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Notebook and Methods

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Notebook

2017-04-03 - 2017-04-09

Technics

Superior experiment: Building a DIHM

Procedure:

  • First team meeting. Getting to know each other. First discussion about probable projects.

    • 2017-04-10 - 2017-04-16

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Further discussion about possible projects and time management. More research is necessary.

    • 2017-04-17 - 2017-04-23

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Discussions lead to the decision for a microscope. For type and functionality research is planned.

    • 2017-04-24 - 2017-04-30

    General Labwork

    Superior experiment: Ordering sequences

    Procedure:

  • Ordering via IDT
    • nodC, nodB, puc and chiA

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Discussion on type of microscope. High resolution wanted with minimum cost. More research is necessary.

    • 2017-05-01 - 2017-05-07

    General Labwork

    Superior experiment: Cloning via GoldenBraid assembly

    Procedure:

  • GoldenBraid assembly
    • Cloning nodC, nodB, puc and chiA into the pUPD vector

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Decision for a holographic microscope. More research about the exact details and what is needed.

    • 2017-05-08 - 2017-05-14

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Ongoing research

    • 2017-05-15 - 2017-05-21

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • First parts like the DVD Pickup are ordered. Ongoing research.

    • 2017-05-22 - 2017-05-28

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Pinhole was sponsored by "Applied semiconductor optics". Ongoing research about detection system and DVD pickup control.

    • 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 XbaI and PstI
    • 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 XbaI and PstI
    • 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 XbaI and PstI
    • Dephosphorylating the backbone pSB1K3
    • Ligation with T4 Ligase
  • Transformation and evaluation
    • Transforming pSB1K3-Anderson-nodB and pUPD-Anderson-puc into Top10 via heat-shock
    • ColonyPCR of pSB1K3-Anderson-nodB and pUPD-Anderson-puc

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Research about possible software starts as well as about micromanipulators. Furthermore continuing research of the last week.

    • 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 NheI and PstI, pSB1C3-AraC with SpeI and PstI
    • 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 XbaI and PstI
    • 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-puc
  • Retransformation
    • Retransformation of pSB1C3-Anderson-nodB and pUPD-Anderson-puc into Top10
  • Sequencing
    • Over-night cultures of pSB1C3-Anderson-nodB
    • Plasmid extraction via Mini-prep-kit and sequencing
      • No insert was found

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Continuing research of last weeks. First ideas of DVD Pickup control and micromanipulators are acquired.

    • 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 XbaI and PstI
    • 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 XbaI and PstI
    • 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 XbaI and PstI
    • Dephosphorylating the backbone pSB1C3
    • PCR clean-up to
    • Ligation with T4 Ligase
  • Transformation and evaluation
    • Transforming pSB1C3-Anderson-nodB into Top10 via heat-shock

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Start working with the 3D printer. Ongoing research.

    • 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-puc in pUPD
  • Transformation
    • Transforming the pUPD-Anderson-puc 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

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • 3D printer crashed and needs repair. Ongoing research.

    • 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-puc via Mini-prep Kit and sequencing
  • Cloning
    • Digesting pUPD-Anderson-nodB with PstI
    • PCR clean-up
    • Digesting pUPD-Anderson-nodB with PstI again
    • Ligation of nodB and pSB1C3 with T4 Ligase

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Still fixing 3D printer. Ongoing research.

    • 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 XbaI and PstI
    • Digesting pSB1K3 with XbaI and PstI
    • Gelelectrophoresis and gelextraction
    • Ligation with T4 Ligase
  • Cloning nodC into pSB1C3
    • Digesting pUPD-Anderson-nodC with XbaI and PstI
    • Digesting pSB1C3 with XbaI and PstI
    • Gelelectrophoresis and gelextraction
    • Ligation with T4 Ligase
  • Cloning nodC into pSB1C3-AraC
    • Digesting pUPD-Anderson-nodC with XbaI and PstI
    • Digesting pSb1C3-AraC with SpeI and PstI
    • 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 puc 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-puc, pSB1C3-Anderson-puc and pSB1K3-Anderson-puc into Top10 via heat-shock
  • Over-night cultures of pSB1C3-puc, pSB1K3-puc, pSB1C3-Anderson-puc and pSB1K3-Anderson-puc
  • DNA-preparation
    • Plasmid extraction of pSB1C3-puc and pSB1K3-puc via Mini-prep Kit
  • Gelelectrophoresis of pSB1C3-puc and pSB1K3-puc
  • Transformation (repeat)
    • Transforming the pSB1C3-nodB into Top10 via heat-shock

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Learning about CAD and working on manipulators. Continuing last week?s work.

    • 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 XbaI and PstI
    • Dephosphorylating the backbone pSB1K3
    • Ligation with T4 Ligase
  • Transformation
    • Transforming the pSB1K3-nodB into Top10

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Continuing last week's work

    • 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-puc, pSB1K3-Anderson-puc and pSB1C3-puc via Mini-prep Kit
    • Restriction digestion and gelelectrophoresis of pSB1C3-Anderson-puc, pSB1K3-Anderson-puc and pSB1C3-puc

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Continuing last week's work

    • 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

    • Technics

    Superior experiment: Building a DIHM

    Procedure:

  • 3D printer is fixed. Deciding on a raspberry pie cam for detecting the hologram. First control for DVD pick up is build, but breaks down after several tests. DIYouware PCB board is investigated. Collaboration with iGEM Team York. We send them one of our pickups.

    • 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 XbaI and PstI
    • 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

    • Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Pi Cam is ordered. Learning PCB boards. Printing parts for adjustment of pinhole and DVD pickup. Ongoing research about software.

    • 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

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • First try of acquiring holograms. Probably intensity to low, further improvement necessary.

    • 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

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Implementing an self-made millimetre sized pinhole to increase intensity. Pictures were taken. Holopy is chosen as suitable software.

    • 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

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • ordering PCB boards and all parts necessary for it. Further work on micromanipulators, learning about flexing bearings. Research about the full functionality of the holopy software is carried out. York do not want to use DVD pickup due to safety reasons.

    • 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

    • Technics

    Superior experiment: Building a DIHM

    Procedure:

  • First flexible bearing manipulators are printed and tested. Discussion about what to see on the first images. Ongoing work with the software.

    • 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

    Chitin Deacetylases

    Superior experiment: Cloning via GoldenBraid assembly, transformation and evaluation

    Procedure:

  • GoldenBraid assembly
    • Cloning cod into pUPD
  • Transformation and evaluation
    • Transforming pUPD-T7-cod into Top10 via heat-shock
    • Over-night cultures of pUPD-T7-cod
    • Plasmid extraction via Mini-prep-kit and sequencing

    Hydrogels

    Superior experiment: How to solve chitosan and first tries to produce a dressing

    Procedure:

  • Chitosan solved with acetic acid
    • Different chitosan/acetic acid concentration were tested
    • in 100 % Milli-Q H2O
    • 1%Chitosan - 0,5 % acetic acid
    • 1%Chitosan - 1 % acetic acid
    • 1%Chitosan - 1,5 % acetic acid
    • 1%Chitosan - 2 % acetic acid
    • 1,5%Chitosan - 0,5 % acetic acid
    • 1,5%Chitosan - 1 % acetic acid
    • 1,5%Chitosan - 1,5 % acetic acid
    • 1,5%Chitosan - 2 % acetic acid
    • 2%Chitosan - 0,5 % acetic acid
    • 2%Chitosan - 1 % acetic acid
    • 2%Chitosan - 1,5 % acetic acid
    • 2%Chitosan - 2 % acetic acid
    • 3%Chitosan - 0,5 % acetic acid
    • 3%Chitosan - 1 % acetic acid
      • It is possible to solve chitosan, but it needs time and mechanical stirring, the more acid the visose the solution gets
    • Chitosan-Agarose Hydrogels
    • Chitosan 1%, Acetic acid 1%, Agarose 1%
    • Chitosan 1%, Acetic acid 1%, Agarose 2%
    • Chitosan 1%, Acetic acid 1%, Agarose 3%
    • Chitosan 2%, Acetic acid 1%, Agarose 1%
    • Chitosan 2%, Acetic acid 1%, Agarose 2%
    • Chitosan 2%, Acetic acid 1%, Agarose 3%
  • Chitosan was dissolved in water and 1% acetic acid, then agarose is added and heated.
    • Chitosan is slighlty soluble.
  • New approach:
    • first Agarose was dissolved in water by heating. Then acetic acid and chitosan were added -> better results, better soluble
    • Chitosan 1%, Acetic acid 1%, Agarose 1%
    • Chitosan 1%, Acetic acid 1%, Agarose 3%
      • With 1%: promising
    • Chitosan-Agar Hydrogels
    • Chitosan 1%, Acetic acid 1%, Agar 1%
    • Chitosan 1%, Acetic acid 1%, Agar 2%
    • Chitosan 1%, Acetic acid 1%, Agar 3%

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Further work on bearings and software.

    • 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
    • DPNI 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

    Chitin Deacetylases

    Superior experiment: Mutagenesis PCR, transformation and evaluation

    Procedure:

  • Mutagenese PCR
    • PCR with His-tag primers for mutagenesis PCR
    • Phosphorylation of mutagenesis PCR batch
    • Ligation with T4 Ligase
    • DPN1 digestion
  • Transformation and evaluation
    • Transforming pSB1C3-NodB-His and pUPD-NodB-His into Top10 via heat-shock
    • ColonyPCR of
    • Gelelectrophoresis
  • Mutagenese PCR (repeat)
    • PCR with His-tag primers for mutagenesis PCR
    • Phosphorylation of mutagenesis PCR batch
    • Ligation with T4 Ligase
    • DPNI digestion
  • Transformation
    • Transforming pSB1C3-NodB-His and pUPD-NodB-His into Top10 via heat-shock

    Hydrogels

    Superior experiment: Testing of different gels

    Procedure:

  • DAB Gel
    • Hydroxyethylcellulose 2,5
    • Glycerol 10%
    • Milli-Q H2O 87,5%
    • no swelling
      • Hydroxyethylcellulose was not "Hydroxyethylcellulose 10000", but with a lower viscosity, so the gel did not form a stable gel
    • Order of mixing was determed;
    • 1) 2% Chitosan, than 97,5% Milli-Q water, than 0,5% acetic acid
    • 2) 2% Chitosan, than 50% Milli-Q water, than 0,5% acetic acid, than 47,5% Milli-Q water
    • 3) 2% Chitosan, than 0,5% acetic acid, than 98% Milli-Q water
    • 4) 97,5% Milli-Q water mixed with 0,5% acetic acid, than 2% Chitosan
  • Chitosan-Agarose Hydrogels
    • With new approach: first dissolving of agarose, then adding chitosan and acetic acid.
    • Chitosan 1%, Acetic acid 1%, Agarose 1%
    • Chitosan 1%, Acetic acid 1%, Agarose 2%
    • Chitosan 1%, Acetic acid 1%, Agarose 3%
    • Chitosan 2%, Acetic acid 1%, Agarose 1%
    • Chitosan 2%, Acetic acid 1%, Agarose 2%
    • Chitosan 2%, Acetic acid 1%, Agarose 3%
  • Chitosan-Gelatine Hydrogels
    • Different concentration of gelatine were dissolved in water
    • 1 % Acetic acid and 1% chitosan were added
    • Chitosan 1%, Acetic acid 1%, Gelatine 1%
    • Chitosan 1%, Acetic acid 1%, Gelatine 2%
    • Chitosan 1%, Acetic acid 1%, Gelatine 3%

    Chemistry

    Superior experiment: Preperation of N-Succinic-Chitosan concentration 1 and 2

    Procedure:

  • Put Succinic Anhydride and Chitosan in acetic acid in a three necked flask

    • Technics

    Superior experiment: Building a DIHM

    Procedure:

  • 3D printer crashed again. Continuing with software and setup design.

    • 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

    • Chitin Deacetylases

    Superior experiment: Cloning T7-cod and cod in pSB1C3

    Procedure:

  • Evaluation
    • Plasmid extraction of pSB1C3-nodB-His and pUPD-nodB-His via Mini-prep-kit and sequencing
  • Cloning T7-cod in pSB1C3
    • Digesting pUPD-T7-cod and pSB1C3 with XbaI and PstI
    • Dephosphorylating the backbone pSB1C3
    • PCR clean-up
    • Ligation with T4 Ligase
  • Cloning cod in pSB1C3
    • Digesting pSB1C3 with XbaI and PstI
    • Digesting pUPD-T7-cod with PstI
    • Dephosphorylating the backbone pSB1C3
    • PCR clean-up
    • Ligation with T4 Ligase
  • Transformation and evaluation
    • Transforming pSB1C3-T7-cod and pSB1C3-cod into Top10 via heat-shock
    • cPCR of pSB1C3-T7-cod and pSB1C3-cod
    • Gelelectrophoresis
    • Over-night cultures of pSB1C3-T7-cod and pSB1C3-cod
    • Plasmid extraction of pSB1C3-cod via Mini-prep-kit and sequencing

    Hydrogels

    Superior experiment: trying to reproduce papers' hydrogels

    Procedure:

  • hydrogel with beta-GP
    • Hydrogels were produced as in [doi: 10.3390/ijms151017765]
    • No solid gel could be archieved, but after autoclaving it showed more stability, but was dropped to to its expensiveness
    • Chitosan 1,8% in 0,2M acetic acid, cooled at 4°C for 20min
    • beta-glycerol-phosphate disodium salt 50% w/v was added dropwise, ratio 9:1 - Chitosan:beta-G.P.
    • Stirred for 20min and poured
      • No solid gel
    • hydrogel basic with less beta-GP
    • 2% chitosan and 1% acetic acid + 8% beta-GP. was placed at 39° C for 15h
      • no changes, still not solid
    • Using glutaraldehyde
    • 1% chitosan + 1% acetic acid + 1% glutaraldehyde
    • 1% chitosan + 1% acetic acid + 3% glutaraldehyde
    • 1% chitosan + 1% acetic acid + 5% glutaraldehyde
    • 2% chitosan + 1% acetic acid + 10% glutaraldehyde
      • no solid gel
    • placed at 55°C for 12 and 24 h
      • still not solid
    • determine and pH-level
    • 0,2 M chitosan - pH: 4,7

    Chemistry

    Superior experiment: Preperation of N-Succinic-Chitosan concentration 3

    Procedure:

  • Put Succinic Anhydride and Chitosan in acetic acid in a three necked flask
  • Centrifugate concentration 1 at 5000 rpm

    • Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Repairing 3D printer. Continuing with software and setup design.

    • 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

    Chitin Deacetylases

    Superior experiment: Detection of NodB via SDS-Page

    Procedure:

  • Transformation
    • Transforming pUPD-T7-cod and pUPD-T7-nodB-His into BL21 via heat-shock
  • Over-night cultures of pUPD-T7-nodB-His, pUPD-T7-cod and empty BL21 cells
  • Growing a main culture
    • Induction with IPTG 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
  • Expression of pUPD-nodB-His by 16 °C and 37°C
  • Sequencing
    • Plasmid extraction of pUPD-cod via Mini-prep-kit and sequencing

    Hydrogels

    Superior experiment: gelantine as chross-linking agent

    Procedure:

  • Chitosan-Gelantine
    • 2%Chitosan - 0,5 % acetic acid - 2% Gelantine:
    • 2%Chitosan - 1 % acetic acid - 2% Gelantine:
    • 3%Chitosan - 0,5 % acetic acid - 2% Gelantine:
    • 3%Chitosan - 0,5 % acetic acid - 3% Gelantine:
  • DAB-Gel #2
    • redone experiments DAB-Gel
    • same results
  • centrifugation of chitosan solution
    • 2%chitosan + 1% acetic acid
    • 5000 rpm for 20 min
    • no supernatent, visually didnt change anything

    Chemistry

    Superior experiment: Preperation of N-Succinic-Chitosan concentration 4

    Procedure:

  • Put Succinic Anhydride and Chitosan in acetic acid in a three necked flask
  • Consultaion of Prof. Schoenherr

    • Technics

    Superior experiment: Building a DIHM

    Procedure:

  • 3D printer is working again. Bearings are excluded due to their degrees of freedom. Idea about replacing the pinhole with a glassfiber comes up, glassfiber is ordered. Discussion about changing the light source start due to the lack of light intensity.

    • 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 XbaI and PstI
    • Digesting pSB1C3 with XbaI and PstI
    • Dephosphorylating the backbone pSB1C3
    • Ligation with T4 Ligase
  • Cloning nodC into pSB1C3-AraC
    • Digesting pUPD-Anderson-nodC with NheI and PstI
    • Digesting pSB1C3-AraC with SpeI and PstI
    • 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

    Chitin Deacetylases

    Superior experiment: SDS-Page, Transformation and evaluation

    Procedure:

  • Glycerol stocks
    • Glycerol stocks of pSB1C3-cod were made
  • Transformation
    • Transforming pUPD-T7-cod into Top10 and pUPD-T7-nodB-His into BL21 via heat-shock
  • Over-night cultures of pUPD-T7-nodB-His, pUPD-T7-cod and empty BL21 cells
  • Growing a main culture
    • Induction with IPTG 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
  • Restriction
    • Digesting pUPD-T7-nodB-His with NheI-Hf
    • Gel-filtration
    • Ligation with T4 ligase
  • Transformation and evaluation
    • Transforming pUPD-T7-nodB-His into Top10 and BL21 via heat-shock
    • Plasmid extraction of pUPD-T7-nodB-His and pUPD-T7-nodB-His (dig. with NheI-Hf) via Mini-prep-kit and sequencing
  • Over-night cultures of pUPD-T7-nodB-His, pUPD-T7-nodB-His (dig. with NheI-Hf) and empty BL21 cells
  • Growing a main culture
    • Induction with IPTG 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

    • Hydrogels

    Superior experiment: Switching back to agar/agarose

    Procedure:

  • Testing of different gels
    • 1%Chitosan - 1 % acetic acid - 1% Gelantine: good
    • 1,5%Chitosan - 0,5 % acetic acid - 2% Agar:
    • 0,5%Chitosan - 0,5 % acetic acid - 2% Agar:
    • 2%Chitosan - 0,5 % acetic acid - 1% Agar:
    • 2%Chitosan - 0,5 % acetic acid - 2% Agar:
    • 2%Chitosan - 0,5 % acetic acid - 3% Agar:
    • 1%Chitosan - 0,5 % acetic acid - 1% Agar:
    • 1,5%Chitosan - 0,5 % acetic acid - 2% Agarose: good
    • 0,5%Chitosan - 0,5 % acetic acid - 2% Agarose: too hard
    • 1%Chitosan - 1 % acetic acid - 1% Agarose: solid
    • 1%Chitosan - 1 % acetic acid - 2% Agarose: solid
    • 1%Chitosan - 1 % acetic acid - 3% Agarose: not solid
    • 2%Chitosan - 1 % acetic acid - 1% Agarose: solid
    • 2%Chitosan - 1 % acetic acid - 2% Agarose: not solid
    • 2%Chitosan - 1 % acetic acid - 3% Agarose: not solid
  • Chitosan-Agarose Hydrogel
    • Dissolving of agarose in water,
    • Simultaneously dissolving of chitosan in 1% acetic acid and stirring.
    • When chitosan is dissolved, mixing of both solutions.
    • Chitosan 1%, Acetic acid 1%, Agarose 1%
    • Chitosan 1%, Acetic acid 1%, Agarose 1%

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • Changed the light source to smartphone LED and LEDs in general. Both deliver enough intensity for the cam and the two micrometres scaled bar becomes visible. Working on a clever manipulator continues. Software make further progress; first pictures are processed.

    • Chemistry

    Superior experiment: Spin Coating and modification of the films

    Procedure:

  • Spin Coat the hydrogels on microscope sildes
  • Modificate the surface with either DMSO and succinic anhydride or Aceton and succinic anhydride

    • 2017-10-09 - 2017-10-15

    Chitin Synthase

    Superior experiment: Verificate the functionality of NodC

    Procedure:

  • Assay
    • Repetition of UDPTM Glycosyltransferase Assay twice

    Chitin Deacetylases

    Superior experiment: Purification

    Procedure:

  • pUPD-NodB-His via Äkta pure
    • Over-night cultures of pUPD-T7-nodB-His
    • Growing a main culture
    • Induction with IPTG at OD=0.6
    • Buffer exchange via PD10 coloumns
  • A SDS-Page was performed
    • A band is visible by a size of 23kDa, accordingly NodB is present

    Hydrogels

    Superior experiment: Quercetin as cheap crosslinker

    Procedure:

  • Gel cintaining quercetin
    • 2% Chitosan solved with 0,5% acetic acid
    • beta-glycerol-phosphate disodium salt was added dropwise (0,1g/mL)
    • quercetin 200mg/mL
    • DMSO was added 0,15%/0,2%/0,5%/1%, mixed into the solution
    • not any of the gels were solid enough

    Technics

    Superior experiment: Building a DIHM

    Procedure:

  • A Fresnel lens in the DVD pickup eluded the team with a possible hologram. Expanding the laser diode from the pick-up and trying to couple its light into a glassfiber was unsatisfying due to low transmission. First idea of own micromanipulator is printed, needs improvement. Software is able to fully process holograms. 3D output will be included.

    • Chemistry

    Superior experiment: Coupling of the peptide (ala-ala-phe-7amc) to the hydrogel layers and prove the concept

    Procedure:

  • Putting the microscope sildes in a EDC/NHS solution
  • Put it afterwards in the peptide solution
  • Verfication of the concept in protease solution

    • 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

    Chitin Deacetylases

    Superior experiment: Acetic Acid Assay

    Procedure:

  • A SDS-Page was performed
  • Enzyme reaction
    • Enzyme reaction with 1mM chitin pentamer, 2,5µM NodB and NH4HCO3
  • Acetic Acid Assay from Megazym
    • The assay was performed two times with 4 replicates

    Hydrogels

    Superior experiment: Swelling chitosan hydrogel testing

    Procedure:

  • Chitosan hydrogel solidified in alginate-quercetin
    • different incubation times were tested;
    • after 12 h: the gel was not solid
    • after 18 h: the gel didn't had the required solidarity
    • as well as chitosan 1g/2g/3g
  • The most promising gel:
    • 2%chitosan
    • 100 % dH2O
    • 6h stirring
    • 0,5 % acetic acid
    • 12h stirring
    • 12h rest at 4degree Celsius
    • prepare alginate 2% + quercetin (in dmso) at 1%
    • pour Chitosan onto frozen Alginate/Quercetin
    • add more Alginate/Quercetin
    • rest at 37°C for 24h
    • it was placed in water and the thicknes was measured as well as the weight
    • after 12 hours: swelling +200%
    • after 24 hours: swelling +600%
  • gel produced for spin-coating (see. chemistry subproject)

    • Chemistry

    Superior experiment: Making dilution series and test some more hydrogel films with protease

    Procedure:

  • Different concentrations of peptide and protease to check detection limit
  • Put finished films in protease solution

    • Technics

    Superior experiment: Building a DIHM

    Procedure:

  • 3D printer crashed again. Another workgroup allows to use theirs. Trying to repair our own. Further work on the setup and the software. More pictures acquired with different smartphones and LEDs.

    • 2017-10-23 - 2017-10-29

    Chitin Synthase

    Superior experiment: Verificate the functionality of NodC

    Procedure:

  • Assay
    • Repetition of UDPTM Glycosyltransferase Assay

    Chitin Deacetylases

    Superior experiment: Acetic Acid Assay and thin-layer chromatographie

    Procedure:

  • Acetic Acid Assay from Megazym
    • The assay was performed
  • A thin-layer chromatographie was performed

    • Hydrogels

    Superior experiment: reproducing our most promising hydrogels

    Procedure:

  • Reproduce
    • we had redone our best hydrogel and produced them in a larger scale

    General labwork

    Superior experiment: DNA submission

    Procedure:

  • Using the DNA submission kit
    • Dispense each 10ul (25ng/ul) of every part in pSB1C3 into a well
    • Dry down the plate in a lab laminar flow hood (over night)
    • Label and ship the DNA

    Chemistry

    Superior experiment: The first realistic prototype

    Procedure:

  • Repeat the dilution series
  • Put Succinic Anhydride and Chitosan in acetic acid in a three necked flask
  • Add EDC/NHS soluion
  • Add peptide
  • Spin coat the hydrogel and test with protease
  • Different concentrations of peptide and protease to check detection limit.
  • Put finished films in protease solution.