iGEM 2012 — Team Kyoto

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# Team:Kyoto/Notebook

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| Contents   * [1 Caution](#Caution) * 2   + [2.1 August 2](#August_2)     - [2.1.1 Mutation of FT](#Mutation_of_FT)   + [2.2 August 13](#August_13)     - [2.2.1 Liquid culture](#Liquid_culture)   + [2.3 August 14](#August_14)     - [2.3.1 Miniprep of FT](#Miniprep_of_FT)     - [2.3.2 Restriction digestion and Electrophoresis](#Restriction_digestion_and_Electrophoresis)     - [2.3.3 Liquid culture](#Liquid_culture_2)   + [2.4 August 15](#August_15)     - [2.4.1 Miniprep of FT](#Miniprep_of_FT_2)     - [2.4.2 Electrophoresis](#Electrophoresis)   + [2.5 August 16](#August_16)     - [2.5.1 Transformation](#Transformation)   + [2.6 August 17](#August_17)     - [2.6.1 Transformation](#Transformation_2)   + [2.7 August 20](#August_20)     - [2.7.1 PCR of FT](#PCR_of_FT)     - [2.7.2 Electrophoresis](#Electrophoresis_2)   + [2.8 August 21](#August_21)     - [2.8.1 Restriction digestion](#Restriction_digestion)     - [2.8.2 Ligation](#Ligation)     - [2.8.3 Liquid culture](#Liquid_culture_3)   + [2.9 August 22](#August_22)     - [2.9.1 Miniprep](#Miniprep)   + [2.10 August 23](#August_23)     - [2.10.1 Ethanol Precipitation](#Ethanol_Precipitation)   + [2.11 August 24](#August_24)     - [2.11.1 Restriction enzyme processing](#Restriction_enzyme_processing)     - [2.11.2 Ligation](#Ligation_2)   + [2.12 August 27](#August_27)     - [2.12.1 Colony PCR](#Colony_PCR)     - [2.12.2 FT(TOPO) PCR(re)](#FT.28TOPO.29_PCR.28re.29)     - [2.12.3 Liquid culture](#Liquid_culture_4)   + [2.13 August 28](#August_28)     - [2.13.1 Mutation of FT (re)](#Mutation_of_FT_.28re.29)     - [2.13.2 Miniprep FT(TOPO)](#Miniprep_FT.28TOPO.29)     - [2.13.3 Tranformation](#Tranformation)   + [2.14 August 29](#August_29)     - [2.14.1 Mutaion of FT(re;re)](#Mutaion_of_FT.28re.3Bre.29)     - [2.14.2 Inverse PCR](#Inverse_PCR)     - [2.14.3 Self-Ligation](#Self-Ligation)   + [2.15 August 30](#August_30)     - [2.15.1 Liquid culture(FT) 4mL x2](#Liquid_culture.28FT.29_4mL_x2)   + [2.16 August 31](#August_31)     - [2.16.1 Miniprep(FT)](#Miniprep.28FT.29)     - [2.16.2 Restriction enzyme processing (Mutation checking)](#Restriction_enzyme_processing_.28Mutation_checking.29)     - [2.16.3 PCR(RBS primer)](#PCR.28RBS_primer.29)     - [2.16.4 PCR(re)](#PCR.28re.29)     - [2.16.5 PCR(re)](#PCR.28re.29_2)   + [2.17 September 2](#September_2)     - [2.17.1 PCR(re;re)](#PCR.28re.3Bre.29)   + [2.18 September 3](#September_3)     - [2.18.1 Restriction enzyme processing](#Restriction_enzyme_processing_2)   + [2.19 September 4](#September_4)   + [2.20 September 5](#September_5)   + [2.21 September 6](#September_6)   + [2.22 September 9](#September_9)   + [2.23 September 10](#September_10)     - [2.23.1 Restriction enzyme processing](#Restriction_enzyme_processing_3)   + [2.24 September 11](#September_11)   + [2.25 September 12](#September_12)   + [2.26 September 13](#September_13)   + [2.27 September 14](#September_14)   + [2.28 September 15](#September_15)   + [2.29 September 16](#September_16)   + [2.30 September 17](#September_17)   + [2.31 September 18](#September_18)   + [2.32 September 19](#September_19)     - [2.32.1 Colony PCR(re)](#Colony_PCR.28re.29)   + [2.33 September 20](#September_20)   + [2.34 September 22](#September_22)   + [2.35 September 23](#September_23)   + [2.36 September 24](#September_24)   + [2.37 September 25](#September_25)   + [2.38 October 12](#October_12)   + [2.39 October 16](#October_16)   + [2.40 October 17](#October_17)   + [2.41 October 20](#October_20)   + [2.42 October 21](#October_21)   + [2.43 October 22](#October_22)   + [2.44 October 23](#October_23)   + [2.45 October 24](#October_24)   + [2.46 October 25](#October_25) * [3](#_2)    + [3.1 February 7](#February_7)     - [3.1.1 Preculture](#Preculture)   + [3.2 February 8](#February_8)     - [3.2.1 Liquid culture](#Liquid_culture_5)     - [3.2.2 Making Competent Cell](#Making_Competent_Cell)     - [3.2.3 Transformation](#Transformation_3)     - [3.2.4 Making Culture Medium Plates](#Making_Culture_Medium_Plates)     - [3.2.5 Transformation](#Transformation_4)   + [3.3 February 9](#February_9)     - [3.3.1 Transformation](#Transformation_5)     - [3.3.2 Liquid culture](#Liquid_culture_6)     - [3.3.3 Making Competent cells](#Making_Competent_cells)   + [3.4 February 11](#February_11)     - [3.4.1 Checking Transformation efficiency](#Checking_Transformation_efficiency)   + [3.5 February 13](#February_13)     - [3.5.1 Transformation](#Transformation_6)     - [3.5.2 Liquid culture](#Liquid_culture_7)   + [3.6 February 14](#February_14)     - [3.6.1 Miniprep](#Miniprep_2)     - [3.6.2 Restrictive Digestion](#Restrictive_Digestion)   + [3.7 February 15](#February_15)     - [3.7.1 Making gel](#Making_gel)     - [3.7.2 Electrophoresis](#Electrophoresis_3)     - [3.7.3 Testing whether restriction enzyme were deactivated or not](#Testing_whether_restriction_enzyme_were_deactivated_or_not)   + [3.8 February 16](#February_16)     - [3.8.1 Electrophoresis](#Electrophoresis_4)     - [3.8.2 Genomic PCR](#Genomic_PCR)     - [3.8.3 Electrophoresis](#Electrophoresis_5)     - [3.8.4 Transformation](#Transformation_7)   + [3.9 February 17](#February_17)     - [3.9.1 PCR](#PCR)     - [3.9.2 Genomic PCR](#Genomic_PCR_2)     - [3.9.3 Electrophoresis](#Electrophoresis_6)     - [3.9.4 Restrictive Digestion](#Restrictive_Digestion_2)     - [3.9.5 Genomic PCR](#Genomic_PCR_3)     - [3.9.6 Electrophoresis](#Electrophoresis_7)     - [3.9.7 Checking of restriction enzyme](#Checking_of_restriction_enzyme)   + [3.10 February 18](#February_18)     - [3.10.1 Miniprep](#Miniprep_3)     - [3.10.2 Making Competent cells](#Making_Competent_cells_2)     - [3.10.3 Transformation](#Transformation_8)   + [3.11 February 20](#February_20)     - [3.11.1 Restrictive Digestion](#Restrictive_Digestion_3)     - [3.11.2 Electrophoresis](#Electrophoresis_8)     - [3.11.3 PCR](#PCR_2)     - [3.11.4 Electrophoresis](#Electrophoresis_9)   + [3.12 February 21](#February_21)     - [3.12.1 PCR (Advantage HF protocol)](#PCR_.28Advantage_HF_protocol.29)     - [3.12.2 Electrophoresis](#Electrophoresis_10)     - [3.12.3 Liquid culture](#Liquid_culture_8)   + [3.13 February 22](#February_22)     - [3.13.1 Gel extraction](#Gel_extraction)     - [3.13.2 PCR purification](#PCR_purification)     - [3.13.3 PCR](#PCR_3)     - [3.13.4 Electrophoresis](#Electrophoresis_11)     - [3.13.5 PCR](#PCR_4)     - [3.13.6 Electrophoresis](#Electrophoresis_12)     - [3.13.7 Checking Dpn1](#Checking_Dpn1)   + [3.14 February 23](#February_23)     - [3.14.1 Colony PCR](#Colony_PCR_2)     - [3.14.2 Electrophoresis](#Electrophoresis_13)     - [3.14.3 Miniprep](#Miniprep_4)     - [3.14.4 Liquid culture](#Liquid_culture_9)     - [3.14.5 Electrophoresis](#Electrophoresis_14)   + [3.15 February 27](#February_27)     - [3.15.1 Test of Dpn1](#Test_of_Dpn1)     - [3.15.2 Colony PCR](#Colony_PCR_3)     - [3.15.3 Electrophoresis](#Electrophoresis_15)     - [3.15.4 Results of liquid culture](#Results_of_liquid_culture)     - [3.15.5 PCR](#PCR_5)   + [3.16 February 28](#February_28)     - [3.16.1 Electrophoresis](#Electrophoresis_16)     - [3.16.2 Restrictive Digestion](#Restrictive_Digestion_4)     - [3.16.3 Electrophoresis](#Electrophoresis_17)     - [3.16.4 PCR and Electrophoresis](#PCR_and_Electrophoresis)     - [3.16.5 Restrictive Digestion](#Restrictive_Digestion_5)     - [3.16.6 PCR purification](#PCR_purification_2)     - [3.16.7 Ligation](#Ligation_3)   + [3.17 February 29](#February_29)     - [3.17.1 Transformation](#Transformation_9)     - [3.17.2 Checking Restriction enzyme](#Checking_Restriction_enzyme)     - [3.17.3 Checking TatABCD](#Checking_TatABCD)     - [3.17.4 PCR](#PCR_6)     - [3.17.5 Electrophoresis](#Electrophoresis_18)   + [3.18 March 1](#March_1)     - [3.18.1 PCR](#PCR_7)     - [3.18.2 Electrophoresis](#Electrophoresis_19)     - [3.18.3 PCR](#PCR_8)     - [3.18.4 Restrictive Digestion](#Restrictive_Digestion_6)     - [3.18.5 PCR Purification](#PCR_Purification_3)     - [3.18.6 Ligation](#Ligation_4)   + [3.19 March 2](#March_2)     - [3.19.1 Ligation](#Ligation_5)     - [3.19.2 Transformation](#Transformation_10)     - [3.19.3 PCR](#PCR_9)     - [3.19.4 Electrophoresis](#Electrophoresis_20)     - [3.19.5 Restrictive Digestion](#Restrictive_Digestion_7)     - [3.19.6 Electrophoresis](#Electrophoresis_21)     - [3.19.7 Restrictive Digestion](#Restrictive_Digestion_8)     - [3.19.8 Restrictive Digestion](#Restrictive_Digestion_9)     - [3.19.9 Checking TMAO](#Checking_TMAO)     - [3.19.10 Electrophoresis](#Electrophoresis_22)     - [3.19.11 Electrophoresis](#Electrophoresis_23)   + [3.20 March 3](#March_3)     - [3.20.1 PCR](#PCR_10)     - [3.20.2 Miniprep](#Miniprep_5)   + [3.21 March 4](#March_4)     - [3.21.1 Sequence of TatABCD](#Sequence_of_TatABCD)     - [3.21.2 Colony PCR of TMAO](#Colony_PCR_of_TMAO)     - [3.21.3 Electrophoresis](#Electrophoresis_24)     - [3.21.4 Restrictive Digestion](#Restrictive_Digestion_10)     - [3.21.5 Electrophoresis](#Electrophoresis_25)     - [3.21.6 Transformation](#Transformation_11)   + [3.22 March 5](#March_5)     - [3.22.1 Restrictive Digestion](#Restrictive_Digestion_11)     - [3.22.2 Ligation](#Ligation_6)     - [3.22.3 Transformation](#Transformation_12)     - [3.22.4 PCR](#PCR_11)     - [3.22.5 Electrophoresis](#Electrophoresis_26)     - [3.22.6 Restrictive Digestion](#Restrictive_Digestion_12)     - [3.22.7 Ligation](#Ligation_7)     - [3.22.8 Transformation](#Transformation_13)     - [3.22.9 Restrictive Digestion](#Restrictive_Digestion_13)     - [3.22.10 Electrophoresis](#Electrophoresis_27)     - [3.22.11 PCR](#PCR_12)     - [3.22.12 electrophoresis](#electrophoresis_28)   + [3.23 March 6](#March_6)     - [3.23.1 Restrictive Digestion](#Restrictive_Digestion_14)     - [3.23.2 PCR](#PCR_13)     - [3.23.3 Restrictive Digestion](#Restrictive_Digestion_15)     - [3.23.4 Ligation](#Ligation_8)   + [3.24 March 7](#March_7)     - [3.24.1 Electrophoresis](#Electrophoresis_29)     - [3.24.2 Ligation](#Ligation_9)     - [3.24.3 Restrictive Digestion](#Restrictive_Digestion_16)     - [3.24.4 Purification](#Purification)     - [3.24.5 Ligation](#Ligation_10)   + [3.25 March 8](#March_8)     - [3.25.1 Restrictive Digestion](#Restrictive_Digestion_17)     - [3.25.2 Ligation](#Ligation_11)     - [3.25.3 Liquid culture](#Liquid_culture_10)     - [3.25.4 Transformation](#Transformation_14)   + [3.26 March 9](#March_9)     - [3.26.1 Restrictive Digestion](#Restrictive_Digestion_18)     - [3.26.2 Miniprep](#Miniprep_6)     - [3.26.3 Colony PCR](#Colony_PCR_4)     - [3.26.4 Ligation](#Ligation_12)     - [3.26.5 Transformation](#Transformation_15)     - [3.26.6 Miniprep](#Miniprep_7)   + [3.27 March 10](#March_10)     - [3.27.1 Screening PCR](#Screening_PCR)     - [3.27.2 Restrictive Digestion](#Restrictive_Digestion_19)     - [3.27.3 Ligation](#Ligation_13)   + [3.28 March 11](#March_11)     - [3.28.1 Miniprep](#Miniprep_8)     - [3.28.2 Screening PCR](#Screening_PCR_2)     - [3.28.3 Electrophoresis](#Electrophoresis_30)   + [3.29 March 12](#March_12)     - [3.29.1 Transformation](#Transformation_16)     - [3.29.2 Restrictive Digestion](#Restrictive_Digestion_20)     - [3.29.3 Ligation](#Ligation_14)   + [3.30 March 13](#March_13)     - [3.30.1 Miniprep](#Miniprep_9)   + [3.31 March 14](#March_14)     - [3.31.1 Restrictive Digestion](#Restrictive_Digestion_21)     - [3.31.2 Ligation](#Ligation_15)     - [3.31.3 Liquid culture](#Liquid_culture_11)   + [3.32 March 15](#March_15)     - [3.32.1 Liquid culture](#Liquid_culture_12)     - [3.32.2 Restrictive Digestion](#Restrictive_Digestion_22)     - [3.32.3 Miniprep](#Miniprep_10)     - [3.32.4 Ligation](#Ligation_16)     - [3.32.5 Transformation](#Transformation_17)     - [3.32.6 Screening PCR](#Screening_PCR_3)   + [3.33 March 16](#March_16)     - [3.33.1 Miniprep](#Miniprep_11)     - [3.33.2 Colony PCR](#Colony_PCR_5)     - [3.33.3 Electrophoresis](#Electrophoresis_31)     - [3.33.4 Checking Transformation Efficiency](#Checking_Transformation_Efficiency_2)     - [3.33.5 Restrictive Digestion](#Restrictive_Digestion_23)     - [3.33.6 Ligation](#Ligation_17)   + [3.34 March 17](#March_17)     - [3.34.1 Miniprep](#Miniprep_12)     - [3.34.2 Checking the Insert](#Checking_the_Insert)   + [3.35 March 19](#March_19)     - [3.35.1 Restrictive Digestion](#Restrictive_Digestion_24)     - [3.35.2 Ligation](#Ligation_18)     - [3.35.3 Restrictive Digestion](#Restrictive_Digestion_25)     - [3.35.4 Ligation](#Ligation_19)     - [3.35.5 Transformation](#Transformation_18)   + [3.36 March 20](#March_20)     - [3.36.1 Screaning PCR](#Screaning_PCR)     - [3.36.2 Restrictive Digestion](#Restrictive_Digestion_26)   + [3.37 March 21](#March_21)     - [3.37.1 Miniprep](#Miniprep_13)     - [3.37.2 Restrictive Digestion](#Restrictive_Digestion_27)     - [3.37.3 Ligation](#Ligation_20)   + [3.38 March 22](#March_22)     - [3.38.1 PCR](#PCR_14)     - [3.38.2 Restrictive Digestion](#Restrictive_Digestion_28)     - [3.38.3 Ligation](#Ligation_21)   + [3.39 March 23](#March_23)     - [3.39.1 Screening PCR](#Screening_PCR_4)     - [3.39.2 Restrictive Digestion](#Restrictive_Digestion_29)     - [3.39.3 Ethanol precipitation](#Ethanol_precipitation_2)     - [3.39.4 Miniprep](#Miniprep_14)     - [3.39.5 Restrictive Digestion](#Restrictive_Digestion_30)     - [3.39.6 Gel Extraction](#Gel_Extraction_2)   + [3.40 March 26](#March_26)     - [3.40.1 Miniprep](#Miniprep_15)     - [3.40.2 Restrictive Digestion](#Restrictive_Digestion_31)     - [3.40.3 Ligation](#Ligation_22)     - [3.40.4 Transformation](#Transformation_19)   + [3.41 March 27](#March_27)     - [3.41.1 Miniprep](#Miniprep_16)     - [3.41.2 Transformation](#Transformation_20)     - [3.41.3 Screening PCR](#Screening_PCR_5)     - [3.41.4 Liquid culture](#Liquid_culture_13)   + [3.42 July 23](#July_23)     - [3.42.1 Transformation](#Transformation_21)   + [3.43 July 24](#July_24)     - [3.43.1 Plating in SOC medium](#Plating_in_SOC_medium)   + [3.44 July 25](#July_25)     - [3.44.1 Transformation](#Transformation_22)   + [3.45 July 27](#July_27)     - [3.45.1 Restrictive Digestion](#Restrictive_Digestion_32)   + [3.46 July 30](#July_30)     - [3.46.1 Ligation](#Ligation_23)   + [3.47 July 31](#July_31)     - [3.47.1 Restriction Enzyme Processing](#Restriction_Enzyme_Processing_4)     - [3.47.2 Liquid Culture](#Liquid_Culture_14)   + [3.48 August 1](#August_1)     - [3.48.1 Restrictive Digestion](#Restrictive_Digestion_33)     - [3.48.2 MIniprep](#MIniprep_17)     - [3.48.3 Liquid culture](#Liquid_culture_15)   + [3.49 August 3](#August_3)     - [3.49.1 Restrictive Digestion](#Restrictive_Digestion_34)     - [3.49.2 DNA purification](#DNA_purification)     - [3.49.3 Ligation](#Ligation_24)     - [3.49.4 Miniprep](#Miniprep_18)   + [3.50 August 10](#August_10)     - [3.50.1 Ligation](#Ligation_25)     - [3.50.2 Transformation](#Transformation_23)   + [3.51 August 13](#August_13_2)     - [3.51.1 Liquid culture](#Liquid_culture_16)   + [3.52 August 14](#August_14_2)     - [3.52.1 Miniprep](#Miniprep_19)     - [3.52.2 Colony PCR](#Colony_PCR_6)   + [3.53 August 15](#August_15_2)     - [3.53.1 Colony PCR](#Colony_PCR_7)   + [3.54 August 16](#August_16_2)     - [3.54.1 Electrophoresis](#Electrophoresis_32)   + [3.55 August 17](#August_17_2)     - [3.55.1 Colony PCR](#Colony_PCR_8)     - [3.55.2 Electrophoresis](#Electrophoresis_33)   + [3.56 August 20](#August_20_2)     - [3.56.1 Restrictive Digestion](#Restrictive_Digestion_35)   + [3.57 August 21](#August_21_2)     - [3.57.1 Colony PCR](#Colony_PCR_9)     - [3.57.2 Restrictive Digestion](#Restrictive_Digestion_36)     - [3.57.3 Electrophoresis](#Electrophoresis_34)     - [3.57.4 Transformation](#Transformation_24)     - [3.57.5 Electrophoresis](#Electrophoresis_35)     - [3.57.6 Colony PCR](#Colony_PCR_10)     - [3.57.7 Transformation](#Transformation_25)     - [3.57.8 PCR](#PCR_15)   + [3.58 August 22](#August_22_2)     - [3.58.1 Restrictive Digestion](#Restrictive_Digestion_37)     - [3.58.2 Gel extraction](#Gel_extraction_3)     - [3.58.3 Transformation(the second time)](#Transformation.28the_second_time.29)     - [3.58.4 Electrophoresis](#Electrophoresis_36)     - [3.58.5 PCR](#PCR_16)     - [3.58.6 Colony PCR](#Colony_PCR_11)     - [3.58.7 Electrophoresis](#Electrophoresis_37)     - [3.58.8 Transformation](#Transformation_26)   + [3.59 August 23](#August_23_2)     - [3.59.1 Colony PCR](#Colony_PCR_12)   + [3.60 August 24](#August_24_2)     - [3.60.1 Purification of PCR products](#Purification_of_PCR_products)     - [3.60.2 Restrictive Digestion](#Restrictive_Digestion_38)     - [3.60.3 Gel extraction](#Gel_extraction_4)     - [3.60.4 Restrictive Digestion](#Restrictive_Digestion_39)     - [3.60.5 Electrophoresis](#Electrophoresis_38)     - [3.60.6 Ligation](#Ligation_26)   + [3.61 August 25](#August_25)   + [3.62 August 26](#August_26)   + [3.63 August 27](#August_27_2)     - [3.63.1 Ligation](#Ligation_27)     - [3.63.2 Colony PCR](#Colony_PCR_13)     - [3.63.3 Purification of PCR products](#Purification_of_PCR_products_2)     - [3.63.4 Restrictive Digestion](#Restrictive_Digestion_40)     - [3.63.5 Purification of PCR products](#Purification_of_PCR_products_3)     - [3.63.6 Restrictive Digestion](#Restrictive_Digestion_41)   + [3.64 August 28](#August_28_2)     - [3.64.1 Colony PCR](#Colony_PCR_14)     - [3.64.2 Transformation](#Transformation_27)     - [3.64.3 Liquid culture](#Liquid_culture_17)   + [3.65 August 29](#August_29_2)     - [3.65.1 Kil assay](#Kil_assay)     - [3.65.2 Liquid culture](#Liquid_culture_18)     - [3.65.3 Restrictive Digestion](#Restrictive_Digestion_42)     - [3.65.4 Colony PCR](#Colony_PCR_15)     - [3.65.5 Gel extraction](#Gel_extraction_5)   + [3.66 August 30](#August_30_2)     - [3.66.1 Ligation](#Ligation_28)     - [3.66.2 Transformation](#Transformation_28)     - [3.66.3 Electrophoresis](#Electrophoresis_39)     - [3.66.4 Miniprep](#Miniprep_20)     - [3.66.5 Colony PCR](#Colony_PCR_16)     - [3.66.6 Restrictive Digestion](#Restrictive_Digestion_43)     - [3.66.7 Electrophoresis](#Electrophoresis_40)   + [3.67 August 31](#August_31_2)     - [3.67.1 Gel extraction](#Gel_extraction_6)     - [3.67.2 Purification](#Purification_2)     - [3.67.3 Ligation](#Ligation_29)     - [3.67.4 Transformation](#Transformation_29)   + [3.68 September 2](#September_2_2)     - [3.68.1 Colony PCR](#Colony_PCR_17)     - [3.68.2 Restrictive Digestion](#Restrictive_Digestion_44)     - [3.68.3 Electrophoresis](#Electrophoresis_41)   + [3.69 September 3](#September_3_2)     - [3.69.1 PCR](#PCR_17)     - [3.69.2 Gel extraction](#Gel_extraction_7)     - [3.69.3 Restrictive Digestion](#Restrictive_Digestion_45)     - [3.69.4 Purification](#Purification_3)     - [3.69.5 Electrophoresis](#Electrophoresis_42)     - [3.69.6 Liquid culture](#Liquid_culture_19)     - [3.69.7 PCR](#PCR_18)     - [3.69.8 Gel extraction](#Gel_extraction_8)     - [3.69.9 Transformation](#Transformation_30)     - [3.69.10 Electrophoresis](#Electrophoresis_43)     - [3.69.11 Colony PCR](#Colony_PCR_18)   + [3.70 September 4](#September_4_2)     - [3.70.1 Liquid culture](#Liquid_culture_20)     - [3.70.2 PCR](#PCR_19)     - [3.70.3 Electrophoresis](#Electrophoresis_44)     - [3.70.4 Liquid culture](#Liquid_culture_21)   + [3.71 September 5](#September_5_2)     - [3.71.1 Colony PCR](#Colony_PCR_19)     - [3.71.2 Gel extraction](#Gel_extraction_9)     - [3.71.3 Miniprep](#Miniprep_21)     - [3.71.4 Restrictive Digestion](#Restrictive_Digestion_46)     - [3.71.5 Electrophoresis](#Electrophoresis_45)     - [3.71.6 Colony PCR](#Colony_PCR_20)     - [3.71.7 Gel extraction](#Gel_extraction_10)     - [3.71.8 Ligation](#Ligation_30)     - [3.71.9 Electrophoresis](#Electrophoresis_46)     - [3.71.10 Ligation](#Ligation_31)     - [3.71.11 Liquid culture](#Liquid_culture_22)   + [3.72 September 6](#September_6_2)     - [3.72.1 Miniprep](#Miniprep_22)     - [3.72.2 Restrictive Digestion](#Restrictive_Digestion_47)     - [3.72.3 Transformation](#Transformation_31)     - [3.72.4 Gel extraction](#Gel_extraction_11)     - [3.72.5 Electrophoresis](#Electrophoresis_47)     - [3.72.6 Restrictive Digestion](#Restrictive_Digestion_48)     - [3.72.7 Gel extraction](#Gel_extraction_12)   + [3.73 September 7](#September_7)     - [3.73.1 Liquid culture](#Liquid_culture_23)     - [3.73.2 Colony PCR](#Colony_PCR_21)     - [3.73.3 Restrictive Digestion](#Restrictive_Digestion_49)     - [3.73.4 Electrophoresis](#Electrophoresis_48)     - [3.73.5 Gel extraction](#Gel_extraction_13)     - [3.73.6 Restrictive Digestion](#Restrictive_Digestion_50)     - [3.73.7 Liquid culture](#Liquid_culture_24)   + [3.74 September 8](#September_8)     - [3.74.1 Check the effect of Amp](#Check_the_effect_of_Amp)       * [3.74.1.1 5.5h after incubating at 37℃](#5.5h_after_incubating_at_37.E2.84.83)     - [3.74.2 Observation through a confocal microscope](#Observation_through_a_confocal_microscope)     - [3.74.3 Gel extraction](#Gel_extraction_14)     - [3.74.4 Restrictive Digestion](#Restrictive_Digestion_51)   + [3.75 September 9](#September_9_2)     - [3.75.1 Restrictive Digestion](#Restrictive_Digestion_52)     - [3.75.2 Electrophoresis](#Electrophoresis_49)     - [3.75.3 Gel extraction](#Gel_extraction_15)     - [3.75.4 Liquid culture](#Liquid_culture_25)   + [3.76 September 10](#September_10_2)     - [3.76.1 Miniprep](#Miniprep_23)     - [3.76.2 Ligation](#Ligation_32)     - [3.76.3 Restrictive Digestion](#Restrictive_Digestion_53)     - [3.76.4 Gel extraction](#Gel_extraction_16)     - [3.76.5 Ligation](#Ligation_33)     - [3.76.6 Transformation](#Transformation_32)   + [3.77 September 11](#September_11_2)     - [3.77.1 Colony PCR](#Colony_PCR_22)     - [3.77.2 Electrophoresis](#Electrophoresis_50)     - [3.77.3 Restrictive Digestion](#Restrictive_Digestion_54)     - [3.77.4 Liquid culture](#Liquid_culture_26)     - [3.77.5 Ligation](#Ligation_34)     - [3.77.6 Colony PCR](#Colony_PCR_23)     - [3.77.7 Gel extraction](#Gel_extraction_17)     - [3.77.8 Electrophoresis](#Electrophoresis_51)     - [3.77.9 Liquid culture](#Liquid_culture_27)     - [3.77.10 Colony PCR](#Colony_PCR_24)   + [3.78 September 12](#September_12_2)     - [3.78.1 Colony PCR](#Colony_PCR_25)     - [3.78.2 Check the effect of Amp](#Check_the_effect_of_Amp_2)     - [3.78.3 Electrophoresis](#Electrophoresis_52)     - [3.78.4 Restrictive Digestion](#Restrictive_Digestion_55)     - [3.78.5 Electrophoresis](#Electrophoresis_53)     - [3.78.6 Transformation](#Transformation_33)     - [3.78.7 Gel extraction](#Gel_extraction_18)     - [3.78.8 Ligation](#Ligation_35)     - [3.78.9 Observation through a confocal microscope](#Observation_through_a_confocal_microscope_2)   + [3.79 September 13](#September_13_2)     - [3.79.1 Miniprep](#Miniprep_24)     - [3.79.2 Restrictive Digestion](#Restrictive_Digestion_56)     - [3.79.3 Transformation](#Transformation_34)     - [3.79.4 Colony PCR](#Colony_PCR_26)   + [3.80 September 14](#September_14_2)     - [3.80.1 Colony PCR](#Colony_PCR_27)     - [3.80.2 Colony PCR](#Colony_PCR_28)   + [3.81 September 15](#September_15_2)     - [3.81.1 Liquid culture](#Liquid_culture_28)     - [3.81.2 Miniprep](#Miniprep_25)     - [3.81.3 Restrictive Digestion](#Restrictive_Digestion_57)     - [3.81.4 Electrophoresis](#Electrophoresis_54)     - [3.81.5 Gel extraction](#Gel_extraction_19)     - [3.81.6 Purification](#Purification_4)     - [3.81.7 Ligation](#Ligation_36)   + [3.82 September 16](#September_16_2)     - [3.82.1 Transformation](#Transformation_35)   + [3.83 September 17](#September_17_2)     - [3.83.1 Colony PCR](#Colony_PCR_29)     - [3.83.2 Restrictive Digestion](#Restrictive_Digestion_58)     - [3.83.3 Electrophoresis](#Electrophoresis_55)     - [3.83.4 Liquid culture](#Liquid_culture_29)     - [3.83.5 Electrophoresis](#Electrophoresis_56)     - [3.83.6 Miniprep](#Miniprep_26)   + [3.84 September 18](#September_18_2)     - [3.84.1 Gel extraction](#Gel_extraction_20)     - [3.84.2 Ligation](#Ligation_37)     - [3.84.3 Transformation](#Transformation_36)     - [3.84.4 Observation through a confocal microscope](#Observation_through_a_confocal_microscope_3)     - [3.84.5 Restrictive Digestion](#Restrictive_Digestion_59)     - [3.84.6 Gel extraction](#Gel_extraction_21)     - [3.84.7 Miniprep](#Miniprep_27)     - [3.84.8 Restrictive Digestion](#Restrictive_Digestion_60)     - [3.84.9 Electrophoresis](#Electrophoresis_57)     - [3.84.10 Restrictive Digestion](#Restrictive_Digestion_61)   + [3.85 September 19](#September_19_2)     - [3.85.1 Gel extraction](#Gel_extraction_22)     - [3.85.2 Ligation](#Ligation_38)     - [3.85.3 Electrophoresis](#Electrophoresis_58)     - [3.85.4 Liquid Culture](#Liquid_Culture_30)     - [3.85.5 Transformation](#Transformation_37)     - [3.85.6 Liquid culture](#Liquid_culture_31)     - [3.85.7 Colony PCR](#Colony_PCR_30)     - [3.85.8 Restrictive Digestion](#Restrictive_Digestion_62)     - [3.85.9 Electrophoresis](#Electrophoresis_59)     - [3.85.10 Ligation](#Ligation_39)     - [3.85.11 Restrictive Digestion](#Restrictive_Digestion_63)     - [3.85.12 PCR](#PCR_20)     - [3.85.13 Electrophoresis](#Electrophoresis_60)     - [3.85.14 Liquid culture](#Liquid_culture_32)     - [3.85.15 PCR purification](#PCR_purification_4)   + [3.86 September 20](#September_20_2)     - [3.86.1 Gel extraction](#Gel_extraction_23)     - [3.86.2 Restrictive Digestion](#Restrictive_Digestion_64)     - [3.86.3 Ligation](#Ligation_40)     - [3.86.4 Transformation](#Transformation_38)     - [3.86.5 Miniprep](#Miniprep_28)     - [3.86.6 Restrictive Digestion](#Restrictive_Digestion_65)     - [3.86.7 Colony PCR](#Colony_PCR_31)     - [3.86.8 Liquid culture](#Liquid_culture_33)     - [3.86.9 Gel extraction](#Gel_extraction_24)     - [3.86.10 Ligation](#Ligation_41)     - [3.86.11 Colony PCR](#Colony_PCR_32)     - [3.86.12 Transformation](#Transformation_39)     - [3.86.13 Liquid culture](#Liquid_culture_34)     - [3.86.14 PCR](#PCR_21)     - [3.86.15 Miniprep](#Miniprep_29)       * [3.86.15.1 Electrophoresis](#Electrophoresis_61)   + [3.87 September 21](#September_21)     - [3.87.1 Gel extraction](#Gel_extraction_25)     - [3.87.2 Electrophoresis](#Electrophoresis_62)     - [3.87.3 Electrophoresis](#Electrophoresis_63)     - [3.87.4 Restrictive Digestion](#Restrictive_Digestion_66)     - [3.87.5 Electrophoresis](#Electrophoresis_64)     - [3.87.6 Colony PCR](#Colony_PCR_33)     - [3.87.7 Electrophoresis](#Electrophoresis_65)   + [3.88 September 22](#September_22_2)     - [3.88.1 Miniprep](#Miniprep_30)     - [3.88.2 Electrophoresis](#Electrophoresis_66)     - [3.88.3 Restrictive Digestion](#Restrictive_Digestion_67)     - [3.88.4 Restrictive Digestion](#Restrictive_Digestion_68)     - [3.88.5 Electrophoresis](#Electrophoresis_67)     - [3.88.6 Gel extraction](#Gel_extraction_26)     - [3.88.7 Purification](#Purification_5)     - [3.88.8 Restrictive Digestion](#Restrictive_Digestion_69)     - [3.88.9 Electrophoresis](#Electrophoresis_68)   + [3.89 September 23](#September_23_2)     - [3.89.1 Gel extraction](#Gel_extraction_27)     - [3.89.2 Restrictive Digestion](#Restrictive_Digestion_70)     - [3.89.3 Ligation](#Ligation_42)     - [3.89.4 Liquid culture](#Liquid_culture_35)     - [3.89.5 Colony PCR](#Colony_PCR_34)     - [3.89.6 Electrophoresis](#Electrophoresis_69)     - [3.89.7 Liquid culture](#Liquid_culture_36)     - [3.89.8 Gel extraction](#Gel_extraction_28)     - [3.89.9 Purification](#Purification_6)     - [3.89.10 Ligation](#Ligation_43)     - [3.89.11 Transformation](#Transformation_40)     - [3.89.12 Electrophoresis](#Electrophoresis_70)   + [3.90 September 24](#September_24_2)     - [3.90.1 Miniprep](#Miniprep_31)     - [3.90.2 Colony PCR](#Colony_PCR_35)     - [3.90.3 Liquid culture](#Liquid_culture_37)     - [3.90.4 Colony PCR](#Colony_PCR_36)     - [3.90.5 Electrophoresis](#Electrophoresis_71)   + [3.91 September 25](#September_25_2)     - [3.91.1 Liquid culture](#Liquid_culture_38)     - [3.91.2 Miniprep](#Miniprep_32)     - [3.91.3 Restrictive Digestion](#Restrictive_Digestion_71)     - [3.91.4 Electrophoresis](#Electrophoresis_72)     - [3.91.5 Liquid culture](#Liquid_culture_39)     - [3.91.6 Restrictive Digestion](#Restrictive_Digestion_72)     - [3.91.7 Electrophoresis](#Electrophoresis_73)     - [3.91.8 Observation through a confocal microscope](#Observation_through_a_confocal_microscope_4)   + [3.92 October 12](#October_12_2)   + [3.93 October 15](#October_15)   + [3.94 October 16](#October_16_2)   + [3.95 October 17](#October_17_2)   + [3.96 October 18](#October_18)   + [3.97 October 19](#October_19)   + [3.98 October 22](#October_22_2)   + [3.99 October 23](#October_23_2)   + [3.100 October 24](#October_24_2)   + [3.101 October 25](#October_25_2) * [4](#_3)    + [4.1 September 6](#September_6_3)     - [4.1.1 PCR and Electrophoresis assay](#PCR_and_Electrophoresis_assay)   + [4.2 September 7](#September_7_2)     - [4.2.1 PCR and Electrophoresis assay of psB1K3](#PCR_and_Electrophoresis_assay_of_psB1K3)   + [4.3 September 10](#September_10_3)     - [4.3.1 PCR and Electrophoresis assay of CFP](#PCR_and_Electrophoresis_assay_of_CFP)     - [4.3.2 PCR and Electrophoresis assay of lacI](#PCR_and_Electrophoresis_assay_of_lacI)   + [4.4 PCR](#PCR_22)   + [4.5 September 11](#September_11_3)     - [4.5.1 PCR and Electrophoresis assay](#PCR_and_Electrophoresis_assay_2)     - [4.5.2 PCR and Electrophoresis assay](#PCR_and_Electrophoresis_assay_3)   + [4.6 September 12](#September_12_3)     - [4.6.1 Purification of PCR product](#Purification_of_PCR_product)     - [4.6.2 Golden Gate Assembly](#Golden_Gate_Assembly)     - [4.6.3 Transformation](#Transformation_41)   + [4.7 September 13](#September_13_3)     - [4.7.1 Transformation](#Transformation_42)   + [4.8 September 14](#September_14_3)     - [4.8.1 Liquid Culture](#Liquid_Culture_40)   + [4.9 September 15](#September_15_3)     - [4.9.1 Miniprep](#Miniprep_33)     - [4.9.2 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion)   + [4.10 September 17](#September_17_3)     - [4.10.1 Electrophoresis assay](#Electrophoresis_assay)   + [4.11 September 18](#September_18_3)     - [4.11.1 Restriction Enzyme Digestion](#Restriction_Enzyme_Digestion_2)     - [4.11.2 Colony PCR](#Colony_PCR_37)   + [4.12 September 19](#September_19_3)     - [4.12.1 DpnI Restriction Enzyme Digestion of psB1K3](#DpnI_Restriction_Enzyme_Digestion_of_psB1K3)     - [4.12.2 Ligation of psB1K3](#Ligation_of_psB1K3)   + [4.13 September 20](#September_20_3)     - [4.13.1 Transformation of psB1K3](#Transformation_of_psB1K3)   + [4.14 September 21](#September_21_2)     - [4.14.1 PCR of psB3C5 and Electrophoresis assay](#PCR_of_psB3C5_and_Electrophoresis_assay)     - [4.14.2 PCR and Electrophoresis assay](#PCR_and_Electrophoresis_assay_4)     - [4.14.3 PCR of DT and Electrophoresis assay](#PCR_of_DT_and_Electrophoresis_assay)   + [4.15 September 22](#September_22_3)     - [4.15.1 Colony PCR (Colony plated in September 20)](#Colony_PCR_.28Colony_plated_in_September_20.29)     - [4.15.2 PCR of Primers and Electrophoresis assay](#PCR_of_Primers_and_Electrophoresis_assay)     - [4.15.3 Ligation](#Ligation_44)   + [4.16 September 23](#September_23_3)     - [4.16.1 Transformation of product of ligation(9/22)](#Transformation_of_product_of_ligation.289.2F22.29)   + [4.17 September 24](#September_24_3)     - [4.17.1 Transformation of product of ligation(9/22)](#Transformation_of_product_of_ligation.289.2F22.29_2)   + [4.18 September 25](#September_25_3)     - [4.18.1 Liquid Culture](#Liquid_Culture_41)     - [4.18.2 Ligation](#Ligation_45)     - [4.18.3 Transformation of product of ligation(9/25)](#Transformation_of_product_of_ligation.289.2F25.29) |

# Caution

|  |
| --- |
| [80px](/File%3ACausion.gif "File:Causion.gif") We devided our team into 2 groups in order to achieve Flower Fairy E.coli, [Florigen](#FlorigenNotebook) and [Secretion](#SecretionNotebook) group. Florigen aims to confirm expression of florigen in *E.coli*, activation of R9 peptide, and function of FT made by *E.coli*. Secretion group aims to make a secretion system without cell death and confirm function of tatABCD and the other proteins. |

# [![Kyoto FlorigenNotebook.png](/wiki/images/a/ab/Kyoto_FlorigenNotebook.png)](/File%3AKyoto_FlorigenNotebook.png)

## August 2

#### Mutation of FT

by Sato
FT gene has two BioBrick restriction enzyme sites, EcoR1 and Pst1 which is next to each other.
So we tried to delete both at once by using two primers with mutation.

Inverse PCR

| 10xBufer | 2mM dNTPs | primer fwd | primer rev | template | polymerase | MilliQ | Total |
| 5 | 5 | 1.5 | 1.5 | 0.5 | 1 | 35.5 | 50 |

94℃ 2min, (98℃ 10sec, 68℃ 4min)x4cycles, 4℃ Hold

Dpn1 Digestion

| PCR product | Dpn1 |
| 50 | 2 |

37℃,1h incubate

Self-ligation

| product | MilliQ | Ligase | T4 Kinase | Total |
| 2 | 7 | 5 | 1 | 15 |

16℃, 1h incubate

Transformation

| competent cell | DNA |
| 20 | 2 |

Cells were stored on ice for 30min.
After 42℃ 60sec heat shock, cells were stored on ice for 2min.
Then cells were precultureed at 37℃ for 1hr, plated to Kanamycin plate.

## August 13

#### Liquid culture

FT at 37°C, for overnight.

## August 14

#### Miniprep of FT

by Sato, Takeuchi
The concentration was 81.5ng/uL

#### Restriction digestion and Electrophoresis

by Sato
To check wheter mutation was succeed, we did restriction enzyme digestion.

| DNA(FT,80ng/uL) | 10xBuferH | EcoR1 | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 5 | 2 | 1 | - | 12 | 20 |
| 5 | 2 | - | 1 | 12 | 20 |

37°C 2h incubate
We did electrophoresis of three FT plasmids, non-cutted, EcoR1 cutted, Pst1 cutted.
However, we couldn't get any bands (data not shown.)

#### Liquid culture

FT (4mL)

## August 15

#### Miniprep of FT

by Sato, Takeuchi, Hyungcheol
We couldn't get enough concentration of plasmids.

#### Electrophoresis

[![IMG 2177.jpg](/wiki/images/c/cd/IMG_2177.jpg)](/File%3AIMG_2177.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2177.jpg "Enlarge")

by Sato
We retried electrophoresis of three samples same as yesterday.
However, we couldn't get any bands as well.

## August 16

#### Transformation

by Takeuchi, Ota

| Name | Well | Sample | Competent Cells | Total | Plate | Colony |
| --- | --- | --- | --- | --- | --- | --- |
| FT | - | 1 µL | 10 | 11 | LB (Kan+) | × |
| pSB1C3 | 1-3-A | 1 | 10 | 11 | LB (CP+) | ○ |
| I719005 | 1-15-N | 1 | 10 | 11 | LB (Amp+) | ○ |

## August 17

#### Transformation

by Takeuchi

| Name | Well | Sample | Competent Cells | MilliQ | Total | Plate | Colony |
| --- | --- | --- | --- | --- | --- | --- | --- |
| FT | - | 2 | 2 | 18 | 22 | LB (Kan+) | × |

We found that mutation of FT was not successful.

## August 20

We decided to do PCR using FT specific primers before mutation.

#### PCR of FT

[![IMG 2178.jpg](/wiki/images/8/8d/IMG_2178.jpg)](/File%3AIMG_2178.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2178.jpg "Enlarge")

by Sato

| 10xBufer | dNTPs | MgSO4 | primer fwd | primer rev | template | polymerase | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1 | 1 | 1(130ng/µL) | 1(KOD plus neo) | 33 | 50 |

94°C 2min, (98°C 10sec, 68°C 15sec)x30cycles, 4°C Hold
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 203ng/µL.

#### Electrophoresis

[![Electrophoresis0821.png](/wiki/images/e/ed/Electrophoresis0821.png)](/File%3AElectrophoresis0821.png)

| Lane | Name | length(bp) |
| --- | --- | --- |
| 1 | 1kb ladder | - |
| 2 | FT | 600 |

## August 21

#### Restriction digestion

by Sato

| DNA(FT,203ng/µL) | 10xBuferM | Xba11 | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 10 | 4 | 1 | 1 | 24 | 40 |

37°C, 5h incubate
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 54,9ng/µL.

#### Ligation

by Sato

| Vector | | Insert | | Ligation High Ver.2 |
| --- | --- | --- | --- | --- |
| pSB1C3 | 1 | FT | 10 | 5.5 |

#### Liquid culture

T7 promoter, pSB1C3 (4mL)

## August 22

#### Miniprep

by Sato

| T7 promoter | pSB1C3 |
| --- | --- |
| 85.3ng/µL | 82.93ng/µL |

## August 23

#### Ethanol Precipitation

diluted in 20µL 79.3ng/µL

## August 24

#### Restriction enzyme processing

[![IMG 2179.jpg](/wiki/images/f/fe/IMG_2179.jpg)](/File%3AIMG_2179.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2179.jpg "Enlarge")

| T7 promoter(85.3ng/µL) | Spel | Pstl | buffer M | MiliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 10 | 1 | 1 | 2 | 6 | 20 |

->purifying column 33.4ng/µL(dissolution 40µL)

| pSB1C3(82.9ng/µL) | Xbal | Spel | buffer M | MiliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 20 | 1 | 1 | 4 | 14 | 40 |

->gene clean2 39.9ng/µL(dissolution 40µL)

#### Ligation

| FT(600bp, 79.3ng/µL) | pSB1C3(2000bp,39.9ng/µL) | Ligation High Ver.2 |
| --- | --- | --- |
| 3µL => 597fmol | 2µL => 60fmol | 2.5µL |

| FT(600bp, 79.3ng/µL) | T7(2100bp,33.4ng/µL) | Ligation High Ver.2 |
| --- | --- | --- |
| 2.4µL => 478fmol | 2µL => 48fmol | 2.2µL |

=> 16℃,1hr incubate

## August 27

#### Colony PCR

[![IMG 2180.jpg](/wiki/images/0/09/IMG_2180.jpg)](/File%3AIMG_2180.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2180.jpg "Enlarge")

| 2X Quick Tag | VF2 | VR | MiliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

Lane1: 1kb ladder
Lane2~16: FT(pSB1C3) about 800bp

#### FT(TOPO) PCR(re)

[![IMG 2181.jpg](/wiki/images/5/55/IMG_2181.jpg)](/File%3AIMG_2181.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2181.jpg "Enlarge")

| buffer | dNTPs | MgSO4 | primer f | primer r | Template(130ng/µL) | KODplus neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |

| 94℃ | 98℃ | 68℃ | cycles |
| --- | --- | --- | --- |
| 2min | 10sec | 15sec | 30 |

#### Liquid culture

by Nobeyama
FT 4ml

## August 28

#### Mutation of FT (re)

[![IMG 2182.jpg](/wiki/images/b/b2/IMG_2182.jpg)](/File%3AIMG_2182.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2182.jpg "Enlarge")

inverse PCR

| MilliQ | buffer | dNTP | primer f | primer r | FT(130ng/µL) | KODplus | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 35.5 | 5 | 5 | 1.5 | 1.5 | 0.5 | 1 | 50 |

| 94℃ | 98℃ | 68℃ | cycles |
| --- | --- | --- | --- |
| 2min | 10sec | 4min | 18 |

Lane1: 1kb ladder
Lane2: FT

#### Miniprep FT(TOPO)

158ng/µL

#### Tranformation

competent cell: 20
BBa.I746902  : 2
(plate 316f)
(GFP generator of pBAD/araC-mut3 GFP:6His-DT)

## August 29

#### Mutaion of FT(re;re)

#### Inverse PCR

first

| MilliQ | buffer | dNTP | primer f | primer r | FT(130ng/µL) | KODplus | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 35.5 | 5 | 5 | 1.5 | 1.5 | 0.5 | 1 | 50 |

second

| MilliQ | buffer | dNTP | primer f | primer r | FT(52ng/µL) | KODplus | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 35 | 5 | 5 | 1.5 | 1.5 | 1 | 1 | 50 |

| 94℃ | 98℃ | 68℃ | cycles |
| --- | --- | --- | --- |
| 2min | 10sec | 4min | 30 |

Lane1: 1kb ladder
Lane2: first Template 130ng/µL
Lane3: second Template 53ng/µL

[![IMG 2183.jpg](/wiki/images/b/b6/IMG_2183.jpg)](/File%3AIMG_2183.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2183.jpg "Enlarge")

| first sample | Dpnl | total |
| --- | --- | --- |
| 45µL | 2µL | 47µL |

in 37℃, 1 hour

#### Self-Ligation

| PCR products | MilliQ | Ligation High | T4 kinase | total |
| --- | --- | --- | --- | --- |
| 2µL | 7µL | 5µL | 1µL | 15µL |

in 16℃, 1.5hour incubate
Transformation
competent cell: 20
DNA  : 2

Liquid culture(I746902): 3mL

## August 30

#### Liquid culture(FT) 4mL x2

## August 31

#### Miniprep(FT)

(1) 64.9ng/µL
(2) 52.6ng/µL

#### Restriction enzyme processing (Mutation checking)

| FT(52.6ng/µL) | bufferH | *E.coli* | Pst1 | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 1µL | 5µL | 0.5µL | 0.5µL | 3.5µL | 10µL |

in 37℃,1.5hour

#### PCR(RBS primer)

| buffer for KODplus neo | dNTPs | MgSO4 | primer f | primer r | Template(52.6ng/µL) | KODplus neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |

[![IMG 2184.jpg](/wiki/images/e/e4/IMG_2184.jpg)](/File%3AIMG_2184.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2184.jpg "Enlarge")

| 94℃ | 98℃ | 68℃ | cycles |
| --- | --- | --- | --- |
| 2min | 10sec | 15sec | 30 |

Lane1: 1kb ladder
Lane2: FT
Lane3: FT(*E.coli*)
Lane4: FT(Pst1)
Lane5: FT PCR

#### PCR(re)

| buffer | dNTPs | MgSO4 | primer f | primer r | Template | KOD neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |

[![IMG 2185.jpg](/wiki/images/8/8b/IMG_2185.jpg)](/File%3AIMG_2185.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2185.jpg "Enlarge")

| 94℃ | 98℃ | 68℃ | cycles |
| --- | --- | --- | --- |
| 2min | 10sec | 15sec | 35 |

Lane1: 1kb ladder
Lane2: FT

#### PCR(re)

| buffer | dNTPs | MgSO4 | primer f | primer r | Template | KOD neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 2 | 1 | 1 | 1 | 1 | 34 | 50 |

| 94℃ | 98℃ | 68℃ | cycles |
| --- | --- | --- | --- |
| 2min | 10sec | 10sec | 30 |

## September 2

#### PCR(re;re)

first

| buffer | dNTPs | MgSO4 | primer f | primer r | Template(1ng/µL) | KODplus neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |

second

| buffer | dNTPs | MgSO4 | primer f | primer r | Template(10ng/µL) | KODplus neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |

[![IMG 2186.jpg](/wiki/images/8/8b/IMG_2186.jpg)](/File%3AIMG_2186.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2186.jpg "Enlarge")

| 94℃ | 98℃ | 68℃ | cycles |
| --- | --- | --- | --- |
| 2min | 10sec | 10sec | 25 |

Lane1: first Template 1ng
Lane2: second Template 10ng
Lane3: 1kb ladder
refine first Template => 132ng/µL

## September 3

#### Restriction enzyme processing

| FT(132ng/µL) | bufferM | EcoRI | SpeI | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 10 | 2 | 1 | 1 | 6 | 20 |

37℃,overnight => refinement 31.6ng/µL (Elution 40µL)

**Ligation**
Insert(FT: 31.6ng/µL, 600bp ): 2µL = 26fmol
Vector(DT: 28.0ng/µL, 3300bp): 3µL = 240fmol
Ligation High ver.2  :2.5µL
=> 16℃, 2 hours

**Transformation**

| competent cell | DNA | plate | colony |
| --- | --- | --- | --- |
| 20µL | FT-DT 2µL | Amp | o |
| 20µL | pT7-6His-R9 2µL | Amp | o |
| 10µL | GFP generator 1µL | Amp | o |

(BBa,I746915,pT7-6His-GFP)

## September 4

**Colony PCR**

[![IMG 2187.jpg](/wiki/images/c/cd/IMG_2187.jpg)](/File%3AIMG_2187.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2187.jpg "Enlarge")

| Quick Tag | VF2 | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

| 94℃ | 98℃ | 55℃ | 68℃ | cycles |
| --- | --- | --- | --- | --- |
| 2min | 30sec | 30sec | 1min | 25 |

Lane1: 1kb ladder
Lane2: FT-DT(about 700bp)

Liquid culture(4ml) 23:30 ~
GFP generator, FT-DT

## September 5

**Miniprep(FT-DT)** by Sato
88.8ng/µL

**Restriction enzyme processing**

| FT-DT(88.8ng/µL) | XbaI | PstI | bufferM | MiliiQ | total |
| --- | --- | --- | --- | --- | --- |
| 20 | 1 | 1 | 4 | 14 | 40 |

at 37℃, 2 hours

**Electrophoresis** by Takeuchi

[![IMG 2188.jpg](/wiki/images/7/7b/IMG_2188.jpg)](/File%3AIMG_2188.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2188.jpg "Enlarge")

**Restriction enzyme processing**by Takeuchi

[![IMG 2189.jpg](/wiki/images/2/21/IMG_2189.jpg)](/File%3AIMG_2189.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2189.jpg "Enlarge")

| FT-DT(88.8ng/µL) | bufferM | XbaI/Spe | MiliiQ | total |
| --- | --- | --- | --- | --- |
| 4 | 1 | 0.5 | 4.5 | 10 |

| FT-DT | bufferH | Pst/Eco | MiliiQ | total |
| --- | --- | --- | --- | --- |
| 4 | 1 | 0.5 | 4.5 | 10 |

at 37℃,1hour 10min

## September 6

**Western blotting**(BBa,I746915)

**Sample making**
SOC(ampt) 50ml + pre culture 1ml x2
OD600 = 0.5~0.7 incubate at 37℃ (OD 0.642)
add IPTG final concentration is 1mM (negative control)
incubate at 37℃,4 hours

**SDS-PAGE**
Do spin down *E.coli* and make suspension put *E.coli* into 1mL 1x sample buffer
95℃,10min
electrophoresis at 500V, 30mA, 50min
Lane1: 10µL (IPTG -)
Lane2: 10µL (IPTG -)
Lane3: 10µL (IPTG +)
Lane4: 5µL (IPTG -)
Lane5: 5µL (IPTG +)
Lane6: 2µL (IPTG -)
Lane7: 2µL (IPTG +)

Blotting at 50V,100mA,30min
Put into blocking buffer and vibrating 30min
Incubate with Anti GFP(1/1000) 10mL at RT,1h
Washing with 10mL TBST (vibrating 10min x2)
Incubate with Anti-mouseAP(1/1000) 10mL at RT, 30min
Washing with 10mL TBST (vibrating 10min x3)
Put NBT,BCIP into dye buffer

## September 9

**Mutaion of FT**

| MilliQ | buffer | dNTP | primer f | primer r | FT(52ng/µL) | KODplus | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 35 | 5 | 5 | 1.5 | 1.5 | 1 | 1 | 50 |

| 94℃ | 98℃ | 68℃ | cycles |
| --- | --- | --- | --- |
| 2min | 10sec | 4min | 20 |

→　GeneClean II　32.6ng/µL

**Dpn1 processing**

[![IMG 2190.jpg](/wiki/images/c/cc/IMG_2190.jpg)](/File%3AIMG_2190.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2190.jpg "Enlarge")

| TA buffer | DNA | Dpn1 | Total |
| --- | --- | --- | --- |
| 3.3 | 31 | 2 | 36.3 |

**Ligation**

| DNA | MiliiQ | Ligation high Ver.2 | T4 kinase | Total |
| --- | --- | --- | --- | --- |
| 2 | 7 | 5 | 1 | 15 |

**Transformation**
competent cell: 10
DNA  : 1

## September 10

**Miniprep of FT**
①116.9ng/µL
② 34.5ng/µL
**Restriction enzyme processing**(Mutation checking)

| FT①/② | bufferHl | EcoRI | PstI | MiliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 4 | 1 | 0.5 | 0 | 4 | 10 |
| 4 | 1 | 0 | 0.5 | 4 | 10 |

**PCR**

| Bufer | dNTPs | MgSO4 | primer(+/-RBS)fwd | primer(+/-RBS)rev | template①/② | KOD plus neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |

| 94℃ | 98℃ | 68℃ | cycles |
| --- | --- | --- | --- |
| 2min | 10sec | 10sec | 25 |

->purifying column 17.4ng/µL

#### Restriction enzyme processing

| FT(RBS+)32.6ng/µL | Xbal | Pstl | buffer M | BSA | Total |
| --- | --- | --- | --- | --- | --- |
| 30 | 1 | 1 | 4 | 4 | 40 |

| T7-His:R9(62.6ng/µL) | SpeI | Pstl | MilliQ | buffer M | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 0.5 | 0.5 | 2 | 2 | 20 |

incubate at 37℃, 3hours
->purifying column 17.4ng/µL

## September 11

**Ligation**
Vector(T7: 33.4ng/µL, 2100bp): 1µL = 24fmol
Insert(FT: 17.4ng/µL, 600bp ): 5µL = 217fmol
Ligation High ver.2  : 3µL
=> 16℃, 1 hours

**Transformation**

| competent cell | DNA |
| --- | --- |
| 20µL | T7-FT 2µL |

**PCR(FT without RBS retry)**

[![IMG 2191.jpg](/wiki/images/c/c6/IMG_2191.jpg)](/File%3AIMG_2191.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2191.jpg "Enlarge")

| Bufer | dNTPs | MgSO4 | primer(-RBS)fwd | primer(-RBS)rev | template | KOD plus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1 | 1 | 1 | 1 | 33 | 50 |

| 94℃ | 98℃ | 65℃ | 68℃ | cycles |
| --- | --- | --- | --- | --- |
| 2min | 15sec | 30sec | 30sec | 25 |

Lane1:FT(RBS-) 600bp
Lane2:Ladder 100bp
=>purifying column 34.6ng/µL

**Restriction enzyme processing**

| FT(RBS-) | XbaI | PstI | bufferM | BSA | total |
| --- | --- | --- | --- | --- | --- |
| 30 | 1 | 1 | 4 | 4 | 40 |

at 37℃, 2 hours
=> 6.2ng/μL

| GFP-DT | XbaI | PstI | bufferM | BSA | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 3 | 7 | 30 |

at 37℃, 2 hours
=> 7.0ng/μL

**Ligation**
Vector(T7-6His-R9: 11.6ng/µL, 2100bp): 1µL = 9fmol
Insert(FT: 6.2ng/µL, 600bp ): 5µL = 79fmol
Ligation High ver.2  : 3µL
=> 16℃, 1 hours

Vector(T7-6His-R9: 11.6ng/µL, 2100bp): 1µL = 9fmol
Insert(GFP-DT: 7.0ng/µL, 1000bp ): 9µL = 95fmol
Ligation High ver.2  : 4µL
=> 16℃, 1 hours

## September 12

**Transformation**

| competent cell | DNA | plate |
| --- | --- | --- |
| 20µL | T7-R9-GFP-DT 2µL | Amp |
| 20µL | T7-R9-FT 2µL | Amp |
| BL21CDE3 10µL | T7-FT 1µL | Amp |

**Miniprep (T7-FT)**
37.1ng/µL

## September 13

**Miniprep**
①T7-R9-GFP-DT 90ng/µL
②T7-R9-FT 160ng/μL

**Restriction enzyme processing**

| T7-R9-GFP-DT (90ng/µL) | EcoRI | PstI | buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 20 | 1 | 1 | 3 | 5 | 30 |

at 37℃, 2 hours
=> 20.7ng/μL

| T7-R9-FT (160ng/µL) | EcoRI | SpeI | buffer M | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 20 | 1 | 1 | 3 | 5 | 30 |

at 37℃, 2 hours
=> 20.1ng/μL

'*Transformation*

| competent cell BL21(DE3) | DNA | plate |
| --- | --- | --- |
| 10µL | T7-R9-GFP-DT 1µL | Amp |
| 10µL | T7-R9-FT 1µL | Amp |

**Restriction enzyme processing**

| Buffer H | BSA | EcoRI | PstI | DpnⅠ | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 0.5 | 0.5 | 0.5 | 13.5 | 25 |

=>We define this solution "2× Master Mix"

| 2× Master Mix | pSB1C3(Linerarized Plasmid Backbone) |
| --- | --- |
| 4 | 4 |

at 37℃, 30 minutes
80℃, 30 minutes

**PCR(Insert His-tag)**

[![IMG 2192.jpg](/wiki/images/b/b0/IMG_2192.jpg)](/File%3AIMG_2192.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2192.jpg "Enlarge")

| MilliQ | Buffer for iPCR | dNTPs | primer fwd | primer rev | template(T7-FT 37.1ng/μL) | KOD plus | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 35 | 5 | 5 | 1.5 | 1.5 | 1 | 1 | 50 |

| 94℃ | 94℃ | 56℃ | 68℃ | cycles |
| --- | --- | --- | --- | --- |
| 2min | 15sec | 30sec | 2.5min | 15 |

Lane1:Ladder 1kb
Lane2:T7-6His:FT about 2700bp

## September 14

**Ligation**
Vector(pSB1C3: 12.5/µL, 2000bp)  : 2µL = 19fmol
Insert(T7-R9-GFP-DT: 20.7ng/µL, 700bp ): 4µL = 180fmol
Ligation High ver.2  : 3µL
=> 16℃, 1 hours

**Dpn1 processing**

| PCR products(9/13) | Dpn1 | Total |
| --- | --- | --- |
| 45 | 2 | 47 |

=>37℃, 1 hour

**Self Ligation**

| PCR products | Ligation High | T4 Kinase | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 2 | 5 | 1 | 7 | 15 |

=>16℃, 1 hour

**Ligation**
Vector(DT: 17.1/µL, 2100bp)  : 2µL = 12fmol
Insert(T7-R9-FT: 20.1ng/µL, 650bp ): 5µL = 496fmol
Ligation High ver.2  : 3.5µL
=> 16℃, 1 hours

## September 15

**Restriction enzyme processing**

| FT without RBS (34.6ng/µL) | EcoRI | PstI | 10× buffer H | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 2 | 7 | 20 |

**Colony PCR**

| Quick Tag | VF | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

| 94℃ | 94℃ | 55℃ | 68℃ | cycles |
| --- | --- | --- | --- | --- |
| 2min | 30sec | 30sec | 1min | 25 |

**Electrophoresis**
Lane1: ladder
Lane2: T7-R9-GFP-DT
Lane3: T7-R9-GFP-DT

Lane4: T7-R9-FT-DT
Lane5: T7-R9-FT-DT
Lane6: T7-R9-FT-DT
Lane7: T7-R9-FT-DT
Lane8: T7-R9-FT-DT
A member of secretion group electrophoresed DNA from Lane9 to Lane12.

[![IMG 2193.jpg](/wiki/images/c/c0/IMG_2193.jpg)](/File%3AIMG_2193.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2193.jpg "Enlarge")

Lane13: T7-His:FT
Lane14: T7-His:FT
Lane15: T7-His:FT
Lane16: T7-His:FT
Lane17: ladder

Liquid culture(3ml) 3:30~
T7-R9-FT-DT×2, T7-His:FT×2, T7-His:FT

## September 16

**Miniprep**
①T7-R9-FT-DT 150ng/µL
②T7-R9-FT-DT 139ng/μL
③T7-R9-GFP-DT 67ng/µL
④T7-His:FT 153ng/μL
⑤T7-His:FT 73ng/μL

## September 17

**Purifying column**
=>FT without RBS :36.4ng/µL

**Ligation**
Vector(PSB1C3: 12.5/µL, 2000bp)  : 3µL = 9fmol
Insert(FT without RBS: 36.4ng/µL, 600bp ): 3µL = 92fmol
Ligation High ver.2  : 3µL
=> 16℃, 1 hours

## September 18

**Transformation** by NAKAGAWA

| competent cell | DNA | plate |
| --- | --- | --- |
| 20µL | PSB1C3 FT without RBS 1µL | CP+ |

**Verification of R9 function** by TAKEUCHI

|  |  |
| --- | --- |
| R9(20µg/µL) | 0.9µL |
| GFP(1.2mg/mL) | 2.23µL |
| RBS | 16.85µL |
| total | 20µL |

X5

Method:
1. Peel cuticles on parafilm by using the head of pencil.(Menasha,wI,54952)
2. Put plant cells into GFP&R9 or GFP for 5~30min.
3. Put plant cells into PBS.
4. Hoechst dyeing.

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | 1 | 2 | 3 | 4 | 5 | 6 |
| R9 | o | o | o | x | o | o |
| cuticle | o | o | o | o | x | x |
| soak in GFP | 5min | 15min | 30min | 5min | 5min | 30min |

## September 19

**Transformation(re)** by NAKAGAWA,TAKEUCHI
competent cell:20µL
DNA(No RBS FT):1µL
plate  :CP+

**Transformation(re;re)** by NAKAGAWA,TAKEUCHI
competent cell:10µL
DNA(No RBS FT):1µL
LB  :100µL
plate  :CP+

**Adjusted GM Agar Medium making** by TAKEUCHI

Ingredient of MS medium(SIGMA M5519): 0.88g
MES  : 0.1g
ion exchanged water  : 200mL
NaOH  : 26µL(adjust to pH5.6)
Agar medium  : 1.6g

Autoclave 120℃

#### Colony PCR(re)

[![IMG 2194.jpg](/wiki/images/3/3e/IMG_2194.jpg)](/File%3AIMG_2194.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2194.jpg "Enlarge")

**gelA**
Lane1: 100bp Ladder
Lane2: No RBS FT colony number 9
Lane3: No RBS FT colony number 10
Lane4: No RBS FT colony number 11
Lane5: No RBS FT colony number 12
Lane6: No RBS FT colony number 13
Lane7: empty
Lane8: empty

[![IMG 2195.jpg](/wiki/images/e/e3/IMG_2195.jpg)](/File%3AIMG_2195.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2195.jpg "Enlarge")

**gelB**

[![IMG 2196.jpg](/wiki/images/7/70/IMG_2196.jpg)](/File%3AIMG_2196.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2196.jpg "Enlarge")

Lane1: 100bp Ladder
Lane2: No RBS FT colony number 14
Lane3: No RBS FT colony number 15
Lane4: No RBS FT colony number 16
Lane5: No RBS FT colony number 17
Lane6: No RBS FT colony number 18
Lane7: No RBS FT colony number 19
Lane8: empty

**gelC**

[![IMG 2197.jpg](/wiki/images/6/68/IMG_2197.jpg)](/File%3AIMG_2197.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2197.jpg "Enlarge")

Lane1: 100bp Ladder
Lane2: No RBS FT colony number 20
Lane3: No RBS FT colony number 21
Lane4: No RBS FT colony number 22
Lane5: No RBS FT colony number 23
Lane6: No RBS FT colony number 24
Lane7: empty
Lane8: empty

## September 20

**Liquid culture** by NAKAGAWA
No RBS FT x8 (by using September 18)
Plate: CP+

**Colony PCR**

[![IMG 2194.jpg](/wiki/images/3/3e/IMG_2194.jpg)](/File%3AIMG_2194.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2194.jpg "Enlarge")

No RBS FT by using September19

Lane1 : 100bp ladder
Lane2 : colony number 1
Lane3 : colony number 2
Lane4 : colony number 3
Lane5 : colony number 4
Lane6 : colony number 5
Lane7 : colony number 6
Lane8 : colony number 7
Lane9 : colony number 8
Lane10: empty
Lane11: empty
Lane12: 100bp ladder
**Liquid culture**
A-11, A-12

**Miniprep**
No RBS FT(by using September20)
1.-10.4µg/mL
2.-4.7µg/mL
3.-3.6µg/mL
4.-7.6µg/mL
5.-4.5µg/mL
6.-8.4µg/mL
7. 1.8µg/mL(average)
8. 18.1µg/mL
**Restriction enzyme processing**

| DNA(No RBS FT)x2 | *E.coli* | BufferH | MilliQ | total |
| --- | --- | --- | --- | --- |
| 30µL | 1µL | 4µL | 5µL | 40µL |

in 37℃,2hours

**Electrophoresis**
DNA(No RBS FT) sample7,8: 10µL
Loading Dye  : 2µL

Lane1: 1kb ladder
Lane2: empty
Lane3: No RBS FT(E) sample7
Lane4: empty
Lane5: No RBS FT(E) sample8
Lane6: empty

[![IMG 2198.jpg](/wiki/images/4/4a/IMG_2198.jpg)](/File%3AIMG_2198.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2198.jpg "Enlarge")

**Electrophoresis(re)**

[![IMG 2199.jpg](/wiki/images/b/be/IMG_2199.jpg)](/File%3AIMG_2199.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2199.jpg "Enlarge")

**RNA Extraction**
5 leaves: 100mg
ISOGEN  : 1mL
Elution : 20µL

**cDNA Synthesis(1/4)**

| gDNA wipeout buffer | template RNA | H2O | Total |
| --- | --- | --- | --- |
| 1µL | 0.5µL | 5.5µL | 7µL |

at 42℃, 2min

| Reverse Transcriptase | RT buffer | primer mix | template | Total |
| --- | --- | --- | --- | --- |
| 0.5µL | 2µL | 0.5µL | 7µL | 10µL |

at 42℃, 2min and 95℃,3min

**RT PCR**

| buffer | dNTPs | MgSO4 | primer f | primer r | Template | KODplus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 2 | 1.5 | 1.5 | 1 | 1 | 34.5 | 50 |

| 94℃ | 94℃ | 54℃ | 68℃ | cycles |
| --- | --- | --- | --- | --- |
| 2min | 15sec | 30sec | 10sec | 30 |

1.TUBULIN
2.FUL
3.SEP3
4.AP1

[![IMG 2200.jpg](/wiki/images/3/3f/IMG_2200.jpg)](/File%3AIMG_2200.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2200.jpg "Enlarge")

## September 22

**mRNA extraction**
FT and R9
Arabidopsis thaliana's leaves : 200mg

| R9 | FT | total |
| --- | --- | --- |
| 1.575μL | 33.425μL | 35μL |

| R9 | GFP |
| --- | --- |
| 1.575 | 33.425 | 35 |

1. Strip cuticles on palafilm by the head of a pencil.
2. Soak plant cells in R9 & FT or R9 & GFP for 2 hours(19:45~).
3. Add PBS (21:45~)

**Microscope**
R9+GFP+Plant Cell ver.3
R9+

| R9 | GFP | PBS | total |
| --- | --- | --- | --- |
| 0.9 | 2.25 | 16.85 | 20μL |

R9-

| R9 | GFP | PBS | total |
| --- | --- | --- | --- |
| 0 | 2.25 | 17.65 | 20 |

same as 9/20, only not on parafilm but on microscope slide from the start.

## September 23

[![Data0923.jpg](/wiki/images/e/e9/Data0923.jpg)](/File%3AData0923.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AData0923.jpg "Enlarge")

**RNA Extraction**
FT- (GFP+)
FT+

| ISOGEN | chloroform | elution | total |
| --- | --- | --- | --- |
| 1 | 200 | 11 | 212 |

**Reverce Transcription**

|  |  |  |
| --- | --- | --- |
| gDNA wipeout buffer | RNA | total |
| 1 | 6 | 7 |

at 42 degree, for 2min.

| Reverce Transcriptase | RT buffer | primer mix | Template | total |
| --- | --- | --- | --- | --- |
| 0.5 | 2 | 0.5 | 7 | 10 |

at 42 degree, for 30min.
at 95 degree, for 3min.

**RT-PCR**

| Buffer | dNTPs | MgSO4 | primer | Template | KOD Plus | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 2 | 1.5 | 1 | 1 | 34.5 |

94℃, 2min
94℃, 15sec
54℃, 30sec
68℃, 10sec

1.NC TUBULIN
2. FUL
3. SEP3
4. AP1
5.FT TUBULIN
6. FUL
7. SEP3
8. AP1

## September 24

**ScreeningPCR & Electrophoresis**

[![ElectrophoresisFFE0924.jpg](/wiki/images/3/3b/ElectrophoresisFFE0924.jpg)](/File%3AElectrophoresisFFE0924.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresisFFE0924.jpg "Enlarge")

Lane
1. 100bp ladder
2. T7-His:FT ① 9.2ng/μL
3. T7-His:FT ② 11.7ng/μL
4. T7-His:FT ③ 23.6ng/μL
5. T7-His:FT ④ 21.6ng/μL
6. T7-His:FT ⑤ 30.6ng/μL
7. T7-His:FT ⑥ 51.5ng/μL
8. T7-His:FT ⑦ 47.1ng/μL
9. T7-His:FT(A4) 20.0ng/μL
10. T7-His:FT(B1) 18.8ng/μL
11. final2(by\_Secretion\_group)
12. blank

**FT Introduce**

| FT | R9 | PBS | total |
| --- | --- | --- | --- |
| 50 | 2.88 | 11.12 | 64 |

| GFP | R9 | PBS | total |
| --- | --- | --- | --- |
| 7.2 | 2.88 | 53.02 | 64 |

We took 1 leaf out of 6 individuals of Arabidopsis thaliana that grow three weeks one by one
Leaves 30mg
1.cut off leaves
2.Inject the juice（by terumo-syringe）（Center tip、tuberculin 1nl)
3.store 20 min, then add PBS 600μL

## September 25

**RNA extraction**
FT- (GFP+) FT+
elution 6µL

**Reverce Transcription**

|  |  |  |
| --- | --- | --- |
| gDNA wipeout buffer | RNA | total |
| 1 | 6 | 7 |

at 42 degree, for 2min.

| Reverce Transcriptase | RT buffer | primer mix | Template | total |
| --- | --- | --- | --- | --- |
| 0.5 | 2 | 0.5 | 7 | 10 |

at 42 degree, for 30min.
at 95 degree, for 3min.

**RT-PCR**

| Buffer | dNTPs | MgSO4 | primer | Template | KOD Plus | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 2 | 1.5 | 1 | 1 | 34.5 |

94℃, 2min
94℃, 15sec
54℃, 30sec
68℃, 10sec

1.TUBULIN NC
2.TUBULIN FT
3.FUL NC
4.FUL FT
5.SEP3 NC
6.SEP3 FT
7.AP1 NC
8.AP1 FT

[![RT-PCR5.png](/wiki/images/d/d8/RT-PCR5.png)](/File%3ART-PCR5.png)

## October 12

**Transformation**

| BL21(DE3) | T7-His-FT | T7-His-GFP-DT | total |
| --- | --- | --- | --- |
| 1μL | 10μL |  | 11μL |
| 1μL |  | 10μL | 11μL |

## October 16

**Liquid culture**
T7-His-FT 3mL

**Transformation (retry)**

| BL21(DE3) | T7-His-GFP-DT | total |
| --- | --- | --- |
| 1μL | 10μL | 11μL |

## October 17

Moved 1mL of Liquid culture of T7-His-FT in 100mL of SOC medium -> 2 samples
Added IPTG 100μL to the medium(final conc. is 1mM) when value of OD600 is 0.649
Incubated at 20℃ for 4 hours.
Collect bacterial cells and freeze-preserved.

## October 20

**Liquid culture**
2mL (T7-His-GFP)

## October 21

Collect bacterial cells same way described above(value of OD600 is 0.8, at 37℃, for 4 hour)

**Purification of GFP**

| Lysis buffer | lysozyme | Triton X-100 | Ni-NTA resin | Elution buffer |
| --- | --- | --- | --- | --- |
| 5mL | 0.1mL | 0.3mL | 1mL | 2.5mL |

fraction1:31.5µM->After consentrating 47.4µM
fraction2:15.8µM->After consentrating 33.4µM
fraction3:20.1µM
(Extinction coefficient=21,890)
**RNA Extraction**
Added 3μL of IPTG to 3mL of E.coli and then incubated 2 hour.

| ISOGEN | chloroform | isopropanol | Elution buffer |
| --- | --- | --- | --- |
| 1mL | 0.2mL | 0.5mL | 11μL |

deluted to 250ng/μL

**Reverce Transcription**

| RNA(250ng/μL) | gDNA wipe out buffer | dH2O | total |
| --- | --- | --- | --- |
| 0.5μL | 1μL | 5.5μL | 7μL |

| Template | RT buffer | Primer mix | RT | total |
| --- | --- | --- | --- | --- |
| 7μL | 2μL | 0.5μL | 0.5μL | 10μL |

**RT-PCR**
Template is 20 times deluted cDNA

| buffer | dNTPs | MgSO4 | primer | primer | Template | KOD-Plus | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 2 | 1.5(His-FT f) | 1.5(FT sequence r) | 1 | 1 | 33 | 50 |
| 5 | 5 | 2 | 1.5(GAPDH) |  | 1 | 1 | 34.5 | 50 |

94℃ 2min
94℃ 15sec
55℃ 30sec
68℃ 30sec
for 30 cycles

lane\_1 : GAPDH
lane\_2 : FT
lane\_3 : 100bp ladder

## October 22

**Restriction**

| T7-His-FT(A4, 20ng/μL) | T7-His-FT(B2, 73ng/μL) | bufferH | EcoR1 | Pst1 | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 10μL |  | 2μL | 0.5μL | 0.5μL | 7μL | 20μL |
|  | 5μL | 2μL | 0.5μL | 0.5μL | 12μL | 20μL |

at 37℃ for 1 hour

lane1 : T7-His-FT(B2)
lane2 : T7-His-FT(B2, EcoR1/Pst1)
lane3 : T7-His-FT(A4)
lane4 : T7-His-FT(A4, EcoR1/Pst1)

**Transformation**

| T7-His-FT(B2) | BL21(DE3) | total |
| --- | --- | --- |
| 1 | 10 | 11 |

## October 23

**RNA Extraction from plant cell**

| ISOGEN | chloroform | isopropanol | High salt solution | Elution buffer |
| --- | --- | --- | --- | --- |
| 1mL | 0.25mL | 0.25mL | 0.25mL | 11μL |

notes: After the homogenization with ISOGEN, sample 1 was incubated at 50℃ for 10min.
Sample1. 855.8ng/µL
Sample2. 812.4ng/µLL

## October 24

Collect bacterial cells(T7-6His:FT) same way described above(value of OD600 is 0.722, at 37℃, for 4 hour)
2mL of culture was used for RNA extraction.
**Purification of FT**

| Lysis buffer | lysozyme | Triton X-100 | Ni-NTA resin | Elution buffer |
| --- | --- | --- | --- | --- |
| 5mL | 0.15mL | 0.3mL | 0.5mL | 1.2mL |

fraction1:65.1µM
fraction2:42.1µM
fraction3:10.9µM
fraction4:6.8µM
(Ectinction coefficient=21,430)

**RNA extraction**

| ISOGEN | chloroform | isopropanol | Elution buffer |
| --- | --- | --- | --- |
| 1mL | 0.2mL | 0.5mL | 11μL |

diluted to 250ng/μL

**Reverce Transcription**

| RNA(250ng/μL) | gDNA wipe out buffer | dH2O | total |
| --- | --- | --- | --- |
| 0.5μL | 1μL | 5.5μL | 7μL |

| Template | RT buffer | Primer mix | RT | total |
| --- | --- | --- | --- | --- |
| 7μL | 2μL | 0.5μL | 0.5μL | 10μL |

**RT-PCR**
Template is 20 times deluted cDNA

| buffer | dNTPs | MgSO4 | primer | primer | Template | KOD-Plus | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 2 | 1.5(His-FT f) | 1.5(FT sequence r) | 1 | 1 | 33 | 50 |
| 5 | 5 | 2 | 1.5(GAPDH) |  | 1 | 1 | 34.5 | 50 |

94℃ 2min
94℃ 15sec
55℃ 30sec
68℃ 30sec
for 30 cycles

lane\_1 : 100bp ladder
lane\_2 : GAPDH
lane\_3 : FT

**Assay of FT activation**

| protein-R9 solution mix | FT | GFP | R9 | PBS |
| --- | --- | --- | --- | --- |
| sample1 (FT-) | - | 200µL | 0.11mg | 100µL |
| sample2 (FT+) | 230µL | - | 0.21mg | 70µL |

100mg of Arabidopsis thaliana leaves were harvested for each samples and injected the solution mix.
incubation at room temperature for 13h in dark

## October 25

[![](/wiki/images/0/02/RNA20121025FT-.jpg)](/File%3ARNA20121025FT-.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ARNA20121025FT-.jpg "Enlarge")

sample1

[![](/wiki/images/d/d5/RNA20121025FT%2B.jpg)](/File%3ARNA20121025FT%2B.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ARNA20121025FT%2B.jpg "Enlarge")

sample2

| ISOGEN | chloroform | isopropanol | High salt solution | Elution buffer |
| --- | --- | --- | --- | --- |
| 1mL | 0.25mL | 0.25mL | 0.25mL | 11μL |

Sample1. 682.7ng/µL
Sample2. 859.3ng/µLL

diluted to 250ng/μL

**Reverce Transcription**

| RNA(250ng/μL) | gDNA wipe out buffer | dH2O | total |
| --- | --- | --- | --- |
| 1μL | 2μL | 11μL | 14μL |

| Template | RT buffer | Primer mix | RT | total |
| --- | --- | --- | --- | --- |
| 14μL | 4μL | 1μL | 1μL | 20μL |

**RT-PCR**
Template is 20 times diluted cDNA

| buffer | dNTPs | MgSO4 | primer | Template | KOD-Plus | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 2 | 1.5 | 1 | 1 | 34.5 | 50 |

94℃ 2min
94℃ 15sec
54℃ 30sec
68℃ 10sec

# ![Kyoto SecretionNotebook.png](/wiki/images/6/65/Kyoto_SecretionNotebook.png)

## February 7

#### Preculture

We started preculture at 12:10.

## February 8

#### Liquid culture

We start culturing with 300mL of LB medium.

| time | OD600 |
| --- | --- |
| 12:00 | start |
| 14:10 | 0.019 |
| 14:45 | 0.154 |
| 15:05 | 0.267 |
| 15:21 | 0.64 |

#### Making Competent Cell

We made competent cells.

#### Transformation

pGEM\_TAP
LacP (BBa\_R0011)
DT (BBa\_B0015)

#### Making Culture Medium Plates

We made 200mL of ampicillin culture, kanamycin culture, and chloramphenicol culture.

#### Transformation

GFP(BBa\_E0040) in pSB1A2
DT(BBa\_B0015) in pSB1AK3
ara(BBa\_I0500) in pSB2K3
LacP(BBa\_R0011) in pSB1A2

## February 9

#### Transformation

BBa-E0040(GFP)(Mr.Fujita)

#### Liquid culture

DT, LacP colony transformed on February 8
colony of competent cell made on February 8
B0040 1.4k pSB1A2 B0034 1.2M pSB1A2(from iGEM parts plate)

#### Making Competent cells

We did preculture for overnight. We put 1.5mL of preculture on 150mL of LB culture.

| time | OD600 |
| --- | --- |
| 11:45 | start |
| 13:30 | 0.048 |
| 14:30 | 0.168 |
| 15:03 | 0.256 |
| 15:20 | 0.405 |
| 15:35 | 0.459 |
| at last | 0.576 |

## February 11

#### Checking Transformation efficiency

Conpetent cell's transformation efficiency is 1.3x10^4colonys/μg

## February 13

#### Transformation

Const promoter J23110, J23109, J23100

| DNA | Competent cell | Total |
| --- | --- | --- |
| 1μL | 20 | 21 |

No colony was there on February 14

#### Liquid culture

LacP, DT, RBS(BBa\_B0034),GFP
start at 20:00
in Plus grow with Ampicilin 3mL

## February 14

#### Miniprep

|  |  |
| --- | --- |
| DNA | concentration[μg/μL] |
| LacP3 | 39.6 |
| LacP4 | 40.8 |
| LacP5 | 28.9 |
| RBS1 | 28.2 |
| RBS2 | 57.4 |
| RBS3 | 13.2 |
| DT3 | 69.7 |
| DT4 | 64.4 |
| DT5 | 61.5 |
| GFP1 | 64.0 |
| GFP2 | 50.5 |
| GFP3 | 66.0 |

#### Restrictive Digestion

Const promoter J23100

| DNA | Spe1 | Pst1 | Buffer2 | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |

at 37℃ for overnight

## February 15

#### Making gel

1% Agarose gel

| Agarose | TAE |
| --- | --- |
| 1.6g | 160mL |

#### Electrophoresis

[![](/wiki/images/0/0b/Electrophoresis021501.JPG)](/File%3AElectrophoresis021501.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis021501.JPG "Enlarge")

No.1

Gel No.1

| Restriction product | loading dye |
| --- | --- |
| 5μL | 1 |

The marker was 1kb ladder
It seemed that this restriction product was not cut.

[![](/wiki/images/8/8e/Electrophoresis021502.JPG)](/File%3AElectrophoresis021502.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis021502.JPG "Enlarge")

No.2

Gel No.2
Lane1 : 1kb ladder
Lane2 : J23100 2μL + 6\*Loading dye 1μL
Lane3 : J23100(Spe1,Pst1) 5μL
Lane4 : J23100(Spe1,Pst1) 2μL

* There were bands on lane\_2 and we cannot identify these bands because the sample of lane\_2 was not cut with any restriction enzyme.
* There must have been bands at 2100bp and 883bp on lane\_3 and lane\_4.

#### Testing whether restriction enzyme were deactivated or not

| DNA(DT) | restriction enzyme | Buffer | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 3 | 0.5 | 16 | 30 |

at 37℃ for Oveernight
Restriction enzyme means Spe1(1, 2) Pst1(1, 2, 3) in this time.

## February 16

#### Electrophoresis

[![Electrophoresis021601.JPG](/wiki/images/d/d8/Electrophoresis021601.JPG)](/File%3AElectrophoresis021601.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis021601.JPG "Enlarge")

1. 1kb ladder
2. DT2
3. DT3
4. DT2 (Spe1-1)
5. DT2 (Spe1-2)
6. DT2 (Pst1-1)
7. DT2 (Pst1-2)
8. DT2 (Pst1-3)
9. DT3 (Pst1-4)
10. 1kb ladder

Pst1-1, Pst1-2, and Pst1-3 did not cut DNAs. They seemed to be deactivated.

#### Genomic PCR

| 10\*Buffer for KOD Plus | 2mM dNTPs | 25mM MgSO4 | 10μM primer-f | 10μM primer-r | 158ng/μL Genomic DNA | KOD plus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 1 | 32 | 50 |

#### Electrophoresis

[![Electrophoresis021602.JPG](/wiki/images/4/4f/Electrophoresis021602.JPG)](/File%3AElectrophoresis021602.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis021602.JPG "Enlarge")

1. 1kb ladder
2. TatABCD (2.5kb)
3. TAMO reductase (2.7kb)
4. Negative control
We got bands of TatABCD but there were nonspecific amplification products.
We failed amplification of TAMO reductase.

#### Transformation

Constitutive promoter (BBa\_J23107, BBA\_J23117)
High copy plasmid (pSB1AT3)

| DNA | competent cell |
| --- | --- |
| 1μL | 10μL |

## February 17

#### PCR

We did PCR to amplify products of PCR that we had done yesterday but we could not amplify TatABCD.

#### Genomic PCR

| Buffer | dNTPs | MgSO4 | primer-f | primer-r | genomic DNA | KOD plus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 2.5 | 5 | 0.75 | 0.75 | 0.5 | 0.5 | 16 | 50 |

Predenature 94℃, 2min
Denature 98℃, 10sec
Annealing 57℃, 30sec
Extension 68℃, 2.5min
(30cycles)

#### Electrophoresis

[![Electrophoresis021701.JPG](/wiki/images/f/f3/Electrophoresis021701.JPG)](/File%3AElectrophoresis021701.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis021701.JPG "Enlarge")

1. 1kb ladder
2. TAMO reductase
3. Negative control

#### Restrictive Digestion

| J23100 | Spe1 | Pst1 | Buffer2 | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |

#### Genomic PCR

|  | Buffer | dNTPs | MgSO4 | primer-f | primer-r | genomic DNA | KOD plus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| TMAO reductase | 2.5 | 2.5 | 3 | 0.75 | 0.75 | 0.5 | 0.5 | 15.5 | 25 |
| TatABCD | 2.5 | 2.5 | 2 | 0.75 | 0.75 | 0.5 | 0.5 | 15.5 | 25 |
| TatABCD | 2.5 | 2.5 | 2 | 0.75 | 0.75 | 5 | 0.5 | 10.5 | 25 |

#### Electrophoresis

[![Electrophoresis021702.JPG](/wiki/images/1/14/Electrophoresis021702.JPG)](/File%3AElectrophoresis021702.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis021702.JPG "Enlarge")

1. 1kb ladder
2.3. TAMO reductase
4. TatABCD
5. TatABCD(10 times amount of genome)

#### Checking of restriction enzyme

| DT | enzyme | Buffer | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 2 | 0.5 | 3 | 0.5 | 24 | 30 |

at 37℃ for overnight
We checked EcoR1 and Xba1.

## February 18

#### Miniprep

|  | μg/mL | 260/280 | 230/260 |
| --- | --- | --- | --- |
| JS3117-1 | 135 | 1.5 | 2.06 |
| JS3117-2 | 75 | 1.6 | 1.63 |
| JS3109-1 | 115 | 1.5 | 1.88 |
| JS3109-2 | 75 | 1.65 | 1.71 |
| pSB1AT3-1 | 70 | 1.66 | 1.83 |
| pSB1AT3-2 | 100 | 1.52 | 1.54 |

diluted to 25 times

#### Making Competent cells

We put 3mL of preculture product on yesterday onto 300mL of LB medium

| time | OD600 |
| --- | --- |
| 10:30 | start |
| 12:10 | 0.118 |
| 13:00 | 0.270 |
| 13:30 | 0.502 |

#### Transformation

| pSB1AT3-2 | competent cell | MilliQ | Total |
| --- | --- | --- | --- |
| 0 | 20 | 10 | 30 |
| 2 | 20 | 8 | 30 |
| 10 | 20 | 0 | 30 |

* Results(on Feb. 19)

| pSB1AT3-2 | number of colony |
| --- | --- |
| 0 | 0 |
| 2 | 177 |
| 10 | 590 |

Transformation efficiency 7.4x10^4 colonys/μg

## February 20

#### Restrictive Digestion

sample 1

| DT plasmid | EcoR1 | Xba1 | Buffer | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 7.5 | 0.5 | 0.5 | 3 | 0.5 | 18 | 30 |

sample2

| GFP plasmid | EcoR1 | Spe1 | Buffer | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |

#### Electrophoresis

* sample1

[![Electrophoresis022001 .JPG](/wiki/images/5/5b/Electrophoresis022001_.JPG)](/File%3AElectrophoresis022001_.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis022001_.JPG "Enlarge")

a. sample1 2μL + MilliQ 3μL + 6×Loading Dye 1μL
b. sample1 5μL + 6×Loading Dye 1μL
lane1. 1kb ladder
lane2. a
lane3. b
lane4. a
lane5. b
lane6. a
lane7. b
lane8. 1kb ladder

* sample2

[![Electrophoresis022302.JPG](/wiki/images/5/5a/Electrophoresis022302.JPG)](/File%3AElectrophoresis022302.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis022302.JPG "Enlarge")

c. sample2 2μL + MilliQ 3μL + 6×Loading Dye 1μL
d. sample2 5μL + 6×Loading Dye 1μL
lane1. 1kb ladder
lane2. c
lane3. d
lane4. 1kb ladder

#### PCR

|  | Buffer | dNTPs | MgSO4 | primer-f | primer-r | genomic DNA | KOD plus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| TatABCD1 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 0.5 | 16 | 25 |
| TatABCD2 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 0.5 | 16 | 25 |
| TMAO reductase1 | 2.5 | 2.5 | 2 | 0.75 | 0.75 | 0.5 | 0.5 | 15.5 | 25 |
| TMAO reductase2 | 2.5 | 2.5 | 2 | 0.75 | 0.75 | 5 | 0.5 | 10.5 | 25 |

Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 2.5min
→30cycles

|  | Buffer | dNTPs | MgSO4 | primer-f | primer-r | PCR products | genomic DNA | KOD plus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| TatABCD1 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0 | 0.5 | 0.5 | 16 | 25 |
| TatABCD2 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 1 | 0 | 0.5 | 15.5 | 25 |
| TMAO reductase1 | 2.5 | 2.5 | 2 | 0.75 | 0.75 | 0 | 0 | 0.5 | 16 | 25 |
| TMAO reductase2 | 2.5 | 2.5 | 2 | 0.75 | 0.75 | 0 | 0 | 0.5 | 16 | 25 |

Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 2.5min
→35cycles

#### Electrophoresis

[![Electrophoresis022003.JPG](/wiki/images/d/d4/Electrophoresis022003.JPG)](/File%3AElectrophoresis022003.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis022003.JPG "Enlarge")

[![Electrophoresis022004.JPG](/wiki/images/f/fc/Electrophoresis022004.JPG)](/File%3AElectrophoresis022004.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis022004.JPG "Enlarge")

lane1.1kb ladder
lane2.TatABCD
lane3.TatABCD
lane4.TAMO1
lane5.TAMO2
lane6.1kb ladder

lane1.1kb ladder
lane2.TatABCD1
lane3.TatABCD2
lane4.TAMO
lane5.TAMO
lane6.1kb ladder

## February 21

#### PCR (Advantage HF protocol)

|  | buffer | dNTPs | primer-f | primer-r | gDNA | PCR products | DW | polymerase | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| TatABCD | 2.5 | 2.5 | 0.75 | 0.75 | 1 | 0 | 17 | 0.5 | 25 |
| TMAO | 2.5 | 2.5 | 0.75 | 0.75 | 0 | 1 | 17 | 0.5 | 25 |

Predenature 94℃ 1min
Denature 94℃ 30sec
Annealing 58℃ 30sec
Extension 68℃ 3min
→25cycles

#### Electrophoresis

[![Electrophoresis0221.JPG](/wiki/images/c/cb/Electrophoresis0221.JPG)](/File%3AElectrophoresis0221.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis0221.JPG "Enlarge")

1. 1kb ladder 2μL
2. TatABCD 5μL + 6×Loading Buffer 1μL
3. TAMO 5μL + 6×Loading Buffer 1μL
4. 1kb ladder 2μL

#### Liquid culture

pSB3C5-1,2
pSB4K5-1,2

## February 22

#### Gel extraction

lane 1 of the gel 45.0μg/mL

#### PCR purification

product 38.2μg/mL

#### PCR

TMAO reductase

|  | Buffer | dNTPs | MgSO4 | prefix primer-f | suffix primer-r | product of gel extract | product of PCR purification(1ng/μL) | KOD plus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 5 | 5 | 4 | 1.5 | 1.5 | 0.5 | 0 | 1 | 32.5 | 50 |
| 2 | 5 | 5 | 4 | 1.5 | 1.5 | 1 | 0 | 1 | 32.5 | 50 |
| 3 | 5 | 5 | 4 | 1.5 | 1.5 | 0 | 0.5 | 1 | 32.5 | 50 |
| 4 | 5 | 5 | 4 | 1.5 | 1.5 | 0 | 1 | 1 | 32.5 | 50 |

94℃, 2min
98℃, 10sec
59℃, 30sec
68℃, 3min
→25cycles

#### Electrophoresis

[![Electrophoresis022201.JPG](/wiki/images/2/21/Electrophoresis022201.JPG)](/File%3AElectrophoresis022201.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis022201.JPG "Enlarge")

1. 1kb ladder
2. TAMO1
3. TAMO2
4. TAMO3
5. TAMO4
6. constructive promoter 1-18C
7. constructive promoter Spe1
8. constructive promoter Pst1
9. 1kb ladder

#### PCR

|  | Buffer | dNTPs | MgSO4 | prefix primer-f | suffix primer-r | product of PCR purification(1ng/μL) | KOD plus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 5 | 5 | 3 | 1.5 | 1.5 | 0.5 | 1 | 32.5 | 50 |
| 2 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 1 | 32 | 50 |
| 3 | 5 | 5 | 3 | 1.5 | 1.5 | 2 | 1 | 31 | 50 |
| 4 | 5 | 5 | 3 | 1.5 | 1.5 | 3 | 1 | 30 | 50 |
| 5 | 5 | 5 | 3 | 1.5 | 1.5 | 10 | 1 | 29 | 50 |
| 6 | 5 | 5 | 3 | 1.5 | 1.5 | 0 | 1 | 33 | 50 |

94℃, 2min
98℃, 10sec
59℃, 30sec
68℃, 3min
→25cycles

#### Electrophoresis

[![Electrophoresis022202.JPG](/wiki/images/3/37/Electrophoresis022202.JPG)](/File%3AElectrophoresis022202.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis022202.JPG "Enlarge")

#### Checking Dpn1

| Buffer | LacP(28.7ng/μL) | Dpn1 | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 2 | 10 | 0.5 | 7.5 | 20 |
| 2 | 10 | 0 | 5 | 17 |

## February 23

#### Colony PCR

TatABCD(2 samples)

| Buffer | dNTPs | MgSO4 | primer-f | primer-r | KOD plus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |

Predenature 94℃ 1min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 3min
→25cycles

#### Electrophoresis

[![Electrophoresis022301.JPG](/wiki/images/9/96/Electrophoresis022301.JPG)](/File%3AElectrophoresis022301.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis022301.JPG "Enlarge")

1. 1kb ladder 2μL
2. TatABCD 1 5μL + 6×Loading Buffer 1μL
3. TatABCD 2 5μL + 6×Loading Buffer 1μL
4. 1kb ladder 2μL

#### Miniprep

pSB4K5 and pSB3C5
deluted it to 25 times and then measured it
pSB4K5 1 : 60.0 μg/ml 1.67(260/280) 1.98(260/230)
pSB4K5 2 : 55.0 μg/ml 1.49(260/280) 1.62(260/230)
pSB3C5-3 : 3.6 μg/ml 1.57(260/280) 3.00(260/230)
pSB3C5-4 : 1.3 μg/ml 1.44(260/280) 1.04(260/230)

#### Liquid culture

pSB3C5-3,4

#### Electrophoresis

[![Electrophoresis022302.JPG](/wiki/images/5/5a/Electrophoresis022302.JPG)](/File%3AElectrophoresis022302.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis022302.JPG "Enlarge")

1. 1kb ladder 2μL
2. pSB3C5-3 5μL, 6×loading dye 1μL
3. pSB3C5-4 5μL, 6×loading dye 1μL
4. 1kb ladder 2μL

## February 27

#### Test of Dpn1

| Buffer2 | GFP2 | BSA | MilliQ | Dpn1 |
| --- | --- | --- | --- | --- |
| 3 | 3 | 0.3 | 23 | 1 |

#### Colony PCR

|  | buffer | dNTPs | MgSO4 | Primer-f | Primer-r | MilliQ | KOD Plus | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Colony PCR(2 samples) | 5 | 5 | 3 | 1.5 | 1.5 | 33 | 1 | 50 |
| Negative control | 5 | 5 | 3 | 1.5 | 1.5 | 34 | 0 | 50 |

Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 59℃ 30sec
Extension 68℃ 3min
→25cycles

#### Electrophoresis

[![Electrophoresis0227.JPG](/wiki/images/f/fd/Electrophoresis0227.JPG)](/File%3AElectrophoresis0227.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis0227.JPG "Enlarge")

| lane | DNA | sample | Loading Dye | MilliQ |
| --- | --- | --- | --- | --- |
| 1 | 1kb ladder | 2 | 0 | 0 |
| 2 | product of PCR1 | 5 | 1 | 0 |
| 3 | product of PCR2 | 5 | 1 | 0 |
| 4 | product of PCR(Negative control) | 5 | 1 | 0 |
| 5 | product of PCR(2/23) | 5 | 1 | 0 |
| 6 | GFP2(DPN1) | 10 | 2 | 0 |
| 7 | GFP2 | 3 | 2 | 7 |
| 8 | 1kb ladder | 2 | 0 | 0 |

#### Results of liquid culture

We measure this after dilute it to 10 times.

| pSB3C5-5 | pSB3C5-6 | pSB3C5-5(1% glucose) | pSB3C5-6(1% glucose) |
| --- | --- | --- | --- |
| 8.5[µg/ml] | -1.8 | -17.9 | -18.2 |

#### PCR

|  | buffer | dNTPs | MgSO4 | Primer-f(prefix) | Primer-r(suffix) | PCR purification product(1ng/µL) | MilliQ | KOD Plus | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 5 | 5 | 3 | 1.5 | 1.5 | 0.2 | 32.8 | 1 | 50 |
| 2 | 5 | 5 | 3 | 1.5 | 1.5 | 0.5 | 32.5 | 1 | 50 |

* PCR purification product was that purification product(75ng/µL) of electrophoresis-3 deluted to 1ng/µL

Predenature 94℃,2min
Denature 98℃,10sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles

## February 28

#### Electrophoresis

[![Electrophoresis022801.JPG](/wiki/images/7/78/Electrophoresis022801.JPG)](/File%3AElectrophoresis022801.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis022801.JPG "Enlarge")

1. 1kb ladder
2. PCR1 →Product of gel extraction : TatABCD with prefix and suffix 105[ng/µL]
3. PCR2

#### Restrictive Digestion

| Buffer2 | plasmid(?) | enzyme | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 2 | 2 | 0.2 | 15.8 | 20 |

incubate 1 hour at 37℃

#### Electrophoresis

[![Electrophoresis022802.JPG](/wiki/images/4/45/Electrophoresis022802.JPG)](/File%3AElectrophoresis022802.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis022802.JPG "Enlarge")

[![Electrophoresis022803.JPG](/wiki/images/5/5f/Electrophoresis022803.JPG)](/File%3AElectrophoresis022803.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis022803.JPG "Enlarge")

1. 1kb ladder
2. Control (without enzymes)
3. EcoR1
4. Xba1 (crystallized)
5. Xba1 (with seal)
6. Spe1
7. Pst1
8. 1kb ladder

#### PCR and Electrophoresis

| Quick Taq Dye Mix | primer-f | primer-r | template | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 25 | 1.0 | 1.0 | 0.5 | 22.5 | 50 |

Predenature 94℃,2min
Denature 94℃,30sec
Annealing 59℃,30sec
Extension 68℃,3min
→25cycles

#### Restrictive Digestion

| BufferH | TatABCD | EcoR1 | Spe1 | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 2 | 5 | 0.2 | 0.2 | 12.6 | 20 |

#### PCR purification

We eluted the product for 30µL MilliQ

#### Ligation

| Insert(TatABCD) | Vector(pSB1C3) | Ligation High | Total |
| --- | --- | --- | --- |
| 10 | 1 | 5 | 16 |

4℃, overnight

## February 29

#### Transformation

| TatABCD | competent cell | Total |
| --- | --- | --- |
| 1 | 10 | 11 |

#### Checking Restriction enzyme

| plasmid seems to be 1-18C promoter | Enzyme | Buffer | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 2 | 0.2 | 2 | 15.8 | 20 |

#### Checking TatABCD

| TatABCD | Hind3 | Buffer | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 5 | 0.2 | 2 | 12.8 | 20 |

#### PCR

|  | buffer | dNTPs | MgSO4 | Primer-f | Primer-r | ColE1(6.5ng/µL) / TMAO | MilliQ | KOD Plus Neo | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Kil | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16 | 0.5 | 25 |
| TMAO | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16 | 0.5 | 25 |

Predenature 94℃,2min
Denature 98℃,10sec
Annealing 60℃,30sec
Extension 68℃,3min
→30cycles

#### Electrophoresis

[![38f7cf5b-20b9-42d0-a497-1885f1f92bbe.jpg](/wiki/images/0/06/38f7cf5b-20b9-42d0-a497-1885f1f92bbe.jpg)](/File%3A38f7cf5b-20b9-42d0-a497-1885f1f92bbe.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A38f7cf5b-20b9-42d0-a497-1885f1f92bbe.jpg "Enlarge")

1. 1kb ladder
2. Kil (649bp)
3. TMAO (2720bp)
4. TMAO (Quick Taq)
5. TatABCD (Quick Taq)
6. TatABCD (Hind3)
7. 1kb ladder

## March 1

#### PCR

* TMAO

Template is gDNA and product of colony PCR gel extraction

|  | Buffer | gNTPs | MgSO4 | Primer-f | Primer-r | KOD Plus Neo | Template gDNA | product of gel extraction | DW | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 0.5 | 0 | 16 | 25 |
| 2 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 0 | 2 | 14.5 | 25 |

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.5min
→25cycles

* Kil

| Buffer | dNTPs | MgSO4 | primer-f | primer-r | colE1(6.5ng/µL) | KOD Plus Neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 16 | 0.5 | 25 |

94℃, 2min
98℃, 10sec
61℃, 30sec
68℃, 1min
→20cycles

#### Electrophoresis

[![Electrophoresis030101.JPG](/wiki/images/b/b6/Electrophoresis030101.JPG)](/File%3AElectrophoresis030101.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis030101.JPG "Enlarge")

1. 1kb ladder
2. TMAO1 (gDNA)
3. TMAO2 (product of gel extraction)
4. Kil

#### PCR

* kil

| Buffer | dNTPs | MgSO4 | primer-f | primer-r | Product of Purification | KOD Plus Neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50 |

[![Electrophoresis030102.JPG](/wiki/images/e/ed/Electrophoresis030102.JPG)](/File%3AElectrophoresis030102.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis030102.JPG "Enlarge")

94℃, 2min
98℃, 10sec
61℃, 30sec
68℃, 30sec
20cycles
→Purification 230ng/µL

#### Restrictive Digestion

| Kil | EcoR1 | Spe1 | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 5 | 0.2 | 0.2 | 2 | 12.6 | 20 |

incubate at 37℃, for 1.5 hours

#### PCR Purification

#### Ligation

| Kil | pSB1C3 | Ligation High | Total |
| --- | --- | --- | --- |
| 5 | 1 | 3 | 9 |

at 4℃, for overnight

## March 2

#### Ligation

| Kil | pSB1C3 | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 5 | 1 | 3 | 9 |

| TatABCD | pSB1C3 | Ligation | Total |
| --- | --- | --- | --- |
| 10 | 1 | 5 | 16 |

at 16℃ for 1 hour

#### Transformation

| Kil | Kil(3/1,Ligation) | TatABCD | competet cell | Total |
| --- | --- | --- | --- | --- |
| 1 | 0 | 0 | 10 | 11 |
| 0 | 1 | 0 | 10 | 11 |
| 0 | 0 | 1 | 10 | 11 |

#### PCR

| Quick Taq | primer-r | primer-f | template | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 0.5 | 22.5 | 50 |

#### Electrophoresis

[![Electrophoresis030227.JPG](/wiki/images/8/80/Electrophoresis030227.JPG)](/File%3AElectrophoresis030227.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis030227.JPG "Enlarge")

#### Restrictive Digestion

| pSB3C5-5 | EcoR1 | Pst1 | BufferH | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |

at 37℃, for 2 hour

#### Electrophoresis

[![Electrophoresis030225.JPG](/wiki/images/c/ce/Electrophoresis030225.JPG)](/File%3AElectrophoresis030225.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis030225.JPG "Enlarge")

1. 1kb ladder 2µL
2. pSB3C5 5µL + 6×Loading Buffer 1µL
・product of gel extraction(about 2700bp)
－30.9µg/mL

#### Restrictive Digestion

GFP1 ,2 ,3

| GFP | EcoR1 | Pst1 | Buffer | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |

at 37℃, for 2.5 hours

#### Restrictive Digestion

| DT | EcoR1 | Xba1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.2 | 0.2 | 3 | 0.3 | 16.3 | 30 |

| Constitutive Promoter | Spe1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 15 | 0.2 | 0.2 | 3 | 0.3 | 11.3 | 30 |

at 37℃, 2 hours

* J23117-1:135ng/µL, J23107-1:115ng/µL
* DT3→PCR Purification
* Promoter→Gel Extraction

#### Checking TMAO

| something seems to be TMAO | Buffer2 | EcoR1 | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 10 | 2 | 0.5 | 7.5 | 20 |

at 37℃, for 1 hour

#### Electrophoresis

[![Electrophoresis030226.JPG](/wiki/images/2/25/Electrophoresis030226.JPG)](/File%3AElectrophoresis030226.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis030226.JPG "Enlarge")

1. 1kb ladder
2. GFP1 that had been cut by restriction enzyme
3. GFP2 that had been cut by restriction enzyme
4. GFP3 that had been cut by restriction enzyme
5. GFP1
6. GFP2
7. GFP3
8. TMAO (control)
9. TMAO (EcoR1)
10. DT (control)
11. DT (EcoR1, Xba1)
12. 1kb ladder

**Checking TatABCD**

| Quick Taq | primer-f | primer-r | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

#### Electrophoresis

[![Electrophoresis030228.JPG](/wiki/images/6/69/Electrophoresis030228.JPG)](/File%3AElectrophoresis030228.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis030228.JPG "Enlarge")

## March 3

#### PCR

|  | template | buffer | dNTPs | MgSO4 | VF | VR | KOD plus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 1 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 50 |
| 2 | 2 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 31 | 50 |

94℃, 2min
98℃, 10sec
50℃, 30sec
68℃, 1min
→30cycles

#### Miniprep

14.2µg/mL, 15.0µg/mL

## March 4

#### Sequence of TatABCD

| Quick Taq | primer-f | promer-r sequence | template | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 1 | 23 | 50 |

| Quick Taq | primer-f sequence | primer-r | template | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 1 | 23 | 50 |

#### Colony PCR of TMAO

| buffer | dNTPs | NgSO4 | primer-f | primerr-r | KOD plus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 4 | 1.5 | 1.5 | 1 | 32 | 50 |

→ethanol precipitation
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 2.5min
→25cycles

#### Electrophoresis

[![34 percent 20~2.JPG](/wiki/images/e/ec/34_percent_20~2.JPG)](/File%3A34_percent_20~2.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A34_percent_20~2.JPG "Enlarge")

1. 1kb ladder
2. TatABCD1
3. TatABCD2
4. TMAO
5. 1kb ladder

#### Restrictive Digestion

| TMAO | EcoR1 | BufferH | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 10 | 0.2 | 3 | 0.3 | 16.5 | 30 |

| TMAO | Xba1 | Pst1 | BudderM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.2 | 0.2 | 3 | 0.3 | 16.3 | 30 |

at 37℃ for 1 hour

#### Electrophoresis

[![34 percent 20~1.JPG](/wiki/images/e/ed/34_percent_20~1.JPG)](/File%3A34_percent_20~1.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A34_percent_20~1.JPG "Enlarge")

#### Transformation

| pSB1C3 | competent cell(made at 2/8) | Total |
| --- | --- | --- |
| 5 | 100 | 105 |

## March 5

#### Restrictive Digestion

| pSB1C3(Xba1, Spe1) | Pst1 | BufferH | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 10 | 0.2 | 2 | 0.2 | 7.6 | 20 |
| pSB1C3(Xba1, Spe1) | EcoR1 | BufferH | BSA | MilliQ | Total |
| 10 | 0.2 | 2 | 0.2 | 7.6 | 20 |

at 37℃ for 1 hour
→Then we did ethanol precipitation

#### Ligation

| Kil(EcoR1, Spe1) | pSB1C3(EcoR1) | Ligation High | Total |
| --- | --- | --- | --- |
| 5 | 1 | 3 | 9 |

at 16℃ for 1 hour

#### Transformation

| Kil | competent cell | Total |
| --- | --- | --- |
| 1 | 10 | 11 |

We used commercially available competent cells in this time.

#### PCR

TMAO

| buffer | dNTPs | MgSO4 | Primer-f | Primer-r | Template | KOD plus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 1 | 32 | 50 |

#### Electrophoresis

[![35percent20~2.JPG](/wiki/images/a/a7/35percent20~2.JPG)](/File%3A35percent20~2.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A35percent20~2.JPG "Enlarge")

#### Restrictive Digestion

|  | LacP | pSB3C5 | EcoR1 | Pst1 | BufferH | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 20 | 0 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |
| 2 | 0 | 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |

at 37℃ for 1 hour
1→Ethanol precipitation　45.4µg/mL
2→Gel extraction　38.7µg/mL

#### Ligation

| LacP | pSB3C5 | Ligation High | Total |
| --- | --- | --- | --- |
| 10 | 2 | 6 | 18 |

at 4℃ for overnight

#### Transformation

| LacP+pSB3C5 | competent cell | Total |
| --- | --- | --- |
| 1 | 10 | 11 |

on ice for 30 mins.
heat shock at 42℃ for 60secs
on ice for 2 mins.
After we incubate with 200µL of SOC culture for 1 hour, we did plating on LB culture with CP

#### Restrictive Digestion

| GFP Plasmid | EcoR1 | Spe1 | Buffer2 | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |

at 37℃ for 2 hours

#### Electrophoresis

1. Ladder 2µL
2. GFP Plasmid (already restricted) 2µL + Loading Dye 2µL + MilliQ 8µL
3. Ladder 2µL

#### PCR

torA signal and pspA
We did Colony PCR to pspA

| Buffer | dNTPs | MgSO4 | Primer-f | Primer-r | template(TMAO) | KOD plus | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 0.5 | 16 | 25 |

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec

#### electrophoresis

1. 100bp Ladder
2. torA signal (272bp)
3. pspA (969bp)
4. 100bp Ladder

## March 6

#### Restrictive Digestion

| GFP | EcoR1 | Spe1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 12 | 0.5 | 0.5 | 3 | 0.5 | 13.5 | 30 |

We did incubate at 37℃ for 1.5hours.
And we did gel extraction on 3/7 and get 40.0μg/mL GFP.

#### PCR

| buffer | dNTPs | MgSO4 | Primer-f | Primer-r | template | KOD plus neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 0.5 | 1 | 32.5 | 50 |

94℃　2min
98℃　10sec
60℃　30sec
68℃　(torA 10sec / pspA 30sec) 25 cycles
We used product of PCR on 3/5 of torA and pspA as template.

#### Restrictive Digestion

| Kil | EcoR1 | Spe1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.3 | 0.3 | 3 | 0.3 | 16.1 | 30 |

| DT | EcoR1 | Xba1 | Buffer2 | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |

at 37℃ for 2 hours
→ purification 37.7ng/μL

#### Ligation

| Kil | pSB1C3 | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 4 | 2 | 3 | 9 |

* Kil : 350fmol
* pSB1C3 : 29fmol

at 16℃ for overnight

## March 7

#### Electrophoresis

[![Electrophoresis0307.JPG](/wiki/images/f/f3/Electrophoresis0307.JPG)](/File%3AElectrophoresis0307.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis0307.JPG "Enlarge")

1. 1kb ladder 2µL
2. pspA (PCR product)2.5µL + Loading Dye 0.5µL
3. GFP 30µL + Loading Dye 6µL
4. 1kb ladder 2µL

* The GFP was gel extracted on 3/6 and concentrated by Vacuum in 150µg/mL

#### Ligation

| VectorDNA | GFP | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 5 | 15 | 10 | 30 |

#### Restrictive Digestion

| torA | EcoR1 | Spe1 | bufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.3 | 0.3 | 3 | 0.3 | 16.1 | 30 |

| pspA | EcoR1 | Spe1 | bufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 5 | 0.3 | 0.3 | 3 | 0.3 | 21.1 | 30 |

at 37℃ for 1.5 hours

#### Purification

torA→31.8ng/µL
pspA→49.3ng/µL

#### Ligation

| torA | pSB1C3 | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 3 | 3 | 3 | 9 |

| pspA | pSB1C3 | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 4 | 2 | 3 | 9 |

at 4℃, for overnight

* torA→31.8ng/µL×3µL=95.4ng=0.529pmol
* pSB1C3→19.4ng/µL×3µL=58.2ng=0.042pmol
* pspA→49.3ng/µL×4µL=197.2ng=0.308pmol
* pSB1C3→19.4ng/µL×2µL=38.8ng=0.029pmol

## March 8

#### Restrictive Digestion

| pSB4K5 | EcoR1 | Spe1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.2 | 0.2 | 3 | 0.2 | 6.4 | 30 |

at 37℃ for 1 hour.
→Purification : 36.6ng/µL

#### Ligation

| Kil | pSB4K5 | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 10 | 1 | 5 | 16 |

at 4℃ for overnight

* Kil→37.7ng/µL×10µL=377ng=879fmol
* pSB4K5→36.6ng/µL×1µL=36.6ng=86fmol

#### Liquid culture

LacP + pSB3C5 -1, 2

#### Transformation

| torA | pspA | competent cell | Total |
| --- | --- | --- | --- |
| 1 | 0 | 10 | 11 |
| 0 | 1 | 10 | 11 |

We use commercially available competent cells in this time.

## March 9

#### Restrictive Digestion

| TatABCD | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.2 | 0.2 | 3 | 0.3 | 16.3 | 30 |

at 37℃ for 1hour
→purification : 75.0μg/mL (1.11 260/280 , 0.81 260/230)

#### Miniprep

LacP + pSB3C5 1 80.5μg/mL (1.78 260/280 , 2.00 260/230)
LacP + pSB3C5 2 107.2μg/mL (1.83 260/280 , 1.90 260/230)

#### Colony PCR

| Quick Taq | VF | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

94℃ 2min
94℃ 30sec
55℃ 30sec
68℃ 6sec
25cycles

#### Ligation

| TatABCD | constP J23107 | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 5 | 1 | 3 | 9 |

TatABCD : 227fmol
constP J23107 : 21fmol

#### Transformation

|  | pspA | torA | Kil | competent cell |
| --- | --- | --- | --- | --- |
| 1 | 1 | 0 | 0 | 10 |
| 2 | 0 | 1 | 0 | 10 |
| 3 | 0 | 0 | 1 | 10 |

#### Miniprep

4mL of plusgrow which had been cultured for overnight.
pSB4K5 : 80.5μg/mL

## March 10

#### Screening PCR

| Quick Taq | VF | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

[![Electrophoresis0310.JPG](/wiki/images/a/a5/Electrophoresis0310.JPG)](/File%3AElectrophoresis0310.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis0310.JPG "Enlarge")

Then we did electrophoresis to confirm.
1. 1kb ladder
2. kil (649bp)
3. 4. 5. pspA (969bp)
6. 1kb ladder

1. 100bp ladder
2. 3. 4. torA signal

#### Restrictive Digestion

| LacP-pSB3C5 | Spe1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.2 | 0.2 | 2 | 0.2 | 7.4 | 20 |

for 2.5 hours at 37℃
→purification 29.0ng/μL

| torA | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.2 | 0.2 | 2 | 0.2 | 7.4 | 20 |

for 2.5 hours at 37℃
→purification 91.8ng/μL

#### Ligation

| torA | LacP-pSB3C5 | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 4 | 2 | 3 | 9 |

torA : 929fmol
LacP-pSB3C5 : 284fmol
for overnight at 4℃

## March 11

#### Miniprep

We used 3μL of plus grow that we had cultured for overnight.
torA : 62.8ng/μL

#### Screening PCR

| Quick Taq | VF | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

#### Electrophoresis

[![Electrophoresis031101.JPG](/wiki/images/5/5a/Electrophoresis031101.JPG)](/File%3AElectrophoresis031101.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis031101.JPG "Enlarge")

[![Electrophoresis031102.JPG](/wiki/images/c/cb/Electrophoresis031102.JPG)](/File%3AElectrophoresis031102.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis031102.JPG "Enlarge")

1. 1kb ladder
2〜11. pspA (969bp)
12. 1kb ladder

The results were shown as photograph in the right.

It seemed that there were shorter sample than expected sample, so we did electrophoresis with pspA which was product of PCR and pspA which had already cut with EcoR1 and Spe1.

1.1kb ladder
2. pspA (PCR product)
3, pspA (Eco, Spe)
4〜6, pspA (colony PCR)
7,1kb ladder

The results were shown as photograph in the right.

## March 12

#### Transformation

| DT(1ng/μL) | DT(0.1ng/μL) | Kil | LacP-torA | MilliQ | competent cell | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 0 | 0 | 0 | 0 | 20 | 21 |
| 0 | 1 | 0 | 0 | 0 | 20 | 21 |
| 0 | 0 | 5 | 0 | 0 | 50 | 51 |
| 0 | 0 | 0 | 5 | 0 | 50 | 51 |
| 0 | 0 | 0 | 0 | 1 | 20 | 21 |

#### Restrictive Digestion

| pspA | EcoR1 | Spe1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 3 | 0.3 | 15.7 | 30 |

at 37℃ for 4 hours
→ We did purification and got 48.3ng/μL pspA.

#### Ligation

| pspA | pSB1C3 | MilliQ | Ligation High Ver.2 | Total |
| --- | --- | --- | --- | --- |
| 4 | 2 | 0 | 3 | 9 |
| 2 | 2 | 0 | 2 | 6 |
| 0 | 2 | 2 | 2 | 6 |

## March 13

#### Miniprep

pSB1C3 74.2µg/mL 1.65 (260/280) 1.31 (260/230)

## March 14

#### Restrictive Digestion

| pSB1C3 | EcoR1 | Spe1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.2 | 0.2 | 4 | 0.4 | 15.2 | 40 |

We did gel extraction and got 47.2ng/µL pSB1C3 but we did not cut out RFP.

#### Ligation

| torA | pSB1C3 | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 4 | 2 | 3 | 9 |
| MilliQ | pSB1C3 | Ligation High Ver.2 | Total |
| 4 | 2 | 3 | 9 |

at 16℃, for 1 hour

* torA : 0.707pmol
* pSB1C3 : 0.068pmol

#### Liquid culture

We cultured LacP-pSB3C5 1,2,3,4,5 (CP tolerance)on culture with Amp.
→Only 4 which did not be cultured succeeded.

## March 15

#### Liquid culture

We cultured LacP-pSB3C5 6,7,8,9,10 on culture with ampicillin.
→6,8,9,10 were succeeded.

#### Restrictive Digestion

| GFP | EcoR1 | Spe1 | Buffer2 | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |

| DT | EcoR1 | Xba1 | Buffer2 | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |

for 2 hours at 37℃.

#### Miniprep

pspA (pSB1C3) 40.5ng/µL

#### Ligation

| pspA | DT | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 5 | 1.5 | 3 | 9.5 |

* pspA : 385fmol
* DT : 36fmol

#### Transformation

| J23107-TatABCD | DT (0.1ng/µL) | DT (0.01ng/µL) | pspA-DT | competent cells on 3/15 | Total |
| --- | --- | --- | --- | --- | --- |
| 2 | 0 | 0 | 0 | 20 | 22 |
| 0 | 2 | 0 | 0 | 20 | 22 |
| 0 | 0 | 2 | 0 | 20 | 22 |
| 0 | 0 | 0 | 2 | 20 | 22 |

#### Screening PCR

Kil, pspA and torA

| Quick Taq | Primer-r | Primer-f | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

## March 16

#### Miniprep

torA (pSB1C3) 68.8ng/µL
Kil (pSB4K5) 92.7ng/µL
torA was red for some reason. We do not know why.

#### Colony PCR

| Quick Taq | Primer-r | Primer-f | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 6sec
→25cycles

#### Electrophoresis

[![Electrophoresis0316.JPG](/wiki/images/5/5f/Electrophoresis0316.JPG)](/File%3AElectrophoresis0316.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis0316.JPG "Enlarge")

The results were shown as photograph in the right.

#### Checking Transformation Efficiency

competent cells that were made on March 15.
DNA : 0.02ng → 668 colonies Transformation Efficiency : 3.3×10^7
DNA : 0.2ng → 1739 colonies Transformation Efficiency : 8.7×10^6

#### Restrictive Digestion

| pSB1C3 | EcoR1 | Spe1 | BSA | BufferM | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.2 | 0.2 | 0.3 | 3 | 0 | 6.3 | 30 |
| 5 | 0.2 | 0 | 0.2 | 0 | 2 | 12.6 | 20 |

at 37℃, for 2 hours.
We did gel extraction for product with EcoR1, Spe1. We got 46.1ng/µL pSB1C3.

| Kil(pSB4K5) | EcoR1 | Pst1 | BSA | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 5 | 0.2 | 0.2 | 0.2 | 2 | 12.4 | 20 |
| 5 | 0.2 | 0 | 0.2 | 2 | 12.6 | 20 |

for overnight at 37℃.
We did this to confirm.

| pspA (pSB1C3) | EcoR1 | Pst1 | BSA | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 5 | 0.2 | 0.2 | 0.2 | 2 | 12.4 | 20 |
| 5 | 0.2 | 0 | 0.2 | 2 | 12.6 | 20 |

for overnight at 37℃.
We did this to confirm.

#### Ligation

| torA | pSB1C3 | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 4 | 2 | 3 | 9 |

| MilliQ | pSB1C3 | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 4 | 2 | 3 | 9 |

at 16℃, for overnight

* torA→767fmol
* pSB1C3→68fmol

## March 17

#### Miniprep

J23107-TatABCD 72.7ng/µL
pspA-DT 50.5ng/µL

#### Checking the Insert

| J21037-TatABCD | EcoR1 | Pst1 | BSA | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 5 | 0.2 | 0.2 | 0.2 | 2 | 12.4 | 20 |
| 5 | 0.2 | 0 | 0.2 | 2 | 12.6 | 20 |

Success.

| pspA-DT | EcoR1 | Pst1 | BSA | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 5 | 0.2 | 0.2 | 0.2 | 2 | 12.4 | 20 |
| 5 | 0.2 | 0 | 0.2 | 2 | 12.6 | 20 |

Failed.

## March 19

#### Restrictive Digestion

| DT | EcoR1 | Xba1 | BSA | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.2 | 0.2 | 0.3 | 3 | 16.3 | 30 |

We did Gel extraction and got 17.2ng/µL of DT.

#### Ligation

| Kil | DT | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 10 | 2 | 6 | 18 |

We did this for an hour at 16℃.

#### Restrictive Digestion

| GFP | EcoR1 | Spe1 | BSA | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 0.5 | 3 | 15.5 | 30 |

We did this for 4 hours at 37℃

#### Ligation

| pspA | DT | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 5 | 5 | 5 | 15 |

| pspA | pSB1C3 | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 4 | 1 | 3 | 8 |

We did these for an hour at 16℃.

* pspA (5µL)→377fmol
* DT→39fmol
* pspA (4µL)→339fmol
* pSB1C3→34fmol

#### Transformation

pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT

## March 20

#### Screaning PCR

| Quick Taq | Primer-R | Primer-F | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

* pspA→○
* pspA-DT→○
* GFP-DT→○
* torA→×
* Kil-DT 6 of 8 sumples→○

| Quick Taq | VR | VF | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

* pspA→○
* pspA-DT→×

#### Restrictive Digestion

| torA | EcoR1 | Spe1 | BSA | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.3 | 0.3 | 0.3 | 3 | 16.1 | 30 |

| DT | EcoR1 | Xba1 | BSA | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.2 | 0.2 | 0.3 | 3 | 16.3 | 30 |

We did this for overnight at 37℃. And we did purification.
torA 34.2ng/µL
DT 28.0ng/µL

## March 21

#### Miniprep

GFP-DT-1 : 77.7ng/µL
GFP-DT-2 : 67.6ng/µL

#### Restrictive Digestion

| pSB1C3 | EcoR1 | Spe1 | BSA | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.2 | 0.2 | 0.3 | 3 | 16.3 | 30 |
| 5 | 0.2 | 0 | 0.2 | 2 | 12.6 | 20 |

We did this for 3 hours at 37℃, and then we did gel extraction. We got 25.1ng/µL pSB1C3

| GFP-DT | EcoR1 | Pst1 | Xba1 | BSA | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 0.2 | 0.2 | 0 | 0.2 | 2 | 12.4 | 20 |
| 5 | 0.2 | 0 | 0 | 0.2 | 2 | 12.6 | 20 |
| 10 | 0.2 | 0 | 0.2 | 0.3 | 3 | 16.3 | 30 |

We did this for 3 hours at 37℃, and then we did Purification. We got 30.7ng/µL GFP-DT.

#### Ligation

|  | torA | pSB1C3 | pspA | DT | GFP-DT | Ligation High Ver.2 | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 4 | 1 | 0 | 0 | 0 | 3 | 8 |
| 2 | 0 | 1 | 7 | 0 | 0 | 4 | 12 |
| 3 | 0 | 0 | 5 | 3 | 0 | 4 | 12 |
| 4 | 3 | 0 | 0 | 0 | 5 | 4 | 12 |

We did this for an hour at16℃.

## March 22

#### PCR

We did PCR to amplify torA\_signal that was product of PCR at March 5 with redesigned primers.

| Buffer | dNTPs | MgSO4 | Primer-F | Primer-R | Template | MilliQ | KOD plus neo | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 0.5 | 32.5 | 1 | 50 |

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles
→Purification 110.7ng/µL

#### Restrictive Digestion

| torA | EcoR1 | Spe1 | BSA | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.2 | 0.2 | 0.3 | 3 | 16.3 | 30 |

#### Ligation

|  | torA | pSSB1C3 | pspA | DT | GFP-DT | Ligation high | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 4 | 3 | 0 | 0 | 0 | 4 | 11 |
| 2 | 3 | 0 | 0 | 0 | 3 | 3 | 9 |
| 3 | 0 | 0 | 5 | 5 | 0 | 5 | 15 |

* torA (4µL)→512fmol
* pSB1C3→54fmol
* torA (3µL)→384fmol
* GFP-DT→36fmol
* pspA→377fmol
* DT→65fmol

## March 23

#### Screening PCR

torA (pSB1C3), torA-GFP-DT and pspA-DT

| Quick Taq | VF | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

*E.coli* in liquid culture that had pspA(pSB1C3) also expressed RFP.

#### Restrictive Digestion

| pspA | EcoR1 | Spe1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 5 | 0.3 | 0.3 | 3 | 0.3 | 21.1 | 30 |

And then we did ethanol precipitation

#### Ethanol precipitation

pspA 11.5ng/µL.

#### Miniprep

LacP+pSB3C5-8 77.9µg/mL
LacP+pSB3C5-10 69.0µg/mL
Kil+DT-4 58.0µg/mL
Kil+DT-7 15.2µg/mL

#### Restrictive Digestion

| LacP+pSB3C5-8 | Spe1 | Pst1 | Buffer2 | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |

| Kil+DT-4 | Xba1 | Pst1 | Buffer2 | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |

at 37℃ for 1.5 hours
And then we did Gel extraction.

#### Gel Extraction

LacP+pSB3C5-8 40.4µg/mL
Kil+DT-4 26.8µg/mL

## March 26

#### Miniprep

torA(pSB1C3) 18.5[ng/µL]
torA-GFP-DT 20.0[ng/µL]

#### Restrictive Digestion

| torA(pSB1C3) | EcoR1 | Pst1 | BufferH | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 5 | 0.2 | 0.2 | 2 | 0.2 | 12.4 | 20 |
| 5 | 0.2 | 0 | 2 | 0.2 | 12.6 | 20 |

| torA-GFP-DT | EcoR1 | Xba1 | Pst1 | BufferH | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 0.2 | 0 | 0.2 | 2 | 0 | 0.2 | 12.4 | 20 |
| 5 | 0.2 | 0 | 0 | 2 | 0 | 0.2 | 12.6 | 20 |
| 20 | 0 | 0.2 | 0.2 | 0 | 3 | 0.3 | 6.3 | 30 |

We did Gel extraction and then got ??? 28.7[ng/µL]

#### Ligation

| LacP (pSB3C5) | torA-GFP-DT | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 1 | 5 | 3 | 9 |

* LacP : 22fmol
* torA-GFP-DT : 197fmol

| pspA | pSB1C3 | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 10 | 1 | 5 | 16 |

| pspA | DT | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 10 | 1 | 5 | 16 |

* pspA : 180fmol
* pSB1C3 : 18fmol
* DT : 16fmol

| LacP(pSB3C5) | Kil-DT | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 1 | 5 | 3 | 9 |

for 2 hours at 16℃

#### Transformation

| LacP-Kil-DT | competent cell | Total |
| --- | --- | --- |
| 1 | 10 | 11 |

## March 27

#### Miniprep

We retryed miniprep of torA(pSB1C3).
We got torA and its concentration was 39.3[ng/µL].

#### Transformation

| Name | Well | Sample | Competent Cells | Total | Plate | Colony |
| --- | --- | --- | --- | --- | --- | --- |
| BBa\_K117004 | 14J(2011 plate2) | 5 | 20 | ? | ? | ? |

We added 100[µL] of culture medium before we started culturing the *E.coli*.

#### Screening PCR

| Quick Taq | VF2 | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

LacP-torA-GFP-DT 1, 3, 5, 6, 8, 9 was successful.
pspA-DT was failed.
*E.coli* that had pspA(pSB1C3) did not make any colony.
LacP-Kil-DT 1,2,3,4,5,6,7,8 was failed.

#### Liquid culture

LacP-torA-GFP-DT

## July 23

#### Transformation

| DT(64.4ng/μl) | Competent cell | Plating in SOC medium | Total |
| --- | --- | --- | --- |
| 1 | 20 | 100 | 121 |

## July 24

#### Plating in SOC medium

## July 25

#### Transformation

* BBa\_J23113 from iGEM Kit
* LacP from iGEM Kit

## July 27

#### Restrictive Digestion

| Kil +DT (44.0 ng/μl) | 10xBuffer2 | BSA | Xbal | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 13.6 | 3.0 | 0.5 | 0.5 | 11.9 | 30.0 |

| LacP(28.7ng/μl) | 10xBuffer2 | BSA | Spe1 | Pst1 | Total |
| --- | --- | --- | --- | --- | --- |
| 20.9 | 3.0 | 0.5 | 0.5 | 4.6 | 30.0 |

## July 30

#### Ligation

| kil+DT(Xba1,Pst1) | LacP(Spe1,Pst1) | Ligation High | Total |
| --- | --- | --- | --- |
| 30 | 6 | 36 | 72 |

## July 31

#### Restriction Enzyme Processing

| torA-GFP+DT | 10×M Buffer | BSA | MilliQ | Xba1 | Pst1 | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 3.0 | 0.5 | 23.4 | 0.3 | 0.3 | 30.0 |

#### Liquid Culture

J23113(backbone J61002)

## August 1

#### Restrictive Digestion

| LacP+kil+DT | BSA | 10×H Buffer | EcoR1 | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 5.0 | 0.5 | 3.0 | 0.5 | 0.5 | 20.5 | 30.0 |

37℃ 2h

| LacP middle copy | BSA | 10×H Buffer | EcoR1 | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10.0 | 0.5 | 3.0 | 0.5 | 0.5 | 15.5 | 30.0 |

37℃ 2h

#### MIniprep

J23113(backbone J61002)

#### Liquid culture

J23113(backboneJ61002)
Three test tubes of 4 mL LB medium with ampicillin
37℃ overnight

## August 3

#### Restrictive Digestion

| pSB4K5 | BSA | 10×H Buffer | EcoR1 | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 8.0 | 0.5 | 3.0 | 0.5 | 0.5 | 17.5 | 30.0 |

| B0034 | BSA | 10×H Buffer | Spe1 | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 12.0 | 0.5 | 3.0 | 0.5 | 0.5 | 13.5 | 30.0 |

37℃ 2h

#### DNA purification

#### Ligation

| LacP+kil+DT | pSB4K5 | ligation high | Total |
| --- | --- | --- | --- |
| 15 | 15 | 15 | 45 |

16℃ 1h

#### Miniprep

* J23113(backbone J61002) 218.0μg/mL
* J23113(backbone J61002) 252.5μg/mL
* J23113(backbone J61002) 201.0μg/mL
* pSB3C5 45.0μg/ml 1.60 260/280 1.80 260/230
* pSB4C5 213.0μg/mL 1.77 260/280 4.40 260/230

## August 10

#### Ligation

| B0034 Spe1 Pst1 | GFP+DT Xba1 Pst1 | ligation high | Total |
| --- | --- | --- | --- |
| 2 | 3 | 4 | 9 |

16℃ 1h

#### Transformation

* RBS+GFP+DT
* LacP+kil+DT
* RBS（for control）

:   To put 2ng DNA in 20μL competent cell and leave it on ice for 30min
:   Heat shock for 60s at 42℃
:   To leave on ice for 2min
:   To spread on ampicillin LB plate

## August 13

#### Liquid culture

B0034 3mL ×3

:   37℃　overnight

## August 14

#### Miniprep

:   B0034 0μg/mL
:   B0034 235.5μg/mL
:   B0034 76.5μg/mL

#### Colony PCR

LacP+kil+DT ×5

| 2×Quick Taq | VF | VF | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

25cycles
pspA

| 10×Buffer for KOD-Plus-Ver2 | 2mM dNTPs | 25mM MgSO4 | Primer PsPA f-p | Primer PsPA r-s | KOD-Plus- | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |

## August 15

#### Colony PCR

pspA

| 2×Quick Taq | pspA r-s | pspA f-p | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

94℃, 2min
94℃, 30sec
56℃, 30sec
68℃, 1min
→35cycles

## August 16

#### Electrophoresis

[![Electrophoresis081601.jpg](/wiki/images/f/ff/Electrophoresis081601.jpg)](/File%3AElectrophoresis081601.jpg)

[![0814.jpg](/wiki/images/6/6c/0814.jpg)](/File%3A0814.jpg)

positive control(GFP+DT)
negative control(MilliQ)
① ～⑥ colony PCR LacP+kil+DT

①1kb ladder
②Positive control (GFP+DT)
③Negative control (MilliQ)
④～⑨LacP+kil+DT　①～⑥
⑩～⑫pspA 10μL ,6×buffer 2μL
⑬1kb ladder

## August 17

#### Colony PCR

pspA

| 10×Buffer for KOD-Plus-Ver2 | 2mM dNTPs | 25mM MgSO4 | Primer PsPA f | Primer PsPA r | KOD-Plus- | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 3 | 3 | 1.5 | 1.5 | 1 | 35 | 50 |
| 5 | 3 | 5 | 1.5 | 1.5 | 1 | 33 | 50 |

negative control(MilliQ 50μL)
94℃, 2min
94℃, 15sec
55℃, 30sec
68℃, 1min
→35cycles

#### Electrophoresis

[![0820.JPG](/wiki/images/7/75/0820.JPG)](/File%3A0820.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A0820.JPG "Enlarge")

① 1kb ladder
②, ③ MgSO4 3μL, pspA
④, ⑤ MgSO4 5μL, pspA
⑥ negative control(MilliQ)
⑧ 1kb ladder

## August 20

#### Restrictive Digestion

| J23113(201.0ng/μL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 1 | 1 | 3 | 0.5 | 14.5 | 30 |

## August 21

#### Colony PCR

pspA

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | Primer PsPA f | Primer PsPA r | KOD Plus Neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 3 | 5 | 1.5 | 1.5 | 1 | 33 | 50 |
| 5 | 3 | 7 | 1.5 | 1.5 | 1 | 31 | 50 |
| 5 | 3 | 10 | 1.5 | 1.5 | 1 | 28 | 50 |

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→25cycles

#### Restrictive Digestion

| J23113(218ng/μL) | Spe1 | Pst1 | BufferH | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 1 | 1 | 3 | 0.5 | 14.5 | 30 |

| torA-GFP-DT(20ng/μL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 5 | 0.6 | 0.6 | 3 | 0.5 | 20.3 | 30 |

#### Electrophoresis

[![0821e.jpg](/wiki/images/0/0b/0821e.jpg)](/File%3A0821e.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A0821e.jpg "Enlarge")

① 1kb ladder
②, ③　torA-GFP-DT(Xba1,Pst1)
④, ⑤　J23113(Spe1,Pst1)

#### Transformation

* LacP-torA-GFP-DT(Backbone pSB3C5)
* BBa\_K11704(Backbone pSB1A2)

#### Electrophoresis

[![0821r.jpg](/wiki/images/f/f2/0821r.jpg)](/File%3A0821r.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A0821r.jpg "Enlarge")

laneA-C. pspA
laneD. pspA(without primer)

#### Colony PCR

* pspA

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | Primer PsPA f | Primer PsPA r | KOD Plus Neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |

* negative control for pspA

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | Primer PsPA f-p | Primer PsPA r-s | KOD Plus Neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |

* GFP-DT(positive control)

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec

#### Transformation

* LacP-torA-GFP-DT(Backbone pSB3C5)
* J23107-TatABCD

#### PCR

kil, torA-GFP

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | DNA | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50 |

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→25cycles

## August 22

#### Restrictive Digestion

| pSB3C5 | EcoR1 | Pst1 | BufferH | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 13.3 | 0.5 | 0.5 | 3 | 0.5 | 12.2 | 30 |

#### Gel extraction

pSB3C5(EcoR1, Pst1)
27.2μg/mL

#### Transformation(the second time)

* LacP-torA-GFP-DT(Backbone pSB3C5)
* J23107-TatABCD

#### Electrophoresis

[![Electrophoresis082202.JPG](/wiki/images/6/67/Electrophoresis082202.JPG)](/File%3AElectrophoresis082202.JPG)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis082202.JPG "Enlarge")

See "August 21 -Colony PCR-"
1. 1kb ladder
2. pspA(Primer pspA f&r)
3. negative control for 2
4. GFP-DT
5. pspA(Primer pspA f-p&r-s)
6. pspA(Primer pspA f-p&r-s)
7. negative control for 5&6

#### PCR

LacP-GFP-DT(a kit of biological parts)

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | DNA | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50 |

#### Colony PCR

pspA

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | Primer pspA f | Primer pspA r | KOD Plus Neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | Primer pspA f-p | Primer pspA r-s | KOD Plus Neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→30cycles

#### Electrophoresis

[![Electrophoresis082201.jpg](/wiki/images/0/0f/Electrophoresis082201.jpg)](/File%3AElectrophoresis082201.jpg)

1. 1kb ladder
2. pspA(Primer pspA f&r)
3. pspA(Primer pspA f-p&r-s)
4. negative control for pspA(Primer pspA f&r)
5. negative control for pspA(Primer pspA f-p&r-s)

#### Transformation

Kil

## August 23

#### Colony PCR

J23107-TatABCD
VF and VR were used for amplifying approximately 2800bp

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | DNA | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32.5 | 0.5 | 50 |

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 90sec
→25cycles

## August 24

#### Purification of PCR products

pspA(Primer pspA f-p&r-s)
84.1μg/mL
→See "August 22 -Colony PCR-"

#### Restrictive Digestion

| pspA | EcoR1 | Pst1 | BufferH | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |

| pspA | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |

at 37℃ for 1h

#### Gel extraction

* pspA(EcoR1, Pst1) 30.7μg/mL
* pspA(Xba1, Pst1) 44.0μg/mL

#### Restrictive Digestion

| pSB1C3(82.9μg/mL) | EcoR1 | Pst1 | BufferH | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |

→Gel extraction

#### Electrophoresis

[![Electrophoresis082401.jpg](/wiki/images/d/d9/Electrophoresis082401.jpg)](/File%3AElectrophoresis082401.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis082401.jpg "Enlarge")

1. 1kb ladder
2. J23107-TatABCD →See "August 23 -PCR-"
3. negative control for J23107-TatABCD
4. pSB1C3(EcoR1, Pst1)

#### Ligation

| pSB3C5(EcoR1, Pst1) | pspA(EcoR1, Pst1) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 1 | 5 | 3 | 9 |

16℃, overnight

## August 25

## August 26

## August 27

#### Ligation

| pSB1C3(EcoR1, Pst1) | pspA(EcoR1, Pst1) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 1 | 5 | 3 | 9 |

16℃, overnight

#### Colony PCR

Kil (1-7)

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→30cycles

[![0827c.jpg](/wiki/images/e/e7/0827c.jpg)](/File%3A0827c.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A0827c.jpg "Enlarge")

#### Purification of PCR products

torA-GFP →See "August 21 -PCR-"

#### Restrictive Digestion

| LacP(Backbone:pSB3C5) | Spe1 | Pst1 | BufferH | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |

at 37℃ for 1h
→Gel extraction

| torA-GFP | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |

at 37℃ for 1h
→Purification

#### Purification of PCR products

J23107-TatABCD
42.0μg/mL
→See "August 23 -PCR-"

#### Restrictive Digestion

| J23107-TatABCD | Spe1 | EcoR1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |

## August 28

#### Colony PCR

Kil (1-16)

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |

[![IMG 2207.jpg](/wiki/images/b/ba/IMG_2207.jpg)](/File%3AIMG_2207.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2207.jpg "Enlarge")

#### Transformation

* pspA-pSB1C3
* pspA-pSB3C5

#### Liquid culture

* Kil-3 →Miniprep 100.4μg/mL
* Kil-6
* Kil-7 →Miniprep 77.7μg/mL
* LacP-torA-GFP-1 →Miniprep 88.3μg/mL
* LacP-torA-GFP-2 →Miniprep 85.0μg/mL
* LacP-torA-GFP-3 →Miniprep +++

## August 29

#### Kil assay

After culturing for 18hr at 37℃, we eliminated the supernatant using a centrifuge, and diluted it until OD600=0.1. Then we dispensed it. The dispense volume was 3mL. We added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600.

| IPTG | 0 | 0.001mM | 0.01mM | 0.1mM | 1mM |
| --- | --- | --- | --- | --- | --- |
| 0.5h | 0.202 | 0.207 | 0.201 | 0.207 | 0.200 |
| 1h | 0.406 | 0.428 | 0.424 | 0.402 | 0.421 |
| 1.5h | 0.796 | 0.813 | 0.779 | 0.751 | 0.802 |
| 2h | 1.107 | 1.129 | 1.141 | 1.092 | 1.124 |
| 2.5h | 1.546 | 1.565 | 1.541 | 1.532 | 1.578 |
| 3h | 1.912 | 1.933 | 1.890 | 1.883 | 1.940 |
| 3.5h | 2.300 | 2.295 | 2.238 | 2.247 | 2.259 |
| 4h | 2.546 | 2.545 | 2.528 | 2.490 | 2.520 |
| 4.5h | 2.688 | 2.663 | 2.603 | 2.633 | 2.666 |
| 5h | 2.699 | 2.826 | 2.787 | 2.593 | 2.673 |
| 5.5h | 2.863 | 2.742 | 2.754 | 2.741 | 2.756 |
| 6h | 2.831 | 2.876 | 2.758 | 2.759 | 2.728 |
| 20h | 2.671 | 2.706 | 2.680 | 2.606 | 2.619 |

#### Liquid culture

* pspA-pSB1C3(1-3)
* pspA-pSB3C5(1-3)

#### Restrictive Digestion

| LacP-Kil-DT(134.7ng/μL) | EcoR1 | Spe1 | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 10 | 1 | 1 | 3 | 15 | 30 |

37℃, overnight

#### Colony PCR

* LacP-torA-GFP-DT (1-5)
* pspA-pSB1C3 (1-5)
* pspA-pSB3C5 (1-6)

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 40sec
→30cycles

#### Gel extraction

J23107-TatABCD(EcoR1, Pst1) -5.9μg/mL

[![0829.jpg](/wiki/images/8/84/0829.jpg)](/File%3A0829.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A0829.jpg "Enlarge")

## August 30

#### Ligation

| J23107-TatABCD(EcoR1, Spe1)22.2μg/mL | DT(EcoR1, Xba1)37.5μg/mL | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 10 | 1 | 9 | 20 |

at 16℃ for 1h

#### Transformation

J23107-TatABCD-DT (Amp resistance)

#### Electrophoresis

[![0830e.jpg](/wiki/images/0/01/0830e.jpg)](/File%3A0830e.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A0830e.jpg "Enlarge")

1. LacP-torA-GFP-DT-1
2. LacP-torA-GFP-DT-2
3. LacP-torA-GFP-DT-3
4. LacP-torA-GFP-DT-4
5. LacP-torA-GFP-DT-5
6. pspA-pSB1C3-1
7. pspA-pSB1C3-2
8. pspA-pSB1C3-3
9. pspA-pSB1C3-4
10.pspA-pSB1C3-5
11.pspA-pSB3C5-1
12.pspA-pSB3C5-2
13.pspA-pSB3C5-3
14.pspA-pSB3C5-4
15.pspA-pSB3C5-5
16.pspA-pSB3C5-6

#### Miniprep

* pspA-pSB3C5-1 176μg/mL
* pspA-pSB3C5-2 214μg/mL
* pspA-pSB3C5-3 461μg/mL
* pspA-pSB1C3-2 79μg/mL

#### Colony PCR

LacP-torA-GFP-DT (6-13)

| 2×Quick Taq | VF | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

94℃, 2min
94℃, 30sec
56℃, 30sec
68℃, 1min
→30cycles

#### Restrictive Digestion

| pspA-pSB1C3 | EcoR1 | Spe1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 15 | 0.5 | 0.5 | 5 | 0.5 | 28.5 | 50 |

at 37℃ for 1h

#### Electrophoresis

[![0830r.jpg](/wiki/images/5/5a/0830r.jpg)](/File%3A0830r.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A0830r.jpg "Enlarge")

1. 1kb ladder
3. pspA-pSB1C3(EcoR1, Spe1)

## August 31

#### Gel extraction

[![0831e.jpg](/wiki/images/0/0b/0831e.jpg)](/File%3A0831e.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A0831e.jpg "Enlarge")

pspA-pSB1C3(EcoR1, Spe1) 8.0μg/mL

#### Purification

LacP-GFP-DT -0.7μg/mL

#### Ligation

| pspA(EcoR1, Spe1) | DT(EcoR1, Xba1) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 25 | 1 | 13 | 39 |

at 16℃ for 1h

#### Transformation

pspA-DT (Amp resistance)

## September 2

#### Colony PCR

J23107-TatABCD-DT (1-8)

| 2×Quick Taq | VF | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 3min
→25cycles

pspA-DT (2-8)

| 2×Quick Taq | VF | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

Extension, 10sec
→25cycles

#### Restrictive Digestion

| LacP-torA-GFP-DT | EcoR1 | Spe1 | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 10 | 1 | 1 | 2 | 6 | 20 |

at 4℃ for overnight

#### Electrophoresis

[![0902.jpg](/wiki/images/0/08/0902.jpg)](/File%3A0902.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A0902.jpg "Enlarge")

1. 1kb ladder
3. J23107-TatABCD-DT-1
4. J23107-TatABCD-DT-3
5. J23107-TatABCD-DT-4
6. J23107-TatABCD-DT-5
7. J23107-TatABCD-DT-6
8. J23107-TatABCD-DT-7
9. J23107-TatABCD-DT-8

[![0902-2.jpg](/wiki/images/7/78/0902-2.jpg)](/File%3A0902-2.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A0902-2.jpg "Enlarge")

1. 1kb ladder
3. J23107-TatABCD-DT-1
4. J23107-TatABCD-DT-3
5. J23107-TatABCD-DT-4
6. J23107-TatABCD-DT-5
7. J23107-TatABCD-DT-6
8. J23107-TatABCD-DT-7
9. J23107-TatABCD-DT-8
11. LacP-pSB3C5(Spe1, Pst1)

[![0902-3.jpg](/wiki/images/5/57/0902-3.jpg)](/File%3A0902-3.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A0902-3.jpg "Enlarge")

1. 1kb ladder
3. pspA-DT-2
4. pspA-DT-3
5. pspA-DT-4
6. pspA-DT-5
7. pspA-DT-6
8. pspA-DT-7
9. pspA-DT-8

## September 3

#### PCR

TatABCD-DT

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | DNA | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50 |

#### Gel extraction

[![0903.jpg](/wiki/images/6/66/0903.jpg)](/File%3A0903.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A0903.jpg "Enlarge")

LacP-torA-GFP-DT →See "September 5 -Gel extraction-"

#### Restrictive Digestion

| torA-GFP-DT | Xba1 | Pst1 | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 20 | 2 | 2 | 4 | 12 | 40 |

#### Purification

J23107-TatABCD 29.6μg/mL 1.56(260/280) 1.60(260/230)

#### Electrophoresis

[![0903-2.jpg](/wiki/images/e/e6/0903-2.jpg)](/File%3A0903-2.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3A0903-2.jpg "Enlarge")

1. 1kb ladder
2. J23107-TatABCD

#### Liquid culture

* LacP-kil-DT(1)
* pspA-DT(4, 7)

#### PCR

J23107-TatABCD

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | DNA | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 29 | 4 | 50 |

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles

#### Gel extraction

[![IMG 2135.jpg](/wiki/images/f/fb/IMG_2135.jpg)](/File%3AIMG_2135.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2135.jpg "Enlarge")

LacP-torA-GFP-DT 31μg/mL 1.28(260/280) 0.49(260/230)
LacP-torA-GFP-DT 8μg/mL 1.32(260/280) 0.83(260/230)

#### Transformation

J23107-TatABCD (Amp resistance)
J23113-kil-DT (Amp resistance)
J23100-kil-DT (CP resistance)
J23101-kil-DT (CP resistance)
J23106-kil-DT (CP resistance)
J23115-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.
J23105-kil-DT (CP resistance)→We didn't transform because of lack of culture medium.

#### Electrophoresis

[![IMG 2136.jpg](/wiki/images/d/de/IMG_2136.jpg)](/File%3AIMG_2136.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2136.jpg "Enlarge")

1. 1kb ladder
2-7. LacP-torA-GFP-DT

#### Colony PCR

LacP-torA-GFP-DT (14-21)
J23113-kil-DT (Amp resistance, 1-4)
J23113-kil-DT (CP resistance, 1-4)

| 2×Quick Taq | primer-f | primer-r | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1min
→25cycles

## September 4

#### Liquid culture

* LacP-kil-DT(4, 5)(Amp resistance)
* pspA-DT(3, 6, 7)(Amp resistance)

#### PCR

We did PCR 10 more cycles to amplify products of PCR that we had done yesterday because we could not amplify J23107-TatABCD on September 3.
94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.5min
→30cycles

#### Electrophoresis

[![IMG 2137.jpg](/wiki/images/f/f7/IMG_2137.jpg)](/File%3AIMG_2137.jpg)
1-8. LacP-torA-GFP-DT

[![IMG 2138.jpg](/wiki/images/3/3c/IMG_2138.jpg)](/File%3AIMG_2138.jpg)
1-4. J23113-kil-DT (Amp resistance)
5-8. J23113-kil-DT (CP resistance)

#### Liquid culture

* LacP-torA-GFP-DT
* J23113-kil-DT
* LacP-kil-DT(with IPTG)
* LacP-kil-DT(without IPTG)

After 20 hour, we quantified OD600.

| plasmid | OD600 |
| --- | --- |
| LacP-kil-DT(with IPTG) | 2.116 |
| LacP-kil-DT(without IPTG) | 2.320 |

We cultured CP tolerance plasmid and give an antibiotic(Amp or Kan or none) after 2 hours.
We quantify OD600.

| time | 1 | 2 | 3 |
| --- | --- | --- | --- |
| 2hours | 0.131 | 0.100 | 0.606 |
| add an antibiotic | Amp | Kan | none |
| 7hours | 0.491 | 0.988 | 2.634 |
| 19hours | 2.253 | 0.815 | 2.742 |

## September 5

#### Colony PCR

J23107-TatABCD (1-7)

| buffer | 2mM dNTPs | 25mM MgSO4 | primer-f | primer-r | KOD Plus Neo | DW | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 1.25min
→30cycles

#### Gel extraction

by Kato
See "September 3 -Gel extraction-"
LacP-torA-GFP-DT 15.9μg/mL 1.29(260/280) 0.38(260/230)
LacP-torA-GFP-DT 17.2μg/mL 1.23(260/280) 0.48(260/230)

#### Miniprep

* J23113-kil-DT 85.7μg/mL 1.76(260/280) 1.97(260/230)
* LacP-torA-GFP-DT 126.6μg/mL 1.81(260/280) 2.09(260/230)
* LacP-kil-DT-4 62.5μg/mL 1.79(260/280) 1.99(260/230)
* LacP-kil-DT-5 56.0μg/mL 1.79(260/280) 2.01(260/230)
* pspA-DT-3 322μg/mL 1.45(260/280) 1.42(260/230)
* pspA-DT-6 147.6μg/mL 1.25(260/280) 1.37(260/230)
* pspA-DT-7 145.5μg/mL 1.24(260/280) 1.27(260/230)

#### Restrictive Digestion

| pspA-DT-6 | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |

at 37℃ for 1h

#### Electrophoresis

[![IMG 2139.jpg](/wiki/images/0/0b/IMG_2139.jpg)](/File%3AIMG_2139.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2139.jpg "Enlarge")

J23107-TatABCD (1-7)

#### Colony PCR

J23107-TatABCD (8-23)

| 2×Quick Taq | VF2 | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1.5min
→30cycles

#### Gel extraction

pspA-DT 10.2μg/mL 1.42(260/280) 0.76(260/230)

#### Ligation

| pspA-DT(Xba1, Pst1) | LacP(pSB3C5)(Spe1, Pst1) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 5 | 1 | 3 | 9 |

at 16℃

#### Electrophoresis

[![IMG 2140.jpg](/wiki/images/0/06/IMG_2140.jpg)](/File%3AIMG_2140.jpg)
LacP-torA-GFP-DT(8-15)

[![IMG 2141.jpg](/wiki/images/2/2a/IMG_2141.jpg)](/File%3AIMG_2141.jpg)
LacP-torA-GFP-DT(9-23)

#### Ligation

| LacP-torA-GFP-DT(EcoR1, Spe1) | DT 17.2μg/mL(Xba1, Pst1) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 15 | 25 | 20 | 60 |

#### Liquid culture

* J23107-TatABCD-8
* J23107-TatABCD-20
* LacP-torA-GFP-DT-21

## September 6

#### Miniprep

* J23107-TatABCD-8 82.4μg/mL 2.06(260/280) 2.95(260/230)
* J23107-TatABCD-20 102.9μg/mL 2.04(260/280) 2.94(260/230)

#### Restrictive Digestion

| J23107-TatABCD 102.9μg/mL | Spe1 | Pst1 | BufferH | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |

at 37℃ for 1h

#### Transformation

LacP-pspA-DT(pSB3C5)

| DNA | Competent cell | Total |
| --- | --- | --- |
| 2 | 20 | 22 |

#### Gel extraction

[![IMG 2142.jpg](/wiki/images/1/1a/IMG_2142.jpg)](/File%3AIMG_2142.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2142.jpg "Enlarge")

J23107-TatABCD(Spe1, Pst1)
→We failed gel extraction because we mistook steps of experiment.

#### Electrophoresis

[![IMG 2143.jpg](/wiki/images/9/9d/IMG_2143.jpg)](/File%3AIMG_2143.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2143.jpg "Enlarge")

LacP-torA-GFP-DT-pspA-DT

#### Restrictive Digestion

by Terasaka

| J23107-TatABCD | Spe1 | Pst1 | BufferH | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |

#### Gel extraction

[![IMG 2144.jpg](/wiki/images/4/4a/IMG_2144.jpg)](/File%3AIMG_2144.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2144.jpg "Enlarge")

J23107-TatABCD(Spe1, Pst1)
→See "September 7 -Gel extraction-"

## September 7

#### Liquid culture

* LacP-torA-GFP-DT-1 (CP resistance)
* LacP-torA-GFP-DT-21 (CP resistance)

preculture for 1 hour

#### Colony PCR

LacP-torA-GFP-DT (1-8)
LacP-pspA-DT (1-8)

| 2×Quick Taq | VF2 | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 1.5min
→30cycles

#### Restrictive Digestion

by Terasaka

| J23107-TatABCD | EcoR1 | Spe1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |

| GFP-DT | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 15 | 0.5 | 0.5 | 3 | 0.5 | 10.5 | 30 |

#### Electrophoresis

[![IMG 2145.jpg](/wiki/images/5/52/IMG_2145.jpg)](/File%3AIMG_2145.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2145.jpg "Enlarge")

① -⑧. LacP-pspA-DT
1-8. LacP-torA-GFP-DT

#### Gel extraction

[![IMG 2146.jpg](/wiki/images/9/9c/IMG_2146.jpg)](/File%3AIMG_2146.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2146.jpg "Enlarge")

* J23107-TatABCD(EcoR1, Spe1)
* J23107-TatABCD(Pst1, Spe1) ①57.6μg/mL ②23.4μg/mL
* GFP-DT(Xba1, Pst1)

#### Restrictive Digestion

| pspA-DT(145.5μg/mL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 20 | 0.5 | 0.5 | 3 | 0.5 | 5.5 | 30 |

37℃, overnight

#### Liquid culture

* LacP-torA-GFP-DT-7 (LB 2mL)

+IPTG 0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM, 0.3mM

* LacP-torA-GFP-DT-7 (LB 5mL)

## September 8

#### Check the effect of Amp

We used 5mL of the liquid culture medium with LacP-torA-GFP-DT-7.
→See "September 7 -Liquid culture-"
We diluted it with LB until OD600=0.444, and dispensed it.
The dispense volume was 1.3mL.
We added 0/13/130μL of Amp(50mg/mL) to it.

##### 5.5h after incubating at 37℃

| Amp | 0μL | 13μL | 130μL |
| --- | --- | --- | --- |
| OD600 | 2.457 | 2.696 | 0.310 |

#### Observation through a confocal microscope

We used the liquid culture media with LacP-torA-GFP-DT + 13μL of Amp(50mg/mL) + IPTG 0/0.1/0.15mM.
Their OD600 was 0.47.
3h after incubating at 37℃, we eliminated each supernatant using a centrifuge, and diluted them with LB to which Amp was added.
2.5h after incubating at 37℃, we observed them through a confocal microscope.
We failed to observe it.

#### Gel extraction

[![IMG 2147.jpg](/wiki/images/f/f4/IMG_2147.jpg)](/File%3AIMG_2147.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2147.jpg "Enlarge")

pspA-DT -5.5μg/mL

#### Restrictive Digestion

| pspA-DT-3(322μg/mL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 1 | 1 | 3 | 3 | 12 | 30 |

at 37℃ for 1h

## September 9

#### Restrictive Digestion

| J23107-TatABCD(102.9μg/mL) | EcoR1 | Spe1 | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 10 | 1 | 1 | 3 | 15 | 30 |

#### Electrophoresis

[![IMG 2148.jpg](/wiki/images/0/01/IMG_2148.jpg)](/File%3AIMG_2148.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2148.jpg "Enlarge")

1. J23107-TatABCD(EcoR1, Spe1)
2. J23107-TatABCD(before restriction)
3. pspA-DT(Xba1, Pst1)
4. pspA-DT(before restriction)

#### Gel extraction

[![IMG 2149.jpg](/wiki/images/7/77/IMG_2149.jpg)](/File%3AIMG_2149.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2149.jpg "Enlarge")

* J23107-TatABCD(EcoR1, Spe1) 26μg/mL
* pspA-DT(Xba1, Pst1) 199μg/mL

#### Liquid culture

* J23100/J23101/J23106-1
* pspA-DT-5 ×2
* LacP-torA-GFP-DT-21
* GFP generator-2
* LacP-pspA-DT-6

## September 10

#### Miniprep

* J23100-1 105.5μg/mL
* J23101-1 76.0μg/mL
* J23106-1 90.9μg/mL
* pspA-DT-5 ①104.1μg/mL ②80.0μg/mL
* LacP-torA-GFP-DT-21 79.6μg/mL
* GFP generator-2 84.1μg/mL
* LacP-pspA-DT-6 99.8μg/mL

#### Ligation

* J23107-TatABCD-DT

| J23107-TatABCD(EcoR1, Spe1) | DT(EcoR1, Xba1) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 10 | 5 | 7.5 | 22.5 |

* Negative control for J23107-TatABCD-DT

| DT(EcoR1, Xba1) | Ligation High Ver.2 | Total |
| --- | --- | --- |
| 5 | 5 | 10 |

* J23107-TatABCD-pspA-DT

| J23107-TatABCD(Spe1, Pst1) | pspA-DT(Xba1, Pst1) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 3 | 6 | 9 | 18 |

* Negative control for J23107-TatABCD-pspA-DT

| J23107-TatABCD(Spe1, Pst1) | Ligation High Ver.2 | Total |
| --- | --- | --- |
| 3 | 3 | 6 |

#### Restrictive Digestion

[![IMG 2151.jpg](/wiki/images/e/e3/IMG_2151.jpg)](/File%3AIMG_2151.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2151.jpg "Enlarge")

| DNA | Spe1 | Pst1 | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 10 | 30 |

DNA=J23100(105.5μg/mL), J23101(76.0μg/mL), J23106(90.9μg/mL)
at 37℃

#### Gel extraction

[![IMG 2152.jpg](/wiki/images/4/44/IMG_2152.jpg)](/File%3AIMG_2152.jpg)
[![IMG 2153.jpg](/wiki/images/0/06/IMG_2153.jpg)](/File%3AIMG_2153.jpg)
J23100 27.1μg/mL
J23101 50.2μg/mL
J23106 16.1μg/mL

#### Ligation

* J23100/J23101/J23106-kil-DT

| J23100/J23101/J23106(Spe1, Pst1) | kil-DT(Xba1, Pst1) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 1 | 5 | 3 | 9 |

* Negative control for J23100/J23101/J23106-kil-DT

| J23100/J23101/J23106(Spe1, Pst1) | MilliQ | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 1 | 5 | 3 | 9 |

#### Transformation

J23100/J23101/J23106-kil-DT

## September 11

#### Colony PCR

* J23107-TatABCD-DT (1-8)
* J23107-TatABCD-pspA-DT (1-8)

| 2×Quick Taq | VF2 | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 3.5min
→30cycles

#### Electrophoresis

* J23107-TatABCD-DT (1-8)

[![IMG 2156.jpg](/wiki/images/9/9a/IMG_2156.jpg)](/File%3AIMG_2156.jpg)

* J23107-TatABCD-pspA-DT (1-8)

[![IMG 2155.jpg](/wiki/images/1/17/IMG_2155.jpg)](/File%3AIMG_2155.jpg)

#### Restrictive Digestion

[![IMG 2154.jpg](/wiki/images/3/34/IMG_2154.jpg)](/File%3AIMG_2154.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2154.jpg "Enlarge")

| GFP generator(84.1μg/mL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 3 | 7 | 30 |

| LacP-torA-GFP-DT(85.0μg/mL) | Spe1 | Pst1 | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 10 | 30 |

| LacP-pspA-DT(99.8μg/mL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 3 | 7 | 30 |

#### Liquid culture

pSB1C3(self-ligation) ×5 in LB 3mLCP 3μL
When OD600=approximately 0.6, we added 0/30/60/150/300μL of Amp(50mg/mL) to each liquid culture medium.
1h and 5h after incubating at 37℃, we measured OD600.

|  | 1h | 5h |
| --- | --- | --- |
| 0 | -0.020 | 0.403 |
| 30 | -0.046 | 0.544 |
| 60 | 0.263 | 0.007 |
| 150 | 0.061 | 0.839 |
| 300 | 0.093 | 1.441 |

#### Ligation

| pSB3C5(EcoR1, Pst1) | pspA-DT(Xba1, Pst1) | J23107-TatABCD(EcoR1, Spe1) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- | --- |
| 1 | 3 | 5 | 4.5 | 13.5 |

4℃, overnight

#### Colony PCR

* J23100-kil-DT (1-6)
* J23101-kil-DT (1-7)
* J23106-kil-DT (1-2)

| 2×Quick Taq | VF | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 23 | 1 | 1 | 25 | 50 |

#### Gel extraction

[![IMG 2159.jpg](/wiki/images/a/a2/IMG_2159.jpg)](/File%3AIMG_2159.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2159.jpg "Enlarge")

* GFP generator (Xba1, Pst1) 14.1μg/mL
* LacP-torA-GFP-DT (Spe1, Pst1) 42.9μg/mL 1.13(260/280) 1.19(260/230)
* LacP-pspA-DT (Xba1, Pst1) 20.0μg/mL 1.01(260/280) 1.30(260/230)

#### Electrophoresis

[![IMG 2160.jpg](/wiki/images/f/f3/IMG_2160.jpg)](/File%3AIMG_2160.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2160.jpg "Enlarge")

* J23107-TatABCD-DT (1-8)
* J23106-kil-DT (1, 2)
* J23100-kil-DT (1-4)

#### Liquid culture

* LacP-torA-GFP-DT
* pSB1C3

#### Colony PCR

We did PCR 10 more cycle to amplify products of PCR on 9/11 of J23107-TatABCD-pspA-DT and J23107-TatABCD-DT.

## September 12

#### Colony PCR

J23107-TatABCD-pspA-DT (9-15)
94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→26cycles

#### Check the effect of Amp

We cultured pSB1C3 for overnight.
→See "September 7 -Liquid culture-"
We diluted it with LB, and dispensed it.
We added Amp(50mg/mL) to it until density of Amp was 1/10, 1/30, 1/50 .

| Amp | start | 5 hours |
| --- | --- | --- |
| 1/10 | 0.557 | 0.077 |
| 1/30 | 0.628 | 0.166 |
| 1/50 | 0.472 | 0.055 |
| 0 | 0.594 | 2.585 |

#### Electrophoresis

[![IMG 2162.jpg](/wiki/images/d/dd/IMG_2162.jpg)](/File%3AIMG_2162.jpg)

* J23107-TatABCD-pspA-DT

[![IMG 2161.jpg](/wiki/images/9/9c/IMG_2161.jpg)](/File%3AIMG_2161.jpg)

* J23101-kil-DT (1-7)

#### Restrictive Digestion

| pspA(pSB3C5) (106μg/mL) | EcoR1 | Pst1 | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 10 | 30 |

| pspA-DT-3 | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 3 | 7 | 30 |

| J23107-TatABCD (102.4μg/mL) | EcoR1 | Spe1 | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 10 | 30 |

#### Electrophoresis

[![IMG 2165.jpg](/wiki/images/2/21/IMG_2165.jpg)](/File%3AIMG_2165.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2165.jpg "Enlarge")

* pspA(pSB3C5)(EcoR1, Pst1)
* pspA-DT(Xba1, Pst1)
* J23107-TatABCD(EcoR1, Spe1)
* pSB1C3(EcoR1, Spe1)

#### Transformation

J23107-TatABCD-pspA-DT(pSB3C5)

| DNA | Competent cell | Total |
| --- | --- | --- |
| 2 | 20 | 22 |

on ice for 30 minites
heatshock at 42℃ for 1 hour
on ice 2 minites
then add SOC medium 200μl
preculture at 37℃ for 1 hour
incubate CP culture plate

#### Gel extraction

[![IMG 2166.jpg](/wiki/images/4/4f/IMG_2166.jpg)](/File%3AIMG_2166.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2166.jpg "Enlarge")

* pspA(pSB3C5)(EcoR1, Pst1) 26.8μg/mL 1.27(260/280) 0.32(260/230)
* pspA-DT(Xba1, Pst1) 26.1μg/mL 1.13(260/280) 0.55(260/230)
* J23107-TatABCD(EcoR1, Spe1) 1.5μg/mL 1.99(260/280) 0.06(260/230)

#### Ligation

| pSB3C5(EcoR1, Pst1) 26.8μg/mL | pspA-DT(Xba1, Pst1) 26.1μg/mL | J23107-TatABCD(EcoR1, Spe1) 1.5μg/mL | Ligation High Ver.2 | Total |
| --- | --- | --- | --- | --- |
| 1 | 3 | 6 | 5 | 15 |

| pSB3C5(EcoR1, Pst1) 26.8μg/mL | MilliQ | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 1 | 9 | 5 | 15 |

| LacP(pSB3C5)(Spe1, Pst1) 10μg/mL | GFP-DT(Xba1, Pst1) 7.0μg/mL | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 1 | 5 | 3 | 9 |

| LacP(pSB3C5)(Spe1, Pst1) 10μg/mL | MilliQ | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 1 | 5 | 3 | 9 |

4℃, overnight

#### Observation through a confocal microscope

left:*E.coli* sample stained with Hoechstunder and observed with 352nm wavelength
right:*E.coli* having Lacp-torA-GFP-DT and observed with 489nm wavelength

|  |
| --- |
| [![光る大腸菌.jpg](/wiki/images/3/3b/%E5%85%89%E3%82%8B%E5%A4%A7%E8%85%B8%E8%8F%8C.jpg)](/File%3A%E5%85%89%E3%82%8B%E5%A4%A7%E8%85%B8%E8%8F%8C.jpg) |

## September 13

#### Miniprep

* J23107-TatABCD-pspA-DT(pSB3C5)-1 130μg/mL 1.37(260/280) 0.79(260/230)

#### Restrictive Digestion

| J23107-TatABCD-pspA-DT(pSB3C5) (130μg/mL) | Spe1 | Pst1 | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 10 | 1 | 1 | 3 | 15 | 30 |

#### Transformation

* LacP-GFP generater

* LacP
* J23107-TatABCD-pspA-DT(pSB3C5)
* J23107-TatABCD

#### Colony PCR

J23107-TatABCD-pspA-DT

→See "September 14 -Colony PCR-"

## September 14

#### Colony PCR

[![IMG 2169.jpg](/wiki/images/3/32/IMG_2169.jpg)](/File%3AIMG_2169.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2169.jpg "Enlarge")

See "September 13 -Colony PCR-"
J23107-TatABCD-pspA-DT

#### Colony PCR

* J23107-TatABCD-pspA-DT (1-6)

| 2×Quick Taq | VF | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 23 | 1 | 1 | 25 | 50 |

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→30cycles

## September 15

#### Liquid culture

* J23107-TatABCD-pspA-DT-1
* J23107-TatABCD-pspA-DT-2
* J23107-TatABCD-pspA-DT-3

#### Miniprep

* J23107-TatABCD-pspA-DT-1 22.9μg/mL
* J23107-TatABCD-pspA-DT-2 51.8μg/mL 1.57(260/280) 1.01(260/230)
* J23107-TatABCD-pspA-DT-3 28.5μg/mL 1.57(260/280) 0.94(260/230)

#### Restrictive Digestion

| J23107-TatABCD-pspA-DT (51.8μg/mL) | EcoR1 | Spe1 | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 0.5 | 0.5 | 3 | 11 | 30 |

| LacP-torA-GFP-DT (79.6μg/mL) | EcoR1 | Xba1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 0.5 | 0.5 | 3 | 0.5 | 15.5 | 30 |

| LacP-kil-DT (56.0μg/mL) | EcoR1 | Xba1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 15 | 0.5 | 0.5 | 3 | 0.5 | 10.5 | 30 |

| J23107-TatABCD-pspA-DT (51.8μg/mL) | EcoR1 | Pst1 | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 0.5 | 0.5 | 3 | 11 | 30 |

37℃, 2 hours

#### Electrophoresis

[![IMG 2170.jpg](/wiki/images/6/6c/IMG_2170.jpg)](/File%3AIMG_2170.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2170.jpg "Enlarge")

①. 1kb ladder
②. J23107-TatABCD-pspA-DT
③. J23107-TatABCD-pspA-DT(EcoR1, Spe1)
④. J23107-TatABCD-pspA-DT(EcoR1, Pst1)
⑤. LacP-kil-DT
⑥. LacP-kil-DT(EcoR1, Xba1)
⑦. LacP-torA-GFP-DT
⑧. LacP-torA-GFP-DT(EcoR1, Xba1)

#### Gel extraction

[![IMG 2171.jpg](/wiki/images/b/ba/IMG_2171.jpg)](/File%3AIMG_2171.jpg)
[![IMG 2172.jpg](/wiki/images/2/2c/IMG_2172.jpg)](/File%3AIMG_2172.jpg)
①. 1kb ladder
②. J23107-TatABCD-pspA-DT(EcoR1, Spe1)
③. J23107-TatABCD-pspA-DT(EcoR1, Spe1)
④. J23107-TatABCD-pspA-DT(EcoR1, Pst1)
⑤. J23107-TatABCD-pspA-DT(EcoR1, Pst1)
⑥. 1kb ladder

* J23107-TatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mL 1.28(260/280) 0.54(260/230)
* J23107-TatABCD-pspA-DT(EcoR1, Pst1) 7.3μg/mL 1.55(260/280) 0.38(260/230)

#### Purification

* LacP-torA-GFP-DT(EcoR1, Xba1) 7.7μg/mL 1.41(260/280) 0.99(260/230)
* LacP-kil-DT(EcoR1, Xba1) →failed

#### Ligation

| LacP-torA-GFP-DT(EcoR1, Xba1) | J23107-TatABCD-pspA-DT(EcoR1, Spe1) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 4 | 10 | 7 | 21 |

| pSB1C3(EcoR1, Pst1) | J23107-TatABCD-pspA-DT(EcoR1, Pst1) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 4 | 10 | 7 | 21 |

at 16℃ for overnight

## September 16

#### Transformation

LacP-torA-GFP-DT-J23107-TatABCD-pspA-DT
J23107-TatABCD-pspA-DT(pSB1C3)

## September 17

#### Colony PCR

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)
J23107-TatABCD-pspA-DT(pSB1C3) (1-6)
J23107-kil-DT (6-10)

#### Restrictive Digestion

| LacP-torA-GFP-DT (126μg/mL) | EcoR1 | Xba1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 1 | 1 | 3 | 3 | 12 | 30 |

| LacP-torA-GFP-DT (126μg/mL) | Xba1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 3 | 1 | 3 | 3 | 20 | 30 |

#### Electrophoresis

[![IMG 2173.jpg](/wiki/images/e/ed/IMG_2173.jpg)](/File%3AIMG_2173.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2173.jpg "Enlarge")

A1-A6. J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT (1-6)
B1-B6. J23107-TatABCD-pspA-DT(pSB1C3) (1-6)

#### Liquid culture

* J23107-TatABCD-pspA-DT(pSB1C3)
* J23107-TatABCD-pspA-DT(pSB3C5)
* LacP-torA-GFP-DT(pSB3C5)

#### Electrophoresis

[![IMG 2174.jpg](/wiki/images/b/b7/IMG_2174.jpg)](/File%3AIMG_2174.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AIMG_2174.jpg "Enlarge")

lane1. 1kb ladder
lane2. J23101-kil-DT-6
lane3. J23101-kil-DT-7
lane4. J23101-kil-DT-8
lane5. J23101-kil-DT-10
lane6. LacP-torA-GFP-DT
lane7. LacP-torA-GFP-DT(EcoR1, Xba1)
lane8. LacP-torA-GFP-DT(Xba1)

#### Miniprep

* J23106-kil-DT 11.7μg/mL 1.93(260/280) 1.46(260/230)

## September 18

#### Gel extraction

* LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL 1.37(260/280) 0.82(260/230)

#### Ligation

| J23107-TatABCD-pspA-DT(EcoR1, Spe1) 14.2μg/mL | LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 8 | 1.5 | 9.5 | 19 |

| LacP-torA-GFP-DT(EcoR1, Xba1) 36.9μg/mL | Ligation High Ver.2 | Total |
| --- | --- | --- |
| 1.5 | 1.5 | 3 |

at 4℃ for 1 hour

#### Transformation

* J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT
* LacP-torA-GFP-DT(EcoR1, Xba1)

#### Observation through a confocal microscope

We made sample for observation through a confocal microscope.
We incubated sample with 0mM/0.5mM IPTG for 1.5 hours.
Then, we incubated onto medium without IPTG for 2.5 hours.

| IPTG(mM) | medium |
| --- | --- |
| 0 | LB |
| 0.1 | LB |
| 0.1 | LB with 0.1mM IPTG (positive control) |
| 0 | LB with 2 percent glucose |
| 0.1 | LB with 2 percent glucose |
| 0 | LB with 5 percent glucose |
| 0.1 | LB with 5 percent glucose |
| 0 | LB with 2 percent glucose (make medium new after incubate for 1.5 hours) |
| 0 | LB with 2 percent glucose (make medium new after incubate for 1.5 hours) |

#### Restrictive Digestion

| pSB4K5 | EcoR1 | Pst1 | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 10 | 1 | 1 | 3 | 15 | 30 |

| pSB4K5 | EcoR1 | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 3 | 0.5 | 1 | 5.5 | 30 |

| pSB4K5 | Pst1 | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 3 | 0.5 | 1 | 5.5 | 10 |

| LacP-torA-GFP-DT | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 1 | 1 | 3 | 3 | 12 | 30 |

| LacP-torA-GFP-DT | Xba1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 2 | 1 | 1 | 1 | 5 | 10 |

| LacP-torA-GFP-DT | Pst1 | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 2 | 1 | 1 | 6 | 10 |

37℃, 2 hours

#### Gel extraction

* LacP-torA-GFP-DT(Xba1, Pst1) 9.7μg/mL 1.34(260/280) 0.62(260/230)
* pSB4K5(EcoR1, Pst1) 9.6μg/mL 1.28(260/280) 0.32(260/230)

#### Miniprep

* J23107-TatABCD-psp-DT(pSB1C3) 78μg/mL
* J23107-TatABCD-psp-DT(pSB1C3) 48μg/mL
* LacP-torA-GFP-DT 29μg/mL
* J23101-kil-DT 25μg/mL

#### Restrictive Digestion

| J23101-Kil-DT 25µg/ml | BufferM | Xba1 | Pst1 | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 40 | 6 | 1 | 1 | 6 | 6 | 60 |

| J23101-Kil-DT 25µg/ml | BufferM | Xba1 | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 3 | 1 | 0.5 | 1 | 4.5 | 10 |

| J23101-Kil-DT 25µg/ml | BufferH | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 3 | 1 | 0.5 | 5.5 | 60 |

at 37℃ for 2 hours

#### Electrophoresis

[![Electrophoresis091801.jpg](/wiki/images/5/5b/Electrophoresis091801.jpg)](/File%3AElectrophoresis091801.jpg)

①. pSB4K5
②. pSB4K5(Pst1)
③. pSB4K5(EcoR1)
④. pSB4K5(EcoR1, Pst1)
⑤. LacP-torA-GFP-DT
⑥. LacP-torA-GFP-DT(Xba1)
⑦. LacP-torA-GFP-DT(Pst1)
⑧. LacP-torA-GFP-DT(Xba1, Pst1)

[![Electrophoresis091802.jpg](/wiki/images/7/7d/Electrophoresis091802.jpg)](/File%3AElectrophoresis091802.jpg)

①. 1kb ladder
②. J23101-Kil-DT(Xba1, Pst1)
③. J23101-Kil-DT(Xba1, Pst1)
④. J23101-Kil-DT(Xba1, Pst1)
⑤. J23101-Kil-DT(Xba1, Pst1)
⑥. 1kb ladder

#### Restrictive Digestion

| J23101-Kil-DT | BufferM | Xba1 | Pst1 | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 15 | 3 | 1 | 1 | 3 | 7 | 30 |

| J23101-Kil-DT | BufferM | Xba1 | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 3 | 1 | 0.5 | 1 | 4.5 | 10 |

| J23101-Kil-DT | BufferH | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 3 | 1 | 0.5 | 5.5 | 10 |

37℃

## September 19

#### Gel extraction

lane1. 1kb ladder
lane2. pSB4K5(EcoR1, Pst1)
lane3. pSB4K5(EcoR1, Pst1)
lane4. empty
lane5. LacP-torA-GFP-DT(Xba1, Pst1)
lane6. LacP-torA-GFP-DT(Xba1, Pst1)
[![GelEx0919.jpg](/wiki/images/4/44/GelEx0919.jpg)](/File%3AGelEx0919.jpg)
[![GelEx091901.jpg](/wiki/images/9/95/GelEx091901.jpg)](/File%3AGelEx091901.jpg)

#### Ligation

| LacP-torA-GFP-DT(Xba1,Pst1) 9.7μg/mL | pSB4K5(EcoR1,Pst1) 9.6μg/mL | J23107-TatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mL | Ligation High Ver.2 | Total |
| --- | --- | --- | --- | --- |
| 7 | 2 | 7 | 8 | 24μL |

| pSB4K5(EcoR1,Pst1) 9.6μg/mL | Ligation High Ver.2 | MilliQ | Total |
| --- | --- | --- | --- |
| 2 | 1 | 21 | 24μL |

incubate at 37℃ for 2h

#### Electrophoresis

[![CheckingElectrophoresis0919.jpg](/wiki/images/b/b3/CheckingElectrophoresis0919.jpg)](/File%3ACheckingElectrophoresis0919.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ACheckingElectrophoresis0919.jpg "Enlarge")

J23101-kil-DT (Xba1,Pst1), (Xba1), (Pst1)
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: (Xba1)
Lane5: (Pst1)
Lane6: (Xba1, Pst1)

#### Liquid Culture

We did liquid culture J23106-kil-DT(9/10, Amp)'s 6,7 at LB mediumAmp.

#### Transformation

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT
pSB4K5 negative control

#### Liquid culture

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT:1~5

#### Colony PCR

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT

| VF2 | VR | Quick tag | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 1 | 1 | 25 | 23 | 50 |

Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles

#### Restrictive Digestion

| LacP-kil-DT(4) 62.5μg/mL | Xba1 | Pst1 | 10xBSA | MilliQ | BufferM | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 10 | 1 | 1 | 3 | 12 | 3 | 30 |

| LacP-kil-DT(4) | EcoR1 | Pst1 | MilliQ | BufferH | Total |
| --- | --- | --- | --- | --- | --- |
| 10 | 1 | 1 | 15 | 3 | 30 |

| LacP-kil-DT | Xba1 | 10xBSA | MilliQ | BufferM | Total |
| --- | --- | --- | --- | --- | --- |
| 2 | 0.5 | 1 | 5.5 | 1 | 10 |

| LacP-kil-DT | EcoR1 | MilliQ | BufferH | Total |
| --- | --- | --- | --- | --- |
| 2 | 0.5 | 6.5 | 1 | 10 |

| LacP-kil-DT | Pst1 | MilliQ | BufferH | Total |
| --- | --- | --- | --- | --- |
| 2 | 0.5 | 6.5 | 1 | 10 |

at 37℃, 2 hours

#### Electrophoresis

[![Electrophoresis0919.jpg](/wiki/images/8/89/Electrophoresis0919.jpg)](/File%3AElectrophoresis0919.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis0919.jpg "Enlarge")

LacP-kil-DT
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: (EcoR1)
Lane5: (Xba1)
Lane6: (Pst1)
Lane7: (EcoR1, Pst1)
Lane8: (Xba1, Pst1)

[![Electrophoresis091901.jpg](/wiki/images/a/a7/Electrophoresis091901.jpg)](/File%3AElectrophoresis091901.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis091901.jpg "Enlarge")

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(1-5)
Lane1: ladder
Lane2: 1
Lane3: 2
Lane4: 3
Lane5: 4
Lane6: 5

#### Ligation

| LacP-torA-GFP-DT(Xba1,Pst1) 9.7μg/mL | pSB4K5(EcoR1,Pst1) 9.6μg/mL | J23107-TatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mL | Ligation High Ver.2 | Total |
| --- | --- | --- | --- | --- |
| 7 | 4 | 7 | 9 | 27μL |

(Negative control)

| pSB4K5(EcoR1,Pst1) 9.6μg/mL | Ligation High Ver.2 | MilliQ | Total |
| --- | --- | --- | --- |
| 4 | 9 | 14 | 27μL |

#### Restrictive Digestion

| torA-R9 101.8μg/mL | bufferH | MilliQ | EcoR1 | Pst1 | Total |
| --- | --- | --- | --- | --- | --- |
| 10 | 3 | 15 | 1 | 1 | 30 |

| torA-R9 101.8μg/mL | bufferH | MilliQ | EcoR1 | Pst1 | Total |
| --- | --- | --- | --- | --- | --- |
| 3 | 1 | 5.5 | 0.5 | 0 | 10 |

| torA-R9 101.8μg/mL | bufferH | MilliQ | EcoR1 | Pst1 | Total |
| --- | --- | --- | --- | --- | --- |
| 3 | 1 | 5.5 | 0 | 0.5 | 10 |

#### PCR

| torA-R9 | MilliQ | 10xPCR buffer for KOD plus neo | 2mM dNTP3 | 25mM MgSO4 | M13 | KOD plus neo | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 0.5 | 34 | 5 | 5 | 3 | 1.5 | 1 | 50 |

Predenature 94℃ 2min
Denature 98℃ 10sec
Annealing 60℃ 30sec
Extension 68℃ 20sec
→35cycles

#### Electrophoresis

[![CheckingElectropjoresis091901.jpg](/wiki/images/2/2f/CheckingElectropjoresis091901.jpg)](/File%3ACheckingElectropjoresis091901.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ACheckingElectropjoresis091901.jpg "Enlarge")

torA-R9 plasmid, (EcoR1), (Pst1), (EcoR1,Pst1)
Lane1: 1kb ladder
Lane2: empty
Lane3: plasmid
Lane4: EcoR1
Lane5: Pst1
Lane6: EcoR1,Pst1
Lane7: empty
Lane8: 1kb ladder

#### Liquid culture

LacP-TatA-GFP-DT (9/4)

#### PCR purification

torA-R9 68.0μg/mL

## September 20

#### Gel extraction

LacP-kil-DT (EcoR1,Pst1) (Xba1,Pst1)

#### Restrictive Digestion

| torA-R9 68.0μg/mL | bufferM | Spe1 | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 20 | 3 | 1 | 6 | 30 |

at 37℃, 2h

#### Ligation

| LacP-kil-DT(Xba1,Pst1) 12.4μg/mL | pSB1C3(EcoR1,Pst1) 34.2μg/mL | J23107-TatABCD-PsPA-DT(EcoR1,Spe1) 14.2μg/mL | Ligation High Ver.2 | Total |
| --- | --- | --- | --- | --- |
| 7 | 4 | 7 | 9 | 27μL |

16℃, 1h

(Negative control)

| pSB1C3(EcoR1,Pst1) | Ligation High Ver.2 | MilliQ | Total |
| --- | --- | --- | --- |
| 4 | 9 | 14 | 27μL |

#### Transformation

Using above Ligation and Negative control's product and torA-R9.

#### Miniprep

J23106-kil-DT 6; 12.0μg/mL, (260/280): 1.78, (260/230): 1.73
J23106-kil-DT 7; 13.6μg/mL, (260/280): 1.85, (260/230): 1.73

#### Restrictive Digestion

| GFP-DT 84.1μg/mL | bufferM | 10xBSA | BbaI | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 3 | 3 | 1 | 8 | 30 |

at 37℃, 2 hours

#### Colony PCR

We made J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT's 1~8 samples.

| Quick tag | Tat f primer | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 54℃ 30sec
Extension 68℃ 4min
→30cycles

[![Electrophoresis091902.jpg](/wiki/images/7/70/Electrophoresis091902.jpg)](/File%3AElectrophoresis091902.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis091902.jpg "Enlarge")

[![Electrophoresis091903.jpg](/wiki/images/8/8a/Electrophoresis091903.jpg)](/File%3AElectrophoresis091903.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis091903.jpg "Enlarge")

J23701-TatABCD-pspA-DT-LacP-torA-GFP-DT
Lane1: 1kb ladder
Lane2: 1
Lane3: 2
Lane4: 3
Lane5: 4
Lane6: 5
Lane7: 6
Lane8: 7
Lane9: 8
Lane10: 1kb ladder
Lane11: empty
Lane12: empty

→Bands didn't appear so we do PCR for 10 more cycles.

#### Liquid culture

We did liquid culture J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT's 1~4 samples.

#### Gel extraction

torA-R9 (EcoR1,Pst1): 31μg/mL, (280/280): 0.80, (280/230): 0.39
pSB1C3(EcoR1,Pst1): 22μg/mL, (280/280):1.33, (280/230):0.6
torA-R9 (Spe1): 56μg/mL, (280/280): 1.11, (280/230): 0.69
GFP-DT(Xba1): 45μg/mL, (280/280): 1.15, (280/230): 0.88

#### Ligation

| torA-R9 (EcoR1,Pst1) | pSB1C3(EcoR1,Pst1) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 8 | 2 | 5 | 15μL |

| torA-R9 (Spe1) | pSB1C3(Xba1) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 3 | 3 | 3 | 9μL |

at 16℃, 1hour

#### Colony PCR

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT (4k5)
numbering 9~15

#### Transformation

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT (4k5)
J23107-TatABCD-pspA-DT-LacP-torA-kil-DT(pSB1C3)
Negative control (pSB1C3)
Negative control(pSB4K5)
torA-R9(pSB1C3)

#### Liquid culture

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT: colony PCR(9~15)

| 2xQuick tag | VF | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

Predenature 94℃ 2min
Denature 94℃ 30sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles

#### PCR

| torA-R9-GFP-DT | MilliQ | KOD plus ver.2 buffer | dNTP3 | MgSO4 | M13 | VR | KOD plus | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 35 | 5 | 5 | 2 | 1.5 | 1.5 | 1 | 52 |

Predenature 94℃ 2min
Denature 94℃ 15sec
Annealing 55℃ 30sec
Extension 68℃ 5min
→30cycles

#### Miniprep

J23701-TatABCD-pspA-DT-LacP-torA-GFP-DT:2,3
2: 10.4μg/mL, (250/280): 1.45, (260/230): 0.95
3: 6.0μg/mL, (250/280): 1.32, (260/230): 0.79
(\*)100μL

##### Electrophoresis

[![Electrophoresis0920.jpg](/wiki/images/c/c2/Electrophoresis0920.jpg)](/File%3AElectrophoresis0920.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis0920.jpg "Enlarge")

torA-R9-GFP-DT(after PCR)

## September 21

#### Gel extraction

torA-R9-GFP-DT(after PCR): 10.3μg/mL, (260/280): 1.28, (220/230): 0.49
[![Electrophoresis0921.jpg](/wiki/images/4/48/Electrophoresis0921.jpg)](/File%3AElectrophoresis0921.jpg)
[![Electrophoresis092101.jpg](/wiki/images/d/df/Electrophoresis092101.jpg)](/File%3AElectrophoresis092101.jpg)

#### Electrophoresis

[![Electrophoresis092102.jpg](/wiki/images/6/67/Electrophoresis092102.jpg)](/File%3AElectrophoresis092102.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis092102.jpg "Enlarge")

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(kan): 9~15

#### Electrophoresis

[![Electrophoresis092103.jpg](/wiki/images/0/06/Electrophoresis092103.jpg)](/File%3AElectrophoresis092103.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis092103.jpg "Enlarge")

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT: 1,2
Lane1: empty
Lane2: empty
Lane3: 2
Lane4: 3
Lane5: 1kb ladder

#### Restrictive Digestion

| LacP-kil-DT(3) | Xba1 | Pst1 | 10xBSA | MilliQ | BufferM | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 7 | 3 | 30 |

| LacP-torA-GFP-DP(9/5) | Xba1 | Pst1 | 10xBSA | MilliQ | BufferM | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 7 | 3 | 30 |

| LacP-kil-DT | Xba1 | 10xBSA | MilliQ | BufferM | Total |
| --- | --- | --- | --- | --- | --- |
| 2 | 0.5 | 1 | 5.5 | 1 | 10 |

| LacP-torA-GFP-DP | Xba1 | 10xBSA | MilliQ | BufferM | Total |
| --- | --- | --- | --- | --- | --- |
| 2 | 0.5 | 1 | 5.5 | 1 | 10 |

| LacP-kil-DT | Pst1 | MilliQ | BufferH | Total |
| --- | --- | --- | --- | --- |
| 2 | 0.5 | 6.5 | 1 | 10 |

| LacP-torA-GFP-DP | Xba1 | Pst1 | MilliQ | BufferH | Total |
| --- | --- | --- | --- | --- | --- |
| 2 | 0.5 | 1 | 6.5 | 1 | 10 |

| J23107-TatABCD-pspA-DT | Spe1 | Pst1 | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 20 | 1 | 1 | 4 | 14 | 40 |

| J23107-TatABCD-pspA-DT | Spe1 | BufferM | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 2 | 0.5 | 1 | 6.5 | 10 |

| J23107-TatABCD-pspA-DT | Pst1 | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 2 | 0.5 | 1 | 6.5 | 10 |

at 37℃, 2hours

#### Electrophoresis

[![Electrophoresis092104.jpg](/wiki/images/3/39/Electrophoresis092104.jpg)](/File%3AElectrophoresis092104.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis092104.jpg "Enlarge")

lane1. 1kb ladder
lane2. empty
lane3. LacP-kil-DT plasmid
lane4. LacP-kil-DT(Xba1)
lane5. LacP-kil-DT(Pst1)
lane6. LacP-kil-DT(Xba1, Pst1)
lane7. empty
lane8. LacP-torA-GFP-DP plasmid
lane9. LacP-torA-GFP-DP(Xba1)
lane10. LacP-torA-GFP-DP(Pst1)
lane11. LacP-torA-GFP-DP(Xba1, Pst1)
lane12. 1kb ladder

[![Electrophoresis092105.jpg](/wiki/images/1/1d/Electrophoresis092105.jpg)](/File%3AElectrophoresis092105.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis092105.jpg "Enlarge")

lane1. 1kb ladder
lane2. empty
lane3. J23107-TatABCD-pspA-DT plasmid
lane4. J23107-TatABCD-pspA-DT(Spe1)
lane5. J23107-TatABCD-pspA-DT(Pst1)
lane6. J23107-TatABCD-pspA-DT(Spe1, Pst1)

#### Colony PCR

* torA-R9(pSB1C3) (1-4)

| 10×Buffer | 2mM dNTPs | 25mM MgSO4 | VF | VR | KOD Plus Neo | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 5 | 5 | 3 | 1.5 | 1.5 | 1 | 33 | 50 |

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 30sec
→30cycles

#### Electrophoresis

[![Electrophoresis092107.jpg](/wiki/images/b/b5/Electrophoresis092107.jpg)](/File%3AElectrophoresis092107.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis092107.jpg "Enlarge")

lane1. 1kb ladder
lane2. empty
lane3. torA-R9-1
lane4. torA-R9-2
lane5. torA-R9-3
lane6. torA-R9-4
lane7. empty
lane8. empty

## September 22

#### Miniprep

torA-R9(Amp)1: 113.7μg/mL, (260/280): 1.96, (280/230): 2.42
torA-R9(Amp)2: 97.8μg/mL, (260/280): 1.67, (280/230): 1.72
torA-R9(Amp)3: 104.2μg/mL, (260/280): 1.93, (280/230): 2.67
LacP-torA-GFP-DT 6: 37.4μg/mL, (260/280): 2.24, (280/230): 3.27

#### Electrophoresis

[![Electrophoresis092108.jpg](/wiki/images/d/d0/Electrophoresis092108.jpg)](/File%3AElectrophoresis092108.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis092108.jpg "Enlarge")

LaneA: J23107-TatABCD (9/6)
LaneB: J23107-TatABCD-pspA-DT pSB3C5 (9/13)
LaneC: J23107-TatABCD-pspA-DT 1C5 (9/15)
LaneD: J23107-TatABCD-pspA-DT (9/15)
LaneE: J23107-TatABCD-pspA-DT(pSB1C3) (9/18)
LaneF: J23107-TatABCD-pspA-DT(pSB3C5) (9/18)
LaneG: J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT:2 (9/20)
LaneH: J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT:3 (9/20)
LaneA~H are (EcoR1,Pst1)

#### Restrictive Digestion

| J23107-TatABCD-pspA-DT-LacPtorA-GFP-DT:2 | BufferH | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 15 | 3 | 1 | 12 | 30 |

| J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT:3 | BufferH | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 15 | 3 | 1 | 12 | 30 |

#### Restrictive Digestion

| LacP-torA-GFP-DT(37.4μg/mL) | BufferH | EcoR1 | Pst1 | Total |
| --- | --- | --- | --- | --- |
| 25 | 3 | 1 | 1 | 30 |

#### Electrophoresis

①: J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT:2
②: LacP-torA-GFP-DT(EcoR1,Pst1) (9/22)
③: J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT:3
[![Electrophoresis092202.jpg](/wiki/images/1/1c/Electrophoresis092202.jpg)](/File%3AElectrophoresis092202.jpg)
[![Electrophoresis092201.jpg](/wiki/images/8/8c/Electrophoresis092201.jpg)](/File%3AElectrophoresis092201.jpg)

#### Gel extraction

LacP-torA-GFP-DT(EcoR1,Pst1) 5.5μg/ml 1.21(260/280) 0.37(260/230)

#### Purification

LacP-torA-GFP-DT 78.4μg/ml 1.62(260/280) 1.03(260/230)

#### Restrictive Digestion

| J23107-TatABCD-pspA-DT(pSB1C3) 78μg/ml | BufferH | Spe1 | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 3 | 1 | 1 | 10 | 30 |

| J23107-TatABCD-pspA-DT(pSB1C3) 78μg/ml | BufferH | Spe1 | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 3 | 1 | 0.5 | 5.5 | 10 |

| J23107-TatABCD-pspA-DT(pSB1C3) 78μg/ml | BufferH | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 3 | 1 | 0.5 | 5.5 | 10 |

| LacP-torA-GFP-DT 78.4μg/ml | BufferM | BSA | Xba1 | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 15 | 3 | 3 | 1 | 1 | 7 | 30 |

| LacP-torA-GFP-DT 78.4μg/ml | BufferM | BSA | Xba1 | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 3 | 1 | 1 | 0.5 | 4.5 | 10 |

| LacP-torA-GFP-DT 78.4μg/ml | BufferH | Pst1 | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 3 | 1 | 0.5 | 5.5 | 10 |

at 37℃ for 2 hours

#### Electrophoresis

[![Electrophoresis092205.jpg](/wiki/images/0/02/Electrophoresis092205.jpg)](/File%3AElectrophoresis092205.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis092205.jpg "Enlarge")

Lane1: 1kb ladder
Lane2: empty
Lane3: J23107-TatABCD-pspA-DT(pSB1C3)
Lane4: J23107-TatABCD-pspA-DT(Spe1, Pst1)
Lane5: J23107-TatABCD-pspA-DT(Spe1)
Lane6: J23107-TatABCD-pspA-DT(Pst1) (9/18)
Lane7: empty
Lane8: LacP-torA-GFP-DT
Lane9: LacP-torA-GFP-DT(Xba1, Pst1)
Lane10: LacP-torA-GFP-DT(Xba1)
Lane11: LacP-torA-GFP-DT(Pst1)
Lane12: empty
Lane13: 1kb ladder

## September 23

#### Gel extraction

[![Electrophoresis092301.jpg](/wiki/images/7/77/Electrophoresis092301.jpg)](/File%3AElectrophoresis092301.jpg)
[![Electrophoresis092302.jpg](/wiki/images/7/79/Electrophoresis092302.jpg)](/File%3AElectrophoresis092302.jpg)

* LacP-torA-GFP-DT(EcoR1, Pst1)

lane1 1kb ladder
lane3 DNA
lane4 DNA
lane5 DNA
→failed

[![Electrophoresis092303.jpg](/wiki/images/b/be/Electrophoresis092303.jpg)](/File%3AElectrophoresis092303.jpg)
[![Electrophoresis092304.jpg](/wiki/images/1/14/Electrophoresis092304.jpg)](/File%3AElectrophoresis092304.jpg)
lane1 1kb ladder
lane3 J23107-TatABCD-pspA-DT(pSB1C3)(Spe1, Pst1)
lane5 LacP-torA-GFP-DT(Xba1, Pst1)
→failed

#### Restrictive Digestion

| LacP-torA-GFP-DT (78.4μg/mL) | Xba1 | Pst1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 3 | 7 | 30 |

| LacP-torA-GFP-DT (78.4μg/mL) | Xba1 | BufferM | BSA | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 3 | 0.5 | 1 | 1 | 4.5 | 10 |

| J23107-TatABCD-pspA-DT(pSB1C3) (78μg/mL) | Spe1 | Pst1 | BufferH | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 9 | 1 | 1 | 2 | 7 | 20 |

#### Ligation

| LacP-torA-GFP-DT(EcoR1, Pst1) (5.5μg/mL) | pSB4K5 (9.6μg/mL) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 8 | 4 | 6 | 18 |

at 16℃ for 1 hour

#### Liquid culture

* LacP-TatABCD-pspA-DT (2, 3, 5, 6)

#### Colony PCR

LacP-TatABCD-pspA-DT (1-6)

| 2×Quick Taq | VF2 | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 4min
→30cycles

#### Electrophoresis

[![Electrophoresis092305.jpg](/wiki/images/4/45/Electrophoresis092305.jpg)](/File%3AElectrophoresis092305.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis092305.jpg "Enlarge")

①, LacP-torA-GFP-DT
②, LacP-torA-GFP-DT(Xba1, Pst1)
③, LacP-torA-GFP-DT(Xba1)
④, LacP-torA-GFP-DT(Pst1)
⑤, J23107-TatABCD-pspA-DT(Spe1, Pst1)

#### Liquid culture

* LacP-torA-GFP-DT (4, 7)
* LacP-GFP generater (1-3)
* T7-6His-GFP-DT (1, 2)

#### Gel extraction

* LacP-torA-GFP-DT (Xba1, Pst1) 6.8μg/mL 1.27(260/280) 0.40(260/230)

[![Electrophoresis092306.jpg](/wiki/images/3/38/Electrophoresis092306.jpg)](/File%3AElectrophoresis092306.jpg)
[![Electrophoresis092307.jpg](/wiki/images/1/19/Electrophoresis092307.jpg)](/File%3AElectrophoresis092307.jpg)

#### Purification

* J23107-TatABCD-pspA-DT(Spe1, Pst1) 15.1μg/mL 1.49(260/280) 0.80(260/230)

#### Ligation

| J23107-TatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL) | LacP-torA-GFP-DT (Xba1, Pst1) (6.8μg/mL) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 10 | 4 | 7 | 21 |

| J23107-TatABCD-pspA-DT(Spe1, Pst1) (15.1μg/mL) | LacP-kil-DT (Xba1, Pst1) (12.4μg/mL) | Ligation High Ver.2 | Total |
| --- | --- | --- | --- |
| 8 | 2 | 5 | 15 |

at 16℃ for 1 hour

#### Transformation

LacP-torA-GFP-DT(pSB4K5)
J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT
J23107-TatABCD-pspA-DT-LacP-kil-DT

| DNA | competent cell | Total |
| --- | --- | --- |
| 2 | 10 | 12 |

preculture at 37℃ for 1 hour
culture at 37℃ for 16 hours

#### Electrophoresis

[![Electrophoresis092308.jpg](/wiki/images/4/4e/Electrophoresis092308.jpg)](/File%3AElectrophoresis092308.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis092308.jpg "Enlarge")

J23107-TatABCD-pspA-DT(pSB1C3) (1-6)
lane1 1kb ladder
lane2~7 DNA
lane8 1kb ladder

## September 24

#### Miniprep

* J23107-TatABCD-pspA-DT-2 139.4μg/ml 1.69(260/280) 1.83(260/230)
* J23107-TatABCD-pspA-DT-3 134.7μg/ml 1.61(260/280) 1.69(260/230)
* J23107-TatABCD-pspA-DT-5 139.3μg/ml 1.55(260/280) 1.45(260/230)
* J23107-TatABCD-pspA-DT-6 138.5μg/ml 1.58(260/280) 1.40(260/230)
* LacP-torA-GFP-DT-4
* LacP-torA-GFP-DT-7
* T7-6His-GFP-DT-1
* T7-6His-GFP-DT-2

#### Colony PCR

LacP-GFP generater (1-3)

| 2×Quick Taq | VF2 | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

[![Electrophoresis092309.jpg](/wiki/images/a/a7/Electrophoresis092309.jpg)](/File%3AElectrophoresis092309.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis092309.jpg "Enlarge")

94℃, 2min

94℃, 30sec
55℃, 30sec
68℃, 1min
→30cycles

①-③, LacP-GFP generater (1-3)

#### Liquid culture

* J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT (1-4)
* J23107-TatABCD-pspA-DT-LacP-kil-DT (1-3)

#### Colony PCR

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT (1-4)
J23107-TatABCD-pspA-DT-LacP-kil-DT (1-3)

| 2×Quick Taq | VF2 | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 5min
→30cycles

#### Electrophoresis

[![Electrophoresis0924.jpg](/wiki/images/c/cf/Electrophoresis0924.jpg)](/File%3AElectrophoresis0924.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis0924.jpg "Enlarge")

lane1, 1kb ladder
lane2-5, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (1-4)
lane6-8, J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (1-3)

## September 25

#### Liquid culture

* J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB4K5)-2

#### Miniprep

J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3)-3: 26.2μg/mL, (260/280): 1.50, (280/230): 0.65
J23107-TatABCD-pspA-DT-LacP-torA-GFP(pSB1C3)-3: 24.8μg/mL, (260/280): 1.56, (280/230): 0.76
LacP-GFP generater-1: 42.0μg/mL, (260/280): 1.58, (280/230): 0.94

#### Restrictive Digestion

| J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL) | EcoRl | Spel | buffer M | MiliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 10 | 30 |

| J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL) | EcoRl | buffer M | MiliQ | Total |
| --- | --- | --- | --- | --- |
| 2 | 0.5 | 1 | 6.5 | 10 |

| J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (26.2ng/µL) | Spel | buffer M | MiliQ | Total |
| --- | --- | --- | --- | --- |
| 2 | 0.5 | 1 | 6.5 | 10 |

| J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL) | EcoRl | Pstl | buffer H | MiliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 10 | 30 |

| J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL) | Pstl | buffer H | MiliQ | Total |
| --- | --- | --- | --- | --- |
| 2 | 0.5 | 1 | 6.5 | 10 |

| J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (24.8ng/µL) | EcoRl | buffer H | MiliQ | Total |
| --- | --- | --- | --- | --- |
| 2 | 0.5 | 1 | 6.5 | 10 |

at 37℃ for 2 hours

#### Electrophoresis

[![Electrophoresis092501.jpg](/wiki/images/c/c1/Electrophoresis092501.jpg)](/File%3AElectrophoresis092501.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis092501.jpg "Enlarge")

lane1, 1kb ladder
lane2, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)
lane3, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)
lane4, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl)
lane5, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (Spel)
lane6, J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3)
lane7, J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl, Pstl)
lane8, J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl)
lane9, J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (Pstl)
lane10, LacP-torA-GFP-DT(EcoR1,Xba1)(9/15)
lane11, LacP-torA-GFP-DT(EcoR1,Xba1)(9/17)
lane12, 1kb ladder

→The bands didn't appear, so we did one more electrophoresis.

[![Electrophoresis092502.jpg](/wiki/images/4/4b/Electrophoresis092502.jpg)](/File%3AElectrophoresis092502.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis092502.jpg "Enlarge")

lane1, 1kb ladder
lane2, LacP-torA-GFP-DT(EcoR1,Xba1)(9/17)
lane3, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)
lane4, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)
lane5, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl)
lane6, J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (Spel)

#### Liquid culture

* LacP-torA-GFP-DT(pSB4K5) (1-4)

#### Restrictive Digestion

| J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) | EcoRl | Spel | buffer M | MiliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 10 | 30 |

| J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) | EcoRl | Pstl | buffer H | MiliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 1 | 1 | 3 | 10 | 30 |

at 37℃ for 1 hour

#### Electrophoresis

J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3) (EcoRl, Spel)
J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3) (EcoRl, Pstl)
→The bands didn't appear.

#### Observation through a confocal microscope

① J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)
② J23107-TatABCD-pspA-DT-LacP-torA-GFP-DT(pSB1C3)
③ J23107-TatABCD-pspA-DT-LacP-kil-DT(pSB1C3)

→We observed nothing.

## October 12

**Transformation**

| conpetent cell(3/15) | J23107-TatABCD-PspA-DT (9/13) (pSB3C5) | J23107-TatABCD-PspA-DT(9/18)(pSB3C5) | LacP-torA-GFP-DT(9/10) | total |
| --- | --- | --- | --- | --- |
| 20μL | 2μL |  |  | 22μL |
| 20μL |  | 2μL |  | 22μL |
| 20μL |  |  | 2μL | 22μL |

preculture in SOC medium for 1 hour at 25 degree

**Checking Colony PCR(negative control)**

[![Electrophoresis1012.jpg](/wiki/images/f/f7/Electrophoresis1012.jpg)](/File%3AElectrophoresis1012.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis1012.jpg "Enlarge")

| Quick Taq | VF | VR | MilliQ | total |
| --- | --- | --- | --- | --- |
| 25μL | 1μL | 1μL | 23μL | 50μL |

94℃ 2min
94℃ 30sec
55℃ 30sec
68℃ 5min
30 cycles

->Electrophoresis

## October 15

**Restrictive Digestion & Electrophoresis**

[![Electrophoresis1015.jpg](/wiki/images/8/88/Electrophoresis1015.jpg)](/File%3AElectrophoresis1015.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis1015.jpg "Enlarge")

| pSB4C5 | Xba1 | Pst1 | BSA | BufferM | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 10μL | 1μL | 1μL | 3μL | 3μL | 12μL | 30μL |

at 37℃ for 1 hour

**Transformation**
retry of lab work that we did on 10/12

| J23107-TatABCD-PspA-DT | LacP-torA-GFP-DT | competent cell | total |
| --- | --- | --- | --- |
| 2μL |  | 20μL | 22μL |
|  | 2μL | 20μL | 22μL |

preculture in 100μL of SOC medium

## October 16

**Colony PCR**

* 4 colonies of J23107-TatABCD-PspA-DT(9/18)

| Quick Taq | VF2 | VR | MilliQ | total |
| --- | --- | --- | --- | --- |
| 25μL | 1μL | 1μL | 23μL | 50μL |

94℃ 2min
94℃ 30sec
55℃ 30sec
68℃ 4min
30 cycles

* 4 colonies of LacP-torA-GFP-DT(10/15)

| Quick Taq | VF2 | VR | MilliQ | total |
| --- | --- | --- | --- | --- |
| 25μL | 1μL | 1μL | 23μL | 50μL |

94℃ 2min
94℃ 30sec
55℃ 30sec
68℃ 1.5min
30 cycles

## October 17

**Electrophoresis**

[![](/wiki/images/9/97/Electrophoresis101701.jpg)](/File%3AElectrophoresis101701.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis101701.jpg "Enlarge")

fig.1

[![](/wiki/images/4/43/Electrophoresis101702.jpg)](/File%3AElectrophoresis101702.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis101702.jpg "Enlarge")

fig.2

[![](/wiki/images/b/b4/Electrophoresis101703.jpg)](/File%3AElectrophoresis101703.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis101703.jpg "Enlarge")

fig.3

[![](/wiki/images/7/73/Electrophoresis101704.jpg)](/File%3AElectrophoresis101704.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis101704.jpg "Enlarge")

fig.4

product of colony PCR on 10/16

* fig.1

lane\_1 : 1kb ladder
lane\_2-5 : J23107-TatABCD-PspA-DT
lane\_6-9 : LacP-torA-GFP-DT
lane\_10 : 1kb ladder

we retried this because something is wrong with 1 kb ladder

* fig.2

lane\_1 : 1kb ladder
lane\_2 : J23107-TatABCD-PspA-DT(lane\_3)

**Liquid culture**
J23017-TatABCD-PspA-DT(10/15)

**Colony PCR**

* 8 colonies of LacP-torA-GFP-DT(10/15)

| Quick Taq | VF2 | VR | MilliQ | total |
| --- | --- | --- | --- | --- |
| 25μL | 1μL | 1μL | 23μL | 50μL |

94℃ 2min
94℃ 30sec
55℃ 30sec
68℃ 1.5min
30 cycles

->Electrophoresis
we named 8 colonies [5]~[12] because on 10/16, we did colony PCR using 4 colonies([1]~[4]) of J23107-TatABCD-PspA-DT.

* fig.3

lane\_1 : 1kb ladder
lane\_2 : [5]
lane\_3 : [6]
lane\_4 : [7]
lane\_5 : [8]

* fig.4

lane\_1 : 1kb ladder
lane\_2 : [9]
lane\_3 : [10]
lane\_4 : [11]
lane\_5 : [12]

**Liquid culture**
[5] and [7]

## October 18

**Miniprep**

| gene | ng/μL |
| --- | --- |
| J23107-TatABCD-PspA-DT (10/17) | 134.0 |
| LacP-torA-GFP-DT [5] (10/17) | 94.7 |

## October 19

**Restrictive Digestion**

[![](/wiki/images/4/4d/Electrophoresis101901.jpg)](/File%3AElectrophoresis101901.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis101901.jpg "Enlarge")

fig.1

[![](/wiki/images/7/79/Electrophoresis101902.jpg)](/File%3AElectrophoresis101902.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis101902.jpg "Enlarge")

fig.2

[![](/wiki/images/6/6c/Electrophoresis101903.jpg)](/File%3AElectrophoresis101903.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis101903.jpg "Enlarge")

fig.3

* sample\_1

| J23107-TatABCD-PspA-DT | EcoR1 | Spe1 | BufferM | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 15μL | 1μL | 1μL | 3μL | 10μL | 30μL |

* sample\_2

| LacP-torA-GFP-DT | Xba1 | Pst1 | BSA | BufferM | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 10μL | 1μL | 1μL | 3μL | 3μL | 12μL | 30μL |

* sample\_3

| LacP-kil-DT | Xba1 | Pst1 | BSA | BufferM | MilliQ | total |
| --- | --- | --- | --- | --- | --- | --- |
| 12μL | 1μL | 1μL | 3μL | 3μL | 10μL | 30μL |

**Electrophoresis**
lane\_1 : 1kb ladder
lane\_2 : J23107-TatABCD-PspA-DT
lane\_3 : LacP-torA-GFP-DT
lane\_4 : LacP-kil-DT

**Restrictive Digestion**

| T7 | Spe1 | Pst1 | BufferH | MilliQ | total |
| --- | --- | --- | --- | --- | --- |
| 10μL | 1μL | 1μL | 2μL | 6μL | 20μL |

at 37℃ for 1 hour

**Eledtrophoresis**
lane\_1 : 1kb ladder
lane\_2 : sample 1
lane\_3 : blank
lane\_4 : sample 2
lane\_5 : blank
lane\_6 : sample 3
Though this figure did not indicate, there was a 1000 bp band that seems to be product of LacP-Kil-DT

## October 22

**Gel extraction**

[![](/wiki/images/c/c2/Electrophoresis102201.jpg)](/File%3AElectrophoresis102201.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis102201.jpg "Enlarge")

fig.1

[![](/wiki/images/4/4d/Electrophoresis102202.jpg)](/File%3AElectrophoresis102202.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis102202.jpg "Enlarge")

fig.2

[![](/wiki/images/e/e3/Electrophoresis102203.jpg)](/File%3AElectrophoresis102203.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis102203.jpg "Enlarge")

fig.3

[![](/wiki/images/d/d2/Electrophoresis102204.jpg)](/File%3AElectrophoresis102204.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis102204.jpg "Enlarge")

fig.4

[![](/wiki/images/a/a2/Electrophoresis102205.jpg)](/File%3AElectrophoresis102205.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis102205.jpg "Enlarge")

fig.5

[![](/wiki/images/9/9a/Electrophoresis102206.jpg)](/File%3AElectrophoresis102206.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis102206.jpg "Enlarge")

fig.6

* LacP-torA-GFP-DT
* LacP-kil-DT

| Gene | ng/µL | 260/280 | 260/230 |
| --- | --- | --- | --- |
| LacP-torA-GFP-DT | 4,4 | 2,02 | 0,22 |
| LacP-kil-DT | 3,8 | 1,54 | 0,11 |

**Restrictive Digestion**

| LacP-torA-GFP-DT(pSB3C5, 9/10) | EcoR1 | Pst1 | Buffer M | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 10µL | 1µL | 1µL | 2µL | 6µL | 20µL |

At 37℃ for 1 hour

**Electrophoresis**

* fig.1

Lane\_1:1kb ladder
Lane\_2:empty
Lane\_3:T7(S,P)

* fig.2

Lane\_1:1kb ladder
Lane\_2:LacP-torA-GFP-DT(E,P)

**Gel Extraction**

* fig.3, 4

Lane\_1:1kb ladder
Lane\_2:blank
Lane\_3:T7(S,P)

| Gene | ng/µL | 260/280 | 260/230 |
| --- | --- | --- | --- |
| T7(S,P) | 5,4 | 1,82 | 0,23 |

* fig.5, 6

Lane\_1:1kb ladder
Lane\_2: LacP-torA-GFP-DT(E,P)
Lane\_3:pSB4C5(X,P)

| Gene | ng/µL | 260/280 | 260/230 |
| --- | --- | --- | --- |
| LacP-torA-GFP-DT(E,P) | 4,9 | 1,66 | 0,28 |
| pSB4C5 | 23,9 | 1,88 | 0,88 |

**Ligation**

| LacP-torA-GFP-DT(X,P) | T7(S,P) | Ligation high Ver.2 | Total |
| --- | --- | --- | --- |
| 10µL | 2µL | 5,5µL | 17,5µL |

| LacP-torA-GFP-DT(E,P) | pSB4K5(E,P) | Ligation high Ver.2 | Total |
| --- | --- | --- | --- |
| 10µL | 2µL | 5,5µL | 17,5µL |

| LacP-torA-GFP-DT(X,P) | pSB4C5(X,P) | Ligation high Ver.2 | Total |
| --- | --- | --- | --- |
| 10µL | 2µL | 5,5µL | 17,5µL |

At 4℃ overnight

## October 23

**Transformation**

* T7-LacP-kil-DT
* LacP-torA-GFP-DT(pSB4K5)
* LacP-torA-GFP-DT(pSB4C5)

transformed competent cells were plated on two plates respectively.(plate a and plate b)

**Ligation**

| pSB4C5(X,P) | LacP-GFP-DT(X,P) | Ligation high Ver.2 | Total |
| --- | --- | --- | --- |
| 2µL | 10µL | 6µL | 18µL |

At 4℃ overnight

## October 24

**Colony PCR**

* T7-LacP-kil-DT(a1~a8)
* LacP-torA-GFP-DT(pSB4C5)(a1~a4,b1~b4)
* LacP-torA-GFP-DT(pSB4K5)(a1~a4,b1,b2)

| Quick Taq | VF2 | VR | MilliQ | Total |
| --- | --- | --- | --- | --- |
| 25µL | 1µL | 1µL | 23µL | 50µL |

94℃ 2min
94 30sec
55 30sec
68 1min
30cycles

**Transformation**

* LacP-GFP-DT(pSB4C5)

**Electrophoresis**
Products of colony PCR

[![](/wiki/images/b/b2/Electrophoresis102401.jpg)](/File%3AElectrophoresis102401.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis102401.jpg "Enlarge")

fig.1

* fig.1

Lane\_1 :1kb ladder
Lane\_2 :empty
Lane\_3 : T7-LacP-kil-DT(a1)
Lane\_4 : T7-LacP-kil-DT(a2)
Lane\_5 : LacP-torA-GFP-DT(pSB4C5)(a1)
Lane\_6 : LacP-torA-GFP-DT(pSB4C5)(a2)
Lane\_7 : LacP-torA-GFP-DT(pSB4C5)(b1)
Lane\_8 : LacP-torA-GFP-DT(pSB4C5)(b2)
Lane\_9 : LacP-torA-GFP-DT(pSB4K5)(a1)
Lane\_10 : LacP-torA-GFP-DT(pSB4K5)(a2)
Lane\_11 : LacP-torA-GFP-DT(pSB4K5)(b1)
Lane\_12 : LacP-torA-GFP-DT(pSB4K5)(b2)

[![](/wiki/images/d/d4/Electrophoresis102402.jpg)](/File%3AElectrophoresis102402.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AElectrophoresis102402.jpg "Enlarge")

fig.2

* fig.2

Lane\_1 :1kb ladder
Lane\_2 : T7-LacP-kil-DT(a3)
Lane\_3 : T7-LacP-kil-DT(a4)
Lane\_4 : T7-LacP-kil-DT(a5)
Lane\_5 : T7-LacP-kil-DT(a6)
Lane\_6 : T7-LacP-kil-DT(a7)
Lane\_7 : T7-LacP-kil-DT(a8)
Lane\_8 : LacP-torA-GFP-DT(pSB4K5)(a3)
Lane\_9 : LacP-torA-GFP-DT(pSB4K5)(a4)
Lane\_10 :1kb ladder

**Liquid culture**

* LacP-torA-GFP-DT(pSB4C5)(a1,a2)
* T7-LacP-kil-DT(a6)

## October 25

**Observation through a confocal microscope**

* LacP-torA-GFP-DT(pSB4C5)

We observed nothing.

# ![Kyoto GoldenGateAssemblyNotebook.png](/wiki/images/a/ae/Kyoto_GoldenGateAssemblyNotebook.png)

## September 6

#### PCR and Electrophoresis assay

[![Epa001.jpg](/wiki/images/f/fb/Epa001.jpg)](/File%3AEpa001.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AEpa001.jpg "Enlarge")

①psB1K3
②lacI
③GFP
④GFP
⑤RFP
⑥RFP
⑦CFP
⑧DT

| Quick Taq | primer F | Primer R | DNA(①-⑧) | milliQ | total |
| --- | --- | --- | --- | --- | --- |
| 12.5 | 0.5 | 0.5 | 1 | 10.5 | 25 |

94℃ 2min, (94℃ 30sec, 50℃ 30sec)x25cycles, 68℃ 50sec

## September 7

#### PCR and Electrophoresis assay of psB1K3

[![Epa002.jpg](/wiki/images/c/c8/Epa002.jpg)](/File%3AEpa002.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AEpa002.jpg "Enlarge")

| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(psB1K3) | KOD plus | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min

## September 10

#### PCR and Electrophoresis assay of CFP

[![Epa002.jpg](/wiki/images/c/c8/Epa002.jpg)](/File%3AEpa002.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AEpa002.jpg "Enlarge")

| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(CFP) | KOD plus | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |

94℃ 2min, (94℃ 15sec, 48℃ 30sec)x30cycles, 68℃ 45sec

#### PCR and Electrophoresis assay of lacI

[![Epa003.jpg](/wiki/images/2/25/Epa003.jpg)](/File%3AEpa003.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AEpa003.jpg "Enlarge")

| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(lacI) | KOD plus | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 30sec

## PCR

①PCR product of psB1K3
②psB1K3
③psB1C3
④psB1C3
⑤PCR product of CFP

| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(①-⑤) | KOD plus | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min20sec

## September 11

#### PCR and Electrophoresis assay

[![Epa004.jpg](/wiki/images/9/92/Epa004.jpg)](/File%3AEpa004.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AEpa004.jpg "Enlarge")

①DT
②PCR product of DT
③PCR product of CFP
④CFP(2011 plate)

| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(①-④) | KOD plus | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |

94℃ 2min, (94℃ 15sec, 48℃ 30sec)x30cycles, 68℃ 1min

#### PCR and Electrophoresis assay

[![Epa005.jpg](/wiki/images/b/b9/Epa005.jpg)](/File%3AEpa005.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AEpa005.jpg "Enlarge")

①DT 92.1ng/µl
②DT 28.0ng/µl
③DT

| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(①-③) | KOD plus | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |

94℃ 2min, (94℃ 15sec, 50℃ 30sec)x30cycles, 68℃ 30sec

## September 12

#### Purification of PCR product

psB1K3(9/10④) 55µg/ml
lacI(9/10) 90µg/ml
GFP(9/6) 42µg/ml
RFP(9/6) 71µg/ml
DT(9/11) 81µg/ml
GFP 37µg/ml
RFP 57µg/ml

#### Golden Gate Assembly

| psB1K3 | lacI | GFP(2'-3) | RFP(3'-5) | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1.8 | 0.1 | 1.0 | 0.6 | 0.1 | 1.5 | 0.15 | 1.0 | 1.0 | 8.15 | 15 |

| psB1K3 | lacI | GFP(2'-5) | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1.8 | 0.1 | 1.0 | 0.1 | 1.5 | 0.15 | 1.0 | 1.0 | 8.75 | 15 |

| psB1K3 | lacI | GFP(2'-3) | RFP(3'-5) | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0 | 0.2 | 1.0 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 6.95 | 15 |

| psB1K3 | lacI | GFP(2'-3) | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0 | 0.2 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 7.95 | 15 |

#### Transformation

①lacI-GFP(2'-3)-RFP(3'-5)-DT
②lacI-GFP(2'-5)-DT
③lacI-GFP(2'-3)-RFP(3'-5)-DT
④lacI-GFP(2'-5)-DT

## September 13

#### Transformation

①lacI-GFP(2'-3)-RFP(3'-5)-DT
②lacI-GFP(2'-5)-DT
③lacI-GFP(2'-3)-RFP(3'-5)-DT
④lacI-GFP(2'-5)-DT

## September 14

#### Liquid Culture

①lacI-GFP(2'-3)-RFP(3'-5)-DT
②lacI-GFP(2'-5)-DT
③lacI-GFP(2'-3)-RFP(3'-5)-DT
④lacI-GFP(2'-5)-DT

## September 15

#### Miniprep

①lacI-GFP(2'-3)-RFP(3'-5)-DT
②lacI-GFP(2'-5)-DT
③lacI-GFP(2'-3)-RFP(3'-5)-DT
④lacI-GFP(2'-5)-DT

①4.0 µg/ml
②17 µg/ml
③15 µg/ml
④8.0 µg/ml

#### Restriction Enzyme Digestion

①lacI-GFP(2'-3)-RFP(3'-5)-DT
②lacI-GFP(2'-5)-DT
③lacI-GFP(2'-3)-RFP(3'-5)-DT
④lacI-GFP(2'-5)-DT

| DNA(①-④) | 10x NEB Bufer3 | BSA | EcoR1 | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 9 | 2 | 0.5 | 0.5 | 8 | 20 |

## September 17

#### Electrophoresis assay

[![Epa006.jpg](/wiki/images/4/42/Epa006.jpg)](/File%3AEpa006.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AEpa006.jpg "Enlarge")

①lacI-GFP(2'-3)-RFP(3'-5)-DT
②lacI-GFP(2'-5)-DT
③lacI-GFP(2'-3)-RFP(3'-5)-DT
④lacI-GFP(2'-5)-DT

## September 18

#### Restriction Enzyme Digestion

③lacI-GFP(2'-3)-RFP(3'-5)-DT
④lacI-GFP(2'-5)-DT

| DNA(③,④) | 10x H Bufer | BSA | EcoR1 | MilliQ | Total |
| --- | --- | --- | --- | --- | --- |
| 15 | 3 | 0.5 | 0.5 | 11 | 30 |

#### Colony PCR

[![Epa007.jpg](/wiki/images/9/95/Epa007.jpg)](/File%3AEpa007.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AEpa007.jpg "Enlarge")

①lacI-GFP(2'-3)-RFP(3'-5)-DT
②lacI-GFP(2'-5)-DT
③lacI-GFP(2'-3)-RFP(3'-5)-DT
④lacI-GFP(2'-5)-DT

| 2x Quick Tag Dye Mix | VF primer | VR primer | MiliQ | Total |
| --- | --- | --- | --- | --- |
| 25 | 1 | 1 | 23 | 50 |

## September 19

#### DpnI Restriction Enzyme Digestion of psB1K3

| psB1K3 | DpnI | Total |
| --- | --- | --- |
| 10 | 0.2 | 10.2 |

37℃ 1h incubate

#### Ligation of psB1K3

| psB1K3 | Ligation High | MiliQ | TH Kinase | Total |
| --- | --- | --- | --- | --- |
| 2 | 5 | 7 | 7 | 15 |

16.0℃　1.5h

## September 20

#### Transformation of psB1K3

## September 21

#### PCR of psB3C5 and Electrophoresis assay

[![Epa008.jpg](/wiki/images/c/cc/Epa008.jpg)](/File%3AEpa008.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AEpa008.jpg "Enlarge")

| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(psB3C5) | KOD plus | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min

#### PCR and Electrophoresis assay

[![Epa009.jpg](/wiki/images/5/56/Epa009.jpg)](/File%3AEpa009.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AEpa009.jpg "Enlarge")

①psB3C5
②psB1K3

| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(①,②) | KOD plus | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5.0 | 5.0 | 2.0 | 1.5 | 1.5 | 1.0 | 1.0 | 33 | 50 |

94℃ 2min, (94℃ 15sec, 58℃ 30sec, 68℃ 2min)x30cycles

#### PCR of DT and Electrophoresis assay

[![Epa011.jpg](/wiki/images/4/41/Epa011.jpg)](/File%3AEpa011.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AEpa011.jpg "Enlarge")

| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(DT) | KOD plus | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5.0 | 5.0 | 2.0 | 1.5 | 1.5 | 1.0 | 1.0 | 33 | 50 |

94℃ 2min, (94℃ 15sec, 50℃ 30sec, 68℃ 30sec)x30cycles

## September 22

#### Colony PCR (Colony plated in September 20)

[![Epa012.jpg](/wiki/images/e/e1/Epa012.jpg)](/File%3AEpa012.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AEpa012.jpg "Enlarge")

| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA | KOD plus | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 5.0 | 5.0 | 2.0 | 1.5 | 1.5 | 1.0 | 1.0 | 33 | 50 |

94℃ 2min, (94℃ 15sec, 58℃ 30sec, 68℃ 2min20sec)x25cycles

#### PCR of Primers and Electrophoresis assay

[![Epa013.jpg](/wiki/images/c/c2/Epa013.jpg)](/File%3AEpa013.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3AEpa013.jpg "Enlarge")

①J23106(1'-2)
②J23106(2'-3)
③J23106(3'-4)
④J23106(4'-5)
⑤J23106(3'-5)
⑥J23106(2'-5)
⑦J23106(1'-5)
⑧araC(1'-6)
⑨araC(5'-6)

| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA | KOD plus | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |

94℃ 2min, (94℃ 15sec, 56℃ 30sec)x30cycles, 68℃ 20sec

①J23106(1'-2) 60µg/ml
②J23106(2'-3) 63µg/ml
③J23106(3'-4) 48µg/ml
④J23106(4'-5) 60µg/ml
⑤J23106(3'-5) 62µg/ml
⑥J23106(2'-5) 40µg/ml
⑦J23106(1'-5) 29µg/ml
⑧araC(1'-6) 60µg/ml
⑨araC(5'-6) 62µg/ml

#### Ligation

4 promoters

| psB3C5 | ①J23106 (1'-2) | ②J23106 (2'-3) | ③J23106 (3'-4) | ④J23106 (4'-5) | ⑨araC (5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0 | 2.0 | 2.0 | 2.5 | 2.0 | 2.0 | 3.0 | 3.0 | 1.5 | 0.15 | 1.0 | 1.0 | 7.85 | 30 |

3 promoters

| psB3C5 | ①J23106 (1'-2) | ②J23106 (2'-3) | ⑤J23106 (3'-5) | ⑨araC (5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0 | 2.0 | 2.0 | 2.5 | 2.0 | 3.0 | 3.0 | 1.5 | 0.15 | 1.0 | 1.0 | 9.85 | 30 |

2 promoters

| psB3C5 | ①J23106(1'-2) | ⑥J23106(2'-5) | ⑨araC(5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0 | 2.0 | 2.5 | 2.0 | 3.0 | 3.0 | 1.5 | 0.15 | 1.0 | 11.85 | 30 |

1 promoter

| psB3C5 | ⑦J23106(1'-5) | ⑨araC(5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0 | 2.5 | 2.0 | 3.0 | 3.0 | 1.5 | 0.15 | 1.0 | 1.0 | 13.85 | 30 |

0 promoter

| psB3C5 | ⑨araC(5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0 | 2.0 | 3.0 | 3.0 | 1.5 | 0.15 | 1.0 | 1.0 | 16.35 | 30 |

## September 23

#### Transformation of product of ligation(9/22)

## September 24

#### Transformation of product of ligation(9/22)

## September 25

#### Liquid Culture

colony1,2,3 of plate2,3

#### Ligation

4 promoters

| psB3C5 | ① J23106 (1'-2) | ② J23106 (2'-3) | ③ J23106 (3'-4) | ④ J23106 (4'-5) | ⑨ araC (5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0 | 0.2 | 0.3 | 0.2 | 0.2 | 0.2 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 7.05 | 15 |

3 promoters

| psB3C5 | ①J23106 (1'-2) | ②J23106 (2'-3) | ⑤J23106 (3'-5) | ⑨araC (5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0 | 0.2 | 0.3 | 0.2 | 0.2 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 7.25 | 15 |

2 promoters

| psB3C5 | ①J23106 (1'-2) | ⑥J23106 (2'-5) | ⑨araC (5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0 | 0.2 | 0.3 | 0.2 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 7.45 | 15 |

1 promoter

| psB3C5 | ⑦J23106 (1'-5) | ⑨araC (5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0 | 0.6 | 0.2 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 7.35 | 15 |

0 promoter

| psB3C5 | ⑨araC (5'-6) | GFP | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 2.0 | 0.2 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 7.95 | 15 |

(37℃ 3min, 16℃ 4min)x25cycles,

#### Transformation of product of ligation(9/25)

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