ChiTUcare
Notebook and Methods
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Notebook
2017-04-03 - 2017-04-09
Technics
Superior experiment: Building a DIHM
Procedure:
2017-04-10 - 2017-04-16
Technics
Superior experiment: Building a DIHM
Procedure:
2017-04-17 - 2017-04-23
Technics
Superior experiment: Building a DIHM
Procedure:
2017-04-24 - 2017-04-30
General Labwork
Superior experiment: Ordering sequences
Procedure:
- nodC, nodB, puc and chiA
Technics
Superior experiment: Building a DIHM
Procedure:
2017-05-01 - 2017-05-07
General Labwork
Superior experiment: Cloning via GoldenBraid assembly
Procedure:
- Cloning nodC, nodB, puc and chiA into the pUPD vector
Technics
Superior experiment: Building a DIHM
Procedure:
2017-05-08 - 2017-05-14
Technics
Superior experiment: Building a DIHM
Procedure:
2017-05-15 - 2017-05-21
Technics
Superior experiment: Building a DIHM
Procedure:
2017-05-22 - 2017-05-28
Technics
Superior experiment: Building a DIHM
Procedure:
2017-05-29 - 2017-06-04
Chitinase A1
Superior experiment: Cloning chiA into pSB1C3
Procedure:
- 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:
- Digesting pUPD-Anderson-nodC and pSB1K3 with XbaI and PstI
- Dephosphorylating the backbone pSB1K3
- Ligation with T4 Ligase
- 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:
- Digesting pUPD-Anderson-nodB and pSB1K3 with XbaI and PstI
- Dephosphorylating the backbone pSB1K3
- Ligation with T4 Ligase
- 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:
2017-06-05 - 2017-06-11
Chitinase A1
Superior experiment: Cloning AraC-chiA into pSB1C3, transformation into Top10, evaluation
Procedure:
- Digesting pUPD-Anderson-chiA with NheI and PstI, pSB1C3-AraC with SpeI and PstI
- Dephosphorylating the backbone pSB1C3-AraC
- Ligation with T4 Ligase
- 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:
- Digesting pUPD-Anderson-nodC and pSB1C3 with XbaI and PstI
- Dephosphorylating the backbone pSB1K3
- Ligation with T4 Ligase
- 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:
- Gelelectrophoresis of colonyPCR product
- Repeating the colonyPCR of pSB1C3-Anderson-nodB and pUPD-Anderson-puc
- Retransformation of pSB1C3-Anderson-nodB and pUPD-Anderson-puc into Top10
- 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:
2017-06-12 - 2017-06-18
Chitinase A1
Superior experiment: Cloning chiA into pSB1C3
Procedure:
- Digesting pUPD-Anderson-chiA and pSB1C3 with XbaI and PstI
- Dephosphorylating the backbone pSB1C3
- Ligation with T4 Ligase
- 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 were made of positive pSB1C3-Anderson-nodC colonies
- Glycerol stocks were made of positive pSB1C3-AraC-nodC colonies
Chitin Deacetylases
Superior experiment: Cloning nodB in pSB1C3, transformation into Top10
Procedure:
- Digesting pUPD-Anderson-nodB and pSB1C3 with XbaI and PstI
- Dephosphorylating the backbone pSB1C3
- PCR clean-up to
- Ligation with T4 Ligase
- 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
- Transforming pSB1C3-Anderson-nodB into Top10 via heat-shock
Technics
Superior experiment: Building a DIHM
Procedure:
2017-06-19 - 2017-06-25
Chitinase A1
Superior experiment: Evaluation of transformation
Procedure:
- 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:
- Several colonyPCRs were done
- Several gelelectrophoresis
- They showed that the colonyPCRs did not work properly
- GoldenBrain assembly
- Cloning Anderson-puc in pUPD
- Transforming the pUPD-Anderson-puc into Top10 via heat-shock
- Plasmid extraction of pSB1C3-Anderson-nodB and pSB1K3-Anderson-nodB via Mini-prep Kit
Technics
Superior experiment: Building a DIHM
Procedure:
2017-06-26 - 2017-07-02
Chitinase A1
Superior experiment: Gaining more of the construct pSB1C3-AraC-chiA
Procedure:
- 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
Chitin Deacetylases
Superior experiment: Sequencing and cloning nodB in pSB1C3 without promotor
Procedure:
- Plasmid extraction of pUPD-Anderson-puc via Mini-prep Kit and sequencing
- 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:
2017-07-03 - 2017-07-09
Chitinase A1
Superior experiment: Transformation of pSB1C3-AraC-chiA into BL21
Procedure:
- 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:
- Digesting pUPD-Anderson-nodC with XbaI and PstI
- Digesting pSB1K3 with XbaI and PstI
- Gelelectrophoresis and gelextraction
- Ligation with T4 Ligase
- Digesting pUPD-Anderson-nodC with XbaI and PstI
- Digesting pSB1C3 with XbaI and PstI
- Gelelectrophoresis and gelextraction
- Ligation with T4 Ligase
- Digesting pUPD-Anderson-nodC with XbaI and PstI
- Digesting pSb1C3-AraC with SpeI and PstI
- Gelelectrophoresis and gelextraction
- Ligation with T4 Ligase
- 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:
- Transforming the pSB1C3-nodB into Top10 via heat-shock
- ColonyPCR of pSB1C3-nodB
- Over-night cultures of pSB1C3-nodB
- Plasmid extraction of pSB1C3-nodB via Mini-prep-kit and sequencing
- Transforming the pSB1C3-CDApuc, pSB1K3-puc, pSB1C3-Anderson-puc and pSB1K3-Anderson-puc into Top10 via heat-shock
- Plasmid extraction of pSB1C3-puc and pSB1K3-puc via Mini-prep Kit
- Transforming the pSB1C3-nodB into Top10 via heat-shock
Technics
Superior experiment: Building a DIHM
Procedure:
2017-07-10 - 2017-07-16
Chitinase A1
Superior experiment: Detection of Chitinase A1 via SDS-Page
Procedure:
- 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:
- 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:
- Digesting pSB1C3-nodB and pSB1K3 with XbaI and PstI
- Dephosphorylating the backbone pSB1K3
- Ligation with T4 Ligase
- Transforming the pSB1K3-nodB into Top10
Technics
Superior experiment: Building a DIHM
Procedure:
2017-07-17 - 2017-07-23
Chitinase A1
Superior experiment: Detection of Chitinase A1 via SDS-Page
Procedure:
- 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
- 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:
Chitin Deacetylases
Superior experiment: Evaluation
Procedure:
- 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:
2017-07-24 - 2017-07-30
Chitinase A1
Superior experiment: Transformation in BL21, detection of Chitinase A1 via SDS-Page
Procedure:
- 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
- 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
- It showed the expected band at 46,5 kDa
Chitin Synthase
Superior experiment: Transformation in BL21, detection of Chitin synthase via SDS-Page
Procedure:
- 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
Technics
Superior experiment: Building a DIHM
Procedure:
Chitin Deacetylases
Superior experiment: Evaluation of ColonyPCR of pSB1K3-Anderson-nodB and detection of Chitin Deacetylase NodB via SDS-Page
Procedure:
- Plasmid extraction via Mini-prep kit and sequencing,
- Glycerol stocks of pSB1K3-Anderson-nodB were made
- 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:
- Digesting pUPD-Anderson-chiA and pSB1C3 with XbaI and PstI
- Dephosphorylating the backbone pSB1C3
- Ligation with T4 Ligase
- Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock
Chitin Synthase
Superior experiment: Detection of Chitin synthase NodC via SDS-Page
Procedure:
Chitin Deacetylases
Superior experiment: Detection of Chitin Deacetylase NodB via SDS-Page
Procedure:
Technics
Superior experiment: Building a DIHM
Procedure:
2017-08-07 - 2017-08-13
Chitinase A1
Superior experiment: Evaluation of transformation
Procedure:
- ColonyPCR of pSB1C3-Anderson-chiA
- Gelelectrophoresis
- Over-night cultures of pSB1C3-Anderson-chiA
- Plasmid extraction via Mini-prep-kit and sequencing
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
- 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
Chitin Deacetylases
Superior experiment: Detection of Chitin Deacetylase NodB via SDS-Page and transformation into BL21
Procedure:
- 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:
2017-08-14 - 2017-08-20
Chitinase A1
Superior experiment: Cloning chiA into pSB1C3-Anderson in order to delete the point mutation
Procedure:
- Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1C3
- Ligation with T4 Ligase
- 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
Chitin Synthase
Superior experiment: Detection of Chitin synthase via SDS-Page
Procedure:
- 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
Chitin Deacetylases
Superior experiment: Detection of Chitin Deacetylase NodB via SDS-Page
Procedure:
- 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
- It showed the expected band at 23 kDa
Technics
Superior experiment: Building a DIHM
Procedure:
2017-08-21 - 2017-08-27
Chitinase A1
Superior experiment: Cloning chiA into pSB1C3-Anderson, transformation in Top10
Procedure:
- Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1C3
- Ligation with T4 Ligase
- 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:
- 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
- Media tested: LB, TB and SOC
- Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
Technics
Superior experiment: Building a DIHM
Procedure:
2017-08-28 - 2017-09-03
Chitinase A1
Superior experiment: Cloning chiA into pSB1C3-Anderson, transformation into Top10, evaluation and sequencing
Procedure:
- Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
- Gelelektrophoresis and gelextraction
- Ligation with T4 Ligase
- Transforming pSB1C3-Anderson-chiA into Top10
- ColonyPCR
- Gelelectrophoresis
- Over-night cultures of pSB1C3-Anderson-chiA
- Plasmid extraction via Mini-prep-kit and sequencing
Chitin Synthase
Superior experiment: Cell-growth test and SDS-Page
Procedure:
- Media tested: LB, TB and SOC
- Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
- tracing cell-growth over 48 hours
- 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
Technics
Superior experiment: Building a DIHM
Procedure:
2017-09-04 - 2017-09-10
Chitin Synthase
Superior experiment: SDS-Page and mutagenesis PCR
Procedure:
- 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:
- Cloning cod into pUPD
- 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:
- 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 is slighlty soluble.
- 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:
2017-09-11 - 2017-09-17
Chitin Synthase
Superior experiment: Mutagenesis PCR, SDS-Page, retransformation of Site-directed mutagenesis products into BL21
Procedure:
- Phosphorylation of mutagenesis PCR batch
- Ligation with T4 Ligase
- DPNI digestion
- Gelelectrophoresis
- 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
- 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:
- PCR with His-tag primers for mutagenesis PCR
- Phosphorylation of mutagenesis PCR batch
- Ligation with T4 Ligase
- DPN1 digestion
- Transforming pSB1C3-NodB-His and pUPD-NodB-His into Top10 via heat-shock
- ColonyPCR of
- Gelelectrophoresis
- PCR with His-tag primers for mutagenesis PCR
- Phosphorylation of mutagenesis PCR batch
- Ligation with T4 Ligase
- DPNI digestion
- Transforming pSB1C3-NodB-His and pUPD-NodB-His into Top10 via heat-shock
Hydrogels
Superior experiment: Testing of different gels
Procedure:
- 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
- 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%
- 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:
Technics
Superior experiment: Building a DIHM
Procedure:
2017-09-18 - 2017-09-24
Chitin Synthase
Superior experiment: Preparation of SDS-Page
Procedure:
Chitin Deacetylases
Superior experiment: Cloning T7-cod and cod in pSB1C3
Procedure:
- Plasmid extraction of pSB1C3-nodB-His and pUPD-nodB-His via Mini-prep-kit and sequencing
- Digesting pUPD-T7-cod and pSB1C3 with XbaI and PstI
- Dephosphorylating the backbone pSB1C3
- PCR clean-up
- Ligation with T4 Ligase
- Digesting pSB1C3 with XbaI and PstI
- Digesting pUPD-T7-cod with PstI
- Dephosphorylating the backbone pSB1C3
- PCR clean-up
- Ligation with T4 Ligase
- 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:
- 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:
Technics
Superior experiment: Building a DIHM
Procedure:
2017-09-25 - 2017-10-01
Chitinase A1
Superior experiment: Mutagenesis PCR, transformation and evaluation via sequencing
Procedure:
- PCR with primers for mutagenesis PCR
- Phosphorylation of mutagenesis PCR batch
- Ligation with T4 Ligase
- DPN1 digestion
- 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
Chitin Synthase
Superior experiment: Protein purification
Procedure:
- Disruption of cells via high pressure
- ultracentrifuge to separate cell components
- Purification via ÄKTA
- A SDS-PAGE of the fractions was performed
Chitin Deacetylases
Superior experiment: Detection of NodB via SDS-Page
Procedure:
- Transforming pUPD-T7-cod and pUPD-T7-nodB-His into BL21 via heat-shock
- 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
- Plasmid extraction of pUPD-cod via Mini-prep-kit and sequencing
Hydrogels
Superior experiment: gelantine as chross-linking agent
Procedure:
- 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:
- redone experiments DAB-Gel
- same results
- 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:
Technics
Superior experiment: Building a DIHM
Procedure:
2017-10-02 - 2017-10-08
Chitinase A1
Superior experiment: Mutagenesis PCR, transformation and evaluation via sequencing
Procedure:
- PCR with primers for mutagenesis PCR
- Ligation with T4 Ligase
- DPN1 digestion
- 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
Chitin Synthase
Superior experiment: Verificate the functionality of NodC
Procedure:
- Buffer exchange via PD10 columns
- UPDTM Glycosyltransferase Assay from Promega
- Digesting pUPD-Anderson-nodC with XbaI and PstI
- Digesting pSB1C3 with XbaI and PstI
- Dephosphorylating the backbone pSB1C3
- Ligation with T4 Ligase
- Digesting pUPD-Anderson-nodC with NheI and PstI
- Digesting pSB1C3-AraC with SpeI and PstI
- Dephosphorylating the backbone pSB1C3-AraC
- Ligation with T4 Ligase
- 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 of pSB1C3-cod were made
- Transforming pUPD-T7-cod into Top10 and pUPD-T7-nodB-His into BL21 via heat-shock
- 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
- Digesting pUPD-T7-nodB-His with NheI-Hf
- Gel-filtration
- Ligation with T4 ligase
- 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
- 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
Hydrogels
Superior experiment: Switching back to agar/agarose
Procedure:
- 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
- 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:
Chemistry
Superior experiment: Spin Coating and modification of the films
Procedure:
2017-10-09 - 2017-10-15
Chitin Synthase
Superior experiment: Verificate the functionality of NodC
Procedure:
- Repetition of UDPTM Glycosyltransferase Assay twice
Chitin Deacetylases
Superior experiment: Purification
Procedure:
- 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 band is visible by a size of 23kDa, accordingly NodB is present
Hydrogels
Superior experiment: Quercetin as cheap crosslinker
Procedure:
- 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:
Chemistry
Superior experiment: Coupling of the peptide (ala-ala-phe-7amc) to the hydrogel layers and prove the concept
Procedure:
2017-10-16 - 2017-10-22
Chitin Synthase
Superior experiment: Verificate the functionality of NodC
Procedure:
- Repetition of UDPTM Glycosyltransferase Assay
- A thin-layer-chromatography for verification of chitin
Chitin Deacetylases
Superior experiment: Acetic Acid Assay
Procedure:
- Enzyme reaction with 1mM chitin pentamer, 2,5µM NodB and NH4HCO3
- The assay was performed two times with 4 replicates
Hydrogels
Superior experiment: Swelling chitosan hydrogel testing
Procedure:
- 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
- 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%
Chemistry
Superior experiment: Making dilution series and test some more hydrogel films with protease
Procedure:
Technics
Superior experiment: Building a DIHM
Procedure:
2017-10-23 - 2017-10-29
Chitin Synthase
Superior experiment: Verificate the functionality of NodC
Procedure:
- Repetition of UDPTM Glycosyltransferase Assay
Chitin Deacetylases
Superior experiment: Acetic Acid Assay and thin-layer chromatographie
Procedure:
- The assay was performed
Hydrogels
Superior experiment: reproducing our most promising hydrogels
Procedure:
- we had redone our best hydrogel and produced them in a larger scale
General labwork
Superior experiment: DNA submission
Procedure:
- 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: