTCTACGTTAAAGGTAAAAGAGGTCGTATCGTACATCGCAATGATGTTACCGCATTCGGTGAAGAAGGCTTTCCAGAGGGGATCAAATAA
hao:
ATGAGAATAGGGGAGTGGATGAGGGGTTTATTGCTCTGTGCGGGGTTGATGATGTGCGGGGTGGTTCATGCGGACATATCGACGGTACCGGACGAGACGTATGATGCGTTGAAGCTGGATCGAGGTAAGGCGACCCCGAAAGAGACGTATGAAGCGCTGGTCAAGCGTTACAAGGACCCGGCGCATGGAGCGGGGAAAGGGACGATGGGAGATTACTGGGAGCCGATAGCGATCAGTATATATATGGATCCCAATACTTTTTACAAACCACCGGTATCTCCCAAAGAAGTTGCGGAACGCAAAGACTGCGTGGAATGCCACTCGGACGAGACCCCGGTATGGGTAAGAGCCTGGAAGCGCAGCACCCACGCCAATCTTGACAAGATTCGCAACCTCAAGTCGGACGATCCCCTGTATTACAAGAAAGGGAAGCTGGAAGAAGTTGAAAACAACCTGCGCTCGATGGGCAAGCTTGGGGAAAAAGAAACCCTCAAGGAAGTAGGCTGTATCGACTGTCACGTTGACGTCAACAAAAAAGACAAAGCGGATCATACCAAAGACATCCGCATGCCGACGGCGGACACCTGCGGAACCTGTCACTTAAGAGAATTTGCCGAACGTGAATCGGAACGGGACACCATGGTCTGGCCGAACGGACAATGGCCTGCCGGACGCCCCTCCCACGCACTGGACTACACAGCCAACATCGAGACCACTGTTTGGGCAGCGATGCCGCAACGTGAAGTTGCCGAAGGTTGCACCATGTGTCACACCAACCAGAACAAATGTGACAACTGTCATACCCGTCATGAATTCTCGGCGGCGGAATCGCGCAAACCGGAAGCCTGTGCCACCTGCCACAGCGGGGTGGATCACAACAACTGGGAAGCGTACACCATGTCCAAGCATGGCAAACTTGCCGAAATGAACCGTGACAAATGGAACTGGGAAGTCCGTCTCAAGGACGCCTTCTCCAAAGGTGGACAGAATGCACCGACCTGTGCAGCCTGCCACATGGAATACGAAGGAGAATACACCCACAACATCACCCGCAAGACTCGCTGGGCGAACTACCCGTTCGTTCCGGGCATTGCAGAGAACATCACCAGCGACTGGTCGGAAGCCCGTCTGGACTCCTGGGTACTGACCTGCACCCAGTGTCACTCGGAACGGTTTGCCCGCTCCTACCTGGATCTGATGGACAAAGGCACCCTGGAAGGGCTGGCCAAATACCAGGAAGCGAACGCAATCGTACACAAAATGTATGAAGACGGCACCCTGACCGGACAGAAGACCAACCGTCCGAATCCGCCGGAACCGGAGAAACCTGGATTTGGTATCTTCACCCAGCTGTTCTGGTCGAAAGGCAACAACCCTGCCTCACTCGAACTGAAAGTTCTGGAAATGGCAGAAAACAACCTGGCCAAGATGCACGTTGGACTGGCTCACGTCAATCCGGGTGGCTGGACCTACACTGAAGGCTGGGGTCCGATGAACCGTGCCTATGTTGAAATCCAGGACGAATACACCAAAATGCAGGAACTGTCAGCCCTGCAGGCCCGTGTAAACAAACTGGAAGGCAAGCAGACCAGTCTGCTCGACCTCAAGGGAACCGGAGAGAAAATCTCCCTGGGAGGCTTGGGTGGTGGTATGTTGCTGGCAGGAGCCCTTGCTCTGATCGGCTGGCGCAAGCGTAAGCAGACCCGAGCTTGA
T7 promoter
AATACGACTCACTATAGG
Lac operator
GGAATTGTGAGCGGATAACAA
AmpR promoter
ACTCTTCCTTTTTCAATCATGATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA
ERα-421F:
CATATGTCTAAGAAGAACTCTTTGGCTTTGTCTTTGACTGCTGATCAAATGGTTTCTGCTTTGTTGGATGCTGAACCTCCTATTTTGTATTCTGAATATGATCCAACTAGACCATTTTCCGAAGCTTCAATGATGGGTTTGTTGACTAATTTGGCTGATAGAGAATTGGTTCACATGATTAATTGGGCTAAGAGAGTTCCTGGTTTTGTTGATTTGACTTTGCATGATCAAGTTCATTTGTTGGAATGTGCTTGGTTGGAAATTTTGATGATTGGTTTGGTTTGGAGATCTATGGAACATCCTGGTAAATTGTTGTTTGCTCCTAATTTGTTGTTGGATAGAAATCAAGGTAAGTGTGTTGAAGGTTTTGTTGAAATTTTCGATATGTTGTTGGCTACTTCTTCTAGATTTAGAATGATGAATTTGCAAGGTGAAGAATTTGTTTGTTTGAAATCTATTATTTTGTTGAACTCCGGTGTTTATACTTTTTTGTCTTCTACTTTGAAGTCCTTGGAAGAAAAAGATCATATTCATAGAGTTTTGGATAAGATTACCGATACTTTGATTCATTTGATGGCTAAGGCTGGTTTGACTTTGCAACAACAACATCAAAGATTGGCTCAATTGTTGTTGATTTTGTCTCATATTAGACACATGTCTAACAAAGGTATGGAACATTTGTACTCTATGAAATGTAAGAACGTTGTTCCATTGTACGATTTATTATTGGAAATGTTGGATGCTCATAGATTGCATGCTCCAACTTAAGGATCC
GRIP1:
AATTCATGATTGCTGTTTCTTTCAAGTGTAGATGTCAAATTTTGAGAAGATTGACTAAGGATGAATCCCCATACACAAAATCCGCTTCCCAAACCAAGCCACCAGATGGTGCTTTGGCTGTTAGAAGACAATCTATTCCAGAAGAATTTAAGGGTTCTACTGTTGTTGAATTGATGAAAAAGGAAGGTACTACTTTGGGTTTGACTGTTTCTGGTGGTATTGATAAGGATGGTAAACCAAGAGTTTCAAATTTGAGACAAGGTGGTATTGCTGCTAGATCTGATCAATTGGATGTTGGTGATTATATTAAGGCTGTTAATGGTATTAACTTGGCTAAATTTAGACATGATGAAATTATTTCCTTGTTGAAGAACGTTGGTGAAAGAGTTGTTTTGGAAGTTGAATATGAATTGCCACCAGTTTCTGTTCAAGGTTCTTCTGTTATTTTTAGAACTGTTGAAGTTACCTTGCATAAAGAAGGTAATACCTTCGGTTTTGTTATTAGAGGTGGTGCTCATGATGATAGAAATAAATCCAGACCAGTTGTTATTACTTGTGTTAGACCAGGTGGTCCAGCTGATAGAGAAGGTACAATTAAACCAGGTGATAGATTGTTATCTGTTGATGGTATTAGATTGTTGGGTACAACACATGCAGAAGCTATGTCAATTTTGAAACAATGTGGTCAAGAAGCTGCTTTGTTGATTGAATATGATGTTAGTGTTATGGATTCCGTTGCTACTGCTTCCGGTCCTTTGTTGGTTGAAGTTGCTAAGACTCCAGGTGCTTCTTTGGGTGTT
GCTTTGACTACTTCTATGTGTTGTAATAAGCAAGTTATTGTTATTGATAAGATTAAGTCCGCTTCTATTGCTGATAGATGTGGTGCTTTGCATGTTGGTGATCATATTTTGTCTATTGATGGTACTTCTATGGAATATTGTACTTTGGCTGAAGCTACTCAATTTTTGGCTAACACTACTGATCAAGTTAAATTGGAAATTTTGCCTCATCATCAAACTAGATTGGCTTTGAAAGGTCCAGATCATGCTGCTTTGGTTTCTTCTTCTTTTTCTCCAACTTCCATGTCTGCATACTCCTTGTCTTCTTTGAATATGGGTACTTTGCCAAGATCCTTGTATTCTACTTCTCCTAGAGGTACCATGATGAGAAGAAGATTGAAAAAGAAAGATTTCAAGTCCTCTTTGTCTTTGGCTTCTTCTACTGTTGGTTTGGCTGGTCAAGTTGTTCATACTGAAACTACAGAAGTTGTTTTGACTGCTGATCCAGTTACTGGTTTTGGTATTCAATTGCAAGGTTCTGTTTTTGCTACTGAAACTTTGTCTTCTCCTCCATTGATTTCTTATATTGAAGCTGATTCTCCAGCTGAAAGATGTGGTGTTTTACAAATTGGTGATAGAGTTATGGCTATTAATGGTATTCCTACTGAAGATTCTACTTTTGAAGAAGCTTCTCAATTGTTGAGAGATTCTTCTATTACTTCTAAGGTTACTTTGGAAATTGAATTTGATGTTGCTGAATCTGTTATTCCTTCTTCTGGTACTTTTCATGTTAAATTGCCAAAAAAACATAATGTTGAATTGGGTATTACTATTTCTTCTCCATCTTCTAGAAAACCAGGTGATCCATTGGTTATTTCTGATATTAAAAAAGGTTCTGTTGCTCATAGAACTGGTACTTTGGAATTGGGTGATAAATTGTTGGCTATTGATAATATTAGATTGGATAATTGTTCTATGGAAGATGCTGTTCAAATTTTGCAACAATGTGAAGATTTGGTTAAATTGAAAATTAGAAAAGATGAAGATAATTCTGATGAACAAGAATCTTCTGGTGCTATTATTTATACTGTTGAATTGAAAAGATATGGTGGTCCATTGGGTATTACTATTTCTGGTACTGAAGAACCATTTGATCCAATTATTATTTCTTCTTTGACTAAAGGTGGTTTGGCTGAAAGAACTGGTGCTATTCATATTGGTGATAGAATTTTGGCTATTAATTCTTCTTCTTTGAAAGGTAAACCATTGTCTGAAGCTATTCATTTGTTGCAAATGGCTGGTGAAACTGTTACTTTGAAAATTAAAAAACAAACTGATGCTCAATCTGCTTCTTCTCCAAAAAAATTTCCAATTTCTTCTCATTTGTCTGATTTGGGTGATGTTGAAGAAGATTCTTCTCCAGCTCAAAAACCAGGTAAATTGTCTGATATGTATCCATCTACTGTTCCATCTGTTGATTCTGCTGTTGATTCTTGGGATGGTTCTGCTATTGATACTTCTTATGGTACTCAAGGTACTTCTTTTCAAGCTTCTGGTTATAATTTTAATACTTATGATTGGAGATCTCCAAAACAAAGAGGTTCTTTGTCTCCAGTTACTAAACCAAGATCTCAAACTTATCCAGATGTTGGTTTGTCTTATGAAGATTGGGATAGATCTACTGCTTCTGGTTTTGCTGGTGCTGCTGATTCTGCTGAAACTGAACAAGAAGAAAATTTTTGGTCTCAAGCTTTGGAAGATTTGGAAACTTGTGGTCAATCTGGTATTTTGAGAGAATTGGAAGCTACTATTATGTCTGGTTCTACTATGTCTTTGAATCATGAAGCTCCAACTCCAAGATCTCAATTGGGTAGACAAGCTTCTTTTCAAGAAAGATCTTCTTCTAGACCACATTATTCTCAAACTACTAGATCTAATACTTTGCCATCTGATGTTGGTAGAAAATCTGTTACTTTGAGAAAAATGAAACAAGAAATTAAAGAAATTATGTCTCCAACTCCAGTTGAATTGCATAAAGTTACTTTGTATAAAGATTCTGATATGGAAGATTTTGGTTTTTCTGTTGCTGATGGTTTGTTGGAAAAAGGTGTTTATGTTAAAAATATTAGACCAGCTGGTCCAGGTGATTTGGGTGGTTTGAAACCATATGATAGATTGTTGCAAGTTAATCATGTTAGAACTAGAGATTTTGATTGTTGTTTGGTTGTTCCATTGATTGCTGAATCTGGTAATAAATTGGATTTGGTTATTTCTAGAAATCCATTGGCTTCTCAAAAATCTATTGATCAACAATCTTTGCCAGGTGATTGGTCTGAACAAAATTCTGCTTTTTTTCAACAACCATCTCATGGTGGTAATTTGGAAACTAGAGAACCAACTAATACTTTGTAAG
The pH was controlled in the range of 7.5~8.0 by manual addition of filter sterilized 0.1 M NaOH solution. Glucose, NH4Cl and 17α-Ethinyl Estradiol were sterilized by filtration and then added into sterilized medium.
Solution B (NaFeEDTA Solution): 100mg NaFeEDTA( molecular formula C10H12FeN2NaO8·3H2O) dissolved in 100mL double distilled water.
Solution C(microelement): 10mg Na2MoO4·H2O, 17.2mg MnSO4·H2O, 0.4mgCoCl2·6H2O, 12mg ZnSO4·7H2O, 50mg CuSO4·5H2O, dissolved in 100mL double distilled water.
Media was sterilized in an autoclave. The pH was controlled in the range of 6.8~7.8 by manual addition of filter sterilized 2 M KHCO3 solution.
