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Revision as of 09:25, 28 October 2017

MainPage

Notebook and Methods

Notebook

2017-04-24 - 2017-04-30

Generally Labwork

Superior experiment: Ordering sequences

Procedure:

  • Ordering via IDT
    • NodC, NodB, CDApuc and ChiA

    2017-05-01 - 2017-05-07

    Generelly Labwork

    Superior experiment: Cloning via GoldenBraid assembly

    Procedure:

  • GoldenBraid assembly
    • Cloning NodC, NodB, CDApuc and ChiA into the pUPD vector

    2017-05-29 - 2017-06-04

    Chitinase A1

    Superior experiment: Cloning chiA into pSB1C3

    Procedure:

  • Cloning chiA into pSB1C3
    • Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
    • Dephosphorylating the backbone pSB1C3
    • Ligation with T4 Ligase

    Chitin Synthase

    Superior experiment: Cloning nodC into pSB1K3

    Procedure:

  • Cloning nodC into pSB1K3
    • Digesting pUPD-Anderson-nodC and pSB1K3 with Xba1 and Pst1
    • Dephosphorylating the backbone pSB1K3
    • Ligation with T4 Ligase
  • Transformation and evaluation
    • Transforming pSB1K3-Anderson-nodC into Top10
    • Over-night cultures of pSB1K3-Anderson-nodC
    • Plasmid extraction via Mini-prep-kit

    Chitin Deacetylases

    Superior experiment: Cloning nodB into pSB1K3, transformation into Top10, evaluation

    Procedure:

  • Cloning nodB into pSB1K3
    • Digesting pUPD-Anderson-nodB and pSB1K3 with Xba1 and Pst1
    • Dephosphorylating the backbone pSB1K3
    • Ligation with T4 Ligase
  • Transformation and evaluation
    • Transforming pSB1K3-Anderson-nodB and pUPD-Anderson-CDApuc into Top10 via heat-shock
    • ColonyPCR of pSB1K3-Anderson-nodB and pUPD-Anderson-CDApuc

    2017-06-05 - 2017-06-11

    Chitinase A1

    Superior experiment: Cloning AraC-chiA into pSB1C3, transformation into Top10, evaluation

    Procedure:

  • Cloning chiA into pSB1C3-AraC
    • Digesting pUPD-Anderson-chiA with Nhe1 and Pst1, pSB1C3-AraC with Spe1 and Pst1
    • Dephosphorylating the backbone pSB1C3-AraC
    • Ligation with T4 Ligase
  • Transformation and evaluation
    • Transforming pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA into Top10 via heat-shock
    • colonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA
    • Gelelectrophoresis
    • Over-night cultures of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA
    • Plasmid extraction via Mini-prep-kit and sequencing

    Chitin Synthase

    Superior experiment: Cloning nodC into pSB1C3 and pSB1C3-AraC

    Procedure:

  • Cloning nodC into pSB1C3-Anderson and pSB1C3-AraC
    • Digesting pUPD-Anderson-nodC and pSB1C3 with Xba1 and Pst1
    • Dephosphorylating the backbone pSB1K3
    • Ligation with T4 Ligase
  • Transformation and evaluation
    • Transforming pSB1K3-Anderson-nodC into Top10
    • ColonyPCR
    • Gelelectrophoresis
    • Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
    • Plasmid extraction via Mini-prep-kit

    Chitin Deacetylases

    Superior experiment: Evaluation of transformation, retransformation

    Procedure:

  • Evaluation
    • Gelelectrophoresis of colonyPCR product
    • Repeating the colonyPCR of pSB1C3-Anderson-nodB and pUPD-Anderson-CDApuc
  • Retransformation
    • Retransformation of pSB1C3-Anderson-nodB and pUPD-Anderson-CDApuc into Top10
  • Sequencing
    • Over-night cultures of pSB1C3-Anderson-nodB
    • Plasmid extraction via Mini-prep-kit and sequencing
      • No insert was found

    2017-06-12 - 2017-06-18

    Chitinase A1

    Superior experiment: Cloning chiA into pSB1C3

    Procedure:

  • Cloning pSB1C3-Anderson-chiA
    • Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
    • Dephosphorylating the backbone pSB1C3
    • Ligation with T4 Ligase
  • Transformation
    • Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock

    Chitin Synthase

    Superior experiment: Sequencing of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC

    Procedure:

    • Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
    • Plasmid extraction via Mini-prep-kit and sequencing
  • Glycerol stocks of pSB1C3-Anderson-nodC
    • Glycerol stocks were made of positive pSB1C3-Anderson-nodC colonies
  • Glycerol stocks of pSB1C3-AraC-nodC
    • Glycerol stocks were made of positive pSB1C3-AraC-nodC colonies

    Chitin Deacetylases

    Superior experiment: Cloning nodB in pSB1C3, transformation into Top10

    Procedure:

  • Cloning nodB into pSB1K3
    • Digesting pUPD-Anderson-nodB and pSB1C3 with Xba1 and Pst1
    • Dephosphorylating the backbone pSB1C3
    • PCR clean-up to
    • Ligation with T4 Ligase
  • Transformation and evaluation
    • Transforming pSB1C3-Anderson-nodB into Top10 via heat-shock
      • No colony was successful
    • Cloning nodB into pSB1K3
    • Digesting pUPD-Anderson-nodB and pSB1C3 with Xba1 and Pst1
    • Dephosphorylating the backbone pSB1C3
    • PCR clean-up to
    • Ligation with T4 Ligase
  • Transformation and evaluation
    • Transforming pSB1C3-Anderson-nodB into Top10 via heat-shock

    2017-06-19 - 2017-06-25

    Chitinase A1

    Superior experiment: Evaluation of transformation

    Procedure:

  • Evaluation
    • ColonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA
    • Gelelectrophoresis
    • Over-night cultures of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA
    • Plasmid extraction via Mini-prep-kit and sequencing

    Chitin Deacetylases

    Superior experiment: Evaluation of transformation, GoldenBraid assembly and DNA-preparation

    Procedure:

  • Evaluation
    • Several colonyPCRs were done
    • Several gelelectrophoresis
      • They showed that the colonyPCRs did not work properly
    • GoldenBrain assembly
    • Cloning Anderson-CDApuc in pUPD
  • Transformation
    • Transforming the pUPD-Anderson-CDApuc into Top10 via heat-shock
  • Over-night cultures of pSB1C3-Anderson-nodB and pSB1K3-Anderson-nodB
  • DNA-preparation
    • Plasmid extraction of pSB1C3-Anderson-nodB and pSB1K3-Anderson-nodB via Mini-prep Kit

    2017-06-26 - 2017-07-02

    Chitinase A1

    Superior experiment: Gaining more of the construct pSB1C3-AraC-chiA

    Procedure:

  • Retransformation and evaluation
    • Retransforming pSB1C3-AraC-chiA into Top10 via heat-shock
    • ColonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA
    • Gelelectrophoresis
    • Over-night cultures
    • Plasmid extraction via Mini-prep-kit
  • Detection of a point mutation in pSB1C3-Anderson-ChiA

    • Chitin Deacetylases

    Superior experiment: Sequencing and cloning nodB in pSB1C3 without promotor

    Procedure:

  • DNA-preparation
    • Plasmid extraction of pUPD-Anderson-CDApuc via Mini-prep Kit and sequencing
  • Cloning
    • Digesting pUPD-Anderson-nodB with Pst1
    • PCR clean-up
    • Digesting pUPD-Anderson-nodB with Pst1 again
    • Ligation of nodB and pSB1C3 with T4 Ligase

    2017-07-03 - 2017-07-09

    Chitinase A1

    Superior experiment: Transformation of pSB1C3-AraC-chiA into BL21

    Procedure:

  • Transformation and evaluation
    • Transforming pSB1C3-AraC-chiA into BL21 via heat-shock
    • ColonyPCR of pSB1C3-AraC-chiA
    • Gelelectrophoresis

    Chitin Synthase

    Superior experiment: Cloning nodC into pSb1K3, pSB1C3 and pSB1C3-AraC, transformation in Top10

    Procedure:

  • Cloning nodC into pSB1K3
    • Digesting pUPD-Anderson-nodC with Xba1 and Pst1
    • Digesting pSB1K3 with Xba1 and Pst1
    • Gelelectrophoresis and gelextraction
    • Ligation with T4 Ligase
  • Cloning nodC into pSB1C3
    • Digesting pUPD-Anderson-nodC with Xba1 and Pst1
    • Digesting pSB1C3 with Xba1 and Pst1
    • Gelelectrophoresis and gelextraction
    • Ligation with T4 Ligase
  • Cloning nodC into pSB1C3-AraC
    • Digesting pUPD-Anderson-nodC with Xba1 and Pst1
    • Digesting pSb1C3-AraC with Spe1 and Pst1
    • Gelelectrophoresis and gelextraction
    • Ligation with T4 Ligase
  • Transformation
    • Transforming pSB1K3-Anderson-nodC, pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into Top10 via heat-shock

    Chitin Deacetylases

    Superior experiment: Transformation pSB1C3-nodB, evaluation and cloning CDApuc into pSB1C3 and pSB1K3 without promotor

    Procedure:

  • Transformation
    • Transforming the pSB1C3-nodB into Top10 via heat-shock
  • Evaluation
    • ColonyPCR of pSB1C3-nodB
    • Over-night cultures of pSB1C3-nodB
    • Plasmid extraction of pSB1C3-nodB via Mini-prep-kit and sequencing
  • Cloning CDApuc into pSB1C3 and pSB1K3 with and without Anderson-promotor
  • Transformation
    • Transforming the pSB1C3-CDApuc, pSB1K3-CDApuc, pSB1C3-Anderson-CDApuc and pSB1K3-Anderson-CDApuc into Top10 via heat-shock
  • Over-night cultures of pSB1C3-CDApuc, pSB1K3-CDApuc, pSB1C3-Anderson-CDApuc and pSB1K3-Anderson-CDApuc
  • DNA-preparation
    • Plasmid extraction of pSB1C3-CDApuc and pSB1K3-CDApuc via Mini-prep Kit
  • Gelelectrophoresis of pSB1C3-CDApuc and pSB1K3-CDApuc
  • Transformation (repeat)
    • Transforming the pSB1C3-nodB into Top10 via heat-shock

    2017-07-10 - 2017-07-16

    Chitinase A1

    Superior experiment: Detection of Chitinase A1 via SDS-Page

    Procedure:

  • Over-night cultures of pSB1C3-AraC-chiA and empty BL21 cells
  • Growing a main culture
    • Induction with arabinose at OD=0.6
      • Taking sample1 before induction
      • Taking sample2 three hours after induction
      • Taking sample3 24 hours after induction
    • Store samples at -20 degree Celsius

    Chitin Synthase

    Superior experiment: Evaluation

    Procedure:

  • Evaluation of transformations
    • colonyPCR of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
    • Gelelectrophoresis
    • Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21
    • Plasmid extraction via Mini-prep-kit and sequencing

    Chitin Deacetylases

    Superior experiment: Cloning nodB in pSB1K3

    Procedure:

  • Cloning nodB in pSB1K3
    • Digesting pSB1C3-nodB and pSB1K3 with Xba1 and Pst1
    • Dephosphorylating the backbone pSB1K3
    • Ligation with T4 Ligase
  • Transformation
    • Transforming the pSB1K3-nodB into Top10

    2017-07-17 - 2017-07-23

    Chitinase A1

    Superior experiment: Detection of Chitinase A1 via SDS-Page

    Procedure:

  • Over-night cultures of pSB1C3-AraC-chiA and empty BL21 cells
  • Growing a main culture
    • Induction with arabinose at OD=0.6
      • Taking sample1 before induction
      • Taking sample2 three hours after induction
      • Taking sample3 24 hours after induction
    • Store samples at -20 degree Celsius
  • SDS-Page
    • A SDS-Page was performed
  • Transformation and evaluation
    • Transforming pSB1C3-AraC-chiA into Top10 via heat-shock
    • ColonyPCR of pSB1C3-AraC-chiA
    • Gelelectrophoresis

    Chitin Synthase

    Superior experiment: Detection of Chitin-Synthase NodC via SDS-Page

    Procedure:

  • Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
  • Growing a main culture

    • Chitin Deacetylases

    Superior experiment: Evaluation

    Procedure:

  • Evaluation
    • ColonyPCR of pSB1K3-Anderson-nodB
    • Plasmid extraction of pSB1C3-Anderson-CDApuc, pSB1K3-Anderson-CDApuc and pSB1C3-CDApuc via Mini-prep Kit
    • Restriction digestion and gelelectrophoresis of pSB1C3-Anderson-CDApuc, pSB1K3-Anderson-CDApuc and pSB1C3-CDApuc

    2017-07-24 - 2017-07-30

    Chitinase A1

    Superior experiment: Transformation in BL21, detection of Chitinase A1 via SDS-Page

    Procedure:

  • Transformation and evaluation
    • Transforming pSB1C3-AraC-chiA into BL21 via heat-shock
    • ColonyPCR of pSB1C3-AraC-chiA
    • Gelelectrophoresis
    • Over-night cultures of pSB1C3-AraC-chiA and empty BL21 cells
  • Growing a main culture
    • Induction with arabinose at OD=0.6
      • Taking sample1 before induction
      • Taking sample2 three hours after induction
      • Taking sample3 24 hours after induction
    • Store samples at -20 degree Celsius
  • A SDS-Page was performed
    • It showed the expected band at 46,5 kDa

    Chitin Synthase

    Superior experiment: Transformation in BL21, detection of Chitin synthase via SDS-Page

    Procedure:

  • Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into BL21 via heat-shock
  • Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21 cells
  • Growing a main culture
    • Induction with arabinose at OD=0.6
      • Taking sample1 before induction
      • Taking sample2 three hours after induction
      • Taking sample3 six hours after induction
      • Taking sample4 the next day
    • Store samples at -20 degree Celsius
  • A SDS-Page was performed

    • Chitin Deacetylases

    Superior experiment: Evaluation of ColonyPCR of pSB1K3-Anderson-nodB and detection of Chitin Deacetylase NodB via SDS-Page

    Procedure:

  • After consultation with an expert the CDApuc was deemed incompatible with the project
  • Evaluation
    • Plasmid extraction via Mini-prep kit and sequencing,
  • Glycerol stocks
    • Glycerol stocks of pSB1K3-Anderson-nodB were made
  • Over-night cultures of pSB1C3-Anderson-nodB, pSB1C3-Anderson-nodB and empty Top10
  • Growing a main culture
    • No induction needed as the Anderson-promotor is constitutive
    • Taking sample1 at OD=0.6
    • Taking sample2 three hours later
    • Taking sample3 six hours later
    • Taking sample4 24 hours later
    • Store samples at -20 degree Celsius

    2017-07-31 - 2017-08-06

    Chitinase A1

    Superior experiment: Cloning chiA into pSB1C3-Anderson in order to delete the point mutation

    Procedure:

  • Cloning chiA into pSB1C3
    • Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
    • Dephosphorylating the backbone pSB1C3
    • Ligation with T4 Ligase
  • Transformation
    • Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock

    Chitin Synthase

    Superior experiment: Detection of Chitin synthase NodC via SDS-Page

    Procedure:

  • Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
  • Growing a main culture

    • Chitin Deacetylases

    Superior experiment: Detection of Chitin Deacetylase NodB via SDS-Page

    Procedure:

  • A SDS-Page was performed

    • 2017-08-07 - 2017-08-13

    Chitinase A1

    Superior experiment: Evaluation of transformation

    Procedure:

  • Evaluation of transformation
    • ColonyPCR of pSB1C3-Anderson-chiA
    • Gelelectrophoresis
    • Over-night cultures of pSB1C3-Anderson-chiA
    • Plasmid extraction via Mini-prep-kit and sequencing
  • The mutation still remains in the construct

    • Chitin Synthase

    Superior experiment: Transformation in BL21, detection of Chitin-Synthase via SDS-Page

    Procedure:

  • Transformation
    • Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into BL21 via heat-shock
  • Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21 cells
  • Growing a main culture
    • Induction with arabinose at OD=0.6
      • Taking sample1 before induction
      • Taking sample2 three hours after induction
      • Taking sample3 six hours after induction
      • Taking sample4 the next day
    • Store samples at -20 degree Celsius
  • A SDS-Page was performed

    • Chitin Deacetylases

    Superior experiment: Detection of Chitin Deacetylase NodB via SDS-Page and transformation into BL21

    Procedure:

  • A SDS-Page was performed
  • Transformation
    • Transforming pUPD-nodB into BL21 cells
      • The pUPD vector does contain the T7-promotor sequence, which was not used before
        • pUPD-T7-nodB is used for detection because the T7-promotor is inducible with IPTG

    2017-08-14 - 2017-08-20

    Chitinase A1

    Superior experiment: Cloning chiA into pSB1C3-Anderson in order to delete the point mutation

    Procedure:

  • Cloning Anderson-chiA into pSB1C3
    • Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
    • Dephosphorylating the backbone pSB1C3
    • Ligation with T4 Ligase
  • Transformation and evaluation
    • Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock
    • ColonyPCR of pSB1C3-Anderson-chiA
    • Gelelectrophoresis
    • Over-night cultures of pSB1C3-Anderson-chiA
    • Plasmid extraction via Mini-prep-kit and sequencing
  • The mutation still remains in the construct

    • Chitin Synthase

    Superior experiment: Detection of Chitin synthase via SDS-Page

    Procedure:

  • Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21 cells
  • Growing a main culture
    • Induction with arabinose at OD=0.6
      • Taking sample1 before induction
      • Taking sample2 three hours after induction
      • Taking sample3 six hours after induction
      • Taking sample4 the next day
    • Store samples at -20 degree Celsius
  • A SDS-Page was performed but showed no satisfactory results
  • Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into BL21 via heat-shock

    • Chitin Deacetylases

    Superior experiment: Detection of Chitin Deacetylase NodB via SDS-Page

    Procedure:

  • Over-night cultures of pUPD-T7-nodB and empty BL21 cells
  • Growing a main cultur
    • Induction with IPTG at OD=0.6
      • Taking sample1 before induction
      • Taking sample2 three hours later
      • Taking sample3 six hours later
      • Taking sample4 24 hours later
    • Store samples at -20 degree Celsius
  • A SDS-Page was performed and repeated
    • It showed the expected band at 23 kDa

    2017-08-21 - 2017-08-27

    Chitinase A1

    Superior experiment: Cloning chiA into pSB1C3-Anderson, transformation in Top10

    Procedure:

  • Cloning Anderson-chiA into pSB1C3
    • Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
    • Dephosphorylating the backbone pSB1C3
    • Ligation with T4 Ligase
  • Transformation and evaluation
    • Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock
    • ColonyPCR of pSB1C3-Anderson-chiA
    • Gelelektrophoresis

    Chitin Synthase

    Superior experiment: Detection of Chitin synthase via SDS-Page

    Procedure:

  • Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and BL21 cells
  • Growing a main culture
    • Induction with arabinose at OD=0.6
      • Taking sample1 before induction
      • Taking sample2 three hours after induction
      • Take sample3 six hours after induction
      • Take sample4 the next day
    • Store samples at -20 degree Celsius
  • A SDS-Page was performed
  • Determinating the best medium
    • Media tested: LB, TB and SOC
    • Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC

    2017-08-28 - 2017-09-03

    Chitinase A1

    Superior experiment: Cloning chiA into pSB1C3-Anderson, transformation into Top10, evaluation and sequencing

    Procedure:

  • Cloning pSB1C3-Anderson-chiA
    • Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
    • Gelelektrophoresis and gelextraction
    • Ligation with T4 Ligase
  • Transformation and evaluation
    • Transforming pSB1C3-Anderson-chiA into Top10
    • ColonyPCR
    • Gelelectrophoresis
    • Over-night cultures of pSB1C3-Anderson-chiA
    • Plasmid extraction via Mini-prep-kit and sequencing
  • The mutation still remains in the construct

    • Chitin Synthase

    Superior experiment: Cell-growth test and SDS-Page

    Procedure:

  • Determinating the best medium
    • Media tested: LB, TB and SOC
    • Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
    • tracing cell-growth over 48 hours
  • Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21 cells
  • Growing a main culture
    • Induction with arabinose at OD=0.6
      • Taking sample1 before induction
      • Taking sample2 three hours after induction
      • Taking sample3 six hours after induction
      • Taking sample4 the next day
    • Store samples at -20 degree Celsius
  • A SDS-Page was performed

    • 2017-09-04 - 2017-09-10

    Chitin Synthase

    Superior experiment: SDS-Page and mutagenesis PCR

    Procedure:

  • A SDS-Page was performed
  • Mutagenesis PCR for the purpose of purification
    • Site-directed-mutagenesis: His-Tag to pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC

    2017-09-11 - 2017-09-17

    Chitin Synthase

    Superior experiment: Mutagenesis PCR, SDS-Page, retransformation of Site-directed mutagenesis products into BL21

    Procedure:

  • Mutagenesis PCR
    • Phosphorylation of mutagenesis PCR batch
    • Ligation with T4 Ligase
    • DPN1 digestion
    • Gelelectrophoresis
  • Transformation into Top10 and evaluation
    • Transforming pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His into Top10 via heat-shock
    • ColonyPCR
    • Gelelectrophoresis
    • Over-night cultures of pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His
    • Plasmid extraction via Mini-prep-kit
  • Transformation and evaluation
    • Transforming pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His into BL21 via heat-shock
    • colonyPCR of pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His
    • Gelelectrophoresis
    • Over-night cultures of pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His
    • Plasmid extraction via Mini-prep-kit and sequencing

    2017-09-18 - 2017-09-24

    Chitin Synthase

    Superior experiment: Preparation of SDS-Page

    Procedure:

  • Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
  • Growing a main culture

    • 2017-09-25 - 2017-10-01

    Chitinase A1

    Superior experiment: Mutagenesis PCR, transformation and evaluation via sequencing

    Procedure:

  • Mutagenese PCR
    • PCR with primers for mutagenesis PCR
    • Phosphorylation of mutagenesis PCR batch
    • Ligation with T4 Ligase
    • DPN1 digestion
  • Transformation and evaluation
    • Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock
    • ColonyPCR of pSB1C3-Anderson-chiA
    • Gelelectrophoresis
    • Over-night culture of pSB1C3-Anderson-chiA
    • Plasmid extraction via Mini-prep-kit and sequencing
  • The mutation still remains in the construct

    • Chitin Synthase

    Superior experiment: Protein purification

    Procedure:

  • Preparation
    • Disruption of cells via high pressure
    • ultracentrifuge to separate cell components
  • Purification
    • Purification via ÄKTA
  • Verification
    • A SDS-PAGE of the fractions was performed

    2017-10-02 - 2017-10-08

    Chitinase A1

    Superior experiment: Mutagenesis PCR, transformation and evaluation via sequencing

    Procedure:

  • Mutagenese PCR
    • PCR with primers for mutagenesis PCR
    • Ligation with T4 Ligase
    • DPN1 digestion
  • Transformation and evaluation
    • Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock
    • ColonyPCR of pSB1C3-Anderson-chiA
    • Gelelectrophoresis
    • Overnightculture of pSB1C3-Anderson-chiA
    • Plasmid extraction via Mini-prep-kit and sequencing
  • The mutation still remains in the construct

    • Chitin Synthase

    Superior experiment: Verificate the functionality of NodC

    Procedure:

  • Verification
    • Buffer exchange via PD10 columns
    • UPDTM Glycosyltransferase Assay from Promega
  • Cloning nodC into pSB1C3
    • Digesting pUPD-Anderson-nodC with Xba1 and Pst1
    • Digesting pSB1C3 with Xba1 and Pst1
    • Dephosphorylating the backbone pSB1C3
    • Ligation with T4 Ligase
  • Cloning nodC into pSB1C3-AraC
    • Digesting pUPD-Anderson-nodC with Nhe1 and Pst1
    • Digesting pSB1C3-AraC with Spe1 and Pst1
    • Dephosphorylating the backbone pSB1C3-AraC
    • Ligation with T4 Ligase
  • Transformation and evaluation
    • Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into Top10 via heat-shock
    • Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
    • Plasmid extraction via Mini-prep-kit and sequencing

    2017-10-09 - 2017-10-15

    Chitin Synthase

    Superior experiment: Verificate the functionality of NodC

    Procedure:

  • Assay
    • Repetition of UDPTM Glycosyltransferase Assay twice

    2017-10-16 - 2017-10-22

    Chitin Synthase

    Superior experiment: Verificate the functionality of NodC

    Procedure:

  • Verification
    • Repetition of UDPTM Glycosyltransferase Assay
    • A thin-layer-chromatography for verification of chitin