iGEM 2013 — Team Kyoto

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# Team:Kyoto/Notebook

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| Contents   * [1 Calender](#Calender) * [2 Aug 8](#Aug_8)   + [2.1 Transformation](#Transformation) * [3 Aug 9](#Aug_9)   + [3.1 Transformation](#Transformation_2) * [4 Aug 10](#Aug_10)   + [4.1 Colony PCR](#Colony_PCR)   + [4.2 Electrophoresis](#Electrophoresis)   + [4.3 Liquid Culture](#Liquid_Culture)   + [4.4 LB Medium Plate(+Ampicillin)](#LB_Medium_Plate.28.2BAmpicillin.29)   + [4.5 Transformation](#Transformation_3) * [5 Aug 11](#Aug_11)   + [5.1 Miniprep](#Miniprep)   + [5.2 Colony PCR](#Colony_PCR_2)   + [5.3 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion)   + [5.4 Electrophoresis](#Electrophoresis_2) * [6 Aug 15](#Aug_15)   + [6.1 Liquid Culture](#Liquid_Culture_2)   + [6.2 Transformation](#Transformation_4)   + [6.3 Miniprep](#Miniprep_2) * [7 Aug 16](#Aug_16)   + [7.1 Colony PCR](#Colony_PCR_3)   + [7.2 Electrophoresis](#Electrophoresis_3)   + [7.3 Master Plate](#Master_Plate)   + [7.4 Transformation](#Transformation_5)   + [7.5 Liquid Culture](#Liquid_Culture_3) * [8 Aug 17](#Aug_17)   + [8.1 Colony PCR](#Colony_PCR_4)   + [8.2 Electrophoresis](#Electrophoresis_4)   + [8.3 Liquid Culture](#Liquid_Culture_4)   + [8.4 Master Plate](#Master_Plate_2)   + [8.5 Miniprep](#Miniprep_3)   + [8.6 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_2)   + [8.7 Electrophoresis](#Electrophoresis_5)   + [8.8 Miniprep](#Miniprep_4) * [9 Aug 18](#Aug_18)   + [9.1 Gel Extraction](#Gel_Extraction)   + [9.2 Ligation](#Ligation)   + [9.3 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_3)   + [9.4 Electrophoresis](#Electrophoresis_6)   + [9.5 Colony PCR](#Colony_PCR_5)   + [9.6 Liquid Culture](#Liquid_Culture_5)   + [9.7 LB Medium Plate](#LB_Medium_Plate)   + [9.8 Transformation](#Transformation_6)   + [9.9 Electrophoresis](#Electrophoresis_7) * [10 Aug 19](#Aug_19)   + [10.1 Colony PCR](#Colony_PCR_6)   + [10.2 Gel Extraction](#Gel_Extraction_2)   + [10.3 LB Medium Plate](#LB_Medium_Plate_2)   + [10.4 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_4)   + [10.5 Ligation](#Ligation_2)   + [10.6 Electrophoresis](#Electrophoresis_8)   + [10.7 Transformation](#Transformation_7)   + [10.8 Gel Extraction](#Gel_Extraction_3)   + [10.9 Liquid Culture](#Liquid_Culture_6)   + [10.10 Master Plate](#Master_Plate_3) * [11 Aug 20](#Aug_20)   + [11.1 Miniprep](#Miniprep_5)   + [11.2 LB Medium Plate](#LB_Medium_Plate_3)   + [11.3 Ristriction Enzyme Digestion](#Ristriction_Enzyme_Digestion)   + [11.4 Transformation](#Transformation_8)   + [11.5 Electrophoresis](#Electrophoresis_9) * [12 Aug 21](#Aug_21)   + [12.1 Plusgrow Medium](#Plusgrow_Medium)   + [12.2 Liquid culture](#Liquid_culture_7)   + [12.3 Gel Extraction](#Gel_Extraction_4)   + [12.4 Ristriction Enzyme Digestion](#Ristriction_Enzyme_Digestion_2)   + [12.5 Electrophoresis](#Electrophoresis_10)   + [12.6 Gel Extraction](#Gel_Extraction_5)   + [12.7 Electrophoresis](#Electrophoresis_11)   + [12.8 Ligation](#Ligation_3)   + [12.9 Transformation](#Transformation_9)   + [12.10 LB Medium Plate](#LB_Medium_Plate_4)   + [12.11 Gel Extraction](#Gel_Extraction_6)   + [12.12 Miniprep](#Miniprep_6) * [13 Aug 22](#Aug_22)   + [13.1 Liquid culture](#Liquid_culture_8)   + [13.2 Colony PCR](#Colony_PCR_7)   + [13.3 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_5)   + [13.4 Electrophoresis](#Electrophoresis_12)   + [13.5 Miniprep](#Miniprep_7)   + [13.6 Electrophoresis](#Electrophoresis_13) * [14 Aug 23](#Aug_23)   + [14.1 Electrophoresis](#Electrophoresis_14)   + [14.2 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_6)   + [14.3 Electrophoresis](#Electrophoresis_15)   + [14.4 Gel Extraction](#Gel_Extraction_7) * [15 Aug 24](#Aug_24)   + [15.1 Gel Extraction](#Gel_Extraction_8)   + [15.2 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_7)   + [15.3 Master Plate](#Master_Plate_4)   + [15.4 Electrophoresis](#Electrophoresis_16) * [16 Aug 26](#Aug_26)   + [16.1 Gel Extraction](#Gel_Extraction_9)   + [16.2 Ligation](#Ligation_4)   + [16.3 Transformation](#Transformation_10) * [17 Aug 27](#Aug_27)   + [17.1 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_8)   + [17.2 Electrophoresis](#Electrophoresis_17)   + [17.3 Colony PCR](#Colony_PCR_8)   + [17.4 Liquid Culture](#Liquid_Culture_9)   + [17.5 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_9)   + [17.6 Electrophoresis](#Electrophoresis_18)   + [17.7 Gel Extraction](#Gel_Extraction_10)   + [17.8 Transformation](#Transformation_11)   + [17.9 Liquid culture](#Liquid_culture_10)   + [17.10 Genome PCR](#Genome_PCR)   + [17.11 Ligation](#Ligation_5) * [18 Aug 28](#Aug_28)   + [18.1 Electrophoresis](#Electrophoresis_19)   + [18.2 Miniprep](#Miniprep_8)   + [18.3 Ligation](#Ligation_6)   + [18.4 Gel Extraction](#Gel_Extraction_11)   + [18.5 Colony PCR](#Colony_PCR_9)   + [18.6 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_10)   + [18.7 Electrophoresis](#Electrophoresis_20)   + [18.8 Gel Extraction](#Gel_Extraction_12)   + [18.9 Colony PCR](#Colony_PCR_10)   + [18.10 Colony PCR](#Colony_PCR_11)   + [18.11 Liquid Culture](#Liquid_Culture_11)   + [18.12 Ligation](#Ligation_7)   + [18.13 Liquid Culture](#Liquid_Culture_12) * [19 Aug 29](#Aug_29)   + [19.1 Colony PCR](#Colony_PCR_12)   + [19.2 Electrophoresis](#Electrophoresis_21)   + [19.3 Miniprep](#Miniprep_9)   + [19.4 Liquid Culture](#Liquid_Culture_13)   + [19.5 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_11)   + [19.6 Transformation](#Transformation_12)   + [19.7 Gel Extraction](#Gel_Extraction_13) * [20 Aug 30](#Aug_30)   + [20.1 Gel Extraction](#Gel_Extraction_14)   + [20.2 Miniprep](#Miniprep_10)   + [20.3 Colony PCR](#Colony_PCR_13)   + [20.4 Ethanol Precipitation](#Ethanol_Precipitation)   + [20.5 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_12)   + [20.6 Electrophoresis](#Electrophoresis_22)   + [20.7 Liquid Culture](#Liquid_Culture_14) * [21 Aug 31](#Aug_31)   + [21.1 Gel Extraction](#Gel_Extraction_15)   + [21.2 Ligation](#Ligation_8)   + [21.3 Miniprep](#Miniprep_11) * [22 Sep 1](#Sep_1)   + [22.1 Transformation](#Transformation_13)   + [22.2 ligation production PCR](#ligation_production_PCR)   + [22.3 Ligation](#Ligation_9)   + [22.4 Liquid Culture](#Liquid_Culture_15)   + [22.5 M9 Medium](#M9_Medium)   + [22.6 Miniprep](#Miniprep_12)   + [22.7 Kanamycin applicaton](#Kanamycin_applicaton)   + [22.8 Plating](#Plating)   + [22.9 Liquid Culture](#Liquid_Culture_16) * [23 Sep 2](#Sep_2)   + [23.1 Colony PCR](#Colony_PCR_14)   + [23.2 Electrophoresis](#Electrophoresis_23)   + [23.3 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_13)   + [23.4 Liquid Culture](#Liquid_Culture_17)   + [23.5 Master Plate](#Master_Plate_5)   + [23.6 Electrophoresis](#Electrophoresis_24)   + [23.7 Gel Extraction](#Gel_Extraction_16)   + [23.8 Colony PCR](#Colony_PCR_15)   + [23.9 Electrophoresis](#Electrophoresis_25)   + [23.10 Master Plate](#Master_Plate_6)   + [23.11 Liquid Culture](#Liquid_Culture_18)   + [23.12 Transformation](#Transformation_14) * [24 Sep 3](#Sep_3)   + [24.1 Electrophoresis](#Electrophoresis_26)   + [24.2 Miniprep](#Miniprep_13)   + [24.3 Liquid Culture](#Liquid_Culture_19)   + [24.4 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_14)   + [24.5 PCR](#PCR)   + [24.6 Mutatation PCR](#Mutatation_PCR)   + [24.7 Colony PCR](#Colony_PCR_16)   + [24.8 Electrophoresis](#Electrophoresis_27)   + [24.9 Electrophoresis](#Electrophoresis_28)   + [24.10 Gel Extraction](#Gel_Extraction_17)   + [24.11 Liquid Culture](#Liquid_Culture_20) * [25 Sep 4](#Sep_4)   + [25.1 Ligation](#Ligation_10)   + [25.2 Transformation](#Transformation_15)   + [25.3 Miniprep](#Miniprep_14)   + [25.4 Master Plate](#Master_Plate_7)   + [25.5 Liquid Culture](#Liquid_Culture_21)   + [25.6 PCR](#PCR_2)   + [25.7 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_15)   + [25.8 Transformation](#Transformation_16)   + [25.9 Electrophoresis](#Electrophoresis_29)   + [25.10 Gel Extraction](#Gel_Extraction_18)   + [25.11 Gel Extraction](#Gel_Extraction_19)   + [25.12 Master Plate](#Master_Plate_8)   + [25.13 Liquid Culture](#Liquid_Culture_22)   + [25.14 Colony PCR](#Colony_PCR_17) * [26 Sep 5](#Sep_5)   + [26.1 Electrophoresis](#Electrophoresis_30)   + [26.2 Miniprep](#Miniprep_15)   + [26.3 Ligation](#Ligation_11)   + [26.4 Electrophoresis](#Electrophoresis_31)   + [26.5 Gel Extraction](#Gel_Extraction_20)   + [26.6 Colony PCR](#Colony_PCR_18)   + [26.7 Liquid Culture](#Liquid_Culture_23)   + [26.8 Transformation](#Transformation_17)   + [26.9 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_16)   + [26.10 Electrophoresis](#Electrophoresis_32)   + [26.11 5x M9 Medium (+EDTA)](#5x_M9_Medium_.28.2BEDTA.29)   + [26.12 Gel Extraction](#Gel_Extraction_21)   + [26.13 DNA Purification](#DNA_Purification)   + [26.14 Miniprep](#Miniprep_16)   + [26.15 Colony PCR](#Colony_PCR_19)   + [26.16 Liquid Culture](#Liquid_Culture_24) * [27 Sep 6](#Sep_6)   + [27.1 Electrophoresis](#Electrophoresis_33)   + [27.2 Colony PCR](#Colony_PCR_20)   + [27.3 Liquid Culture](#Liquid_Culture_25)   + [27.4 Electrophoresis](#Electrophoresis_34)   + [27.5 Miniprep](#Miniprep_17)   + [27.6 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_17)   + [27.7 Electrophoresis](#Electrophoresis_35)   + [27.8 Gel Extraction](#Gel_Extraction_22)   + [27.9 Liquid Culture](#Liquid_Culture_26)   + [27.10 Colony PCR](#Colony_PCR_21) * [28 Sep 7](#Sep_7)   + [28.1 Miniprep](#Miniprep_18)   + [28.2 Electrophoresis](#Electrophoresis_36)   + [28.3 Colony PCR](#Colony_PCR_22)   + [28.4 Ligation](#Ligation_12)   + [28.5 Electrophoresis](#Electrophoresis_37)   + [28.6 Transformation](#Transformation_18)   + [28.7 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_18)   + [28.8 Electrophoresis](#Electrophoresis_38)   + [28.9 Liquid Culture](#Liquid_Culture_27) * [29 Sep 8](#Sep_8)   + [29.1 Gel Extraction](#Gel_Extraction_23)   + [29.2 Miniprep](#Miniprep_19)   + [29.3 Ligation](#Ligation_13)   + [29.4 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_19)   + [29.5 Transformation](#Transformation_19)   + [29.6 Electrophoresis](#Electrophoresis_39)   + [29.7 Gel Extraction](#Gel_Extraction_24)   + [29.8 Ligation](#Ligation_14)   + [29.9 PCR](#PCR_3)   + [29.10 Gel Extraction](#Gel_Extraction_25) * [30 Sep 9](#Sep_9)   + [30.1 PCR](#PCR_4)   + [30.2 Gel Extraction](#Gel_Extraction_26)   + [30.3 Miniprep](#Miniprep_20)   + [30.4 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_20)   + [30.5 Transformation](#Transformation_20)   + [30.6 Ligation](#Ligation_15)   + [30.7 M9 medium](#M9_medium_2)   + [30.8 Colony PCR](#Colony_PCR_23)   + [30.9 Gel Extraction](#Gel_Extraction_27)   + [30.10 Ligation](#Ligation_16)   + [30.11 PCR](#PCR_5)   + [30.12 Liquid Culture](#Liquid_Culture_28)   + [30.13 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_21)   + [30.14 PCR](#PCR_6)   + [30.15 Liquid Culture](#Liquid_Culture_29)   + [30.16 Gel Extraction](#Gel_Extraction_28)   + [30.17 Electrophoresis](#Electrophoresis_40) * [31 Sep 10](#Sep_10)   + [31.1 Ligation](#Ligation_17)   + [31.2 Miniprep](#Miniprep_21)   + [31.3 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_22)   + [31.4 Electrophoresis](#Electrophoresis_41)   + [31.5 Colony PCR](#Colony_PCR_24)   + [31.6 Electrophoresis](#Electrophoresis_42)   + [31.7 Gel Extraction](#Gel_Extraction_29)   + [31.8 Liquid Culture](#Liquid_Culture_30)   + [31.9 Gel Extraction](#Gel_Extraction_30)   + [31.10 Liquid Culture](#Liquid_Culture_31)   + [31.11 Colony PCR](#Colony_PCR_25)   + [31.12 Electrophoresis](#Electrophoresis_43)   + [31.13 Transformation](#Transformation_21)   + [31.14 Electrophoresis](#Electrophoresis_44) * [32 Sep 11](#Sep_11)   + [32.1 Electrophoresis](#Electrophoresis_45)   + [32.2 PCR](#PCR_7)   + [32.3 Electrophoresis](#Electrophoresis_46)   + [32.4 colony PCR](#colony_PCR_26)   + [32.5 Electrophoresis](#Electrophoresis_47)   + [32.6 colony PCR](#colony_PCR_27)   + [32.7 Gel Extraction](#Gel_Extraction_31)   + [32.8 Ligation](#Ligation_18)   + [32.9 Transformation](#Transformation_22)   + [32.10 Electrophoresis](#Electrophoresis_48)   + [32.11 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_23)   + [32.12 Liquid Culture](#Liquid_Culture_32)   + [32.13 PCR](#PCR_8)   + [32.14 Liquid Culture](#Liquid_Culture_33) * [33 Sep 12](#Sep_12)   + [33.1 Miniprep](#Miniprep_22)   + [33.2 Electrophoresis](#Electrophoresis_49)   + [33.3 Liquid Culture](#Liquid_Culture_34)   + [33.4 Electrophoresis](#Electrophoresis_50)   + [33.5 Gel Extraction](#Gel_Extraction_32)   + [33.6 Gel Extraction](#Gel_Extraction_33)   + [33.7 Electrophoresis](#Electrophoresis_51)   + [33.8 Colony PCR](#Colony_PCR_28)   + [33.9 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_24)   + [33.10 Colony PCR](#Colony_PCR_29)   + [33.11 Transformation](#Transformation_23)   + [33.12 Electrophoresis](#Electrophoresis_52)   + [33.13 Electrophoresis](#Electrophoresis_53)   + [33.14 Liquid Culture](#Liquid_Culture_35)   + [33.15 Colony PCR](#Colony_PCR_30)   + [33.16 Electrophoresis](#Electrophoresis_54)   + [33.17 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_25)   + [33.18 Electrophoresis](#Electrophoresis_55)     - [33.18.1 Observation through a confocal microscope](#Observation_through_a_confocal_microscope)   + [33.19 Liquid Culture](#Liquid_Culture_36) * [34 Sep 13](#Sep_13)   + [34.1 Miniprep](#Miniprep_23)   + [34.2 Gel Extraction](#Gel_Extraction_34)   + [34.3 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_26)   + [34.4 M9 (EDTA)](#M9_.28EDTA.29)   + [34.5 Gel Extraction](#Gel_Extraction_35)   + [34.6 Liquid Culture](#Liquid_Culture_37)   + [34.7 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_27)   + [34.8 Electrophoresis](#Electrophoresis_56)   + [34.9 Ligation](#Ligation_19)   + [34.10 PCR](#PCR_9)   + [34.11 PCR](#PCR_10)   + [34.12 Gel Extraction](#Gel_Extraction_36)   + [34.13 Transformation](#Transformation_24) * [35 Sep 14](#Sep_14)   + [35.1 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_28)   + [35.2 Liquid Culture](#Liquid_Culture_38)   + [35.3 Electrophoresis](#Electrophoresis_57)   + [35.4 Sequence PCR](#Sequence_PCR)   + [35.5 Gel Extraction](#Gel_Extraction_37)   + [35.6 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_29)   + [35.7 Ligation](#Ligation_20)   + [35.8 Colony PCR](#Colony_PCR_31)   + [35.9 Electrophoresis](#Electrophoresis_58)   + [35.10 Liquid Culture](#Liquid_Culture_39)   + [35.11 Transformation](#Transformation_25)   + [35.12 Sequence PCR](#Sequence_PCR_2) * [36 Sep 15](#Sep_15)   + [36.1 Miniprep](#Miniprep_24)   + [36.2 Liquid Culture](#Liquid_Culture_40)   + [36.3 Ligation](#Ligation_21)   + [36.4 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_30)   + [36.5 Electrophoresis](#Electrophoresis_59)   + [36.6 Gel Extraction](#Gel_Extraction_38)   + [36.7 Ligation](#Ligation_22)   + [36.8 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_31)   + [36.9 Liquid Culture](#Liquid_Culture_41) * [37 Sep 16](#Sep_16)   + [37.1 Liquid Culture](#Liquid_Culture_42)   + [37.2 Colony PCR](#Colony_PCR_32)   + [37.3 Gel Extraction](#Gel_Extraction_39)   + [37.4 Miniprep](#Miniprep_25)   + [37.5 PCR](#PCR_11)   + [37.6 Electrophoresis](#Electrophoresis_60)   + [37.7 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_32)   + [37.8 Ligation](#Ligation_23)   + [37.9 Transformation](#Transformation_26)   + [37.10 Miniprep](#Miniprep_26)   + [37.11 PCR](#PCR_12) * [38 Sep 17](#Sep_17)   + [38.1 Liquid Culture](#Liquid_Culture_43)   + [38.2 PCR](#PCR_13)   + [38.3 Electrophoresis](#Electrophoresis_61)   + [38.4 Ligation](#Ligation_24)   + [38.5 Gel Extraction](#Gel_Extraction_40)   + [38.6 Mutation PCR](#Mutation_PCR) * [39 Sep 18](#Sep_18)   + [39.1 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_33)   + [39.2 Electrophoresis](#Electrophoresis_62)   + [39.3 Electrophoresis](#Electrophoresis_63)   + [39.4 Gel Extraction](#Gel_Extraction_41) * [40 Sep 19](#Sep_19) * [41 Sep 20](#Sep_20)   + [41.1 Ligation](#Ligation_25)   + [41.2 Transformation](#Transformation_27)   + [41.3 PCR](#PCR_14)   + [41.4 Electrophoresis](#Electrophoresis_64) * [42 Sep 21](#Sep_21)   + [42.1 Electrophoresis](#Electrophoresis_65)   + [42.2 Liquid Culture](#Liquid_Culture_44)   + [42.3 Master Plate](#Master_Plate_9)   + [42.4 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_34)   + [42.5 Electrophoresis](#Electrophoresis_66)   + [42.6 PCR](#PCR_15)   + [42.7 Gel Extraction](#Gel_Extraction_42) * [43 Sep 22](#Sep_22)   + [43.1 Colony PCR](#Colony_PCR_33)   + [43.2 Electrophoresis](#Electrophoresis_67)   + [43.3 Miniprep](#Miniprep_27)   + [43.4 Colony PCR](#Colony_PCR_34)   + [43.5 Gel Extraction](#Gel_Extraction_43)   + [43.6 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_35)   + [43.7 Gel Extraction](#Gel_Extraction_44)   + [43.8 Colony PCR](#Colony_PCR_35)   + [43.9 Colony PCR](#Colony_PCR_36)   + [43.10 Ligation](#Ligation_26)   + [43.11 Liquid Culture](#Liquid_Culture_45)   + [43.12 digestion](#digestion)   + [43.13 Gel Extraction](#Gel_Extraction_45)   + [43.14 Transformation](#Transformation_28)   + [43.15 PCR](#PCR_16)   + [43.16 Electrophoresis](#Electrophoresis_68) * [44 Sep 23](#Sep_23)   + [44.1 Transformation](#Transformation_29)   + [44.2 Gel Extraction](#Gel_Extraction_46)   + [44.3 PCR](#PCR_17)   + [44.4 Ligation](#Ligation_27)   + [44.5 PCR](#PCR_18)   + [44.6 Miniprep](#Miniprep_28)   + [44.7 Electrophoresis](#Electrophoresis_69)   + [44.8 Gel Extraction](#Gel_Extraction_47)   + [44.9 Electrophoresis](#Electrophoresis_70)   + [44.10 Transformation](#Transformation_30)   + [44.11 Ligation](#Ligation_28)   + [44.12 Colony PCR](#Colony_PCR_37) * [45 Sep 24](#Sep_24)   + [45.1 Electrophoresis](#Electrophoresis_71)   + [45.2 Transformation](#Transformation_31)   + [45.3 Liquid Culture](#Liquid_Culture_46)   + [45.4 Restriction Enzyme Digestion(PCR product)](#Restriction_Enzyme_Digestion.28PCR_product.29)   + [45.5 Column Refining](#Column_Refining)   + [45.6 Transformation](#Transformation_32)   + [45.7 Liquid Culture](#Liquid_Culture_47)   + [45.8 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_36)   + [45.9 Electrophoresis](#Electrophoresis_72)   + [45.10 Miniprep](#Miniprep_29)   + [45.11 Gel Extraction](#Gel_Extraction_48)   + [45.12 Colony PCR](#Colony_PCR_38)   + [45.13 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_37)   + [45.14 Ligation](#Ligation_29)   + [45.15 colony PCR](#colony_PCR_39)   + [45.16 Miniprep](#Miniprep_30)   + [45.17 Electrophoresis](#Electrophoresis_73)   + [45.18 Gel Extraction](#Gel_Extraction_49)   + [45.19 Electrophoresis](#Electrophoresis_74)   + [45.20 Ligation](#Ligation_30)   + [45.21 PCR](#PCR_19) * [46 Sep 25](#Sep_25)   + [46.1 Electrophoresis](#Electrophoresis_75)   + [46.2 Colony PCR](#Colony_PCR_40)   + [46.3 Electrophoresis](#Electrophoresis_76)   + [46.4 Transformation](#Transformation_33)   + [46.5 Ligation](#Ligation_31)   + [46.6 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_38)   + [46.7 Liquid Culture](#Liquid_Culture_48)   + [46.8 Gel Extraction](#Gel_Extraction_50)   + [46.9 Electrophoresis](#Electrophoresis_77)   + [46.10 Gel Extraction](#Gel_Extraction_51)   + [46.11 Liquid Culture](#Liquid_Culture_49)   + [46.12 Ligation](#Ligation_32)   + [46.13 PCR](#PCR_20)   + [46.14 Transformation](#Transformation_34)   + [46.15 Electrophoresis](#Electrophoresis_78)   + [46.16 Column Refining](#Column_Refining_2)   + [46.17 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_39)   + [46.18 Liquid Culture](#Liquid_Culture_50)   + [46.19 Plating](#Plating_2)   + [46.20 Colony PCR](#Colony_PCR_41)   + [46.21 RNA Extraction](#RNA_Extraction)   + [46.22 cDNA　Synthesis](#cDNA.E3.80.80Synthesis)   + [46.23 Electrophoresis](#Electrophoresis_79)   + [46.24 Ligation](#Ligation_33) * [47 Sep 26](#Sep_26)   + [47.1 Electrophoresis](#Electrophoresis_80)   + [47.2 Ligasion(Golden Gate Assenbly)](#Ligasion.28Golden_Gate_Assenbly.29)   + [47.3 Liquid Culture](#Liquid_Culture_51)   + [47.4 BSAI Digestion](#BSAI_Digestion)   + [47.5 Colony PCR](#Colony_PCR_42)   + [47.6 Electrophoresis](#Electrophoresis_81)   + [47.7 Electrophoresis](#Electrophoresis_82)   + [47.8 Liquid Culture](#Liquid_Culture_52)   + [47.9 Colony PCR](#Colony_PCR_43)   + [47.10 Ligasion(Golden Gate Assenbly)](#Ligasion.28Golden_Gate_Assenbly.29_2)   + [47.11 RNA Extraction](#RNA_Extraction_2)   + [47.12 Liquid Culture](#Liquid_Culture_53)   + [47.13 Colony PCR](#Colony_PCR_44)   + [47.14 Electrophoresis](#Electrophoresis_83)   + [47.15 Plating](#Plating_3)   + [47.16 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_40)   + [47.17 Electrophoresis](#Electrophoresis_84) * [48 Sep 27](#Sep_27)   + [48.1 Gel Extraction](#Gel_Extraction_52)   + [48.2 Gel Extraction](#Gel_Extraction_53)   + [48.3 Electrophoresis](#Electrophoresis_85)   + [48.4 Liquid Culture](#Liquid_Culture_54)   + [48.5 Ligation](#Ligation_34)   + [48.6 Liquid Culture](#Liquid_Culture_55)   + [48.7 RNA Extraction](#RNA_Extraction_3)   + [48.8 Electrophoresis](#Electrophoresis_86)   + [48.9 Miniprep](#Miniprep_31)   + [48.10 Transformation](#Transformation_35)   + [48.11 Miniprep](#Miniprep_32)   + [48.12 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_41)   + [48.13 RNA Extraction](#RNA_Extraction_4)   + [48.14 DNA sythesis](#DNA_sythesis)   + [48.15 Electrophoresis](#Electrophoresis_87)   + [48.16 Liquid Culture](#Liquid_Culture_56)   + [48.17 RT PCR](#RT_PCR)   + [48.18 Electrophoresis](#Electrophoresis_88)   + [48.19 Digesting cut check](#Digesting_cut_check)   + [48.20 Gel Extraction](#Gel_Extraction_54)   + [48.21 RNA Extraction](#RNA_Extraction_5)   + [48.22 cDNA Synthesis](#cDNA_Synthesis) * [49 Sep 28](#Sep_28)   + [49.1 Electrophoresis](#Electrophoresis_89)   + [49.2 Colony PCR](#Colony_PCR_45)   + [49.3 Electrophoresis](#Electrophoresis_90) |

## Calender

| August | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  | | | | 1 | 2 | 3 |
| 4 | 5 | 6 | 7 | [8](#Aug_8) | [9](#Aug_9) | [10](#Aug_10) |
| [11](#Aug_11) | 12 | 13 | 14 | [15](#Aug_15) | [16](#Aug_16) | [17](#Aug_17) |
| [18](#Aug_18) | [19](#Aug_19) | [20](#Aug_20) | [21](#Aug_21) | [22](#Aug_22) | [23](#Aug_23) | [24](#Aug_24) |
| 25 | [26](#Aug_26) | [27](#Aug_27) | [28](#Aug_28) | [29](#Aug_29) | [30](#Aug_30) | [31](#Aug_31) |

| September | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| [1](#Sep_1) | [2](#Sep_2) | [3](#Sep_3) | [4](#Sep_4) | [5](#Sep_5) | [6](#Sep_6) | [7](#Sep_7) |
| [8](#Sep_8) | [9](#Sep_9) | [10](#Sep_10) | [11](#Sep_11) | [12](#Sep_12) | [13](#Sep_13) | [14](#Sep_14) |
| [15](#Sep_15) | [16](#Sep_16) | [17](#Sep_17) | [18](#Sep_18) | [19](#Sep_19) | [20](#Sep_20) | [21](#Sep_21) |
| [22](#Sep_22) | [23](#Sep_23) | [24](#Sep_24) | [25](#Sep_25) | [26](#Sep_26) | [27](#Sep_27) | [28](#Sep_28) |
| 29 | 30 |  | | | | |

## Aug 8

### Transformation

Inoue and Nakamoto

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| BBa\_J44000 | 2 µL | 20 µL (7/30) | 22 µL | LB (+Amp) |
| BBa\_J44000 | 2 µL | 20 µL (XL10-gold) | 22 µL | LB (+Amp) |
| BBa\_K747028 | 2 µL | 20 µL (XL10-gold) | 22 µL | LB (+CP) |
| BBa\_K747028 | 2 µL | 20 µL (7/30) | 22 µL | LB (+CP) |
| BBa\_J23100 | 2 µL | 20 µL (XL10-gold) | 22 µL | LB (+Amp) |
| BBa\_J23106 | 2 µL | 20 µL (XL10-gold) | 22 µL | LB (+Amp) |
| BBa\_E2030 | 2 µL | 20 µL (XL10-gold) | 22 µL | LB (+Kan) |

## Aug 9

### Transformation

Kojima and Nakamoto

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| BBa\_J44000 | 2 µL | 20 µL | 22 µL | LB (+Amp) |
| BBa\_K747028 | 2 µL | 20 µL | 22 µL | LB (+CP) |
| BBa\_J23100 | 2 µL | 20 µL | 22 µL | LB (+Amp) |
| BBa\_J23106 | 2 µL | 20 µL | 22 µL | LB (+Amp) |
| BBa\_E2030 | 2 µL | 20 µL | 22 µL | LB (+Kan) |

## Aug 10

### Colony PCR

Nakamoto

| Sample |
| --- |
| 8/9 BBa\_J44000 -(1) |
| 8/9 BBa\_J44000 -(2) |
| NC |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94 °C | 94 °C | 55 °C | 68 °C | -- |
| 5 min | 30 sec | 30 sec | 30 sec | 30 cycles |

| Sample |
| --- |
| 8/9 BBa\_J23106 |
| 8/9 BBa\_J23100 -(1) |
| 8/9 BBa\_J23100 -(2) |
| NC |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94 °C | 94 °C | 55 °C | 68 °C | -- |
| 5 min | 30 sec | 30 sec | 1 min | 30 cycles |

### Electrophoresis

Tatsui and Nakamoto

| Lane | Sample |
| --- | --- |
|  | 100bp ladder |
| 4 | 8/9 BBa\_J44000 -1 |
| 5 | 8/9 BBa\_J44000 -2 |
| 6 | NC |

[![Igku Aug10 Electrophoresis BBa 1.jpg](/wiki/images/4/48/Igku_Aug10_Electrophoresis_BBa_1.jpg)](/File%3AIgku_Aug10_Electrophoresis_BBa_1.jpg)

| Lane | Sample |
| --- | --- |
|  | 1kbp ladder |
| 1 | 8/9 BBa\_J23106 |
| 2 | 8/9 BBa\_J23100 -1 |
| 3 | 8/9 BBa\_J23100 -2 |
| 4 | NC |

[![Igku Aug10electrophoresis1.jpg](/wiki/images/7/74/Igku_Aug10electrophoresis1.jpg)](/File%3AIgku_Aug10electrophoresis1.jpg)

### Liquid Culture

Nakamoto

| Sample | medium |
| --- | --- |
| 8/9 BBa-J23106 | LB(+Amp) |
| 8/9 BBa-J23100-1 | LB(+Amp) |
| 8/9 BBa-J23100-2 | LB(+Amp) |
| 8/9 BBa-J44000-1 | LB(+Amp) |
| 8/9 BBa-J44000-2 | LB(+Amp) |

### LB Medium Plate(+Ampicillin)

Nakamoto & Tatsui

| volume | 200ml |
| --- | --- |
| Bacto T2ypton | 2g |
| Bacto yeast extract | 1g |
| NaCl | 1g |
| 0.4M NaOHaq | 500µL |
| Agarose Pouder | 2g |
| Ampicillin | 40µL |

### Transformation

Hirano

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| tetR | 3µL | 19µL | 22µL | Kan |
| RBS | 3µL | 19µL | 22µL | Amp |
| lacZα | 3µL | 19µL | 22µL | CP |
| lacI | 3µL | 19µL | 22µL | CP |
| Plac | 3µL | 19µL | 22µL | CP |

## Aug 11

### Miniprep

Nakamoto

| DNA | concentration[µg/mL] |
| --- | --- |
| 8/9 BBa\_J44000 -(2) | 100 |
| 8/9 BBa\_J23106 | 154 |
| 8/9 BBa\_J23100 | 144 |
| 8/10 T7-His-FT | 92 |

### Colony PCR

Kojima and Yasuda

| Sample |
| --- |
| BBa\_B0034 -(2) |
| BBa\_B0034 -(1) |
| BBa\_R0011 -(2) |
| BBa\_R0011 -(1) |
| NC |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94 °C | 94 °C | 55 °C | 68 °C | -- |
| 5 min | 30 s | 30 s | 20 s | 30 cycles |

| Sample |
| --- |
| BBa\_D0412 -(1) |
| BBa\_D0412 -(2) |
| NC |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94 °C | 94 °C | 55 °C | 68 °C | -- |
| 5 min | 30 s | 30 s | 1 min 30 s | 30 cycles |

### Restriction Enzyme Digestion

Kojima anda Yasuda

| 8/9 BBa\_J44000 2 | EcoRI | PstI | 10x buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 2 | 0.2 | 0.2 | 1 | 6.6 | 10 |
| 2 | 0.2 | -- | 1 | 6.8 | 10 |
| 2 | -- | 0.2 | 1 | 6.8 | 10 |
| 2 | -- | -- | 1 | 7.0 | 10 |

| 8/9 BBa\_J23106 | EcoRI | PstI | 10x buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1.3 | 0.2 | 0.2 | 1 | 7.3 | 10 |
| 1.3 | 0.2 | -- | 1 | 7.5 | 10 |
| 1.3 | -- | 0.2 | 1 | 7.5 | 10 |
| 1.3 | -- | -- | 1 | 7.7 | 10 |

| 8/9 BBa\_J23100 2 | EcoRI | PstI | 10x buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1.3 | 0.2 | 0.2 | 1 | 7.3 | 10 |
| 1.3 | 0.2 | -- | 1 | 7.5 | 10 |
| 1.3 | -- | 0.2 | 1 | 7.5 | 10 |
| 1.3 | -- | -- | 1 | 7.7 | 10 |

* incubated at 37 °C for 1 hour.

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | 8/9 BBa\_J44000 | EcoRI | PstI |
| 3 | 8/9 BBa\_J44000 | EcoRI | -- |
| 4 | 8/9 BBa\_J44000 | -- | PstI |
| 5 | 8/9 BBa\_J44000 | -- | -- |
| 6 | 8/9 BBa\_J23106 | EcoRI | PstI |
| 7 | 8/9 BBa\_J23106 | EcoRI | -- |
| 8 | 8/9 BBa\_J23106 | -- | PstI |
| 9 | 8/9 BBa\_J23106 | -- | -- |
| 10 | 8/9 BBa\_J23100 | EcoRI | PstI |
| 11 | 8/9 BBa\_J23100 | EcoRI | -- |
| 12 | 8/9 BBa\_J23100 | -- | PstI |
| 13 | 8/9 BBa\_J23100 | -- | -- |

[![Igku Aug11electrophoresis1.jpg](/wiki/images/4/46/Igku_Aug11electrophoresis1.jpg)](/File%3AIgku_Aug11electrophoresis1.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | BBa\_B0034 (1) |
| 3 | BBa\_B0034 (2) |
| 4 | BBa\_R0011 (1) |
| 5 | BBa\_R0011 (2) |
| 6 | NC |
| 7 | 100bp ladder |

[![11 electrophoresis2.jpg](/wiki/images/0/08/11_electrophoresis2.jpg)](/File%3A11_electrophoresis2.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 1kb ladder |
| 2 | BBa\_P0412 (1) |
| 3 | BBa\_P0412 (2) |
| 4 | NC |

[![Igku Aug11electrophoresis3.jpg](/wiki/images/2/28/Igku_Aug11electrophoresis3.jpg)](/File%3AIgku_Aug11electrophoresis3.jpg)

## Aug 15

### Liquid Culture

Nakamoto

| Sample | medium |
| --- | --- |
| 8/10 BBa\_B0034(1) | LB (+Amp) |
| 8/10 BBa\_B0034(2) | LB (+Amp) |
| 8/10 BBa\_R0011(1) | LB (+CP) |
| 8/10 BBa\_R0011(2) | LB (+CP) |
| 8/10 BBa\_P0412(1) | LB (+CP) |
| 8/10 BBa\_P0412(2) | LB (+CP) |

### Transformation

Nakamoto

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| BBa\_I0500(Pbad/araC) | 2 | 17 | 19 | LB (+Kan) |
| BBa\_F1610(RBS-luxI-DT) | 2 | 17 | 19 | LB (+Amp) |
| BBa\_R0061(Plux) | 2 | 17 | 19 | LB (+CP) |
| BBa\_I13504(RBS-GFP-DT) | 2 | 17 | 19 | LB (+CP) |
| BBa\_I0462(RBS-luxR-DT) | 2 | 17 | 19 | LB (+Amp) |
| BBa\_K117000(lysis1) | 2 | 17 | 19 | LB (+CP) |
| BBa\_K112806(lysis2) | 2 | 17 | 19 | LB (+CP) |
| BBa\_K748002(lysis3) | 2 | 17 | 19 | LB (+CP) |
| BBa\_B0015(DT) | 2 | 17 | 19 | LB (+CP) |
| BBa\_Q04400(RBS-tetR-DT-Ptet) | 2 | 17 | 19 | LB (+Kan) |
| BBa\_I732020(RBS-lacZα-DT) | 2 | 17 | 19 | LB (+CP) |
| BBa\_J23100 | 2 | 17 | 19 | LB (+Amp) |

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| BBa\_B0034(1) | 84.1 | 2.11 | 2.01 |
| BBa\_B0034(2) | 86.9 | 2.03 | 2.02 |
| BBa\_R0011(1) | 78.3 | 2.26 | 2.02 |
| BBa\_R0011(2) | 88.9 | 2.24 | 2.02 |
| BBa\_P0412(1) | 15.61 | 2.10 | 1.93 |
| BBa\_P0412(2) | 12.93 | 2.18 | 1.91 |

## Aug 16

### Colony PCR

Tatsui and Ashida

| Sample | base pair |
| --- | --- |
| 8/15 BBa\_I13504(RBS-GFP-DT)(1) | 1174 |
| 8/15 BBa\_I13504(RBS-GFP-DT)(2) | 1174 |
| 8/15 BBa\_I0462(RBS-luxR-DT)(1) | 1174 |
| 8/15 BBa\_I0462(RBS-luxR-DT)(2) | 1174 |
| 8/15 BBa\_K748002(lysis3) | 1053 |
| NC | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min | 30cycles |

| Sample | base pair |
| --- | --- |
| 8/15 BBa\_R0061(Plux) | 344 |
| 8/15 BBa\_K117000(lysis1)(1) | 456 |
| 8/15 BBa\_K117000(lysis1)(2) | 456 |
| 8/15 BBa\_B0015(DT) | 443 |
| 8/15 BBa\_I732020(RBS-lacZα-DT) | 703 |
| NC | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 40s | 30cycles |

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | BBa\_I13504(RBS-GFP-DT) -(1) |
| 3 | BBa\_I13504(RBS-GFP-DT) -(2) |
| 4 | BBa\_I0462(RBS-luxR-DT) -(1) |
| 5 | BBa\_I0462(RBS-luxR-DT) -(2) |
| 6 | BBa\_K748002(lysis3) |
| 7 | NC |
| 8 | 100bp ladder |

[![Igku 8161.jpg](/wiki/images/1/17/Igku_8161.jpg)](/File%3AIgku_8161.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | BBa\_R0061(Plux) |
| 3 | BBa\_K117000(lysis1) -(1) |
| 4 | BBa\_K117000(lysis1) -(1) |
| 5 | BBa\_B0015(DT) |
| 6 | BBa\_I732020(RBS-lacZα-DT) |
| 7 | NC |
| 8 | 100bp ladder |

[![Igku 8162.jpg](/wiki/images/1/14/Igku_8162.jpg)](/File%3AIgku_8162.jpg)

### Master Plate

Tatsui

| Number | Use LB plate (+CP) |
| --- | --- |
| 1 | BBa\_I13504(RBS-GFP-DT)(1) |
| 2 | BBa\_I13504(RBS-GFP-DT)(2) |
| 3 | BBa\_K748002(lysis3) |
| 4 | BBa\_R0061(Plux) |
| 5 | BBa\_K117000(lysis1)(1) |
| 6 | BBa\_K117000(lysis1)(2) |
| 7 | BBa\_B0015(DT) |
| 8 | BBa\_I732020(RBS-lacZα-DT) |

| Number | Use LB plate (+Amp) |
| --- | --- |
| 1 | BBa\_I0462(RBS-luxR-DT)(1) |
| 2 | BBa\_I0462(RBS-luxR-DT)(2) |

### Transformation

Hirano

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| pBR322 | 1µL | 100µL(XL-10gold) | 101µL | Amp |
| pBR322 | 1µL | 50µL(takara) | 51µL | Amp |
| Pbad/araC | 2µL | 20µL | 22µL | Kan |
| RBS-luxI-DT | 2µL | 20µL | 22µL | Kan |

### Liquid Culture

Yoshdia

| Sample | medium |
| --- | --- |
| 8/15 BBa\_I13504(RBS-GFP-DT))(1) | Plusgrow medium (+CP) |
| 8/15 BBa\_I13504(RBS-GFP-DT)(2) | Plusgrow medium (+CP) |
| 8/15 BBa\_I0462(RBS-luxR-DT)(2) | Plusgrow medium (+Amp) |
| 8/15 BBa\_K748002(lysis3) | Plusgrow medium (+CP) |
| 8/15 BBa\_R0061(Plux) | Plusgrow medium (+CP) |
| 8/15 BBa\_K117000(lysis1)(1) | Plusgrow medium (+CP) |
| 8/15 BBa\_K117000(lysis1)(2) | Plusgrow medium (+CP) |
| 8/15 BBa\_B0015(DT) | Plusgrow medium (+CP) |
| 8/15 BBa\_I732020(RBS-lacZα-DT) | Plusgrow medium (+CP) |

## Aug 17

### Colony PCR

Nakamoto

| Sample | base pair |
| --- | --- |
| 8/15 BBa\_K112806(lysis2) -(1) | 828 |
| NC | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min | 30cycles |

| Sample | base pair |
| --- | --- |
| 8/10 BBa\_Q0400 -(1) | 3995 |
| NC | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 30s | 30cycles |

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 1kbp ladder |
| 2 | K112806(lysis2) -1 |
| 3 | NC |
| 4 | Q04400(tetR) -1 |
| 5 | NC |
| 6 | 1kbp ladder |

[![Igku Aug17colonyPCR.jpg](/wiki/images/1/1f/Igku_Aug17colonyPCR.jpg)](/File%3AIgku_Aug17colonyPCR.jpg)

### Liquid Culture

Nakamoto

| Sample | medium |
| --- | --- |
| 8/15 BBa\_K112806(lysis2) -(1) | Plusgrow medium(+CP) |
| 8/15 BBa\_Q04400(tetR) -(1) | Plusgrow medium(+Amp) |
| 8/16 pBR322 -(1) | Plusgrow medium(+Amp) |

### Master Plate

Nakamoto

| Number | Use LB plate (+CP) |
| --- | --- |
| 1 | BBa\_K112806(lysis2) -(1) |

| Number | Use LB plate (+Kana) |
| --- | --- |
| 1 | BBa\_Q04400(tetR) -(1) |

### Miniprep

Nakamoto and Murakami

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| K748002(lysis3) -(1) | 330 | 1.66 | 2.08 |
| B0015(DT) -(1) | 188 | 1.73 | 1.82 |
| R0061(Plux) -(1) | 76 | 1.64 | 1.30 |
| K117000(lysis1) -(1) | 286 | 1.71 | 2.01 |
| K117000(lysis1) -(2) | 248 | 1.69 | 1.78 |
| I732020(RBS-lacZα-DT) -(1) | 198 | 1.71 | 1.93 |
| I13504(RBS-GFP-DT) -(1) | 248 | 1.69 | 2.03 |
| I13504(RBS-GFP-DT) -(2) | 246 | 1.55 | 1.73 |
| I0462(RBS-luxR-DT) -(1) | 326 | 1.65 | 1.74 |

### Restriction Enzyme Digestion

Murata, Murakami, Nakamoto, Ashida, Matsumoto and Yoshida

|  | 8/11 J23100 -(2) | SpeI | PstI | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 4.0µL | 1.0µL | 1.0µL | 3.0µL | 21µL | 30µL |
| 1 cut | 1.3µL | 0.2µL | -- | 1.0µL | 7.5µL | 10µL |
| 1 cut | 1.3µL | -- | 0.2µL | 1.0µL | 7.5µL | 10µL |
| NC | 1.3µL | -- | -- | 1.0µL | 7.7µL | 10µL |

|  | 8/15 B0034(RBS) -(2) | SpeI | PstI | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 6.0µL | 1.0µL | 1.0µL | 3.0µL | 19µL | 30µL |
| 1 cut | 2.0µL | 0.2µL | -- | 1.0µL | 6.8µL | 10µL |
| 1 cut | 2.0µL | -- | 0.2µL | 1.0µL | 6.8µL | 10µL |
| NC | 2.0µL | -- | -- | 1.0µL | 7.0µL | 10µL |

|  | 8/17 K117000(lysis1) -(1) | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.0µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 20µL | 30µL |
| 1 cut | 1.0µL | 0.2µL | -- | 1.0µL | 1.0µL | 6.8µL | 10µL |
| 1 cut | 1.0µL | -- | 0.2µL | 1.0µL | 1.0µL | 6.8µL | 10µL |
| NC | 1.0µL | -- | -- | 1.0µL | 1.0µL | 7.0µL | 10µL |

|  | 8/17 K748002(lysis3) -(1) | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.0µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 20µL | 30µL |
| 1 cut | 1.0µL | 0.2µL | -- | 1.0µL | 1.0µL | 6.8µL | 10µL |
| 1 cut | 1.0µL | -- | 0.2µL | 1.0µL | 1.0µL | 6.8µL | 10µL |
| NC | 1.0µL | -- | -- | 1.0µL | 1.0µL | 7.0µL | 10µL |

|  | 8/17 I13504(RBS-GFP-DT) -(1) | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.0µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 20µL | 30µL |
| 1 cut | 1.0µL | 0.2µL | -- | 1.0µL | 1.0µL | 6.8µL | 10µL |
| 1 cut | 1.0µL | -- | 0.2µL | 1.0µL | 1.0µL | 6.8µL | 10µL |
| NC | 1.0µL | -- | -- | 1.0µL | 1.0µL | 7.0µL | 10µL |

|  | 8/17 I0462(RBS-luxR-DT) -(1) | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.0µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 20µL | 30µL |
| 1 cut | 1.0µL | 0.2µL | -- | 1.0µL | 1.0µL | 6.8µL | 10µL |
| 1 cut | 1.0µL | -- | 0.2µL | 1.0µL | 1.0µL | 6.8µL | 10µL |
| NC | 1.0µL | -- | -- | 1.0µL | 1.0µL | 7.0µL | 10µL |

|  | 8/17 I732020(RBS-lacZα-DT) -(1) | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.0µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 20µL | 30µL |
| 1 cut | 1.0µL | 0.2µL | -- | 1.0µL | 1.0µL | 6.8µL | 10µL |
| 1 cut | 1.0µL | -- | 0.2µL | 1.0µL | 1.0µL | 6.8µL | 10µL |
| NC | 1.0µL | -- | -- | 1.0µL | 1.0µL | 7.0µL | 10µL |

|  | 8/11 R6061 -(1) | SpeI | PstI | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 8.0µL | 1.0µL | 1.0µL | 3.0µL | 17µL | 30µL |
| 1 cut | 3.0µL | 0.2µL | -- | 1.0µL | 5.8µL | 10µL |
| 1 cut | 3.0µL | -- | 0.2µL | 1.0µL | 5.8µL | 10µL |
| NC | 3.0µL | -- | -- | 1.0µL | 6.0µL | 10µL |

* incubate at 37 °C, for 1 h

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | J23100 -② | SpeI | PstI |
| 2 | J23100 -② | SpeI | -- |
| 3 | J23100 -② | -- | PstI |
| 4 | J23100 -② | -- | -- |
| 5 | B0034(RBS) -(2) | SpeI | PstI |
| 6 | B0034(RBS) -(2) | SpeI | -- |
| 7 | B0034(RBS) -(2) | -- | PstI |
| 8 | B0034(RBS) -(2) | -- | -- |
| 9 | 1kb ladder | -- | -- |
| 10 | K11700(lysis1) -(1) | XbaI | PstI |
| 11 | K11700(lysis1) -(1) | XbaI | -- |
| 12 | K11700(lysis1) -(1) | -- | PstI |
| 13 | K11700(lysis1) -(1) | -- | -- |
| 14 | K748002(lysis3) -(1) | XbaI | PstI |
| 15 | K748002(lysis3) -(1) | XbaI | -- |
| 16 | K748002(lysis3) -(1) | -- | PstI |
| 17 | K748002(lysis3) -(1) | -- | -- |

[![Igku Aug17 Electrophoresis(1).jpg](/wiki/images/2/28/Igku_Aug17_Electrophoresis%281%29.jpg)](/File%3AIgku_Aug17_Electrophoresis%281%29.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 18 | I13504(RBS-GFP-DT) -(1) | XbaI | PstI |
| 19 | I13504(RBS-GFP-DT) -(1) | XbaI | -- |
| 20 | I13504(RBS-GFP-DT) -(1) | -- | PstI |
| 21 | I13504(RBS-GFP-DT) -(1) | -- | -- |
| 22 | I0462(RBS-luxR-DT) -(1) | XbaI | PstI |
| 23 | I0462(RBS-luxR-DT) -(1) | XbaI | -- |
| 24 | I0462(RBS-luxR-DT) -(1) | -- | PstI |
| 25 | I0462(RBS-luxR-DT) -(1) | -- | -- |
| 26 | 1kb ladder | -- | -- |
| 27 | I732020(RBS-lacZα-DT) -(1) | XbaI | PstI |
| 28 | I732020(RBS-lacZα-DT) -(1) | XbaI | -- |
| 29 | I732020(RBS-lacZα-DT) -(1) | -- | PstI |
| 30 | I732020(RBS-lacZα-DT) -(1) | -- | -- |
| 31 | R0061(Plux) -(1) | SpeI | PstI |
| 32 | R0061(Plux) -(1) | SpeI | -- |
| 33 | R0061(Plux) -(1) | -- | PstI |
| 34 | R0061(Plux) -(1) | -- | -- |

[![Igku Aug17 Electrophoresis(2).jpg](/wiki/images/5/59/Igku_Aug17_Electrophoresis%282%29.jpg)](/File%3AIgku_Aug17_Electrophoresis%282%29.jpg)

### Miniprep

Murata

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/17 PBR322(TaKaRa) -(1) | 47 | 1.68 | 1.55 |
| 8/17 K112806(lysis2) -(1) | 64 | 1.67 | 1.70 |

## Aug 18

### Gel Extraction

Yoshida and Kojima

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100kb ladder | -- |
| 2 | 8/11 J23100 -② | -- |
| 3 | 8/11 J23100 -② | -- |
| 4 | -- | -- |
| 5 | 8/17 K117000(lysis1) -1 | -- |
| 6 | 8/17 K117000(lysis1) -1 | -- |
| 7 | -- | -- |
| 8 | 8/17 K748002(lysis3) -1 | -- |
| 9 | 8/17 K748002(lysis3) -1 | -- |
| 10 | -- | -- |
| 11 | 8/17 I13504(RBS-GFP-DT) -1 | -- |
| 12 | 8/17 I13504(RBS-GFP-DT) -1 | -- |

[![Igku Aug18 Gel Extraction before.jpg](/wiki/images/c/c6/Igku_Aug18_Gel_Extraction_before.jpg)](/File%3AIgku_Aug18_Gel_Extraction_before.jpg)
[![Igku Aug18 Gel Extraction1-2.jpg](/wiki/images/4/48/Igku_Aug18_Gel_Extraction1-2.jpg)](/File%3AIgku_Aug18_Gel_Extraction1-2.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 13 | 1kb ladder | -- |
| 14 | 8/17 I0462(RBS-luxR-DT) -1 | -- |
| 15 | 8/17 I0462(RBS-luxR-DT) -1 | -- |
| 16 | -- | -- |
| 17 | 8/17 I732020(RBS-lacZα-DT) -1 | -- |
| 18 | 8/17 I732020(RBS-lacZα-DT) -1 | -- |

[![Igku Aug18 Gel Extraction2 before.jpg](/wiki/images/3/35/Igku_Aug18_Gel_Extraction2_before.jpg)](/File%3AIgku_Aug18_Gel_Extraction2_before.jpg)
[![Igku Aug18 Gel Extraction2-2.jpg](/wiki/images/e/ef/Igku_Aug18_Gel_Extraction2-2.jpg)](/File%3AIgku_Aug18_Gel_Extraction2-2.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/14 J231000 | 1.7 | 1.61 | 0.04 |
| 8/17 K748002(lysis3) -1 | 5.4 | 2.39 | 0.45 |
| 8/17 I13504(RBS-GFP-DT) -1 | 3.4 | 3.33 | 0.19 |
| 8/17 I0462(RBS-luxR-DT) -1 | 4.4 | 2.07 | 0.37 |
| 8/17 I732020(RBS-lacZα-DT) -1 | 2.9 | 2.17 | 0.29 |

### Ligation

Ashida

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | J23100 | 9.0 | RBS-lacZα-DT | 8.6 | 3.5 |
| experiment | J23100 | 9.0 | RBS-GFP-DT | 7.3 | 3.5 |
| experiment | RBS | 1 | lysis3 | 4.6 | 2.8 |
| experiment | Plux | 1 | GFP | 7.3 | 3.5 |
| experiment | J23100 | 9.0 | -- | -- | 3.5 |
| experiment | RBS | 1.0 | -- | -- | 0.5 |
| experiment | Plux | 1.0 | -- | -- | 0.5 |

incubate 16°C 1hour

### Restriction Enzyme Digestion

Murata and Okazaki

|  | 8/17K112806(lysis2)-1 | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.0µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 3.0µL | 30 µL |
| 1 cuts | 1.0&mic6/8ro;L | 0.2µL | -- | 1.0µL | 1.0µL | 6.8µL | 10 µL |
| 1 cuts | 1.0µL | -- | 0.2µL | 1.0µL | 1.0µL | 6.8µL | 10 µL |

### Electrophoresis

Okazaki and Honda

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | lysis1 | XbaI | PstI |
| 3 | lysis1 | XbaI | -- |
| 4 | lysis1 | -- | PstI |
| 5 | lysis1 | -- | -- |
| 6 | lysis2 | XbaI | PstI |
| 7 | lysis2 | XbaI | -- |
| 8 | lysis2 | -- | PstI |
| 9 | lysis2 | -- | -- |
| 10 | 100bp ladder | -- | -- |

[![Igku Aug18electrophoresis.jpg](/wiki/images/e/e5/Igku_Aug18electrophoresis.jpg)](/File%3AIgku_Aug18electrophoresis.jpg)

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 8/8 LBKana | -- |
| 8/16 Pbad araC | -- |
| NC | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min | 30 |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 7/8 LB Kana | Plusgrow(+Kana) |
| 8/16 Pbad araC | Plusgrow(+Kana) |

### LB Medium Plate

No name

| volume | 200ml |
| --- | --- |
| Bacto Trypton | 2g |
| Bacto yeasy extract | 1g |
| NaCl | 1g |
| Agar Powder | 2g |

### Transformation

No name

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| 8/18 J23100+RBS-LacZα-DT | 2µL | 20µL | 22µL | Amp |
| 8/18 J23100+RBS-GFP-DT | 2µL | 20µL | 22µL | Amp |
| 8/18 J23100+RBS-luxR-DT | 2µL | 20µL | 22µL | Amp |
| RBS+lysis3 | 2µL | 20µL | 22µL | Amp |
| Plux+GFP | 2µL | 20µL | 22µL | CP |
| J23100 | 2µL | 20µL | 22µL | Amp |
| RBS | 2µL | 20µL | 22µL | Amp |
| Plux | 2µL | 20µL | 22µL | CP |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | 8/16Pbad araC-1 | -- | -- |
| 3 | 8/16Pbad araC-2 | -- | -- |
| 4 | NC | -- | -- |

[![Igku Aug18electrophoresis2.jpg](/wiki/images/3/30/Igku_Aug18electrophoresis2.jpg)](/File%3AIgku_Aug18electrophoresis2.jpg)

## Aug 19

### Colony PCR

Kojima

| Sample | base pair |
| --- | --- |
| 8/18 J23100 control -(1) | 1142 |
| 8/18 J23100-RBS-GFP-DT -(1) | 1156 |
| 8/18 J23100-RBS-GFP-DT -(2) | 1156 |
| 8/18 J23100-RBS-luxR-DT -(1) | 1271 |
| 8/18 J23100-RBS-luxR-DT -(2) | 1271 |
| 8/18 J23100-RBS-lacZα-DT -(1) | 670 |
| 8/18 J23100-RBS-lacZα-DT -(2) | 670 |
| 8/18 RBS-lysis3 -(1) | 997 |
| 8/18 RBS-lysis3 -(2) | 997 |
| 8/18 P0440(RBS-tetR-DT) -(1) | 1154 |
| 8/18 P0440(RBS-tetR-DT) -(2) | 1154 |
| negative control | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min | 30cycles |

[![Igku Aug19electrophoresis1(colony PCR).png](/wiki/images/2/2d/Igku_Aug19electrophoresis1%28colony_PCR%29.png)](/File%3AIgku_Aug19electrophoresis1%28colony_PCR%29.png)

| Lane | DNA |
| --- | --- |
| 1 | 100bp ladder |
| 2 | 8/18 NC J23100(1) |
| 3 | 8/18 J23100-RBS-GFP-DT-(1) |
| 4 | 8/18 J23100-RBS-GFP-DT-(2) |
| 5 | 8/18 J23100-RBS-luxR-DT-(1) |
| 6 | 8/18 J23100-RBS-luxR-DT-(2) |
| 7 | 8/18 J23100-RBS-lacZα-DT-(1) |
| 8 | 8/18 J23100-RBS-lacZα-DT-(2) |
| 9 | 8/18 RBS-lysis3-(1) |
| 10 | 8/18 RBS-lysis3-(2) |
| 11 | 8/18 R0400(RBS-tetR-DT)-(1) |
| 12 | 8/18 R0400(RBS-tetR-DT)-(2) |
| 13 | NC |
| 14 | 100bp ladder |

[![Igku Aug19electrophoresis1(Colony PCR).png](/wiki/images/2/23/Igku_Aug19electrophoresis1%28Colony_PCR%29.png)](/File%3AIgku_Aug19electrophoresis1%28Colony_PCR%29.png)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/19 lysis1 ① (XbaI & PstI) | 87.8 | 1.06 | 0.82 |

| Sample | base pair |
| --- | --- |
| 8/18 RBS control -(1) | -- |
| 8/18 R0040(Ptet) -(1) | 686 |
| 8/18 R0040(Ptet) -(2) | 686 |
| negative control | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 30s | 30cycles |

### Gel Extraction

Ashida

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 3 | 8/18 K117000(lysis2) -1 | XbaI & PstI |
| 5 | 8/18 K117000(lysis2) -1 | XbaI & PstI |

[![Igku Aug19electrophoresis3(Gel Extraction).png](/wiki/images/2/29/Igku_Aug19electrophoresis3%28Gel_Extraction%29.png)](/File%3AIgku_Aug19electrophoresis3%28Gel_Extraction%29.png)
[![Igku Aug19electrophoresis4(Gel Extraction).png](/wiki/images/2/20/Igku_Aug19electrophoresis4%28Gel_Extraction%29.png)](/File%3AIgku_Aug19electrophoresis4%28Gel_Extraction%29.png)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/18 lysis2-1(XbaI & PstI) | 3.1 | 3.64 | 0.19 |
| 8/18 lysis2-1(XbaI & PstI) | 3.4 | 1.89 | 0.13 |

### LB Medium Plate

Nakamoto

| volume | 200ml |
| --- | --- |
| Bacto T2ypton | 2g |
| Bacto yeast extract | 1g |
| NaCl | 1g |
| Agar Pouder | 2g |

### Restriction Enzyme Digestion

Tatsui

|  | lysis2-1 | XbaI | PstI | 10xbuffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 10µL | 1µL | 1µL | 3µL | 3µL | 12µL | 30µL |
| 1 cut | 2µL | 0.2µL | -- | 1µL | 1µL | 5.8µL | 10µL |
| 1 cut | 2µL | -- | 0.2µL | 1µL | 1µL | 5.8µL | 10µL |
| NC | 2µL | -- | -- | 1µL | 1µ | 6µL | 10µL |

* at 37°C, for 1h

Murakami and Okazaki

|  | 8/19 lysis1-1 | XbaI | PstI | 10xbuffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2µL | 0.5µL | 0.8µL | 2µL | 2µL | 13µL | 20µL |
| 1 cut | 0.5µL | 0.2µL | -- | 1µL | 1µL | 7.3µL | 10µL |
| 1 cut | 0.5µL | -- | 0.2µL | 1µL | 1µL | 7.3µL | 10µL |
| NC | 0.5µL | -- | -- | 1µL | 1µL | 7.5µL | 10µL |

* at 37°C, for 1h

[![Igku Aug19 Restriction Enzyme Digestion(lysis2-1(1)).jpg](/wiki/images/2/2d/Igku_Aug19_Restriction_Enzyme_Digestion%28lysis2-1%281%29%29.jpg)](/File%3AIgku_Aug19_Restriction_Enzyme_Digestion%28lysis2-1%281%29%29.jpg)

### Ligation

No name

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | RBS | 0.3 | lysis2 | 7.4 | 4 |
| NC | RBS | 0.3 | MilliQ | 7.4 | 4 |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | 8/19 lysis1 | XbaI | PstI |
| 3 | 8/19 lysis1 | XbaI | -- |
| 4 | 8/19 lysis1 | -- | PstI |
| 5 | 8/19 lysis1 | -- | -- |
| 6 | 8/19 lysis1(Gel Extraction Product) | XbaI | PstI |

[![Igku Aug19 Electrophoresis lysis1.jpg](/wiki/images/8/8f/Igku_Aug19_Electrophoresis_lysis1.jpg)](/File%3AIgku_Aug19_Electrophoresis_lysis1.jpg)

### Transformation

Murata and Kojima

| Name | Sample | Competent Cells | Plate |
| --- | --- | --- | --- |
| 8/18 Plux+RBS-GFP-DT | 2µL | 20µL | CP |
| 8/18 Plux(NC) | 2µL | 20µL | CP |
| 8/19 RBS+lysis2 | 2µL | 20µL | Amp |
| 8/15 Pbad-araC | 2µL | 20µL | Kan |

* Incubate at 37°C

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | 8/19 lysis1 ① | XbaI & PstI |

[![Igku Aug19 Gel Extraction(lysis①-1)before.jpg](/wiki/images/0/02/Igku_Aug19_Gel_Extraction%28lysis%E2%91%A0-1%29before.jpg)](/File%3AIgku_Aug19_Gel_Extraction%28lysis%E2%91%A0-1%29before.jpg)
[![Igku Aug19 Gel Extraction(lysis①-1)after.jpg](/wiki/images/1/17/Igku_Aug19_Gel_Extraction%28lysis%E2%91%A0-1%29after.jpg)](/File%3AIgku_Aug19_Gel_Extraction%28lysis%E2%91%A0-1%29after.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/19 lysis1 ① (XbaI & PstI) | 87.8 | 1.06 | 0.82 |

### Liquid Culture

Ashida

| Sample | medium |
| --- | --- |
| 8/18 Ptet -1 | Plusgrow medium(+CP) |
| 8/18 Ptet -2 | Plusgrow medium(+CP) |
| 8/18 RBS-tetR-DT -1 | Plusgrow medium(+CP) |
| 8/18 RBS-tetR-DT -2 | Plusgrow medium(+CP) |
| 8/18 J23100-GFP -1 | Plusgrow medium(+Amp) |
| 8/18 J23100-luxR -1 | Plusgrow medium(+Amp) |
| 8/18 J23100-luxR -2 | Plusgrow medium(+Amp) |
| 8/18 J23100-lacZα -1 | Plusgrow medium(+Amp) |
| 8/18 RBS-lysis3 -1 | Plusgrow medium(+Amp) |
| 8/18 RBS-lysis3 -2 | Plusgrow medium(+Amp) |

### Master Plate

Ashida

| Number | Use LB plate(+Amp) |
| --- | --- |
| 1 | 8/18 J23100-GFP -1 |
| 2 | 8/18 J23100-luxR -1 |
| 3 | 8/18 J23100-luxR -2 |
| 4 | 8/18 J23100-lacZα -1 |
| 5 | 8/18 RBS-lysis3 -1 |
| 6 | 8/18 RBS-lysis3 -2 |

| Number | Use LB plate(+CP) |
| --- | --- |
| 1 | 8/18 Ptet -1 |
| 1 | 8/18 Ptet -2 |
| 3 | 8/18 RBS-tetR-DT -1 |
| 4 | 8/18 RBS-tetR-DT -2 |

## Aug 20

### Miniprep

Kojima

| DNA | concentration[µg/µL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/18 Ptet -1 | 220 | 1.69 | 1.87 |
| 8/18 Ptet -2 | 210 | 1.69 | 1.71 |
| 8/18 RBS-tetR-DT -1 | 236 | 1.63 | 1.69 |
| 8/18 RBS-tetR-DT -2 | 182 | 1.65 | 1.98 |
| 8/18 J23100-RBS-GFP-DT -1 | 334 | 1.71 | 2.12 |
| 8/18 J23100-RBS-luxR-DT -1 | 440 | 1.67 | 1.60 |
| 8/18 J23100-RBS-luxR-DT -2 | 344 | 1.68 | 1.83 |
| 8/18 J23100-RBS-lacZα-DT -1 | 280 | 1.70 | 1.81 |
| 8/18 RBS-lysis3 -1 | 282 | 1.67 | 1.76 |
| 8/18 RBS-lysis3 -2 | 212 | 1.30 | 1.35 |

### LB Medium Plate

Okazaki

| volume | 200mL |
| --- | --- |
| Bacto Trypton | 2g |
| Bacto yeast extract | 1g |
| Nacl | 1g |
| Agar Powder | 2g |
| Amp | 40µl |

1. Measuring reagents and put they in the Erlenmeyer flask.
2. diluting in measuring cylinder to 200ml total.
3. autoclaving.
4. adding ampicirine 40ml.

### Ristriction Enzyme Digestion

Kojima and Ashida

|  | 8/20 Ptet -1(220µg/mL) | EcoRI | XbaI | 10x BSA | 10x buffer M | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 4.5 | 1 | 1 | 3 | 3 | 17.5 | 30 |
| 1 cut | 0.5 | 0.2 | 0 | 1 | 1 | 7.3 | 10 |
| 1 cut | 0.5 | 0 | 0.2 | 1 | 1 | 7.3 | 10 |
| NC | 0.5 | 0 | 0 | 1 | 1 | 7.5 | 10 |

|  | 8/20 RBS-tetR-DT -2(182µg/mL) | EcoRI | SpeI | 10x buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.5 | 1 | 1 | 3 | 19.5 | 30 |
| 1 cut | 0.6 | 0.2 | 0 | 1 | 8.2 | 10 |
| 1 cut | 0.6 | 0 | 0.2 | 1 | 8.2 | 10 |
| NC | 0.6 | 0 | 0 | 1 | 8.4 | 10 |

|  | 8/20 J23100-RBS-GFP-DT(334µg/ml) | EcoRI | SpeI | 10x buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3.0 | 1 | 1 | 3 | 22 | 30 |
| 1 cut | 0.3 | 0.2 | 0 | 1 | 8.5 | 10 |
| 1 cut | 0.3 | 0 | 0.2 | 1 | 8.5 | 10 |
| NC | 0.3 | 0 | 0 | 1 | 8.7 | 10 |

|  | 8/20 J23100-RBS-luxR-DT -2(344µg/mL) | EcoRI | XbaI | BSA | 10x buffer M | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.9 | 1 | 1 | 3 | 3 | 19.1 | 30 |
| 1 cut | 0.3 | 0.2 | 0 | 1 | 1 | 7.5 | 10 |
| 1 cut | 0.3 | 0 | 0.2 | 1 | 1 | 7.5 | 10 |
| NC | 0.3 | 0 | 0 | 1 | 1 | 7.7 | 10 |

|  | 8/20 RBS-lysis3 -1(282µg/mL) | EcoRI | SpeI | 10x buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3.5 | 1 | 1 | 3 | 21.5 | 30 |
| 1 cut | 0.4 | 0.2 | 0 | 1 | 8.4 | 10 |
| 1 cut | 0.4 | 0 | 0.2 | 1 | 8.4 | 10 |
| NC | 0.4 | 0 | 0 | 1 | 8.6 | 10 |

|  | 8/17 DT -1(188µg/mL) | EcoRI | XbaI | 10x BSA | 10x buffer M | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.3 | 1 | 1 | 3 | 3 | 16.7 | 30 |
| 1 cut | 0.3 | 0.2 | 0 | 1 | 1 | 7.5 | 10 |
| 1 cut | 0.3 | 0 | 0.2 | 1 | 1 | 7.5 | 10 |
| NC | 0.3 | 0 | 0 | 1 | 1 | 7.7 | 10 |

### Transformation

Nakamoto

| Name | Sample | Competent Cells(XL10-gold) | Total | Plate |
| --- | --- | --- | --- | --- |
| tRNA-Spinach-tRNA | 1µL | 10µL | 11µL | Amp |
| tetR-aptamer 12\_PC(076) | 1µL | 10µL | 11µL | Amp |
| tetR-aptamer 12\_1R(113) | 1µL | 10µL | 11µL | Amp |
| tetR-aptamer 12\_1M(105) | 1µL | 10µL | 11µL | Amp |
| pT181 attenuator | 1µL | 10µL | 11µL | Amp |
| Fusin1 attenuator | 1µL | 10µL | 11µL | Amp |
| Fusion3m2 attenuator | 1µL | 10µL | 11µL | Amp |
| pT181 antisense | 1µL | 10µL | 11µL | Amp |
| Fusion1 antisense | 1µL | 10µL | 11µL | Amp |
| Fusion6 antisense | 1µL | 10µL | 11µL | Amp |

incubate 37°C overnight 20:34~

### Electrophoresis

Kojima

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 8/20 Ptet -1 | EcoRI | XbaI |
| 2 | 8/20 Ptet -1 | EcoRI | -- |
| 3 | 8/20 Ptet -1 | -- | XbaI |
| 4 | 8/20 Ptet -1 | -- | -- |
| 5 | 8/20 RBS-tetR-DT -2 | EcoRI | SpeI |
| 6 | 8/20 RBS-tetR-DT -2 | EcoRI | -- |
| 7 | 8/20 RBS-tetR-DT -2 | -- | SpeI |
| 8 | 8/20 RBS-tetR-DT -2 | -- | -- |
| 9 | 1kbp ladder | -- | -- |
| 10 | 8/20 J23100-RBS-GFP-DT -1 | EcoRI | SpeI |
| 11 | 8/20 J23100-RBS-GFP-DT -1 | EcoRI | -- |
| 12 | 8/20 J23100-RBS-GFP-DT -1 | -- | SpeI |
| 13 | 8/20 J23100-RBS-GFP-DT -1 | -- | -- |
| 14 | 8/20 J23100-RBS-luxR-DT -1 | EcoRI | XbaI |
| 15 | 8/20 J23100-RBS-luxR-DT -1 | EcoRI | -- |
| 16 | 8/20 J23100-RBS-luxR-DT -1 | -- | XbaI |
| 17 | 8/20 J23100-RBS-luxR-DT -1 | -- | -- |
| 18 | 1kbp ladder | -- | -- |
| 19 | 8/20 RBS-lysis3 -1 | EcoRI | SpeI |
| 20 | 8/20 RBS-lysis3 -1 | EcoRI | -- |
| 21 | 8/20 RBS-lysis3 -1 | -- | SpeI |
| 22 | 8/20 RBS-lysis3 -1 | -- | -- |
| 23 | 1kbp ladder | -- | -- |
| 24 | 8/17 DT | EcoRI | XbaI |
| 25 | 8/17 DT | EcoRI | -- |
| 26 | 8/17 DT | -- | XbaI |
| 27 | 8/17 DT | -- | -- |

[![Igku Aug20Electrophoresis(Ptet E,X).jpg](/wiki/images/2/20/Igku_Aug20Electrophoresis%28Ptet_E%2CX%29.jpg)](/File%3AIgku_Aug20Electrophoresis%28Ptet_E%2CX%29.jpg)
[![Igku Aug20 Electrophoresis2(RBS‐lysis3-1).jpg](/wiki/images/6/61/Igku_Aug20_Electrophoresis2%28RBS%E2%80%90lysis3-1%29.jpg)](/File%3AIgku_Aug20_Electrophoresis2%28RBS%E2%80%90lysis3-1%29.jpg)
[![Igku Aug20Electrophoresis3(DT・E＋X).jpg](/wiki/images/9/90/Igku_Aug20Electrophoresis3%28DT%E3%83%BBE%EF%BC%8BX%29.jpg)](/File%3AIgku_Aug20Electrophoresis3%28DT%E3%83%BBE%EF%BC%8BX%29.jpg)

## Aug 21

### Plusgrow Medium

Tatsui

* material

:   * 8/17 Plusgrow medium 50ml
    * 7/22 5000x Ampicillin 10µl

1. Measuring materials and putting them in a 50ml tube

### Liquid culture

Tatsui

| Sample | medium |
| --- | --- |
| 8/20 tRNA-spinach -(1) | 8/21 Plusgrow medium(+Amp) |
| 8/20 tRNA-spinach -(2) | 8/21 Plusgrow medium(+Amp) |
| 8/20 TetR-aptamer 12\_P(1076) -(1) | 8/21 Plusgrow medium(+Amp) |
| 8/20 TetR-aptamer 12\_P(1076) -(2) | 8/21 Plusgrow medium(+Amp) |
| 8/20 TetR-aptamer 12\_1R(113) -(1) | 8/21 Plusgrow medium(+Amp) |
| 8/20 TetR-aptamer 12\_1R(113) -(2) | 8/21 Plusgrow medium(+Amp) |
| 8/20 TetR-aptamer 12\_1M(105) -(1) | 8/21 Plusgrow medium(+Amp) |
| 8/20 TetR-aptamer 12\_1M(105) -(2) | 8/21 Plusgrow medium(+Amp) |
| 8/20 PT181 attenuator -(1) | 8/21 Plusgrow medium(+Amp) |
| 8/20 PT181 attenuator -(2) | 8/21 Plusgrow medium(+Amp) |
| 8/20 Fusion1 attenuator -(1) | 8/21 Plusgrow medium(+Amp) |
| 8/20 Fusion1 attenuator -(2) | 8/21 Plusgrow medium(+Amp) |
| 8/20 Fusion3m2 attenuator -(1) | 8/21 Plusgrow medium(+Amp) |
| 8/20 Fusion3m2 attenuator -(2) | 8/21 Plusgrow medium(+Amp) |
| 8/20 PT181 antisense -(1) | 8/21 Plusgrow medium(+Amp) |
| 8/20 PT181 antisense -(2) | 8/21 Plusgrow medium(+Amp) |
| 8/20 Fusion1 antisense -(1) | 8/21 Plusgrow medium(+Amp) |
| 8/20 Fusion1 antisense -(2) | 8/21 Plusgrow medium(+Amp) |
| 8/20 Fusion6 antisense -(1) | 8/21 Plusgrow medium(+Amp) |
| 8/20 Fusion6 antisense -(2) | 8/21 Plusgrow medium(+Amp) |

* incubate 37°C 10hour

### Gel Extraction

Nakamoto and Tatsui

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 2 | 8/20 Ptet -(1) | EcoRI & XbaI |
| 3 |
| 5 | 8/20 RBS-tetR-DT -(2) | EcoRI & SpeI |
| 6 |
| 8 | 8/20 Pconst-RBS-GFP-DT -(1) | EcoRI & SpeI |
| 9 |
| 11 | 8/20 RBS-lysis3 -(1) | EcoRI & SpeI |
| 12 |

[![Igku Aug21 Gel Extraction(Ptet‐(1))before.jpg](/wiki/images/0/0c/Igku_Aug21_Gel_Extraction%28Ptet%E2%80%90%281%29%29before.jpg)](/File%3AIgku_Aug21_Gel_Extraction%28Ptet%E2%80%90%281%29%29before.jpg)
[![Igku Aug21 Gel Extraction 2-2.jpg](/wiki/images/f/f4/Igku_Aug21_Gel_Extraction_2-2.jpg)](/File%3AIgku_Aug21_Gel_Extraction_2-2.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Ptet (EcoRI & XbaI) | 21 | 1.55 | 0.78 |
| RBS-tetR-DT (EcoRI & SpeI) | 12 | 1.45 | 0.62 |
| Pconst-RBS-GFP-DT (EcoRI & SpeI) | 11 | 1.36 | 0.60 |
| RBS-lysis3 (EcoRI & SpeI) | 10 | 1.26 | 0.50 |

### Ristriction Enzyme Digestion

Kojima and Nakamoto

|  | 8/20 Ptet-(1) | EcoRI | XbaI | 10x BSA | 10x buffer M | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 4.5 | 1 | 1 | 3 | 3 | 17.5 | 30 |
| 1 cut | 0.5 | 0.2 | 0 | 1 | 1 | 7.3 | 10 |
| 1 cut | 0.5 | 0 | 0.2 | 1 | 1 | 7.3 | 10 |
| NC | 0.5 | 0 | 0 | 1 | 1 | 7.5 | 10 |

|  | 8/20 Pconst-RBS-luxR-DT-(2) | EcoRI | XbaI | 10x BSA | 10x buffer M | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.9 | 1 | 1 | 3 | 3 | 19.1 | 30 |
| 1 cut | 0.3 | 0.2 | 0 | 1 | 1 | 7.5 | 10 |
| 1 cut | 0.3 | 0 | 0.2 | 1 | 1 | 7.5 | 10 |
| NC | 0.3 | 0 | 0 | 1 | 1 | 7.7 | 10 |

|  | 8/17 DT | EcoRI | XbaI | 10x BSA | 10x buffer M | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.3 | 1 | 1 | 3 | 3 | 16.7 | 30 |
| 1 cut | 0.5 | 0.2 | 0 | 1 | 1 | 7.3 | 10 |
| 1 cut | 0.5 | 0 | 0.2 | 1 | 1 | 7.3 | 10 |
| NC | 0.5 | 0 | 0 | 1 | 1 | 7.5 | 10 |

* incubate at 37°C for 1h

### Electrophoresis

Kojima and Nakamoto

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 1kbp ladder | -- | -- |
| 2 | 8/20 Ptet -(1) | EcoRI | XbaI |
| 3 | 8/20 Ptet -(1) | EcoRI | -- |
| 4 | 8/20 Ptet -(1) | -- | XbaI |
| 5 | 8/20 Ptet -(1) | -- | -- |
| 6 | 8/20 J23100-RBS-luxR-DT -(2) | EcoRI | XbaI |
| 7 | 8/20 J23100-RBS-luxR-DT -(2) | EcoRI | -- |
| 8 | 8/20 J23100-RBS-luxR-DT -(2) | -- | XbaI |
| 9 | 8/20 J23100-RBS-luxR-DT -(2) | -- | -- |
| 10 | 8/20 DT | EcoRI | XbaI |
| 11 | 8/20 DT | EcoRI | -- |
| 12 | 8/20 DT | -- | XbaI |
| 13 | 8/20 DT | -- | -- |
| 14 | 1kbp ladder | -- | -- |
| 15 | 8/20 Ptet -(1) | EcoRI | XbaI |

* The reason why lane 2 and 15 were same is that the well of lane 2 might be broken.

[![Igku Aug21 Electorophoresis Rtet E+X.jpg](/wiki/images/d/d4/Igku_Aug21_Electorophoresis_Rtet_E%2BX.jpg)](/File%3AIgku_Aug21_Electorophoresis_Rtet_E%2BX.jpg)
Kojima

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 1kbp ladder | -- | -- |
| 2 | 8/20 J23100-RBS-luxR-DT -(2) | EcoRI | XbaI |
| 3 | 8/20 DT | EcoRI | XbaI |

[![Igku Aug21 Electrophoresis(J23100‐RBS,DT-E+X)bykojima.jpg](/wiki/images/5/55/Igku_Aug21_Electrophoresis%28J23100%E2%80%90RBS%2CDT-E%2BX%29bykojima.jpg)](/File%3AIgku_Aug21_Electrophoresis%28J23100%E2%80%90RBS%2CDT-E%2BX%29bykojima.jpg)

### Gel Extraction

Honda

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kb ladder | -- |
| 2 | 8/21 J23100-RBS-luxR-DT 25µL | EcoRI+XbaI |
| 3 | 8/21 J23100-RBS-luxR-DT 25µL | EcoRI+XbaI |
| 4 | -- | -- |
| 5 | 8/21 Ptet 25µL | EcoRI+XbaI |
| 6 | 8/21 Ptet 25µL | EcoRI+XbaI |

[![Igku Aug21 Gel Extraction(J23100,Ptet①)before.jpg](/wiki/images/7/78/Igku_Aug21_Gel_Extraction%28J23100%2CPtet%E2%91%A0%29before.jpg)](/File%3AIgku_Aug21_Gel_Extraction%28J23100%2CPtet%E2%91%A0%29before.jpg)
[![Igku Aug21 Gel Extraction(J23100,Ptet)after.jpg](/wiki/images/4/41/Igku_Aug21_Gel_Extraction%28J23100%2CPtet%29after.jpg)](/File%3AIgku_Aug21_Gel_Extraction%28J23100%2CPtet%29after.jpg)

### Electrophoresis

Honda

| Lane | Sample | EcoRI | XbaI |
| --- | --- | --- | --- |
| 1 | 1kb ladder | -- | -- |
| 2 | 8/21 DT | + | + |
| 3 | 8/21 DT | + | -- |
| 4 | 8/21 DT | -- | + |
| 5 | 8/21 DT | -- | -- |

[![Igku Aug21electrophoresis.jpg](/wiki/images/e/e1/Igku_Aug21electrophoresis.jpg)](/File%3AIgku_Aug21electrophoresis.jpg)

### Ligation

No name

| state | Vector | | Inserter | | Ligation High ver.2 | total |
| --- | --- | --- | --- | --- | --- | --- |
| experiment | 8/21 Pcn-RBS-luxR-DT(+Amp) | 2.3 | 8/21 Pcn-RBS-GFP-DT | 1.7 | 2 | 6 |
| NC | 8/21 Pcn-RBS-luxR-DT(+Amp) | 2.3 | MilliQ | 1.7 | 2 | 6 |
| experiment | 8/18 Plux(+CP) | 1.1 | 8/19 RBS-GFP-DT | 5 | 3.1 | 9.2 |
| NC | 8/18 Plux(+CP) | 1.1 | MilliQ | 5 | 3.1 | 9.2 |
| experiment | 8/18 RBS(+Amp) | 2.2 | 8/19 lysis1 | 6.0 | 4.1 | 12.3 |
| experiment | 8/18 RBS(+Amp) | 2.2 | 8/19 lysis2 | 3.6 | 2.9 | 8.7 |
| NC | 8/18 RBS(+Amp) | 2.2 | MilliQ | 3.6 | 2.9 | 8.7 |
| experiment | 8/21 Ptet | 1.5 | 8/20 RBS-tetR-DT (2) | 2.1 | 1.8 | 5.4 |
| NC | 8/21 Ptet | 1.5 | MilliQ | 2.1 | 1.8 | 5.4 |

### Transformation

Okazaki

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| 8/21 Pcon-RBS-GFP-DT+Pcon-RBS-GFP-DT | 1µL | 10µL | 11µL | Amp |
| 8/21 Pcon-RBS-GFP-DT NC | 1µL | 10µL | 11µL | Amp |
| 8/18 RBS+8/19 lysis1 | 1µL | 10µL | 11µL | Amp |
| 8/18 RBS+8/19 lysis2 | 1µL | 10µL | 11µL | Amp |
| 8/18 RBS NC | 1µL | 10µL | 11µL | Amp |
| 8/18 Plux+8/18 RBS-GFP-DT | 1µL | 10µL | 11µL | CP |
| 8/18 Plux+8/18 RBS-GFP-DT | 1µL | 10µL | 11µL | CP |
| 8/21 Ptet(pm)+8/20 RBS-tetR-DT(2) | 1µL | 10µL | 11µL | CP |
| 8/21 Ptet(pm)+8/20 RBS-tetR-DT(2) NC | 1µL | 10µL | 11µL | CP |
| 8/21 pSB1C3 | 1µL | 10µL | 11µL | CP |

### LB Medium Plate

### Gel Extraction

Okazaki

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kb ladder | -- |
| 2 | -- | -- |
| 3 | 8/21 DT | EcoRI & XbaI |
| 4 |

### Miniprep

Tatsui, Kojima, and Nakamoto

| DNA | concentration [µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| tRNA-spinach-(1) | 234 | 1.64 | 2.14 |
| tRNA-spinach-(2) | 440 | 1.60 | 1.91 |
| tetR-aptamer 12-p(1076)-(1) | 150 | 1.61 | 2.03 |
| tetR-aptamer 12-p(1076)-(2) | 374 | 1.45 | 1.66 |
| tetR-aptamer 12-1R(113)-(1) | 304 | 1.68 | 2.21 |
| tetR-aptamer 12-1R(113)-(2) | 382 | 1.47 | 1.66 |
| tetR-aptamer 12-1M(105)-(1) | 374 | 1.68 | 2.20 |
| tetR-aptamer 12-1M(105)-(2) | 286 | 1.31 | 1.43 |
| PT181 attenuator-(1) | 330 | 1.66 | 2.11 |
| PT181 attenuator-(2) | 270 | 1.68 | 2.18 |
| Fusion1 attenuator-(1) | 306 | 1.67 | 2.20 |
| Fusion1 attenuator-(2) | 282 | 1.60 | 1.94 |
| Fusion3m2 attenuator-(1) | 332 | 1.68 | 2.25 |
| Fusion3m2 attenuator-(2) | 326 | 1.67 | 2.10 |
| PT181 antisense-(1) | 178 | 1.64 | 1.68 |
| PT181 antisense-(2) | 116 | 1.63 | 1.95 |
| Fusion1 antisense-(1) | 300 | 1.67 | 2.21 |
| Fusion1 antisense-(2) | 164 | 1.42 | 1.46 |
| Fusion6 antisense-(1) | 336 | 1.65 | 2.18 |
| Fusion6 antisense-(2) | 192 | 1.65 | 2.01 |

## Aug 22

### Liquid culture

Nakamoto

| Sample | Medium |
| --- | --- |
| 8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (1) | 8/21 Plusgrow medium (+Amp) |
| 8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (2) | 8/21 Plusgrow medium (+Amp) |
| 8/21 Pcon-RBS-GFP-DT control (1) | 8/21 Plusgrow medium (+Amp) |
| 8/21 Pcon-RBS-GFP-DT control (2) | 8/21 Plusgrow medium (+Amp) |
| 8/21 RBS-lysis1 (1) | 8/21 Plusgrow medium (+Amp) |
| 8/21 RBS-lysis1 (2) | 8/21 Plusgrow medium (+Amp) |
| 8/21 RBS control (1) | 8/21 Plusgrow medium (+Amp) |
| 8/21 RBS control (2) | 8/21 Plusgrow medium (+Amp) |
| 8/21 Ptet(pm)-RBS-tetR-DT (1) | 8/21 Plusgrow medium (+Amp) |
| 8/21 Ptet(pm)-RBS-tetR-DT (2) | 8/21 Plusgrow medium (+Amp) |
| 8/21 Ptet(pm) control (1) | 8/21 Plusgrow medium (+Amp) |
| 8/21 Ptet(pm) control (2) | 8/21 Plusgrow medium (+Amp) |
| 8/21 Plux-RBS-GFP-DT (1) | 8/21 Plusgrow medium (+Amp) |
| 8/21 pSB1C3(BBa\_J04450) (1) | 8/21 Plusgrow medium (+Amp) |
| 8/21 pSB1C3(BBa\_J04450) (2) | 8/21 Plusgrow medium (+Amp) |

incubate 37°C 10hour

### Colony PCR

Kojima

| Sample | base pair |
| --- | --- |
| 8/21 RBS-lysis1(1) | 400 |
| 8/21 RBS-lysis1(2) | 400 |
| 8/21 RBS control(1) | -- |
| 8/21 RBS control(2) | -- |
| negative control | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 30s | 30cycles |

| Sample | base pair |
| --- | --- |
| 8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (1) | 2143 |
| 8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (2) | 2143 |
| 8/21 Pcon-RBS-luxR control(1) | -- |
| 8/21 Pcon-RBS-luxR control(2) | -- |
| negative control | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 2min | 30cycles |

| Sample | base pair |
| --- | --- |
| 8/21 Ptet-RBS-tetR-DT (1) | 1216 |
| 8/21 Ptet-RBS-tetR-DT (2) | 1216 |
| 8/21 Ptet control (1) | -- |
| 8/21 Ptet control (2) | -- |
| 8/21 Plux-RBS-GFP-DT (1) | 1227 |
| 8/21 pSB1C3(BBa\_J04450) (1) | 1353 |
| 8/21 pSB1C3(BBa\_J04450) (2) | 1353 |
| negative control | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min | 30cycles |

### Restriction Enzyme Digestion

Tatsui, Kojima, Honda and Ashida

|  | Fusion1 antisense (1) (300 µg/mL) | EcoRI | SpeI | buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3.3 | 1 | 1 | 3 | 21.7 | 30 |
| 1 cut | 0.7 | 0.2 | 0 | 1 | 8.1 | 10 |
| 1 cut | 0.7 | 0 | 0.2 | 1 | 8.1 | 10 |
| NC | 0.7 | 0 | 0 | 1 | 8.3 | 10 |

|  | Fusion1 antisense (1) (300 µg/mL) | XbaI | PstI | buffer M | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3.3 | 1 | 1 | 3 | 3 | 18.7 | 30 |
| 1 cut | 0.7 | 0.2 | 0 | 1 | 1 | 7.1 | 10 |
| 1 cut | 0.7 | 0 | 0.2 | 1 | 1 | 7.1 | 10 |
| NC | 0.7 | 0 | 0 | 1 | 1 | 7.3 | 10 |

|  | Fusion6 antisense (1) (336 µg/mL) | EcoRI | SpeI | buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3 | 1 | 1 | 3 | 22 | 30 |
| 1 cut | 0.6 | 0.2 | 0 | 1 | 8.2 | 10 |
| 1 cut | 0.6 | 0 | 0.2 | 1 | 8.2 | 10 |
| NC | 0.6 | 0 | 0 | 1 | 8.4 | 10 |

|  | Fusion6 antisense (1) (336 µg/mL) | XbaI | PstI | buffer M | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3 | 1 | 1 | 3 | 3 | 19 | 30 |
| 1 cut | 0.6 | 0.2 | 0 | 1 | 1 | 7.2 | 10 |
| 1 cut | 0.6 | 0 | 0.2 | 1 | 1 | 7.2 | 10 |
| NC | 0.6 | 0 | 0 | 1 | 1 | 7.4 | 10 |

|  | pT181 antisense (1) (178 µg/mL) | EcoRI | SpeI | buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.6 | 1 | 1 | 3 | 19.4 | 30 |
| 1 cut | 1.1 | 0.2 | 0 | 1 | 7.7 | 10 |
| 1 cut | 1.1 | 0 | 0.2 | 1 | 7.7 | 10 |
| NC | 1.1 | 0 | 0 | 1 | 7.9 | 10 |

|  | pT181 antisense (1) (178 µg/mL) | XbaI | PstI | buffer M | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.6 | 1 | 1 | 3 | 3 | 16.4 | 30 |
| 1 cut | 1.1 | 0.2 | 0 | 1 | 1 | 6.7 | 10 |
| 1 cut | 1.1 | 0 | 0.2 | 1 | 1 | 6.7 | 10 |
| NC | 1.1 | 0 | 0 | 1 | 1 | 6.9 | 10 |

|  | pT181 attenuator (1) | EcoRI | SpeI | buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3 | 1 | 1 | 3 | 22 | 30 |
| 1 cut | 0.6 | 0.2 | 0 | 1 | 8.2 | 10 |
| 1 cut | 0.6 | 0 | 0.2 | 1 | 8.2 | 10 |
| NC | 0.6 | 0 | 0 | 1 | 8.4 | 10 |

|  | pT181 attenuator (1) | XbaI | PstI | buffer M | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3 | 1 | 1 | 3 | 3 | 19 | 30 |
| 1 cut | 0.6 | 0.2 | 0 | 1 | 1 | 7.2 | 10 |
| 1 cut | 0.6 | 0 | 0.2 | 1 | 1 | 7.2 | 10 |
| NC | 0.6 | 0 | 0 | 1 | 1 | 7.4 | 10 |

|  | Fusion1 attenuator (1) | EcoRI | SpeI | buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3.3 | 1 | 1 | 3 | 21.7 | 30 |
| 1 cut | 0.7 | 0.2 | 0 | 1 | 8.1 | 10 |
| 1 cut | 0.7 | 0 | 0.2 | 1 | 8.1 | 10 |
| NC | 0.7 | 0 | 0 | 1 | 8.3 | 10 |

|  | Fusion1 attenuator (1) | XbaI | PstI | buffer M | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3.3 | 1 | 1 | 3 | 3 | 18.7 | 30 |
| 1 cut | 0.7 | 0.2 | 0 | 1 | 1 | 7.1 | 10 |
| 1 cut | 0.7 | 0 | 0.2 | 1 | 1 | 7.1 | 10 |
| NC | 0.7 | 0 | 0 | 1 | 1 | 7.3 | 10 |

|  | Fusion3m2 attenuator (1) | EcoRI | SpeI | buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3 | 1 | 1 | 3 | 22 | 30 |
| 1 cut | 0.6 | 0.2 | 0 | 1 | 8.2 | 10 |
| 1 cut | 0.6 | 0 | 0.2 | 1 | 8.2 | 10 |
| NC | 0.6 | 0 | 0 | 1 | 8.4 | 10 |

|  | Fusion3m2 attenuator (1) | XbaI | PstI | buffer M | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3 | 1 | 1 | 3 | 3 | 19 | 30 |
| 1 cut | 0.6 | 0.2 | 0 | 1 | 1 | 7.2 | 10 |
| 1 cut | 0.6 | 0 | 0.2 | 1 | 1 | 7.2 | 10 |
| NC | 0.6 | 0 | 0 | 1 | 1 | 7.4 | 10 |

|  | Spinach (2) | EcoRI | SpeI | buffer B | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.3 | 0.5 | 0.5 | 3 | 0.3 | 23.4 | 30 |
| 1 cut | 0.5 | 0.1 | 0 | 1 | 0.1 | 8.3 | 10 |
| 1 cut | 0.5 | 0 | 0.1 | 1 | 0.1 | 8.3 | 10 |
| NC | 0.5 | 0 | 0 | 1 | 0.1 | 8.4 | 10 |

|  | tetR aptamer 12\_P (2) | EcoRI | SpeI | buffer B | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.7 | 0.5 | 0.5 | 3 | 0.3 | 23 | 30 |
| 1 cut | 0.5 | 0.1 | 0 | 1 | 0.1 | 8.3 | 10 |
| 1 cut | 0.5 | 0 | 0.1 | 1 | 0.1 | 8.3 | 10 |
| NC | 0.5 | 0 | 0 | 1 | 0.1 | 8.4 | 10 |

|  | tetR aptamer 12\_1R (2) | EcoRI | SpeI | buffer B | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.6 | 0.5 | 0.5 | 3 | 0.3 | 23.1 | 30 |
| 1 cut | 0.5 | 0.1 | 0 | 1 | 0.1 | 8.3 | 10 |
| 1 cut | 0.5 | 0 | 0.1 | 1 | 0.1 | 8.3 | 10 |
| NC | 0.5 | 0 | 0 | 1 | 0.1 | 8.4 | 10 |

|  | tetR aptamer 12\_1M (1) | EcoRI | SpeI | buffer B | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.7 | 0.5 | 0.5 | 3 | 0.3 | 23 | 30 |
| 1 cut | 0.5 | 0.1 | 0 | 1 | 0.1 | 8.3 | 10 |
| 1 cut | 0.5 | 0 | 0.1 | 1 | 0.1 | 8.3 | 10 |
| NC | 0.5 | 0 | 0 | 1 | 0.1 | 8.4 | 10 |

|  | J23100 | SpeI | PstI | buffer B | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 7 | 0.5 | 0.5 | 3 | 0.3 | 18.7 | 30 |
| 1 cut | 1.4 | 0.1 | 0 | 1 | 0.1 | 7.4 | 10 |
| 1 cut | 1.4 | 0 | 0.1 | 1 | 0.1 | 7.4 | 10 |
| NC | 1.4 | 0 | 0 | 1 | 0.1 | 7.5 | 10 |

|  | Plac (2) | SpeI | PstI | buffer B | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 11.2 | 0.5 | 0.5 | 3 | 0.3 | 14.5 | 30 |
| 1 cut | 2.2 | 0.1 | 0 | 1 | 0.1 | 6.6 | 10 |
| 1 cut | 2.2 | 0 | 0.1 | 1 | 0.1 | 6.6 | 10 |
| NC | 2.2 | 0 | 0 | 1 | 0.1 | 6.7 | 10 |

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | RBS-lysis1 -(1) |
| 3 | RBS-lysis1 -(2) |
| 4 | RBS-lysis2 -(1) |
| 5 | RBS-lysis2 -(2) |
| 6 | RBS(NC) -(1) |
| 7 | RBS(NC) -(2) |
| 8 | NC |

[![Igku Aug22 Electrophoresis(ColoP)-1.jpg](/wiki/images/d/da/Igku_Aug22_Electrophoresis%28ColoP%29-1.jpg)](/File%3AIgku_Aug22_Electrophoresis%28ColoP%29-1.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 1kb ladder |
| 2 | Ptet-RBS-tetR-DT -(1) |
| 3 | Ptet-RBS-tetR-DT -(2) |
| 4 | Ptet(NC) -(1) |
| 5 | Ptet(NC) -(2) |
| 6 | Plux-RBS-GFP-DT -(1) |
| 7 | pSB1C3 -(1) |
| 8 | pSB1C3 -(2) |
| 9 | NC |

[![Igku Aug22 Electrophoresis(ColoP)-2.jpg](/wiki/images/7/7d/Igku_Aug22_Electrophoresis%28ColoP%29-2.jpg)](/File%3AIgku_Aug22_Electrophoresis%28ColoP%29-2.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 1kb ladder |
| 2 | Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT -(1) |
| 3 | Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT -(2) |
| 4 | Pcon-RBS-luxR -(1) |
| 5 | Pcon-RBS-luxR -(2) |
| 6 | NC |
| 7 | 1kb ladder |

[![Igku Aug22 Electrophoresis(ColoP)-3.jpg](/wiki/images/a/a7/Igku_Aug22_Electrophoresis%28ColoP%29-3.jpg)](/File%3AIgku_Aug22_Electrophoresis%28ColoP%29-3.jpg)

### Miniprep

Ashida

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/22 RBS-lysis (1) | 128 | 1.77 | 2.29 |
| 8/22 RBS-lysis (2) | 96 | 1.80 | 2.07 |
| 8/22 pSB1C3 (1) | 108 | 1.55 | 1.45 |
| 8/22 pSB1C3 (2) | 130 | 1.69 | 1.76 |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp | -- | -- |
| 2 | 8/22 pT181 attenuator (1) | EcoRI | SpeI |
| 3 | 8/22 pT181 attenuator (1) | EcoRI | -- |
| 4 | 8/22 pT181 attenuator (1) | -- | SpeI |
| 5 | 8/22 pT181 attenuator (1) | -- | -- |
| 6 | 8/22 pT181 attenuator (1) | XbaI | PstI |
| 7 | 8/22 pT181 attenuator (1) | XbaI | -- |
| 8 | 8/22 pT181 attenuator (1) | -- | PstI |
| 9 | 8/22 pT181 attenuator (1) | -- | -- |
| 10 | 8/22 Fusion6 antisense (1) | EcoRI | SpeI |
| 11 | 8/22 Fusion6 antisense (1) | EcoRI | -- |
| 12 | 8/22 Fusion6 antisense (1) | -- | SpeI |
| 13 | 8/22 Fusion6 antisense (1) | -- | -- |
| 14 | 100bp | -- | -- |

[![Igku Aug22electrophoresis.jpg](/wiki/images/f/f8/Igku_Aug22electrophoresis.jpg)](/File%3AIgku_Aug22electrophoresis.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp | -- | -- |
| 2 | 8/22 Fusion6 antisense (1) | XbaI | PstI |
| 3 | 8/22 Fusion6 antisense (1) | XbaI | -- |
| 4 | 8/22 Fusion6 antisense (1) | -- | PstI |
| 5 | 8/22 Fusion6 antisense (1) | -- | -- |
| 6 | 8/22 Fusion1 antisense (1) | EcoRI | SpeI |
| 7 | 8/22 Fusion1 antisense (1) | EcoRI | -- |
| 8 | 8/22 Fusion1 antisense (1) | -- | SpeI |
| 9 | 8/22 Fusion1 antisense (1) | -- | -- |
| 10 | 8/22 Fusion1 antisense (1) | XbaI | PstI |
| 11 | 8/22 Fusion1 antisense (1) | XbaI | -- |
| 12 | 8/22 Fusion1 antisense (1) | -- | PstI |
| 13 | 8/22 Fusion1 antisense (1) | -- | -- |
| 14 | 100bp | -- | -- |

[![Igku Aug22electrophoresis2.jpg](/wiki/images/b/b7/Igku_Aug22electrophoresis2.jpg)](/File%3AIgku_Aug22electrophoresis2.jpg)

## Aug 23

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | pT181 antisense | EcoRI | SpeI |
| 2 | pT181 antisense | EcoRI | -- |
| 3 | pT181 antisense | -- | SpeI |
| 4 | pT181 antisense | -- | -- |
| 5 | pT181 antisense | XbaI | PstI |
| 6 | pT181 antisense | XbaI | -- |
| 7 | pT181 antisense | -- | PstI |
| 8 | pT181 antisense | -- | -- |
| 9 | 100bp ladder | -- | -- |
| 10 | Fusion1 attenuator | EcoRI | SpeI |
| 11 | Fusion1 attenuator | EcoRI | -- |
| 12 | Fusion1 attenuator | -- | SpeI |
| 13 | Fusion1 attenuator | -- | -- |
| 14 | Fusion1 attenuator | XbaI | PstI |
| 15 | Fusion1 attenuator | XbaI | -- |
| 16 | Fusion1 attenuator | -- | PstI |
| 17 | Fusion1 attenuator | -- | -- |

[![Igku Aug23electrophoresis.jpg](/wiki/images/3/3c/Igku_Aug23electrophoresis.jpg)](/File%3AIgku_Aug23electrophoresis.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | Fusion3m2 attenuator | EcoRI | SpeI |
| 2 | Fusion3m2 attenuator | EcoRI | -- |
| 3 | Fusion3m2 attenuator | -- | SpeI |
| 4 | Fusion3m2 attenuator | -- | -- |
| 5 | Fusion3m2 attenuator | XbaI | PstI |
| 6 | Fusion3m2 attenuator | XbaI | -- |
| 7 | Fusion3m2 attenuator | -- | PstI |
| 8 | Fusion3m2 attenuator | -- | -- |
| 9 | 100bp ladder | -- | -- |
| 10 | tetR aptamer 12\_1M | EcoRI | SpeI |
| 11 | tetR aptamer 12\_1M | EcoRI | -- |
| 12 | tetR aptamer 12\_1M | -- | SpeI |
| 13 | tetR aptamer 12\_1M | -- | -- |
| 14 | tetR aptamer 12\_P | EcoRI | SpeI |
| 15 | tetR aptamer 12\_P | EcoRI | -- |
| 16 | tetR aptamer 12\_P | -- | SpeI |
| 17 | tetR aptamer 12\_P | -- | -- |

[![Igku Aug23electrophoresis2.jpg](/wiki/images/9/90/Igku_Aug23electrophoresis2.jpg)](/File%3AIgku_Aug23electrophoresis2.jpg)
Kojima and Honda

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | Plac | SpeI | PstI |
| 2 | Plac | SpeI | -- |
| 3 | Plac | -- | PstI |
| 4 | Plac | -- | -- |
| 5 | Spinach | EcoRI | SpeI |
| 6 | Spinach | EcoRI | -- |
| 7 | Spinach | -- | SpeI |
| 8 | Spinach | -- | -- |
| 9 | 100bp ladder | -- | -- |
| 10 | J23100 | SpeI | PstI |
| 11 | J23100 | SpeI | -- |
| 12 | J23100 | -- | PstI |
| 13 | J23100 | -- | -- |
| 14 | tetR aptamer 12\_1R | EcoRI | SpeI |
| 15 | tetR aptamer 12\_1R | EcoRI | -- |
| 16 | tetR aptamer 12\_1R | -- | SpeI |
| 17 | tetR aptamer 12\_1R | -- | -- |

[![Igku Aug23 Electrophoresis(N2).jpg](/wiki/images/e/e0/Igku_Aug23_Electrophoresis%28N2%29.jpg)](/File%3AIgku_Aug23_Electrophoresis%28N2%29.jpg)

### Restriction Enzyme Digestion

Nakamoto and Kojima

|  | Fusion6 antisense (1) | EcoRI | SpeI | buffer B | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3 | 0.5 | 0.5 | 3 | 0.3 | 22.7 | 30 |
| 1 cut | 0.6 | 0.1 | 0 | 1 | 0.1 | 8.2 | 10 |
| 1 cut | 0.6 | 0 | 0.1 | 1 | 0.1 | 8.2 | 10 |
| NC | 0.6 | 0 | 0 | 1 | 0.1 | 8.3 | 10 |

|  | Fusion6 antisense (1) | XbaI | PstI | buffer D | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3 | 0.5 | 0.5 | 3 | 0.3 | 22.7 | 30 |
| 1 cut | 0.6 | 0.1 | 0 | 1 | 0.1 | 8.2 | 10 |
| 1 cut | 0.6 | 0 | 0.1 | 1 | 0.1 | 8.2 | 10 |
| NC | 0.6 | 0 | 0 | 1 | 0.1 | 8.3 | 10 |

|  | Fusion1 antisense (1) | EcoRI | SpeI | buffer B | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3.3 | 0.5 | 0.5 | 3 | 0.3 | 22.4 | 30 |
| 1 cut | 0.7 | 0.1 | 0 | 1 | 0.1 | 8.1 | 10 |
| 1 cut | 0.7 | 0 | 0.1 | 1 | 0.1 | 8.1 | 10 |
| NC | 0.7 | 0 | 0 | 1 | 0.1 | 8.2 | 10 |

|  | pT181 antisense (1) | EcoRI | SpeI | buffer B | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.6 | 0.5 | 0.5 | 3 | 0.3 | 20.1 | 30 |
| 1 cut | 1.1 | 0.1 | 0 | 1 | 0.1 | 7.7 | 10 |
| 1 cut | 1.1 | 0 | 0.1 | 1 | 0.1 | 7.7 | 10 |
| NC | 1.1 | 0 | 0 | 1 | 0.1 | 7.8 | 10 |

|  | pT181 antisense (1) | XbaI | PstI | buffer D | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.6 | 0.5 | 0.5 | 3 | 0.3 | 20.1 | 30 |
| 1 cut | 1.1 | 0.1 | 0 | 1 | 0.1 | 7.7 | 10 |
| 1 cut | 1.1 | 0 | 0.1 | 1 | 0.1 | 7.7 | 10 |
| NC | 1.1 | 0 | 0 | 1 | 0.1 | 7.8 | 10 |

* incubate at 37°C from 17:29.

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp | -- | -- |
| 2 | pT181 attenuator | EcoRI | SpeI |
| 3 | pT181 attenuator | XbaI | PstI |
| 4 | Fusion3m2 attenuator | EcoRI | SpeI |
| 5 | Fusion1 attenuator | XbaI | PstI |
| 6 | Fusion1 attenuator | EcoRI | SpeI |
| 7 | Fusion3m2 attenuator | XbaI | PstI |
| 8 | pT181 antisense | EcoRI | SpeI |
| 9 | pT181 antisense | XbaI | PstI |
| 10 | Fusion1 antisense | EcoRI | SpeI |
| 11 | Fusion1 antisense | XbaI | PstI |
| 12 | Fusion6 antisense | EcoRI | SpeI |
| 13 | Fusion6 antisense | XbaI | PstI |
| 14 | tetR aptamer 12\_1M | EcoRI | SpeI |
| 15 | tetR aptamer 12\_P | EcoRI | SpeI |
| 16 | tetR aptamer 12\_1R | EcoRI | SpeI |
| 17 | 100bp | -- | -- |

[![Igku Aug23 Electrophoresis (N3).jpg](/wiki/images/2/29/Igku_Aug23_Electrophoresis_%28N3%29.jpg)](/File%3AIgku_Aug23_Electrophoresis_%28N3%29.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp | -- | -- |
| 2 | Spinach | EcoRI | SpeI |
| 3 | J23100 | SpeI | PstI |
| 4 | Plac | SpeI | PstI |
| 5 | 100bp | -- | -- |
| 6 | J23100 | SpeI | PstI |

[![Igku Aug23 Electrophoresis(N3)-2.jpg](/wiki/images/e/e7/Igku_Aug23_Electrophoresis%28N3%29-2.jpg)](/File%3AIgku_Aug23_Electrophoresis%28N3%29-2.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp | -- | -- |
| 2 | Fusion6 antisense | EcoRI | SpeI |
| 3 | Fusion6 antisense | EcoRI | -- |
| 4 | Fusion6 antisense | -- | SpeI |
| 5 | Fusion6 antisense | -- | -- |
| 6 | pT181 antisense | XbaI | PstI |
| 8 | 100bp | -- | -- |
| 9 | 100bp | -- | -- |
| 10 | Fusion6 antisense | XbaI | PstI |
| 11 | Fusion6 antisense | XbaI | -- |
| 12 | Fusion6 antisense | -- | PstI |
| 13 | Fusion6 antisense | -- | -- |
| 14 | pT181 antisense | XbaI | -- |
| 16 | 100bp | -- | -- |

[![Igku Aug23electrophoresis4.jpg](/wiki/images/9/9c/Igku_Aug23electrophoresis4.jpg)](/File%3AIgku_Aug23electrophoresis4.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp | -- | -- |
| 2 | Fusion1 antisense | EcoRI | SpeI |
| 3 | Fusion1 antisense | EcoRI | -- |
| 4 | Fusion1 antisense | -- | SpeI |
| 5 | Fusion1 antisense | -- | -- |
| 6 | pT181 antisense | -- | PstI |
| 8 | 100bp | -- | -- |
| 9 | 100bp | -- | -- |
| 10 | pT181 antisense | EcoRI | SpeI |
| 11 | pT181 antisense | EcoRI | -- |
| 12 | pT181 antisense | -- | SpeI |
| 13 | pT181 antisense | -- | -- |
| 14 | pT181 antisense | -- | -- |
| 16 | 100bp | -- | -- |

[![Igku Aug23electrophoresis5.jpg](/wiki/images/c/cd/Igku_Aug23electrophoresis5.jpg)](/File%3AIgku_Aug23electrophoresis5.jpg)

### Gel Extraction

Nakamoto, Ashida, Kojima, Okazaki & Matsumoto

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | pT181 attenuator | XbaI & PstI |
| 3 |
| 5 | Fusion3m2 attenuator | EcoRI & SpeI |
| 6 |
| 8 | Fusion1 attenuator | XbaI & PstI |
| 9 |
| 11 | Fusion1 attenuator | EcoRI & SpeI |
| 12 |

[![Igku Aug23Gel Extraction.jpg](/wiki/images/b/b0/Igku_Aug23Gel_Extraction.jpg)](/File%3AIgku_Aug23Gel_Extraction.jpg)
[![Igku Aug23 Gel Extraction(N6)-1.2.jpg](/wiki/images/3/3b/Igku_Aug23_Gel_Extraction%28N6%29-1.2.jpg)](/File%3AIgku_Aug23_Gel_Extraction%28N6%29-1.2.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | Fusion3m2 attenuator | XbaI & PstI |
| 3 |
| 5 | pT181 antisense | EcoRI & SpeI |
| 6 |
| 8 | pT181 antisense | XbaI & PstI |
| 9 |
| 11 | Fusion1 antisense | XbaI & PstI |
| 12 |

[![Igku Aug23 Gel Extraction(N7)-2.jpg](/wiki/images/4/4c/Igku_Aug23_Gel_Extraction%28N7%29-2.jpg)](/File%3AIgku_Aug23_Gel_Extraction%28N7%29-2.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | Fusion6 attenuator | XbaI & PstI |
| 3 |
| 5 | aptamer 12\_1M | EcoRI & SpeI |
| 6 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | aptamer 12\_P | EcoRI & SpeI |
| 3 |
| 5 | aptamer 12\_1R | EcoRI & SpeI |
| 6 |

[![Igku Aug23 Gel Extraction(N6)-2.4.jpg](/wiki/images/6/67/Igku_Aug23_Gel_Extraction%28N6%29-2.4.jpg)](/File%3AIgku_Aug23_Gel_Extraction%28N6%29-2.4.jpg)
[![Igku Aug23 Gel Extraction(N6)-3-2.jpg](/wiki/images/e/ef/Igku_Aug23_Gel_Extraction%28N6%29-3-2.jpg)](/File%3AIgku_Aug23_Gel_Extraction%28N6%29-3-2.jpg)
Ashida

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | Fusion6 antisense | EcoRI & SpeI |
| 3 |
| 5 | Fusion6 attenuator | XbaI & PstI |
| 6 |
| 8 | Fusion1 antisense | EcoRI & SpeI |
| 9 |
| 11 | pT181 antisense | EcoRI & SpeI |
| 12 |

[![Igku Aug23 Gel Extracton(N8)-1.jpg](/wiki/images/7/75/Igku_Aug23_Gel_Extracton%28N8%29-1.jpg)](/File%3AIgku_Aug23_Gel_Extracton%28N8%29-1.jpg)
[![Igku Aug23 Gel Extraction(N8)-1.2.jpg](/wiki/images/0/03/Igku_Aug23_Gel_Extraction%28N8%29-1.2.jpg)](/File%3AIgku_Aug23_Gel_Extraction%28N8%29-1.2.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 3 | pT181 antisense | XbaI & PstI |
| 4 |

[![Igku Aug23 Gel Extraction(N7)-1.jpg](/wiki/images/4/49/Igku_Aug23_Gel_Extraction%28N7%29-1.jpg)](/File%3AIgku_Aug23_Gel_Extraction%28N7%29-1.jpg)
[![Igku Aug23 Gel Extraction(N7)1 2.jpg](/wiki/images/9/97/Igku_Aug23_Gel_Extraction%28N7%291_2.jpg)](/File%3AIgku_Aug23_Gel_Extraction%28N7%291_2.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 3 | Plac | SpeI & PstI |
| 4 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 3 | J23100 | SpeI & PstI |
| 5 | Spinach | EcoRI & SpeI |
| 6 |

[![Igku Aug23 Gel Extraction(N9)-1-1.jpg](/wiki/images/4/4f/Igku_Aug23_Gel_Extraction%28N9%29-1-1.jpg)](/File%3AIgku_Aug23_Gel_Extraction%28N9%29-1-1.jpg)
[![Igku Aug23 Gel Extraction(N9)-1-2.jpg](/wiki/images/a/ae/Igku_Aug23_Gel_Extraction%28N9%29-1-2.jpg)](/File%3AIgku_Aug23_Gel_Extraction%28N9%29-1-2.jpg)

## Aug 24

### Gel Extraction

Ashida

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/22 aptamer 12\_P (EcoRI & SpeI) | 88 | 21.3 | -- |
| 8/22 aptamer 12\_1R (EcoRI & SpeI) | 63 | 23.1 | -- |
| 8/22 aptamer 12\_1M (EcoRI & SpeI) | 40 | 22.0 | -- |
| 8/23 Fusion6 antisense (EcoRI & SpeI) | 6.0 | 1.60 | 0.17 |
| 8/23 Fusion6 antisense (XbaI & PstI) | 6.0 | 1.71 | 0.25 |
| 8/23 pT181 antisense (EcoRI & SpeI) | 6.0 | 2.22 | 0.15 |
| 8/23 pT181 antisense (XbaI & PstI) | 6.0 | 2.10 | 0.04 |
| 8/22 Plac (SpeI & PstI) | 6.0 | 1.47 | 0.09 |
| 8/22 Fusion1 antisense (EcoRI & SpeI) | 10 | 2.16 | 0.01 |
| 8/22 J23100 (SpeI & PstI) | 6.0 | 1.52 | 0.35 |

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/22 Spinach (EcoRI & SpeI) | 8.0 | 1.15 | 0.77 |
| 8/22 Fusion6 antisense (XbaI & PstI) | 44 | 1.07 | 0.96 |
| 8/22 Fusion1 antisense (XbaI & PstI) | 2 | -- | 1.00 |
| 8/22 pT181 antisense (XbaI & PstI) | 3. | 2.01 | 0.40 |
| 8/22 pT181 antisense (EcoRI & SpeI) | 8 | 1.13 | 0.82 |
| 8/22 Fusion3m2 attenuator (XbaI & PstI) | 5 | 2.27 | 1.50 |
| 8/22 Fusion3m2 attenuator (EcoRI & SpeI) | 34 | 1.16 | 1.02 |
| 8/22 Fusion1 attenuator (XbaI & PstI) | 14 | 1.40 | 0.62 |
| 8/22 Fusion1 attenuator (EcoRI & SpeI) | 16 | 1.27 | 0.61 |
| 8/22 pT181 attenuator (XbaI & PstI) | 5 | -- | 0.88 |

* These samples were performed ethanol ethanl precipitation.

### Restriction Enzyme Digestion

No name

|  | tetR aptamer 12\_P(2) | EcoRI | SpeI | 10x Buffer B | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.7µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 23µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1µL | 0.1µL | 8.6µL | 10µL |

|  | tetR aptamer 12\_1R(2) | EcoRI | SpeI | 10x Buffer B | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.6µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 23.1µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1µL | 0.1µL | 8.6µL | 10µL |

|  | tetR aptamer 12\_M(1) | EcoRI | SpeI | 10x Buffer B | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.7µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 23µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1µL | 0.1µL | 8.6µL | 10µL |

### Master Plate

No name

| Number | Use LB plate(+Kan) |
| --- | --- |
| 1 | entA- #1 |
| 2 | entA- #1 |
| 3 | entA- #1 |
| 4 | entA- #2 |
| 5 | entA- #2 |
| 6 | entA- #2 |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | - | - |
| 2 | tetR aptamer 12\_P | EcoRI | SpeI |
| 3 | tetR aptamer 12\_P | - | - |
| 4 | tetR aptamer 12\_1R | EcoRI | SpeI |
| 5 | tetR aptamer 12\_1R | - | - |
| 6 | tetR aptamer 12\_1M | EcoRI | SpeI |
| 7 | tetR aptamer 12\_1M | - | - |
| 8 | 100bp ladder | - | - |

[![Igku Aug24 Electrophoresis(N1)-pic.jpg](/wiki/images/1/19/Igku_Aug24_Electrophoresis%28N1%29-pic.jpg)](/File%3AIgku_Aug24_Electrophoresis%28N1%29-pic.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | - | - |
| 2 | tetR aptamer 12\_P | EcoRI | SpeI |
| 3 | tetR aptamer 12\_1R | EcoRI | SpeI |
| 4 | tetR aptamer 12\_1M | EcoRI | SpeI |
| 5 | 100bp ladder | - | - |

[![Igku Aug24 Electrophoresis(N2)-pic2.jpg](/wiki/images/2/26/Igku_Aug24_Electrophoresis%28N2%29-pic2.jpg)](/File%3AIgku_Aug24_Electrophoresis%28N2%29-pic2.jpg)

## Aug 26

### Gel Extraction

Matsumoto and Ashida

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | - |
| 2 | 8/24 tetR aptamer 12\_P | EcoRI & SpeI |
| 4 | 8/24 tetR aptamer 12\_1R | EcoRI & SpeI |
| 6 | 8/24 tetR aptamer 12\_M | EcoRI & SpeI |

[![Igku Aug26 Gel Extraction(N①)before.jpg](/wiki/images/2/26/Igku_Aug26_Gel_Extraction%28N%E2%91%A0%29before.jpg)](/File%3AIgku_Aug26_Gel_Extraction%28N%E2%91%A0%29before.jpg)
[![Igku Aug26 Gel Extraction(N②)after.jpg](/wiki/images/b/b4/Igku_Aug26_Gel_Extraction%28N%E2%91%A1%29after.jpg)](/File%3AIgku_Aug26_Gel_Extraction%28N%E2%91%A1%29after.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| tetR aptamer 12\_P (EcoRI & SpeI) | 2.3 | 0.38 | 0.13 |
| tetR aptamer 12\_1R (EcoRI & SpeI) | 12.0 | 0.63 | 1.19 |
| tetR aptamer 12\_M (EcoRI & SpeI) | 2.0 | 6.43 | 0.03 |

### Ligation

Ashida

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 8/21 DT | 5.0 | 8/26 tetR aptamer 12\_1R | 2.0 | 3.5 |
| experiment | 8/21 DT | 5.0 | 8/22 pT181 atteniator | 5.0 | 5.0 |
| experiment | 8/21 DT | 5.0 | 8/22 pT181 antisense | 3.0 | 4.0 |
| experiment | 8/21 DT | 5.0 | 8/22 Spinach | 3.0 | 4.0 |
| experiment | 8/22 Plac | 4.0 | 8/22 pT181 attenuator | 5.0 | 4.5 |
| experiment | 8/22 J23100 | 4.0 | 8/22 pT181 attenuator | 5.0 | 4.5 |
| experiment | 8/22 Plac | 4.0 | 8/22 pT181 antisense | 8.0 | 6.0 |
| experiment | 8/22 J23100 | 4.0 | 8/22 pT181 antisense | 8.0 | 6.0 |

### Transformation

No name

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| 8/26 tetR aptamer 12\_1R-DT | 1µL | 10µL | 11µL | CP |
| 8/26 pT181 attenuator-DT | 1µL | 10µL | 11µL | CP |
| 8/26 pT181 antisense-DT | 1µL | 10µL | 11µL | CP |
| 8/26 Spinach-DT | 1µL | 10µL | 11µL | CP |
| 8/26 Plac-pT181 attenuator | 1µL | 10µL | 11µL | CP |
| 8/26 Pcon-pT181 attenuator | 1µL | 10µL | 11µL | Amp |
| 8/26 Plac-pT181 antisense | 1µL | 10µL | 11µL | CP |
| 8/26 Pcon-pT181 antisense | 1µL | 10µL | 11µL | Amp |
| Pλ-RBS-luxI-DT | 1µL | 10µL | 11µ | Amp |

## Aug 27

### Restriction Enzyme Digestion

No name

|  | 8/21 pT181 attenuator-1(330µg/mL) | EcoRI | SpeI | 10x BufferB | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3.0µL | 0.5µL | 0.5µL | 3.0µL | 0.3µL | 22.7µL | 30µL |
| NC | 0.6µL | 0µL | 0µL | 1µL | 0.1µL | 8.3µL | 10µL |

|  | 8/17 DT-1(188µg/mL) | EcoRI | XbaI | 10x BufferB | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3.0µL | 0.5µL | 0.5µL | 3.0µL | 0.3µL | 22.7µL | 30µL |
| NC | 1.0µl | 0µL | 0µL | 1µL | 0.1µL | 7.9µL | 10µL |

|  | 8/20 Pcon-RBS-luxR-DT-2(344µg/mL) | EcoRI | SpeI | 10x BufferB | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.9µL | 0.5µL | 0.5µL | 3.0µL | 0.3µL | 22.8µL | 30µL |
| NC | 0.6µl | 0µL | 0µL | 1µL | 0.1µL | 8.3µL | 10µL |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | 8/21 pT181 attenuator | EcoI | SpeI |
| 3 | 8/21 pT181 atteniator | -- | -- |
| 4 | 8/17 DT-1 | EcoRI | XbaI |
| 5 | 8/17 DT-1 | -- | -- |
| 6 | 8/20 Pcon-RBS-luxR-DT-2 | EcoI | SpeI |
| 7 | 8/20 Pcon-RBS-luxR-DT-2 | -- | -- |
| 8 | 100bp ladder | -- | -- |

[![Igku Aug27 Electrophoresis(N1)-1.jpg](/wiki/images/f/fb/Igku_Aug27_Electrophoresis%28N1%29-1.jpg)](/File%3AIgku_Aug27_Electrophoresis%28N1%29-1.jpg)

### Colony PCR

Kojima and Yoshida

| Sample | base pair |
| --- | --- |
| 8/26 Pλ-luxI(1) |  |
| 8/26 Pλ-luxI(2) |  |
| NC |  |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 3min | 30cycles |

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Pλ-luxI-(1) |
| 3 | Pλ-luxI-(2) |
| 4 | NC |

[![Igku Aug27 Electrophoresis(ColoP)(N3)-2.jpg](/wiki/images/b/b5/Igku_Aug27_Electrophoresis%28ColoP%29%28N3%29-2.jpg)](/File%3AIgku_Aug27_Electrophoresis%28ColoP%29%28N3%29-2.jpg)

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 8/26 Pλ-RBS-luxI-DT-1 | Plusgrow medium (+Amp) |
| 8/26 Pλ-RBS-luxI-DT-2 | Plusgrow medium (+Amp) |

### Restriction Enzyme Digestion

No name

|  | 8/21 pT181 attenuator-1(330µg/mL) | EcoRI | SpeI | 10x BufferB | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3.0µL | 0.5µL | 0.5µL | 3.0µL | 0.3µL | 22.7µL | 30µL |
| NC | 0.6µL | 0µL | 0µL | 1µL | 0.1µL | 8.3µL | 10µL |

|  | 8/17 DT-1(188µg/mL) | EcoRI | XbaI | 10x BufferB | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3.0µL | 0.5µL | 0.5µL | 3.0µL | 0.3µL | 22.7µL | 30µL |
| NC | 1.0µl | 0µL | 0µL | 1µL | 0.1µL | 7.9µL | 10µL |

|  | 8/20 Pcon-RBS-luxR-DT-2(344µg/mL) | EcoRI | SpeI | 10x BufferB | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2.9µL | 0.5µL | 0.5µL | 3.0µL | 0.3µL | 22.8µL | 30µL |
| NC | 0.6µl | 0µL | 0µL | 1µL | 0.1µL | 8.3µL | 10µL |

### Electrophoresis

Hirano

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
|  | 100bp ladder | -- | -- |
| 1 | pT181 attenuator | EcoRI | SpeI |
| 2 | pT181 attenuator | -- | -- |
| 3 | DT-(1) | EcoRI | XbaI |
| 4 | DT-(1) | -- | -- |
| 5 | Pcon-RBS-luxR-DT-(2) | EcoRI | SpeI |
| 6 | Pcon-RBS-luxR-DT-(2) | -- | -- |
|  | 100bp ladder | -- | -- |

[![Igku Aug27 Electrophoresis(N2)-2.jpg](/wiki/images/1/1a/Igku_Aug27_Electrophoresis%28N2%29-2.jpg)](/File%3AIgku_Aug27_Electrophoresis%28N2%29-2.jpg)

### Gel Extraction

Inoue

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bpladder | -- |
| 2 | pT181 attenuator-1 | EcoRI&SpeI |
| 3 | pT181 attenuator-1 | EcoRI&SpeI |
| 4 | DT-1 | EcoRI&XbaI |
| 5 | DT-1 | EcoRI&XbaI |
| 6 | Pcon-RBS-luxR-DT-2 | EcoRI&SpeI |
| 7 | Pcon-RBS-luxR-DT-2 | EcoRI&SpeI |
| 8 | 100bpladder | -- |

[![Igku Aug27 Gel Extraction(N3)-1-3.jpg](/wiki/images/b/b5/Igku_Aug27_Gel_Extraction%28N3%29-1-3.jpg)](/File%3AIgku_Aug27_Gel_Extraction%28N3%29-1-3.jpg)
[![Igku Aug27 Gel Extraction(N4)pic(1).jpg](/wiki/images/f/fa/Igku_Aug27_Gel_Extraction%28N4%29pic%281%29.jpg)](/File%3AIgku_Aug27_Gel_Extraction%28N4%29pic%281%29.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pT181 attenuator-1(EcoRI&SpeI) | 4.2 | 1.62 | 0.29 |
| DT-1(EcoRI&XbaI) | 17 | 1.88 | 0.88 |

### Transformation

Kojima and Hirano

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| 8/26 tetR aptamer 12\_1R-DT | 1µL | 10µL | 11µL | CP |
| 8/26 pT181 attenuator-DT | 1µL | 10µL | 11µL | CP |
| 8/26 pT181 antisense-DT | 1µL | 10µL | 11µL | CP |
| 8/26 Spinach-DT | 1µL | 10µL | 11µL | CP |
| 8/26 Plac-pT181 attenuator | 1µL | 10µL | 11µL | CP |
| 8/26 Pcon-pT181 attenuator | 1µL | 10µL | 11µL | Amp |
| 8/26 Plac-pT181 antisense | 1µL | 10µL | 11µL | CP |
| 8/26 Pcon-pT181 antisense | 1µL | 10µL | 11µL | Amp |

### Liquid culture

No name

| Sample | medium |
| --- | --- |
| 8/16 Plux | Plusgrow medium(+CP) |

### Genome PCR

Yoshdia and Nakagawa

| genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | SasA\_fwd primer | SasA\_rev primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.5 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16 | 25 |

| genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | RpaA\_fwd primer | RpaA\_rev primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.5 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16 | 25 |

| genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | RpaB\_fwd primer | RpaB\_rev primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.5 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16 | 25 |

| genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | PkaiBC\_fwd primer | PkaiBC\_rev primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.5 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 50°C | 68°C | -- |
| 2 min | 10 sec | 30 sec | 38 sec | 30 cycles |

### Ligation

No name

| state | Vector | | Inserter | |
| --- | --- | --- | --- | --- |
| experiment | 8/27 DT (EcoRI & XbaI) | 2.8 | 8/21 RBS-lysis3 (EcoRI & SpeI) |
| experiment | 8/27 DT (EcoRI & XbaI) | 2.8 | 8/27 pT181 attenuator (EcoRI & SpeI) |

* Samples were evaporeted used evaporator into about 3 µL.

| sample | MilliQ | Ligation High | total |
| --- | --- | --- | --- |
| 3 | 4 | 3.5 | 10.5 |

* incubate overnight at 4 °C

## Aug 28

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 100bp |
| 2 | SasA |
| 3 | RpaA |
| 4 | RpaB |
| 5 | PkaiBC |
| 6 | PCR NC |
| 7 | 100bp |

[![Igku Aug28 Electrophoresis(N①-1).jpg](/wiki/images/2/29/Igku_Aug28_Electrophoresis%28N%E2%91%A0-1%29.jpg)](/File%3AIgku_Aug28_Electrophoresis%28N%E2%91%A0-1%29.jpg)

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/27 Plux | 173.8 | 1.95 | 1.45 |

### Ligation

No name

| state | Vector | | Inserter | |
| --- | --- | --- | --- | --- |
| experiment | 8/17 DT-1 | 3.0 | 8.0 |

* Samples were evaporeted used evaporator into about 3 µL.

| sample | MilliQ | Ligation High | total |
| --- | --- | --- | --- |
| 3 | 4 | 3.5 | 10.5 |

* incubate one hour at 16 °C

### Gel Extraction

No name

| Lane | DNA |
| --- | --- |
| 1 | 100bpladder |
| 3 | SasA(8/27 Genome PCR production) |
| 5 | RpaA(8/27 Genome PCR production) |
| 7 | RpaB(8/27 Genome PCR production) |
| 9 | PkaiBC(8/27 Genome PCR production) |

[![Igku Aug28 Gel Extraction(N②pic)before.jpg](/wiki/images/c/c8/Igku_Aug28_Gel_Extraction%28N%E2%91%A1pic%29before.jpg)](/File%3AIgku_Aug28_Gel_Extraction%28N%E2%91%A1pic%29before.jpg)
[![Igku Aug28 Gel Extraction(N①pic)after.jpg](/wiki/images/a/ae/Igku_Aug28_Gel_Extraction%28N%E2%91%A0pic%29after.jpg)](/File%3AIgku_Aug28_Gel_Extraction%28N%E2%91%A0pic%29after.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| RpaA | 15.4 | 1.88 | 0.03 |
| SasA | 21.6 | 1.66 | 0.79 |
| PkaiBC | 16.3 | 1.79 | 0.68 |
| RpaB | 26.7 | 1.77 | 0.82 |

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 8/21 RBS-lysis2-(3) | 772 |
| 8/21 RBS-lysis2-(4) | 772 |
| 8/21 RBS-lysis2-(5) | 772 |
| 8/21 RBS-lysis2-(6) | 772 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 48s | 30cycles |

[![Igku Aug28 Colony PCR(N③pic).jpg](/wiki/images/c/c3/Igku_Aug28_Colony_PCR%28N%E2%91%A2pic%29.jpg)](/File%3AIgku_Aug28_Colony_PCR%28N%E2%91%A2pic%29.jpg)

### Restriction Enzyme Digestion

No name

|  | 8/22 RBS-lysis1-(1) | EcoRI | SpeI | BufferB | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 9µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 16.7µL | 30µL |
| NC | 1µL | 0µL | 0µL | 1µL | 0.1µL | 7.9µL | 10µL |

|  | 8/17 RBS-GFP-DT-(2) | XbaI | PstI | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 4µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 21.7µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 1µL | 0.1µL | 8.4µL | 10µL |

|  | 8/20 Pcon-RBS-GFP-DT-(1) | EcoRI | SpeI | BufferB | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 22.7µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1µL | 0.1µL | 8.6µL | 10µL |

|  | 8/20 Pcon-RBS-luxR-DT-(1) | EcoRI | XbaI | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 2µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 23.7µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1µL | 0.1µL | 8.6µL | 10µL |

No name

|  | 8/28 Plux | SpeI | PstI | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 6µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 19.7µL | 30µL |
| NC | 1µL | 0µL | 0µL | 1µL | 0.1µL | 7.9µL | 10µL |

|  | 8/20 RBS-tetR-DT-(1) | XbaI | PstI | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 20.7µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 1µL | 0.1µL | 8.4µL | 10µL |

* incubated 37°C 1hour

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 8/22 RBS-lysis1-(1) | EcoRI | SpeI |
| 2 | 8/22 RBS-lysis1-(1) | -- | -- |
| 3 | 8/17 RBS-GFP-DT-(2) | XbaI | PstI |
| 4 | 8/17 RBS-GFP-DT-(2) | -- | -- |
| 5 | 8/20 Pcon-RBS-GFP-DT-(1) | EcoRI | SpeI |
| 6 | 8/20 Pcon-RBS-GFP-DT-(1) | -- | -- |
| 7 | 100bp ladder | -- | -- |
| 8 | 8/20 Pcon-RBS-luxR-DT-(1) | EcoRI | XbaI |
| 9 | 8/20 Pcon-RBS-luxR-DT-(1) | -- | -- |
| 10 | 8/28 Plux | SpeI | PstI |
| 11 | 8/28 Plux | -- | -- |
| 12 | 8/20 RBS-tetR-DT | XbaI | PstI |
| 13 | 8/20 RBS-tetR-DT | -- | -- |

[![Igku Aug28 Electrophoresis(ColoP)(N4)pic.jpg](/wiki/images/7/77/Igku_Aug28_Electrophoresis%28ColoP%29%28N4%29pic.jpg)](/File%3AIgku_Aug28_Electrophoresis%28ColoP%29%28N4%29pic.jpg)

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | 8/20 RBS-lysis1-(1) | EcoRI & SpeI |
| 3 |
| 4 | -- | -- |
| 5 | 8/28 RBS-GFP-DT-(2) | XbaI & PstI |
| 6 |

[![Igku Aug28 Gel Extraction(N⑤-1,2)before.jpg](/wiki/images/0/00/Igku_Aug28_Gel_Extraction%28N%E2%91%A4-1%2C2%29before.jpg)](/File%3AIgku_Aug28_Gel_Extraction%28N%E2%91%A4-1%2C2%29before.jpg)
[![Igku Aug28 Gel Extraction4-2.jpg](/wiki/images/3/38/Igku_Aug28_Gel_Extraction4-2.jpg)](/File%3AIgku_Aug28_Gel_Extraction4-2.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | 8/28 Plux | SpeI & PstI |
| 3 |
| 4 | -- | -- |
| 5 | 8/20 RBS-tetR-DT-(1) | XbaI & PstI |
| 6 |

[![Igku Aug28 Gel Extraction(N⑤-3).jpg](/wiki/images/7/7e/Igku_Aug28_Gel_Extraction%28N%E2%91%A4-3%29.jpg)](/File%3AIgku_Aug28_Gel_Extraction%28N%E2%91%A4-3%29.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-RBS-GFP-DT (EcoRI&SpeI) | 7.1 | 1.88 | 0.07 |
| Pcon-RBS-luxR-DT (EcoRI&XbaI) | 15.3 | 1.93 | 0.70 |
| Plux (SpeI&PstI) | 4.5 | 1.79 | 0.31 |
| RBS-GFP-DT (XbaI&PstI) | 6.0 | 2.16 | 0.03 |
| RBS-tetR-DT (XbaI&PstI) | 7.6 | 1.91 | 0.38 |

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 8/27 pT181 attenuator-DT-1 | 738 |
| 8/27 pT181 attenuator-DT-2 | 738 |
| 8/27 pT181 attenuator-DT-3 | 738 |
| 8/27 TetR-aptamer 12\_1R-DT-1 | 521 |
| 8/27 TetR-aptamer 12\_1R-DT-2 | 521 |
| 8/27 pT181 antisense-DT-1 | 542 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 30s | 30cycles |

[![Igku Aug28 c(N⑤-1,2)before.jpg](/wiki/images/d/d2/Igku_Aug28_c%28N%E2%91%A4-1%2C2%29before.jpg)](/File%3AIgku_Aug28_c%28N%E2%91%A4-1%2C2%29before.jpg)

| Sample | base pair |
| --- | --- |
| 8/27 Pλ-luxI-1 | -- |
| 8/27 Pλ-luxI | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 30min | 3cycles |

[![Igku Aug28 ColonyPCR(N⑦-2).jpg](/wiki/images/1/1d/Igku_Aug28_ColonyPCR%28N%E2%91%A6-2%29.jpg)](/File%3AIgku_Aug28_ColonyPCR%28N%E2%91%A6-2%29.jpg)

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 8/21 RBS-lysis2-9 | 772 |
| 8/21 RBS-lysis2-10 | 772 |
| 8/21 RBS-lysis2-11 | 772 |
| 8/21 RBS-lysis2-12 | 772 |
| 8/21 RBS-lysis2-13 | 772 |
| 8/21 RBS-lysis2-14 | 772 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 48s | 30cycles |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 8/16 Master Plate(CP)-6 DT | Plusgrow medium(+CP) |
| J23100 Master Plate-1 | Plusgrow medium(+Amp) |
| Plac Master Plate-1 | Plusgrow medium(+CP) |

### Ligation

No name

| state | Vector | | Inserter | |
| --- | --- | --- | --- | --- |
| experiment | 8/27 DT (EcoRI & XbaI) | 2.9 | 8/28 RBS-lysis1 (EcoRI & SpeI) | 9.5 |
| experiment | 8/28 Plux (SpeI & PstI) | 2.3 | 8/28 RBS-GFP-DT (XbaI & PstI) | 18 |
| experiment | 8/28 Pcon-RBS-luxR-DT (EcoRI & XbaI) | 3.3 | 8/28 Pcon^RBS-GFP-DT-(1) (EcoRI & SpeI) | 28 |
| experiment | 8/27 DT (EcoRI & XbaI) | 2.7 | 8/24 Spinach (EcoRI & SpeI) | 2.4 |

* Samples were evaporeted used evaporator into about 1 µL.

| sample | MilliQ | Ligation High | total |
| --- | --- | --- | --- |
| 1 | 3 | 4 | 9 |

* incubate overnight at 4 °C

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 8/26 Pλ-RBS-luxI-DT-1 | Plusgrow medium (+Amp) |
| 8/26 Pλ-RBS-luxI-DT-2 | Plusgrow medium (+Amp) |

* incubate 37°C 10hour

## Aug 29

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 8/27 Pcon-pT181 antisense-(1) | 542 |
| 8/26 pT181 attenuator-DT-(1) | 738 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 30s | 30cycles |

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | 8/27 Pcon-pT181antisense-1 |
| 3 | 8/26 pT181attenuator-DT-1 |
| 4 | 100bp ladder |

[![Igku Aug29 Electrophoresis(ColoP)(N1)pic.jpg](/wiki/images/0/03/Igku_Aug29_Electrophoresis%28ColoP%29%28N1%29pic.jpg)](/File%3AIgku_Aug29_Electrophoresis%28ColoP%29%28N1%29pic.jpg)

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | 8/21 RBS-lysis2-9(Colony PCR prodution) |
| 3 | 8/21 RBS-lysis2-8(Colony PCR prodution) |
| 4 | 8/21 RBS-lysis2-7(Colony PCR prodution) |
| 5 | 8/21 RBS-lysis2-10(Colony PCR prodution) |
| 6 | 8/21 RBS-lysis2-11(Colony PCR prodution) |
| 7 | 8/21 RBS-lysis2-12(Colony PCR prodution) |
| 8 | 8/21 RBS-lysis2-13(Colony PCR prodution) |
| 9 | 8/21 RBS-lysis2-14(Colony PCR prodution) |

[![Igku Aug29 Electrophoresis1(N②).jpg](/wiki/images/5/59/Igku_Aug29_Electrophoresis1%28N%E2%91%A1%29.jpg)](/File%3AIgku_Aug29_Electrophoresis1%28N%E2%91%A1%29.jpg)

### Miniprep

No Name

| DNA | concentration [µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Plac | 153.7 | 2.03 | 2.10 |
| Pλ-luxⅠ-(1) | 76.5 | 2.07 | 2.06 |
| Pλ-luxⅠ-(2) | 86.4 | 2.08 | 2.10 |
| DT | 166.2 | 1.65 | 2.12 |
| J23100 | 190.1 | 1.68 | 2.34 |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 8/21 RBS-Lysis2-9 | Plusgrow medium(+Amp) |
| 8/21 RBS-Lysis2-12 | Plusgrow medium(+Amp) |
| 8/21 RBS-Lysis2-14 | Plusgrow medium(+Amp) |

### Restriction Enzyme Digestion

No name

|  | 8/22 psB1C3-(2) | EcoRI | SpeI | XbaI | PstI | BufferB | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 8µL | 0.5µL | 0.5µL | 0µL | 0µL | 3µL | 0µL | 0.3µL | 17.7µL | 30µL |
| NC | 1µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 0.1µL | 7.9µL | 10µL |
| 2 cuts | 8µL | 0µL | 0µL | 0.5µL | 0.5µL | 0µL | 3µL | 0.3µL | 17.7µL | 30µL |
| NC | 1µL | 0µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0.1µL | 7.9µL | 10µL |

|  | 8/21 tRMA-spinach(1) | XbaI | SpeI | BufferB | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 4µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 21.7µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 1µL | 0.1µL | 8.4µL | 10µL |

|  | 8/21 tetR aptamer12-1R(1) | EcoRI | SpeI | BufferB | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3µL | 0.5µL | 0.5µL | 3µL | 0.3µL | 22.7µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 0.1µL | 8.5µL | 10µL |

|  | 8/21 PT181 attenuator-(2) | EcoRI | SpeI | XbaI | PstI | BufferB | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 4µL | 0.5µL | 0.5µL | 0µL | 0µL | 3µL | 0µL | 0.3µL | 21.7µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 0.1µL | 8.4µL | 10µL |
| 2 cuts | 4µL | 0µL | 0µL | 0.5µL | 0.5µL | 0µL | 3µL | 0.3µL | 21.7µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0.1µL | 8.4µL | 10µL |

|  | 8/21 PT181 antisense-(2) | EcoRI | SpeI | XbaI | PstI | BufferB | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 10µL | 0.5µL | 0.5µL | 0µL | 0µL | 3µL | 0µL | 0.3µL | 15.7µL | 30µL |
| NC | 1µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 0.1µL | 7.9µL | 10µL |
| 2 cuts | 10µL | 0µL | 0µL | 0.5µL | 0.5µL | 0µL | 3µL | 0.3µL | 15.7µL | 30µL |
| NC | 1µL | 0µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0.1µL | 7.9µL | 10µL |

* incubated 37°C 1hour

[![Igku Aug29 Restriction Enzyme Digestion(N③).jpg](/wiki/images/0/0c/Igku_Aug29_Restriction_Enzyme_Digestion%28N%E2%91%A2%29.jpg)](/File%3AIgku_Aug29_Restriction_Enzyme_Digestion%28N%E2%91%A2%29.jpg)

### Transformation

No Name

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| 8/27 pT181 attenuator-DT | 2µL | 20µL | 22µL | CP |
| 8/27 RBS-lysis3-DT | 2µL | 20µL | 22µL | CP |
| 8/28 RBS-lysis3-DT | 2µL | 20µL | 22µL | CP |
| 8/28 Pcon-RBS-GFP-DT-Pcon-RBS-LuxR-DT | 2µL | 20µL | 22µL | Amp |
| 8/28 RBS-lysis1-DT | 2µL | 20µL | 22µL | CP |
| 8/28 Plux-RBS-GFP-DT | 2µL | 20µL | 22µL | CP |
| 8/28 Spinach-DT | 2µL | 20µL | 22µL | CP |

### Gel Extraction

No name

| Lane | DNA | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
|  | 100bp ladder | -- | -- |
| 9 | 8/29 pT181attenuator(2) | EcoRI | SpeI |
| 10 | 8/29 pT181attenuator(2) | EcoRI | SpeI |
| 11 | 8/29 pT181attenuator(2) | XbaI | PstI |
| 12 | 8/29 pT181attenuator(2) | XbaI | PstI |
| 13 | pT181antisense(2) | EcoRI | SpeI |
| 14 | pT181antisense(2) | EcoRI | SpeI |
| 15 | pT181antisense(2) | XbaI | PstI |
| 16 | pT181antisense(2) | XbaI | PstI |
|  | 100bp ladder | -- | -- |

[![Igku Aug29 Transformation1(N④-1).jpg](/wiki/images/e/e6/Igku_Aug29_Transformation1%28N%E2%91%A3-1%29.jpg)](/File%3AIgku_Aug29_Transformation1%28N%E2%91%A3-1%29.jpg)
[![Igku Aug29 Transformation(N④-2).jpg](/wiki/images/0/07/Igku_Aug29_Transformation%28N%E2%91%A3-2%29.jpg)](/File%3AIgku_Aug29_Transformation%28N%E2%91%A3-2%29.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
|  | 100bp ladder | -- | -- |
| 1 | 8/29 pSB1C3 | EcoRI | SpeI |
| 2 | 8/29 pSB1C3 | EcoRI | SpeI |
| 3 | 8/29 pSB1C3 | XbaI | PstI |
| 4 | 8/29 pSB1C3 | XbaI | PstI |
| 5 | 8/29 spinach | EcoRI | SpeI |
| 6 | 8/29 spinach | EcoRI | SpeI |
| 7 | 8/29 tetRaptarmer12-1R(1) | EcoRI | SpeI |
| 8 | 8/29 tetRaptarmer12-1R(1) | EcoRI | SpeI |
|  | 100bp ladder | -- | -- |

[![Igku Aug29 Transformation(N⑤).jpg](/wiki/images/7/75/Igku_Aug29_Transformation%28N%E2%91%A4%29.jpg)](/File%3AIgku_Aug29_Transformation%28N%E2%91%A4%29.jpg)
[![Igku Aug29 Gel Extraction4-2.jpg](/wiki/images/a/ae/Igku_Aug29_Gel_Extraction4-2.jpg)](/File%3AIgku_Aug29_Gel_Extraction4-2.jpg)

## Aug 30

### Gel Extraction

No Name

| Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- | --- |
| pT181 attenuator(2) (EcoRI & SpeI) | 305.3 | 2.2 | 1.66 | 0.36 |
| pT181 attenuator(2) (XbaI & PstI) | 217.7 | 3.9 | 1.73 | 0.04 |
| pT181 antisense(2) (EcoRI & SpeI) | 347.7 | 3.1 | 1.88 | 0.31 |
| pT181 antisense(2) (XbaI & PstI) | 376.5 | 5.9 | 0.49 | 2.36 |
| pSB1C3 (EcoRI & SpeI) | 436.6 | 30.7 | 1.03 | 1.28 |
| pSB1C3 (XbaI & PstI) | 457.4 | 7.9 | 1.65 | 0.42 |
| Spinach (EcoRI & SpeI) | 490.5 | 3.0 | 1.87 | 0.26 |
| tetR aptamer 12\_1R (EcoRI & SpeI) | 451.7 | -12.8 | 5.56 | -0.04 |

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| RBS-lysis2 9 | 264.6 | 1.97 | 2.21 |
| RBS-lysis2 12 | 220.0 | 1.89 | 2.46 |
| RBS-lysis2 14 | 195.3 | 1.88 | 1.53 |

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT(8/20 LB Amp) (1)~(4) | 2143 |
| 8/29 Plux-RBS-GFP-DT(8/27 LB-CP) (1)~(2) | 1227 |
| 8/29 Pbad/araC-RBS-RFP{BBa\_I13516} (8/21 LB-CP) (1)~(2) | 2256 |
| 8/29 RBS-lysis3-DT-(1) | 1210 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 2min | 30cycle |

[![Igku Aug30 colonyPCR N1.jpg](/wiki/images/c/c9/Igku_Aug30_colonyPCR_N1.jpg)](/File%3AIgku_Aug30_colonyPCR_N1.jpg)

### Ethanol Precipitation

No name

| DNA |
| --- |
| pT181 attenuator-(2) (EcoRI & SpeI) |
| pT181 attenuator-(2) (XbaI & PstI) |
| pT181 antisense-(2) (EcoRI & SpeI) |
| pT181 antisense-(2) (XbaI & PstI) |
| pSB1C3 (EcoRI & SpeI) |
| pSB1C3 (XbaI & PstI) |
| Spinach (EcoRI & SpeI) |
| tetR aptamer 12\_1R (EcoRI & SpeI) |

### Restriction Enzyme Digestion

No name

|  | 8/30 RBS-lysis2 (9) | EcoRI | SpeI | 10x bufferB | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 7.5 µL | 0.5 µL | 0.5 µL | 3 µL | 0.3 µL | 18.2 µL | 30 µL |
| NC | 0.5 µL | 0 µL | 0 µL | 1 µL | 0.1 µL | 8.4 µL | 10 µL |

|  | 8/22 pSB1C3-(2) | EcoRI | SpeI | 10x Buffer | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 8 µL | 0.5 µL | 0.5 µL | 3 µL | 0.3 µL | 17.7 µL | 30 µL |
| NC | 1 µL | 0 µL | 0 µL | 1 µL | 0.1 µL | 7.9 µL | 10 µL |

|  | 8/22 pSB1C3-(2) | XbaI | PstI | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 8 µL | 0.5 µL | 0.5 µL | 3 µL | 0.3 µL | 17.7 µL | 30 µL |
| NC | 1 µL | 0 µL | 0 µL | 1 µL | 0.1 µL | 7.9 µL | 10 µL |

|  | 8/21 Spinach (1) | EcoRI | SpeI | 10x BufferB | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 4 µL | 0.5 µL | 0.5 µL | 3 µL | 0.3 µL | 21.9 µL | 30 µL |
| NC | 0.5 µL | 0 µL | 0 µL | 1 µL | 0.1 µL | 8.4 µL | 10 µL |

|  | 8/21 tetR aptamer 12\_1R (1) | EcoRI | SpeI | 10xBufferB | 100xBSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 3 µL | 0.5 µL | 0.5 µL | 3 µL | 0.3 µL | 22.7 µL | 30 µL |
| NC | 0.4 µL | 0 µL | 0 µL | 1 µL | 0.1 µL | 8.4 µL | 10 µL |

|  | 8/21 pT181 attenuator-(2) | EcoRI | SpeI | XbaI | PstI | 10x BufferB | 10x BufferD | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 4 µL | 0.5 µL | 0.5 µL | 0 µL | 0 µL | 3 µL | 0 µL | 0.3 µL | 21.7 µL | 30 µL |
| NC | 0.5 µL | 0 µL | 0 µL | 0 µL | 0 µL | 1 µL | 0 µL | 0.1 µL | 8.4 µL | 10 µL |
| 2 cuts | 4 µL | 0 µL | 0 µL | 0.5 µL | 0.5 µL | 0 µL | 3 µL | 0.3 µL | 21.7 µL | 30 µL |
| NC | 0.5 µL | 0 µL | 0 µL | 0 µL | 0 µL | 0 µL | 1 µL | 0.1 µL | 8.4 µL | 10 µL |

|  | 8/21 pT181 antisense-(2) | EcoRI | SpeI | XbaI | PstI | 10x BufferB | 10x BufferD | 100x BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 9 µL | 0.5 µL | 0.5 µL | 0 µL | 0 µL | 3 µL | 0 µL | 0.3 µL | 16.7 µL | 30 µL |
| NC | 1 µL | 0 µL | 0 µL | 0 µL | 0 µL | 1 µL | 0 µL | 0.1 µL | 7.9 µL | 10 µL |
| 2 cuts | 9 µL | 0 µL | 0 µL | 0.5 µL | 0.5 µL | 0 µL | 3 µL | 0.3 µL | 16.7 µL | 30 µL |
| NC | 1 µL | 0 µL | 0 µL | 0 µL | 0 µL | 0 µL | 1 µL | 0.1 µL | 7.9 µL | 10 µL |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | RBS-lysis2-(9) | EcoRI | SpeI |
| 3 | RBS-lysis2-(9) | -- | -- |
| 4 | pSB1C3-(2) | EcoRI | SpeI |
| 5 | pSB1C3-(2) | -- | -- |
| 6 | pSB1C3-(2) | XbaI | PstI |
| 7 | pSB1C3-(2) | -- | -- |
| 8 | tRNA-Spinach-(1) | EcoRI | SpeI |
| 9 | tRNA-Spinach-(1) | -- | -- |
| 10 | tetR aptamer12\_1R | EcoRI | SpeI |
| 11 | tetR aptamer12\_1R | -- | -- |
| 12 | pT181 attenuator | EcoRI | SpeI |
| 13 | pT181 attenuator | EcoRI | SpeI |
| 14 | 100bp ladder | -- | -- |

[![Igku Aug30 Electrophoresis(N2-1).jpg](/wiki/images/6/6b/Igku_Aug30_Electrophoresis%28N2-1%29.jpg)](/File%3AIgku_Aug30_Electrophoresis%28N2-1%29.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | pT181 antisense | EcoRI | SpeI |
| 3 | pT181 antisense | -- | -- |
| 4 | pT181 antisense | XbaI | PstI |
| 5 | pT181 antisense | -- | -- |
| 6 | pT181 attenuator | XbaI | PstI |
| 7 | pT181 attenuator | -- | -- |
| 8 | 100bp ladder | -- | -- |

[![Igku Aug30 Electrophoresis(N2-2).jpg](/wiki/images/8/86/Igku_Aug30_Electrophoresis%28N2-2%29.jpg)](/File%3AIgku_Aug30_Electrophoresis%28N2-2%29.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 1kb ladder |
| 2 | 8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)(Colony PCR product) |
| 3 | 8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)(Colony PCR product) |
| 4 | 8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)(Colony PCR product) |
| 5 | 8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)(Colony PCR product) |
| 6 | Pbad/araC-RBS-RFP-DT-(1)(Colony PCR product) |
| 7 | Pbad-araC-RBS-RFP-DT-(2)(Colony PCR product) |

[![Igku Aug30 Electrophoresis(N2-3).jpg](/wiki/images/e/ea/Igku_Aug30_Electrophoresis%28N2-3%29.jpg)](/File%3AIgku_Aug30_Electrophoresis%28N2-3%29.jpg)

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1) | Plusgrow medium(+Amp) |
| 8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2) | Plusgrow medium(+Amp) |
| 8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3) | Plusgrow medium(+Amp) |
| 8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4) | Plusgrow medium(+Amp) |
| 8/29 Plux-RBS-GFP-DT-(1) | Plusgrow medium(+CP) |
| 8/29 Plux-RBS-GFP-DT-(2) | Plusgrow medium(+CP) |
| 8/29 Pbad/araC-RBS-RFP-DT-(1) | Plusgrow medium(+CP) |
| 8/29 Pbad-araC-RBS-RFP-DT-(2) | Plusgrow medium(+CP) |
| RBS-lysis3-DT-(1) | Plusgrow medium(+CP) |

## Aug 31

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | -- | -- |
| 3 | RBS-lysis2-(9) | EcoRI & SpeI |
| 4 |
| 5 | -- | -- |
| 6 | pSB1C3(1) | EcoRI & SpeI |
| 7 |
| 8 | -- | -- |
| 9 | pSB1C3(1) | XbaI & PstI |
| 10 |

[![Igku Aug30 Gel Extraction(N1).jpg](/wiki/images/a/a8/Igku_Aug30_Gel_Extraction%28N1%29.jpg)](/File%3AIgku_Aug30_Gel_Extraction%28N1%29.jpg)
[![Igku Aug30 Gel Extraction(N2-1).jpg](/wiki/images/f/fc/Igku_Aug30_Gel_Extraction%28N2-1%29.jpg)](/File%3AIgku_Aug30_Gel_Extraction%28N2-1%29.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | tRNA-Spinach | EcoRI & SpeI |
| 3 |
| 4 | -- | -- |
| 5 | pT181 attenuator-(2) | EcoRI & SpeI |
| 6 |
| 7 | pT181 attenuator-(2) | XbaI & PstI |
| 8 |
| 9 | -- | -- |
| 10 | pT181 antisense-(2) | EcoRI & SpeI |
| 11 |

[![Igku Aug31 Gel Extraction(N2-2).jpg](/wiki/images/b/bd/Igku_Aug31_Gel_Extraction%28N2-2%29.jpg)](/File%3AIgku_Aug31_Gel_Extraction%28N2-2%29.jpg)
[![Igku Aug31 Gel Extraction(N2-3).jpg](/wiki/images/4/48/Igku_Aug31_Gel_Extraction%28N2-3%29.jpg)](/File%3AIgku_Aug31_Gel_Extraction%28N2-3%29.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | pT181 antisense | XbaRI & PstI |
| 3 |
| 4 | -- | -- |
| 5 | tetR aptamer 12\_1R-(1) | EcoRI & SpeI |
| 6 |

[![Igku Aug31 Gel Extraction(N3-1).jpg](/wiki/images/c/cc/Igku_Aug31_Gel_Extraction%28N3-1%29.jpg)](/File%3AIgku_Aug31_Gel_Extraction%28N3-1%29.jpg)
[![Igku Aug31 Gel Extraction(N3-2).jpg](/wiki/images/5/58/Igku_Aug31_Gel_Extraction%28N3-2%29.jpg)](/File%3AIgku_Aug31_Gel_Extraction%28N3-2%29.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| RBS-lysis2(EcoRI&SpeI) | 4.0 | 2.69 | 0.24 |
| pSB1C3(EcoRI&SpeI) | 6.0 | 2.48 | 0.05 |
| pSB1C3(XbaI&PstI) | -- | -- | -- |
| tRNA-Spinach | -- | -- | -- |
| pT181 attenuator-(2)(EcoRI&SpeI) | -- | -- | -- |
| pT181 anttenuator-(2)(SbaI&PstI) | -- | -- | -- |
| pT181 antisense-(2)(EcoRI&SpeI) | -- | -- | -- |
| pT181 antisense-(2)(XbaI&PstI) | -- | -- | -- |
| tetR aptamer 12\_1R(EcoRI&SpeI) | -- | -- | -- |

### Ligation

Nakamoto and Hirano

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 8/30 pSB1C3 (EcoRI & SpeI) | 6.6 | 8/30 tRNA-Spinach (EcoRI & SpeI) | 12.5 | 3.5 |
| experiment | 8/30 pSB1C3 (EcoRI & SpeI) | 6.6 | 8/30 pT181 antisense (EcoRI & SpeI) | 12.5 | 3.5 |
| experiment | 8/30 pSB1C3 (XbaI & Pst) | 12.5 | 8/30 pT181 attenuator (XbaI & PstI) | 25 | 3.5 |
| experiment | 8/30 pSB1C3 (XbaI & Pst) | 12.5 | 8/30 pT181 antisense (XbaI & PstI) | 25 | 3.5 |
| experiment | 8/20 DT (EcoRI & XbaI) | 6.6 | 8/30 pT181 attenuator(2) (EcoRI & SpeI) | 12.5 | 3.5 |
| experiment | 8/20 DT (EcoRI & XbaI) | 6.6 | 8/30 pT181 antisense (EcoRI & SpeI) | 12.5 | 3.5 |
| experiment | 8/27 DT (EcoRI & XbaI) | 3.0 | 8/30 tRNA-Spinach (EcoRI & SpeI) | 12.5 | 3.5 |
| experiment | 8/20 DT (EcoRI & XbaI) | 6.6 | 8/30 RBS-lysis2 (EcoRI & SpeI) | 12.5 | 3.5 |
| experiment | 8/22 J23100 (SpeI & PstI) | -- | RBS-tetR-DT (XbaI & PstI) | -- | 3.5 |
| experiment | 8/22 Plac (SpeI & PstI) | 8.0 | 8/18 RBS-lacZα-DT (XbaI & PstI) | 10 | 3.5 |

Samples were evaporeted used evaporator into about 7 µL

### Miniprep

Kojima and Nakamoto

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/30 Plux-RBS-GFP-DT-(1) | 124 | 1.77 | 1.87 |
| 8/30 Plux-RBS-GFP-DT-(2) | 213 | 1.75 | 1.52 |
| 8/30 Pbad/araC-RBS-RFP | 427 | 1.70 | 1.56 |
| 8/30 Pbad/araC-RBS-RFP | 670 | 1.30 | 0.83 |
| 8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1) | 332 | 1.73 | 2.04 |
| 8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2) | 478 | 1.74 | 2.09 |
| 8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3) | 146 | 1.77 | 2.11 |
| 8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4) | 454 | 1.75 | 2.19 |

## Sep 1

### Transformation

No name

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| 8/31 tRNA-Spinach(pSB1C3) | 2µL | 20µL | 22µL | CP |
| 8/31 pT181 antisense(pSB1C3) | 2µL | 20µL | 22µL | CP |
| 8/31 pT181 attenuator(pSB1C3) | 2µL | 20µL | 22µL | CP |
| 8/31 pT181 antisense(pSB1C3) | 2µL | 20µL | 22µL | CP |
| 8/31 pT181 attenuator(2)-DT | 2µL | 20µL | 22µL | CP |
| 8/31 pT181 antisense-DT | 2µL | 20µL | 22µL | CP |
| 8/31 tRNA-Spniach-DT | 2µL | 20µL | 22µL | CP |
| 8/31 RBS-lysis2-DT | 2µL | 20µL | 22µL | CP |
| 8/31 Pconst-RBS-tetR-DT | 2µL | 20µL | 22µL | Amp |
| 8/31 Plac-RBS-lacZα-DT | 2µL | 20µL | 22µL | CP |
| 8/31 Ptet-RBS-lacZα-DT | 2µL | 20µL | 22µL | CP |
| 8/31 | 2µL | 20µL | 22µL | CP |

### ligation production PCR

No name

| Sample | base pair |
| --- | --- |
| 8/31 tRNA Spinach(pSB1C3) | 459 |
| 8/31 pT181 antisense(2)(pSB1C3)(EcoRI+SpeI) | 415 |
| 8/31 pT181 attenuator(2)(pSB1C3)(XbaI+PstI) | 601 |
| 8/31 pT181 antisense(2)(pSB1C3)(XbaI+PstI) | 415 |
| 8/31 pT181 attenuator(2)-DT | 754 |
| 8/31 pT181 antisense(2)-DT | 577 |
| 8/31 tRNA Spinach-DT | 596 |
| 8/22 pSB1C3(2)(controll) | 1383 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 30min | 30cycle |

[![Igku Sep1 ligation production PCR.jpg](/wiki/images/2/2b/Igku_Sep1_ligation_production_PCR.jpg)](/File%3AIgku_Sep1_ligation_production_PCR.jpg)

### Ligation

Nakamoto

| state | Vector | | Inserter | |
| --- | --- | --- | --- | --- |
| experiment | DT(EcoRI+SpeI) | 5.5 | 8/28 RBS-lysis1(EcoRI+SpeI)2.4ng/µL | 2.4 |

1. Samples were evaporeted used evaporator into about 7 µL.
2. Add Ligation High 3.5&micro:L and incubate 16°C 1hour.

### Liquid Culture

Tatsui,Stephane,Nakamoto

| Sample | medium |
| --- | --- |
| 8/21 pSB1C3(BBa\_J04450)-(1) | Plusgrow medium(+CP) |
| 8/21 pSB1C3(BBa\_J04450)-(2) | Plusgrow medium(+CP) |
| RBS-lysis3-DT | Plusgrow medium(+CP) |

### M9 Medium

No name
5xM9 liquid medium

| volume | 100mL |
| --- | --- |
| NaHPO4 | 30g |
| KH2PO | 3g |
| Nacl | 0.25g |
| NH4Cl | 0.5g |
| 3.75% agar solution | 400mL |
| 1M MgSO4 | 0.5mL |
| 2M Glucose | 2.8mL |
| 1% Vitamin B1 | 0.5mL |
| 1M CaCl2 | 0.05mL |

1. mix 5xM9 medium and 3.75%Agar solution 400mL
2. Add 1M MgSO4, 2M Glucose and 1 VitaminB1 0.5mL
3. Add CaCl2 while shaking Erlenmeyer flask.

### Miniprep

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/21 pSB1C3(1) | 309.2 | 1.69 | 1.72 |
| 8/21 pSB1C3(2) | 201.6 | 1.65 | 1.76 |

### Kanamycin applicaton

Hirano

Add 20µL Kanamycin into 80µL MilliQ and apply it on a 9/1 M9 plate

### Plating

Hirano

Apply entA-colony liquid culture (100µL) on M9(Km)plate and incubate 37°C

2 fold diluted OD 600 2.107

### Liquid Culture

Hirano

| Sample | medium |
| --- | --- |
| 9/1 entA- Master Plate-(4) | SOB Medium(+Km) |

## Sep 2

### Colony PCR

Tatsui

| Sample | base pair |
| --- | --- |
| 9/1 RBS-lysis2-DT-(1) | 985 |
| Ptet-RBS-lacZα-DT-(1) | 765 |
| Plac-RBS-lacZα-DT-(1) | 765 |
| Plac-RBS-lacZα-DT-(2) | 765 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min | 30cycles |

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | 9/1 RBS-lysis2+DT -(1) |
| 3 | 9/1 Ptet+RBS-lacZα-DT -(1) |
| 4 | 9/1 Plac+RBS-lacZα-DT -(1) |
| 5 | 9/1 Plac+RBS-lacZα-DT -(2) |
| 6 | 100bp ladder |

[![Igku Sep2 ColonyPCR (N1-2).jpg](/wiki/images/5/52/Igku_Sep2_ColonyPCR_%28N1-2%29.jpg)](/File%3AIgku_Sep2_ColonyPCR_%28N1-2%29.jpg)

### Restriction Enzyme Digestion

Nakamoto

|  | pSB1C3-(1) | EcoRI | SpeI | XbaI | PstI | BufferB | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts(EcoRI+SpeI) | 7µL | 1µL | 1µL | 0µL | 0µL | 3µL | 0µL | 0.3µL | 17.7µL | 30µL |
| NC(EcoRI+SpeI) | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 0.1µL | 8.6µL | 10µL |
| 2 cuts(XbaI+PstI) | 7µL | 0µL | 0µL | 1µL | 1µL | 0µL | 3µL | 0.3µL | 17.7µL | 30µL |
| NC(XbaI+PstI) | 0.3µL | 0µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0.1µL | 8.6µL | 10µL |

|  | 8/21 tRMA-spinach(1) | EcoRI | SpeI | BufferB | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 8µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 17.6µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 1µL | 0.1µL | 8.4µL | 10µL |

|  | 8/21 tetR aptamer12\_1R-(1) | EcoRI | SpeI | BufferB | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 6µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 18.7µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 0.1µL | 8.5µL | 10µL |

|  | 8/21 pT181 attenuator-(2) | EcoRI | SpeI | XbaI | PstI | BufferB | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts(EcoRI+SpeI) | 8µL | 1µL | 1µL | 0µL | 0µL | 3µL | 0µL | 0.3µL | 16.3µL | 30µL |
| NC(EcoRI+SpeI) | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 0.1µL | 8.4µL | 10µL |
| 2 cuts(XbaI+PstI) | 8µL | 0µL | 0µL | 1µL | 1µL | 0µL | 3µL | 0.3µL | 16.3µL | 30µL |
| NC(XbaI+PstI) | 0.5µL | 0µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0.1µL | 8.4µL | 10µL |

[![Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg](/wiki/images/c/c1/Igku_Sep2_Restriction_Enzyme_Digestion_%28N1-1%29.jpg)](/File%3AIgku_Sep2_Restriction_Enzyme_Digestion_%28N1-1%29.jpg)

### Liquid Culture

Kojima

| Sample | medium |
| --- | --- |
| tRNA-Spinach-1 | Plusgrow medium(+CP) |
| tetR aptamaer12\_1R-1 | Plusgrow medium(+CP) |
| pT181 attenuator-1 | Plusgrow medium(+CP) |
| pT181 antisense-1 | Plusgrow medium(+CP) |

### Master Plate

Kojima

| Number | Use LB plate(+CP) |
| --- | --- |
| 1 | tRNA Spinach-1 |
| 2 | tetR aptamaer12\_1R |
| 3 | tetR aptamaer12\_P |
| 4 | tetR aptamaer12\_1M |
| 5 | pT181 attenuator-2 |
| 6 | Fusion1 attenuator-1 |
| 7 | Fusion3m2 attenuator-1 |
| 8 | pT181 antisense-1 |
| 9 | Fusion1 antisense-1 |
| 10 | Fuaion6 antisense-1 |

### Electrophoresis

Nakamoto & Tatsui

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | pSB1C3 -(1) | EcoRI | SpeI |
| 3 | pSB1C3 -(1) | -- | -- |
| 5 | pSB1C3 -(1) | XbaI | PstI |
| 6 | pSB1C3 -(1) | -- | -- |
| 7 | pT181 attenuator (2) | EcoRI | SpeI |
| 8 | pT181 attenuator (2) | -- | -- |
| 9 | pT181 attenuator (2) | XbaI | SpeI |
| 10 | pT181 attenuator (2) | -- | -- |
| 11 | tetR-aptamer 12\_1R | EcoRI | SpeI |
| 12 | tetR-aptamer 12\_1R | -- | -- |
| 13 | tRNA Spinach (1) | EcoRI | SpeI |
| 14 | 100bp ladder | -- | -- |
| 15 | tRNA Spinach (1) | -- | -- |

[![Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg](/wiki/images/c/c1/Igku_Sep2_Restriction_Enzyme_Digestion_%28N1-1%29.jpg)](/File%3AIgku_Sep2_Restriction_Enzyme_Digestion_%28N1-1%29.jpg)

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | - |
| 2 | pSB1C3(EcoRI+SpeI) | EcoRI&SpeI |
| 3 |
| 4 | -- | -- |
| 5 | 8/24 tetR aptamer 12\_1R | EcoRI&SpeI |
| 6 |

[![Igku Sep1 Gel Extraction(N2-1).jpg](/wiki/images/7/7a/Igku_Sep1_Gel_Extraction%28N2-1%29.jpg)](/File%3AIgku_Sep1_Gel_Extraction%28N2-1%29.jpg)
[![Igku Sep1 Gel Extraction(N2-2).jpg](/wiki/images/f/fe/Igku_Sep1_Gel_Extraction%28N2-2%29.jpg)](/File%3AIgku_Sep1_Gel_Extraction%28N2-2%29.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | - |
| 2 | pT181 attenuator(2)(EcoRI+SpeI) | EcoRI&SpeI |
| 3 |
| 4 | -- | -- |
| 5 | pT181 attenuator(2)(XbaI+PstI) | EcoRI&SpeI |
| 6 |
| 7 | -- | -- |
| 8 | pT181 attenuator(2)(XbaI+PstI) | EcoRI&SpeI |
| 9 |
| 10 | -- | -- |
| 11 | Spinach | EcoRI&SpaI |
| 12 |

[![Igku Sep1 Gel Extraction(N3-1).jpg](/wiki/images/2/29/Igku_Sep1_Gel_Extraction%28N3-1%29.jpg)](/File%3AIgku_Sep1_Gel_Extraction%28N3-1%29.jpg)
[![Igku Sep1 Gel Extraction(N3-2).jpg](/wiki/images/6/66/Igku_Sep1_Gel_Extraction%28N3-2%29.jpg)](/File%3AIgku_Sep1_Gel_Extraction%28N3-2%29.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pSB1C3 (EcoRI&SpeI) | 3.0 | 2.18 | 0.31 |
| pSB1C3 (XbaI&PstI) | 4.7 | 2.25 | 0.36 |
| pT181 attenuator-(2)(EcoR&SpeI) | 8.6 | 2.74 | 0.01 |
| pT181 attenuator-(2) (XbaI&PstI) | 16.5 | 2.46 | 0.03 |
| Spinach (EcoRI&SpeI) | 2.8 | 2.98 | 0.27 |
| tetR aptamer12\_1R (EcoRI&SpeI) | 50.4 | 28.07 | 1.97 |

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 9/1 Spinach(pSB1C3) | about 450 |
| 9/1 Spinach-DT | 596 |
| 9/1 RBS-lysis1-DT | 613 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 40s | 30cycles |

| Sample | base pair |
| --- | --- |
| 9/1 Ptet-RBS-lacZα-DT-2 | 738 |
| 9/1 Ptet-RBS-lacZα-DT-3 | 738 |
| 9/1 Ptet-RBS-lacZα-DT-4 | 738 |
| 9/1 Ptet-RBS-lacZα-DT-5 | 738 |
| 9/1 Ptet-RBS-lacZα-DT-6 | 738 |
| 9/1 RBS-lysis2-DT-2 | 985 |
| 9/1 RBS-lysis2-DT-3 | 985 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min | 30cycles |

[![Igku Sep3 ColonyPCR (N1).jpg](/wiki/images/5/53/Igku_Sep3_ColonyPCR_%28N1%29.jpg)](/File%3AIgku_Sep3_ColonyPCR_%28N1%29.jpg)

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Spinach(pSB1C3) |
| 3 | Spinach-DT |
| 4 | RBS-lysis1-DT |
| 5 | 100bp ladder |

[![Igku Sep2 ColonyPCR (N4).jpg](/wiki/images/0/02/Igku_Sep2_ColonyPCR_%28N4%29.jpg)](/File%3AIgku_Sep2_ColonyPCR_%28N4%29.jpg)

### Master Plate

No name

| Number | Use LB plate(+CP) |
| --- | --- |
| 1 | 9/1 Spinach(pSB1C3) |
| 2 | 9/1 Spinach-DT-(1) |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| pT181 attenuator | Plusgrow medium (+CP) |
| pT181 antisense | Plusgrow medium (+CP) |
| Spinach | Plusgrow medium (+CP) |

* incubated at 37 °C for 1 hour

### Transformation

No name

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| 8/28 RBS-lysis3-DT | 3µL | 30µL | 33µL | CP |

## Sep 3

### Electrophoresis

Kojima

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | pT181 attenuator(2)-DT2(738) |
| 3 | Ptet-RBS-lacZα-DT-2(756) |
| 4 | Ptet-RBS-lacZα-DT-3 |
| 5 | Ptet-RBS-lacZα-DT-4 |
| 6 | Ptet-RBS-lacZα-DT-5 |
| 7 | Ptet-RBS-lacZα-DT-6 |
| 8 | RBS-lysis2-DT-2(985) |
| 9 | RBS-lysis2-DT-3 |
| 10 | 100bp ladder |

[![Igku Sep3 Electrophoresis(N1).jpg](/wiki/images/b/b5/Igku_Sep3_Electrophoresis%28N1%29.jpg)](/File%3AIgku_Sep3_Electrophoresis%28N1%29.jpg)

### Miniprep

Tatsui

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/2 14:00 pT181 attenuator | 418.4 | 1.82 | 1.80 |
| 9/2 14:00 pT181 antisense | 362.2 | 1.92 | 1.96 |
| 9/2 14:00 Spinach | 438.3 | 1.86 | 1.99 |
| 9/2 14:00 tetR-apt 12\_1R | 567.5 | 1.90 | 1.99 |
| 9/2 21:00 pT181 attenuator | 320.9 | 1.90 | 2.15 |
| 9/2 21:00 Spinach | 457.0 | 1.88 | 2.24 |

### Liquid Culture

Kojima

| Sample | medium |
| --- | --- |
| Spinach-DT(9/2 master plate) | plusgrow(CP)4ml |

### Restriction Enzyme Digestion

Kojima

|  | DNA | EcoRI | SpeI | XbaI | PstI | 10xBuffer B(EcoRI&SpeI) | 10xBuffer D(XbaI&PstI) | 100xBSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 9/3 pT181attenuator 14:00 418.4ng/µL | 4.8µL | 1.0µL | 1.0µL | 0µL | 0µL | 3µL | 0µL | 0.3µL | 19.9µL | 30µL |
| 9/3 pT181attenuator 14:00 418.4ng/µL | 0.2µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 0.1µL | 8.7µL | 10µL |
| 9/3 pT181attenuator 14:00 418.4ng/µL | 4.8µL | 0µL | 0µL | 1.0µL | 1.0µL | 0µL | 3µL | 0.3µL | 19.9µL | 30µL |
| 9/3 pT181attenuator 14:00 418.4ng/µL | 0.2µL | 0µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0.1µL | 8.7µL | 10µL |

|  | DNA | EcoRI | SpeI | XbaI | PstI | 10xBuffer B(EcoRI&SpeI) | 10xBuffer D(XbaI&PstI) | 100xBSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 9/3 pT181antisense 14:00 362.2ng/µL | 5.5µL | 1.0µL | 1.0µL | 0µL | 0µL | 3µL | 0µL | 0.3µL | 19.2µL | 30µL |
| 9/3 pT181attenuator 14:00 418.4ng/µL | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 0.1µL | 8.6µL | 10µL |
| 9/3 pT181attenuator 14:00 418.4ng/µL | 5.5µL | 0µL | 0µL | 1.0µL | 1.0µL | 0µL | 3µL | 0.3µL | 19.2µL | 30µL |
| 9/3 pT181attenuator 14:00 418.4ng/µL | 0.3µL | 0µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0.1µL | 8.6µL | 10µL |

|  | DNA | EcoRI | SpeI | 10xBuffer B | 100xBSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/3 apt 12\_1R 14:00 507.5ng/µL | 3.9µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 20.8µL | 30µL |
| 9/3 apt 12\_1R 14:00 507.5ng/µL | 0.2µL | 0µL | 0µL | 1µL | 0.1µL | 8.7µL | 10µL |

|  | DNA | EcoRI | XbaI | 10xBuffer D | 100xBSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 8/29 DT 166.2ng/µL | 12µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 12.7µL | 30µL |
| 8/29 DT 166.2ng/µL | 0.6µL | 0µL | 0µL | 1µL | 0.1µL | 8.3µL | 10µL |

|  | DNA | EcoRI | SpeI | 10xBuffer B | 100xBSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 8/22 RBS-lysis1(2) 96ng/µL | 21µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 3.7µL | 30µL |
| 8/22 RBS-lysis1(2) 96ng/µL | 1.0µL | 0µL | 0µL | 1µL | 0.1µL | 7.9µL | 10µL |

|  | DNA | EcoRI | SpeI | 10xBuffer B | 100xBSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 8/30 RBS-lysis2 220ng/µL | 9.1µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 15.6µL | 30µL |
| 8/30 RBS-lysis2 220ng/µL | 0.5µL | 0µL | 0µL | 1µL | 0.1µL | 8.4µL | 10µL |

|  | DNA | EcoRI | SpeI | 10xBuffer B | 100xBSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 8/20 RBS-lysis3(1) 282ng/µL | 7.1µL | 1.0µL | 1.0µL | 3µL | 0.3µL | 17.6µL | 30µL |
| 8/20 RBS-lysis3(1) 282ng/µL | 0.4µL | 0µL | 0µL | 1µL | 0.1µL | 8.5µL | 10µL |

### PCR

Hirano

| Plac(1) plasmid 153.7ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer (KaiA\_BsaI\_Mut\_fwd) | primer (KaiA\_BsaI\_Mut\_rer) | KOD plus-ver.2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.13 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16.37 | 25 |

| KaiB (plasmid) 20.2ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer (GG2\_RBS\_KaiB\_fwd) | primer (KaiB\_GG3\_rer) | KOD plus-ver.2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.5 | 25 |

| KaiC (plasmid) 4.4ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer | primer | KOD plus-ver.2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.5 | 25 |

| DT-1 (plasmid) 153.7ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer | primer | KOD plus-ver.2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.1 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16.4 | 25 |

| PKaiBC(PCR product) 16.3ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer | primer | KOD plus-ver.2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.7 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.8 | 25 |

| SasA(PCR product) 21.6ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer | primer | KOD plus-ver.2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16 | 25 |

| RpaA(PCR product) 15.4ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer | primer | KOD plus-ver.2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.6 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.9 | 25 |

| RpaB(PCR product) 26.7ng/µL | 10x buffer KOD plus-ver.2 | 2mM dNTPs | 25mM MgSO4 | primer | primer | KOD plus-ver.2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.4 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16.1 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C |  | 68°C | -- |
| 5min | 10sec | 30sec |  | 30 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C |  | 68°C | -- |
| 5min | 10sec | 30sec |  | 30 |

### Mutatation PCR

No name

| KaiABC(12.6pg/µL) | Primer(Kai\_BsaIMut\_fwd) | Primer(Kai\_BsaIMut\_rer) | Prime STAR Max Premix | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1 | 1 | 1 | 25 | 22 | 50 |

|  | Primer(Kai\_BsaIMut\_fwd) | Primer(Kai\_BsaIMut\_rer) | Prime STAR Max Premix | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
|  | 1 | 1 | 25 | 23 | 50 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 65°C | 72°C | -- |
| 5min | 10sec | 15sec | 40sec | 30 |

### Colony PCR

| Sample | base pair |
| --- | --- |
| 9/1 pT181antisense(pSB1C3) | 400bp |
| 9/1 pT181antisense-DT | 547bp |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5m | 30s | 30s | 30s | 30 |

### Electrophoresis

Kojima

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | 9/1 pT181antisense(pSB1C3) |
| 3 | 9/1 pT181antisense |

[![Igku Sep3 Electrophoresis (N2).jpg](/wiki/images/5/56/Igku_Sep3_Electrophoresis_%28N2%29.jpg)](/File%3AIgku_Sep3_Electrophoresis_%28N2%29.jpg)

### Electrophoresis

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | attenuator EcoRI&SpeI |
| 3 | attenuator negative EcoRI&SpeI |
| 4 | attenuator XbaI&PstI |
| 5 | attenuator negative XbaI&PstI |
| 6 | antisense EcoRI&SpeI |
| 7 | antisense negative EcoRI&SpeI |
| 8 | 100bp ladder |

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | antisense XbaI&PstI |
| 3 | antisense negative XbaI&PstI |
| 4 | apt EcoRI&SpeI |
| 5 | apt negative EcoRI&SpeI |
| 6 | DT EcoRI&XbaI |
| 7 | DT negative EcoRI&XbaI |
| 8 | 100bp ladder |

[![Igku Sep3 Electrophoresis(N3 2).jpg](/wiki/images/7/7b/Igku_Sep3_Electrophoresis%28N3_2%29.jpg)](/File%3AIgku_Sep3_Electrophoresis%28N3_2%29.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | RBS-lysis1 EcoRI&SpeI |
| 3 | RBS-lysis1 negative |
| 4 | RBS-lysis2 EcoRI&SpeI |
| 5 | RBS-lysis2 negative |
| 6 | RBS-lysis3 EcoRI&SpeI |
| 7 | RBS-lysis3 negative |
| 8 | 100bp ladder |

[![Igku Sep3 Electrophoresis(N3 3).jpg](/wiki/images/8/8b/Igku_Sep3_Electrophoresis%28N3_3%29.jpg)](/File%3AIgku_Sep3_Electrophoresis%28N3_3%29.jpg)

### Gel Extraction

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 3 | pT181 attenuator | EcoRI&SpeI |
| 4 | pT181 attenuator | EcoRI&SpeI |
| 6 | pT181 attenuator | XbaI&PstI |
| 7 | pT181 attenuator | XbaI&PstI |
| 9 | pT181 antisense | EcoRI&SpeI |
| 10 | pT181 antisense | EcoRI&SpeI |

[![Igku Sep3 Electrophoresis(N4 1).jpg](/wiki/images/b/b1/Igku_Sep3_Electrophoresis%28N4_1%29.jpg)](/File%3AIgku_Sep3_Electrophoresis%28N4_1%29.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 2 | pT181 antisense | XbaI&PstI |
| 3 | pT181 antisense | XbaI&PstI |
| 5 | apt12\_1R | EcoRI&SpeI |
| 6 | apt12\_1R | EcoRI&SpeI |

[![Igku Sep3 Electrophoresis(N4 2).jpg](/wiki/images/6/68/Igku_Sep3_Electrophoresis%28N4_2%29.jpg)](/File%3AIgku_Sep3_Electrophoresis%28N4_2%29.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 2 | DT | EcoRI&XbaI |
| 3 | DT | EcoRI&XbaI |
| 5 | RBS-lysis1 | EcoRI&SpeI |
| 6 | RBS-lysis1 | EcoRI&SpeI |

[![Igku Sep3 Electrophoresis(N5 3).jpg](/wiki/images/b/b0/Igku_Sep3_Electrophoresis%28N5_3%29.jpg)](/File%3AIgku_Sep3_Electrophoresis%28N5_3%29.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 2 | RBS-lysis2 | EcoRI&SpeI |
| 3 | RBS-lysis2 | EcoRI&SpeI |
| 5 | RBS-lysis3 | EcoRI&SpeI |
| 6 | RBS-lysis3 | EcoRI&SpeI |

[![Igku Sep3 Electrophoresis(N5 4).jpg](/wiki/images/f/f9/Igku_Sep3_Electrophoresis%28N5_4%29.jpg)](/File%3AIgku_Sep3_Electrophoresis%28N5_4%29.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pT181 attenuator(EcoRI&SpeI) | 10.0 | 1.86 | 0.39 |
| pT181 attenuator(XbaI&PstI) | 11.7 | 1.65 | 0.36 |
| pT181 antisense(EcoRI&SpeI) | 4.2 | 1.95 | 0.25 |
| pT181 antisense(XbaI&PstI) | 6.6 | 1.78 | 0.33 |
| tRNA 12\_1R apt(EcoRI&SpeI) | 5.7 | 1.73 | 0.29 |
| DT(EcoRI&XbaI) | 16.6 | 1.99 | 0.76 |
| RBS-lysis1(EcoRI&SpeI) | 8.8 | 1.65 | 0.32 |
| RBS-lysis2(EcoRI&SpeI) | 13.4 | 1.61 | 0.48 |
| RBS-lysis3(EcoRI&SpeI) | 10.2 | 2.19 | 0.51 |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 8/16master plate7(DT) |  |
| 8/21RBS-lysis1 |  |

## Sep 4

### Ligation

No name

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/3 DT(EcoRI&XbaI) | 3.2µL | 9/3 RBS-lysis1 | 2.4µL | 2.7µL |
| experiment | 9/3 DT(EcoRI&XbaI) | 3.0µL | 9/3 RBS-lysis2 | 4.7µL | 4.0µL |
| experiment | 9/3 DT(EcoRI&XbaI) | 3.0µL | 9/3 RBS-lysis3 | 8.8µL | 4.0µL |
| experiment | 9/3 DT(EcoRI&XbaI) | 3.0µL | 9/3 aptamer12\_1R(EcoRI&SpeI) | 1.9µL | 2.45µL |
| experiment | 9/2 pSB1C3(XbaI&PstI) | 10.6µL | 9/3 pT181attenuator (XbaI& PstI) | 3.2µL | 4.0µL |
| experiment | 9/2 pSB1C3(XbaI&PstI) | 9.6µL | 9/3 pT181antisense (XbaI& PstI) | 2.1µL | 4.0µL |
| experiment | 9/2 pSB1C3(EcoRI&SpeI) | 16.7µL | 9/3 aptamer12\_1R(EcoRI&SpeI) | 2.0µL | 4.0µL |

incubate 16 °C 1 hour

### Transformation

Tatsui

| Name | Sample | Competent Cells | Plate |
| --- | --- | --- | --- |
| 9/4 RBS-lysis1-DT | 2µL | 20µL | Amp |
| 9/4 RBS-lysis2-DT | 2µL | 20µL | Amp |
| 9/4 RBS-lysis3-DT | 2µL | 20µL | Amp |
| 9/4 aptamer12\_1R-DT | 2µL | 20µL | Amp |
| 9/4 pT181attenuator –pSB1C3 | 2µL | 20µL | Amp |
| 9/4 pT181antisense-pSB1C3 | 2µL | 20µL | Amp |
| 9/4 aptamer12\_1R –pSB1C3 | 2µL | 20µL | Amp |
| 9/4 pSB4K5(2013plate5 56) | 2µL | 20µL | Amp |
| 9/3 Mutagenesis product 1(KaiABC) | 2µL | 20µL | Amp |
| 9/3 Mutagenesis product 2(NC) | 2µL | 20µL | Amp |

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| DT | 193.1 | 1.93 | 1.81 |
| Spinach-DT | 373.3 | 1.92 | 1.69 |
| RBS-lysis1 | 332.0 | 1.95 | 1.62 |

### Master Plate

Hirano

| Number | Use LB plate (+CP) |
| --- | --- |
| 1 | 8/10 Plac(CP)-2 |
| 2 | 8/10 Plac(CP)-2 |
| 3 | 8/10 Plac(CP)-2 |

| Number | Use LB plate (+Amp) |
| --- | --- |
| 1 | 8/9 J23100(Amp)-1 |
| 2 | 8/9 J23100(Amp)-1 |
| 3 | 8/9 J23100(Amp)-1 |

### Liquid Culture

Hirano

| Sample | medium |
| --- | --- |
| 8/10 Plac(CP)-2 | Plusgrow medium(+CP) |
| 8/9 J23100(Amp)-1 | Plusgrow medium(+Amp) |

### PCR

Hirano

| 8/29 Pλ-RBS-luxI-DT | KOD plus | 10x buffer | dNTP | MgSO4 | F primer (RBS-luxI-DT-cloning-fwd) | R primer(RBS-luxI-DT-cloning-rev) | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.3µL | 0.5µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 16.2µL | 25µL |

| 8/31 Pbad/araC-RBS-RFP(1) | KOD plus | 10x buffer | dNTP | MgSO4 | F primer (Pbad/araC-pSB1C3-cloning-fond) | R primer(Pbad/araC-cloning-rex) | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.3µL | 0.5µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 16.2µL | 25µL |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 65°C | 68°C | -- |
| 5min | 10s | 30s | 3min10s | 30cycles |

### Restriction Enzyme Digestion

No name

|  | 8/21 Pconst | SpeI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 10.5µL | 1.0µL | 1.0µL | 3.0µL | 0.3µL | 14.2µL | 30 µL |
| NC | 0.5µL | 0µL | 0µ | 1.0µL | 0.1µL | 8.4µL | 10 µL |

|  | 8/29 Plac | SpeI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 13.0µL | 1.0µL | 1.0µL | 3.0µL | 0.3µL | 11.7µL | 30 µL |
| NC | 0.7µL | 0µL | 0µ | 1.0µL | 0.1µL | 8.2µL | 10 µL |

|  | 8/20 Ptet | SpeI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 9.0µL | 1.0µL | 1.0µL | 3.0µL | 0.3µL | 15.7µL | 30 µL |
| NC | 0.5µL | 0µL | 0µ | 1.0µL | 0.1µL | 8.4µL | 10 µL |

|  | 8/20 RBS-tetR-DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 8.5µL | 1.0µL | 1.0µL | 3.0µL | 0.3µL | 16.2µL | 30 µL |
| NC | 0.5µL | 0µL | 0µ | 1.0µL | 0.1µL | 8.4µL | 10 µL |

|  | spinach -DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.4µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 16.6µL | 30 µL |
| NC | 0.3µL | 0µL | 0µ | 1.0µL | 1.0µL | 7.7µL | 10 µL |

|  | 8/17 RBS-lacZα-DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 11µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 11µL | 30 µL |
| NC | 0.5µL | 0µL | 0µ | 1.0µL | 1.0µL | 7.5µL | 10 µL |

### Transformation

No name

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| Ptrc KaiC | 1µL | 10µL | 11µL | Amp |
| Ptrc KaiB | 1µL | 10µL | 11µL | Amp |
| pSΩ | 1µL | 10µL | 11µL | Amp |

### Electrophoresis

No name
1

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | Pconst | SpeI | PstI |
| 3 | Pconst NC | -- | -- |
| 4 | Plac | SpeI | PstI |
| 5 | Plac NC | -- | -- |
| 6 | 100bp ladder | -- | -- |

2

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | Ptet | SpeI | PstI |
| 3 | Ptet NC | -- | -- |
| 4 | RBS-tetR-DT | XbaI | PstI |
| 5 | RBS-tetR-DT NC | -- | -- |
| 6 | 100bp ladder | -- | -- |

3

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | Spinach-DT | XbaI | PstI |
| 3 | Spinach-DT NC | -- | -- |
| 4 | RBS-lacZα-DT | XbaI | PstI |
| 5 | RBS-lacα-DT NC | -- | -- |
| 6 | 100bp ladder | -- | -- |

[![Igku Sep4 Electrophoresis(N1).jpg](/wiki/images/d/d6/Igku_Sep4_Electrophoresis%28N1%29.jpg)](/File%3AIgku_Sep4_Electrophoresis%28N1%29.jpg)
[![Igku Sep4 Electrophoresis N2 3.jpg](/wiki/images/e/ed/Igku_Sep4_Electrophoresis_N2_3.jpg)](/File%3AIgku_Sep4_Electrophoresis_N2_3.jpg)

### Gel Extraction

No name

1

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | Pcon | SpeI&PstI |
| 3 | Pcon | SpeI&PstI |
| 4 | -- | -- |
| 5 | Plac | SpeI&PstI |
| 6 | Plac | SpeI&PstI |

2

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | Ptet | SpeI&PstI |
| 3 | Ptet | SpeI&PstI |
| 4 | -- | -- |
| 5 | RBS-tetR-DT | XbaI&PstI |
| 6 | RBS-tetR-DT | XbaI&PstI |

[![Igku Sep4 Electrophoresis(N2).jpg](/wiki/images/5/5e/Igku_Sep4_Electrophoresis%28N2%29.jpg)](/File%3AIgku_Sep4_Electrophoresis%28N2%29.jpg)
[![Igku Sep4 Gel Extraction(N2) 2.jpg](/wiki/images/5/5f/Igku_Sep4_Gel_Extraction%28N2%29_2.jpg)](/File%3AIgku_Sep4_Gel_Extraction%28N2%29_2.jpg)

| Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- | --- |
| Pcon | -- | 13.2 | 1.99 | -1.64 |
| Plac | -- | 15.3 | 2.07 | -0.39 |
| Ptet | -- | 30.1 | 1.97 | -0.97 |
| RBS-tetR-DT | -- | 3.0 | 2.44 | 0.88 |

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | spinach-DT | XbaI&PstI |
| 3 | spinach-DT | XbaI&PstI |
| 4 | -- | -- |
| 5 | RBS-lacZα-DT | XbaI&PstI |
| 6 | RBS-lacZα-DT | XbaI&PstI |

[![Igku Sep4 Gel Extraction(N3) 1.jpg](/wiki/images/4/41/Igku_Sep4_Gel_Extraction%28N3%29_1.jpg)](/File%3AIgku_Sep4_Gel_Extraction%28N3%29_1.jpg)
[![Igku Sep4 Gel Extraction(N3) 2.jpg](/wiki/images/a/ae/Igku_Sep4_Gel_Extraction%28N3%29_2.jpg)](/File%3AIgku_Sep4_Gel_Extraction%28N3%29_2.jpg)

| Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- | --- |
| spinach-DT | -- | 2.1 | 2.10 | -0.05 |
| RBS-lacZα-DT | -- | 3.3 | 2.59 | -0.07 |

### Master Plate

No name

| Number | Use LB plate (+Amp) |
| --- | --- |
| 4 | 8/9 BBa\_J23100-3 |
| 5 | 8/9 BBa\_J23100-4 |
| 6 | 8/9 BBa\_J23100-5 |

| Number | Use LB plate |
| --- | --- |
| 1 | JM109-1 |
| 2 | JM109-2 |
| 3 | JM109-3 |
| 4 | JM109-4 |

### Liquid Culture

Hirano

| Sample | medium |
| --- | --- |
| 8/9 BBa\_J23100-3 | Plusgrow medium(+Amp) |
| JM109-1(Master plate) | LB medium |

### Colony PCR

Hirano

| Sample | base pair |
| --- | --- |
| 8/9 BBa\_J23100 3 | -- |
| 8/9 BBa\_J23100 4 | -- |
| 8/9 BBa\_J23100 5 | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 30s | 30cycles |

## Sep 5

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | J23100-3 | -- | -- |
| 3 | J23100-4 | -- | -- |
| 4 | J23100-5 | -- | -- |
| 5 | 100bp ladder | -- | -- |

[![Igku Sep5 electrophoresis N1.jpg](/wiki/images/3/3b/Igku_Sep5_electrophoresis_N1.jpg)](/File%3AIgku_Sep5_electrophoresis_N1.jpg)

### Miniprep

Nakamoto

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/3 J23100-3 | 349.1 | 1.69 | 1.851 |
| 8/10 Plac-2 | 331.3 | 1.84 | 1.42 |

### Ligation

Kojima

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | Pconst | 13.2 | spinach-DT | 2.1 | 3.5 |
| experiment | Plac | 15.3 | spinach-DT | 2.1 | 3.5 |
| experiment | Pconst | 13.2 | pT181 antisense (XbaI & PstI) | 6.6 | 3.5 |
| experiment | Plac | 15.3 | pT181 antisense (XbaI & PstI) | 6.6 | 3.5 |
| experiment | Ptet | 30.1 | pT181 antisense (XbaI & PstI) | 6.6 | 3.5 |
| experiment | Pconst | 13.2 | pT181 attenuator | 11.7 | 3.5 |
| experiment | Plac | 15.3 | pT181 attenuator | 11.7 | 3.5 |
| experiment | Ptet | 30.1 | RBS-lacZα-DT | 3.0 | 3.5 |

incubate 16 °C 1 hour

### Electrophoresis

Tatsui

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | RBS-luxI-DT |
| 3 | Pbad/araC |
| 4 | NC |
| 5 | 100bp ladder |

[![Igku Sep5 electrophorsis N2 1.jpg](/wiki/images/9/9c/Igku_Sep5_electrophorsis_N2_1.jpg)](/File%3AIgku_Sep5_electrophorsis_N2_1.jpg)
[![Igku Sep5 electrophorsis N2 2.jpg](/wiki/images/1/12/Igku_Sep5_electrophorsis_N2_2.jpg)](/File%3AIgku_Sep5_electrophorsis_N2_2.jpg)

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder |  |
| 2 | RBS-luxI-DT |  |
| 3 | RBS-luxI-DT |  |
| 4 |  |  |
| 5 | Pbad/araC |  |
| 6 | Pbad/araC |  |

[![Igku Sep5 Gel Extraction(N3) 1.jpg](/wiki/images/b/b5/Igku_Sep5_Gel_Extraction%28N3%29_1.jpg)](/File%3AIgku_Sep5_Gel_Extraction%28N3%29_1.jpg)
[![Igku Sep5 Gel Extraction(N3) 2.jpg](/wiki/images/e/ec/Igku_Sep5_Gel_Extraction%28N3%29_2.jpg)](/File%3AIgku_Sep5_Gel_Extraction%28N3%29_2.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| RBS-luxI-DT | 23.2 | 0.83 | 7.89 |
| Pbad/araC | 51.8 | 1.20 | 0.11 |

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 9/4 aptamer 12\_1R(pSB1C3)-1 | 384 |
| 9/4 aptamer 12\_1R(pSB1C3)-2 | 384 |
| 9/4 pT181 antisense(pSB1C3)-1 | 415 |
| 9/4 pT181 antisense(pSB1C3)-2 | 415 |
| 9/4 pT181 antisense(pSB1C3)-3 | 415 |
| 9/4 pT181 antisense(pSB1C3)-4 | 415 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 30s | 30cycles |

| Sample | base pair |
| --- | --- |
| 9/4 aptamer 12\_1R-DT-1 | 521 |
| 9/4 aptamer 12\_1R-DT-2 | 521 |
| 9/4 aptamer 12\_1R-DT-3 | 521 |
| 9/4 aptamer 12\_1R-DT-4 | 521 |
| 9/4 pT181 attenuator(pSB1C3) | 601 |
| 9/4 pT181 attenuator(pSB1C3) | 601 |
| 9/4 RBS-lysis1-DT | 613 |
| 9/4 RBS-lysis1-DT | 613 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 36s | 30cycles |

| Sample | base pair |
| --- | --- |
| 9/4 RBS-lysis2-DT-1 | 985 |
| 9/4 RBS-lysis2-DT-2 | 985 |
| 9/4 RBS-lysis2-DT-3 | 985 |
| 9/4 RBS-lysis2-DT-4 | 985 |
| 9/4 RBS-lysis3-DT-1 | 1210 |
| 9/4 RBS-lysis3-DT-2 | 1210 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 72s | 30cycles |

### Liquid Culture

Nakamoto

| Sample | medium |
| --- | --- |
| 8/9 J23100-5 | Plusgrow medium(+Amp) |

37°C

### Transformation

No name

| Name | Sample(µL) | Competent Cells(µL) | Total(µL) | Plate |
| --- | --- | --- | --- | --- |
| 9/5 Pconst+spinach-DT | 2 | 20 | 22 | -- |
| 9/5 Plac+spinach-DT | 2 | 20 | 22 | -- |
| 9/5 Pconst+pT181 antisense | 2 | 20 | 22 | -- |
| 9/5 Plac+pT181 antisense | 2 | 20 | 22 | -- |
| 9/5 Ptet+pT181 antisense | 2 | 20 | 22 | -- |
| 9/5 Plux+pT181 attenuator | 2 | 20 | 22 | -- |
| 9/5 Ptet+pT181 attenuator | 2 | 20 | 22 | -- |
| 9/5 Ptet+pT181 attenuator | 2 | 20 | 22 | -- |
| 9/5 Pconst+RBS-tetR+DT | 2 | 20 | 22 | -- |
| 9/16(2012) T7-His-FT | 2 | 20 | 22 | -- |
| 9/16(2012) pBr322 | 2 | 20 | 22 | -- |

### Restriction Enzyme Digestion

No name

|  | 9/5 Pbad/araC | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 4µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 18µL | 30µL |

|  | 9/5 RBS-luxI-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 4µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 18µL | 30µL |

|  | 8/28 Plux | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 12µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 10µL | 30µL |
| NC | 0.6µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.4µL | 10µL |

|  | 9/5 Pconst(J23100) | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.7µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 16.3µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |

|  | 9/5 Plac | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 6.0µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 16µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |

|  | 8/31 Plux-RBS-GFP-DT-1 | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 9.4µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 12.6µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |

|  | 8/20 Pconst-RBS-luxR-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 4.5µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 13.5µL | 30µL |
| NC | 0.2µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.8µL | 10µL |

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/5 RBS-luxI-DT | 7.6 | 1.56 | -1.42 |
| 9/5 Pbad/araC | 7.5 | 1.54 | 3.84 |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | aptamer 12\_1R (pSB1C3)-1 | -- | -- |
| 3 | aptamer 12\_1R (pSB1C3)-2 | -- | -- |
| 4 | pT181 antisense(pSB1C3)-1 | -- | -- |
| 5 | pT181 antisense(pSB1C3)-2 | -- | -- |
| 6 | pT181 antisense(pSB1C3)-3 | -- | -- |
| 7 | pT181 antisense(pSB1C3)-4 | -- | -- |
| 8 | 100bp ladder | -- | -- |

[![Igku Sep5 Electrophoresis(N4).jpg](/wiki/images/3/3a/Igku_Sep5_Electrophoresis%28N4%29.jpg)](/File%3AIgku_Sep5_Electrophoresis%28N4%29.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | aptamer 12\_1R-DT-1 | -- | -- |
| 3 | aptamer 12\_1R-DT-2 | -- | -- |
| 4 | aptamer 12\_1R-DT-3 | -- | -- |
| 5 | aptamer 12\_1R-DT-4 | -- | -- |
| 6 | pT181 attenuator(pSB1C3)-1 | -- | -- |
| 7 | pT181 attenuator(pSB1C3)-2 | -- | -- |
| 8 | RBS-lysis1-DT-1 | -- | -- |
| 9 | RBS-lysis1-DT-2 | -- | -- |
| 10 | 100bp ladder | -- | -- |

[![Igku Sep5 Electrophoresis(N6) 2.jpg](/wiki/images/b/b2/Igku_Sep5_Electrophoresis%28N6%29_2.jpg)](/File%3AIgku_Sep5_Electrophoresis%28N6%29_2.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | RBS-lysis2-DT-1 | -- | -- |
| 3 | RBS-lysis2-DT-2 | -- | -- |
| 4 | RBS-lysis2-DT-3 | -- | -- |
| 5 | RBS-lysis2-DT-4 | -- | -- |
| 6 | RBS-lysis3-DT-1 | -- | -- |
| 7 | RBS-lysis3-DT-2 | -- | -- |
| 8 | 100bp ladder | -- | -- |

[![Igku Sep5 Electrophoresis(N5).jpg](/wiki/images/f/f6/Igku_Sep5_Electrophoresis%28N5%29.jpg)](/File%3AIgku_Sep5_Electrophoresis%28N5%29.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 1kbp ladder | -- | -- |
| 2 | Pconst | SpeI | PstI |
| 3 | Pconst NC | -- | -- |
| 4 | Plac | SpeI | PstI |
| 5 | Plac NC | -- | -- |
| 6 | Plux | SpeI | PstI |
| 7 | Plux NC | -- | -- |
| 8 | Pconst-RBS-luxR-DT | EcoRI | XbaI |
| 9 | Pconst-RBS-luxR-DT NC | -- | -- |
| 10 | Plux-RBS-GFP-DT | EcoRI | SpeI |
| 11 | Plux-RBS-GFP-DT NC | -- | -- |
| 12 | 1kbp ladder | -- | -- |

[![Igku Sep5 Electrophoresis(N6) 3.jpg](/wiki/images/b/b9/Igku_Sep5_Electrophoresis%28N6%29_3.jpg)](/File%3AIgku_Sep5_Electrophoresis%28N6%29_3.jpg)

### 5x M9 Medium (+EDTA)

Hirano

| volume | 10ml |
| --- | --- |
| Na2HPO4 | 60mg |
| KH2PO4 | 30mg |
| NaCl | 5mg |
| NH4Cl | 10mg |
| Fe(III)-EDTA | 1263.27mg |
| MilliQ | up to 10 mL |

* autoclave at 121 °C for 20 min

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder |  |
| 2 | Pconst | SpeI & PstI |
| 3 | Pconst | SpeI & PstI |

[![Igku Sep5 gel extraction N7 1.jpg](/wiki/images/9/90/Igku_Sep5_gel_extraction_N7_1.jpg)](/File%3AIgku_Sep5_gel_extraction_N7_1.jpg)
[![Igku Sep5 gel extraction N7 2.jpg](/wiki/images/5/5f/Igku_Sep5_gel_extraction_N7_2.jpg)](/File%3AIgku_Sep5_gel_extraction_N7_2.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pconst | 5.3 | 1.98 | 0.06 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 2 | Plac | SpeI & PstI |
| 3 | Plac | SpeI & PstI |
| 5 | Plux | SpeI & PstI |
| 6 | Plux | SpeI & PstI |

[![Igku Sep5 gel extraction N7 3.jpg](/wiki/images/3/32/Igku_Sep5_gel_extraction_N7_3.jpg)](/File%3AIgku_Sep5_gel_extraction_N7_3.jpg)
[![Igku Sep5 gel extraction N7 4.jpg](/wiki/images/d/d5/Igku_Sep5_gel_extraction_N7_4.jpg)](/File%3AIgku_Sep5_gel_extraction_N7_4.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Plac (SpeI & PstI) | 5.2 | 1.82 | 0.36 |
| Plux(SpeI & PstI) | 8.0 | 1.87 | 0.25 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder |  |
| 2 | Pconst-RBS-luxR-DT | EcoRI & XbaI |
| 3 | Pconst-RBS-luxR-DT | EcoRI & XbaI |
| 5 | Plux-RBS-GFP-DT | EcoRI & SpeI |
| 6 | Plux-RBS-GFP-DT | EcoRI & SpeI |

[![Igku Sep5 gel extraction N7 5.jpg](/wiki/images/1/10/Igku_Sep5_gel_extraction_N7_5.jpg)](/File%3AIgku_Sep5_gel_extraction_N7_5.jpg)
[![Igku Sep5 gel extraction N7 6.jpg](/wiki/images/7/7c/Igku_Sep5_gel_extraction_N7_6.jpg)](/File%3AIgku_Sep5_gel_extraction_N7_6.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pconst-RBS-luxR-DT(EcoRI & XbaI) | 30.6 | 1.84 | 1.16 |
| Plux-RBS-GFP-DT (EcoRI & SpeI) | 21.8 | 1.87 | 0.98 |

### DNA Purification

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/5 RBS-luxI-DT | 7.6 | 1.56 | -1.42 |
| 9/5 Pbad/araC | 7.5 | 1.54 | 3.84 |

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/9 J23100-5 | 243.0 | 1.90 | 1.81 |

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 9/4 RBS-lysis3-DT-3 | 1210 |
| 9/4 RBS-lysis3-DT-4 | 1210 |
| 9/4 RBS-lysis3-DT-5 | 1210 |
| 9/4 RBS-lysis1-DT-3 | 613 |
| 9/4 Ptrc KaiC-1 | -- |
| 9/4 pSB4K5 | 1370 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min 25s | 30cycles |

### Liquid Culture

Hirano

| Sample | medium |
| --- | --- |
| 9/4 aptamer 12\_1R-DT-1 | Plusgrow medium (+CP) |
| 9/4 pT181 attenuator(pSB1C3)-1 | Plusgrow medium (+CP) |
| 9/4 RBS-lysis2-DT-1 | Plusgrow medium (+CP) |
| 9/4 Ptrc-KaiC -1 | Plusgrow medium (+Amp) |
| 9/4 pSB4K5 -1 | Plusgrow medium (+Kan) |

## Sep 6

### Electrophoresis

Hirano

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | 9/4 RBS-lysis3-DT -3 |
| 3 | 9/4 RBS-lysis3-DT -4 |
| 4 | 9/4 RBS-lysis3-DT -5 |
| 5 | 9/4 RBS-lysis1-DT -3 |
| 6 | 9/4 Ptrc-KaiC -1 |
| 7 | 9/4 pSB4K5 -1 |
| 8 | 100bp ladder |

[![Igku Sep6 ColonyPCRN1.jpg](/wiki/images/9/9b/Igku_Sep6_ColonyPCRN1.jpg)](/File%3AIgku_Sep6_ColonyPCRN1.jpg)

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| Pconst-spinach-DT-(1~4) | 563 |
| Plac-spinach-DT-(1~4) | 659 |
| Pcon-pT181 antisense-(1~4) | 468 |
| Ptet-pT181 antisense-(1~4) | 604 |
| Pconst-pT181 attenuator-(1~4) | 568 |
| Pconst+RBS-tetR-DT-(1~4) | 1121 |
| Ptet+RBS-lacZα-DT-(1~4) | 1162 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 40s | 30cycles |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 9/4 RBS-Lysis3+DT3 | Plusgrow medium(+CP) |
| 9/4 RBS-Lysis1+DT3 | Plusgrow medium(+CP) |
| 9/4 pSB4K5 | Plusgrow medium(+Kan) |

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Pconst-spinach-DT -(1) |
| 3 | Pconst-spinach-DT -(2) |
| 4 | Pconst-spinach-DT -(3) |
| 5 | Pconst-spinach-DT -(4) |
| 6 | Plac-spinach-DT -(1) |
| 7 | Plac-spinach-DT -(2) |
| 8 | Plac-spinach-DT -(3) |
| 9 | Plac-spinach-DT -(4) |
| 10 | Pcon-pT181 antisense -(1) |
| 11 | Pcon-pT181 antisense -(2) |
| 12 | Pcon-pT181 antisense -(3) |
| 13 | Pcon-pT181 antisense -(4) |

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Ptet-pT181 antisense -(1) |
| 3 | Ptet-pT181 antisense -(2) |
| 4 | Ptet-pT181 antisense -(3) |
| 5 | Ptet-pT181 antisense -(4) |
| 6 | Plac-pT181 attenuator -(1) |
| 7 | Plac-pT181 attenuator -(2) |
| 8 | Plac-pT181 attenuator -(3) |
| 9 | Plac-pT181 attenuator -(4) |

[![Igku Sep6 COlonyPCRN1 1a.jpg](/wiki/images/5/5b/Igku_Sep6_COlonyPCRN1_1a.jpg)](/File%3AIgku_Sep6_COlonyPCRN1_1a.jpg)
[![Igku Sep6 colonyPCR N2 1b.jpg](/wiki/images/8/88/Igku_Sep6_colonyPCR_N2_1b.jpg)](/File%3AIgku_Sep6_colonyPCR_N2_1b.jpg)

### Miniprep

Honda and Kojima

| DNA | concentration [µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pT181 antisense-(1) | 238.7 | 1.68 | 1.44 |
| pT181 antisense-(2) | 248.8 | 1.83 | 1.77 |
| pT181 antisense-(3) | 223.2 | 1.78 | 1.61 |
| pT181 antisense-(4) | 220.8 | 1.89 | 1.92 |
| tetR aptamer-(1) | 252.5 | 1.89 | 1.90 |
| tetR aptamer-(2) | 211.2 | 1.90 | 1.99 |
| DT181 attenuator | 102.2 | 1.98 | 2.17 |
| 121R aptamer-DT | 251.7 | 1.92 | 2.01 |
| pSB4K5 | 212.9 | 1.80 | 1.90 |
| RBS-lacZα-DT | 185.7 | 1.78 | 1.79 |
| RBS-lysis2-DT-(1) | 84.8 | 1.85 | 2.24 |

### Restriction Enzyme Digestion

Tatsui

|  | 9/6 RBS-lysis2-DT (78.3 µg/mL) | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 22 µL | 1 µL | 1 µL | 3 µL | 3 µL | 0 µL | 30 µL |
| NC | 1.3 µL | 0 µL | 0 µL | 1 µL | 1 µL | 6.7 µL | 10 µL |

|  | aptamer-DT (251.7 µg/mL) | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 0.4 µL | 1 µL | 1 µL | 3 µL | 3 µL | 14.1 µL | 30 µL |
| NC | 0.4 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.6 µL | 10 µL |

|  | Pλ-luxI (86.4 µg/mL) | EcoRI | SpeI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 23.1 µL | 1 µL | 1 µL | 3 µL | 3 µL | 1.9 µL | 30 µL |
| NC | 1.2 µL | 0 µL | 0 µL | 1 µL | 1 µL | 6.8 µL | 10 µL |

* at 37 °C, for 1h

### Electrophoresis

Hirano

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Pconst+RBS-tetR-DT -(1) |
| 3 | Pconst+RBS-tetR-DT -(2) |
| 4 | Pconst+RBS-tetR-DT -(3) |
| 5 | Pconst+RBS-tetR-DT -(4) |
| 6 | Ptet+RBS-lacZα-DT -(1) |
| 7 | Ptet+RBS-lacZα-DT -(2) |
| 8 | 100bp ladder |
| 9 | Ptet+RBS-lacZα-DT -(3) |
| 10 | Ptet+RBS-lacZα-DT -(4) |
| 11 | 100bp ladder |

[![Igku Sep6 electrophoresis N3.jpg](/wiki/images/e/e2/Igku_Sep6_electrophoresis_N3.jpg)](/File%3AIgku_Sep6_electrophoresis_N3.jpg)

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | RBS-lysis2-DT | XbaI & PstI |
| 3 |
| 4 | -- | -- |
| 5 | aptamer 12\_1R-DT | XbaI & PstI |
| 6 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | Pλ-luxI | EcoRI & SpeI |
| 3 |

[![Igku Sep6 gel extraction N4 2.jpg](/wiki/images/7/77/Igku_Sep6_gel_extraction_N4_2.jpg)](/File%3AIgku_Sep6_gel_extraction_N4_2.jpg)
[![Igku Sep6 gel extraction N4 3.jpg](/wiki/images/4/40/Igku_Sep6_gel_extraction_N4_3.jpg)](/File%3AIgku_Sep6_gel_extraction_N4_3.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| RBS-lysis2-DT (XbaI & PstI) | 7.1 | 1.88 | 0.07 |
| aptamer 12\_1R-DT (XbaI & PstI) | 15.3 | 1.93 | 0.70 |
| Pλ-luxI (EcoRI & SpeI) | 4.5 | 1.79 | 0.31 |

[![Igku Sep6 gel extraction N5 1.jpg](/wiki/images/4/4f/Igku_Sep6_gel_extraction_N5_1.jpg)](/File%3AIgku_Sep6_gel_extraction_N5_1.jpg)
[![Igku Sep6 gel extraction N5 2.jpg](/wiki/images/e/e6/Igku_Sep6_gel_extraction_N5_2.jpg)](/File%3AIgku_Sep6_gel_extraction_N5_2.jpg)

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 9/5 Pconst-spinach-DT-(1) | Plusgrow medium(+Amp&CP) |
| 9/5 Ptet-pT181 antisense-(1) | Plusgrow medium(+CP) |
| 9/5 Pconst+RBS-tetR-DT-(1) | Plusgrow medium(+Amp&CP) |
| 9/5 Ptet+RBS-lacZα-DT-(1) | Plusgrow medium(+CP) |
| 9/5 Pcon-pT181 attenuator-(1) | Plusgrow medium(+CP) |
| 9/4 pT181 antisense-(1) | Plusgrow medium(+CP) |
| 9/4 aptamer 12\_1R-(1) | Plusgrow medium(+CP) |

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 9/5 Plac-spinach-DT-(5~12) | 563 |
| 9/5 Pcon-antisense-(5~12) | 468 |
| 9/5 Plac-attenuator-(5~12) | 664 |
| 9/5 pT181 antisense+Plac-(1) | - |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 42s | 30cycles |

## Sep 7

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| RBS-lysis1-DT | 195.3 |  |  |
| RBS-lysis3-DT | 255.5 | 1.91 | 2.04 |
| Ptet-pT18 | 183.6 | 1.85 | 1.48 |
| Pcon-RBS-tetR-DT | 134.3 | 1.94 | 2.20 |
| Ptet-RBS-lacZα-DT | 67.7 | 1.92 | 1.79 |
| Pcon-pT181 attenuator | 232.3 | 1.95 | 1.90 |
| pSB4K5 | 237.0 | 1.61 | 1.50 |
| pT181 antisense(pSB1C3) | 256.4 | 1.63 | 1.78 |
| apt12\_1R(pSB1C3) | 230.0 | 1.98 | 2.28 |

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Plac-Spinach-DT -5 |
| 3 | Plac-Spinach-DT -6 |
| 4 | Plac-Spinach-DT -7 |
| 5 | Plac-Spinach-DT -8 |
| 6 | Plac-Spinach-DT -9 |
| 7 | Plac-Spinach-DT -10 |
| 8 | 100bp ladder |
| 1 | 100bp ladder |
| 2 | Plac-Spinach-DT -11 |
| 3 | Plac-Spinach-DT -12 |
| 4 | Pcon-antisense -5 |
| 5 | Pcon-antisense -6 |
| 6 | Pcon-antisense -7 |
| 7 | Pcon-antisense -8 |
| 8 | 100bp ladder |

[![Igku Sep7electrophoresis1.jpg](/wiki/images/7/78/Igku_Sep7electrophoresis1.jpg)](/File%3AIgku_Sep7electrophoresis1.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Pcon-antisense -9 |
| 3 | Pcon-antisense -10 |
| 4 | Pcon-antisense -11 |
| 5 | Pcon-antisense -12 |
| 6 | Plac-attenuator -5 |
| 7 | Plac-attenuator -6 |
| 8 | 100bp ladder |
| 1 | 100bp ladder |
| 2 | Plac-attenuator -7 |
| 3 | Plac-attenuator -8 |
| 4 | Plac-attenuator -9 |
| 5 | Plac-attenuator -10 |
| 6 | Plac-attenuator -11 |
| 7 | Plac-attenuator -12 |
| 8 | 100bp ladder |

[![Igku Sep7electrophoresis2.jpg](/wiki/images/b/b9/Igku_Sep7electrophoresis2.jpg)](/File%3AIgku_Sep7electrophoresis2.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Plac-pT181 antisense |

[![Igku Sep7electrophoresis3.jpg](/wiki/images/e/e3/Igku_Sep7electrophoresis3.jpg)](/File%3AIgku_Sep7electrophoresis3.jpg)

### Colony PCR

No name

| Sample |
| --- |
| Plac-spinach-DT -13~20 |
| Plac-pT181 attenuator -13~20 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94 °C | 94 °C | 55 °C | 68°C | -- |
| 5 min | 30 s | 30 s | 42 sec | 30 cycles |

### Ligation

No Name

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/6 Plux (SpeI&PstI) | 2 µL | 9/7 RBS-lysis2-DT (EcoRI&XbaI) | µL | 3.5 µL |
| experiment | 9/6 Plac (SpeI&PstI) | µL | 9/7 RBS-lysis2-DT (EcoRI&XbaI) | µL | 3.5 µL |
| experiment | 9/6 Pcon-RBS-luxR-DT (EcoRI&XbaI) | 1.9 µL | 9/6 Plux-RBS-GFP-DT(SpeI&PstI) | 2.2 µL | 3.5 µL |
| experiment | 9/6 Pbed/araC (SpeI&PstI) | 1.6 µL | 9/5 RBS-luxI-DT (EcoRI&XbaI) | 16 µL | 3.5 µL |
| experiment | 9/6 Plac (SpeI&PstI) | 1.5 µL | 9/5 pT181 antisense (EcoRI&XbaI) | 2.0 µL | 3.5 µL |
| experiment | 9/6 Pcon (SpeI&PstI) | 3.0 µL | 9/5 RBS-luxI-DT (EcoRI&XbaI) | 10 µL | 3.5 µL |

### Electrophoresis

Nakamoto

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Plac-Spinach-DT -13 |
| 3 | Plac-Spinach-DT -14 |
| 4 | Plac-Spinach-DT -15 |
| 5 | Plac-Spinach-DT -16 |
| 6 | Plac-Spinach-DT -17 |
| 7 | Plac-Spinach-DT -18 |
| 8 | Plac-Spinach-DT -19 |
| 9 | Plac-Spinach-DT -20 |
| 10 | Plac-pT181 attenuator -13 |
| 11 | Plac-pT181 attenuator -14 |
| 12 | 100bp ladder |

[![Igku 974.jpg](/wiki/images/f/ff/Igku_974.jpg)](/File%3AIgku_974.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Plac-pT181 attenuator -15 |
| 3 | Plac-pT181 attenuator -16 |
| 4 | Plac-pT181 attenuator -17 |
| 5 | Plac-pT181 attenuator -18 |
| 6 | Plac-pT181 attenuator -19 |
| 7 | Plac-pT181 attenuator -20 |
| 8 | 100bp ladder |

[![Igku 975.jpg](/wiki/images/f/fa/Igku_975.jpg)](/File%3AIgku_975.jpg)

### Transformation

Tatsui

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT | 2µL | 20µL | 22µL |  |
| Pbed/araC+RBS-luxI-DT | 2µL | 20µL | 22µL |  |
| Plac+pT181 antisense | 2µL | 20µL | 22µL |  |
| Pcon+RBS-luxI-DT | 2µL | 20µL | 22µL |  |

### Restriction Enzyme Digestion

No Name

|  | DNA | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/7 RBS-lysis1-DT(XbaI & PstI) | 10 µL | 1 µL | 1 µL | 3 µL | 3 µL | 12 µL | 30 µL |
| NC | 0.5 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.5 µL | 10 µL |

|  | DNA | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/7 RBS-lysis3-DT (XbaI & PstI) | 7.8 µL | 1 µL | 1 µL | 3 µL | 3 µL | 14.2 µL | 30 µL |
| NC | 0.4 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.6 µL | 10 µL |

|  | DNA | SpeI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/5 Plac (SpeI & PstI) | 6.0 µL | 1 µL | 1 µL | 3 µL | 3 µL | 16.0 µL | 30 µL |
| NC | 0.3 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.7 µL | 10 µL |

|  | DNA | EcorI | SpeI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/7 pSB4K5 (EcoRI & SpeI) | 8.4 µL | 1 µL | 1 µL | 3 µL | 3 µL | 16.0 µL | 30 µL |
| NC | 0.4 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.7 µL | 10 µL |

|  | DNA | restricion enzyme | restriction enzyme | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Pcon-pT181 attenuator (SpeI & PstI) | 8.6 µL | 1 µL | 1 µL | 3 µL | 3 µL | 13.4 µL | 30 µL |
| Pcon-pT181 attenuator (EcorI & SpeI) | 8.6 µL | 1 µL | 1 µL | 3 µL | 3 µL | 13.4 µL | 30 µL |
| NC | 0.4 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.6 µL | 10 µL |

|  | DNA | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/5 Spinach-DT (XbaI & PstI) | 5.4 µL | 1 µL | 1 µL | 3 µL | 3 µL | 16.6 µL | 30 µL |
| NC | 0.3 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.7 µL | 10 µL |

|  | DNA | SpeI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Ptet-anticodone (SpeI & PstI) | 11 µL | 1 µL | 1 µL | 3 µL | 3 µL | 11 µL | 30 µL |
| NC | 0.5 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.5 µL | 10 µL |

|  | DNA | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/6 RBS-lacZα-DT (XbaI & PstI) | 11 µL | 1 µL | 1 µL | 3 µL | 3 µL | 11 µL | 30 µL |
| NC | 0.5 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.5 µL | 10 µL |

|  | DNA | EcoRI | SpeI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| pSB1C3 (1) (EcoRI & SpeI) | 6.6 µL | 1 µL | 1 µL | 3 µL | 3 µL | 15.4 µL | 30 µL |
| NC | 0.3 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.7 µL | 10 µL |

|  | DNA | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| pSB1C3 (3) (XbaI & PstI) | 6.6 µL | 1 µL | 1 µL | 3 µL | 3 µL | 15.4 µL | 30 µL |
| NC | 0.3 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.7 µL | 10 µL |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | RBS-lysis1-DT (9/7) | XbaI | PstI |
| 3 | RBS-lysis1-DT (9/7) | -- | -- |
| 4 | RBS-lysis3-DT (9/7) | XbaI | PstI |
| 5 | RBS-lysis3-DT (9/7) | -- | -- |
| 6 | Pcon-pT181 attenuator | EcoRI | SpeI |
| 7 | Pcon-pT181 attenuator | -- | -- |
| 8 | 9/5 Spinach-DT | XbaI | PstI |
| 9 | 9/5 Spinach-DT | -- | -- |
| 10 | 100bp ladder | -- | -- |
| 11 | 100bp ladder | -- | -- |
| 12 | RBS-lacZα-DT | XbaI | PstI |
| 13 | RBS-lacZα-DT | -- | -- |

[![Igku 971.jpg](/wiki/images/9/91/Igku_971.jpg)](/File%3AIgku_971.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | Ptet-antisense | SpeI | PstI |
| 3 | Plac (NC) | -- | -- |
| 4 | pSB4K5 | EcoRI | SpeI |
| 5 | pSB4K5 | -- | -- |
| 6 | Pcon-pT181 attenuator | SpeI | PstI |
| 7 | 1kbp ladder | -- | -- |

[![Igku 972.jpg](/wiki/images/1/13/Igku_972.jpg)](/File%3AIgku_972.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | Plac | SpeI | PstI |
| 3 | Ptet-antisense | -- | -- |
| 4 | pSB1C3 | EcoRI | SpeI |
| 5 | pSB1C3 | -- | -- |
| 6 | pSB1C3 | XbaI | PstI |
| 7 | pSB1C3 | -- | -- |
| 8 | 1kbp ladder | -- | -- |

[![Igku 973.jpg](/wiki/images/3/3c/Igku_973.jpg)](/File%3AIgku_973.jpg)

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| entA- strain | SOB medium (+Kan) |
| 9/15 Pcon-pT181 antisense | Plusgrow (+Amp) |

## Sep 8

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 2 | RBS-lysis1-DT | XbaI & PstI |
| 3 | RBS-lysis1-DT | XbaI & PstI |
| 5 | RBS-lysis3-DT | XbaI & PstI |
| 6 | RBS-lysis3-DT | XbaI & PstI |

[![Igku 981b.png](/wiki/images/e/e9/Igku_981b.png)](/File%3AIgku_981b.png)
[![Igku 981a.png](/wiki/images/c/cb/Igku_981a.png)](/File%3AIgku_981a.png)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| RBS-lysis1-DT (XbaI & PstI) | 5.3 | 1.98 | 0.06 |
| RBS-lysis3-DT (XbaI & PstI) | 5.3 | 1.98 | 0.06 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 3 | Pcon-pT181 attenuator | EcoRI & SpeI |
| 4 | Pcon-pT181 attenuator | EcoRI & SpeI |
| 6 | Spinach-DT | XbaI & PstI |
| 7 | Spinach-DT | XbaI & PstI |
| 9 | RBS-lacZα-DT | XbaI & PstI |
| 10 | RBS-lacZα-DT | XbaI & PstI |

[![Igku 982b.png](/wiki/images/5/53/Igku_982b.png)](/File%3AIgku_982b.png)
[![Igku 982a.png](/wiki/images/f/fc/Igku_982a.png)](/File%3AIgku_982a.png)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-pT181 attenuator (EcoRI & SpeI) | 5.2 | 1.82 | 0.36 |
| Spinach-DT (XbaI & PstI) | 8.0 | 1.87 | 0.25 |
| RBS-lacZα-DT (XbaI & PstI) | 10.3 | 1.71 | 0.54 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder |  |
| 2 | Ptet-pT181 antisense | EcoRI & PstI |
| 3 | Ptet-pT181 antisense | EcoRI & PstI |
| 5 | pSB4K5 | EcoRI & SpeI |
| 6 | pSB4K5 | EcoRI & SpeI |

[![Igku 983b.png](/wiki/images/9/9b/Igku_983b.png)](/File%3AIgku_983b.png)
[![Igku 983a.png](/wiki/images/4/40/Igku_983a.png)](/File%3AIgku_983a.png)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Ptet-pT181 antisense(EcoRI & PstI) | 30.6 | 1.84 | 1.16 |
| pSB4K5 (EcoRI & SpeI) | 21.8 | 1.87 | 0.98 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder |  |
| 2 | Pcon-pT181 attenuator | SpeI & PstI |
| 3 | Pcon-pT181 attenuator | SpeI & PstI |
| 5 | Plac | SpeI & PstI |
| 6 | Plac | SpeI & PstI |
| 8 | pSB1C3 | EcoRI & SpeI |
| 9 | pSB1C3 | EcoRI & SpeI |
| 11 | pSB1C3 | XbaI & PstI |
| 12 | pSB1C3 | XbaI & PstI |

[![Igku 984b.png](/wiki/images/e/e9/Igku_984b.png)](/File%3AIgku_984b.png)
[![Igku 984a.png](/wiki/images/b/b6/Igku_984a.png)](/File%3AIgku_984a.png)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-pT181 attenuator (SpeI & PstI) | 14.5 | 1.91 | 1.09 |
| Plac (SpeI & PstI) | 11.6 | 1.74 | 1.19 |
| pSB1C3 (EcoRI & SpeI) | 5.7 | 1.74 | 0.34 |
| pSB1C3 (XbaI & PstI) | 5.6 | 2.00 | 0.37 |

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-pT181 anti | 243.0 | 1.90 | 1.81 |
| Pcon-Spinach-DT | 253.7 | 1.57 | 1.51 |

### Ligation

Kojima

| state | Vector(µL) | | Inserter(µL) | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/8 Plac SpeI+PstI | 4.3 | 9/3 pT181 attenuator | 3.1 | 3.5 |
| experiment | 9/8 Plac SpeI+PstI | 4.3 | 9/7 aptamer12-1R-DT XbaI+PstI | 2.1 | 3.2 |
| experiment | 9/8 Plac SpeI+PstI | 4.3 | 9/8 spinach-DT XbaI+PstI | 4.6 | 3.5 |
| experiment | 9/6 Plac SpeI+PstI | 2 | 9/7 RBS-lysis2-DT XbaI+PstI | 10 | 3.5 |
| experiment | 9/8 Plac SpeI+PstI | 4.3 | 9/7 RBS-lysis2-DT XbaI+PstI | 10 | 3.5 |
| experiment | 9/8 Ptet-pT181 anti | 1.6 | 9/8 spinach-DT XbaI+PstI | 4.3 | 3.0 |
| experiment | 9/6 Pcon SpeI+PstI | 1.4 | 9/8 RBS-laczα-DT XbaI+PstI | 4.8 | 3.1 |
| experiment | 9/8 Pcon-pT181 attenuator SpeI+PstI | 3.4 | 9/7 aptamer12-1R-DT XbaI+PstI | 1.9 | 2.7 |
| experiment | 9/6 Pcon SpeI+PstI | 1.4 | 9/7 aptamer12-1R-DT XbaI+PstI | 2.1 | 1.3 |
| experiment | 9/8 DT | 3.0 | 9/8 Pcon-pT181 attenuator EcoRI+SpeI | 7.9 | 3.5 |
| experiment | 9/6 Plux SpeI+PstI | 2 | 9/7 RBS-lysis1-DT XbaI+PstI | 7.2 | 3.5 |
| experiment | 9/8 Plac SpeI+PstI | 4.3 | 9/7 RBS-lysis1-DT XbaI+PstI | 7.2 | 3.5 |
| experiment | 9/4 Plac SpeI+PstI | 1.7 | 9/8 RBS-laczα-DT XbaI+PstI | 4.8 | 3.3 |

### Restriction Enzyme Digestion

Kojima

|  | 9/8 Pcon-pT181 anti | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 8.2µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 13.8µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 9/8 Pcon-spinach-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 7.9µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 14.1µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 8/21 F1 attenuator | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 6.5µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 15.5µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |

|  | 8/21 F3m2 attenuator | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 6.0µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 16µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |

|  | 8/21 F1 anti | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 6.7µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 15.3µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |

|  | 8/21 F6 anti | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 6.0µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 16µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |

|  | 8/21 tetR aptamer 12\_1M | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.3µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 16.7µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |

|  | 8/21 tetR aptamer 12\_P | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 13.3µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 8.7µL | 30µL |
| NC | 0.7µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.3µL | 10µL |

|  | 9/4 spinach-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.4µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 16.6µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |

|  | 9/7 RBS-lysis1-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 10µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 12µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |

|  | 9/6 RBS-lysis2-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 13µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 9µL | 30µL |
| NC | 0.7µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.3µL | 10µL |

|  | 9/7 RBS-lysis3-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 10µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 12µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |

|  | 8/20 J23100 RBS-lacZα-DT-(1) | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 7µL | 0.5µL | 0.5µL | 0µL | 0µL | 3µL | 3µL | 16µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |

|  | 8/20 RBS-lacZα-DT | EcoRI | SpeI | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 10µL | 0.5µL | 0.5µL | 0µL | 0µL | 3µL | 3µL | 13µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |

### Transformation

Nakamoto & Ashida

| Name | Sample(µL) | Competent Cells(µL) | Total(µL) | Plate |
| --- | --- | --- | --- | --- |
| 9/8 Pcont+aptamer 12-1R-DT | 1 | 10 | 11 | Amp |
| 9/8 Pcon+RBS-lacZα-DT | 1 | 10 | 11 | Amp |
| 9/8 Pcon-pT181 attenuator+aptamer 12-1R-DT | 1 | 10 | 11 | Amp |
| 9/8 Plac+RBS-lysis1-DT | 2 | 20 | 22 | CP |
| 9/8 Plac+RBS-lysis2-DT | 2 | 20 | 22 | CP |
| 9/8 Plac+aptamer 12-1R-DT | 2 | 20 | 22 | CP |
| 9/8 Plac+spinach | 2 | 20 | 22 | CP |
| 9/8 Plac+pT181 attenuator | 2 | 20 | 22 | CP |
| 9/8 Plux+RBS-lysis1-DT | 1 | 10 | 11 | CP |
| 9/8 Plux+RBS-lysis2-DT | 1 | 10 | 11 | CP |
| 9/8 Ptet+RBS-lacZα-DT | 1 | 10 | 11 | CP |
| 9/8 Ptet-pT181 anti+spinach-DT | 1 | 10 | 11 | CP |
| 9/8 Pcon-pT181 attenuator+DT | 1 | 10 | 11 | CP |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder |  |  |
| 2 | Pcon-spinach-DT | EcoRI | SpeI |
| 3 | Pcon-spinach-DT |  |  |
| 4 | Fusion1 attenuator | XbaI | PstI |
| 5 | Fusion1 attenuator |  |  |
| 6 | Fusion3m2 attenuator | XbaI | PstI |
| 7 | Fusion3m2 attenuator |  |  |
| 8 | Fusion1 anti | XbaI | PstI |
| 9 | Fusion1 anti |  |  |
| 10 | Fusion6 anti | XbaI | PstI |
| 11 | Fusion6 anti |  |  |
| 12 | aptamer 12-1M | XbaI | PstI |
| 13 | aptamer 12-1M |  |  |
| 14 | aptamer 12-P | XbaI | PstI |
| 15 | aptamer 12-P |  |  |
| 16 | spinach-DT | XbaI | PstI |

[![Igku 985.png](/wiki/images/d/d9/Igku_985.png)](/File%3AIgku_985.png)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | spinach-DT |  |  |
| 2 | J23100-RBS-lacZα-DT | EcoRI | SpeI |
| 3 | J23100-RBS-lacZα-DT |  |  |
| 4 | 100bp ladder |  |  |
| 5 | RBS-lacZα-DT | EcoRI | SpeI |
| 6 | RBS-lacZα-DT |  |  |
| 7 | RBS-lysis1-DT | XbaI | PstI |
| 8 | RBS-lysis1-DT |  |  |

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder |  |  |
| 2 | RBS-lysis2-DT | XbaI | PstI |
| 3 | RBS-lysis2-DT |  |  |
| 4 | RBS-lysis3-DT | XbaI | PstI |
| 5 | RBS-lysis3-DT |  |  |
| 6 | Pcon pT181 anti | SpeI | PstI |
| 7 | Pcon pT181 anti |  |  |

[![Igku 986.png](/wiki/images/4/44/Igku_986.png)](/File%3AIgku_986.png)

### Gel Extraction

Ashida and Nakamoto

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 2 | Pcon-spinach-DT | EcoRI+SpeI |
| 3 | Pcon-Spinach-DT | EcoRI+SpeI |
| 5 | Fusion 1 attenuator | XbaI+PstI |
| 6 | Fusion 1 attenuator | XbaI+PstI |
| 8 | Fusion3m2 attenuator | XbaI+PstI |
| 9 | Fusion3m2 attenuator | XbaI+PstI |
| 11 | Fusion 1 antisense | XbaI+PstI |
| 12 | Fusion 1 antisense | XbaI+PstI |

[![Igku 987.png](/wiki/images/e/e5/Igku_987.png)](/File%3AIgku_987.png)

| Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- | --- |
| Pcon-spinach-DT(EcoRI+SpeI) |  | 10.5 | 1.86 | 0.39 |
| Fusion 1 attenuator(XbaI+PstI) |  | 7.0 | 1.71 | 0.27 |
| Fusion3m2 attenuator(XbaI+PstI) |  | 8.6 | 1.75 | 0.47 |
| Fusion 1 antisense(XbaI+PstI) |  | 7.6 | 1.87 | 0.43 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 2 | Fusion6 antisense | XbaI+PstI |
| 3 | Fusion6 antisense | XbaI+PstI |
| 5 | aptamer12\_1M | XbaI+PstI |
| 6 | aptamer12\_1M | XbaI+PstI |

[![Igku 988b.png](/wiki/images/5/58/Igku_988b.png)](/File%3AIgku_988b.png)
[![Igku 988a.png](/wiki/images/7/79/Igku_988a.png)](/File%3AIgku_988a.png)

| Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- | --- |
| Fusion6 antisense(XbaI+PstI) |  | 5.0 | 1.76 | 0.20 |
| aptamer12\_1M(XbaI+PstI) |  | 3.3 | 2.31 | 0.04 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 2 | aptamer12\_P | XbaI+PstI |
| 3 | aptamer12\_P | XbaI+PstI |
| 5 | spinach-DT | EcoRI+SpeI |
| 6 | spinach-DT | EcoRI+SpeI |

[![Igku 989b.png](/wiki/images/a/a8/Igku_989b.png)](/File%3AIgku_989b.png)
[![Igku 989a.png](/wiki/images/3/3c/Igku_989a.png)](/File%3AIgku_989a.png)

| Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- | --- |
| aptamer12\_P(XbaI+PstI) |  | 3.4 | 1.72 | 0.26 |
| spinach-DT(EcoRI+SpeI) |  | 10.1 | 1.57 | 0.47 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 2 | J23100-RBS-laczα-DT | EcoRI+SpeI |
| 3 | J23100-RBS-laczα-DT | EcoRI+SpeI |
| 5 | RBS-laczα-DT | EcoRI+SpeI |
| 6 | RBS-laczα-DT | EcoRI+SpeI |
| 8 | RBS-lysis1-DT | XbaI+PstI |
| 9 | RBS-lysis1-DT | XbaI+PstI |
| 11 | RBS-lysis2-DT | XbaI+PstI |
| 12 | RBS-lysis2-DT | XbaI+PstI |

[![Igku 9810b.png](/wiki/images/7/72/Igku_9810b.png)](/File%3AIgku_9810b.png)
[![Igku 9810a.png](/wiki/images/f/fa/Igku_9810a.png)](/File%3AIgku_9810a.png)

| Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- | --- |
| Pcon-RBS-laczα-DT(EcoRI+SpeI) |  | 5.6 | 2.03 | 0.25 |
| RBS-laczα-DT(EcoRI+SpeI) |  | 4.4 | 2.04 | 0.33 |
| RBS-lysis1-DT(XbaI+PstI) |  | 5.9 | 2.22 | 0.53 |
| RBS-lysis2-DT(XbaI+PstI) |  | 5.1 | 1.67 | 0.48 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 2 | RBS-lysis3-DT | XbaI+PstI |
| 3 | RBS-lysis3-DT | XbaI+PstI |
| 5 | Pcon-pT181 antisense | SpeI+PstI |
| 6 | Pcon-pT181 antisense | SpeI+PstI |

[![Igku 9811b.png](/wiki/images/8/8c/Igku_9811b.png)](/File%3AIgku_9811b.png)
[![Igku 9811a.png](/wiki/images/f/f4/Igku_9811a.png)](/File%3AIgku_9811a.png)

| Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- | --- |
| RBS-lysis3-DT(XbaI+PstI) |  | 23.4 | 1.72 | 0.88 |
| Pcon-pT181 antisense(SpeI+PstI) |  | 31.3 | 1.86 | 0.92 |

### Ligation

No name

| state | Vector | | Inserter | |
| --- | --- | --- | --- | --- |
| experiment | Pcon-spinach-DT(EcoRI&SpeI) | 10.5µg/mL | pSB6A1(EcoRI&SpeI) | 8.9µg/mL |
| experiment | Pcon-spinach-DT(EcoRI&SpeI) | 10.5µg/mL | pSB4K5(EcoRI&SpeI) | 21.8µg/mL |
| experiment | Pcon-pT181 anti(SpeI+PstI) | 31.3µg/mL | spinach-DT(XbaI+PstI) | 10.1µg/mL |
| experiment | Plac(SpeI+PstI) | 11.6µg/mL | RBS-lysis3-DT | 23.4µg/mL |
| experiment | Plux(SpeI+PstI) | 25µg/mL | RBS-lysis3-DT | 23.4µg/mL |
| experiment | Ptet-pT181 anti(SpeI+PstI) | 30.6µg/mL | spinach-DT(XbaI+PstI) | 10.1µg/mL |
| experiment | Pcon-pT181 attenuator(SpeI+PstI) | 14.5µg/mL | RBS-laczα-DT(XbaI+PstI) | 4.4µg/mL |

### PCR

No name

| KaiA(plasmid) | KOD plus | 10x buffer | dNTP | MgSO4 | Primer(661-RBS-KaiA-fwd) | Primer(kaiA-662-rev-ver2) | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 28s | 30cycles |

| KaiB(plasmid) | KOD plus | 10x buffer | dNTP | MgSO4 | Primer(662-RBS-KaiB-fwd-ver2) | Primer(kaiB-663-rev-ver2) | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 28s | 30cycles |

| DT(plasmid) | KOD plus | 10x buffer | dNTP | MgSO4 | Primer(664-DT-fwd-ver2) | Primer(DT-suffix-660-rev-ver2) | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.1 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.4 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 28s | 30cycles |

| P-KaiB(PCR prpduct) | KOD plus | 10x buffer | dNTP | MgSO4 | Primer(prefix-PkaiBC-fwd-ver2) | Primer(PkaiBC-suffix--662-rev-ver2) | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.7 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.8 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 28s | 30cycles |

| Plac(1)(plasmid) | KOD plus | 10x buffer | dNTP | MgSO4 | Primer(GG0-pSBIC3-fwd-ver2) | Primer(Plac-GG1-rev-ver2) | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.13 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.37 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 60s | 30cycles |

| KaiC(plasmid) | KOD plus | 10x buffer | dNTP | MgSO4 | Primer(GG3-RBS-KaiC-fwd-ver2) | Primer(KaiC-GG4-rev-ver2) | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 60s | 30cycles |

### Gel Extraction

Yoshida

| Lane | DNA |
| --- | --- |
| 1 | 100bp ladder |
| 2 | KaiA |
| 3 | KaiA |
| 4 | KaiB |
| 5 | KaiB |
| 6 | DT |
| 7 | DT |
| 8 | P-KaiBC |
| 9 | P-KaiBC |
| 10 | nega |
| 11 | 100bp ladder |

[![9898.jpg](/wiki/images/e/eb/9898.jpg)](/File%3A9898.jpg)
[![9897.jpg](/wiki/images/0/05/9897.jpg)](/File%3A9897.jpg)

| Lane | DNA |
| --- | --- |
| 1 | Plac |
| 2 | Plac |
| 3 | KaiC |
| 4 | KaiC |
| 5 | nega |
| 6 | 1kb ladder |

[![9899.jpg](/wiki/images/2/2c/9899.jpg)](/File%3A9899.jpg)
[![991.jpg](/wiki/images/d/dc/991.jpg)](/File%3A991.jpg)

## Sep 9

### PCR

Nakagawa

| KaiA(plasmid) | 10x buffer | dNTPs | MgSO4 | Primer(661-RBS-KaiA-fwd) | Primer(kaiA-662-rev-ver2) | Kod-plus ver2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1.0 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 58.5°C | 68°C | -- |
| 2min | 10s | 30s | 30s | 30cycles |

| KaiB(plasmid) | 10x buffer | dNTPs | MgSO4 | Primer(661-RBS-KaiA-fwd-ver2) | Primer(kaiB-663-rev-ver2) | Kod-plus ver2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1.0 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 58.5°C | 68°C | -- |
| 2min | 10s | 30s | 30s | 30cycles |

| P-KaiBC(PCR products) | 10x buffer | dNTPs | MgSO4 | Primer(prefix-P-kaiBC-fwd-ver2) | Primer(P-KaiBC-suffix-rev-verw) | Kod-plus ver2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.7 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.8 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 58.5°C | 68°C | -- |
| 2min | 10s | 30s | 30s | 30cycles |

| DT(plasmid) | 10x buffer | dNTPs | MgSO4 | Primer(664-DT-fwd-ver2) | Primer(DT-suffix-660-rev-ver2) | Kod-plus ver2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1.0 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 58.5°C | 68°C | -- |
| 2min | 10s | 30s | 30s | 30cycles |

### Gel Extraction

Yoshida

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | KaiA | -- |
| 3 |
| 4 | KaiB | -- |
| 5 |
| 6 | P-KaiBC | -- |
| 7 |
| 8 | DT | -- |
| 9 |
| 10 | NC | -- |
| 11 | 100bp ladder | -- |

[![Igku 992.jpg](/wiki/images/3/38/Igku_992.jpg)](/File%3AIgku_992.jpg)
[![Igku 993.jpg](/wiki/images/3/34/Igku_993.jpg)](/File%3AIgku_993.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| KaiC | 28.5 | 1.26 | 11.92 |
| Plac | 53.9 | 1.82 | 1.61 |
| KaiB | 59.6 | 1.61 | 2.20 |
| KaiB | 76.5 | 1.86 | 1.44 |
| DT | 98.7 | 1.47 | 1.43 |

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/8 pSB1C3 | 83.2 | 1.45 | 0.76 |
| 9/8 RBS-lysis2-DT | 72.8 | 1.67 | 0.97 |
| 9/8 spinach-DT | 152.6 | 1.88 | 1.97 |
| Nuclease-Free water | -0.4 | -14.30 | 0.25 |

### Restriction Enzyme Digestion

No name

|  | 9/9 pSB1C3 | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 24µL | 1µL | 1µL | 3µL | 3µL | 22µL | 30µL\* |
| NC | 1.2µL | 0µL | 0µL | 1µL | 1µL | 6.8 µL | 10µL |

|  | 9/9 pSB1C3 | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 27.4µL | 1µL | 1µL | 3µL | 3µL | 22µL | 30µL\* |
| NC | 1.3µL | 0µL | 0µL | 1µL | 1µL | 6.7µL | 10µL |

|  | 9/9 spinach-DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 10.8µL | 1µL | 1µL | 3µL | 3µL | 22µL | 30µL\* |
| NC | 0.5µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |

* "\*"=be evaporated

### Transformation

No name

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| Pcon+RBS-luxI-DT | 2µL | 20µL | 22µL | Amp |
| Pcon-RBS-luxR-DT+Plux-GFP | 2µL | 20µL | 22µL |  |
| Ptet-PT181 antisense+spinach-DT-Pbad/araC+RBS-lux-DT | 2µL | 20µL | 22µL |  |

### Ligation

No Name

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/8 F1-attenuator(XbaI&PstI) | 5.3µg | 9/8 pSB1C3(XbaI&PstI) | 8.9µg | 3.5µg |
| experiment | 9/8 F3m2-attenuator(XbaI&PstI) | 4.5µg | 9/8 pSB1C3(XbaI&PstI) | 8.9µg | 3.5µg |
| experiment | 9/8 F1-antisense(XbaI&PstI) | 1.7µg | 9/8 pSB1C3(XbaI&PstI) | 8.9µg | 3.5µg |
| experiment | 9/8 F6-antisense(XbaI&PstI) | 2.9µg | 9/8 pSB1C3(XbaI&PstI) | 8.9µg | 3.5µg |
| experiment | 9/8 aptamer12-P(EcoRI&SpeI) | 3.1µg | 9/8 pSB1C3(EcoRI&SpeI) | 8.8µg | 3.5µg |
| experiment | 9/8 aptamer12-1M(EcoRI&SpeI) | 3.1µg | 9/8 pSB1C(EcoRI&SpeI)3 | 8.8µg | 3.5µg |

### M9 medium

Shimizu & Hirano

| EDTA consenlation | Na2HPO4 | KH2PO4 | NaCl | NH4Cl | Fe(Ⅲ)-EDTA | distilled water | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1mMol | 300mg | 150mg | 25mg | 50mg | 21mg | up to 10mL | 10mL |
| 10mMol | 300mg | 150mg | 25mg | 50mg | 210.5mg | up to 10mL | 10mL |
| 50mMol | 300mg | 150mg | 25mg | 50mg | 1052.7mg | up to 10mL | 10mL |

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 9/8 Plac-spinach-DT-(1) | 659 |
| 9/8 Plac-spinach-DT-(2) | 659 |
| 9/8 Pcon-pt181attenuator-DT-(1) | 584 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 40s | 30cycles |

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | Spinach-DT | XbaI+PstI |
| 3 |
| 5 | RBS-lacZα-DT | XbaI+PstI |
| 6 |

[![Igku 995.jpg](/wiki/images/3/3f/Igku_995.jpg)](/File%3AIgku_995.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 3 | pSB1C3 | XbaI+PstI |
| 4 |

[![Igku 996.jpg](/wiki/images/5/57/Igku_996.jpg)](/File%3AIgku_996.jpg)

### Ligation

No name

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | pSB1C3(XbaI+PstI) | xx µL | 9/8 F1-attenuator(XbaI+PstI) | xx µL | xx µL |
| experiment | pSB1C3(XbaI+PstI) | xx µL | 9/8 F3m2-attenuator(XbaI+PstI) | xx µL | xx µL |

### PCR

Tatsui and Yoshida

| KaiC | 10x buffer KOD plus ver2 | 2M dNTP | MgSO4 | Primer Mix(F&R) | Kod-plus ver2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 0.5 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16.0 | 25.0 |
| 1.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.5 | 25.0 |
| 2.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.5 | 25.0 |
| 4.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 12.5 | 25.0 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 2min | 30cycles |

| Plac(100pg) | 10x buffer KOD plus ver2 | 2M dNTP | MgSO4 | Primer Mix(F&R) | Kod-plus ver2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 0.3 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16.2 | 25.0 |
| 0.6 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.9 | 25.0 |
| 1.2 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.3 | 25.0 |
| 2.4 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.1 | 25.0 |
| 0.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16.5 | 25.0 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 2min | 30cycles |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| pSB2C3(master)-2 | Plusgrow medium(+CP) |

### Restriction Enzyme Digestion

No name

|  | 8/17 lysis1-2 | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 8.1µL | 1µL | 1µL | 3µL | 3µL | 13.9µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 8/17 lysis3-1 | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 6.1µL | 1µL | 1µL | 3µL | 3µL | 15.9µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |

|  | 9/15 Plac | XbaI | PstI | BSA | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 cuts | 12.1µL | 0µL | 1µL | 3µL | 3µL | 10.9µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |

### PCR

Yoshida

| KaiA(KaiABC) 100pg/µL | 10x buffer KOD plus ver2 | dNTPs | MgSO4 | Primer Mix(Fwd&Rev) | KOD plus ver2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.5 | 25 |
| 4.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 12.5 | 25 |
| 8.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 8.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 60°C | 68°C | -- |
| 2min | 10s | 30s | 1min | 30cycles |

| PKaiBC 400pg/µL | 10x buffer KOD plus ver2 | dNTPs | MgSO4 | Primer Mix(Fwd&Rev) | KOD plus ver2 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 0.5 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16.0 | 25 |
| 1.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.5 | 25 |
| 2.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 60°C | 68°C | -- |
| 2m | 10s | 30s | 1m | 30cycles |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 9/2 Spinach-DT | Plusgrow medium(+CP) |
| 8/29 Plux-RBS-GFP-DT | Plusgrow medium |
| 9/6 master plate | Plusgrow medium(+Amp) |
| 3 Pcon-PT181 attenuator | Plusgrow medium |

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 3 | lysis1 | XbaI+PstI |
| 4 |
| 5 |
| 6 |

[![Igku 9910.jpg](/wiki/images/2/2e/Igku_9910.jpg)](/File%3AIgku_9910.jpg)
[![Igku 9911.jpg](/wiki/images/d/df/Igku_9911.jpg)](/File%3AIgku_9911.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 3 | lysis3 | XbaI+PstI |
| 4 |
| 5 |
| 6 |

[![Igku 9912.jpg](/wiki/images/8/82/Igku_9912.jpg)](/File%3AIgku_9912.jpg)
[![Igku 9913.jpg](/wiki/images/f/f9/Igku_9913.jpg)](/File%3AIgku_9913.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| （´ω｀） |  |  |  |
| （ΦωΦ^） |  |  |  |

### Electrophoresis

Yoshida

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 1kb ladder | -- | -- |
| 2 | Plac 100pg | -- | -- |
| 3 | Plac 200pg | -- | -- |
| 4 | Plac 400pg | -- | -- |
| 5 | Plac 800pg | -- | -- |
| 6 | NC | -- | -- |
| 7 | 1kbp ladder | -- | -- |

[![Igku 9914.jpg](/wiki/images/d/dc/Igku_9914.jpg)](/File%3AIgku_9914.jpg)

## Sep 10

### Ligation

Nakamoto

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/6 Pcon-RBS-luxR-DT | 1.9µL | 9/6Plux-RBS-GFP-DT | 22 µL | 3.5µL |
| experiment | 9/6 Plux | 2µL | 9/8 RBS-lysis1-DT | 6.6µL | 3.5µL |
| experiment | 9/6 Plux | 2µL | 9/8 RBS-lysis2-DT | 6.3µL | 3.5µL |
| experiment | 9/8 Ptet-PT181 antisense | 1.6µL | 9/8 spinach-DT | 4.4µL | 3.0µL |
| experiment | 9/8 Pcon-PT181 attenuator | 3.4µL | 9/8 apz 12-1R-PT | 2.1µL | 2.8µL |
| experiment | 9/8 Pcon | 3.4µL | 9/8 apz 12-1R-PT | 2.3µL | 2,9µL |
| experiment | 9/8 DT | 3.0µL | 9/8 Pcon-PT181 attenuator | 7.9µL | 3.5µL |
| experiment | 9/10 pSB1C3 | 1.8µL | 9/8 F3m2 attenuator | 4.6µL | 3.2µL |
| experiment | 9/10 pSB1C3 | 1.8µL | 9/8 F1 attenuator | 3.4µL | 2.6µL |

### Miniprep

Nakamoto

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| spinach-DT | 202.4 | 1.97 | 1.93 |
| Plux-RBS-GFP-DT | 164.2 | 2.80 | 2.06 |
| Pcon-DT attenuator | 205.4 | 1.81 | 2.39 |
| RSBJC3-2 | 240.8 | 1.73 | 1.43 |
| Plaz-1 | 240.8 | 1.73 | 1.43 |

### Restriction Enzyme Digestion

Nakamoto

|  | 9/10　Plux-RBS-GFP-DT | EcoRI | SpeI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 12.1µL | 1µL | 1µL | 3µL | 3µL | 9.9µL | 30µL |
| NC | 0.6µL | 0µL | 0µL | 1µL | 1µL | 7.4µL | 10µL |

|  | 9/10　Spinach-DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 9.9µL | 1µL | 1µL | 3µL | 3µL | 12.1µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |

|  | 9/6 apt12-1R -DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 8.0µL | 1µL | 1µL | 3µL | 3µL | 14.0µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 9/10　Pcon-pt81 attenuator | EcoRI | SpeI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 9.8µL | 1µL | 1µL | 3µL | 3µL | 12.2µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | Plux-RBS-GEP-DT | EcoRI | SpeI |
| 3 | Plux-RBS-GEP-DT | -- | -- |
| 4 | Spinach-DT | XbaI | PstI |
| 5 | Spinach-DT | -- | -- |
| 6 | aptamer 121R-DT | XbaI | PstI |
| 7 | aptamer 121R-DT | -- | -- |
| 8 | Pcon-pt181 attenuator | EcoRI | SpeI |
| 9 | Pcon-pt181 attenuator | -- | -- |
| 10 | 100bp ladder | -- | -- |

[![Igku 9104.jpg](/wiki/images/9/9c/Igku_9104.jpg)](/File%3AIgku_9104.jpg)

### Colony PCR

Tatsui

| Sample | base pair |
| --- | --- |
| 9/9 Pcon-pT181 antisense | -- |
| 9/8 J223100(entA) | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min10sec | 30 |

### Electrophoresis

Tatsui

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Pcon Pt181 antisense1+spinach-DT |
| 3 | Pcon Pt181 antisense2+spinach-DT |
| 4 | Pcon Pt181 antisense3+spinach-DT |
| 5 | Pcon Pt181 antisense4+spinach-DT |
| 6 | Pcon Pt181 antisense5+spinach-DT |
| 7 | Pcon Pt181 antisense6+spinach-DT |
| 8 | Pcon Pt181 antisense7+spinach-DT |
| 9 | Pcon Pt181 antisense8+spinach-DT |
| 10 | Pcon Pt181 antisense9+spinach-DT |
| 11 | Pcon Pt181 antisense10+spinach-DT |
| 12 | Pcon Pt181 antisense11+spinach-DT |
| 13 | Pcon Pt181 antisense12+spinach-DT |
| 14 | 100bp ladder |

[![Igku 0910 E2.jpg](/wiki/images/8/81/Igku_0910_E2.jpg)](/File%3AIgku_0910_E2.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 1kbp ladder |
| 2 | Pcon entA1 |
| 3 | Pcon entA2 |
| 4 | Pcon entA3 |
| 5 | 1kbp ladder |

[![Igku 0910 E3.jpg](/wiki/images/a/aa/Igku_0910_E3.jpg)](/File%3AIgku_0910_E3.jpg)

### Gel Extraction

Tatsui

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 2 | KaiA(200pg) | -- |
| 3 | KaiA(200pg) | -- |
| 4 | -- | -- |
| 5 | KaiA(400pg) | -- |
| 6 | KaiA(400pg) | -- |
| 7 | -- | -- |
| 8 | KaiA(800pg | -- |
| 9 | KaiA(800pg) | -- |
| 10 | 1kbp ladder | -- |

[![Igku 9101.jpg](/wiki/images/9/99/Igku_9101.jpg)](/File%3AIgku_9101.jpg)
[![Igku 9102.jpg](/wiki/images/3/35/Igku_9102.jpg)](/File%3AIgku_9102.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 2 | PKaiBC(200pg) | -- |
| 3 | PKaiBC(200pg) | -- |
| 4 | -- | -- |
| 5 | PKaiBC(400pg) | -- |
| 6 | PKaiBC(400pg) | -- |
| 7 | -- | -- |
| 8 | PKaiBC(800pg | -- |
| 9 | PKaiBC(800pg) | -- |
| 10 | 1kbp ladder | -- |

[![Igku 0910 G3.jpg](/wiki/images/f/ff/Igku_0910_G3.jpg)](/File%3AIgku_0910_G3.jpg)

### Liquid Culture

Hirano

| Sample | medium |
| --- | --- |
| 9/4 J23100①(JM109) | LB(Amp) |
| 9/8 J23100① (entA) | LB(Amp) |

### Gel Extraction

Tatsui

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 2 | KaiC | -- |
| 3 | KaiC | -- |
| 4 | KaiC | -- |
| 5 | -- | -- |
| 6 | 1kbp ladder | -- |
| 7 | 1kbp ladder | -- |
| 8 | KaiC | -- |
| 9 | KaiC | -- |
| 10 | KaiC | -- |
| 11 | -- | -- |
| 12 | 1kbp ladder | -- |

[![Igku 9105.jpg](/wiki/images/b/b6/Igku_9105.jpg)](/File%3AIgku_9105.jpg)
[![Igku 9106.jpg](/wiki/images/5/52/Igku_9106.jpg)](/File%3AIgku_9106.jpg)

### Liquid Culture

Tatsui

| Sample | medium |
| --- | --- |
| 9/10 Pcon-pt181 antisense-Spinach-DT | Plusgrow |

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 9/9 Phad larac-1 RBS-lux1-DT | 2331 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 2min | 30 |

| Sample | base pair |
| --- | --- |
| 9/9 Pcon-lacZα (psB4KS) | 712 |
| 9/9 Pcon-Spinach-DT(psB4KS) | 605 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 42s | 30 |

| Sample | base pair |
| --- | --- |
| 9/9 Plac+RBS-lysis3}-DT | 1273 |
| 9/9 Pcon+RBS-lux1-DT | 1079 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min12s | 30 |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | 9/9 Pcou-RBS-lacZα-DT(p3B4KS9)-1 | -- | -- |
| 3 | 9/9 Pcou-RBS-lacZα-DT(p3B4KS9)-2 | -- | -- |
| 4 | 9/9 Pcou-Spinach-DT(pB4KS)-1 | -- | -- |
| 5 | 9/9 Pcou-Spinach-DT(pB4KS)-2 | -- | -- |
| 6 | 100bp ladder | -- | -- |

[![Igku 91014.jpg](/wiki/images/8/86/Igku_91014.jpg)](/File%3AIgku_91014.jpg)

### Transformation

Nakamoto

| Name | Plate |
| --- | --- |
| F1 attenuator +p3B1C3 | CP |
| F3n2 attenuator +p3B1C3 | CP |
| F1 antisense +p3B1C3 | CP |
| F6 antisense+p3B1C3 | CP |
| apt12-P +p3B1C3 | CP |
| apt12-1M +p3B1C3 | CP |
| Pcon-luxR+Plux-GFP | Amp |
| Plux+RBS-lysis1-DT | CP |
| Plux+RBS-lysis2-DT | CP |
| Ptet-pT181antisense+Spinach-DT | CP |
| Pcon-pT181 attenuator+apt121R-DT | Amp |
| Pcon-1 apt12-1R-DT | Amp |
| DT+Pcon-pT181 attenuator | CP |

### Electrophoresis

Okazaki

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | Spinach-DT | XbaI | PstI |
| 3 | Spinach-DT | XbaI | PstI |
| 4 | Spinach-DT | XbaI | PstI |
| 5 | Spinach-DT | XbaI | PstI |
| 6 | 100bp ladder | -- | -- |

[![Igku 9108.jpg](/wiki/images/7/7a/Igku_9108.jpg)](/File%3AIgku_9108.jpg)
[![Igku 9109.jpg](/wiki/images/2/26/Igku_9109.jpg)](/File%3AIgku_9109.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | Pcon-pT181 attenuator | EcoRI | SpeI |
| 3 | Pcon-pT181 attenuator | EcoRI | SpeI |
| 4 | Pcon-pT181 attenuator | EcoRI | Spei |
| 5 | Pcon-pT181 attenuator | EcoRI | SpeI |
| 6 | 100bp ladder | -- | -- |

[![Igku 91010.jpg](/wiki/images/e/e0/Igku_91010.jpg)](/File%3AIgku_91010.jpg)
[![Igku 91011.jpg](/wiki/images/3/39/Igku_91011.jpg)](/File%3AIgku_91011.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | Plux-RBS-GFP-DT | EcoRI | SpeI |
| 2 | Plux-RBS-GFP-DT | EcoRI | SpeI |
| 3 | Plux-RBS-GFP-DT | EcoRI | SpeI |
| 4 | Plux-RBS-GFP-DT | EcoRI | SpeI |
| 5 | 100bp ladder | -- | -- |
| 6 | apt12-1R-DT | XbaI | PstI |
| 7 | apt12-1R-DT | XbaI | PstI |
| 8 | apt12-1R-DT | XbaI | PstI |
| 9 | apt12-1R-DT | XbaI | PstI |

[![Igku 91012.jpg](/wiki/images/c/c6/Igku_91012.jpg)](/File%3AIgku_91012.jpg)
[![Igku 91013.jpg](/wiki/images/a/a1/Igku_91013.jpg)](/File%3AIgku_91013.jpg)

## Sep 11

### Electrophoresis

| Lane | Sample |
| --- | --- |
| 1 | 1kb ladder |
| 2 | Pbad/araC+RBS-luxI-DT-1 |
| 3 | Pbad/araC+RBS-luxI-DT-1 |
| 4 | Plac+RBS-lysis-DT |
| 5 | Pcon+RBS-luxI-DT |
| 6 | 1kb ladder |

[![Igku 9111.jpg](/wiki/images/9/94/Igku_9111.jpg)](/File%3AIgku_9111.jpg)

### PCR

Tatsui

| pKaiBC(200p) | 10\*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 0.5 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 60°C | 68°C | 30 |
| 2min | 10sec | 30sec | 30sec |  |

| pKaiBC(400p) | 10\*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 60°C | 68°C | 30 |
| 2min | 10sec | 30sec | 30sec |  |

| pKaiBC(800p) | 10\*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 60°C | 68°C | 30 |
| 2min | 10sec | 30sec | 30sec |  |

| pKaiBC(1ng) | 10\*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 60°C | 68°C | 30 |
| 2min | 10sec | 30sec | 30sec |  |

### Electrophoresis

Tatsui

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | pKaiBC 800pg 60°C |
| 3 | pKaiBC 1ng 60°C |
| 4 | pKaiBC 800pg 61°C |
| 5 | pKaiBC 1ng 61°C |
| 6 | pKaiBC 800pg 62°C |
| 7 | pKaiBC 1ng 62°C |
| 8 | 100bp ladder |

[![Igku 9112.jpg](/wiki/images/8/89/Igku_9112.jpg)](/File%3AIgku_9112.jpg)

### colony PCR

Tatsui

| Sample | base pair |
| --- | --- |
| 9/9Pcon-lacZα(p3B4k5) | 712bp |
| 9/9Pcon-spinach-DT(p3B4k5) | 605bp |

Tatsui

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 42s | 30 |

| Sample | base pair |
| --- | --- |
| Pbad\araC-RBS-luxI-DT |  |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 2min | 30 |

### Electrophoresis

Tatsui

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Pcon-lacZα(pSB4K5)3 |
| 3 | Pcon-lacZα(pSB4K5)4 |
| 4 | Pcon-lacZα(pSB4K5)5 |
| 5 | Pcon-lacZα(pSB4K5)6 |
| 6 | Pcon-spinach-DT(pSB4K5)3 |
| 7 | Pcon-spinach-DT(pSB4K5)4 |
| 8 | Pcon-spinach-DT(pSB4K5)5 |
| 9 | Pcon-spinach-DT(pSB4K5)6 |
| 10 | 100bp ladder |

[![Igku 9113.jpg](/wiki/images/a/ab/Igku_9113.jpg)](/File%3AIgku_9113.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 1kbp ladder |
| 2 | Pbad/araC-RBS-luxI-DT3 |
| 3 | Pbad/araC-RBS-luxI-DT4 |
| 4 | Pbad/araC-RBS-luxI-DT5 |
| 5 | Pbad/araC-RBS-luxI-DT6 |
| 6 | 1kbp ladder |

[![Igku 9114.jpg](/wiki/images/0/07/Igku_9114.jpg)](/File%3AIgku_9114.jpg)

### colony PCR

| Sample | base pair |
| --- | --- |
| 9/9Pcon+RBS-luxI-DT | 1079bp |
| 9/10Pcon+sptamer12\_1R-DT | 584bp |
| 9/10DT+Pcon-pT181 attenuator 1 | 781bp |
| 9/10Fusion1 attenuator(p3B1C3)-1 | 638bp |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 50sec | 30sec | 30sec | 1min | 30 |

### Gel Extraction

[![Igku 9115.jpg](/wiki/images/3/33/Igku_9115.jpg)](/File%3AIgku_9115.jpg)
[![Igku 9116.jpg](/wiki/images/a/a1/Igku_9116.jpg)](/File%3AIgku_9116.jpg)
[![Igku 9117.jpg](/wiki/images/4/4c/Igku_9117.jpg)](/File%3AIgku_9117.jpg)
[![Igku 9118.jpg](/wiki/images/8/8e/Igku_9118.jpg)](/File%3AIgku_9118.jpg)

### Ligation

Okazaki

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/8pSB4K5(EcoRI&SpeI)21.8ng/µL | 2.3 µL | 9/8Pcon-Spinach-DT(EcoRI&SpeI)10.5ng/µL | 1.7 µL | 2.0 µL |
| experment | 9/8pSB4K5(EcoRI&SpeI)21.8ng/µL | 2.3 µL | 9/8RBS-lacZα-DT(EcoRI&SpeI)4.4ng/µL | 6.7µL | 3.5 µL |
| experiment | 9/8pSB4K5(EcoRI&SpeI)21.8ng/µL | 2.3 µL | 9/8Pcon-RBS-lacZα-DT(EcoRI&SpeI)5.6ng/µL | 1.9 µL | 2.1 µL |
| experiment | 9/6Plux 25ng/µL | 2 µL | 9/8 RBS-lysis1-DT(XbaI&PstI) | 6.6 µL | 3.5 µL |
| experiment | 9/6Plux 25ng/µL | 2 µL | 9/8 RBS-lysis3-DT(XbaI&PstI) | 4.7 µL | 3.4 µL |
| experiment | 9/10Pcon-pT181 attenuator(SpeI&PstI) | 3.4µL | 9/8RBS-lacZα-DT(EcoRI&SpeI) | 4.5 µL | 3.5 µL |
| experiment | 9/6Pcon-RBS laxR-DT(EcoRI&XbaI) 26.7ng/µL | 1.9 µL | Plax-RBS-GFP-DT | 5.5 µL | 3.5 µL |
| experiment | 9/9 Pcon-pT181 attenuator(SpeI&PstI) | 3.4 µL | 9/10 aptamer12\_1R-DT(XbaI&PstI) | 1.7 µL | 2.6 µL |

### Transformation

Nakamoto Tatsui

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| 9/9 Fusion1 antisense(pSB2C3) | 2µL | 20µL | 22µL | CP |
| 9/9 Fusion6 antisense(pSB2C3) | 2µL | 20µL | 22µL | CP |
| 9/9 aptamer 12\_P(pSB2C3) | 2µL | 20µL | 22µL | CP |
| 9/9 aptamer 12\_1&micro(pSB2C3) | 2µL | 20µL | 22µL | CP |
| 9/9 Fusion2 attenuator(pSB2C3) | 2µL | 20µL | 22µL | CP |
| 9/11 Pcon-spinach-DT(pSB4K5) | 2µL | 20µL | 22µL | K |
| 9/11 RBS-lacZα-DT(pSB4K5) | 2µL | 20µL | 22µL | K |
| 9/11 Pcon-RBS-lacZα-DT(pSB4K5) | 2µL | 20µL | 22µL | K |
| Plux+RBS-lysis1-DT | 2µL | 20µL | 22µL | CP |
| Plux+RBS-lysis3-DT | 2µL | 20µL | 22µL | CP |
| Pcon-pT181attenuator+RBS-lacZα-DT | 2µL | 20µL | 22µL | Amp |
| Pcon-iuxR+Plux-GFP | 2µL | 20µL | 22µL | Amp |
| Pcon-pT181attenuator+apt12\_1R-DT | 2µL | 20µL | 22µL | Amp |
| Plac(BBa\_R0011) | 2µL | 20µL | 22µL | Amp |  |

### Electrophoresis

Tatsui

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Pcon+RBS luxI-DT2 |
| 3 | Pcon+RBS luxI-DT3 |
| 4 | Pcon+RBS luxI-DT4 |
| 5 | Pcon+RBS luxI-DT5 |
| 6 | Pcon+RBS luxI-DT6 |
| 7 | Pcon+apF12\_1R-DT1 |
| 8 | Pcon+apF12\_1R-DT2 |
| 9 | Pcon+apF12\_1R-DT3 |
| 10 | Pcon+apF12\_1R-DT4 |
| 11 | DT+Pcon-pT181attenuator1 |
| 12 | F1attenuator(pSB1C3) |
| 13 | 100bp ladder |

[![Igku 9119.jpg](/wiki/images/a/a2/Igku_9119.jpg)](/File%3AIgku_9119.jpg)

### Restriction Enzyme Digestion

Nakamoto

|  | DNA | EcoRI | XbaI | SpeI | PstI | buffer | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| pSB4K5(EcoRI+SpeI) | 9.4µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 12.6µL | 30 µL |
| pSB4K5(EcoRI+SpeI) negative | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |
| RBS-lacZα-DT(EcoRI+SpeI) | 10µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 12µL | 30µL |
| RBS-lacZα-DT(EcoRI+SpeI) negative | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |
| Ptet-pT181 antisense(SteI+PstI) | 10.8µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 12µL | 30µL |
| Ptet-pT181 antisense(SteI+PstI) negative | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |
| Pcon-RBS-lacZα-DT(EcoRI+SpeI) | 7.1µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 14.9µL | 30µL |
| Pcon-RBS-lacZα-DT(EcoRI+SpeI) negative | 0.4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |
| RBS-lysis2-DT(XbaI+PstI) | 22µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 0µL | 30µL |
| RBS-lysis2-DT(XbaI+PstI) negative | 1.4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 6.6µL | 10µL |

### Liquid Culture

Nakamoto

| Sample | medium |
| --- | --- |
| 8/16 Master4 Plax |  |
| 8/25 Pλ-laxI-2 |  |

### PCR

Hirano

| 8/29 Pλ-RBS-luxI-DT(76.5ng/µL) | 10x buffer for KOD plus-ver.2 | KOD-plus | dNTP | MgSO4 | F primer(RBS-luxI-DT-cloning-fwd) | R primer(RBS-luxI-DT-cloning-rev) | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.3 | 2.5 | 0.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.2 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 65°C | 68°C | -- |
| 5min | 30sec | 30sec | 1min | 30 |

| 8/31 Pbad/araC-RBS-RFP(1)(67.6ng/µL) | 10x buffer for KOD plus-ver.2 | KOD-plus | dNTP | MgSO4 | F primer(Pbad/araC(pSBlc3)-cloning-fwd) | R primer(Pbad/araC(pSBlc3)-cloning-rev) | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.3 | 2.5 | 0.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.2 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 54°C | 68°C | -- |
| 5min | 30sec | 30sec | 3min 20sec | 30 |

### Liquid Culture

Hirano

| Sample | medium |
| --- | --- |
| 9/4 RBS-lysis1+DT 3 | plusgrow(+CP) 3ml 37°C |
| 9/4 RBS-lysis2+DT 1 | plusgrow(+CP) 3ml 37°C |
| 9/4 RBS-lysis3+DT 3 | plusgrow(+CP) 3ml 37°C |

## Sep 12

### Miniprep

Nakamoto

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-pT181attenuator-DT | 293.6 | 1.88 | 1.84 |
| Pcon-apt12-1R-DT | 303.8 | 1.75 | 1.64 |
| Fusion1attenuator(pSB1c3) | 172.0 | 1.96 | 2.31 |
| RBS-lisis1-DT | 235.1 | 1.59 | 1.60 |
| RBS-lisis2-DT | 387.7 | 1.91 | 2.01 |
| RBS-lisis3-DT | 387.9 | 1.91 | 2.09 |
| Plux | 166.7 | 1.98 | 1.63 |

### Electrophoresis

Nakamoto

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | 9/11 P-RBS-lisis1-DT(Colony PCR prodution) |

[![Igku 9121.png](/wiki/images/9/9c/Igku_9121.png)](/File%3AIgku_9121.png)

### Liquid Culture

Nakamoto

| Sample | medium |
| --- | --- |
| 9/10 Pcon-apt12-1R-DT1 | Plusgrow medium(+Amp) |
| 9/10 DT-Pcon-pT181attenuator1 | Plusgrow medium(+CP) |
| 9/10 Fusion1-attenuator(pSB1c3) | Plusgrow medium(+CP) |

### Electrophoresis

Tatsui

| Lane | Sample |
| --- | --- |
| 1 | 1kb ladder |
| 2 | pSB4K5(EcoRI&SpeI) |
| 3 | pSB4K5 NC(EcoRI&SpeI) |
| 4 | RBS-lacz-DT(EcoRI&SpeI) |
| 5 | EcoRI&SpeI NC( EcoRI&SpeI) |
| 6 | Pcon-RBS-lacz-DT( EcoRI&SpeI) |
| 7 | Pcon-RBS-lacz-DT NC( EcoRI&SpeI) |
| 8 | 1kb ladder |

[![Igku 9122.png](/wiki/images/9/99/Igku_9122.png)](/File%3AIgku_9122.png)

2

| Lane | Sample |
| --- | --- |
| 1 | 1kb ladder |
| 2 | Ptet-pT181antisense(SpeI&PstI) |
| 3 | Ptet-pT181antisense NC(SpeI&PstI) |
| 4 | RBS-lysis2-DT(XbaI&PstI) |
| 5 | EcoRI&SpeI NC( EcoRI&SpeI) |
| 6 | -- |
| 7 | -- |
| 8 | 1kb ladder |

[![Igku 9123.png](/wiki/images/d/d6/Igku_9123.png)](/File%3AIgku_9123.png)

### Gel Extraction

Tatsui

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kb ladder | -- |
| 2 | RBS-lacZα-DT | EcoRI&SpeI |
| 3 | RBS-lacZα-DT | EcoRI&SpeI |
| 4 | RBS-lacZα-DT | EcoRI&SpeI |
| 5 | -- | -- |
| 6 | Pcon-RBS-lacZα-DT | EcoRI&SpeI |
| 7 | Pcon-RBS-lacZα-DT | EcoRI&SpeI |
| 8 | Pcon-RBS-lacZα-DT | EcoRI&SpeI |
| 9 | 1kb ladder | -- |

[![Igku 9129.png](/wiki/images/3/38/Igku_9129.png)](/File%3AIgku_9129.png)
[![Igku 91210.png](/wiki/images/f/fd/Igku_91210.png)](/File%3AIgku_91210.png)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| RBS-lacZα-DT(EcoRI&SpeI) | 9.8 | 1.50 | 0.39 |
| Pcon-RBS-lacZα-DT(EcoRI&SpeI) | 11.2 | 1.47 | 0.08 |

### Gel Extraction

Tatsui

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kb ladder | -- |
| 2 | RBS-lysis2-DT | XbaI&PstI |
| 3 | RBS-lysis2-DT | XbaI&PstI |
| 4 | RBS-lysis2-DT | XbaI&PstI |
| 5 | -- | -- |
| 6 | 1kb ladder | -- |

[![Igku 9127.png](/wiki/images/4/44/Igku_9127.png)](/File%3AIgku_9127.png)
[![Igku 9128.png](/wiki/images/7/7e/Igku_9128.png)](/File%3AIgku_9128.png)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| RBS-lysis2-DT(XbaI&PstI) | 5.4 | 1.43 | 0.77 |

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 1kb ladder |
| 2 | Pλ-RBS-luxI-DT(PCR product) |
| 3 | Pbad/araC-RBS-RFP(PCR product) |

[![Igku 9124.png](/wiki/images/1/11/Igku_9124.png)](/File%3AIgku_9124.png)

### Colony PCR

Honda

| Sample | base pair |
| --- | --- |
| 9/11 apt12-1M(pSB1C3) 1 | 513 |
| 9/11 apt12-1M(pSB1C3) 2 | 513 |
| 9/11 Fusion3m2a09ttenuator(pSB1C3) 1 | 609 |
| 9/11 Fusion6 antisense(pSB1C3)1 | 431 |
| 9/11 aptamer 12-P(pSB1C3)1 | 515 |
| 9/11 Fusion1 antisense(pSB1C3) 1 | 420 |
| 9/11 Fusion1 antisense(pSB1C3) 2 | 420 |
| 9/11 Plac(BBa-R0011) 1 | 293 |
| 9/11 Plac(BBa-R0011) 2 | 293 |
| 9/11 Plac(BBa-R0011) 3 | 293 |
| 9/11 Plac(BBa-R0011) 4 | 293 |
| 9/11 Pcon-luxR-Plux-GFP 1 | 2138 |
| 9/11 Pcon-RBS-lacZα-DT(pSB4K5) 1 | 712 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 2min12s | 30cycles |

### Restriction Enzyme Digestion

Tatsui

|  | 9/5 Pcon | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1cuts(PstI) | 5.7 | 0µL | 0µL | 0µL | 1µL | 3µL | 3µL | 17.3µL | 30µL |
| NC(PstI) | 1.9 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 6.1µL | 10µL |

|  | 8/17 DT | EcoRI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 1cut(EcoRI) | 10.6 | 1µL | 3µL | 3µL | 12.4µL | 30µL |
| NC(EcoRI) | 3.5 | 0µL | 1µL | 1µL | 4.5µL | 10µL |

### Colony PCR

Nakamoto and Tatsui

| Sample | base pair |
| --- | --- |
| 9/11 Pcon-pT181attenuator-RBS-lacZα-DT | 965 |
| 9/11 Plux-PBS-lysis1-DT 1 | 613 |
| 9/11 Pcon-pT181attenuator-aptamer12-1R-DT 1 | 859 |
| 9/11 Pcon-pT181attenuator-aptamer12-1R-DT 2 | 859 |
| 9/11 Plux-RBS-lysis3-DT 1 | 1210 |
| 9/11 Plux-RBS-lysis3-DT 2 | 1210 |
| Pcon-Spinach-DT(pSB4K5) 1 | 605 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min12s | 30cycles |

### Transformation

Tatsui

| Name | Sample | Competent Cells | Plate |
| --- | --- | --- | --- |
| 9/7 pSB4K5 | 2µL | 20µL | Amp |

### Electrophoresis

No name

1

| Lane | Sample |
| --- | --- |
| 1 | Fusion6 antisense |
| 2 | Fusion1 antisense-1 |
| 3 | Fusion1 antisense-2 |
| 4 | 100bp ladder |
| 5 | Plac(BBa-R0011) 1 |
| 6 | Plac(BBa-R0011) 2 |
| 7 | Plac(BBa-R0011) 3 |
| 8 | Plac(BBa-R0011) 4 |

[![Igku 91211.png](/wiki/images/9/9b/Igku_91211.png)](/File%3AIgku_91211.png)

2

| Lane | Sample |
| --- | --- |
| 1 | 1kb ladder |
| 2 | aptamer12-1M 1 |
| 3 | aptamer12-1M 2 |
| 4 | Fusion3m2 attenuator |
| 5 | aptamer12-P1 |
| 6 | Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT |
| 7 | Pcon-RBS-lacZα=DT |
| 8 | 1kb ladder |

[![Igku 91212.png](/wiki/images/b/b9/Igku_91212.png)](/File%3AIgku_91212.png)

### Electrophoresis

No name

| Lane | Sample | Enzyme |
| --- | --- | --- |
| 1 | Pcon-pT181attenuator-RBS-lacZα-DT 1 | -- |
| 2 | Pcon-pT181attenuator-RBS-lacZα-DT 2 | -- |
| 3 | Plux-RBS-lysis1-DT 1 | -- |
| 4 | Pcon-pT181attenuator-aptamer12-1R-DT 1 | -- |
| 5 | Pcon-pT181attenuator-aptamer12-1R-DT 2 | -- |
| 6 | Plux-RBS-lysis3-DT 1 | -- |
| 7 | 1kbp ladder | -- |
| 8 | Plux-RBS-lysis3-DT 2 | -- |
| 9 | Pcon-spinach-DT(pSB4K5) 1 | -- |
| 10 | 9/5 Pcon | PstI |
| 11 | 9/5 Pcon NC | -- |
| 12 | 8/17 DT | EcoRI |
| 13 | 8/17 DT | -- |

[![Igku 91213.png](/wiki/images/a/a4/Igku_91213.png)](/File%3AIgku_91213.png)

### Liquid Culture

Nakamoto

| Sample | medium |
| --- | --- |
| 9/11Fusion6 antisense-1 | Plusgrow medium(+CP) |
| Plac(BBa-R0011)-3 | Plusgrow medium(+Amp) |
| Plac(BBa-R0011)-4 | Plusgrow medium(+Amp) |
| Fusion3m2 attenuator -1 | Plusgrow medium(+CP) |
| Pλ-luxI-2 | Plusgrow medium(+Amp) |
| 9/11 aptamer12-1M-2 | Plusgrow medium(+CP) |

### Colony PCR

Nakamoto

| Sample | base pair |
| --- | --- |
| 9/11 Fusion1 antisense 3 | 394 |
| 9/11 Fusion1 antisense 4 | 394 |
| 9/11 aptamer12-P 2 | 378 |
| 9/11 aptamer12-P 3 | 378 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 36s | 30cycles |

### Electrophoresis

Nakamoto

| Lane | Sample | Enzyme |
| --- | --- | --- |
| 1 | Fusion1 antisense 2 | -- |
| 2 | Fusion1 antisense 3 | -- |
| 3 | 1kb ladder | -- |
| 4 | apt12-P 2 | -- |
| 5 | apt12-P 3 | -- |

[![Igku 91214.png](/wiki/images/c/c5/Igku_91214.png)](/File%3AIgku_91214.png)

### Restriction Enzyme Digestion

Tatsui

|  | 9/12 RBS-lysis2-DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.2 | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 16.8µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1.0µL | 1.0µL | 7.7µ | 10µL |

|  | 9/12 RBS-lysis3-DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.2 | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 16.8µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1.0µL | 1.0µL | 7.7µ | 10µL |

|  | 9/11 RBS-lysis1-DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 8.5 | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 13.5µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1.0µL | 1.0µL | 7.6µ | 10µL |

|  | 8/20 J23100-RBS-luxR -DT | EcoRI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 1cut | 5.8 | 1.0µL | 3.0µL | 3.0µL | 17.2µL | 30µL |
| NC | 0.3µL | 0µL | 1.0µL | 1.0µL | 7.7µ | 10µL |

|  | 9/10 pSB1C3 | EcoRI | SpeI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 8.1 | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 13.9µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1.0µL | 1.0µL | 7.6µ | 10µL |

|  | 9/12 Plux | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 1cut | 12.0 | 1.0µL | 3.0µL | 3.0µL | 11.0micro;L | 30µL |
| NC | 0.6µL | 0µL | 1.0µL | 1.0µL | 7.4µ | 10µL |

|  | 9/10 pSB1C3 | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 8.1 | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 13.9µL | 30µL |

### Electrophoresis

Tatsui

| Lane | Sample | Enzyme1 | Enzymel2 |
| --- | --- | --- | --- |
| 1 | 9/12 RBS-lysis2-DT | XbaI | PstI |
| 2 | 9/12 RBS-lysis2-DT NC | -- | -- |
| 3 | 9/11 RBS-lysis1-DT | XbaI | PstI |
| 4 | 9/11 RBS-lysis1-DT NC | -- | -- |
| 5 | 9/11 RBS-lysis3-DT | XbaI | PstI |
| 6 | 9/11 RBS-lysis3-DT NC1 | -- | -- |
| 7 | 1kb ladder | -- | -- |
| 8 | 100bp ladder | -- | -- |
| 9 | J23100-RBS-luxR-DT | EcoRI | -- |
| 10 | J23100-RBS-luxR-DT | -- | -- |
| 11 | 9/10 pSB1C3 | EcoRI | SpeI |
| 12 | 9/10 pSB1C3 NC | -- | --- |
| 13 | 9/10 pSB1C3 | XbaI | PstI |
| 14 | 9/12 Plux | PstI | -- |
| 15 | 9/12 Plux NC | -- | -- |

[![Igku 91215.png](/wiki/images/a/ac/Igku_91215.png)](/File%3AIgku_91215.png)

#### Observation through a confocal microscope

We used the liquid culture media with 200μL 9/10Pcon-pT181antisense-spinach-DT& 200μL 9/10 Pcon-spinach-DT& 200μL 9/10 JM109(overnight culture).

We translated them into each 1.5mL tube.

We elminated each supernatant using a 5000xg centrifuge for 1minute,.
Then,we resuspended with 100µL M9(distilled water) and eliminated each supernatant using a 5000xg centrifuge for 1 minute.x2

Adding 100µL M9(in 20µM DFHBI), we resuspended the pret.

15min after incubating in shield light, we eliminated each supernatant using a 5000xg centrifuge for 1 minute.

We looked at the fluorescence of the pret.

We failed to observe it.

### Liquid Culture

Hirano

| Sample | medium |
| --- | --- |
| 9/10 Pcon-pT181antisense-spinach-DT | Plusgrow medium |
| 9/10 Pcon-spinach-DT | Plusgrow medium |
| 9/1 spinach-DT(RNA master plate) | Plusgrow medium |

## Sep 13

### Miniprep

Nakamoto

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/12 Fusion6 antisense | 245.9 | 1.88 | 1.41 |
| 9/12 Plux-3 | 115.5 | 1.58 | 1.32 |
| 9/12 Plux-4 | 125.3 | 1.93 | 1.44 |
| 9/12 Fusion3n2 attenuator-1 | 231.9 | 1.81 | 1.31 |

### Gel Extraction

Tatsui,Ashida

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kb ladder | -- |
| 2 | RBS-lysis1-DT | XbaI&PstI |
| 3 | RBS-lysis1-DT | XbaI&PstI |
| 4 | RBS-lysis1-DT | XbaI&PstI |

[![Igku 9131.jpg](/wiki/images/4/4a/Igku_9131.jpg)](/File%3AIgku_9131.jpg)
[![Igku 9132.jpg](/wiki/images/c/c4/Igku_9132.jpg)](/File%3AIgku_9132.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kb ladder | -- |
| 3 | RBS-lysis2-DT | XbaI&PstI |
| 4 | RBS-lysis2-DT | XbaI&PstI |
| 5 | RBS-lysis2-DT | XbaI&PstI |

[![Igku 9133.jpg](/wiki/images/5/53/Igku_9133.jpg)](/File%3AIgku_9133.jpg)
[![Igku 9134.jpg](/wiki/images/1/1f/Igku_9134.jpg)](/File%3AIgku_9134.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 3 | RBS-lysis3-DT | XbaI&PstI |
| 4 | RBS-lysis3-DT | XbaI&PstI |
| 5 | RBS-lysis3-DT | XbaI&PstI |
| 6 | 1kb ladder | -- |

[![Igku 9135.jpg](/wiki/images/0/02/Igku_9135.jpg)](/File%3AIgku_9135.jpg)
[![Igku 9136.jpg](/wiki/images/9/98/Igku_9136.jpg)](/File%3AIgku_9136.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 2 | 1kb ladder | -- |
| 3 | pSB1C3 | EcoRI&SpeI |
| 4 | pSB1C3 | EcoRI&SpeI |
| 5 | pSB1C3 | EcoRI&SpeI |

[![Igku 9137.jpg](/wiki/images/4/43/Igku_9137.jpg)](/File%3AIgku_9137.jpg)
[![Igku 9138.jpg](/wiki/images/e/eb/Igku_9138.jpg)](/File%3AIgku_9138.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kb ladder | -- |
| 2 | pSB1C3 | XbaI&PstI |
| 3 | pSB1C3 | XbaI&PstI |
| 4 | pSB1C3 | XbaI&PstI |

[![Igku 9139.jpg](/wiki/images/a/af/Igku_9139.jpg)](/File%3AIgku_9139.jpg)
[![Igku 91310.jpg](/wiki/images/7/7e/Igku_91310.jpg)](/File%3AIgku_91310.jpg)

### Restriction Enzyme Digestion

Nakamoto

|  | 9/13 Plac | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1cuts(PstI) | 16 | 0µL | 0µL | 0µL | 1µL | 3µL | 3µL | 7µL | 30µL |
| NC | 0.8 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.2µL | 10µL |

|  | 9/10 Pcon attenuator | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1cut(PstI) | 10 | 0µL | 0µL | 0µL | 1µL | 3µL | 3µL | 13µL | 30µL |
| NC | 0.5 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |

|  | 9/12 Plux | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1cuts(PstI) | 12 | 0µL | 0µL | 0µL | 1µL | 3µL | 3µL | 11µL | 30µL |
| NC | 0.6 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.4µL | 10µL |

|  | 9/12 DT-E | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1cut(XbaI) | 23.7 | 0µL | 0µL | 1µL | 0µL | 3µL | 3µL | 0µL | 30.7µL |
| NC | 1.2 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 6.8µL | 10µL |

|  | 9/12 Pcon-RBS-luxR-bTE | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1cuts(XbaI) | 22.4 | 0µL | 0µL | 1µL | 0µL | 3µL | 3µL | 0.6µL | 30µL |
| NC | 2.2 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 5.8µL | 10µL |

|  | 9/12 Pcon-P | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1cut(SpeI) | 21 | 0µL | 1µL | 0µL | 0µL | 3µL | 3µL | 12.0µL | 30µL |
| NC | 2.1 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 5.7µL | 10µL |

### M9 (EDTA)

Kanbayashi

1mM EDTA

| volume | 10ml |
| --- | --- |
| Na2HPO4 | 300mg |
| KH2PO4 | 150mg |
| NaCl | 25mg |
| NH4Cl | 50mg |
| Fe(3)-EDTA | 21mg |

5mM EDTA

| volume | 10ml |
| --- | --- |
| Na2HPO4 | 300mg |
| KH2PO4 | 150mg |
| NaCl | 25mg |
| NH4Cl | 50mg |
| Fe(3)-EDTA | 105mg |

### Gel Extraction

Nakamoto

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kb ladder | -- |
| 3 | Pcon-P | SpeI |
| 4 | Pcon-P | SpeI |
| 5 | Pcon-P | SpeI |

[![Igku 91311.jpg](/wiki/images/f/fd/Igku_91311.jpg)](/File%3AIgku_91311.jpg)
[![Igku 91312.jpg](/wiki/images/b/b2/Igku_91312.jpg)](/File%3AIgku_91312.jpg)

### Liquid Culture

Tatsui

| Sample | medium |
| --- | --- |
| 9/11 aptamer12\_1M(pSB1C3)-1 | -- |
| 9/11 aptamer 12\_P1 | -- |
| 9/11 Fusaion1 antisense-4 | -- |
| 9/12 pSB4K5-1 | -- |
| 9/12 pSB4K5-2 | -- |

### Restriction Enzyme Digestion

Nakamoto

|  | 9/13 Plac-P | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1cuts(SpeI) | 23.5 | 0µL | 0.5µL | 0µL | 0µL | 3µL | 3µL | 0µL | 30µL |
| NC | 8 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 0µL | 10µL |

|  | 9/13 Pcon-pT181 attenuator-P | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1cut(SpeI) | 23.5 | 0µL | 0.5µL | 0µL | 0µL | 3µL | 3µL | 0µL | 30µL |
| NC | 02.3 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 5.7µL | 10µL |

|  | 9/13 Plux-P | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1cuts(SpeI) | 23.8 | 0µL | 0.2µL | 0µL | 1µL | 3µL | 3µL | 0µL | 30µL |
| NC | 8 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 0µL | 10µL |

|  | 9/5 RBS-lacZα-DT | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cut(XbaI&PstI) | 10.8 | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 11.2µL | 30µL |
| NC | 0.5 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |

### Electrophoresis

Hirano

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | RBS-lacZα-DT(XbaI&PstI) |
| 3 | RBS-lacZα-DT(NC) |

[![Igku 91313.jpg](/wiki/images/2/25/Igku_91313.jpg)](/File%3AIgku_91313.jpg)

### Ligation

No Name

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/13 Plac (SpeI&PstI) | 8.5µL | 9/13 pT181 antisense (XbaI&PstI) | 2.1 µL | 5.3 µL |
| experiment | 9/13 Plac (SpeI&PstI) | 8.5 µL | 9/10 aptamer 12\_1R-DT (XbaI&PstI) | 1.8 µL | 5.15µL |
| experiment | 9/13 Plac (SpeI&PstI) | 8.5 µL | 9/2 pT181 attenuator(XbaI&PstI) | 2.3 µL | 10.4 µL |
| experiment | 9/13 Plux (SpeI&PstI) | 11.4 µL | 9/13 RBS-lysis1-DT (XbaI&PstI) | 5.0 µL | 8.2 µL |
| experiment | 9/13 Plux (SpeI&PstI) | 11.4 µL | 9/13 RBS-lysis2-DT (XbaI&PstI) | 7.0 µL | 9.2 µL |
| experiment | 9/13 Plux (SpeI&PstI) | 7.2 µL | 9/13 RBS-lysis3-DT (XbaI&PstI) | 6.0 µL | 6.6 µL |
| experiment | 9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI) | 2.0 µL | 9/4 Plux-RBS-GFP-DT (EcoRI&SpeI) | 5.2 µL | 3.6 µL |
| experiment | 9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI) | 2.0 µL | 9/10 aptamer 12\_1R-DT (XbaI&PstI) | 1.6 µL | 1.8 µL |
| experiment | 9/12 Ptet-pT181 antisense (--) | 1.0 µL | 9/10 spinach-DT (--) | 2.4 µL | 1.7 µL |

### PCR

No name

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/7 tetR aptamer 12\_1R(pSB1C3) | 0.5 | 6.05 | 1.75 | 1.7 | 0.5 | 0 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/6 pT181 antisense-1(pSB1C3) | 0.5 | 6.05 | 1.75 | 1.7 | 0.5 | 0 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/6 pT181 attenuator (pSB1C3) | 0.5 | 3.85 | 1.75 | 3.9 | 0.5 | 0 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 8/27 Pλ-RBS-luxI-DT-1 | 0.5 | 2.55 | 1.75 | 5.2 | 0.5 | 0 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 8/31 Pbad/araC-RBS-RFP-2 | 0.5 | 3.75 | 1.75 | 4 | 0.5 | 0 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/13 Plac(pSB1A2) | 0.5 | 4.2 | 1.75 | 3.5 | 0.5 | 0 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 8/20 J23100-RBS-lacZα-DT | 0.5 | 6.35 | 1.75 | 1.4 | 0.5 | 0 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/7 Ptet-RBS-lacZα-DT | 0.5 | 6.05 | 1.75 | 1.7 | 0.5 | 0 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/8 J23100-spinach-DT | 0.5 | 5.75 | 1.75 | 2 | 0.5 | 0 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/11 RBS-lysis1-DT | 0.5 | 6.05 | 1.75 | 1.7 | 0.5 | 0 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/12 RBS-lysis2-DT | 0.5 | 6.75 | 1.75 | 1.0 | 0.5 | 0 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/12 RBS-lysis3-DT | 0.5 | 6.75 | 1.75 | 1.0 | 0.5 | 0 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/8 J23100-spinach-DT | 0.5 | 6.35 | 1.75 | 1.4 | 0 | 0.5 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 8/27 Pλ-RBS-luxI-DT-1 | 0.5 | 2.55 | 1.75 | 5.2 | 0 | 0.5 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/12 RBS-lysis2-DT | 0.5 | 6.75 | 1.75 | 1.0 | 0 | 0.5 | 10.5 |

| genome DNA | Big Dye | MilliQ | 5x buffer | temp(400mg) | SasA\_fwd primer | SasA\_rev primer | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/11 RBS-lysis1-DT | 0.5 | 6.05 | 1.75 | 1.7 | 0 | 0.5 | 10.5 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 96°C | 96°C | 50°C | 68°C | -- |
| 2m | 10s | 5s | 4m | 30 |

### PCR

Yoshida

| genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | Prefix-RBS-RpaB\_fwd primer | RpaB-GG4-suffix\_rev primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| RpaB | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.5 | 25 |

| genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | Prefix-PkaiBC\_fwd primer ver.3 | PkaiBC-suffix\_rev primer ver.3 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| RpaB | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 58°C | 68°C | -- |
| 2m | 10s | 30s | 30s | 30 |

### Gel Extraction

Nakamoto

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 3 | RBS-lacZα-DT | XbaI&PstI |
| 4 | RBS-lacZα-DT | XbaI&PstI |
| 5 | RBS-lacZα-DT | XbaI&PstI |
| 6 | RBS-lacZα-DT | XbaI&PstI |

[![Igku 91318.jpg](/wiki/images/3/3e/Igku_91318.jpg)](/File%3AIgku_91318.jpg)
[![Igku 91319.jpg](/wiki/images/e/e9/Igku_91319.jpg)](/File%3AIgku_91319.jpg)

### Transformation

No name

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| 9/13 Plac+pT181 antisense | 2µL | 20µL | 22µL | Amp |
| 9/13 Plac+apt12\_1R | 2µL | 20µL | 22µL | Amp |
| 9/13 Plac+pT181 attenuator | 2µL | 20µL | 22µL | Amp |
| 9/13 Plux+pT181 antisense | 2µL | 20µL | 22µL | CP |
| 9/13 Plux+RBS-lysis2-DT | 2µL | 20µL | 22µL | CP |
| 9/13 Plux+RBS-lysis2-DT | 2µL | 20µL | 22µL | CP |
| 9/13 Pcon-RBS-lacR-DT+Plux-RBS-GFP-DT | 2µL | 20µL | 22µL | Amp |
| 9/13 Pcon-pT181 antisense+apt12\_1R-DT | 2µL | 20µL | 22µL | Amp |
| 9/13 Ptet-pT181antisense+spinach-DT | 2µL | 20µL | 22µL | CP |
| Pλ-luxI-1 | 2µL | 20µL | 22µL | Amp |

## Sep 14

### Restriction Enzyme Digestion

Hirano

|  | 9/14 pSB4K5S | EcoRI | XbaI | SpeI | PstI | BufferB | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 10µL | 1µL | 0µL | 1µL | 0µL | 3µL | 0µL | 3µL | 12µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 0µL | 1µL | 7.5µL | 10µL |

|  | 9/13 Plac | XbaI | PstI | BufferH | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 cut | 9µL | 0µL | 1µL | 3µL | 3µL | 14µL | 30µL |
| NC | 1µL | 0µL | 0µL | 1µL | 1µL | 7µL | 10µL |

|  | 9/14 PKaiBC | EcoRI | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 11µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 11µL | 30µL |
| NC | 4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 4µL | 10µL |

|  | 9/14 RpaB | EcoRI | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 13µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 9µL | 30µL |
| NC | 2µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 5µL | 10µL |

|  | 8/17 RBS-GFP-DT | EcoRI | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1cut | 9µL | 0µL | 1µL | 0µL | 0µL | 3µL | 3µL | 14µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |

* incubated 37°C 1hour

### Liquid Culture

Hirano

| Sample | medium |
| --- | --- |
| 9/11 Plac(BBa-R0011)-(3) | Plusgrow medium(+Amp) |

### Electrophoresis

Hirano

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 1kb ladder | -- | -- |
| 2 | pSB4K5(1) | EcoRI | SpeI |
| 3 | pSB4K5(1) | -- | -- |
| 4 | Plac(1A2) | PstI | -- |
| 5 | Plac(1A2) | -- | -- |
| 6 | PKaiBC | EcoRI | SpeI |
| 7 | PKaiBC | -- | -- |
| 8 | RpaB(1) | XbaI | PstI |
| 9 | RpaB(1) | -- | -- |
| 10 | RBS-GFP-DT | XbaI | -- |
| 11 | RBS-GFP-DT | -- | -- |
| 12 | 1kb ladder | -- | -- |

[![Igku 0914 E1.jpg](/wiki/images/0/01/Igku_0914_E1.jpg)](/File%3AIgku_0914_E1.jpg)

| Lane | Sample | Enzyme |
| --- | --- | --- |
| 1 | 1kb ladder | -- |
| 2 | Plac(pSB1A2) | PstI |
| 3 | Plac(pSB1A2) | -- |

[![Igku 0914 E2.jpg](/wiki/images/5/5b/Igku_0914_E2.jpg)](/File%3AIgku_0914_E2.jpg)

### Sequence PCR

No name

| genome DNA | Big Dye | 5x buffer | temp(400mg) | primer | milliQ |
| --- | --- | --- | --- | --- | --- |
| Pcon-RBS-luxR-DT | 0.5 | 1.75 | 0.9 | 0.5 | 6.85 |
| Pcon-RBS-luxR-DT | 0.5 | 1.75 | 0.4 | 0.5 | 7.35 |
| Pcon-RBS-tetR-DT | 0.5 | 1.75 | 3 | 0.5 | 5.25 |
| Pcon-RBS-tetR-DT | 0.5 | 1.75 | 3 | 0.5 | 5.25 |
| Plux | 0.5 | 1.75 | 2.4 | 0.5 | 5.35 |
| Plux-RBS-GFP-DT | 0.5 | 1.75 | 1.9 | 0.5 | 5.85 |
| Plux-RBS-GFP-DT | 0.5 | 0.5 | 1.9 | 0.5 | 5.85 |
| Pcon-RBS-GFP-DT | 0.5 | 0.5 | 1.2 | 0.5 | 6.55 |
| Pcon-RBS-GFP-DT | 0.5 | 0.5 | 1.2 | 0.5 | 6.55 |
| RBS-lysis3-DT | 0.5 | 0.5 | 1.0 | 0.5 | 6.75 |
| Pcon-antisense-Spinach-DT | 0.5 | 0.5 | 1.4 | 0.5 | 6.35 |
| Pcon-RBS lacZα-DT | 0.5 | 0.5 | 1.4 | 0.5 | 6.35 |
| Ptet-RBS lacZα-DT | 0.5 | 0.5 | 0.59 | 0.5 | 1.85 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 96°C | 96°C | 50°C | 60°C | -- |
| 2min | 10sec | 5sec | 4min | 30cycle |

### Gel Extraction

Tatsui

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kb ladder | -- |
| 2 | pSB4K5 | EcoRI&SpeI |
| 3 | pSB4K5 | EcoRI&SpeI |
| 4 | pSB4K5 | EcoRI&SpeI |

[![Igku 0914 G8.jpg](/wiki/images/b/bf/Igku_0914_G8.jpg)](/File%3AIgku_0914_G8.jpg)
[![Igku 0914 G9.jpg](/wiki/images/8/8d/Igku_0914_G9.jpg)](/File%3AIgku_0914_G9.jpg)

### Restriction Enzyme Digestion

No name

|  | 9/14 Plac(1A2) | EcoRI | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 23µL | 0µL | 0µL | 0.3µL | 0µL | 3µL | 3µL | 0µL | 30µL |

|  | 9/14 RBS-GFP-DT | EcoRI | XbaI | SpeI | PstI | BufferH | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 23µL | 1µL | 0µL | 0µL | 0µL | 3µL | 3µL | 0µL | 30µL |

### Ligation

Nakamoto and Hirano

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/13 Pcon-PT181attenuator(SpeI&PstI) | 1.7 | 9/13 RBS-lacZα-DT (XbaI & PstI) | 12.5 | 5 |
| experiment | 9/13 pSB2C3 (XbaI & PstI) | 1.9 | 9/14 RpaB (XbaI & PstI) | 16.3 | 10.5 |
| experiment | 9/13 pSB2C3 (XbaI & PstI) | 4.7 | 9/14 PkaiBC (EcoRI & SpeI) | 6.7 | 5.7 |
| experiment | 9/14 RBS-GFP-DT (PstI&XbaI) | 0.7 | 9/14 PkaiBC (EcoRI & SpeI) | 2.6 | 1.6 |
| experiment | 9/14 Plac (SpeI&PstI) | 2.4 | 9/14 RBS-lysis1-DT (XbaI & PstI) | 4.9 | 3.6 |
| experiment | 9/14 Plac (SpeI&PstI) | 2.4 | 9/14 RBS-lysis2-DT (XbaI & PstI) | 7.0 | 4.7 |
| experiment | 9/14 Plac (SpeI&PstI) | 2.4 | 9/14 RBS-lysis3-DT (XbaI & PstI) | 9.2 | 5.8 |

Samples were evaporeted used evaporator into about 7 µL

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 9/13 Plux＋RBS-lysis1-DT (1)~(3) | -- |
| 9/13 Plux＋RBS-lysis2-DT (1)~(3) | - |
| 9/13 Plux＋RBS-lysis3-DT (1)~(2) | - |

| Sample | base pair |
| --- | --- |
| 9/13 Pcon-RBS-luxR-DT＋Plux-RBS-GFP-DT (1)~(4) | -- |
| 9/13 Pcon-PT181attenuator＋aptamer121R-DT (1)~(2) | - |
| 9/13 Plac＋PT181attenuator (1) | - |

| Sample | base pair |
| --- | --- |
| Dλ-lux(1) (1)~(2) | -- |
| NC | -- |

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 1kbp ladder |
| 2 | 9/13 Plux+RBS-lysis1-DT1 |
| 3 | 9/13 Plux+RBS-lysis1-DT2 |
| 4 | 9/13 Plux+RBS-lysis1-DT3 |
| 5 | 9/13 Plux+RBS-lysis2-DT1 |
| 6 | 9/13 Plux+RBS-lysis2-DT2 |
| 7 | 9/13 Plux+RBS-lysis2-DT3 |
| 8 | 9/13 Plux+RBS-lysis3-DT1 |
| 9 | 9/13 Plux+RBS-lysis3-DT2 |
| 10 | 9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT1 |
| 11 | 9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT2 |
| 12 | 9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT3 |
| 13 | 9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT4 |
| 14 | 9/13 Ptet-pT181 antisense+Spinach-DT1 |
| 15 | 9/13 Pcon-pT181 attenuator+aptamer12-1R-PT1 |
| 16 | 9/13 Pcon-pT181 attenuator+aptamer12-1R-PT1 |
| 17 | 9/13 Plac+pT181 attenuator |

[![Igku 0914 E5.jpg](/wiki/images/8/83/Igku_0914_E5.jpg)](/File%3AIgku_0914_E5.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 1kbp ladder |
| 2 | Pλ-lux(1)1 |
| 3 | Pλ-lux(1)1 |
| 4 | NC |

[![Igku 0914 E6.jpg](/wiki/images/7/74/Igku_0914_E6.jpg)](/File%3AIgku_0914_E6.jpg)

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 9/13 Pλ-luxI(1)-1 | Plusgrow medium(+Amp) |

### Transformation

Nakamoto

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| 9/13 RBS-lysisZα-DT(XbaI&PstI)＋9/13 Pcon-PT181 attenuator(SpeI&PstI) | 2µL | 20µL | 22µL | Amp |
| 9/13 SB2C3＋9/14 PzαB | 2µL | 20µL | 22µL | CP |
| 9/13 pSB2C3＋9/14 PkaiBC | 2µL | 20µL | 22µL | CP |
| 9/13 RBS-GFP-DT＋9/14 PkaiBC | 2µL | 20µL | 22µL | CP |
| Plac＋RBS-lysis1-DT | 2µL | 20µL | 22µL | Amp |
| Plac＋RBS-lysis2-DT | 2µL | 20µL | 22µL | Amp |
| Plac＋RBS-lysis2-DT | 2µL | 20µL | 22µL | Amp |

### Sequence PCR

Tatsui

| genome DNA | temp | Big Dye | 5x buffer | primer | MilliQ |
| --- | --- | --- | --- | --- | --- |
| 9/6 PT181 attenuator(VR) | 3.9 | 0.5 | 1.75 | 0.5 | 3.85 |
| 8/17 RBS lacZα-DT | 2.1 | 0.5 | 1.75 | 0.5 | 5.65 |
| 9/6 PT181 attenuator(VR) | 3.9 | 0.5 | 1.75 | 0.5 | 3.85 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 96°C | 50°C | 60°C | 4°C | -- |
| 10sec | 5sec | 4min | 1min | 30cycle |

## Sep 15

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pλ-luxI | 225 | 1.59 | 0.83 |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 9/13 Pλ-luxI-1 |  |
| 9/7 pSB4K5-1 |  |
| 9/11 Plac-3 |  |

### Ligation

No name

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/13 Pcon(SpeI & PstI) | 3.6 µL | 9/12 RBS-lacZα-DT(XbaI & PstI) | 4.7 µL | 8 µL |

### Restriction Enzyme Digestion

No Name

|  | DNA | EcorI | SpeI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/7Pcon-RBS-tetR-DT (EcoRI & SpeI) | 15 µL | 1 µL | 1 µL | 3 µL | 3 µL | 6 µL | 30 µL |
| NC | 0.7 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.3 µL | 10 µL |

|  | DNA | EcoRI | SpeI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/12 Pcon-pT181 attenuator-DT (EcoRI & SpeI) | 6.8 µL | 1 µL | 1 µL | 3 µL | 3 µL | 15.2 µL | 30 µL |
| NC | 0.3 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.7 µL | 10 µL |

|  | DNA | EcoRI | SpeI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/12 Pcon-apt12\_1R-DT(EcoRI & SpeI) | 6.6 µL | 1 µL | 1 µL | 3 µL | 3 µL | 15.4 µL | 30 µL |
| NC | 0.3 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.7 µL | 10 µL |

|  | DNA | EcoRI | SpeI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/8 Pcon-spinach-DT (EcoRI & SpeI) | 7.6 µL | 1 µL | 1 µL | 3 µL | 3 µL | 15.4 µL | 30 µL |
| NC | 0.3 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.7 µL | 10 µL |

|  | DNA | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/11 Pcon-pT181 anticodone-spinach-DT(XbaI & PstI) | 7.2 µL | 1 µL | 1 µL | 3 µL | 3 µL | 14.8 µL | 30 µL |
| NC | 0.4 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.6 µL | 10 µL |

|  | DNA | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 8/17 RBS-lacZα-DT (XbaI & PstI) | 5.3 µL | 1 µL | 1 µL | 3 µL | 3 µL | 16.7 µL | 30 µL |
| NC | 0.5 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.5 µL | 10 µL |

|  | DNA | EcoRI | XbaI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/7 Ptet-RBS-LacZα-DT (EcoRI & XbaI) | 2.9 µL | 1 µL | 1 µL | 3 µL | 3 µL | 18.5 µL | 30 µL |
| NC | 1.5 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7 µL | 10 µL |

### Electrophoresis

No Name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1,14 | 100bp ladder |  |  |
| 2 | Pcon-RBS-tetR-DT | EcoRI | SpeI |
| 3 | Pcon-RBS-tetR-DT |  |  |
| 4 | Pcon-pT181 attenuator-DT | EcoRI | SpeI |
| 5 | Pcon-pT181 attenuator-DT |  |  |
| 6 | Pcon-apt12-1R-DT | EcoRI | SpeI |
| 7 | Pcon-apt12-1R-DT |  |  |
| 8 | Pcon-spinach-DT | EcoRI | SpeI |
| 9 | Pcon-spinach-DT |  |  |
| 10 | Pcon-pT181 antisense-spinach-DT | XbaI | PstI |
| 11 | Pcon-pT181 antisense-spinach-DT |  |  |
| 12 | RBS-lacZα-DT | XbaI | PstI |
| 13 | RBS-lacZα-DT |  |  |
| 15 | Ptet-RBS-lacZα-DT | EcoRI | XbaI |
| 16 | Ptet-RBS-lacZα-DT |  |  |
| 17 | 1kbp ladder |  |  |

[![Igku 9151.jpg](/wiki/images/a/a6/Igku_9151.jpg)](/File%3AIgku_9151.jpg)

### Gel Extraction

No Name
1

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 2~4 | Pcon-pT181 attenuator-DT | EcoRI & SpeI |
| 6~8 | Pcon-apt12\_1R-DT |  |
| 10~12 | Pcon-spinach-DT |  |

[![Igku 9152.jpg](/wiki/images/8/88/Igku_9152.jpg)](/File%3AIgku_9152.jpg)
[![Igku 9153.jpg](/wiki/images/8/85/Igku_9153.jpg)](/File%3AIgku_9153.jpg)
2

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder |  |
| 2~4 | Pcon-pT181 anticodone-spinach-DT | XbaI & PstI |
| 6~8 | RBS-lacZα-DT | XbaI & PstI |
| 10~12 | Pcon-RBS-tetR-DT | EcoRI & SpeI |

[![Igku 9154.jpg](/wiki/images/9/97/Igku_9154.jpg)](/File%3AIgku_9154.jpg)
[![Igku 9155.jpg](/wiki/images/3/32/Igku_9155.jpg)](/File%3AIgku_9155.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/15 Ptet-RBS-lacZα-DT (EcoRI & SpeI) | 1.9 | 1.08 | 0.77 |
| 9/15 Pcon-RBS-tetR-DT (EcoRI & SpeI) | 5.9 | 1.30 | 0.17 |
| 9/15 RBS-lacZα-DT (XbaI & PstI) | 1.0 | 0.85 | 0.44 |
| Pcon-pT181 anticodone-spinach-DT (XbaI & PstI) | 1.3 | 0.95 | 0.28 |
| Pcon-spinach-DT | 0.9 | 1.37 | 0.05 |
| Pcon-apt12\_1R-DT | 1.7 | 1.42 | 0.29 |
| Pcon-pT181 attenuator-DT (EcoRI & SpeI) | 2.1 | 1.53 | 0.46 |

### Ligation

No Name

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/13 pSB1C3 EcoRI & SpeI | 4.7 µL | 9/15 Pcon-RBS-tetR-DT EcoRI & SpeI | 1.9 µL | xx µL |
| experiment | 9/13 pSB1C3 EcoRI & SpeI | 4.7 µL | 9/15 Pcon-spinach-DT EcoRI & SpeI | 4.7 µL | xx µL |
| experiment | 9/13 pSB1C3 EcoRI & SpeI | 4.7 µL | 9/15 Pcon-apt12\_1R-DT EcoRI & SpeI | 2.0 µL | xx µL |
| experiment | 9/13 pSB1C3 EcoRI & SpeI | 4.7 µL | 9/15 Pcon-pT181 attenuator-DT EcoRI & SpeI | 3.0 µL | xx µL |
| experiment | 9/13 pSB1C3 XbaI & PstI | 1.9 µL | 9/15 Pcon-pT181 anticodone-spinach-DT XbaI & PstI | 4.4 µL | xx µL |
| experiment | 9/13 pSB1C3 EcoRI & SpeI | 4.7 µL | 9/3 pT181 attenuator EcoRI & SpeI | 3.8 µL | xx µL |
| experiment | 9/6 Pcon SpeI & PstI | 1.4 µL | 9/15 RBS-lacZα-DT XbaI & PstI | 2.0 µL | xx µL |
| experiment | 9/14 pSB 4k5 EcoRI & SpeI | µL | 9/15 Pcon-spinach-DT EcoRI & SpeI | 9.4 µL | xx µL |
| experiment | 9/15 Peet-RBS-lacZα-DT EcoRI & XbaI | 2.6 µL | Pcon-RBS-tetR-DT EcoRI & SpeI | 1.5 µL | xx µL |

### Restriction Enzyme Digestion

No Name

|  | DNA | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/13 pT181 attenuator XbaI & PstI | 6.3 µL | 1 µL | 1 µL | 3 µL | 3 µL | 15.7 µL | 30 µL |
| NC | 0.3 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.7 µL | 10 µL |

|  | DNA | EcoRI | SpeI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/10 pSB1C3 EcoRI & SpeI | 8.1 µL | 1 µL | 1 µL | 3 µL | 3 µL | 13.9 µL | 30 µL |
| NC | 0.4 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.6 µL | 10 µL |

|  | DNA | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/14 pSB4k5 XbaI & PstI | 9.5 µL | 1 µL | 1 µL | 3 µL | 3 µL | 12.5 µL | 30 µL |
| NC | 0.5 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.5 µL | 10 µL |

|  | DNA | SpeI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/11 DT SpeI & PstI | 10.4 µL | 1 µL | 1 µL | 3 µL | 3 µL | 11.6 µL | 30 µL |
| NC | 0.5 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.5 µL | 10 µL |

|  | DNA | SpeI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/6 apt12\_1R-DT SpeI & PstI | 7.9 µL | 1 µL | 1 µL | 3 µL | 3 µL | 14.1 µL | 30 µL |
| NC | 0.4 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.6 µL | 10 µL |

|  | DNA | SpeI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/10 spinach-DT SpeI & PstI | 9.9 µL | 1 µL | 1 µL | 3 µL | 3 µL | 12.1 µL | 30 µL |
| NC | 0.5 µL | 0 µL | 0 µL | 1 µL | 1 µL | 7.5 µL | 10 µL |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| pSB1C3 |  |
| DT |  |
| Spinach-DT |  |

## Sep 16

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| pSB1C3+P-KaiBC① | Plusgrow medium(+CP) |
| pSB1C3+P-KaiBC② | Plusgrow medium(+CP) |

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| pSB1C3+P-KaiBC① | 283 |
| pSB1C3+P-KaiBC② | 283 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 2min | 30s | 30s | 18s | 30cycles |

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kb ladder |  |
| 2 | pT181 attenuator | XbaI+PstI |
| 3 | pT181 attenuator | XbaI+PstI |
| 4 | pT181 attenuator | XbaI+PstI |
| 6 | pSB1C3 | EcoRI+SpeI |
| 7 | pSB1C3 | EcoRI+SpeI |
| 8 | pSB1C3 | EcoRI+SpeI |
| 10 | pSB4K5 | XbaI+PstI |
| 11 | pSB4K5 | XbaI+PstI |
| 12 | pSB4K5 | XbaI+PstI |

[![Igku 9161.png](/wiki/images/6/6e/Igku_9161.png)](/File%3AIgku_9161.png)
[![Igku 9161a.png](/wiki/images/3/31/Igku_9161a.png)](/File%3AIgku_9161a.png)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pT181 attenuator(XbaI+PstI) | 8.2 | 1.62 | 0.11 |
| pSB1C3(EcoRI+SpeI) | 49.5 | 1.48 | 0.18 |
| pSB4K5(XbaI+PstI) | 18.1 | 1.73 | 0.57 |
| DT(SpeI+PstI) | 70.4 | 1.36 | 1.02 |
| aptamer12-1R-DT(SpeI+PstI) | 42.0 | 1.82 | 0.47 |
| spinach-DT(SpeI+PstI) | 36.8 | 1.79 | 0.61 |

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Dx-lux1 | 136.7 | 1.46 | 1.28 |
| RBS-laczα-DT | 237.6 | 1.88 | 1.70 |
| pSB4K5 | 148.4 | 1.55 | 1.09 |
| Pcon-KBS-tetR-DT | 126.6 | 1.73 | 1.36 |
| 9/11Plac1 | 160.0 | 1.78 | 1.28 |
| 9/15Plac2 | 144.0 | 1.73 | 1.81 |
| 9/14Plac | 332.5 | 1.51 | 2.33 |
| Ptet-RBS-laczα-DT | 231.3 | 1.84 | 1.57 |

### PCR

No name

| DNA | 9/6pT181 antisense psB1C3(1) | KOD plus | 10x buffer | dNTP | MgSO4 | GG1\_1C3\_fwd primer | GG2\_1C3\_rev primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 400pg | 1 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |
| 800pg | 2 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 14.5 | 25 |
| 1000pg | 2.5 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 14 | 25 |
| nega | 0 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 30s | 30cycles |

| DNA | 9/6 12-1R aptamer-DT | KOD plus | 10x buffer | dNTP | MgSO4 | GG1\_1C3\_fwd primer | GG1\_1C3\_rev primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 400pg | 1 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |
| 800pg | 2 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 14.5 | 25 |
| 1000pg | 2.5 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 14 | 25 |
| NC | 0 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 16.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 30s | 30cycles |

### Electrophoresis

Yoshida

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | antisense 1000pg |
| 3 | antisense 800pg |
| 4 | antisense 400pg |
| 5 | tet aptamer-DT 1000pg |
| 6 | tet aptamer-DT 800pg |
| 7 | tet aptamer-DT 400pg |
| 8 | NC |

[![Igku 9162.png](/wiki/images/6/60/Igku_9162.png)](/File%3AIgku_9162.png)

### Restriction Enzyme Digestion

Tatsui

|  | 9/16 Pcon-RBS-tetR-DT(µL) | EcoRI(µL) | SpeI(µL) | XbaI(µL) | PstI(µL) | Buffer(µL) | BSA(µL) | MilliQ(µL) | total(µL) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 15.8 | 0 | 0 | 1 | 1 | 3 | 3 | 6.2 | 30 |
| NC | 0.8 | 0 | 0 | 0 | 0 | 1 | 1 | 7.2 | 10 |

|  | 9/16 pSB4K5(µL) | EcoRI(µL) | SpeI(µL) | XbaI(µL) | PstI(µL) | Buffer(µL) | BSA(µL) | MilliQ(µL) | total(µL) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 13.5 | 0 | 0 | 1 | 1 | 3 | 3 | 8.5 | 30 |
| NC | 0.7 | 0 | 0 | 0 | 0 | 1 | 1 | 7.3 | 10 |

|  | 8/22 pSB1C3(µL) | EcoRI(µL) | SpeI(µL) | XbaI(µL) | PstI(µL) | Buffer(µL) | BSA(µL) | MilliQ(µL) | total(µL) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 18.5 | 1 | 0 | 0 | 1 | 3 | 3 | 3.5 | 30 |
| NC | 0.9 | 0 | 0 | 0 | 0 | 1 | 1 | 7.1 | 10 |

|  | 8/22 pSB1C3(µL) | EcoRI(µL) | SpeI(µL) | XbaI(µL) | PstI(µL) | Buffer(µL) | BSA(µL) | MilliQ(µL) | total(µL) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 18.5 | 1 | 1 | 0 | 0 | 3 | 3 | 3.5 | 30 |
| NC | 0.9 | 0 | 0 | 0 | 0 | 1 | 1 | 7.1 | 10 |

|  | 8/21 pT181 attenuator2(µL) | EcoRI(µL) | SpeI(µL) | XbaI(µL) | PstI(µL) | Buffer(µL) | BSA(µL) | MilliQ(µL) | total(µL) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 7.4 | 1 | 1 | 0 | 0 | 3 | 3 | 14.6 | 30 |
| NC | 0.4 | 0 | 0 | 0 | 0 | 1 | 1 | 7.6 | 10 |

### Ligation

Tatsui

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | Pcon-pT181antisense-spinach-DT(XbaI+PstI) | 5.0µg/mL | pSB4K5(XbaI+PstI) | 18.1µg/mL | xx µg/mL |
| experiment | pC181 attenuator(XbaI+PstI) | 8.2µg/mL | pSB1C3(XbaI+PstI) | 26.0µg/mL | xx µg/mL |

### Transformation

tatsui

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| Pcon+RBS-laczα-DT | µL | µL | µL |  |
| Pcon-pT181 attenuator-DT+pSB1C3 | µL | µL | µL |  |
| pT181 attenuator(EcoRI&SpeI)+pSB1C3 | µL | µL | µL |  |
| Pcon-aptamer12\_1R-DT(EcoRI&SpeI)+pSB1C3 | µL | µL | µL |  |
| Pcon-spinach-DT(EcoRI&SpeI)+pSB1C3 | µL | µL | µL |  |
| Pcon-RBS-tetR-DT(EcoRI&SpeI)+pSB1C3 | µL | µL | µL |  |
| Pcon-pT181 antisense-spinach-DT(XbaI&PstI)+pSB1C3 | µL | µL | µL |  |

### Miniprep

tatsui

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/15 pSBiC3 | 111.7 | 1.79 | 1.36 |
| 9/15 spinach-DT | 113.9 | 1.57 | 1.38 |
| 9/15 DT | 77.0 | 2.05 | 1.92 |

### PCR

tatsui

| 9/14 aptamer12-P(VF) | Big Dye | 5×Buffer | primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1.5 | 0.5 | 1.75 | 0.5 | 5.75 | 10.5 |

| 9/14 aptamer12-1M(VF) | Big Dye | 5×Buffer | primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1.7 | 0.5 | 1.75 | 0.5 | 6.05 | 10.5 |

| 9/14 Fusion1 attenuator(VF) | Big Dye | 5×Buffer | primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 2.3 | 0.5 | 1.75 | 0.5 | 5.45 | 10.5 |

| 9/14 Fusion1 antisense(VF) | Big Dye | 5×Buffer | primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1.3 | 0.5 | 1.75 | 0.5 | 6.45 | 10.5 |

| 9/13 Fusion3m2 attenuator(VF) | Big Dye | 5×Buffer | primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1.7 | 0.5 | 1.75 | 0.5 | 6.05 | 10.5 |

| 9/13 Fusion6 antisense(VF) | Big Dye | 5×Buffer | primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1.6 | 0.5 | 1.75 | 0.5 | 6.15 | 10.5 |

| 9/12 plux(VR) | Big Dye | 5×Buffer | primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 2.4 | 0.5 | 1.75 | 0.5 | 5.35 | 10.5 |

| 9/9 Ptet antisense(VF) | Big Dye | 5×Buffer | primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 2.2 | 0.5 | 1.75 | 0.5 | 5.55 | 10.5 |

| 9/12 Pcon-pT181 attenuator-DT(VF) | Big Dye | 5×Buffer | primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1.4 | 0.5 | 1.75 | 0.5 | 6.35 | 10.5 |

| 9/12 Pcon-pT181 attenuator-DT(VR) | Big Dye | 5×Buffer | primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1.4 | 0.5 | 1.75 | 0.5 | 6.35 | 10.5 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 96°C | 96°C | 50°C | 60°C | -- |
| 2min | 10s | 5s | 4min | 30cycles |

## Sep 17

### Liquid Culture

Tatsui and Hirano

| Sample | medium |
| --- | --- |
| Spinach-DT | LB medium(+CP) |
| Pcon-Spinach-DT | LB medium(+Amp) |
| Pcon-pT181 antisense-Spinach-DT | LB medium(+Amp) |

### PCR

Hirano

| Pcon-tetR aptamer-DT(VF) | BigDye | 5x buffer | primer(VF) | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1.3 | 0.5 | 1.75 | 0.5 | 0.45 | 10.5 |

| Pcon-tetR aptamer-DT(VR) | BigDye | 5x buffer | primer(VR) | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1.3 | 0.5 | 1.75 | 0.5 | 0.45 | 10.5 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 96 °C | 96 °C | 50 °C | 60 °C | -- |
| 2 min | 10 sec | 5 sec | 4 min | 30 cycles |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 1kbp ladder | -- | -- |
| 2 | Pcon-RBS-tetR-DT | XbaI | PstI |
| 3 | Pcon-RBS-tetR-DT | -- | -- |
| 4 | pSB4K5 | XbaI | PstI |
| 5 | pSB4K5 | -- | -- |
| 6 | pSB1C3 | EcoRI | SpeI |
| 7 | pSB1C3 | EcoRI | PstI |
| 8 | pSB1C3 | -- | -- |
| 9 | pT181 attenuator | EcoRI | SpeI |
| 10 | pT181 attenuator | -- | -- |
| 11 | 1kbp ladder | -- | -- |

[![Igku 9171.jpg](/wiki/images/2/24/Igku_9171.jpg)](/File%3AIgku_9171.jpg)

### Ligation

No Name

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 8/28 Plux (SpeI & PstI) | 5.0 µL | 9/13 RBS-lysis1-DT (XbaI & PstI) | 3.5 µL | 3.5 µL |
| experiment | 8/28 Plux (SpeI & PstI) | 5.0 µL | 9/13 RBS-lysis2-DT (XbaI & PstI) | 3.6 µL | 3.5 µL |
| experiment | 8/28 Plux (SpeI & PstI) | 11.0 µL | 9/13 RBS-lysis3-DT (XbaI & PstI) | 9.5 µL | 3.5 µL |
| experiment | 9/14 Plac (SpeI & PstI) | 2.4 µL | 9/16 pT181 attenuator-DT (XbaI & PstI) | 4.8 µL | 3.5 µL |
| experiment | 9/14 Plac (SpeI & PstI) | 2.4 µL | 9/3 pT181 antisense (XbaI & PstI) | 2.0 µL | 2.2 µL |
| experiment | 9/14 Plac (SpeI & PstI) | 2.4 µL | 9/10 aptamer 12\_1R-DT (XbaI & PstI) | 1.8 µL | 2.1 µL |
| experiment | 9/13 pSB1C3 (XbaI & PstI) | 2.0 µL | 9/15 Pcon-PT181 antisense-Spinach-DT (XbaI & PstI) | 12.0 µL | 3.5 µL |
| experiment | 9/16 pSB1C3 (EcoRI & SpeI) | 1.0 µL | 9/15 Pcon-Spinach-DT (EcoRI & SpeI) | 4.1 µL | 2.5 µL |
| experiment | 9/16 pSB1C3 (EcoRI & SpeI) | 1.0 µL | 9/15 Pcon-RBS-tetR-DT (EcoRI & SpeI) | 2.0 µL | 1.5 µL |
| experiment | 9/16 pSB1C3 (EcoRI & SpeI) | 1.0 µL | 9/15 Pcon-PT181 attenuator-DT (EcoRI & SpeI) | 2.4 µL | 1.7 µL |
| experiment | 9/16 pSB1C3 (EcoRI & SpeI) | 1.0 µL | 9/15 Pcon-aptamer 12\_1R-DT (EcoRI & SpeI) | 2.0 µL | 1.5 µL |
| experiment | 9/13 pSB1C3 (XbaI & PstI) | 2.0 µL | 9/16 Pcon-pT181-attenuator (XbaI & PstI) | 4.4 µL | 3.2 µL |

### Gel Extraction

No Name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 2 | pT181 attenuator -(2) | EcoRI & SpeI |
| 3 |
| 4 |

[![Igku 9172.jpg](/wiki/images/c/c5/Igku_9172.jpg)](/File%3AIgku_9172.jpg)
[![Igku 9173.jpg](/wiki/images/4/4b/Igku_9173.jpg)](/File%3AIgku_9173.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pT181 attenuator (EcoI & SpeI) | 9.2 | 1.15 | 0.12 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 2 | pSB1C3 | EcoRI & SpeI |
| 3 |
| 4 |

[![Igku 9174.jpg](/wiki/images/e/e7/Igku_9174.jpg)](/File%3AIgku_9174.jpg)
[![Igku 9175.jpg](/wiki/images/7/78/Igku_9175.jpg)](/File%3AIgku_9175.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pSB1C3 | 10.0 | 1.84 | 0.40 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 2 | pSB4K5 | XbaI & PstI |
| 3 |
| 4 |

[![Igku 9176.jpg](/wiki/images/d/d5/Igku_9176.jpg)](/File%3AIgku_9176.jpg)
[![Igku 9177.jpg](/wiki/images/d/da/Igku_9177.jpg)](/File%3AIgku_9177.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pSB4K5 | 7.6 | 1.81 | 0.32 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 2 | Pcon-RBS-tetR-DT | XbaI & PstI |
| 3 |
| 4 |

[![Igku 9178.jpg](/wiki/images/e/e2/Igku_9178.jpg)](/File%3AIgku_9178.jpg)
[![Igku 9179.jpg](/wiki/images/b/b4/Igku_9179.jpg)](/File%3AIgku_9179.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-RBS-tetR-DT | 8.0 | 2.40 | 1.14 |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 2 | pSB1C3 | EcoRI & PstI |
| 3 |
| 4 |

[![Igku 91710.jpg](/wiki/images/2/29/Igku_91710.jpg)](/File%3AIgku_91710.jpg)
[![Igku 91711.jpg](/wiki/images/d/df/Igku_91711.jpg)](/File%3AIgku_91711.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pSB1C3 | 9.1 | 2.17 | 0.35 |

### Mutation PCR

Kinose

| pC KaiABC(100pg/µL) | Prime STAR(2x) | Primer(KaiA-BsaI MuT-Fwd) | Primer(KaiA-BsaI MuT-Rev) | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1.0 | 25.0 | 1.0 | 1.0 | 22.0 | 50.0 |
| 1.5 | 25.0 | 1.0 | 1.0 | 21.5 | 50.0 |
| 2.0 | 25.0 | 1.0 | 1.0 | 21.0 | 50.0 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 96 °C | 98 °C | 65 °C | 72 °C | -- |
| 5 min | 10 s | 15 s | 40 s | 30 cycles |

## Sep 18

### Restriction Enzyme Digestion

No name

|  | 9/18Plux | SpeI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 3.2µL | 1µL | 1µL | 3µL | 3µL | 18.8µL | 30µL |
| NC | 1.7µL | 0µL | 0µL | 1µL | 1µL | 6.3µL | 10µL |

|  | 9/18Pcon-pT181antisense-spinach-DT② | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 7.5µL | 1µL | 1µL | 3µL | 3µL | 14.5µL | 30µL |
| NC | 1.4icro;L | 0µL | 0µL | 1µL | 1µL | 6.6µL | 10µL |

|  | 9/16 Plac② | SpeI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 13.8µL | 1µL | 1µL | 3µL | 3µL | 14.5µL | 30µL |
| NC | 0.7µL | 0µL | 0µL | 1µL | 1µL | 7.3µL | 10µL |

|  | 9/16 Spinach-DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 17.6µL | 1µL | 1µL | 3µL | 3µL | 4.4µL | 30µL |
| NC | 0.9µL | 0µL | 0µL | 1µL | 1µL | 7.1µL | 10µL |

|  | 9/8 Pcon J23100 | SpeI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 5.7µL | 1µL | 1µL | 3µL | 3µL | 16.3µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |

|  | 9/16 RBS-lacZα-DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 8.4µL | 1µL | 1µL | 3µL | 3µL | 13.6µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 9/11 Pcon-pT181antisense-spinach-DT&#9312: | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 7.2µL | 1µL | 1µL | 3µL | 3µL | 14.8µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 9/6 apt12-1R-DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 7.9µL | 1µL | 1µL | 3µL | 3µL | 14.1µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 9/3 pT181antisense | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 6.3µL | 1µL | 1µL | 3µL | 3µL | 15.7µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1µL | 1µL | 7.7µL | 10µL |

|  | 9/14 pSB4K5 | EcoRI | SpeI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 7.3µL | 1µL | 1µL | 3µL | 3µL | 14.7µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 8/31 Plux-RBS-GFP-DT | EcoRI | SpeI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 9.2µL | 1µL | 1µL | 3µL | 3µL | 12.8µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL |

|  | 9/12 RBS-lysis3-DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 5.2µL | 1µL | 1µL | 3µL | 3µL | 16.8µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 9/18 Pcon-pT181attenuator | EcoRI | SpeI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 14.3µL | 1µL | 1µL | 3µL | 3µL | 7.7µL | 30µL |
| NC | 0.7µL | 0µL | 0µL | 1µL | 1µL | 7.3µL | 10µL |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | Plae② | SpeI | PstI |
| 2 | RBS-lysis3-DT | XbaI | PstI |
| 3 | Plux-RBS-GFP-DT | EcoRI | SpeI |
| 4 | PSB4K5 | EcoRI | SpeI |
| 5 | Pcon | SpeI | PstI |
| 6 | Plux | SpeI | PstI |
| 7 | 1kbp ladder | -- | -- |
| 8 | Plae② | -- | -- |
| 9 | RBS-lysis3-DT | -- | -- |
| 10 | Plux-RBS-GFP-DT | -- | -- |
| 11 | PSB4K5 | -- | -- |
| 12 | Pcon | -- | -- |
| 13 | Plux | -- | -- |

[![Igku 9183.jpg](/wiki/images/6/6b/Igku_9183.jpg)](/File%3AIgku_9183.jpg)

### Electrophoresis

Okazaki

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | Pcon pT181antisense spinach-DT② | XbaI | PstI |
| 3 | Spinach-DT | XbaI | PstI |
| 4 | RBS-lacZα-DT | XbaI | PstI |
| 5 | Pcon pT181antisense-Spinach-DT① | XbaI | PstI |
| 6 | aptamer12-1R-DT | XbaI | PstI |
| 7 | pT181attenuator | XbaI | PstI |
| 8 | Pcon attenuator | EcoRI | SpeI |

[![Igku 9181.jpg](/wiki/images/b/b4/Igku_9181.jpg)](/File%3AIgku_9181.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | Pcon pT181antisense spinach-DT② | XbaI | PstI |
| 3 | Spinach-DT | XbaI | PstI |
| 4 | RBS-lacZα-DT | XbaI | PstI |
| 5 | Pcon pT181antisense-Spinach-DT① | XbaI | PstI |
| 6 | apt12-1R-DT | XbaI | PstI |
| 7 | pT181attenuator | XbaI | PstI |
| 8 | Pcon attenuator | EcoRI | SpeI |

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | Pcon pT181antisense spinach-DT② | -- | -- |
| 3 | Spinach-DT | -- | -- |
| 4 | RBS-lacZα-DT | -- | -- |
| 5 | Pcon pT181antisense-Spinach-DT① | -- | -- |
| 6 | apt12-1R-DT | -- | -- |
| 7 | pT181attenuator | -- | -- |
| 8 | Pcon attenuator | -- | -- |

[![Igku 9182.jpg](/wiki/images/5/5e/Igku_9182.jpg)](/File%3AIgku_9182.jpg)

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ldder | -- |
| 2 | Plac② | SpeI+PstI |
| 3 | Plac② | SpeI+PstI |
| 4 | -- | -- |
| 5 | RBS-lysis3-DT | XbaI+PstI |
| 6 | RBS-lysis3-DT | XbaI+PstI |
| 7 | -- | -- |
| 8 | Plux-RBS-GFP-DT | EcoRI+SpeI |
| 9 | Plux-RBS-GFP-DT | EcoRi+SpeI |
| 10 | -- | -- |
| 11 | PSB4KS | EcoRI+SpeI |
| 12 | PSB4KS | EcoRI+SpeI |

[![Igku 9188.jpg](/wiki/images/9/9c/Igku_9188.jpg)](/File%3AIgku_9188.jpg)
[![Igku 9189.jpg](/wiki/images/1/18/Igku_9189.jpg)](/File%3AIgku_9189.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ldder | -- |
| 2 | Pcon | SpeI+PstI |
| 3 | Pcon | SpeI+PstI |
| 4 | -- | -- |
| 5 | Plux | SpeI+PStI |
| 6 | Plux | SpeI+PStI |

[![Igku 9184.jpg](/wiki/images/9/9b/Igku_9184.jpg)](/File%3AIgku_9184.jpg)
[![Igku 9185.jpg](/wiki/images/4/40/Igku_9185.jpg)](/File%3AIgku_9185.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | Pcon PT181 antisense spinach-DT | XbaI+PstI |
| 2 | Pcon PT181 antisense Spinach-DT | XbaI+PstI |
| 3 | 100bp ladder | -- |
| 4 | Pcon PT181 antisense Spinach-DT | XbaI+PstI |
| 5 | Pcon PT181 antisense Spinach-DT | XbaI+PstI |
| 6 | 1kbp ladder | -- |

[![Igku 9186.jpg](/wiki/images/c/c9/Igku_9186.jpg)](/File%3AIgku_9186.jpg)
[![Igku 9187.jpg](/wiki/images/f/f2/Igku_9187.jpg)](/File%3AIgku_9187.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | apt12-1R-DT | XbaI+PstI |
| 3 | apt12-1R-DT | XbaI+PstI |
| 4 | -- | -- |
| 5 | PT181 attenuator | XbaI+PstI |
| 6 | PT181 attenuator | XbaI+PstI |

[![Igku 91810.jpg](/wiki/images/e/eb/Igku_91810.jpg)](/File%3AIgku_91810.jpg)
[![Igku 91811.jpg](/wiki/images/c/c6/Igku_91811.jpg)](/File%3AIgku_91811.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | Pcoa attenuator | EcoRI+PstI |
| 3 | Pcoa attenuator | EcoRI+PstI |
| 4 | -- | -- |
| 5 | RBS-lacZα-DT | XbaI+PstI |
| 6 | RBS-lacZα-DT | XbaI+PstI |

[![Igku 91812.jpg](/wiki/images/3/31/Igku_91812.jpg)](/File%3AIgku_91812.jpg)
[![Igku 91813.jpg](/wiki/images/b/be/Igku_91813.jpg)](/File%3AIgku_91813.jpg)

## Sep 19

## Sep 20

### Ligation

Okazaki, Kanaoka, Tatsui and Hirano

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/18 Plac (SpeI & PstI) | 3.0 µL | 9/18 RBS-lysis3-DT (XbaI & PstI) | 12.4 µL | 3.5 µL |
| experiment | 9/12 Ptet-pT181 antisense | 1.0 µL | 9/18 spinach-DT | 11.9 µL | 3.5 µL |
| experiment | 9/13 Pcon-RBS-luxR-DT | 2.1 µL | 9/10 Plux-RBS-GFP-DT | 5.6 µL | 3.5 µL |
| experiment | 9/13 Pcon-pT181 attenuator (SpeI & PstI) | 2.0 µL | 9/18 aptamer 12-1R-DT (XbaI & PstI) | 6.3 µL | 3.5 µL |
| experiment | 9/18 Plac ②(SpeI & PstI) | 3.0 µL | 9/18 aptamer 12-1R-DT(XbaI & PstI) | 4.5µL | x3.5 µL |

### Transformation

Tatsui

| Name | Sample | Competent Cells | Plate |
| --- | --- | --- | --- |
| 9/17 pSB1C3-Pcon-pT181 attenuator-DT | 2 µL | 20 µL | CP |
| 9/17 pSB1C3-Pcon-aptamer 12\_1R-DT | 2 µL | 20 µL | CP |
| 9/17 pSB1C3-Pcon-antisense-spinach-DT | 2 µL | 20 µL | CP |
| 9/17 pSB1C3-Pcon-RBS-tetR-DT x2 | 2 µL | 20 µL | CP |
| 9/14 pSB1C3-PkaiBC | 2 µL | 20 µL | CP |
| 9/14 pSB1C3-RpaB | 2 µL | 20 µL | CP |
| 9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) | 2 µL | 20 µL | CP |
| 9/17 pSB1C3-pT181 attenuator(XbaI&PstI) | 2 µL | 20 µL | CP |
| 9/17 pSB1C3-Pcon-spinach-DT | 2 µL | 20 µL | CP |
| 9/14 RBS-GFP-DT-PkaiBC | 2 µL | 20 µL | CP |
| Plux-RBS-lysis3-DT | 2 µL | 20 µL | CP |
| 9/20 Ptet-pT181 antisense-spinach-DT | 2 µL | 20 µL | CP |
| 9/20 Pcon-spinach-DT(pSB4K5) | 2 µL | 20 µL | Kan |
| 9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) | 2 µL | 20 µL | Kan |
| 9/20 KaiABC | 2 µL | 20 µL | Kan |
| 9/20 KaiABC | 2 µL | 20 µL | Kan |
| 9/20 Plac-RBS-lysis3-DT | 2 µL | 20 µL | Amp |
| 9/20 Plac-aptamer 12\_1R | 2 µL | 20 µL | Amp |
| 9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT | 2 µL | 20 µL | Amp |
| Plac-aptamer12\_1R-DT | 2 µL | 20 µL | Amp |
| 9/20 Plac-aptamer12\_1R-DT | 2 µL | 20 µL | Amp |
| 9/20Pcon-pT181attenuator-aptamer-DT | 2 µL | 20 µL | Amp |
| 9/17 Plac-pT181-attenuatorx2 | 2 µL | 20 µL | Amp |
| 9/15 Pcon-RBS-lacZα-DT | 2 µL | 20 µL | Amp |
| 9/14 Pcon-pT181attenuator-RBS-lacZα-DT | 2 µL | 20 µL | Amp |
| PUC19 | 2 µL | 20 µL | Amp |

### PCR

Tatsui

| Sample | base pair |
| --- | --- |
| pT181 attenuator(pSB1C3) | -- |
| Pcon-RBS-lacZα-DT | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94 °C | 94 °C | 55 °C | 68 °C | -- |
| 5 min | 30 s | 30 s | 3 min | 30 cycles |

### Electrophoresis

No name

| Lane | Sample | Enzyme |
| --- | --- | --- |
| 1 | Pcon-RBS-lacZα-DT | -- |
| 2 | 1kb ladder | -- |
| 3 | pSB1C3-pT181attenuator | -- |

## Sep 21

### Electrophoresis

no name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | Pcon-RBS-lacZα-DT | -- | -- |
| 2 | 1kbp ladder | -- | -- |
| 3 | pSB1C3+pT181 attenuator | -- | -- |

[![Igku 9211.jpg](/wiki/images/c/cc/Igku_9211.jpg)](/File%3AIgku_9211.jpg)

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 9/2 spinach-DT-2 | -- |
| 8/16 Plux-4 | -- |
| 8/27 Plux-RBS-GFP-DT-1 | -- |
| 9/5 Pcon-pT181 attenuator-1 | -- |

### Master Plate

No name

| Number | Use LB plate |
| --- | --- |
| 1 | 8/27 Pλ-luxI-1(+Amp) |
| 2 | 9/10 Plac(BBa-R0011)-3(+Amp) |
| 3 | 9/5 Pcon-RBS-tetR-DT-1(+Amp) |
| 4 | 9/5 Pcon-pT181 antisense-1(+Amp) |
| 5 | 9/15 Pcon-pT181 attenuator(+Amp) |
| 6 | 9/- Pcon-apt12\_1R-DT-1(+Amp) |
| 7 | 9/5 Pcon-spinach-DT-3(+Amp) |
| 8 | 8/27 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT(+Amp) |

| Number | Use LB plate |
| --- | --- |
| 1 | 9/4 pT181 antisense(pSB1C3)-1(+CP) |
| 2 | 9/11 Fusion1 antisense(pSB1C3)-4(+CP) |
| 3 | 9/11 Fusion6 antisense(pSB1C3)-1(+CP) |
| 4 | 9/4 pT181 attenuator(pSB1C3)-1(+CP) |
| 5 | 9/10 Fusion1 attenuator(pSB1C3)-1(+CP) |
| 6 | 9/11 Fusion3n2 attenuator(pSB1C3)-1(+CP) |
| 7 | 9/4 aptamer12\_1R(pSB1C3)-1(+CP) |
| 8 | 9/11 aptamer12\_P(pSB1C3)-3(+CP) |
| 9 | 9/11 aptamer12\_1M(pSB1C3)-1(+CP) |

| Number | Use LB plate |
| --- | --- |
| 1 | 8/29 Pbad/araC-RBS-RFP-DT-1(+CP) |
| 2 | 9/4 RBS-lysis3-DT-3(+CP) |
| 3 | 9/10 Pcon-pT181 attenuator-DT-1(+CP) |
| 4 | 9/5 Ptet-pT181 antisense-1(+CP) |
| 5 | 9/4aptamer 12\_1R-DT-1(+CP) |
| 6 | 9/5 Ptet-RBS-lacZα-DT-4(+CP) |
| 7 | 8/10 RBS-lacI-DT-2(+CP) |
| 8 | 8/29 Plux-RBS-GFP-DT-1(+CP) |

### Restriction Enzyme Digestion

Noname

|  | 8/17 RBS-DT | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts(XbaI&PstI) | 8.1 | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 13.9µL | 30µL |
| NC | 0.4 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 8/17 RBS-DT | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cut(EcoRI&XbaI) | 16.2 | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 5.8µL | 30µL |

|  | 9/15 Pcon-pT181attenuator | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts(EcoRI&PstI) | 12 | 1µL | 0µL | 0µL | 1µL | 3µL | 3µL | 10µL | 30µL |
| NC | 0.6 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.4µL | 10µL |

|  | 8/20 Pcon=RBS-GFP-DT-1 | EcoRI | SpeI | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cut(EcoRI&XbaI) | 6 | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 0µL | 14µL |
| NC | 0.3 | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.3µL | 10µL |

incubate 37°C 1hour

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | RBS-GFP-DT | XbaI | PstI |
| 2 | RBS-GFP-DT | -- | -- |
| 3 | RBS-GFP-DT | EcoRI | XbaI |
| 4 | 1kbp ladder | -- | -- |
| 5 | Pcon-pT181attenuator | SpeI | PstI |
| 6 | Pcon-pT181attenuator | -- | -- |
| 7 | Pcon-RBs-GFP-DT | EcoRI | XbaI |
| 8 | Pcon-RBs-GFP-DT | -- | -- |

[![Igku 9212.jpg](/wiki/images/5/50/Igku_9212.jpg)](/File%3AIgku_9212.jpg)

### PCR

Nakamoto

| Sample | base pair |
| --- | --- |
| Pcon-apt12\_1R-DT(E-1A) | 50 |
| Pcon-spinach-DT(E-1A) | 402 |
| Pcon-pT181 attenuator(E-1A) | 400 |
| DT(1-3) | 65 |
| tetR aptamer 12\_1R-DT(1-3) | 283 |
| Pcon-pT181 attenuator(1-SA) | 303 |
| spinach-DT(2-1) | 338 |
| Pcon-pT181 attenuator(E-0) | 403 |
| Pcon-pT181 attenuator(2-SA) | 403 |
| spinach-DT(3-2) | 395 |
| Ptet-PT181 anisense | 400 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 35s | 30cycles |

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kb ladder | -- |
| 3 | RBS-GFP-DT | XbaI&PstI |
| 4 | RBS-GFP-DT | XbaI&PstI |
| 5 | RBS-GFP-DT | XbaI&PstI |

[![Igku 9213.jpg](/wiki/images/3/33/Igku_9213.jpg)](/File%3AIgku_9213.jpg)
[![Igku 9214.jpg](/wiki/images/4/4b/Igku_9214.jpg)](/File%3AIgku_9214.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| RBS-GFP-DT(XbaI&PstI) | 14.8 | 1.74 | 0.47 |
| RBS-GFP-DT(EcoRI&SpeI) | 88.4 | 1.85 | 1.49 |
| Pcon-RBS-GFP-DT(EcoRI&XbaI) | 47.5 | 1.82 | 1.12 |

## Sep 22

### Colony PCR

Tatsui

| Sample | base pair |
| --- | --- |
| 9/20 Plac-PT181 attenuator(1~4) | 664 |
| 9/20 Plac-PT181 antisense(1~6) | 369 |
| 9/20 Pcon-PT181 attenuator-aptamer-DT(1~6) | 859 |
| 9/20 Pcon-PT181 antisense-spinach-DT(1~8) | 723 |

[![Igku 0903 E1.jpg](/wiki/images/2/22/Igku_0903_E1.jpg)](/File%3AIgku_0903_E1.jpg)
[![Igku 0922 E2.jpg](/wiki/images/c/c9/Igku_0922_E2.jpg)](/File%3AIgku_0922_E2.jpg)

### Electrophoresis

Nakamoto

| Lane | Sample |
| --- | --- |
| 1 | Pcon-apt12\_1R-DT(E-1A) |
| 2 | Pcon-spinach-DT(E-1A) |
| 3 | Pcon-pT181 attenuator(E-1A) |
| 4 | DT(1-3) |
| 5 | tetR aptamer 12\_1R-DT(1-3) |
| 6 | 100bp ladder |
| 7 | Pcon-pT181 attenuator(1-SA) |
| 8 | spinach-DT(2-1) |
| 9 | Pcon-pT181 attenuator(E-0) |
| 10 | Pcon-pT181 attenuator(2-SA) |
| 11 | spinach-DT(3-2) |
| 12 | Ptet-PT181 anisense |

[![Igku 0922 E3.jpg](/wiki/images/b/be/Igku_0922_E3.jpg)](/File%3AIgku_0922_E3.jpg)
[![Igku 0922 E4.jpg](/wiki/images/5/5e/Igku_0922_E4.jpg)](/File%3AIgku_0922_E4.jpg)

### Miniprep

Honda

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/21 spinach-DT | 197.9 | 1.67 | 1.54 |
| 9/21 Plux | 125.9 | 1.71 | 1.27 |
| 9/21 Plux-RBS-GFP-DT-(1) | 168.0 | 1.62 | 1.73 |
| 9/21 Pcon-PT181 attenuator-(1) | 368.3 | 1.57 | 1.62 |
| 9/21 RBS-GFP-DT | 532.8 | 1.75 | 1.78 |
| 9/21 Pcon-RBS-GFP-DT | 845.2 | 2.02 | 1.70 |

### Colony PCR

Honda

| Sample | base pair |
| --- | --- |
| 9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1~2) | 283 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 2min10s | 30cycles |

[![Igku 0922 E5.jpg](/wiki/images/5/54/Igku_0922_E5.jpg)](/File%3AIgku_0922_E5.jpg)
[![Igku 0922 E6.jpg](/wiki/images/4/4c/Igku_0922_E6.jpg)](/File%3AIgku_0922_E6.jpg)

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2~4 | Pcon-aptamer12-1R-DT(E-1A) | -- |
| 6~8 | Pcon-spinach-DT(E-1A) | -- |
| 10~12 | Pcon-attenuator PT181(E-1A) | -- |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2~4 | DT(1-3) | -- |
| 6~8 | aptamer12-1RDT(1-3) | -- |
| 10~12 | Pcon-PT181 attenuator(1-5A) | -- |

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-aptamer12-1R-DT(E-1A) | 31.2 | 1.87 | 0.30 |
| Pcon-spinach-DT(E-1A) | 25.6 | 1.88 | 0.99 |
| Pcon-attenuator PT181(E-1A) | 25.3 | 1.99 | 0.86 |
| DT(1-3) | 22.1 | 1.82 | 0.20 |
| aptamer12-1RDT(1-3) | 33.5 | 1.99 | 0.05 |
| Pcon-PT181 attenuator(1-5A) | 36.9 | 1.12 | 0.96 |

[![Igku 0922 G1.jpg](/wiki/images/e/e2/Igku_0922_G1.jpg)](/File%3AIgku_0922_G1.jpg)
[![Igku 0922 G2.jpg](/wiki/images/d/d7/Igku_0922_G2.jpg)](/File%3AIgku_0922_G2.jpg)
[![Igku 0922 G3.jpg](/wiki/images/7/7d/Igku_0922_G3.jpg)](/File%3AIgku_0922_G3.jpg)
[![Igku 0922 G4.jpg](/wiki/images/c/c0/Igku_0922_G4.jpg)](/File%3AIgku_0922_G4.jpg)

### Restriction Enzyme Digestion

No name

|  | 9/16 Plac | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 12.5µL | 0µL | 1µL | 1µL | 3µL | 3µL | 9.5µL | 30µL |
| NC | 0.8µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.4µL | 10µL |

|  | 9/22 Plux | XbaI | SpeI | PstI | BufferB | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 16µL | 0µL | 1µL | 1µL | 3µL | 3µL | 14µL | 30µL |
| NC | 0.8µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.2µL | 10µL |

|  | 9/20 RBS-tetR-2R-DT | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 8.5µL | 1µL | 0µL | 1µL | 3µL | 3µL | 13.5µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

[![Igku 0922 E7.jpg](/wiki/images/a/aa/Igku_0922_E7.jpg)](/File%3AIgku_0922_E7.jpg)

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2~4 | Pcon-attenuator PT181(E-0) | -- |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2~4 | Pcon-attenuator PT181(E-0) | -- |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 2 | 100bp ladder | -- |
| 4~6 | Pcon-attenuator PT181(2-SA) | -- |

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 3~5 | spinach-DT(3-2) | -- |
| 8~10 | Ptet-antisense PT181(1-3) | -- |

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-attenuator PT181(E-0) | 2.4 | 1.80 | 0.17 |
| Pcon-attenuator PT181(2-SA) | 10.8 | 1.85 | 0.54 |
| spinach-DT(3-2) | 36.7 | 1.80 | 0.75 |
| Ptet-antisense PT181(1-3) | 16.8 | 1.74 | 0.78 |

[![Igku 0922 G5.jpg](/wiki/images/8/85/Igku_0922_G5.jpg)](/File%3AIgku_0922_G5.jpg)
[![Igku 0922 G6.jpg](/wiki/images/1/1a/Igku_0922_G6.jpg)](/File%3AIgku_0922_G6.jpg)
[![Igku 0922 G7.jpg](/wiki/images/9/9b/Igku_0922_G7.jpg)](/File%3AIgku_0922_G7.jpg)
[![Igku 0922 G8.jpg](/wiki/images/f/f6/Igku_0922_G8.jpg)](/File%3AIgku_0922_G8.jpg)
[![Igku 0922 G9.jpg](/wiki/images/e/ee/Igku_0922_G9.jpg)](/File%3AIgku_0922_G9.jpg)
[![Igku 0922 G10.jpg](/wiki/images/7/7d/Igku_0922_G10.jpg)](/File%3AIgku_0922_G10.jpg)

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 9/20 Pcon-PT181 attenuator-aptamer12-1R-DT-(7~14) | 859 |

### Colony PCR

Tatsui

| Sample | base pair |
| --- | --- |
| PconPT181antisense-spinach-DT(9~15) | 723 |

[![Igku 0922 E8.jpg](/wiki/images/e/ed/Igku_0922_E8.jpg)](/File%3AIgku_0922_E8.jpg)

### Ligation

No name

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/13 Pcon-PT181 attenuator (SpeI & PstI) | 1.7 µL | 9/18 aptamer-1R-DT (XbaI & PstI) | 5.4µL | 3.5µL |
| experiment | 9/12 Plac(SpeI & PstI) | 2.4 µL | 9/18 aptamer-1R-DT (XbaI & PstI) | 6.1µL | 3.5 µL |
| experiment | 9/13 Ptet(SpeI & PstI) | 1.7 µL | 9/10 Plux-RBS-GFP-DT | 7.0 µL | 4.4 µL |
| experiment | 9/21 RBS-GFP-DT (EcoRI & XbaI) | 0.6 µL | 9/18 aptamer 12-1R-DT (XbaI & PstI) | 5.6µL | 3.1 µL |
| experiment | 9/13 PSB1C3(XbaI & PstI) | 1.9 µL | 9/16 PT181 attenuator(XbaI & PstI) | 4.6µL | 3.3 µL |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| 9/20 Plac-antisense-1 | -- |
| 9/20 Plac-attenuator-1 | -- |
| 9/20 Plac-antisense-1 | -- |
| 9/20 Plac-attenuator-1 | -- |

### digestion

No name

|  | 9/8 Pcon-PT181 antisense | EcoRI | SpeI | BufferM | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 8.3µL | 1µL | 1µL | 3µL | 16.7µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 8.3µL | 10µL |

|  | 9/7 Pcon-RBS-tetR-DT | EcoRI | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cut | 14µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 18.2µL | 30µL |
| NC | 0.7µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.3µL | 10µL |

|  | 9/22 RBS-GFP-DT | EcoRI | XbaI | SpeI | PstI | BufferD | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 3.8µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 18.2µL | 30µL |
| NC | 0.2µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.8µL | 10µL |

|  | 9/6 aptamer12-1R-DT | EcoRI | XbaI | SpeI | PstI | BufferD | BSA | MilliQ |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2cut | 8µL | 1µL | 1µL | 0µL | 0µL | 3µL | 3µL | 16µL |
| NC | 0.4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL |

incubate 37°C 1hour

[![Igku 0922 E9.jpg](/wiki/images/d/d8/Igku_0922_E9.jpg)](/File%3AIgku_0922_E9.jpg)

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 3~5 | Pcon-PT181 antisense (EcoRI&SpaI) | -- |

[![Igku 0922 G11.jpg](/wiki/images/7/7f/Igku_0922_G11.jpg)](/File%3AIgku_0922_G11.jpg)
[![Igku 0922 G12.jpg](/wiki/images/d/d0/Igku_0922_G12.jpg)](/File%3AIgku_0922_G12.jpg)

### Transformation

No name

| Name | Sample | Competent Cells | Plate |
| --- | --- | --- | --- |
| 9/20 Plac-aptamer12-1R-DT(XbaI&PstI) | 2 µL | 20 µL | Amp |
| 9/20 pT181 attenuator(1C3)(EcoRI&SpeI) | 2 µL | 20 µL | CP |
| 9/17 pT181 attenuator(1C3)(XbaI&PstI) | 2 µL | 20 µL | CP |
| 9/20 Pcon-pT181 attenuator(SpaI&PstI)＋aptamer12-1R-DT(XbaI&PstI) | 2 µL | 20 µL | Amp |
| kaiABC | 2 µL | 20 µL | Amp |
| 9/22 pSB1C3-RpaB(XbaI&PstI) | 2 µL | 20 µL | Amp |
| 9/22 pSB1C3-pT181 attenuator(XbaI&PstI) | 2 µL | 20 µL | Amp |
| 9/22 Ptet(SpaI&PstI)＋RBS-GFP-DT(XbaI&PstI) | 2 µL | 20 µL | CP |
| 9/22 RBS-GFP-DT＋Pcon-attenuator pT181 (EcoRI&SpeI) | 2 µL | 20 µL | CP |
| 9/22 pT181 attenuator(XbaI&PstI)(1C3) | 2 µL | 20 µL | CP |

### PCR

No name

| Sample | base pair |
| --- | --- |
| Pcon-RBS-tetR-DT(1-2(A)) | -- |
| Pcon-RBS-tetR-DT(3-2(A)) | -- |
| Pcon-RBS-GFP-DT(1-S(A)) | -- |
| Pcon-RBS-GFP-DT(2-S(A)) | -- |
| Pcon-tetR-DT(E-2(A)) | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94 °C | 94 °C | 57 °C | 68 °C | -- |
| 2 min | 10 s | 30 s | 1min5s | 30 cycles |

[![Igku 0922 E10.jpg](/wiki/images/2/2b/Igku_0922_E10.jpg)](/File%3AIgku_0922_E10.jpg)

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 1kbp ladder |
| 2 | Pcon-RBS-tetR-DT(1-2Ⓐ) |
| 3 | Pcon-RBS-tetR-DT(3-2Ⓐ) |
| 4 | Pcon-RBS-GFP-DT(1-SⒶ) |
| 5 | Pcon-RBS-GFP-DT(2-SⒶ) |
| 6 | Pcon-tetR-DT(E-2Ⓐ) |

[![Igku 0922 E10.jpg](/wiki/images/2/2b/Igku_0922_E10.jpg)](/File%3AIgku_0922_E10.jpg)

## Sep 23

### Transformation

No name

| Name | Sample | Competent Cells | Plate |
| --- | --- | --- | --- |
| 9/14 pSB1C3＋PkaiBC | 2 µL | 20 µL | -- |
| 9/14 pSB1C3＋RpaB | 2 µL | 20 µL | -- |
| 9/14 pkaiBC＋Rbs-GFP-DT | 2 µL | 20 µL | -- |

### Gel Extraction

No Name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder |  |
| 3-4 | Pcon-RBS-tetR-PT |  |

[![Igku Sep23 Gel Extraction1.jpg](/wiki/images/0/0a/Igku_Sep23_Gel_Extraction1.jpg)](/File%3AIgku_Sep23_Gel_Extraction1.jpg)
[![Igku Sep23 Gel Extraction2.jpg](/wiki/images/e/e5/Igku_Sep23_Gel_Extraction2.jpg)](/File%3AIgku_Sep23_Gel_Extraction2.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-PT181 anti EcoRI+SpeI | 46.7 | 1.06 | 0.93 |
| Pcon-RBS-tetR-DT EcoRI+XbaI | 119.3 | 0.99 |  |
| RBS-8FP-DT EcoRI+XbaI | 87.7 | 1.00 | 0.98 |
| ap42\_1R-DT EcoRI+XbaI | 68.8 | 1.32 | 1.03 |
| Pcon-RBS-tetR-DT 1-2 A | 203.0 | 1.66 | 1.79 |
| Pcon-RBS-tetR-DT 3-2 A | 67.7 | 2.64 | 1.28 |
| Pcon-RBS-GFP-DT 1-S A | 185.6 | 1.88 | 2.04 |
| Pcon-RBS-GFP-DT 2-S A | 298.6 | 1.68 | 1.55 |
| Pcon-tetR-DT E-2 A | 202.4 | 1.57 | 1.66 |

### PCR

No name

| genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | SasA\_fwd primer | SasA\_rev primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 9/8 Pcon-pT181 anti | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |
| 8/20 Ptet(1) | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |
| 9/16 Plac 2 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94 °C | 98 °C | 57 °C | 68 °C | -- |
| 2min | 10sec | 30sec | 20sec | 30cycles |

### Ligation

Yoshida

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/21 Pcon-RBS-GFP-DT EcoRI+XbaI | 47.5 µg/mL | 9/15 Pcon-RBS-tetR-DT EcoRI+SpeI | 59 µg/mL | 2.3 µg/mL |
| experiment | 9/21 Pcon-RBS-GFP-DT EcoRI+XbaI | 47.5 µg/mL | 9/15 Pcon-apt12\_1R-DT EcoRI+SpeI | 17 µg/mL | 2.4 µg/mL |
| experiment | 9/21 Pcon-RBS-GFRDT EcoRI+XbaI | 2.1 µg/mL | 9/15 Pcon-DT181 attenuator-DT | 21 µg/mL | 3.0 µg/mL |
| experiment | 9/22 apt 12\_1R-DT EcoRI+XbaI | 67.8 µg/mL | 9/18 Pcon-DT181 attenuator EcoRI+SpeI | 5.7 µg/mL | 3.5 µg/mL |
| experiment | 9/22RBS-GFP-DT EcoRI+XbaI | 87.7 µg/mL | 9/18 Pcon-DT18 attenuator EcoRI+SpeI | 6.7 µg/mL | 3.5 µg/mL |
| experiment | 9/13 DT EcoRI+XbaI | 25.4 µg/mL | 9/22 Pcon-DT181 antisense EcoRI+SpeI | 46.8 µg/mL | 2.3 µg/mL |

### PCR

No name

| genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | SasA\_fwd primer | SasA\_rev primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Pcon-attenuator-DT (E-1A) | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 77.5 | 25 |
| Pcon-attenuator-DT (2-1A) | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 77.5 | 25 |
| Pcon-antisense-spinach-DT (1-2A) | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 77.5 | 25 |
| Pcon-antisense-spinach-DT (E-1A) | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 77.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94 °C | 98 °C | 57 °C | 68 °C | -- |
| 2min | 10s | 30s | 45s | 30cycle |

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/22 Plac-DT181 attenuator-1 | 204.0 | 2.00 | 1.95 |
| 9/22 Plac-DT181 attenuator-2 | 195.3 | 1.84 | 1.62 |
| 9/22 Plac-DT181 antisense-1 | 188.2 | 1.87 | 2.15 |
| 9/22 Plac-DT181 antisense-2 | 165.0 | 1.95 | 2.66 |

### Electrophoresis

Hirano

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1,6 | 100bp ladder(Promega) | -- | -- |
| 2 | Pcon-attenuator-DT (E-1A) | -- | -- |
| 3 | Pcon-attenuator-DT (2-1A) | -- | -- |
| 4 | Pcon-antisense-spinach-DT (1-2A) | -- | -- |
| 5 | Pcon-antisense-spinach-DT (E-1A) | -- | -- |

[![Igku 9232.png](/wiki/images/5/5b/Igku_9232.png)](/File%3AIgku_9232.png)

### Gel Extraction

Hirano

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-pT181 attenuator-DT(E-1A) | 166.7 | 1.51 | 0.99 |
| Pcon-pT181 attenuator-DT(2-1A) | 122.1 | 1.75 | 1.20 |
| Pcon-pT181 antisense-spinach-DT(1-2A) | 99.1 | 1.86 | 1.81 |

### Electrophoresis

Hirano

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | 9/8 Pcon-pT181 antisense(E-1A) | -- | -- |
| 3 | 8/20 Ptet(1)(2-S) | -- | -- |
| 4 | 9/16 Plac2(E-1A) | -- | -- |

[![Igku 9231.png](/wiki/images/2/2e/Igku_9231.png)](/File%3AIgku_9231.png)

### Transformation

No Name

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| pSB1C3 | 2µL | 20µL | 22µL |  |

### Ligation

No Name

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/19 Plac SpeI+PstI | 24.6 µg/mL | 9/18 ape12\_1R-DT XbaI+PstI | 4.5 µg/mL | 2 µg/mL |
| experiment | 9/21 RBS-GFP-DT EcoRI+XbaI | 88.4 µg/mL | 9/15 Pcon-pT181 attenuator EcoRI+SpeI | 21 µg/mL | 2 µg/mL |
| experiment | 9/13 PSB1C3 XbaI+PstI | 26.0 µg/mL | 9/16 pT181 attenuator XbaI+PstI | 8.2 µg/mL | 2 µg/mL |

### Colony PCR

Tatsui

| Sample | base pair |
| --- | --- |
| 9/22 Pcon-PT181 attenuator12-1R-DT(1~8) | -- |
| 9/22 Pcon-PT181 attenuator(1~4) | -- |
| 9/20 Plac＋aptamer12-1R-DT(1~4) | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 54s | 30cycle |

## Sep 24

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 9/20 Pcon-pT181aptamer12-1R-DT 1 |
| 2 | 9/20 Pcon-pT181aptamer12-1R-DT 2 |
| 3 | 9/20 Pcon-pT181aptamer12-1R-DT 3 |
| 4 | 9/20 Pcon-pT181aptamer12-1R-DT 4 |
| 5 | 9/20 Pcon-pT181aptamer12-1R-DT 5 |
| 6 | 9/20 Pcon-pT181aptamer12-1R-DT 6 |
| 7 | 9/20 Pcon-pT181aptamer12-1R-DT 7 |
| 8 | 9/20 Pcon-pT181aptamer12-1R-DT 8 |
| 9 | 100bp ladder |
| 10 | 9/22 Pcon-pT181attenuator-aptamer12-1R-DT 1 |
| 11 | 9/22 Pcon-pT181attenuator-aptamer12-1R-DT 2 |
| 12 | 9/22 Pcon-pT181attenuator-aptamer12-1R-DT 3 |
| 13 | 9/22 Pcon-pT181attenuator-aptamer12-1R-DT 4 |
| 14 | 9/20 Plac-aptamer12-1R-DT 1 |
| 15 | 9/20 Plac-aptamer12-1R-DT 2 |
| 16 | 9/20 Plac-aptamer12-1R-DT 3 |
| 17 | 9/20 Plac-aptamer12-1R-DT 4 |

[![Igku 0924 E1.jpg](/wiki/images/2/24/Igku_0924_E1.jpg)](/File%3AIgku_0924_E1.jpg)

### Transformation

No name

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| 9/23 pSB1C3-pT181attenuator | 2 µL | 20 µL | 22 µL | LB (+CP) |
| RBS-GFP-DT+Pcon-pT181attenuator | 2 µL | 20 µL | 22 µL | LB (+CP) |
| 9/23 Plac-+spinach-DT | 2 µL | 20 µL | 22 µL | LB (+Amp) |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT | 2 µL | 20 µL | 22 µL | LB (+Amp) |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT | 2 µL | 20 µL | 22 µL | LB (+Amp) |
| 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT | 2 µL | 20 µL | 22 µL | LB (+Amp) |
| 9/23 Pcon-pT181antisense-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |
| 9/23 Pcon-pT181attenuator-RBS-GFP-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |
| 9/23 Pcon-pT181attenuator-aptamer12-1R-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |
| PUC19 | 2 µL | 20 µL | 22 µL | LB (+Amp) |
| Ptet-RBS-lacZα-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |
| pSB1C3 | 2 µL | 20 µL | 22 µL | LB (+CP) |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| aptamer12-1R-DT(Master plate) | LB(+CP) |

### Restriction Enzyme Digestion(PCR product)

Hirano

Concentrate PCR product by evaporator until volume become less than 1µL, add all sulution and pipetting.

|  | 10\*cut smart buffer | BsaI-HF | MilliQ | total |
| --- | --- | --- | --- | --- |
| 9/22 Pcon-pT181 attenuator (E-0) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 Pcon-pT181 attenuator (2-3A) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 spinach-DT(3-2) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 Ptet-pT181 antisense (1-3) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 Pcon-tet aptamer-DT (E-1A) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 Pcon-spinach-DT (E-1A) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 Pcon-pT181 attenuator (E-1A) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 DT (1-3) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 tet aptamer-DT (1-3) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 Pcon-pT181 attenuator (1-SA) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 Pcon-RBS-tetR-DT (1-2A) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 Pcon-RBS-tetR-DT (3-2A) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 Pcon-RBS-tetR-DT (E-2A) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 Pcon-RBS-GFP-DT (1-2A) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/22 Pcon-RBS-GFP-DT (2-SA) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/23 Pcon-pT181 attenuator-DT (E-1A) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/23 Pcon-pT181 attenuator-DT (0-1A) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/23 Pcon-pT181 antisense-spinach-DT (1-2A) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/23 Pcon-pT181 antisense (1-2A) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/23 Ptet-1 (2-S) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |
| 9/23 Plac-2 (E-1A) | 2.0 µL | 0.3 µL | 17 µL | 19.3µL |

Incubate 37°C 12hour

### Column Refining

Kojima

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/22 Pcon-pT181 attenuator(E-0) | 7.8 | 1.70 | 0.03 |
| 8/22 Pcon-pT181 attenuator(2-SA) | 17.3 | 1.93 | 0.03 |
| 8/22 spinach-DT (3-2) | 22.4 | 1.93 | 0.09 |
| 8/22 Ptet-PT181 anisense (1-3) | 18.3 | 1.58 | 0.14 |
| 8/22 Pcon-aptamer12\_1R-DT (E-1A) | 8.2 | 2.10 | 0.05 |
| 8/22 Pcon-spinach-DT(E-1A) | 5.6 | 0.91 | 0.05 |
| 8/22 Pcon-pT181 attenuator(E-1A) | 18.5 | 1.80 | 0.12 |
| 8/22 DT(1-3) | 7.3 | 1.13 | 0.04 |
| 8/22 tetR aptamer 12\_1R-DT(1-3) | 14.6 | 1.74 | 0.06 |
| 8/22 Pcon-pT181 attenuator(1-SA) | 7.9 | 1.57 | 0.04 |
| 8/22 Pcon-RBS-tetR-DT(1-2Ⓐ) | 41.6 | 1.81 | 0.25 |
| 8/22 Pcon-RBS-tetR-DT(3-2Ⓐ) | 30.2 | 1.75 | 0.15 |
| 8/22 Pcon-RBS-tetR-DT(E-2Ⓐ) | 36.9 | 1.75 | 0.56 |
| 8/22 Pcon-RBS-GFP-DT(1-SⒶ) | 43.5 | 1.83 | 0.33 |
| 8/22 Pcon-RBS-GFP-DT(2-SⒶ) | 64.6 | 1.82 | 0.30 |
| 8/23 Pcon-pT181 attenuator-DT (E-1A) | 31.2 | 1.83 | 0.09 |
| 8/23 Pcon-pT181 attenuator-DT (0-1A) | 64.5 | 1.80 | 0.43 |
| 8/23 Pcon-pT181 antisense-spinach-DT (1-2A) | 36.4 | 1.88 | 0.21 |
| 8/23 Pcon-pT181 antisense-spinach-DT (E-1A) | 27.9 | 1.92 | 0.16 |
| 8/23 Ptet(1)(2-S) | 37.0 | 1.91 | 0.16 |
| 9/23 Plac-2(E-1A) | 21.2 | 1.82 | 0.13 |

### Transformation

No name

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| 9/23 Pcon-attenuator-aptamer12-1R-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |
| 9/23 pT181attenuator(1C3) | 2 µL | 20 µL | 22 µL | LB (+CP) |
| 9/23 Pcon-pT181antisense-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |
| 9/23 Pcon-pT181attenuator-RBS--GFP-DT | 2 µL | 20 µL | 22 µL | LB (+CP) |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| pSB1C3 (Master plate-1) | LB(+CP) |

### Restriction Enzyme Digestion

Kojima

|  | 8/21 pT181attenuator (330 µg/mL) | XbaI | PstI | buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 6.1 | 1 | 1 | 3 | 3 | 15.9 | 30 |
| NC | 0.3 | 0 | 0 | 1 | 1 | 7.7 | 10 |

|  | 9/22 Pcon-pT181attenuator (368.3 µg/mL) | EcoRI | SpeI | buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 5.4 | 1 | 1 | 3 | 0 | 19.6 | 30 |
| NC | 0.3 | 0 | 0 | 1 | 0 | 8.7 | 10 |

|  | 9/16 pSB1C3 | EcoRI | SpeI | buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 18 | 1 | 1 | 3 | 0 | 7.0 | 30 |
| NC | 0.9 | 0 | 0 | 1 | 0 | 8.1 | 10 |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 1kb ladder | - | - |
| 2 | pT181attenuator | XbaI | PstI |
| 3 | pT181attenuator | -- | -- |
| 4 | Pcon-pT181attenuator | EcoRI | SpeI |
| 5 | Pcon-pT181attenuator | -- | -- |
| 6 | pSB1C3 | EcoRI | SpeI |
| 7 | pSB1C3 | -- | -- |

[![Igku 0924 E2.jpg](/wiki/images/d/d5/Igku_0924_E2.jpg)](/File%3AIgku_0924_E2.jpg)

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/24 aptamer12-1R-DT | 144.7 | 1.03 | 1.21 |

### Gel Extraction

Kojima

| Lane | DNA | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100kb ladder | -- | -- |
| 2 | pT181attenuator | XbaI | PstI |
| 3 | pT181attenuator | XbaI | PstI |
| 4 | pT181attenuator | XbaI | PstI |
| 5 | -- | -- | -- |
| 6 | Pcon-pT181attenuator | EcoRI | SpeI |
| 7 | Pcon-pT181attenuator | EcoRI | SpeI |
| 8 | Pcon-pT181attenuator | EcoRI | SpeI |
| 9 | -- | -- | -- |
| 10 | pSB1C3 | EcoRI | SpeI |
| 11 | pSB1C3 | EcoRI | SpeI |
| 12 | pSB1C3 | EcoRI | SpeI |

[![Igku 0924 G1.jpg](/wiki/images/2/29/Igku_0924_G1.jpg)](/File%3AIgku_0924_G1.jpg)
[![Igku 0924 G2.jpg](/wiki/images/a/a7/Igku_0924_G2.jpg)](/File%3AIgku_0924_G2.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pT181attenuator(XbaI&PstI) | 10.1 | 2.02 | 0.43 |
| Pcon-pT181attenuator(EcoRI&SpeI) | 10.3 | 1.81 | 0.32 |
| pSB1C3(EcoRI&SpeI) | 10.0 | 2.81 | 0.02 |

### Colony PCR

Nakamoto

| Sample | base pair |
| --- | --- |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7 | 1414 |
| 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8 | 1414 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7 | 1631 |
| 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8 | 1631 |
| 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1 | 2042 |
| 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2 | 2042 |
| 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3 | 2042 |
| 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4 | 2042 |
| 9/23 Pcon-pT181antisense-DT 1 | 508 |
| 9/23 Pcon-pT181antisense-DT 2 | 508 |
| 9/23 Pcon-pT181antisense-DT 3 | 508 |
| 9/23 Pcon-pT181antisense-DT 4 | 508 |
| 9/23 Pcon-pT181antisense-DT 5 | 508 |
| 9/23 Pcon-pT181antisense-DT 6 | 508 |
| 9/23 Pcon-pT181antisense-DT 7 | 508 |
| 9/23 Pcon-pT181antisense-DT 8 | 508 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94 °C | 94 °C | 55 °C | 68 °C | -- |
| 5 min | 30 sec | 30 sec | 2min | 30 cycles |

| Lane | Sample |
| --- | --- |
| 1 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1 |
| 2 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2 |
| 3 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3 |
| 4 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4 |
| 5 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5 |
| 6 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6 |
| 7 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7 |
| 8 | 9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8 |
| 9 | 1kb ladder |
| 10 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1 |
| 11 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2 |
| 12 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3 |
| 13 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4 |
| 14 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5 |
| 15 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6 |
| 16 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7 |
| 17 | 9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8 |

[![Igku 0924 E3.jpg](/wiki/images/b/bb/Igku_0924_E3.jpg)](/File%3AIgku_0924_E3.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1 |
| 2 | 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2 |
| 3 | 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3 |
| 4 | 9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4 |
| 5 | 9/23 Pcon-pT181antisense-DT 1 |
| 6 | 9/23 Pcon-pT181antisense-DT 2 |
| 7 | 1kb ladder |
| 8 | 9/23 Pcon-pT181antisense-DT 3 |
| 9 | 9/23 Pcon-pT181antisense-DT 4 |
| 10 | 9/23 Pcon-pT181antisense-DT 5 |
| 11 | 9/23 Pcon-pT181antisense-DT 6 |
| 12 | 9/23 Pcon-pT181antisense-DT 7 |
| 13 | 9/23 Pcon-pT181antisense-DT 8 |
| 14 | -- |

[![Igku 0924 E4.jpg](/wiki/images/9/99/Igku_0924_E4.jpg)](/File%3AIgku_0924_E4.jpg)

### Restriction Enzyme Digestion

Nakamoto

|  | 9/24 aptamer12-1R-DT | EcoRI | XbaI | buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 17.5µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 4.5µL | 30µL |
| NC | 0.9µL | 0µL | 0µL | 1.0µL | 1.0µL | 7.1µL | 10µL |

|  | 9/20 Ptet | SpeI | PstI | buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 9.5µL | 1.0µL | 1.0µL | 3.0µL | 0µL | 15.5µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 1.0µL | 0µL | 8.5µL | 10µL |

|  | 9/22 RBS-GFP-DT | EcoRI | SpeI | buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 3.8µL | 1.0µL | 1.0µL | 3.0µL | 0µL | 21.2µL | 30µL |
| NC | 0.2µL | 0µL | 0µL | 1.0µL | 0µL | 8.8µL | 10µL |

|  | 9/22 Pcon-RBS-GFP-DT | EcoRI | XbaI | buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 2.4µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 19.6µL | 30µL |
| NC | 0.1µL | 0µL | 0µL | 1.0µL | 1.0µL | 7.9µL | 10µL |

|  | 9/22 Pcon-pT181attenuator 1 | EcoRI | XbaI | buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 5.4µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 16.6µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1.0µL | 1.0µL | 7.7µL | 10µL |

|  | 9/22 RBS-GFP-DT | XbaI | PstI | buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 3.8µL | 1.0µL | 1.0µL | 3.0µL | 3.0µL | 18.2µL | 30µL |
| NC | 0.2µL | 0µL | 0µL | 1.0µL | 1.0µL | 7.8µL | 10µL |

### Ligation

Nakamoto

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/22 aptamer12-1R-DT(EcoRI&XbaI) | 1.4 µL | 9/24 Pcon-pT181attenuator(EcoRI&SpeI) | 26 µL | 3.5 µL |
| experiment | 9/4 Ptet(SpeI&PstI) | 2.0 µL | 9/21 RBS-GFP-DT(XbaI&PstI) | 9.0 µL | 3.5 µL |

* incubated at 37 °C for 1 hour.

### colony PCR

Kojima

| Sample | base pair |
| --- | --- |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11 | 2042bp |
| 9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12 | 2042bp |
| 9/23 Pcon-pT181antisense-DT 9 | 508bp |
| 9/23 Pcon-pT181antisense-DT 10 | 508bp |
| 9/23 Pcon-pT181antisense-DT 11 | 508bp |
| 9/23 Pcon-pT181antisense-DT 12 | 508bp |
| 9/23 Pcon-pT181antisense-DT 13 | 508bp |
| 9/24 Pcon-pT181attenuator-RBS-GFP-DT 1 | 1527bp |
| 9/24 Pcon-pT181attenuator-RBS-GFP-DT 2 | 1527bp |
| 9/24 Pcon-pT181attenuator-RBS-GFP-DT 3 | 1527bp |
| 9/24 Pcon-pT181attenuator-RBS-GFP-DT 4 | 1527bp |
| 9/24 Pcon-pT181attenuator-aptamer12-1R-DT 1 | 859bp |
| 9/24 Pcon-pT181attenuator-aptamer12-1R-DT 2 | 859bp |
| 9/24 Pcon-pT181attenuator-aptamer12-1R-DT 3 | 859bp |
| 9/24 Pcon-pT181attenuator-aptamer12-1R-DT 4 | 859bp |
| 9/24 pT181attenuator(pSB1C3) 1 | 390bp |
| 9/24 pT181attenuator(pSB1C3) 2 | 390bp |
| 9/24 pT181attenuator(pSB1C3) 3 | 390bp |
| 9/24 pT181attenuator(pSB1C3) 4 | 390bp |

Tatsui

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 2min | 30cycle |

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pSB1C3 | 183.9 | 1.91 | 1.59 |
| Pcon-pT181attenuator-aptamer12-1R-DT | 231.7 | 1.82 | 2.35 |
| Plac-aptamer12-1R-DT | 208.9 | 1.98 | 1.79 |

### Electrophoresis

Nakamoto

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 1kb ladder | -- | -- |
| 2 | 9/24 aptamer12-1R-DT | EcoRI | XbaI |
| 3 | 9/24 aptamer12-1R-DT | -- | -- |
| 4 | 9/24 Ptet | SpeI | PstI |
| 5 | 9/24 Ptet | -- | -- |
| 6 | 9/24 RBS-GFP-DT | EcoRI | SpeI |
| 7 | 9/24 RBS-GFP-DT | -- | -- |
| 8 | 9/24 Pcon-RBS-GFP-DT | EcoRI | XbaI |
| 8 | 9/24 Pcon-RBS-GFP-DT | -- | -- |
| 9 | 9/24 Pcon-RBS-GFP-DT | -- | -- |
| 10 | 9/24 Pcon-pT181attenuator 1 | EcoRI | XbaI |
| 11 | 9/24 Pcon-pT181attenuator 1 | -- | -- |
| 12 | 9/24 RBS-GFP-DT | XbaI | PstI |
| 13 | 9/24 RBS-GFP-DT | -- | -- |
| 14 | 1kb ladder | -- | -- |

[![Igku 0924 E5.jpg](/wiki/images/2/23/Igku_0924_E5.jpg)](/File%3AIgku_0924_E5.jpg)

### Gel Extraction

No name
1

| Lane | DNA | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 1kb ladder | -- | -- |
| 2 | aptamer12-1R-DT | EcoRI | XbaI |
| 3 | aptamer12-1R-DT | EcoRI | XbaI |
| 4 | aptamer12-1R-DT | EcoRI | XbaI |
| 5 | -- | -- | -- |
| 6 | Ptet | SpeI | PstI |
| 7 | Ptet | SpeI | PstI |
| 8 | Ptet | SpeI | PstI |
| 9 | -- | -- | -- |
| 10 | RBS-GFP-DT | EcoRI | SpeI |
| 11 | RBS-GFP-DT | EcoRI | SpeI |
| 12 | RBS-GFP-DT | EcoRI | SpeI |

[![Igku 0924 G3.jpg](/wiki/images/c/cf/Igku_0924_G3.jpg)](/File%3AIgku_0924_G3.jpg)
[![Igku 0924 G4.jpg](/wiki/images/b/bd/Igku_0924_G4.jpg)](/File%3AIgku_0924_G4.jpg)

| Lane | DNA | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 1kb ladder | -- | -- |
| 2 | Pcon-RBS-GFP-DT | EcoRI | XbaI |
| 3 | Pcon-RBS-GFP-DT | EcoRI | XbaI |
| 4 | Pcon-RBS-GFP-DT | EcoRI | XbaI |
| 5 | -- | -- | -- |
| 6 | Pcon-pT181attenuator | EcoRI | XbaI |
| 7 | Pcon-pT181attenuator | EcoRI | XbaI |
| 8 | Pcon-pT181attenuator | EcoRI | XbaI |
| 9 | -- | -- | -- |
| 10 | RBS-GFP-DT | XbaI | PstI |
| 11 | RBS-GFP-DT | XbaI | PstI |
| 12 | RBS-GFP-DT | XbaI | PstI |

[![Igku 0924 G5.jpg](/wiki/images/a/aa/Igku_0924_G5.jpg)](/File%3AIgku_0924_G5.jpg)
[![Igku 0924 G6.jpg](/wiki/images/a/a4/Igku_0924_G6.jpg)](/File%3AIgku_0924_G6.jpg)

### Electrophoresis

No name
1

| Lane | Sample |
| --- | --- |
| 1 | 1kb ladder |
| 2 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5 |
| 3 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6 |
| 4 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7 |
| 5 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8 |
| 6 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9 |
| 7 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10 |
| 8 | 1kb ladder |

2

| Lane | Sample |
| --- | --- |
| 1 | 1kb ladder |
| 2 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11 |
| 3 | Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12 |
| 4 | Pcon-pT181attenuator-RBS-GFP-DT 1 |
| 5 | Pcon-pT181attenuator-RBS-GFP-DT 2 |
| 6 | Pcon-pT181attenuator-RBS-GFP-DT 3 |
| 7 | Pcon-pT181attenuator-RBS-GFP-DT 4 |
| 8 | 1kb ladder |

[![Igku 0924 E6.jpg](/wiki/images/0/0f/Igku_0924_E6.jpg)](/File%3AIgku_0924_E6.jpg)

3

| Lane | Sample |
| --- | --- |
| 1 | Pcon-pT181antisense-DT 9 |
| 2 | Pcon-pT181antisense-DT 10 |
| 3 | Pcon-pT181antisense-DT 11 |
| 4 | Pcon-pT181antisense-DT 12 |
| 5 | Pcon-pT181antisense-DT 13 |
| 6 | 100bp ladder |
| 7 | Pcon-pT181attenuator-aptamer12-1R-DT 1 |
| 8 | Pcon-pT181attenuator-aptamer12-1R-DT 2 |

4

| Lane | Sample |
| --- | --- |
| 1 | Pcon-pT181attenuator-aptamer12-1R-DT 3 |
| 2 | Pcon-pT181attenuator-aptamer12-1R-DT 4 |
| 3 | 100bp ladder |
| 4 | pT181attenuator(pSB1C3) 1 |
| 5 | pT181attenuator(pSB1C3) 2 |
| 6 | pT181attenuator(pSB1C3) 3 |
| 7 | pT181attenuator(pSB1C3) 4 |

[![Igku 0924 E7.jpg](/wiki/images/2/27/Igku_0924_E7.jpg)](/File%3AIgku_0924_E7.jpg)

### Ligation

Hirano

| state | Vector | | Inserter1 | | Inserter2 | | Inserter3 | | Inserter4 | | (NEB)T4ligase | (NEB)Buffer | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| experiment | RBS-GFP-DT(pSB1C3) | 1µL | tetR-aptamer12-1R-DT(1-3) | 1 µL | Ptet(2-S) | 0.3µL | Pcon-tetR-aptamer12-1R-DT(3-2A) | 1.1µL | Plac(E-1A) | 0.5µL | 0.5µL | 0.5µL | 0.2 µL | 5µL |
| experiment | RBS-GFP-DT(pSB1C3) | 1µL | Pcon-pT181attenuator(E-1A) | 1 µL | Pcon-tetR-aptamer12-1R-DT(1-2A) | 1µL | Ptet(2-S) | 0.3µL | -- | -- | 0.5µL | 0.5µL | 0.7 µL | 5µL |
| experiment | RBS-GFP-DT(pSB1C3) | 1µL | Pcon-tetRaptamer12-1R-DT(E-1A) | 2µL | Pcon-tetRaptamer12-1R-DT(1-2A) | 1µL | Ptet(2-S) | 0.3µL | -- | -- | 0.5µL | 0.5µL | 0 µL | 5.3µL |
| experiment | RBS-GFP-DT(pSB1C3) | 1µL | Pcon-spinach-DT(E-1A) | 3µL | Pcon-tetRaptamer12-1R-DT(1-2A) | 1µL | Ptet(2-S) | 0.3µL | -- | -- | 0.5µL | 0.5µL | 0 µL | 6.3µL |
| experiment | RBS-GFP-DT(pSB1C3) | 1µL | Pcon-pT181antisense-DT(E-1A) | 0.5µL | DT(1-3) | 2µL | Ptet(2-S) | 0.3µL | Pcon-tetRaptamer12-1R-DT(3-2A) | 1.1µL | 0.5µL | 0.5µL | 0.1 µL | 5µL |
| experiment | RBS-GFP-DT(pSB1C3) | 1µL | Pcon-pT181attenuator(E-1A) | 1µL | tetRaptamer12-1R-DT(1-3) | 1µL | Ptet(2-S) | 0.3µL | Pcon-tetRaptamer12-1R-DT(3-2A) | 1.1µL | 0.5µL | 0.5µL | 0µL | 5.4µL |
| experiment | RBS-GFP-DT(pSB1C3) | 1µL | Ptet(2-S) | 0.3µL | Pcon-tetRaptamer12-1R-DT(E-2A) | 1µL | -- | -- | -- | -- | 0.5µL | 0.5µL | 1.7µL | 5µL |
| experiment | RBS-GFP-DT(pSB1C3) | 1µL | Pcon-GFP-DT(2-SA) | 0.5µL | 1µL | -- | -- | -- | -- | -- | 0.5µL | 0.5µL | 1.5µL | 5µL |
| experiment | RBS-GFP-DT(pSB1C3) | 1µL | Pcon-tetRaptamer12-1R-DT(E-1A) | 2µL | Pcon-pT181attenuator(1-SA) | 2.1µL | -- | -- | -- | -- | 0.5µL | 0.5µL | 0µL | 6.1µL |
| experiment | RBS-GFP-DT(pSB1C3) | 1µL | Pcon-pT181attenuator-DT(E-1A) | 1µL | Pcon-pT181attenuator(1-SA) | 2.1µL | -- | -- | -- | -- | 0.5µL | 0.5µL | 0µL | 5.1µL |
| experiment | RBS-GFP-DT(pSB1C3) | 1µL | Pcon-tetR12-1R-DT(E-1A) | 2µL | Pcon-GFP-DT(2-SA) | 0.5µL | spinach-DT(3-2) | 1µL | Ptet-pT181antisense(1-3) | 1µL | 0.5µL | 0.5µL | 0µL | 6.5µL |
| experiment | RBS-GFP-DT(pSB1C3) | 1µL | Pcon-tetRaptamer12-1R-DT(E-1A) | 2µL | Pcon-pT181antisense(1-2A) | 0.7µL | Pcon-pT181attenuator(2-SA) | 2.1µL | -- | -- | 0.5µL | 0.5µL | 0µL | 6.8µL |
| experiment | RBS-GFP-DT(pSB1C3) | 1µL | Pcon-tetRaptamer12-1R-DT(E-1A) | 2µL | Pcon-pT181attenuator(2-SA) | 2.1µL | Spinach-DT(3-2) | 1µL | Ptet-pT181antisense(1-3) | 1µL | 0.5µL | 0.5µL | 0µL | 8.1µL |

Concentrate total (more than 5µL) by evaporater until volume become less than 5 µL

### PCR

No name

| genome DNA | KOD plus | 10x buffer | dNTP | MgSO4 | F primer | R primer | KOD-plus | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Pcon antisense(1C3-GGE-fwd,1A2-GG2-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |
| DT-(1C3-GG1-fwd,1C3-GG2-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |
| Spinach-DT-(1C3-GG1-fwd,1C3-GG2-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |
| Pcon-antisense-spinach-DT(1C3-GGE-fwd,1A2-GG1-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |
| Ptet(1C3-GGE-fwd,1C3-GG0-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |
| Pcon attenuator(1C3-GGE-fwd,1C3-GG0-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |
| DT(1C3-GG2-fwd,1C3-GG1-rev) | 1µL | 2.5µL | 2.5µL | 1.5µL | 0.75µL | 0.75µL | 0.5µL | 15.5 | 25µL |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10sec | 30sec | 36sec | 30 |

## Sep 25

### Electrophoresis

nakamoto

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | Pcon-antisense (1C3-66E-fwd A2-662 rev) | -- | -- |
| 3 | DT (1C3-661-fwd 1C3 662 rev) | -- | -- |
| 4 | spinach-DT(1C3-661-fwd 1C3 662 rev) | -- | -- |
| 5 | Pcon-antisense-spinach-DT(1C3-66E fwd A2-661 rev) | -- | -- |
| 6 | 100bp ladder | -- | -- |

[![Igku 9251a.jpg](/wiki/images/b/b0/Igku_9251a.jpg)](/File%3AIgku_9251a.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 2 | 100bp ladder | -- | -- |
| 3 | Ptet (1C3 66E fwd-1C3 660 rev) | -- | -- |
| 4 | Pcon-attenuator(1C3-660 fwd A2-661 rev) | -- | -- |
| 5 | DT(1C3 662 fwd-1C3 661 rev) | -- | -- |
| 6 | Pcon-attenuator-aptamer-DT(1C3 66E fwd-A2-661-rev) | -- | -- |
| 7 | 100bp ladder | -- | -- |

[![Igku 9251b.jpg](/wiki/images/9/94/Igku_9251b.jpg)](/File%3AIgku_9251b.jpg)

### Colony PCR

Nakamoto

| Sample | base pair |
| --- | --- |
| 9/23 Pcon-PT181 attenuator-aptamer12-1R-DT-(5~12) | 859 |
| 9/23 Pcon-PT181 attenuator-RBS-GFP-DT-(5~8) | 1527 |
| 9/23 Pcon-PT181 attenuator-pSB1C3-(5~8) | 601 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94 °C | 94 °C | 55 °C | 68 °C | -- |
| 2 min | 30 s | 30 s | 1min5s | 30 cycles |

### Electrophoresis

no name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | Pcon-PT181 attenuator-aptamer12-1R-DT-5 | -- | -- |
| 2 | Pcon-PT181 attenuator-aptamer12-1R-DT-6 | -- | -- |
| 3 | Pcon-PT181 attenuator-aptamer12-1R-DT-7 | -- | -- |
| 4 | Pcon-PT181 attenuator-aptamer12-1R-DT-8 | -- | -- |
| 5 | Pcon-PT181 attenuator-aptamer12-1R-DT-9 | -- | -- |
| 6 | Pcon-PT181 attenuator-aptamer12-1R-DT-10 | -- | -- |
| 7 | Pcon-PT181 attenuator-aptamer12-1R-DT-11 | -- | -- |
| 8 | Pcon-PT181 attenuator-aptamer12-1R-DT-12 | -- | -- |
| 9 | 1kbp ladder | -- | -- |
| 10 | Pcon-PT181 attenuator-RBS-GFP-DT-5 | -- | -- |
| 11 | Pcon-PT181 attenuator-RBS-GFP-DT-6 | -- | -- |
| 12 | Pcon-PT181 attenuator-RBS-GFP-DT-7 | -- | -- |
| 13 | Pcon-PT181 attenuator-RBS-GFP-DT-8 | -- | -- |
| 14 | Pcon-PT181 attenuator-pSB1C3-5 | -- | -- |
| 15 | Pcon-PT181 attenuator-pSB1C3-6 | -- | -- |
| 16 | Pcon-PT181 attenuator-pSB1C3-7 | -- | -- |
| 17 | Pcon-PT181 attenuator-pSB1C3-8 | -- | -- |

[![Igku 9252.jpg](/wiki/images/3/34/Igku_9252.jpg)](/File%3AIgku_9252.jpg)

### Transformation

kojima and nakamoto

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| Plac+tet aptamer12+Pcon tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | -- |
| Pcon-pT181attenuator-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | -- |
| Pcon-tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | -- |
| Pcon-Spinach-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | -- |
| Pcon-pT181antisense+DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | -- |
| Pcon-pT181attenuator+tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | -- |
| Pcon tetR-DT+Ptet+RBS-GFP-DT | 1µL | 10µL | 11µL | -- |
| Pcon tetR-DT+Pcon-GFP-DT+RBS-GFP-DT | 1µL | 10µL | 11µL | -- |
| Pcon-tet aptamer12-DT+Pcon-pT181attenuator+RBS-GFP-DT | 1µL | 10µL | 11µL | -- |
| Pcon-pT181attenuator-DT+Pcon-pT181attenuator+RBS-GFP-DT | 1µL | 10µL | 11µL | -- |
| Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-RBS-GFP-DT+RBS-GFP-DT | 1µL | 10µL | 11µL | -- |
| Pcon-tet aptamer12-DT+Pcon-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT | 1µL | 10µL | 11µL | -- |
| Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT | 1µL | 10µL | 11µL | -- |

| Name | Sample | Competent Cells | Total | Plate |
| --- | --- | --- | --- | --- |
| aptamer12-DT(EcoRI+XbaI)+Pcon-pT181attenuator(EcoRI+SpeI) | 2µL | 20µL | 22µL | -- |
| Ptet(SpeI+PstI)+RBS-GFP-DT(XbaI+PstI)) | 2µL | 20µL | 22µL | -- |

### Ligation

nakamoto

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/25 aptamer12\_1R-DT(EcoRI&XbaI) | 4.5 µL | Pcon-pT181 attenuator(EcoRI&SpeI) | 7.7 µL | 6.1 µL |
| experiment | 9/17 pSB1C3(EcoRI&SpeI) | 2.0 µL | 9/3 pT181 attenuator(EcoRI&SpeI) | 14 µL | 8 µL |
| experiment | 9/25 Pcon-pT181 attenuator(EcoRI&XbaI) | 4.5 µL | 9/25 RBS-GFP-DT(EcoRI&SpeI) | 19.3 µL | 11.9 µL |
| experiment | 9/25 Pcon-RBS-GFP-DT(EcoRI&XbaI) | 4.2 µL | Pcon-RBS-tetR-DT(EcoRI&SpeI) | 20 µL | 12.1 µL |

### Restriction Enzyme Digestion

nakamoto>

|  | 9/24 Pcon-attenuator-aptamer-DT(µL) | EcoRI(µL) | SpeI(µL) | Buffer(µL) | MilliQ(µL) | total(µL) |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 8.6 | 1 | 1 | 3 | 16.4 | 30 |
| NC | 0.4 | 0 | 0 | 1 | 8.6 | 10 |

|  | 9/11 DT(µL) | EcoRI(µL) | XbaI(µL) | Buffer(µL) | BSA(µL) | MilliQ(µL) | total(µL) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 10.4 | 1 | 1 | 3 | 3 | 11.6 | 30 |
| NC | 0.5 | 0 | 0 | 1 | 1 | 7.5 | 10 |

### Liquid Culture

Kojima

| Sample | medium |
| --- | --- |
| DT | -- |
| pT181attenuator | -- |
| Pcon-RBS-tetR-DT | -- |

### Gel Extraction

Kojima

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 2 | Pcon-pT181antisense | -- |
| 3 |
| 4 |
| 5 | -- | -- |
| 6 | DT | -- |
| 7 |
| 8 |
| 9 | -- | -- |
| 10 | Spinach-DT | -- |
| 11 |
| 12 |

[![Igku 9253.jpg](/wiki/images/b/b3/Igku_9253.jpg)](/File%3AIgku_9253.jpg)
[![Igku 9254.jpg](/wiki/images/1/19/Igku_9254.jpg)](/File%3AIgku_9254.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 100bp ladder | -- |
| 3 | Pcon-antisense-spinach-DT(E-1A) | -- |
| 4 | Pcon-antisense-spinach-DT(E-1A) | -- |
| 5 | Pcon-antisense-spinach-DT(E-1A) | -- |

[![Igku 9255.jpg](/wiki/images/1/12/Igku_9255.jpg)](/File%3AIgku_9255.jpg)
[![Igku 9256.jpg](/wiki/images/0/05/Igku_9256.jpg)](/File%3AIgku_9256.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 7 | 100bp ladder | -- |
| 9 | Ptet(E-0) | -- |
| 10 | Ptet(E-0) | -- |
| 11 | Ptet(E-0) | -- |

[![Igku 9257.jpg](/wiki/images/6/67/Igku_9257.jpg)](/File%3AIgku_9257.jpg)
[![Igku 9258.jpg](/wiki/images/4/42/Igku_9258.jpg)](/File%3AIgku_9258.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 13 | 100bp ladder | -- |
| 15 | Pcon-attenuator(0-1A) | -- |
| 16 | Pcon-attenuator(0-1A) | -- |
| 17 | Pcon-attenuator(0-1A) | -- |

[![Igku 9259.jpg](/wiki/images/c/c7/Igku_9259.jpg)](/File%3AIgku_9259.jpg)
||--
[![Igku 92510.jpg](/wiki/images/4/40/Igku_92510.jpg)](/File%3AIgku_92510.jpg)

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 19 | 100bp ladder | -- |
| 21 | Pcon-attenuator-aptamer-DT(E-1A) | -- |
| 22 | Pcon-attenuator-aptamer-DT(E-1A) | -- |
| 23 | Pcon-attenuator-aptamer-DT(E-1A) | -- |

[![Igku 92511.jpg](/wiki/images/a/ab/Igku_92511.jpg)](/File%3AIgku_92511.jpg)
[![Igku 92512.jpg](/wiki/images/e/e4/Igku_92512.jpg)](/File%3AIgku_92512.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| DT(2-1) | 24.9 | 1.73 | 0.37 |
| Ptet(E-0) | 12.8 | 1.59 | 0.90 |
| Pcon-antisense(E-2A) | 16.5 | 1.72 | 1.07 |
| Pcon-attenuator-aptamer-DT(E-1A) | 22.1 | 1.75 | 0.94 |
| Pcon-antisense-spinach-DT(E-1A) | 31.0 | 1.78 | 1.15 |
| spinach-DT(1-2) | 23.4 | 1.85 | 1.11 |
| Pcon-attenuator(0-1A) | 22.5 | 1.76 | 0.46 |
| DT(1-2) | 25.1 | 1.78 | 1.21 |

### Electrophoresis

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 1kbp ladder | -- | -- |
| 2 | Pcon-pT181 attenuator-aptamer 12\_1R-DT | EcoRI | SpeI |
| 3 | Pcon-pT181 attenuator-aptamer 12\_1R-DT | -- | -- |
| 4 | DT | EcoRI | XbaI |
| 5 | DT | -- | -- |

[![Igku 92513.jpg](/wiki/images/6/64/Igku_92513.jpg)](/File%3AIgku_92513.jpg)

### Gel Extraction

Nakamoto

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | DT | EcoRI+XbaI |
| 2 | DT | EcoRI+XbaI |
| 3 | Pcon-pT181 attenuator-aptamer12\_1R-DT | EcoRI+SpeI |
| 4 | Pcon-pT181 attenuator-aptamer12\_1R-DT | EcoRI+SpeI |
| 5 | Pcon-pT181 attenuator-aptamer12\_1R-DT | EcoRI+SpeI |

[![Igku 92514.jpg](/wiki/images/5/53/Igku_92514.jpg)](/File%3AIgku_92514.jpg)
[![Igku 92515.jpg](/wiki/images/3/36/Igku_92515.jpg)](/File%3AIgku_92515.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-pT181 attenuator-aptamer12\_1R-DT(EcoRI&SpeI) | 11.8 | 1.80 | 0.46 |
| DT(EcoRI+XbaI) | 42.6 | 1.84 | 0.98 |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| Pcon-pT181anntisense-spinach-DT(4k5) | - |

### Ligation

Nakamoto and Tatsui

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/24 pSB1C3(EcoRI+Spel) | 10µL | 9/25 PconpT181attenuator-aptamer12-1R-DT(EcoRI+Spel) | 5.9µL | 8µL |

### PCR

Hirano

| 800pg/μL DT | KOD plus | 10x buffer | dNTP | MgSO4 | 1C3\_GG2\_fwd | iC3\_GG1\_rev | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 0.5 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 57°C | 68°C | -- |
| 2min | 10s | 30s | 24s | 30cycles |

### Transformation

Tatsui

| Name | Plate |
| --- | --- |
| aptamer12-1R-DT | CP |
| pSB1C3(EcoRI+SpeI)+pT181attenuator(EcoRI+Spal) | CP |
| Pcon-pT181attenuator(EcoRI+XbaI)+RBS-GFP-DT(EcoRI+SpeI) | Amp |
| Pcon-RBS-GFP-DT(EcoRI+XbaI)+Pcon-RBS-tetR-DT(EcoRI+SpeI) | Amp |
| pSB1C3(EcoRI+SpeI)+Pcon-pT181attenuator-aptamer12-1R-DT(EcoRI+SpeI) | CP |

### Electrophoresis

Hirano

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | DT(2-1) | -- | -- |

[![Igku 92516.jpg](/wiki/images/5/5e/Igku_92516.jpg)](/File%3AIgku_92516.jpg)

### Column Refining

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/25 DT(2-1) | -- | -- | -- |

### Restriction Enzyme Digestion

Concentrate PCR product by evaporator until volume become less than 3µL, add all sulution and pipetting.

| 9/25 DT(2-1) | 10\*cut smart Buffer | BsaI-HF | MilliQ | total |
| --- | --- | --- | --- | --- |
| 3.0 µL | 2.0 µL | 0.3 µL | 14.7 µL | 20.0 µL |

Incubate 37°C 12hour

### Liquid Culture

Hirano

| Sample | medium |
| --- | --- |
| Pcon-GFP-DT | LB(Amp) |
| RBS-GFP-DT | LB(CP) |
| Pcon-spinach-DT | LB(Amp) |
| Pcon-tetRaptamer-DT | LB(Amp) |
| Pcon-attenuator-DT | LB(Amp) |
| spinach-DT | LB(CP) |
| Pcon-antisense-spinach-DT | LB(Amp) |

### Plating

Hirano

Mix Liquid Culture 1mL and LB(+Amp), incubate 37°C 30min

OD600-1.445

| Sample | Use plate | The number of plate |
| --- | --- | --- |
| Pcon-GFP-DT | M9(+Amp) | 3 |
| Pcon-GFP-DT | LB(+Amp) | 3 |

</div>
incubate 37°C

### Colony PCR

No name

| Sample | base pair |
| --- | --- |
| 9/24 GGA-1(1~8) | 3177 |
| 9/24 GGA-2(1~8) | 3189 |
| 9/24 GGA-3(1~8) | 2989 |
| 9/24 GGA-4(1~8) | 2939 |
| 9/24 GGA-7(1) | 2489 |
| GGA-16(1) | 2717 |
| RBS-GFP-DT | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 3min15s | 30cycles |

### RNA Extraction

Nakamoto
sumple 0.25mL
Add 1mLISOGEN-LS
Lysis or homogenization
Store for 5 min, at room temperature
Add 0.2mL Chloroform
Shake vigorously for 15sec.
Store for 2~3min.at room temperature
Centrifuge 12K\*g for 15min. at 4°C
Extract aqueous phase
Add 0.5mL isopropanol
Store for 5~10min. at room temperature
Centrifuge 12K\*g for 10min. at 4°C
Remove aqueous phase
Add at least 1mL 70% ethanol
Vortex
Centrifuge 7.5K\*g for 5min. at 4°C
Remove aqueous phase
Dry briefly
Add ddH2O, TE(pH8.0) or 0.5% SDS
Dissolve
Total RNA solution

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-RBS-GFP-DT | 2783 | 1.95 | 1.48 |

Add 91.2µL, Store at freezer
Total 250µL</div>

### cDNA　Synthesis

Nakamoto

| volume | 7 µL |
| --- | --- |
| 7\*cDNA Wipcout Buffer | 1 µL |
| Template RNA(9/25 Pcon-RBS-GFP-DT) | 3 µL |
| RNase-frec water | 4 µL |

Incubate 42°C 2min, then on ice immediately
Add

| 5\*Quantiscript Reverse Transcriptase | 0.5 µL |
| --- | --- |
| 5\*Quantiscript RT Buffer | 2 µL |
| RT Primer Mix | 0.5µL |

Incubate 42°C 15min, then incubate 95°C 3min

### Electrophoresis

Nakamoto

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 100bp ladder | -- | -- |
| 2 | Pcon-RBS-GFP-DT(RNA) | -- | -- |

[![Igku 92517.jpg](/wiki/images/8/8d/Igku_92517.jpg)](/File%3AIgku_92517.jpg)

### Ligation

Tatsui

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/13 Pcon-pT181-attenuator(SpeI&PstI) | 3.5µL | 9/25 RBS-GFP-DT(XbaI&PstI) | 13.5µL | 8.5 µL |

## Sep 26

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | GGA1-1 | -- | -- |
| 2 | GGA1-2 | -- | -- |
| 3 | GGA1-3 | -- | -- |
| 4 | GGA1-4 | -- | -- |
| 5 | GGA1-5 | -- | -- |
| 6 | GGA1-6 | -- | -- |
| 7 | GGA1-7 | -- | -- |
| 8 | GGA1-8 | -- | -- |
| 9 | 1kbp ladder | -- | -- |
| 10 | GGA2-1 | -- | -- |
| 11 | GGA2-2 | -- | -- |
| 12 | GGA2-3 | -- | -- |
| 13 | GGA2-4 | -- | -- |
| 14 | GGA2-5 | -- | -- |
| 15 | GGA2-6 | -- | -- |
| 16 | GGA2-7 | -- | -- |
| 17 | GGA2-8 | -- | -- |

[![Igku 0926 E1.jpg](/wiki/images/6/60/Igku_0926_E1.jpg)](/File%3AIgku_0926_E1.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | GGA3-1 | -- | -- |
| 2 | GGA3-2 | -- | -- |
| 3 | GGA3-3 | -- | -- |
| 4 | 1kbp ladder | -- | -- |
| 5 | GGA3-4 | -- | -- |
| 6 | GGA3-5 | -- | -- |
| 7 | GGA3-6 | -- | -- |
| 8 | GGA3-7 | -- | -- |

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | GGA3-8 | -- | -- |
| 2 | GGA4-1 | -- | -- |
| 3 | GGA4-2 | -- | -- |
| 4 | GGA4-3 | -- | -- |
| 5 | 1kbp ladder | -- | -- |
| 6 | GGA4-4 | -- | -- |
| 7 | GGA4-5 | -- | -- |
| 8 | GGA4-6 | -- | -- |

[![Igku 0926 E2.jpg](/wiki/images/1/17/Igku_0926_E2.jpg)](/File%3AIgku_0926_E2.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | GGA4-7 | -- | -- |
| 2 | GGA4-8 | -- | -- |
| 3 | 1kbp ladder | -- | -- |
| 4 | GGA7-1 | -- | -- |
| 5 | GGA16-1 | -- | -- |
| 6 | NC(RBS-GFP-DT) | -- | -- |

[![Igku 0926 E3.jpg](/wiki/images/7/70/Igku_0926_E3.jpg)](/File%3AIgku_0926_E3.jpg)

### Ligasion(Golden Gate Assenbly)

No name

| state | Vector | | Inserter1 | | Inserter2 | | Inserter3 | | Inserter4 | | Inserter5 | | (NEB)T4 ligase | (NEB)10\*T4 ligase | MilliQ |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| experiment | RBS-GFP-DT | 1.0 µL | Pcon-pT181 antisense(E-2A) | 0.6µL | DT(2-1) | 0.5µL | Pcon-pT181 attenuator(1-SA) | 2.1 µL | -- | -- | -- | -- | 0.5 µL | 0.5 µL | up to 5 µL |
| experiment | pSB1C3(EcoRI&SpeI) | 1.8 µL | Pcon-pT181 antisense(E-2A) | 0.9 µL | DT(2-1) | 0.5 µL | Pcon-GFP-DT(1-SA) | 1.5 µL | -- | -- | -- | -- | 0.5 µL | 0.5 µL | up to 5 µL |
| experiment | RBS-GFP-DT | 1.0 µL | Ptet(E-0) | 0.7µL | Pcon pT181 attenuator(0-1A) | 0.3µL | tet aptamer(1-3) | 0.3 µL | Pcon-tetR-DT(3-2A) | 1.1 µL | Ptet(2-5) | 0.3 µL | 0.5 µL | 0.5 µL | up to 5 µL |
| experiment | RBS-GFP-DT | 1.0 µL | Ptet(E-0) | 0.7 µL | Pcon-pT181 attenuator-DT(0-1A) | 1.0 µL | Pcon-tetR-DT(1-2A) | 1.0 µL | Ptet(2-5) | 0.3µL | -- | -- | 0.5 µL | 0.5 µL | up to 5 µL |
| experiment | RBS-GFP-DT | 1.0 µL | Pcon-pT181 attenuator-tetR-DT(E-1A) | 1.0 µL | Pcon antisense-spinach-DT(1-2A) | 0.7µL | Pcon-pT 181 attenuator(2-SA) | 2.1 µL | -- | -- | -- | -- | 0.5 µL | 0.5 µL | up to 5 µL |
| experiment | RBS-GFP-DT | 1.0 µL | Pcon antisense(E-1A) | 0.5 µL | spinach-DT(1-2) | 1.0 µL | Pcon-GFP-DT(1-2) | 0.8 µL | -- | -- | -- | -- | 0.5 µL | 0.5 µL | up to 5 µL |
| experiment | pSB1C3(EcoRI&SpeI) | 1.8 µL | Pcon-pT181 antisense(E-1A) | 0.8µL | spinach-DT(1-2) | 1.0µL | Pcon-GFP-DT(2-SA) | 0.8 µL | -- | -- | -- | -- | 0.5 µL | 0.5 µL | up to 5 µL |
| experiment | pSB1C3(EcoRI&SpeI) | 1.8 µL | Pcon-tet aptamer-DT(E-1A) | 3.0 µL | Ptet-pT181 antisense(1-3) | 1.5 µL | spinach-DT(3-2) | 1.5 µL | Pcon-GFP-DT(2-5A) | 0.8 µL | -- | -- | 0.5 µL | 0.5 µL | up to 5 µL |

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| Pcon-GFP-DT | Amp |
| RBS-GFP-DT | CP |
| Pcon-pT181 attenuator-DT | Amp |
| Pcon-tet aptamer-DT | Amp |
| Pcon-spinach-DT | Amp |
| Pcon-pT181 antisense-spinach-DT | Amp |
| spinach-DT | CP |

### BSAI Digestion

No name

|  | DNA | MilliQ | BSAI-HF | 10\*cut smart | total |
| --- | --- | --- | --- | --- | --- |
| Pcon | 7 µL | 10.7 µL | 0.3 µL | 2 µL | 20 µL |
| RBS-luxR-DT | 10 µL | 7 µL | 0.3 µL | 2 µL | 20 µL |

[![Igku 0926 E4.jpg](/wiki/images/1/18/Igku_0926_E4.jpg)](/File%3AIgku_0926_E4.jpg)

### Colony PCR

Tatsui

| Sample | base pair |
| --- | --- |
| GGA-1 (9~16) | 3177 |
| GGA-2 (9~16) | 3189 |
| GGA-3 (9~21) | 2989 |
| GGA-4 (9~16) | 2939 |
| GGA-6 (1) | 3359 |
| GGA-16(2~4) | 2939 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 3min30s | 30cycles |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | GGA-1 (9) | -- | -- |
| 2 | GGA-1 (10) | -- | -- |
| 3 | GGA-1 (11) | -- | -- |
| 4 | GGA-1 (12) | -- | -- |
| 5 | GGA-1 (13) | -- | -- |
| 6 | GGA-1 (14) | -- | -- |
| 7 | GGA-1 (15) | -- | -- |
| 8 | GGA-1 (16) | -- | -- |
| 9 | 1kbp ladder | -- | -- |
| 10 | GGA-2 (9) | -- | -- |
| 11 | GGA-2 (10) | -- | -- |
| 12 | GGA-2 (11) | -- | -- |
| 13 | GGA-2 (12) | -- | -- |
| 14 | GGA-2 (13) | -- | -- |
| 15 | GGA-2 (14) | -- | -- |
| 16 | GGA-2 (15) | -- | -- |
| 17 | GGA-2 (16) | -- | -- |

[![Igku 0926 E6.jpg](/wiki/images/5/5d/Igku_0926_E6.jpg)](/File%3AIgku_0926_E6.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | GGA-4 (9) | -- | -- |
| 2 | GGA-4 (10) | -- | -- |
| 3 | GGA-4 (11) | -- | -- |
| 4 | GGA-4 (12) | -- | -- |
| 5 | GGA-4 (13) | -- | -- |
| 6 | GGA-4 (14) | -- | -- |
| 7 | GGA-4 (15) | -- | -- |
| 8 | GGA-4 (16) | -- | -- |
| 9 | 1kbp ladder | -- | -- |
| 10 | GGA-6 (1) | -- | -- |
| 11 | GGA-16 (2) | -- | -- |
| 12 | GGA-16 (3) | -- | -- |
| 13 | GGA-16 (4) | -- | -- |

[![Igku 0926 E7.jpg](/wiki/images/a/a6/Igku_0926_E7.jpg)](/File%3AIgku_0926_E7.jpg)

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | GGA-3 (9) | -- | -- |
| 2 | GGA-3 (10) | -- | -- |
| 3 | GGA-3 (11) | -- | -- |
| 4 | GGA-3 (12) | -- | -- |
| 5 | 1kbp ladder | -- | -- |
| 6 | GGA-3 (13) | -- | -- |
| 7 | GGA-3 (15) | -- | -- |
| 8 | GGA-3 (14) | -- | -- |

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | GGA-3 (17) | -- | -- |
| 2 | GGA-3 (18) | -- | -- |
| 3 | GGA-3 (19) | -- | -- |
| 4 | 1kbp ladder | -- | -- |
| 5 | GGA-3 (20) | -- | -- |
| 6 | GGA-3 (21) | -- | -- |
| 7 | GGA-3 (16) | -- | -- |

[![Igku 0926 E8.jpg](/wiki/images/c/c4/Igku_0926_E8.jpg)](/File%3AIgku_0926_E8.jpg)

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | RBS-GFP-DT(RNA) | -- | -- |
| 2 | 1kbp ladder | -- | -- |
| 3 | Pcon-RBS-GFP-DT(RNA) | -- | -- |

[![Igku 0926 E9.jpg](/wiki/images/8/8a/Igku_0926_E9.jpg)](/File%3AIgku_0926_E9.jpg)

### Liquid Culture

Hirano

| Sample | medium |
| --- | --- |
| Pcon-GFP-DT | -- |
| RBS-GFP-DT | -- |
| Pcon-tet aptamer-DT | -- |
| Pcon-spinach-DT | -- |
| Pcon-pT181 antisense-spinach-DT | -- |
| spinach-DT | -- |

### Colony PCR

Hirano

| Sample | base pair |
| --- | --- |
| 9/25 Pcon-pH181 attenuator+RBS-GFP-DT-1 | -- |
| 9/25 Pcon-pH181 attenuator+RBS-GFP-DT-2 | -- |
| 9/25 Pcon-pH181 attenuator+RBS-GFP-DT-3 | -- |
| 9/25 Pcon-pH181 attenuator+RBS-GFP-DT-4 | -- |
| 9/24 Ptet-RBS-GFP-DT-1 | -- |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min40s | 30cycles |

### Ligasion(Golden Gate Assenbly)

Hirano

| state | Vector | Inserter1 | Inserter2 | Inserter3 | Inserter4 | Inserter5 |
| --- | --- | --- | --- | --- | --- | --- |
| experiment | GGA2 | Pcon-attenuator(E-1A) | Pcon-tetR-DT(1-2A) | Ptet(2-S) | RBS-GFP-DT(EcoRI&XbaI) |  |
| experiment | GGA3 | Pcon-tetap-DT(E-1A) | Pcon-tetR-DT(1-2A) | Ptet(2-S) | RBS-GFP-DT(EcoRI&XbaI) |  |
| experiment | GGA4 | Pcon-spi-DT(E-1A) | Pcon-tetR-DT(1-2A) | Ptet(2-S) | RBS-GFP-DT(EcoRI&XbaI) |  |
| experiment | GGA5 | Pcon-antisense(E-1A) | DT(1-3) | Pcon-tetR-DT(3-2A) | Ptet(2-S) | RBS-GFP-DT(EcoRI&XbaI) |
| experiment | GGA6 | Pcon-attenuator(E-1A) | tetap-DT(1-3) | Pcon-tetR-DT(3-2A) | Ptet(2-S) | RBS-GFP-DT(EcoRI&XbaI) |
| experiment | GGA7 | Pcon-tetR-DT(E-2A) | Ptet(2-S) | RBS-GFP-DT(EcoRI&XbaI) |  |  |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 37°C | 16°C | 50°C | 80°C | -- |
| 3min | 4min | 5min | 5min | 25cycles |

### RNA Extraction

Nakamoto

| Sample |
| --- |
| Pcon-Spinach-DT |
| Pcon-tetRaptamer-DT |
| Pcon-attenuator-DT |
| Spinach-DT |
| Pcon-antisense-Spinach-DT |

### Liquid Culture

Kozima

| Sample |
| --- |
| Pcon-attemuater-DT |
| Pcon-aptamer-DT |
| Pcon-spinach-DT |
| Pcon-RBS-tetR-DT |
| Pcon-antisense-spinach-DT(Master 1) |
| Pcon-antisense |

### Colony PCR

Nakamoto Kozima

| Sample | base pair |
| --- | --- |
| 9/25 9/19pSB1C3&9/3PT181 attenuator(1~3) | 601bp |
| 9/25 aptamer12\_1R-DT&9/24Pcon-pT181 attenuator(1~4) | 859bp |
| Pcon-attenuator-aptamer-DT(1) | 859bp |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 65°C | -- |
| 5min | 30sec | 30sec | 54sec | 30 |

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | 1kbp ladder | -- | -- |
| 2 | 9/25 9/23 1C3&9/3attenuator | -- | -- |
| 3 | 9/25 9/23 1C3&9/3attenuator | -- | -- |
| 4 | 9/25 9/23 1C3&9/3attenuator | -- | -- |
| 5 | 9/25aptamer-DT&9/24Pcon-attenuator | -- | -- |
| 6 | 9/25aptamer-DT&9/24Pcon-attenuator | -- | -- |
| 7 | 9/25aptamer-DT&9/24Pcon-attenuator | -- | -- |
| 8 | 9/24 Pcon attenuator-aptamer-DT(1) | -- | -- |
| 9 | 9/25aptamer-DT&9/24Pcon-attenuator | -- | -- |

[![Igku 0926 E10.jpg](/wiki/images/4/4a/Igku_0926_E10.jpg)](/File%3AIgku_0926_E10.jpg)

### Plating

Hirano

| Sample | Use plate |
| --- | --- |
| 9/26 GGA2 | LB(CP) |
| 9/26 GGA3 | LB(CP) |
| 9/26 GGA4 | LB(CP) |
| 9/26 GGA5 | LB(CP) |
| 9/26 GGA6 | LB(CP) |
| 9/26 GGA7 | LB(CP) |
| 9/26 GGA11 | LB(CP) |
| 9/26 GGA312 | LB(CP) |
| 9/26 GGA13 | LB(CP) |
| 9/26 GGA14 | LB(CP) |
| 9/26 GGA15 | LB(CP) |
| 9/26 GGA20 | LB(CP) |
| 9/26 GGA23 | LB(CP) |
| 9/26 GGA24 | LB(CP) |
| 9/26 Ptet+RBS-GFP-DT | LB(CP) |

incubate 37°C

### Restriction Enzyme Digestion

Kojima

|  | 9/24 pSB1C3 | EcoRI | SpeI | buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 11µL | 1µL | 1µL | 3µL | 14µL | 30µL |
| NC | 0.5µL | 0µL | 0µL | 1µL | 8.5µL | 10µL |

|  | 9/24 Pcon-pT181attenuator-aptamer12-1R-DT | EcoRI | SpeI | buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 8.7µL | 1µL | 1µL | 3µL | 16.3µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 8.6µL | 10µL |

|  | 9/24 pT181attenuator | EcoRI | SpeI | buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 3.1µL | 1µL | 1µL | 3µL | 21.9µL | 30µL |
| NC | 0.2µL | 0µL | 0µL | 1µL | 8.8µL | 10µL |

|  | 8/17 RBS-GFP-DT | EcoRI | BSA | buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 1cut | 16.4µL | 1µL | 3µL | 3µL | 6.6µL | 30µL |
| NC | 0.4µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 9/17 pSB4K5 | EcoRI | PstI | BSA | buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 8.5µL | 1µL | 1µL | 3µL | 3µL | 13.5µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 9/14 pSB4K5 | EcoRI | PstI | BSA | buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 7.3µL | 1µL | 1µL | 3µL | 3µL | 14.7µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 9/22 Pcon-pT181attenuator-DT | EcoRI | SpeI | buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 5.4µL | 1µL | 1µL | 3µL | 19.6µL | 30µL |
| NC | 0.3µL | 0µL | 0µL | 1µL | 8.7µL | 10µL |

|  | 8/7 RBS-GFP-DT 1 | XbaI | PstI | BSA | buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 8.1µL | 1µL | 1µL | 3µL | 3µL | 13.9µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

|  | 9/26 RBS-GFP-DT 1 | XbaI | BSA | buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 1cut | 8.1µL | 1µL | 3µL | 3µL | 14.9µL | 30µL |

|  | 9/16 Pλ-luxI | EcoRI | PstI | BSA | buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 14.7µL | 1µL | 1µL | 3µL | 3µL | 7.3µL | 30µL |
| NC | 0.7µL | 0µL | 0µL | 1µL | 1µL | 7.3µL | 10µL |

|  | 9/21 Ptet | EcoRI | SpeI | buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 14.2µL | 1µL | 1µL | 3µL | 10.8µL | 30µL |
| NC | 0.7µL | 0µL | 0µL | 1µL | 8.3µL | 10µL |

|  | 9/24 Pcon-pT181attenuator-aptamer12-1R-DT | XbaI | PstI | BSA | buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2cuts | 8.7µL | 1µL | 1µL | 3µL | 3µL | 13.3µL | 30µL |
| NC | 0.4µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL |

### Electrophoresis

Kojima

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | pSB1C3 | EcoRI | SpeI |
| 2 | pSB1C3 | -- | -- |
| 3 | Pcon-pT181attenuator-aptamer12-1R-DT | EcoRI | SpeI |
| 4 | Pcon-pT181attenuator-aptamer12-1R-DT | -- | -- |
| 5 | 1kb ladder | -- | -- |
| 6 | pT181attenuator | EcoRI | SpeI |
| 7 | pT181attenuator | -- | -- |
| 8 | RBS-GFP-DT | EcoRI | -- |
| 9 | RBS-GFP-DT | -- | -- |

[![Igku Sep26 Electrophoresis(E11) 1.jpg](/wiki/images/c/c1/Igku_Sep26_Electrophoresis%28E11%29_1.jpg)](/File%3AIgku_Sep26_Electrophoresis%28E11%29_1.jpg)

## Sep 27

### Gel Extraction

Kojima

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 2 | pSB1C3 | EcoRI+SpeI |
| 3 |
| 4 |
| 6 | Pcon-attenuator-aptmer-DT | EcoRI+SpeI |
| 7 |
| 8 |
| 10 | Pcon-attenuator-aptmer-DT | EcoRI+SpeI |
| 11 |
| 12 |

[![Igku 9272.jpg](/wiki/images/5/53/Igku_9272.jpg)](/File%3AIgku_9272.jpg)
[![Igku 9273.jpg](/wiki/images/4/45/Igku_9273.jpg)](/File%3AIgku_9273.jpg)

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-attenuator-DT(EcoRI+SpeI) | 6.8 | 1.98 | 0.43 |
| pT181-attenuator(EcoRI+SpeI) | 7.5 | 1.84 | 0.06 |
| pSB1C3(EcoRI+SpeI) | 17.7 | 1.97 | 0.71 |

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 3 | pSB4K5 | EcoRI+PstI |
| 4 | pSB4K5 | EcoRI+PstI |
| 5 | pSB4K5 | EcoRI+PstI |
| 7 | pSB6A1 | EcoRI+PstI |
| 8 | pSB6A1 | EcoRI+PstI |
| 9 | pSB6A1 | EcoRI+PstI |

[![Igku 9274.jpg](/wiki/images/a/ac/Igku_9274.jpg)](/File%3AIgku_9274.jpg)
[![Igku 9275.jpg](/wiki/images/6/69/Igku_9275.jpg)](/File%3AIgku_9275.jpg)

### Electrophoresis

No name

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | pSB4K5 | EcoRI | PstI |
| 2 | pSB4K5 | - | - |
| 3 | 1kbp ladder | - | - |
| 4 | Pcon-attenuator | EcoRI | SpeI |
| 5 | Pcon-attenuator | - | - |

| Lane | Sample | Enzyme1 | Enzyme2 |
| --- | --- | --- | --- |
| 1 | RBS-GFP-DT | XbaI | PstI |
| 2 | RBS-GFP-DT | - | - |
| 3 | RBS-GFP-DT | EcoRI | XbaI |
| 4 | 1kbp ladder | - | - |
| 5 | Ptet | EcoRI | SpeI |
| 6 | Ptet | - | - |

[![Igku 0927 G5.jpg](/wiki/images/f/fd/Igku_0927_G5.jpg)](/File%3AIgku_0927_G5.jpg)

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| Pcon-GFP | Amp |
| RBS-GFP-DT | CP |
| Pcon-attenuator | Amp |
| Pcon-attenuator-aptamer-DT | Amp |
| Pcon-tetR-DT | Amp |

### Ligation

No name

| state | Vector | | Inserter | | Ligation High ver.2 |
| --- | --- | --- | --- | --- | --- |
| experiment | 9/17 pSB1C3 (EcoRI+SpeI) 10.0ng/µL | 10µL | 9/26 pT181 attenuator(EcoRI+SpeI) 7.5ng/µL | 1.3µL | 3.5 µL |
| experiment | 9/25 DT (EcoRI+XbaI) 42.8ng/µL | 2.3µL | 9/22 Pcon-pT181 antisense(EcoRI+SpeI) 46.7ng/µL | 1.5µL | 1.9 µL |
| experiment | 9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL | 7.8µL | 9/10 Pcon-attenuator(EcoRI+SpeI) 13.8ng/µL | 8.0µL | 3.5 µL |
| experiment | 9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL | 7.8µL | 9/25 RBS-GFP-DT(XbaI+PstI) 13.9ng/µL | 12µL | 3.5 µL |
| experiment | 9/8 pSB4K5 (EcoRI+SpeI) 17.6ng/µL | 5.7 µL | 8/21 Pcon-GFP-DT(EcoRI+SpeI) 11ng/µL | 12.6µL | 3.5 µL |

### Liquid Culture

Hirano

| Sample | medium |
| --- | --- |
| Pcon-spinach-DT | plusgrow Amp |
| Pcon-tetRaptamer-DT | plusgrow Amp |
| Pcon-antisense-spinach-DT | plusgrow Amp |
| spinach-DT | plusgrow CP |

### RNA Extraction

Nakamoto

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/26 Pcon-GFP-DT | 423.0 | 1.83 | 1.46 |
| 9/26 Pcon-GFP-DT K | 298.7 | 1.82 | 1.50 |

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 9/27 Pcon-GFP-DT(RNA) |
| 2 | 1kb ladder |
| 3 | 9/27 Pcon-GFP-DT K(RNA) |

[![Igku 9271.jpg](/wiki/images/4/4f/Igku_9271.jpg)](/File%3AIgku_9271.jpg)

### Miniprep

Tatsui

| DNA |
| --- |
| Pcon-aptamer-DT |
| Pcon-spinach-DT |
| Pcon-antisense |
| Pcon-tetR-DT |
| Pcon-attenuator-DT |
| Pcon-antisense-spinach-DT |

### Transformation

Hirano

| Name1 | Name2 | Sample1(µL) | Sample2(µL) | Competent Cells(µL) | Total(µL) |
| --- | --- | --- | --- | --- | --- |
| Pcon-attenuator+RBS-GFP-DT(4K5) | Pcon-attenuator-DT(1A2) | 2 | 1 | 30 | 33 |
| Pcon-attenuator+RBS-GFP-DT(4K5) | Pcon-antisense-spinach-DT(1A2) | 2 | 1 | 30 | 33 |
| Pcon-attenuator+RBS-GFP-DT(4K5) | Pcon-tetR aptamer-DT(1A2) | 2 | 1 | 30 | 33 |
| Pcon-RBS-GFP-DT(4K5) | Pcon-attenuator-DT(1A2) | 2 | 1 | 30 | 33 |
| Pcon-RBS-GFP-DT(4K5) | Pcon-antisense-spinach-DT(1A2) | 2 | 1 | 30 | 33 |
| Pcon-RBS-GFP-DT(4K5) | Pcon-tetR aptamer-DT(1A2) | 2 | 1 | 30 | 33 |
| Pcon-attenuator+RBS-GFP-DT(4K5) | -- | 2 | -- | 20 | 22 |
| Pcon-RBS-GFP-DT(4K5) | -- | 2 | -- | 20 | 22 |
| attenuator(1C3) | -- | 2 | -- | 20 | 22 |

### Miniprep

No name

| DNA | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon-RBS－GFP-DT | 137.6 | 1.95 | 1.78 |
| RBS-GFP-DT | 77.0 | 2.06 | 1.96 |
| Pcon-PT181 attenuator | 231.0 | 1.89 | 1.89 |
| Pcon-antisense-Spinach-DT(2) | 130.0 | 1.56 | 1.62 |
| Pcon-aptamer-DT | 218.8 | 1.92 | 1.72 |
| Pcon-attenuator-DT | 180.7 | 1.95 | 1.78 |
| Pcon-Spinach-DT | 177.6 | 1.96 | 1.06 |
| Pcon-antisense-Spinach-DT(1) | 181.1 | 1.96 | 1.92 |
| Pcon-antisense | 152.9 | 2.02 | 1.96 |
| Pcon-RBS+ezR-DT | 164.1 | 1.93 | 1.91 |

### Restriction Enzyme Digestion

No name

|  | 9/27 RBS-GFP-DT | XbaI | PstI | Buffer | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 12µL | 1µL | 1µL | 3µL | 3µL | 20µL | 40µL |
| NC | 1.3µL | 0µL | 0µL | 1µL | 1µL | 6.7µL | 10µL |

|  | 9/27 Pcon-tetR-DT | EcoRI | PstI | Buffer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 2 cuts | 12.2µL | 1µL | 1µL | 3µL | 12.8µL | 30µL |
| NC | 0.6µL | 0µL | 0µL | 1µL | 8.4µL | 10µL |

### RNA Extraction

Nakamoto

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/27 Pcon-RBS-GFP-DT | 686.6 | 1.99 | 1.34 |
| 9/27 Pcon-RBS-GFP-DT (+High salt) | 127.6 | 1.76 | 0.27 |

### DNA sythesis

No name

| genome DNA | DNA volume | genome DNA wipeout buffer | RNase freewater | RT | RT buffer | Primer Mix | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/27 Pcon-RBS-GFP-DT | 0.8µL | 1µL | 5.2µL | 0.5µL | 2µL | 0.5µL | 10µL |
| 9/27 pcon-RBS-GFP-DT(+high salt) | 2.5µL | 1µL | 3.5µL | 0.5µL | 2µL | 0.5µL | 10µL |
| 9/27 Pcon-RBS-GFP-DT(revised concentration) | 0.5µL | 1µL | 5.5µL | 0.5µL | 2µL | 0.5µL | 10µL |

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 1kb ladder |
| 2 | Pcon-RBS-GFP-DT |
| 3 | Pcon-RBS-GFP-DT |

[![Igku 0927 E2.jpg](/wiki/images/e/ec/Igku_0927_E2.jpg)](/File%3AIgku_0927_E2.jpg)

### Liquid Culture

No name

| Sample | medium |
| --- | --- |
| Pcon spinach DT | plusgrow 2µL(Amp) |
| Pcon aptamer DT | plusgrow 2µL(Amp) |
| Pcon antisense Spinach DT | plusgrow 2µL(Amp) |
| Spinach DT | plusgrow 4µL(CP) |

### RT PCR

No name

| genome DNA | KOD plus | 10x buffer for KOD-plus-ver.2 | 2nM dNTPs | 25mM MgSO4 | F primer | R primer | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.5 | 1 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 15.5 | 25 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 98°C | 49.8°C | 68°C | -- |
| 2min | 10sec | 30sec | 10sec | 30 |

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | Pcon-GFP-DT(GFP) |
| 2 | Pcon-GFP-DT(internal) |
| 3 | Pcon-GFP-DT(HS)(GFP) |
| 4 | Pcon-GFP-DT(internal) |

| Lane | Sample |
| --- | --- |
| 1 | Pcon-GFP-DT(GFP) |
| 2 | Pcon-GFP-DT(internal) |
| 3 | 100bp ladder |

[![Igku 927E4.jpg](/wiki/images/4/44/Igku_927E4.jpg)](/File%3AIgku_927E4.jpg)

### Digesting cut check

No name

| Lane | Sample |
| --- | --- |
| 1 | RBS-GFP-DT(XbaI&PstI) |
| 2 | RBS-GFP-DT NC(XbaI&PstI) |
| 3 | 1kbp ladder |
| 4 | Pcon-RBS-tetR-DT(EcoRI&SpeI) |
| 5 | Pcon-RBS-tetR-DT NC(EcoRI&SpeI) |

[![Igku 927 E3.jpg](/wiki/images/d/d8/Igku_927_E3.jpg)](/File%3AIgku_927_E3.jpg)

### Gel Extraction

No name

| Lane | DNA | Enzyme |
| --- | --- | --- |
| 1 | 1kbp ladder | -- |
| 3 | RBS-GFP-DT | Xbal+PstI |
| 4 | RBS-GFP-DT | Xbal+PstI |
| 5 | RBS-GFP-DT | Xbal+PstI |
| 7 | 1kbp ladder | -- |
| 9 | Pcon-tetR-DT | EcoRI+SpeI |
| 10 | Pcon-tetR-DT | EcoRI+SpeI |
| 11 | Pcon-tetR-DT | EcoRI+SpeI |

[![Igku 0927 G7.jpg](/wiki/images/1/1b/Igku_0927_G7.jpg)](/File%3AIgku_0927_G7.jpg)
[![Igku 0927 G8.jpg](/wiki/images/b/bd/Igku_0927_G8.jpg)](/File%3AIgku_0927_G8.jpg)
[![Igku 0927 G9.jpg](/wiki/images/3/31/Igku_0927_G9.jpg)](/File%3AIgku_0927_G9.jpg)
[![Igku 0927 G10.jpg](/wiki/images/b/b3/Igku_0927_G10.jpg)](/File%3AIgku_0927_G10.jpg)

### RNA Extraction

Nakamoto

|  | 0 | 5min | 15min | 20min | 23min | 28min | 33min | 36min |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| aptamer generator | 0.348 | 0.408 | 0.550 | 0.865 |  |  |  |  |
| Pcon-spinach-DT | 0.250 | 0.240 | 0.305 | 0.426 | 0.471 | 0.554 | 0.668 | 0 |
| GFP generator | 0.389 | 0.420 | 0.516 | 0.884 |  |  |  |  |
| antisense-spinach | 0.371 | 0.438 | 0.537 | 0.733 |  |  |  |  |
| Spinach-DT | 0.084 | 0.110 | 0.134 | 0.433 | 0.269 | 0.326 | 0.409 | 0.742 |

| Name | concentration[µg/mL] | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Spinach-DT | 83.9 | 1.63 | 0.17 |
| Pcon-tetRaptamer-DT | 425.1 | 2.10 | 0.64 |
| Pcon-RBS-GFP-DT | 262.4 | 2.01 | 0.84 |
| Pcon-antisense-Spinach-DT | 489.1 | 2.05 | 0.68 |
| Pcon-Spinach-DT | 266.6 | 2.01 | 0.66 |

### cDNA Synthesis

Nakamoto

|  | 9/27 Spinach-DT | 9/23 Pcon-tetRaptamer-DT | 9/23 Pcon-RBS-GFP-DT | 9/23 Pcon-antisense-spinach-DT | 9/23 Pcon-Spinach-DT |
| --- | --- | --- | --- | --- | --- |
| RNA | 1 | 1 | 1 | 1 | 1 |
| gDNA wipeout | 6 | 1.2 | 1.9 | 1.0 | 1.9 |
| RNase-free water | 0 | 4.8 | 4.1 | 5.0 | 4.1 |
| RT | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| RT Buffer | 2 | 2 | 2 | 2 | 2 |
| Primer mix | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| total | 10µL | 10µL | 10µL | 10µL | 10µL |

## Sep 28

### Electrophoresis

Tatsui

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Spinach-DT+GAPDH+A1 |
| 3 | Spinach-DT+Spinach+C1 |
| 4 | Spinach-DT+gfp+E1 |
| 5 | Spinach-DT+aptamer+G1 |
| 6 | Pcon-tetR aptamer-DT+GADPH+A3 |
| 7 | Pcon-tetR aptamer-DT+Spinach+C3 |
| 8 | Pcon-tetR aptamer-DT+gfp+E3 |
| 9 | Pcon-tetR aptamer-DT+aptamer+G3 |
| 10 | Pcon-RBS-GFP-DT+GADPH+A5 |
| 11 | Pcon-RBS-GFP-DT+Spinach+C5 |
| 12 | Pcon-RBS-GFP-DT+gfp+E5 |
| 13 | Pcon-RBS-GFP-DT+aptamer+G5 |
| 14 | 100bp ladder |

[File:Igku xxxxxx.xxx](/File%3AIgku_xxxxxx.xxx "File:Igku xxxxxx.xxx")

| Lane | Sample |
| --- | --- |
| 1 | Pcon-antisense-Spinach-DT+GAPDH+A7 |
| 2 | Pcon-antisense-Spinach-DT+Spinach+C7 |
| 3 | Pcon-antisense-Spinach-DT+gfp+E7 |
| 4 | Pcon-antisense-Spinach-DT+aptamer+G7 |
| 5 | 100bp ladder |
| 6 | Pcon-Spinach-DT+GAPDH+A9 |
| 7 | Pcon-Spinach-DT+Spinach+C9 |
| 8 | Pcon-Spinach-DT+gfp+E9 |
| 9 | Pcon-Spinach-DT+aptamer+G9 |

[File:Igku xxxxxx.xxx](/File%3AIgku_xxxxxx.xxx "File:Igku xxxxxx.xxx")

### Colony PCR

| Sample | base pair |
| --- | --- |
| 9/27 pT181 attenuatorC(C3)1 | 330 |
| 9/27 pT181 attenuatorC(C3)2 | 330 |
| 9/27 Pcon antisense-DT1 | 372 |
| 9/27 Pcon antisense-DT2 | 372 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 24s |  |

| Sample | base pair |
| --- | --- |
| 9/27 Pcon-RBS-GFP-DT (4&5) | 1158 |
| 9/27 Pcon-RBS-GFP-DT (4&5) | 1158 |
| 9/27 Pcon-RBS-GFP-DT (4&5) | 1158 |
| 9/27 Pcon-RBS-GFP-DT (4&5) | 1158 |

| PreDenature | Denature | Annealing | Extension | cycle |
| --- | --- | --- | --- | --- |
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min24s |  |

### Electrophoresis

No name

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Pcon-tetR aptamer-DT+GADPH+A3 |
| 3 | Pcon-antisense-Spinach-DT+GADPH+A7 |
| 4 | Spinach-DT+GADPH+A1 |
| 5 | Pcon-RBS-GFP-DT+GADPH+A5 |
| 6 | Pcon-Spinach-DT+GADPH+A9 |

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Pcon-tetR aptamer-DT+gfp+E3 |
| 3 | Pcon-antisense-Spinach-DT+gfp+E7 |
| 4 | Spinach-DT+gfp+E1 |
| 5 | Pcon-RBS-GFP-DT+gfp+E5 |
| 6 | Pcon-Spinach-DT+gfp+E9 |

[![Igku 0928 E1.jpg](/wiki/images/b/b4/Igku_0928_E1.jpg)](/File%3AIgku_0928_E1.jpg)

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Pcon-tetR aptamer-DT+Spinach+C3 |
| 3 | Pcon-antisense-Spinach-DT+gfp+C7 |
| 4 | Spinach-DT+Spinach+C1 |
| 5 | Pcon-RBS-GFP-DT+Spinach+C5 |
| 6 | Pcon-Spinach-DT+Spinach+C9 |

| Lane | Sample |
| --- | --- |
| 1 | 100bp ladder |
| 2 | Pcon-tetR aptamer-DT+aptamer+G3 |
| 3 | Pcon-antisense-Spinach-DT+aptamer+G7 |
| 4 | Spinach-DT+aptamer+G1 |
| 5 | Pcon-RBS-GFP-DT+aptamer+G5 |
| 6 | Pcon-Spinach-DT+aptamer+G9 |

[![Igku 0928 E2.jpg](/wiki/images/2/22/Igku_0928_E2.jpg)](/File%3AIgku_0928_E2.jpg)

|  |
|  |

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