Wetlab
General Protocols
Agarose Gel Electrophoresis▼
Introduction
The agarose gel needs to be prepared for the future electrophoresis.
Materials
- 0.6 g Agarose
- 60 ml 1x TAE buffer
- Midori Green DNA Dye
- 250 ml Erlenmeyer flask
- 100 ml measuring cylinder
- Scale
- Weighing spoon
- Agarose electrophoresis chamber (BioRad)
- Power output for electrophoresis
Procedure
- Measure 60 ml of 1x TAE buffer and pour it in the Erlenmeyer flask
- Add 0,6 g of Agarose to the solution
- Microwave until the solution is clear, do not overboil
- Before adding the Midori Green Dye make sure, the solution is not hot. Cool it down by pouring water on the outside of the Erlenmeyer flask with water in the sink
- Midori Green Dye is potentially carcinogenic, handle it cautiously, wear gloves and a mask.
- Add 2µl of Midori Green Dye
- Mix by swirling the Erlenmeyer flask around 5-10 times
- Pour agarose gel and let set until solid
- Load gel and let run
SDS PAGE▼
Materials
- pre-made 10% SDS gel
- SERVA Triple Color Protein Standard III
- 1x SDS runnning buffer
- electrophoresis chamber
- Coomassie Staining colution ("Der Blaue Jonas", GPR)
Procedure
- add 2x lämmli buffer to sample
- heat 95°C for 5-10 min
- load gels and run at 200V for 45min
- rinsed gel with VE water, then incubated 10 min with Coomassie Staining colution ("Der Blaue Jonas", GPR) while shaking manually
- rinsed the gel twice with VE water
- scan image
Plasmid Isolation (commercial kit)▼
Introduction
Plasmid Isolation is a crucial step for the future procedures like cloning, DNA sequencing etc. The aim is to purify the plasmid from the bacteria by removing all the cell components, proteins and chromosomal DNA and RNA molecules.
Materials
- Bacteria culture
- 5 ml LB medium
- (1 x 1000)V Antibiotic
- 250 μl P1 Buffer (resuspension buffer)
- 250 μl P2 Buffer (lysis buffer)
- 350 μl N3 Buffer
- 0.5 ml PB Buffer
- 0.75 ml Buffer PE
- 50 μl Nuclease free water
- QIAprep 2.0 spin column
- Microcentrifuge Tube
Procedure
- Add 1-5ml LB medium to autoclaved glass tubes.
- Add antibiotic corresponding to the resistance of the plasmid that needs to be isolated
- With a pipette tip, take some bacteria from a plate and inoculate the LB medium
- Incubate overnight (12-15h) at 120-150 rpm, 37°C, roughly 45° angle
- Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000rpm (6800 * g) for 3 min at room temperature (15-25°C)
- Resuspend pelleted bacterial cells in 250 μl Buffer P1 (resuspension buffer) and transfer to a microcentrifuge tube
- Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue
- Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. If using LyseBlue reagent, the solution will turn colorless
- Centrifuge for 10 min at 13000rpm (17900*g) in a table-top microcentrifuge
- Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30-60s and discard the flow-through
- Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30-60s and discard the flow-through
- Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30-60s and discard the flow-through
- Centrifuge for 1min to remove residual wash buffer
- Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA add 50 μl Nuclease-Free water to the center of the QIAprep 2.0 spin column, let stand for 1min, then centrifuge for 1min
- Store plasmids at -20°C
Making competent cells▼
Day 1: Pre-culture
- Inoculate 5 ml of LB medium in autoclaved glass tube with correct antibiotic with cells
- Inucubate 13-15h at 37°, 120-150 rpm, 45° tilted
- Autoclave a 250-500ml Erlenmeyer for the next day
Day 2
- Re-inoculate E. coli into fresh LB medium by 1:100 dilution and cultivate at 37o C, 150 rpm (we did it with 120 rpm) for 2.5 - 3 h. Note: 200 mL of fresh culture is necessary to prepare 80-100 aliquots of competent cells (100 µL), leading to a 20-fold concentration.
- Note: Since we want to make around 40 aliquots - starting a culture of 100 ml LB with 1 ml of the Pre-Culture should be enough (in a 250 ml Erlenmeyer flask)
- 3. When OD reaches 0.2 - 0.3, cool down the cells to 4°C and harvest the cells at 4°C, 4200 rpm (max. for our centrifuge) for 15 min. Use 50 ml falcons for that.
- Note: Set the centrifuge to 4°C and make a NOTE on the centrifuge indicating the necessary time for low temperature centrifugation. It takes about 30 min to cool down to 4°C. It is important to keep 4°C from this step and manipulate the cells gently - be careful to balance correctly the centrifuge
From this point on keep the cells on ice at all times
- Discard the supernatant and resuspend the cells with COLD 25 mL 0.1 M CaCl2 by pipetting, leave the cells with CaCl2 on ice for 5 min.
- Note: CaCl2 is stored at 4°C. To further cool down CaCl2 it can be placed at -20°C 10min prior to use.
- Centrifuge at 4200rpm for 10 min at 4°C
- Discard the supernatant and gently resuspend the pellet in 4-5 mL of COLD 0.1 M CaCl2 with 25% glycerol
- If you want to do transformations already today and use the cells fresh - it is recommended to resuspend those aliquots with CaCl2 0.1M without glycerol - therefore you would have to separate the part of the culture at step 4 or earlier.
- Incubate the cells on ice for 10 minutes
- Aliquot 100 µL of the competent cells to ice-cold 1.5 mL Eppendorf tubes (put them on ice previous to pipetting) and store them at -80o C. Note: (1) Some protocols require liquid nitrogen for this final cooling step, but it is NOT necessary. Directly transferring these competent cells from ice to -80o C freezer does not influence the transformation efficiency at all. (2) Fresh competent cells could be used on the same day of preparation by keeping them on ice. (3) Final concentration of glycerol could range from 15% to 25%
Sequencing▼
Introduction
The aim of the procedure is to determine the sequence of a plasmid or another DNA fragment
Materials
- 600-1500 ng Plasmid/DNA
- 3 μl of the 20 µM primer
- Sterile Water
- 1.5 ml Eppendorf tube
Procedure
- Perform the plasmid isolation to isolate the plasmid that needs to be sequenced
- In the 1.5 ml Eppendorf tube mix the plasmid (or other DNA fragment) with the corresponding primer
- Add Sterile Water to the final volume of 15 μl
- Label the tubes and send them to the Sequencing
Microvolume Spectrophotometer▼
Introduction
Microvolume Spectrophotometer is used to quantify the amount of DNA, RNA or Protein in a solution
Materials
- Pipette
- Solution
Procedure
- Pipet a single drop (1 μl) of your DNA, RNA, or protein sample on the pedestal and pull down the arm
- Let the spectrophotometer read the solution
- Write down the concentration shown on the screen
Colony PCR▼
Introduction
Colony PCR to verify presence of PctD-LBD plasmid in BL21 DE3 plysS cells
Materials
- Primer 5' T7 promotor fw 10µM
- Primer 3' T7 terminator reverse 10µM
- Nuclease free water
- LB plate with colonies to be checked for presence of plasmid
- PCR tubes (200µl) and pen for labelling
- Thermocylcer
- Pipettes + Pipette Tips
- Ice for cooling
- When using GoTaq 2x Master Mix green:
- GoTaq Green Master Mix, 2x
- When using GoTaq polymerase (non-master mix)
- OneTaq 5x buffer (green color) (promega)
- OneTaq DNA polymerase (promega)
- dNTP mix 10mM (2.5mM each dNTP)
Procedure
- Prepare PCR tubes according to the number of colonies to be screenedby labelling them and placing them on ice. Keep tubes on ice for the following steps.
- PCR-Table
For using non master mix
Reagent
1x volume for 25 µl reaction (µl)
Go Taq buffer 5x
5
dNTP mix 10mM 0.5
FW primer 10µM 1.25
RV primer 10µM 1.25
Nuclease free water 16.875
Go Taq DNA polymerase (5u/μl) 0.125
25
For using 2x GoTaq Master Mix (green)
Reagent 1x volume for 25 µl reaction (µl) 50x
Go Taq Green Master Mix, 2x 12.5 625
FW primer 10µM 1.25 62.5
RV primer 10µM 1.25 62.5
Nuclease free water 10 500
25 1250
- Under the clean bench: Mix all components of the PCR reaction apart from the colony one 1.5 ml tube. Add Polymerase last. After adding the polymerase, mix by vortexing shortly. On ice.
- Transfer 25 µl of the master mix reaction to prepared PCR tubes.
- Use pipette tip to pick a colony from the agar plate and streak them on the plate a little bit to dilute the amount of bacteria you transfer. Then place pipette tip into the correct PCR tube and swirl around. Discard pipette tip. Repeat for all colonies you want to screen.
- Mix contents of PCR tubes by vortexing, then centrifuge with tabletob centrifuge.
- Transfer PCR tubes to Thermocycler and run the following program:
A Temperature C
Initial Denaturation 95 °C 5 min
30x cycle:
Denaturation 95 °C 30 s
Primer annealing depending on primer pair 30 s
Elongation 72 °C 1min /kb
cylce end
Final elongation 72°C 2x the normal elongation time (minimum 5 min)
Cool to room temperature 20 °C infitnite
Gibson Assembly (commercial kit)▼
Introduction
The goal is to assemble together different DNA fragments of our chimeric receptor with a backbone which harbours the reporter protein GFP
Materials
- Gibson MasterMix 2x
- 0.08 pmol DNA (Vector and inserts)
- Nuclease-free water
- Thermocycler
- Ice
Procedure
- Thaw GeneArt Gibson Assembly HiFi Master Mix on ice
- Vortex GeneArt Gibson Assembly HiFi Master Mix immediately before use
- In a microcentrifuge tube on ice, set up the GeneArt™ Gibson Assembly® cloning reaction as described below (1-3 Inserts Assembly):
- Recommended DNA molar ratio vector:insert = 1:1
- Amount of each fragment 0.08 pmol vector 0.08 pmol each insert X µL
- GeneArt™ Gibson Assembly® HF Master Mix 10 µL
- Deionized water volume (10 – X) µL
- Total volume 20 µL
- Incubation time at 50°C 15 minutes
- Mix the reactions by vortexing, spin down and incubate at 50°C for the recommended time. For Positive Control, use 15 minutes incubation time
- After incubation, place the reaction mix on ice and immediately proceed to the transformation step
Transformation (E.coli)▼
Introduction
The aim is to introduce our plasmid of interest into bacteria cells
Materials
- Chemically competent E.coli
- Plasmid DNA
- Agar plates (with appropiate antibiotic)
- S.O.C. medium
- Incubator
- Heating block
- Ice
Procedure
- Thaw a tube of Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
- Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
- Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
- Place on ice for 5 minutes. Do not mix.
- Pipette 950 µl of room temperature SOC into the mixture.
- Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate. Warm selection plates to 37°C.
- Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
- Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours
Buffers and Media
All concentrations are given as final concentrations unless stated otherwise.
10x PBS▼
10x PBS▼
10x PBS▼
Recipe taken from: https://www.sigmaaldrich.com/GB/en/support/calculators-and-apps/1x-phosphate-buffered-saline
- 1330 mMNaCl
- 27 mM KCl
- 100 mM Na2HPO4
- 18 mM KH2PO4
- HCl for adjusting the pH
- MilliQ H20 to 1 l
5x SDS loading buffer/dye▼
This is the recipe for a 5x SDS loading buffer. Use this 1+4 to create samples for SDS PAGE analysis. Remember to heat samples 95°C 5-10 min before loading.
- 250 mM Tris/HCl pH 6.8
- 10 % SDS
- 3 % glycerol
- 5 % beta-mercaptoethanol
- 0.02 % bromophenol blue
10x SDS running buffer▼
Recipe for 10x SDS. Recipe taken from:
https://www.aatbio.com/resources/buffer-preparations-and-recipes/sds-page-10x-sds-running-buffer Concentrations given are final concentrations. Dilute with MilliQ water 1+9.
- Tris 0.25 M
- Glycine 1.9 M
- Sodiumdodecyl sulfate (SDS) 1 % (w/w)
- MilliQ H20 to 1 l
50x TAE buffer▼
- Tris 2 M
- Glacial acetic acid 1 M
- 0.05M EDTA, pH 8.0
- MilliQ water to 1L
HisTrap IMAC binding buffer▼
- 30 mM Tris/HCl
- 300 mM NaCl
- 10 mM imidazole
- 5% [vol/vol] glycerol
- adjust pH to 8.0 with HCl
- -> filter through 0.45µm filter with peristaltic pump
HisTrapIMAC elution buffer▼
- 30 mM Tris/HCl fo
- 300 mM NaCl
- 500 mM imidazole
- 5% [vol/vol] glycerol
- adjust pH to 8.0 with HCl
- -> filter through 0.45µm filter with peristaltic pump
HisTrapIMAC resuspension buffer▼
- 30 mM Tris/HCl
- 300 mM NaCl
- 5% [vol/vol] glycerol
- MilliQ water
ITC buffer▼
Buffer for ITC measurement of PctD-LBD protein according to https://journals.asm.org/doi/10.1128/mbio.03458-21
- 30 mM Tris,
- 5 mM PIPES [piperazine-N,N9-bis(2-ethanesulfonic acid)] disodium salt monohydrate
- 5 mM MES (morpholineethanesulfonic acid) hydrate
- 150 mM NaCl
- 10% (vol/vol) glycerol
- pH 7.5
LB/LB-agar (Miller) medium (1L)▼
- 10g Tryptone
- 10g NaCl
- 5g Yeast Extract
- 15g agar (only for LB-agar medium for plates)
- 1L Milli-Q or distilled water
M9 minimal medium (100 mL)▼
- M9 salts (5X) - 20 mL
- Glucose (20%; Sigma-Aldrich)a - 2 mL
- MgSO4 (1 M; Fisher Scientific)b - 200 μL
- CaCl2 (1 M; Fisher Scientific)b - 10 μL
- H2O - 78 mL (final volume 100 ml)
- a: Filter-sterilize and store at 4°C.
- b: Autoclave and store at room temperature.
M9 salts 5x (1L)▼
- Na2HPO4•7H2O (M=268,07 g·mol−1), 64 g
- -> we have water-free Na2HPO4 (M= 141,96 g·mol−1 )
- -> use 33.89g instead
- KH2PO4, 15 g
- NaCl, 2.5 g
- NH4Cl, 5.0 g
- deionized H2O, to 1 liter
- Divide the salt solution into 200-ml aliquots and sterilize by autoclaving for 15 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle.
LBD-PctD experiments
Expression of PctD-LBD▼
Preparation
- Isolate plasmid containing PctD-LBD acording to plasmid isolation protocol
- Transform BL21 DE3 PLysS cells with isolated plasmid acording to transformation protocol
- Plate transformed cells on LB agar plates substituted with chloramphenicol and kanamycine
Day 1: Pre-Culture
- Inoculate 100 ml LB medium (medium and erlenmeyer flask autoclaved!) substituted with kanamycine (25 µg/mL) and chloramphenicol (17 µg/ml) with 1 single culture of transformed cells
- incubate overnight at 37°C while shaking
Day 2: Main Culture
- Measure OD600 of pre-culture
- Inoculate 500 ml LB medium (medium and erlenmeyer flask autoclaved!) substituted with kanamycine (25 µg/mL) and chloramphenicol (17 µg/ml) with main culture
- Incubate main culture at 37°C while shaking until OD600 = 0.5
- Take sample for SDS PAGE
- induce expression by addition of 1 mM IPTG
- Grow for 5 hours at 37°C while shaking
- Take sample for SDS PAGE
- Measure OD600
- harvest cells:
- transfer cell suspension to centrifugation containers (250 ml fit in each)
- centrifuge at 20,000 g for 20 min at 4°C
- Freeze cells
Cell lysis▼
Note: keep proteins/cell suspension on ice at all times
- Thaw cell pellet on ice
- Resupend cell pellet
- add 20 ml binding buffer
- dissolve 1 tablet of protease inhibitor
- Add DNAse I to final concentration of 500 ng/ml or 0.2 U/ml (2 U total)
- resuspend cells
- transferred protein suspension to 50 ml falcon tube
- lyse cells by sonication:
- keep cells on ice while sonication
- performe sonication with "4 x 4 x 4 x4" settings:
- 4 cycles
- 4 minutes on, 4 min pause between cylces
- 40 % duty cycle
- power output: 4
- Centrifuge at 20000 x g for 20 min at 4 degrees.
- filter supernatent through 0.45 micrometer filter using a syringe
- Take sample for SDS PAGE
Protein Purification▼
Used for purification: Äkta FPLC
column: 5ml HisTrap, flow rate: 2 ml/min
Buffer A: Binding buffer
Buffer B: Elution buffer
- wash with 15 column volumes buffer A
- Take sample for SDS PAGE
- Apply gradient from 0% buffer B to 100% buffer B over 25 column volumes-> fractionated during elution in 1 ml fractions
- cleaning:
- wash with buffer A
- wash with water
- wash with 20 % ethanol
- Confirm purification success by SDS PAGE
Dialysis▼
- cut piece of semipermeable membrane hose (Dialysemembran Spectra/Por , MWCO 3500 Da)
- soak membrane hose in water for 10 min
- use a clip to close hose from one end
- add protein solution to the hose from the open end with a pipette tip
- close second end of the hose with another clip
- fill ITC buffer into 1l glass beaker
- add stirring flea
- attach clips to a styropor floater ring and place hose into beaker
- place beaker onto a magnetic stirrer in 4 °C room
- stirr over night (at least 16h) with ca. 250 rpm
ITC▼
Materials
- Malvern MicroCal PEAQ-ITC
- 300µl PctD -LBD in ITC buffer (c = 13.5 * Kd)
- 40µl choline in ITC buffer (c = 14* protein concentration)
- ITC buffer for washing
Procedure
- Prepare 300 µl protein solution (c = 13.5 * Kd)
- Prepare 40 µl ligand solution (c = 14* protein concentration)
- Degased all solutions utilizing a vacuum pump.
- Wash the chamber of the ITC instrument with buffer 3x
- Rinse the injection needle with ITC buffer.
- Fill the chamber with protein solution.
- Fill injection needle with ligand.
- Set the following instrument settings:
Temperature (C°) 25
Reference power (µcal/s) 10
Feedback High
Stir speed (rpm) 750
Initial Delay (s) 60
Injection spacing (s) 150
Injection duration (s) 4
Chimeric receptor development
DNA fragments amplification (PCRs)▼
Pctd
DNA polymerase Component Volume [µL] Pfu Pfu buffer 10x 5 Pfu 0.5 Q5 GC enhancer 5x 10 dNTPs Mix 1 Primer IGT_002 2.5 Primer IGT_006 2.5 Nuclease-free H2O 27.5 DNA template 1 Total 50 Q5 Q5 High-fidelity MasterMix 2x 12.5 Q5 GC enhancer 5x 5 Primer IGT_002 1 Primer IGT_006 1 Nuclease-free H2O 4.5 DNA template 1 Total 25
Program (thermocycler):
Step Temperature [°C] Time [sec]
Pfu/Q5 Cycle Initial denaturation 95 120 1 Denaturation 95 30 30 Annealing 58 30 30 Elongation 72 180/90 30 Final elongation 72 300/120 1 Storage 20 ∞
Pctd-HAMP
DNA polymerase Component Volume [µL] Pfu Pfu buffer 10x 5 Pfu 0.5 Q5 GC enhancer 5x 10 dNTPs Mix 1 Primer IGT_006 2.5 Primer IGT_008 2.5 Nuclease-free H2O 27.5 DNA template (linearized pkg116) 1 Total 50 Q5 Q5 High-fidelity MasterMix 2x 12.5 Q5 GC enhancer 5x 5 Primer IGT_006 1 Primer IGT_008 1 Nuclease-free H2O 4.5 DNA template 1 Total 25
Program (thermocycler):
Step Temperature [°C] Time [sec]
Pfu/Q5 Cycle Initial denaturation 95 120 1 Denaturation 95 30 30 Annealing 58 30 30 Elongation 72 210/90 30 Final elongation 72 300/120 1 Storage 20 ∞
Envz
DNA polymerase Component Volume [µL] Pfu Pfu buffer 10x 5 Pfu 0.5 dNTPs Mix 1 Primer IGT_003 2.5 Primer IGT_004 2.5 Nuclease-free H2O 37.5 DNA template 1 Total 50 Q5 Q5 High-fidelity MasterMix 2x 12.5 Primer IGT_003 1 Primer IGT_004 1 Nuclease-free H2O 9.5 DNA template 1 Total 25
Program (thermocycler):
Step Temperature [°C] Time [sec]
Pfu/Q5 Cycle Initial denaturation 95 120 1 Denaturation 95 30 30 Annealing 58 30 30 Elongation 72 120/30 30 Final elongation 72 150/45 1 Storage 20 ∞
EnvZ-Hamp
DNA polymerase Component Volume [µL] Pfu Pfu buffer 10x 5 Pfu 0.5 dNTPs Mix 1 Primer IGT_003 2.5 Primer IGT_004 2.5 Nuclease-free H2O 37.5 DNA template 1 Total 50 Q5 Q5 High-fidelity MasterMix 2x 12.5 Primer IGT_003 1 Primer IGT_004 1 Nuclease-free H2O 9.5 DNA template 1 Total 25
Program (thermocycler):
Step Temperature [°C] Time [sec]
Pfu/Q5 Cycle Initial denaturation 95 120 1 Denaturation 95 30 30 Annealing 58 30 30 Elongation 72 120/30 30 Final elongation 72 150/45 1 Storage 20 ∞
Pkg116
DNA polymerase Component Volume [µL] Pfu Pfu buffer 10x 5 Pfu 0.5 dNTPs Mix 1 Primer IGT_005 2.5 Primer IGT_007 2.5 Nuclease-free H2O 37.5 DNA template (linearized pkg116) 1 Total 50 Q5 Q5 High-fidelity MasterMix 2x 12.5 Primer IGT_005 1 Primer IGT_007 1 Nuclease-free H2O 9.5 DNA template (linearized pkg116) 1 Total 25
Program (thermocycler):
Step Temperature [°C] Time [sec]
Pfu/Q5 Cycle Initial denaturation 95 120 1 Denaturation 95 30 35 Annealing 58 30 35 Elongation 72 540/180 35 Final elongation 72 600/240 1 Storage 20 ∞
Plasmid assembly (Gibson assembly)▼
Following recommended protocol on the manual: Mix an equimolar DNA amount of inserts and vector (0.08pmol each)
Component Volume [µL] - Gibson MasterMix 2x 10 - DNA fragments (insert&backbone) X - Nuclease-free H2O (10-X)*
*Fill up until final volume of 20µL
- Mix all components (vortexing)
- Incubation 15min at 50°C
- Place the reaction mix on ice and proceed to the transformation
Transformation into E.coli strains (DH5a, VS1007)▼
- Thaw a tube of Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
- Add 1µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
- Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at exactly 42°C for 30-40 seconds. Do not mix.
- Place on ice for 5 minutes. Do not mix.
- Pipette 950 µl of room temperature SOC into the mixture.
- Place at 37°C for at least 60 minutes. Shake vigorously (250 rpm) or rotate. Warm selection plates to 37°C.
- Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions and concentration in SOC.
- Spread 100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours
Testing of Biosensor
Bacterial growth curves ▼
Day 1
- Inoculate LB medium (supplemented with strain-specific antibiotics) with E.coli
- Let grow overnight at 37°C while shaking at 200 rpm
Day 2
- Inoculate 12 ml LB medium (supplemented with strain-specific antibiotics) with 250µl overnight culture
- Transfer 1ml of culture into cuvette and measure OD600 with Photospectrometer (t0)
- incubate remaining culture at 37°C while shaking at 200 rpm
- Repeat steps 4 and 5 every hour for at least 6h
Fluorescence microscopy▼
Day 1
- Inoculate M9 medium (supplemented with strain-specific antibiotics) with E.coli
- Let grow overnight at 37°C while shaking at 200 rpm
Day 2
- Transfer 1ml of culture into cuvette and measure OD600 with Photospectrometer
- IF OD600 < 0.5: spin down culture and resuspend cell pellet in less M9 medium
- IF OD600 >= 0.5: add Salicylic acid (effective concentration: 600µM) to induce protein expression
- Incubate at 37 °C while shaking at 200 rpm
At least 1h before fluorescence microscopy:
- Transfer 50 µl of each culture into well of 96-well-plate
- Add ligand to an effective concentration of 200m
Fluorescence microscopy:
- Transfer 1 drop of each culture on to agarose slide
- Cover samples with coverslips
- Image slides with fluorescence microscope
Quantification of Fluorescence in Plate Reader▼
Outline:
- Start 10ml over night cultures of cells in LB medium in glass tubes.
- Measure OD600 the next day with spectrohotometer
- Dilute cultures to an OD600 of 0.1 in polypropylene 15 ml falcons in a final volume of 6 ml in M9 medium.
- Grow until OD600 reaches 0.5. Supplement M9 medium with acetylsalicylic acid and acetycholine/choline.
- Measure green fluorescence and absorbence at 600nm with plate reader
- Incubate over the course of 16h. Take samples to measure absorbance at 600nm and green fluorescence at 2h, 4h, 6h, 8h, 16h
Day 1
- start 10 ml overnight cultures for the strains to be tested in glass tubes with M9 medium supplemented with antibiotics
- incubate at 37°C, 200 rpm overnight
Day 2
- place glass tubes on ice to stop growth
- remove 1ml medium to measure OD600 with spectrophotometer
- measure OD600, then dilute cultures into M9 minimal medium to an OD600 of 0.2 in 15ml polypropylene tubes
- mix by swirling, remove 1 ml medium for control measurement of OD600
- incubate cultures in M9 minimal medium at 37°C, 200 rpm until OD600 reaches 0.5
- place tubes on ice to stop growth
- Measure OD600 again; then add Acetylsalicylic acid (2 mM stock, 4μM final concentration) to all cultures and choline or acetlycholine to designated samples
- add 700 μl choline or acetylcholine of 10x stock solutions (3 different stock solutions for each acetylcholine and choline at 100μM, 1mM, 10mM)
- add700μl MilliQ water, sterile filtered to samples with no choline or acetylcholine added to have the same volume in all cultures
- take samples for measurement with plate reader:
- remove 0.6 ml of each culture and transferre it to 1.5ml tubes on ice
- continued incubation of cultures
- meanwhile: pipet 200 μl of samples for plate reader measurement of absorbance at 600nm and green fluorescence
- Incubate cultures at 37°C, 200 rpm for 16h. Take samples to measure absorbance at 600nm and green fluorescence at 2h, 4h, 6h, 8h, 16h
Figure 1: Plate Layout
Drylab
Binding Site Prediction with SSnet▼
Install SSnet from https://github.com/ekraka/SSnet
Preparation of Protein:
- Download Pctd as .pdb File from NCBI (7PRR)
- Remove Acetylcholine from it with PyMol
Preparation of Ligands:
Use SMILE format
- Anatoxin A = CC(=O)C1=CCCC2CCC1N2
- Acetylcholine = CC(=O)OCC[N+](C)(C)C
- Betaine = C[N+](C)(C)CC(=O)[O-]
- Choline = C[N+](C)(C)CCO
Executing via command line:
SSnet_windows.exe -t 7PRRwithoutACh.pdb -l CC(=O)C1=CCCC2CCC1N2 (for Anatoxin A, and p-value)
SSnet_windows.exe -t 7PRRwithoutACh.pdb -l CC(=O)C1=CCCC2CCC1N2 -m grad (for heatmap of binding site)
The heatmap is then safed in a pdb file an can be visualized in PyMol with the command spectrum b, rainbow, minimum = 0.0, maximum = 100.0
References:
- Verma, N.; Qu, X.; Trozzi, F.; Elsaied, M.; Karki, N.; Tao, Y.; Zoltowski, B.; Larson, E.C.; Kraka, E. SSnet: A Deep Learning Approach for Protein-Ligand Interaction Prediction. Int. J. Mol. Sci. 2021, 22, 1392. https://doi.org/10.3390/ijms22031392
SeeSar affinity prediction▼
Install SeeSar from https://www.biosolveit.de/products/seesar/
Preparation of Protein:
- Download Ptcd as pdb File from NCBI (7PRR) or select straight from inside the app
Preparation of Ligands:
- Download Anatoxin A, Acetylcholine, Betaine and Choline as SDF file from PubChem
Detecting Binding Site:
- Select Protein to work with in Protein Mode, than change to Binding Site Mode and detect all binding sites
- Select binding Site where Acetylcholine is inside
Molecular Docking:
- Change to Docking Mode:
- Select or upload Ligand which should be docked inside pocket
- Change sphere size and number of molecule positions
- Run docking
- You can sort the results by best affinity and download them as pdb file to further inspection
Molecular Dynamic Simulation with GROMACS▼
Install GROMACS as adivced by their installation guide (https://manual.gromacs.org/current/install-guide/index.html)
Either use the Linux subsystem for Windows or install it straight on Linux (in our case Ubuntu 18, but there are some packages missing on it).
Follow the Tutorial from bioinformatics review (https://bioinformaticsreview.com/20191210/tutorial-molecular-dynamics-md-simulation-using-gromacs/).
To view the Results as Video:
- Install VMD for Ubuntu.
- Open VMD and load md_0_1.gro into it.
- Then select a trajectory file, ending with .trr or xtc.
- In Graphics and representation replace all by protein so you can only see the protein. Then press play.
Agarose Gel Electrophoresis▼
Introduction
The agarose gel needs to be prepared for the future electrophoresis.
Materials
- 0.6 g Agarose
- 60 ml 1x TAE buffer
- Midori Green DNA Dye
- 250 ml Erlenmeyer flask
- 100 ml measuring cylinder
- Scale
- Weighing spoon
- Agarose electrophoresis chamber (BioRad)
- Power output for electrophoresis
Procedure
- Measure 60 ml of 1x TAE buffer and pour it in the Erlenmeyer flask
- Add 0,6 g of Agarose to the solution
- Microwave until the solution is clear, do not overboil
- Before adding the Midori Green Dye make sure, the solution is not hot. Cool it down by pouring water on the outside of the Erlenmeyer flask with water in the sink
- Midori Green Dye is potentially carcinogenic, handle it cautiously, wear gloves and a mask.
- Add 2µl of Midori Green Dye
- Mix by swirling the Erlenmeyer flask around 5-10 times
- Pour agarose gel and let set until solid
- Load gel and let run
SDS PAGE▼
Materials
- pre-made 10% SDS gel
- SERVA Triple Color Protein Standard III
- 1x SDS runnning buffer
- electrophoresis chamber
- Coomassie Staining colution ("Der Blaue Jonas", GPR)
Procedure
- add 2x lämmli buffer to sample
- heat 95°C for 5-10 min
- load gels and run at 200V for 45min
- rinsed gel with VE water, then incubated 10 min with Coomassie Staining colution ("Der Blaue Jonas", GPR) while shaking manually
- rinsed the gel twice with VE water
- scan image
Plasmid Isolation (commercial kit)▼
Introduction
Plasmid Isolation is a crucial step for the future procedures like cloning, DNA sequencing etc. The aim is to purify the plasmid from the bacteria by removing all the cell components, proteins and chromosomal DNA and RNA molecules.
Materials
- Bacteria culture
- 5 ml LB medium
- (1 x 1000)V Antibiotic
- 250 μl P1 Buffer (resuspension buffer)
- 250 μl P2 Buffer (lysis buffer)
- 350 μl N3 Buffer
- 0.5 ml PB Buffer
- 0.75 ml Buffer PE
- 50 μl Nuclease free water
- QIAprep 2.0 spin column
- Microcentrifuge Tube
Procedure
- Add 1-5ml LB medium to autoclaved glass tubes.
- Add antibiotic corresponding to the resistance of the plasmid that needs to be isolated
- With a pipette tip, take some bacteria from a plate and inoculate the LB medium
- Incubate overnight (12-15h) at 120-150 rpm, 37°C, roughly 45° angle
- Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000rpm (6800 * g) for 3 min at room temperature (15-25°C)
- Resuspend pelleted bacterial cells in 250 μl Buffer P1 (resuspension buffer) and transfer to a microcentrifuge tube
- Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue
- Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. If using LyseBlue reagent, the solution will turn colorless
- Centrifuge for 10 min at 13000rpm (17900*g) in a table-top microcentrifuge
- Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30-60s and discard the flow-through
- Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30-60s and discard the flow-through
- Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30-60s and discard the flow-through
- Centrifuge for 1min to remove residual wash buffer
- Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA add 50 μl Nuclease-Free water to the center of the QIAprep 2.0 spin column, let stand for 1min, then centrifuge for 1min
- Store plasmids at -20°C
Making competent cells▼
Day 1: Pre-culture
- Inoculate 5 ml of LB medium in autoclaved glass tube with correct antibiotic with cells
- Inucubate 13-15h at 37°, 120-150 rpm, 45° tilted
- Autoclave a 250-500ml Erlenmeyer for the next day
Day 2
- Re-inoculate E. coli into fresh LB medium by 1:100 dilution and cultivate at 37o C, 150 rpm (we did it with 120 rpm) for 2.5 - 3 h. Note: 200 mL of fresh culture is necessary to prepare 80-100 aliquots of competent cells (100 µL), leading to a 20-fold concentration.
- Note: Since we want to make around 40 aliquots - starting a culture of 100 ml LB with 1 ml of the Pre-Culture should be enough (in a 250 ml Erlenmeyer flask)
- 3. When OD reaches 0.2 - 0.3, cool down the cells to 4°C and harvest the cells at 4°C, 4200 rpm (max. for our centrifuge) for 15 min. Use 50 ml falcons for that.
- Note: Set the centrifuge to 4°C and make a NOTE on the centrifuge indicating the necessary time for low temperature centrifugation. It takes about 30 min to cool down to 4°C. It is important to keep 4°C from this step and manipulate the cells gently - be careful to balance correctly the centrifuge
From this point on keep the cells on ice at all times
- Discard the supernatant and resuspend the cells with COLD 25 mL 0.1 M CaCl2 by pipetting, leave the cells with CaCl2 on ice for 5 min.
- Note: CaCl2 is stored at 4°C. To further cool down CaCl2 it can be placed at -20°C 10min prior to use.
- Centrifuge at 4200rpm for 10 min at 4°C
- Discard the supernatant and gently resuspend the pellet in 4-5 mL of COLD 0.1 M CaCl2 with 25% glycerol
- If you want to do transformations already today and use the cells fresh - it is recommended to resuspend those aliquots with CaCl2 0.1M without glycerol - therefore you would have to separate the part of the culture at step 4 or earlier.
- Incubate the cells on ice for 10 minutes
- Aliquot 100 µL of the competent cells to ice-cold 1.5 mL Eppendorf tubes (put them on ice previous to pipetting) and store them at -80o C. Note: (1) Some protocols require liquid nitrogen for this final cooling step, but it is NOT necessary. Directly transferring these competent cells from ice to -80o C freezer does not influence the transformation efficiency at all. (2) Fresh competent cells could be used on the same day of preparation by keeping them on ice. (3) Final concentration of glycerol could range from 15% to 25%
- Discard the supernatant and resuspend the cells with COLD 25 mL 0.1 M CaCl2 by pipetting, leave the cells with CaCl2 on ice for 5 min.
- Note: CaCl2 is stored at 4°C. To further cool down CaCl2 it can be placed at -20°C 10min prior to use.
- Centrifuge at 4200rpm for 10 min at 4°C
- Discard the supernatant and gently resuspend the pellet in 4-5 mL of COLD 0.1 M CaCl2 with 25% glycerol
- If you want to do transformations already today and use the cells fresh - it is recommended to resuspend those aliquots with CaCl2 0.1M without glycerol - therefore you would have to separate the part of the culture at step 4 or earlier.
- Incubate the cells on ice for 10 minutes
- Aliquot 100 µL of the competent cells to ice-cold 1.5 mL Eppendorf tubes (put them on ice previous to pipetting) and store them at -80o C. Note: (1) Some protocols require liquid nitrogen for this final cooling step, but it is NOT necessary. Directly transferring these competent cells from ice to -80o C freezer does not influence the transformation efficiency at all. (2) Fresh competent cells could be used on the same day of preparation by keeping them on ice. (3) Final concentration of glycerol could range from 15% to 25%
Sequencing▼
Introduction
The aim of the procedure is to determine the sequence of a plasmid or another DNA fragment
Materials
- 600-1500 ng Plasmid/DNA
- 3 μl of the 20 µM primer
- Sterile Water
- 1.5 ml Eppendorf tube
Procedure
- Perform the plasmid isolation to isolate the plasmid that needs to be sequenced
- In the 1.5 ml Eppendorf tube mix the plasmid (or other DNA fragment) with the corresponding primer
- Add Sterile Water to the final volume of 15 μl
- Label the tubes and send them to the Sequencing
Microvolume Spectrophotometer▼
Introduction
Microvolume Spectrophotometer is used to quantify the amount of DNA, RNA or Protein in a solution
Materials
- Pipette
- Solution
Procedure
- Pipet a single drop (1 μl) of your DNA, RNA, or protein sample on the pedestal and pull down the arm
- Let the spectrophotometer read the solution
- Write down the concentration shown on the screen
Colony PCR▼
Introduction
Colony PCR to verify presence of PctD-LBD plasmid in BL21 DE3 plysS cells
Materials
- Primer 5' T7 promotor fw 10µM
- Primer 3' T7 terminator reverse 10µM
- Nuclease free water
- LB plate with colonies to be checked for presence of plasmid
- PCR tubes (200µl) and pen for labelling
- Thermocylcer
- Pipettes + Pipette Tips
- Ice for cooling
- When using GoTaq 2x Master Mix green:
- GoTaq Green Master Mix, 2x
- When using GoTaq polymerase (non-master mix)
- OneTaq 5x buffer (green color) (promega)
- OneTaq DNA polymerase (promega)
- dNTP mix 10mM (2.5mM each dNTP)
Procedure
- Prepare PCR tubes according to the number of colonies to be screenedby labelling them and placing them on ice. Keep tubes on ice for the following steps.
- PCR-Table
For using non master mixReagent 1x volume for 25 µl reaction (µl) Go Taq buffer 5x 5 dNTP mix 10mM 0.5 FW primer 10µM 1.25 RV primer 10µM 1.25 Nuclease free water 16.875 Go Taq DNA polymerase (5u/μl) 0.125 25
For using 2x GoTaq Master Mix (green)Reagent 1x volume for 25 µl reaction (µl) 50x Go Taq Green Master Mix, 2x 12.5 625 FW primer 10µM 1.25 62.5 RV primer 10µM 1.25 62.5 Nuclease free water 10 500 25 1250 - Under the clean bench: Mix all components of the PCR reaction apart from the colony one 1.5 ml tube. Add Polymerase last. After adding the polymerase, mix by vortexing shortly. On ice.
- Transfer 25 µl of the master mix reaction to prepared PCR tubes.
- Use pipette tip to pick a colony from the agar plate and streak them on the plate a little bit to dilute the amount of bacteria you transfer. Then place pipette tip into the correct PCR tube and swirl around. Discard pipette tip. Repeat for all colonies you want to screen.
- Mix contents of PCR tubes by vortexing, then centrifuge with tabletob centrifuge.
- Transfer PCR tubes to Thermocycler and run the following program:
A Temperature C Initial Denaturation 95 °C 5 min 30x cycle: Denaturation 95 °C 30 s Primer annealing depending on primer pair 30 s Elongation 72 °C 1min /kb cylce end Final elongation 72°C 2x the normal elongation time (minimum 5 min) Cool to room temperature 20 °C infitnite Gibson Assembly (commercial kit)▼
Introduction
The goal is to assemble together different DNA fragments of our chimeric receptor with a backbone which harbours the reporter protein GFP
Materials
- Gibson MasterMix 2x
- 0.08 pmol DNA (Vector and inserts)
- Nuclease-free water
- Thermocycler
- Ice
Procedure
- Thaw GeneArt Gibson Assembly HiFi Master Mix on ice
- Vortex GeneArt Gibson Assembly HiFi Master Mix immediately before use
- In a microcentrifuge tube on ice, set up the GeneArt™ Gibson Assembly® cloning reaction as described below (1-3 Inserts Assembly):
- Recommended DNA molar ratio vector:insert = 1:1
- Amount of each fragment 0.08 pmol vector 0.08 pmol each insert X µL
- GeneArt™ Gibson Assembly® HF Master Mix 10 µL
- Deionized water volume (10 – X) µL
- Total volume 20 µL
- Incubation time at 50°C 15 minutes
- Mix the reactions by vortexing, spin down and incubate at 50°C for the recommended time. For Positive Control, use 15 minutes incubation time
- After incubation, place the reaction mix on ice and immediately proceed to the transformation step
Transformation (E.coli)▼
Introduction
The aim is to introduce our plasmid of interest into bacteria cells
Materials
- Chemically competent E.coli
- Plasmid DNA
- Agar plates (with appropiate antibiotic)
- S.O.C. medium
- Incubator
- Heating block
- Ice
Procedure
- Thaw a tube of Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
- Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
- Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
- Place on ice for 5 minutes. Do not mix.
- Pipette 950 µl of room temperature SOC into the mixture.
- Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate. Warm selection plates to 37°C.
- Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
- Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours
Buffers and Media
All concentrations are given as final concentrations unless stated otherwise.
10x PBS▼
10x PBS▼
10x PBS▼
Recipe taken from: https://www.sigmaaldrich.com/GB/en/support/calculators-and-apps/1x-phosphate-buffered-saline
- 1330 mMNaCl
- 27 mM KCl
- 100 mM Na2HPO4
- 18 mM KH2PO4
- HCl for adjusting the pH
- MilliQ H20 to 1 l
5x SDS loading buffer/dye▼
This is the recipe for a 5x SDS loading buffer. Use this 1+4 to create samples for SDS PAGE analysis. Remember to heat samples 95°C 5-10 min before loading.
- 250 mM Tris/HCl pH 6.8
- 10 % SDS
- 3 % glycerol
- 5 % beta-mercaptoethanol
- 0.02 % bromophenol blue
10x SDS running buffer▼
Recipe for 10x SDS. Recipe taken from: https://www.aatbio.com/resources/buffer-preparations-and-recipes/sds-page-10x-sds-running-buffer Concentrations given are final concentrations. Dilute with MilliQ water 1+9.
- Tris 0.25 M
- Glycine 1.9 M
- Sodiumdodecyl sulfate (SDS) 1 % (w/w)
- MilliQ H20 to 1 l
50x TAE buffer▼
- Tris 2 M
- Glacial acetic acid 1 M
- 0.05M EDTA, pH 8.0
- MilliQ water to 1L
HisTrap IMAC binding buffer▼
- 30 mM Tris/HCl
- 300 mM NaCl
- 10 mM imidazole
- 5% [vol/vol] glycerol
- adjust pH to 8.0 with HCl
- -> filter through 0.45µm filter with peristaltic pump
HisTrapIMAC elution buffer▼
- 30 mM Tris/HCl fo
- 300 mM NaCl
- 500 mM imidazole
- 5% [vol/vol] glycerol
- adjust pH to 8.0 with HCl
- -> filter through 0.45µm filter with peristaltic pump
HisTrapIMAC resuspension buffer▼
- 30 mM Tris/HCl
- 300 mM NaCl
- 5% [vol/vol] glycerol
- MilliQ water
ITC buffer▼
Buffer for ITC measurement of PctD-LBD protein according to https://journals.asm.org/doi/10.1128/mbio.03458-21
- 30 mM Tris,
- 5 mM PIPES [piperazine-N,N9-bis(2-ethanesulfonic acid)] disodium salt monohydrate
- 5 mM MES (morpholineethanesulfonic acid) hydrate
- 150 mM NaCl
- 10% (vol/vol) glycerol
- pH 7.5
LB/LB-agar (Miller) medium (1L)▼
- 10g Tryptone
- 10g NaCl
- 5g Yeast Extract
- 15g agar (only for LB-agar medium for plates)
- 1L Milli-Q or distilled water
M9 minimal medium (100 mL)▼
- M9 salts (5X) - 20 mL
- Glucose (20%; Sigma-Aldrich)a - 2 mL
- MgSO4 (1 M; Fisher Scientific)b - 200 μL
- CaCl2 (1 M; Fisher Scientific)b - 10 μL
- H2O - 78 mL (final volume 100 ml)
- a: Filter-sterilize and store at 4°C.
- b: Autoclave and store at room temperature.
M9 salts 5x (1L)▼
- Na2HPO4•7H2O (M=268,07 g·mol−1), 64 g
- -> we have water-free Na2HPO4 (M= 141,96 g·mol−1 )
- -> use 33.89g instead
- KH2PO4, 15 g
- NaCl, 2.5 g
- NH4Cl, 5.0 g
- deionized H2O, to 1 liter
- Divide the salt solution into 200-ml aliquots and sterilize by autoclaving for 15 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle.
LBD-PctD experiments
Expression of PctD-LBD▼
Preparation
- Isolate plasmid containing PctD-LBD acording to plasmid isolation protocol
- Transform BL21 DE3 PLysS cells with isolated plasmid acording to transformation protocol
- Plate transformed cells on LB agar plates substituted with chloramphenicol and kanamycine
Day 1: Pre-Culture
- Inoculate 100 ml LB medium (medium and erlenmeyer flask autoclaved!) substituted with kanamycine (25 µg/mL) and chloramphenicol (17 µg/ml) with 1 single culture of transformed cells
- incubate overnight at 37°C while shaking
Day 2: Main Culture
- Measure OD600 of pre-culture
- Inoculate 500 ml LB medium (medium and erlenmeyer flask autoclaved!) substituted with kanamycine (25 µg/mL) and chloramphenicol (17 µg/ml) with main culture
- Incubate main culture at 37°C while shaking until OD600 = 0.5
- Take sample for SDS PAGE
- induce expression by addition of 1 mM IPTG
- Grow for 5 hours at 37°C while shaking
- Take sample for SDS PAGE
- Measure OD600
- harvest cells:
- transfer cell suspension to centrifugation containers (250 ml fit in each)
- centrifuge at 20,000 g for 20 min at 4°C
- Freeze cells
Cell lysis▼
Note: keep proteins/cell suspension on ice at all times
- Thaw cell pellet on ice
- Resupend cell pellet
- add 20 ml binding buffer
- dissolve 1 tablet of protease inhibitor
- Add DNAse I to final concentration of 500 ng/ml or 0.2 U/ml (2 U total)
- resuspend cells
- transferred protein suspension to 50 ml falcon tube
- lyse cells by sonication:
- keep cells on ice while sonication
- performe sonication with "4 x 4 x 4 x4" settings:
- 4 cycles
- 4 minutes on, 4 min pause between cylces
- 40 % duty cycle
- power output: 4
- Centrifuge at 20000 x g for 20 min at 4 degrees.
- filter supernatent through 0.45 micrometer filter using a syringe
- Take sample for SDS PAGE
Protein Purification▼
Used for purification: Äkta FPLC
column: 5ml HisTrap, flow rate: 2 ml/min
Buffer A: Binding buffer
Buffer B: Elution buffer
- wash with 15 column volumes buffer A
- Take sample for SDS PAGE
- Apply gradient from 0% buffer B to 100% buffer B over 25 column volumes-> fractionated during elution in 1 ml fractions
- cleaning:
- wash with buffer A
- wash with water
- wash with 20 % ethanol
- Confirm purification success by SDS PAGE
Dialysis▼
- cut piece of semipermeable membrane hose (Dialysemembran Spectra/Por , MWCO 3500 Da)
- soak membrane hose in water for 10 min
- use a clip to close hose from one end
- add protein solution to the hose from the open end with a pipette tip
- close second end of the hose with another clip
- fill ITC buffer into 1l glass beaker
- add stirring flea
- attach clips to a styropor floater ring and place hose into beaker
- place beaker onto a magnetic stirrer in 4 °C room
- stirr over night (at least 16h) with ca. 250 rpm
ITC▼
Materials
- Malvern MicroCal PEAQ-ITC
- 300µl PctD -LBD in ITC buffer (c = 13.5 * Kd)
- 40µl choline in ITC buffer (c = 14* protein concentration)
- ITC buffer for washing
Procedure
- Prepare 300 µl protein solution (c = 13.5 * Kd)
- Prepare 40 µl ligand solution (c = 14* protein concentration)
- Degased all solutions utilizing a vacuum pump.
- Wash the chamber of the ITC instrument with buffer 3x
- Rinse the injection needle with ITC buffer.
- Fill the chamber with protein solution.
- Fill injection needle with ligand.
- Set the following instrument settings:
Temperature (C°) 25 Reference power (µcal/s) 10 Feedback High Stir speed (rpm) 750 Initial Delay (s) 60 Injection spacing (s) 150 Injection duration (s) 4 Chimeric receptor development
DNA fragments amplification (PCRs)▼
Pctd
DNA polymerase Component Volume [µL] Pfu Pfu buffer 10x 5 Pfu 0.5 Q5 GC enhancer 5x 10 dNTPs Mix 1 Primer IGT_002 2.5 Primer IGT_006 2.5 Nuclease-free H2O 27.5 DNA template 1 Total 50 Q5 Q5 High-fidelity MasterMix 2x 12.5 Q5 GC enhancer 5x 5 Primer IGT_002 1 Primer IGT_006 1 Nuclease-free H2O 4.5 DNA template 1 Total 25
Program (thermocycler):Step Temperature [°C] Time [sec]
Pfu/Q5Cycle Initial denaturation 95 120 1 Denaturation 95 30 30 Annealing 58 30 30 Elongation 72 180/90 30 Final elongation 72 300/120 1 Storage 20 ∞ Pctd-HAMP
DNA polymerase Component Volume [µL] Pfu Pfu buffer 10x 5 Pfu 0.5 Q5 GC enhancer 5x 10 dNTPs Mix 1 Primer IGT_006 2.5 Primer IGT_008 2.5 Nuclease-free H2O 27.5 DNA template (linearized pkg116) 1 Total 50 Q5 Q5 High-fidelity MasterMix 2x 12.5 Q5 GC enhancer 5x 5 Primer IGT_006 1 Primer IGT_008 1 Nuclease-free H2O 4.5 DNA template 1 Total 25
Program (thermocycler):Step Temperature [°C] Time [sec]
Pfu/Q5Cycle Initial denaturation 95 120 1 Denaturation 95 30 30 Annealing 58 30 30 Elongation 72 210/90 30 Final elongation 72 300/120 1 Storage 20 ∞ Envz
DNA polymerase Component Volume [µL] Pfu Pfu buffer 10x 5 Pfu 0.5 dNTPs Mix 1 Primer IGT_003 2.5 Primer IGT_004 2.5 Nuclease-free H2O 37.5 DNA template 1 Total 50 Q5 Q5 High-fidelity MasterMix 2x 12.5 Primer IGT_003 1 Primer IGT_004 1 Nuclease-free H2O 9.5 DNA template 1 Total 25
Program (thermocycler):Step Temperature [°C] Time [sec]
Pfu/Q5Cycle Initial denaturation 95 120 1 Denaturation 95 30 30 Annealing 58 30 30 Elongation 72 120/30 30 Final elongation 72 150/45 1 Storage 20 ∞ EnvZ-Hamp
DNA polymerase Component Volume [µL] Pfu Pfu buffer 10x 5 Pfu 0.5 dNTPs Mix 1 Primer IGT_003 2.5 Primer IGT_004 2.5 Nuclease-free H2O 37.5 DNA template 1 Total 50 Q5 Q5 High-fidelity MasterMix 2x 12.5 Primer IGT_003 1 Primer IGT_004 1 Nuclease-free H2O 9.5 DNA template 1 Total 25
Program (thermocycler):Step Temperature [°C] Time [sec]
Pfu/Q5Cycle Initial denaturation 95 120 1 Denaturation 95 30 30 Annealing 58 30 30 Elongation 72 120/30 30 Final elongation 72 150/45 1 Storage 20 ∞ Pkg116
DNA polymerase Component Volume [µL] Pfu Pfu buffer 10x 5 Pfu 0.5 dNTPs Mix 1 Primer IGT_005 2.5 Primer IGT_007 2.5 Nuclease-free H2O 37.5 DNA template (linearized pkg116) 1 Total 50 Q5 Q5 High-fidelity MasterMix 2x 12.5 Primer IGT_005 1 Primer IGT_007 1 Nuclease-free H2O 9.5 DNA template (linearized pkg116) 1 Total 25
Program (thermocycler):Step Temperature [°C] Time [sec]
Pfu/Q5Cycle Initial denaturation 95 120 1 Denaturation 95 30 35 Annealing 58 30 35 Elongation 72 540/180 35 Final elongation 72 600/240 1 Storage 20 ∞ Plasmid assembly (Gibson assembly)▼
Following recommended protocol on the manual: Mix an equimolar DNA amount of inserts and vector (0.08pmol each)
Component Volume [µL] - Gibson MasterMix 2x 10 - DNA fragments (insert&backbone) X - Nuclease-free H2O (10-X)* *Fill up until final volume of 20µL
- Mix all components (vortexing)
- Incubation 15min at 50°C
- Place the reaction mix on ice and proceed to the transformation
Transformation into E.coli strains (DH5a, VS1007)▼
- Thaw a tube of Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
- Add 1µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
- Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at exactly 42°C for 30-40 seconds. Do not mix.
- Place on ice for 5 minutes. Do not mix.
- Pipette 950 µl of room temperature SOC into the mixture.
- Place at 37°C for at least 60 minutes. Shake vigorously (250 rpm) or rotate. Warm selection plates to 37°C.
- Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions and concentration in SOC.
- Spread 100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours
Testing of Biosensor
Bacterial growth curves ▼
Day 1
- Inoculate LB medium (supplemented with strain-specific antibiotics) with E.coli
- Let grow overnight at 37°C while shaking at 200 rpm
Day 2
- Inoculate 12 ml LB medium (supplemented with strain-specific antibiotics) with 250µl overnight culture
- Transfer 1ml of culture into cuvette and measure OD600 with Photospectrometer (t0)
- incubate remaining culture at 37°C while shaking at 200 rpm
- Repeat steps 4 and 5 every hour for at least 6h
Fluorescence microscopy▼
Day 1
- Inoculate M9 medium (supplemented with strain-specific antibiotics) with E.coli
- Let grow overnight at 37°C while shaking at 200 rpm
Day 2
- Transfer 1ml of culture into cuvette and measure OD600 with Photospectrometer
- IF OD600 < 0.5: spin down culture and resuspend cell pellet in less M9 medium
- IF OD600 >= 0.5: add Salicylic acid (effective concentration: 600µM) to induce protein expression
- Incubate at 37 °C while shaking at 200 rpm
At least 1h before fluorescence microscopy:
- Transfer 50 µl of each culture into well of 96-well-plate
- Add ligand to an effective concentration of 200m
Fluorescence microscopy:
- Transfer 1 drop of each culture on to agarose slide
- Cover samples with coverslips
- Image slides with fluorescence microscope
Quantification of Fluorescence in Plate Reader▼
Outline:
- Start 10ml over night cultures of cells in LB medium in glass tubes.
- Measure OD600 the next day with spectrohotometer
- Dilute cultures to an OD600 of 0.1 in polypropylene 15 ml falcons in a final volume of 6 ml in M9 medium.
- Grow until OD600 reaches 0.5. Supplement M9 medium with acetylsalicylic acid and acetycholine/choline.
- Measure green fluorescence and absorbence at 600nm with plate reader
- Incubate over the course of 16h. Take samples to measure absorbance at 600nm and green fluorescence at 2h, 4h, 6h, 8h, 16h
Day 1
- start 10 ml overnight cultures for the strains to be tested in glass tubes with M9 medium supplemented with antibiotics
- incubate at 37°C, 200 rpm overnight
Day 2
- place glass tubes on ice to stop growth
- remove 1ml medium to measure OD600 with spectrophotometer
- measure OD600, then dilute cultures into M9 minimal medium to an OD600 of 0.2 in 15ml polypropylene tubes
- mix by swirling, remove 1 ml medium for control measurement of OD600
- incubate cultures in M9 minimal medium at 37°C, 200 rpm until OD600 reaches 0.5
- place tubes on ice to stop growth
- Measure OD600 again; then add Acetylsalicylic acid (2 mM stock, 4μM final concentration) to all cultures and choline or acetlycholine to designated samples
- add 700 μl choline or acetylcholine of 10x stock solutions (3 different stock solutions for each acetylcholine and choline at 100μM, 1mM, 10mM)
- add700μl MilliQ water, sterile filtered to samples with no choline or acetylcholine added to have the same volume in all cultures
- take samples for measurement with plate reader:
- remove 0.6 ml of each culture and transferre it to 1.5ml tubes on ice
- continued incubation of cultures
- meanwhile: pipet 200 μl of samples for plate reader measurement of absorbance at 600nm and green fluorescence
- Incubate cultures at 37°C, 200 rpm for 16h. Take samples to measure absorbance at 600nm and green fluorescence at 2h, 4h, 6h, 8h, 16h
Figure 1: Plate Layout
Drylab
Binding Site Prediction with SSnet▼
Install SSnet from https://github.com/ekraka/SSnet
Preparation of Protein:
- Download Pctd as .pdb File from NCBI (7PRR)
- Remove Acetylcholine from it with PyMol
Preparation of Ligands:
Use SMILE format
- Anatoxin A = CC(=O)C1=CCCC2CCC1N2
- Acetylcholine = CC(=O)OCC[N+](C)(C)C
- Betaine = C[N+](C)(C)CC(=O)[O-]
- Choline = C[N+](C)(C)CCO
Executing via command line:
SSnet_windows.exe -t 7PRRwithoutACh.pdb -l CC(=O)C1=CCCC2CCC1N2 (for Anatoxin A, and p-value)
SSnet_windows.exe -t 7PRRwithoutACh.pdb -l CC(=O)C1=CCCC2CCC1N2 -m grad (for heatmap of binding site)
The heatmap is then safed in a pdb file an can be visualized in PyMol with the command spectrum b, rainbow, minimum = 0.0, maximum = 100.0
References:
- Verma, N.; Qu, X.; Trozzi, F.; Elsaied, M.; Karki, N.; Tao, Y.; Zoltowski, B.; Larson, E.C.; Kraka, E. SSnet: A Deep Learning Approach for Protein-Ligand Interaction Prediction. Int. J. Mol. Sci. 2021, 22, 1392. https://doi.org/10.3390/ijms22031392
SeeSar affinity prediction▼
Install SeeSar from https://www.biosolveit.de/products/seesar/
Preparation of Protein:
- Download Ptcd as pdb File from NCBI (7PRR) or select straight from inside the app
Preparation of Ligands:
- Download Anatoxin A, Acetylcholine, Betaine and Choline as SDF file from PubChem
Detecting Binding Site:
- Select Protein to work with in Protein Mode, than change to Binding Site Mode and detect all binding sites
- Select binding Site where Acetylcholine is inside
Molecular Docking:
- Change to Docking Mode:
- Select or upload Ligand which should be docked inside pocket
- Change sphere size and number of molecule positions
- Run docking
- You can sort the results by best affinity and download them as pdb file to further inspection
Molecular Dynamic Simulation with GROMACS▼
Install GROMACS as adivced by their installation guide (https://manual.gromacs.org/current/install-guide/index.html)
Either use the Linux subsystem for Windows or install it straight on Linux (in our case Ubuntu 18, but there are some packages missing on it).
Follow the Tutorial from bioinformatics review (https://bioinformaticsreview.com/20191210/tutorial-molecular-dynamics-md-simulation-using-gromacs/).
To view the Results as Video:
- Install VMD for Ubuntu.
- Open VMD and load md_0_1.gro into it.
- Then select a trajectory file, ending with .trr or xtc.
- In Graphics and representation replace all by protein so you can only see the protein. Then press play.