Team:Aachen/Notebook

Our Lab Notebook

Hitchhiker's guide to the lab! Follow our working process by going on a journey through our Labolatoral System

In our notebook, you can take a look at what we were doing in the laboratory week after week accompanied by many vivid photos. We linked our protocols so you can replicate our experiments yourselves and also get energized by M.A.R.S.

Click on the planets to get information about the activities of the respective month

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Month 1
June

Month 2
July

Month 3
August

Month 4
September

Month 5
October

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Month 1

Week 1

Week 2

Week 3

Week 4

Week 5

Experiments

• Getting used to the equipment in the lab
• LB medium (protocol) and TB medium (protocol) for Escherichia coli

• Plasmid extraction of pET-28a from E.coli BL21 with Macherey-Nagel Plasmid-DNA purification kit "NucleoSpin Plasmid" (pre-cultures prepared from Lilin Feng). Concentration of plasmid measured with nanodrop: 144.7 ng/µL
• Pre-cultures for plasmid extraction of pET-28 a from E. coli BL21

• Plasmid extraction of pET-28 a from E. coli BL21 with Macherey-Nagel Plasmid-DNA purification kit “NucleoSpin Plasmid” Concentration of plasmid measured with nanodrop: 38 ng/µL
• Medium with antifoam for Saccharomyces(protocol)
• Cultures for anchor peptides and centrifugation of Cg-Def, LCI, MBP-1, TA2, eGFP (protocol)

• Medium for Halobacterium (protocol) and agar plates

• Buffer Tris 50 mM
• Cell lysis with ultrasonicator and centrifugation of anchor peptides (protocol) ➤
• Because the anchor peptides are fused to eGFP, the green/yellow colour of eGFP is visible after centrifugation
• Binding tests anchor peptides Cg-Def, LCI, MBP-1, TA2, eGFP with magnetic particles coated with polystyrene (protocol)
• Sodium-Bicine buffer 0.2 M (protocol) and lipid solution 10 mM DMPC

Results

 

• Sequencing of pET-28a to check for restriction sites  ➤
• Cleavage site of EcoRI found in the sequenced plasmid pET-28a   ➤
• Cleavage site of NcoI found in the sequenced plasmid pET-28a   ➤

 

 

• Negative control with eGFP showed no binding ➤
• All anchor peptides showed binding to the magnetic particles, MBP-1 is the most fluorescent and therefore binds the best  
• DMPC/Liposome  ➤

Week 2.

• Sequencing of pET-28a to check for restriction sites
• Sequence of pET-28a with cleavage sites of EcoRI and NcoI marked in red

Week 2.

• Cleavage site of EcoRI found in the sequenced plasmid pET-28a

Week 2.

• Cleavage site of NcoI found in the sequenced plasmid pET-28a

Week 5.

• Anchor peptides after centrifugation

Week 5. Result 2

• Before washing
• After washing

eGFP

LCI

MBP-1

Cg-Def

TA2

Week 5. Result 3

• First try of film rehydration method with lipid 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC). DMPC in Chloroform was dried in a rotary evaporator and rehydrated in Sodium-Bicine buffer

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Month 2

Week 5

Week 6

Week 7

Week 8

Week 9

Experiments

• First own tries of liposome production with rehydration method (protocol)

• Cultures of Saccharomyces cerevisiae CEN.PK113-17A
• Yeast medium
• Yeast medium YPG (protocol)
• Tris-HCl buffer 100mM for purification and storage buffer 20 mM for mitochondria (protocol)
• Liposomes with first lipids 10 mM DMPC

• Cultures of Saccharomyces cerevisiae CEN.PK113-17A
• Yeast medium YPG (protocol)
• Liposomes with rehydration method with BODIPY (protocol link) to get used to fluorescent microcopy.
• Isolation of mitochondria from Saccharomyces cerevisiae CEN.PK113-17A (protocol) ➤

• LB medium and LB agar plates (protocol)
• Pre-cultures of Halobacterium salinarium and plates

• Preparation of Halobacterium medium  ➤
• Main cultures of H. salinarium (protocol)
• Microscopy of old DMPC liposomes from 09.07.  ➤

Results

 

 

 

 

 

Week 7.

• Isolated mitochondria from S. cerevisiae

Week 9.

• Halobacterium salinarium on the agar plate
• Main cultures of H. salinarium

Week 9.

• Liposomes still visible 3 weeks after rehydration

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Month 3

Week 9

Week 10

Week 11

Week 12

Week 13

Experiments

• Sucrose solutions 45%, 40%, 38%, 36%, 16%
• Halobacterium medium (protocol)

• Layering of sucrose solutions  ➤
• Dry lipid film with polar soybean solution

• TE buffer for the resuspension of IDT gBlocks (short DNA, Insert) (protocol)
• Resuspension of gblock DNA ordered from IDT (insert I1-60GFPHis) for transformation and measuring concentration with nanodrop 126.0 ng/µL; Restriction of insert I1-60GFPHis and plasmid pET-28 a with EcoRI and NcoI as well as ligation (resuspension protocol)(transformation protocol)
• Centrifugation and resuspension of H. salinarium  ➤
• Rehydration of soybean-lipids dry lipid film ➤

• LB medium for E. coli
• Second part of centrifugation and resuspension of H. salinarium  ➤
• Transformation of E. coli C43 and E. coli BL21 with last week prepared plasmid with insert (pET-28 a with I1-60GFPHis); Plates of transformed E. colis and after growth on plates liquid pre-culture in tubes. Main cultures and inducing with IPTG.
• LB medium for E. coli (protocol)
• Tris-HCl 20mM buffer (protocol)
• Layering of sucrose solutions and sucrose-density-centrifugation of H. salinarium solutions for bacteriorhodopsin
• First liposome binding tests with anchors
• Precultures of E. coli strains BL 21 and C 43 for each anchor peptide

• Centrifugation of E. coli main cultures to find positive clones which are supposed to be green fluorescent
• TE- buffer for transformation (protocol)
• 2 dry lipid films of polar-soybean-lipids
• Centrifugal filter tube for washing step in binding tests found. Test centrifugation of soybean liposomes in filter tubes destroyed the liposomes.

Results

 

 

 

• Sucrose-density-centrifugation of H. salinarium solutions for bacteriorhodopsin did not work due to wrong rotor in centrifuge. Swinging rotor needed  ➤
• No successful liposome binding tests due to lack of a necessary washing step of the liposomes.

• Centrifugation of E. coli main cultures: no fluorescence detectable
• Centrifugal filter tube for washing step in binding tests found. Test centrifugation of soybean liposomes in filter tubes destroyed the liposomes.

Week 10

• Video of layering of sucrose solutions

Week 11

• Pellet after centrifuged for bacteriorhodopsin isolation
• Resuspended pellet of bacteriorhodopsin isolation

Week 11.

• Soy lipid liposomes

Week 12.

• Pellet after centrifugation for bacteriorhodopsin isolation (bacteriorhodopsin is purple)

Week 12.

• After sucrose density centrifugation: no separation due to wrong rotor

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Month 4

Week 14

Week 15

Week 16

Week 17

Week 18

Experiments

• Micro-extrusion of fresh soybean liposomes with 1 µm filter  ➤

• Pre-cultures, main cultures, inducing and centrifugation of anchor peptides cgDEF, LCI, TA2, MBP-1 and eGFP for negative control (protocol)  ➤
• Dry lipid film and rehydration of soy bean lipids with sodium-bicine buffer
• Test dialysis of soy bean lipid-liposomes with sodium-bicine buffer

• Lipid solution with new lipids 30% DOTAP, 35% DOPC and 35% PC prepared and two dry lipid films

• 30mM Tris buffer (protocol)
• Try liposomes with new lipids: 30% DOTAP, 35% DOPC and 35% PC in 30mM Tris buffer (protocol)
• First try of the positive control for the CAR Reaction in Jülich: didn´t work. We also prepared the stock solutions for more reactions

• 1M sorbitol buffer (protocol)
• Second try of liposome formation with new lipids: 30% DOTAP, 35% DOPC and 35% PC with 1M sorbitol buffer
• Liposomes with polar-soybean-lipids and stained with nil red ➤
• Testing the positive control at FZ Jülich of the CAR reaction. (protocol)

Results

 

• After 48 hours of dialysis were less but still a few liposomes visible  ➤

 

• Try liposomes with new lipids: no liposome formation
• First try of the positive control for the CAR Reaction in Jülich: didn´t work.

• Second try of liposomes with new lipids: no liposome formation
• Testing the positive control at FZ Jülich of the CAR reaction: results for the substrate concentration weren´t as expected.

Week 14.

• Micro-extrusion of fresh soybean liposomes

Before

After

Week 15.

• MBP-1 anchor peptide fused to eGFP and eGFP as negative control after centrifugation. Green/Yellow colour due to eGFP

Week 15.

• After 48 hours of est dialysis of soy bean lipid-liposomes with sodium-bicine buffer were less but still a few liposomes visible

Before

After

Week 17.

• Microscope pictures of soybean-liposomes stained with nile red

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Month 5

Week 18

Week 19

Week 20

Week 21

Week 22

Experiments

• New lipid mixture of 30% DOTAP, 35% DOPC and 35% PC rehydrated with 1M Sorbitol buffer and liposome formation
• Dilution of lipid mixture (1:10 and 1:100) and liposome formation
• Liposomes with only one of the lipids each: DOTAP, DOPC and POPC  ➤
• Transformation of E. coli BL21 with new ordered plasmid with I1-60GFPHis (orderd from Twist Bioscience). No Restriction needed, only steps … in the corresponding protocol. Resuspension of the DNA with 120 µL TE buffer. After transformation growth on plates (protocol)
• Dry lipid film of POPC-lipids

• Cultures of transformed E. coli s with I1-60FPHis from plates to liquid cultures, inducing of main cultures with IPTG and centrifugation  ➤
• Phosphate-solution and solubilisation buffer (protocol)
• TB medium for E. coli (protocol)
• Liposome binding tests with anchor peptides LCI, cgDef and eGFP as negative control. Used buffer was 30mM Tris (protocol)  ➤
• Dry lipid films with POPC lipids and rehydration
• Whole cell approach at FZ Jülich for CAR reaction. (protocol) .
• Calibration for the alcohol in the HPLC
• Positive control again (protocol)

• Dialysis of PC liposomes with 0,5 mM pyranin  ➤
• Rehydration of POPC dry lipid film with 30 mM Tris-HCl buffer with 0.5 mM pyranin. ➤
• Binding tests with anchor peptides and POPC liposomes. Different concentrations of anchor peptides tried: 1 µM, 0.5 µM, 0.1 µM, 0.05 µM, 0.01 µM
• Positive control for the CAR reaction at FZ Jülich (protocol). Data was as expected.
• Liposome formation with a lipid batch of 80 µL POPC, 20 µL DOTAP and 20 µL of the fluorescent labeled DOPE lipid (protocol) ➤

• PBS buffer pH 7.4, 10x PBS buffer (protocol)and Stripping buffer pH 2 for gel electrophoresis
• Two mini gels: Separation gel and stacking gel (protocol), storage in wet environment for next week at 4°C.
• Watched microfluidics with liposomes done by Volkan
• Dry lipid film and rehydration of DOTAP lipids with Tris-HCL 30 mM pH 7.4
• Binding tests with DOTAP liposomes with strep-eGFP,strep-LCI and Met rich
• Protein purification of the anchor peptides TA2 and cg-Def at FZ Jülich (protocol). Preparation of buffer for purification (equilibration, washing, elution and desalting buffer) (equilibration protocol) (elution protocol)(desalting protocol).
• Liposome formation of a lipid batch with 100 µL POPC. Binding tests with anker peptides TA2, cg-Def, Cecropin A, Maquace, eGFP and LCI (protocol)

 

Results

• New lipid mixture: no liposomes visible
• Dilution of lipid mixture: no liposomes visible

• Cultures of transformed E. colis with I1-60GFPHis from plates to liquid cultures, inducing of main cultures with IPTG and centrifugation: no fluorescence visible after centrifugation; made a new kryostock of transformed E. coli
• E. coli carry the plasmid, because they are able to growth on the plates with kanamycin antibiotic (the plasmid contains a kanamycin resistance)
• Liposome binding tests with anchor peptides:Anchors cannot get through the dialysis tube, no fluorescence outside the tube but still inside, even if no liposomes are added
• GUV formation with POPC liposomes and Sorbitol 1 M buffer (protocol), GUV were visible and there were more bigger liposomes  ➤
• Data for the whole cell approach was as expected.
• Data for the positive control of the CAR reaction still not as expected.

• After the liposomes were rehydrated with the fluorescent dye pyranine they appeared as dark spots under the fluorescence microscope  ➤
• Dilution of lipid mixture: no liposomes visible
• After the dialysis step which should remove pyranine from the outer phase the liposomes still appeared darker than the background. This could be because the pH of the buffer increased during the dialysis for unknown reasons from 7.4 to 7.76 and the fluorescence of pyranine is highly pH sensitive
• No fluorescence visible with 0.1 µM, 0.05 µM, 0.01 µM anchor peptides
• Too much fluorescence visible at 1 µM anchor peptides
• Liposomes with fluorescence visible with 0.5 µM
• We experienced some difficulties with liposome formation. Probably our lipids got too old or maybe oxidized.

• Binding tests: no liposomes visible after incubation for binding. Try new tests with less shaking.
• Protein purification of the anchor peptides TA2 and cg-Def at FZ Jülich: concentration of TA2 0.164 mg/mL and for cg-Def 0.163 mg/mL

 

Week 18.

• Liposomes with only one of the lipids each: DOTAP, DOPC and POPC
• No liposomes visible

DOTAP

DOPC

PC

Week 19.

• Agar plate with transformed E. coli s with the I1-60GFP His insert

Week 19.

• GUV´s with POPC lipids stained with nil red

Week 20.

• Dialysis of PC liposomes with 0,5 mM pyranin

Before

 

After

Week 20.

• In Brightfield mode
• In GFP mode (exposure time 781 µs, gain 5.5 dB)

Week 20.

• Liposomes were visible (picture 1) and they showed a higher fluorescence than the background (picture 2)
• With this results more tests should be performed with concentrations of 0.4 µM, 0.5 µM and 0.6 µM

Week 20.

• Liposomes under the confocal microscope

Week 21.

• Pictures of binding tests with every anchor peptide with a concentration of 0.5 µM
• Brightfield mode
• eGFP mode

eGFP

LCI

Met Rich

Cecropin A

MA

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