Overview of Experiments
Team:Goettingen/Experiments
Experiments
1. Production Strains
In order to ensure that the bacteria we are working with are really the ones we want to work with, a 16S rDNA PCR was performed.
As an antibiotic resistance will be used in our later experiments to select transformants, we checked for any native antibiotic resistance against ampicillin, kanamycin, tetracyclin and chloramphenicol in an LB agar plate growth test. It turned out that kanamycin was the only antibiotic, against which none of our strains showed any resistance. For this reason, we decided to use only vectors with kanamycin resistance for our later projects.
After this, we prepared electro competent cells from our strains. All of our strains were grown on LB medium.
2. BioBricks
Our BioBricks were designed using the software SnapGene. The original Genes for our B12 binding proteins, and the torA signal sequence were codon optimized for E. coli.
The DNA containing the Genes were synthesized and friendly provided by IDT Integrated DNA Technologies.
In case of MutB, the length of the gene exceeded the maximum length of 2000 bp from IDT. For this reason, we designed two DNA parts, with a natural occurring HindIII restriction site at both ends. We intended to fuse these both genes by ligation. However, after having diffuculties to fuse the genes by ligation, we decided to fuse them by fusion PCR. In order to multiply the DNA for or cloning experiments, and to equip our genes with restriction sites, PCR was used.
For our purpose, we were searching for a vector with (a) kanamycin resistance and (b) araBAD operon. We thus decided to use the pBAD202 expression system. This vector, however, is equipped with a thioredoxin fusion protein which increases the rate of expression, but prohibits the physiological function of our protein. For this reason, we designed primers for an autarkic synporter protein. In order to test the dependence of the expression system on the B12 export rate, we also decided to use different other vectors.
For vectors ligation, we used a standart ligase reaction, for the pBAD202 expression, which works without ligation but topoisomerase reaction, we followed the manufacturer's protocol.
3. Transformation and Cultivation
For our transformation experiments, we used electroporation for all our self prepared competent cells. For the heat competent E. coli Top10 cells provided with the pBAD202 expression system, we used the manufacturer's heat shock transformation protocol.
For vector multiplication, E. coli cells (Top10 for pBAD202 plasmids, DH5alpha for all other plasmids) were transformed with the constructed plasmids. After the transformation, the cells were cultivated on LB Medium. Transformants were selected, and the respective clones were picked, cultivated in LB medium, and plasmids were extracted.
The Plasmids were then introduced into the production strains: Raoultella planticola ATCC 33531, Shimwellia blattae ATCC 29907, and Salmonella typhimurium TA100.
For the expression experiments, the all cells were cultivated and the expression induced in RM medium under aerobic conditions. As S. blattae is known to produce Vitamin B12 only under anaerobic conditions, the expression was induced under anerobic conditions in RM medium.
The induction was performed using different concentrations of l-arabinose and different induction time spans.
4. Expression Test
To test for the expression of our synporter protein, the cell lysates of the expression cultures we submitted to an SDS-PAGE. From the SDS Gels, Western Blots were performed using anti-Poly-Histidine-tag antibodies.
5. Vitamin B12 Assays
To test the physiological function of our synporter protein, different assays for B12 detection were performed. For the assays, (a) the whole cell filtered medium, and (b) the periplasm fraction of the cells were examined.
The periplasm fraction of the producing cells was gained by ultrazentrifugation.
5.1. Microbial Assay
The microbial B12 assay relies on the growth diameter of the E. coli ATCC 10799 strain on an agar plate, in dependence of the Vitamin B12 (or methionine) concentration in the medium.
For this purpose, agar plates with synthetic Guttman medium (free of B12 and amino acids, 1.5% agarose) were prepared. The E coli detection cells were grown in liquid synthetic Guttman medium supplemented with selected amino acids (no methionine) and Vitmamin B12. Afterwards, the cells were washed with PBS buffer three times, in order to make the cells virtually free of Vitamin B12. The cells were resuspended in PBS buffer, and mixed with warm, liquid Guttman medium (free of B12 and amino acids, 0.8% agarose), and then poured on the prepared agar plates. For the assay, small paper platelets (usually used for antibiotic tests) were placed on the top agar layer, and soaked with differently concentrated calibration solutions of Vitamin B12. The plates were incubated overnight, and the diameters measured after 16 h. From the different diameters, dependent of the Vitamin B12 concentration, a calibration curve was calculated.
In order to determine the B12 content of our medium and periplasm fraction, the assay plates were prepared exactly as described above, and paper platelets were soaked with the respective solution. Comparing to the calibration curve, the diameter indicates the B12 amount.
5.1. Photometric Assay
Protocols
Media
LB Medium
| Component | Amount | |
|---|---|---|
| tryptone | 10 g | |
| NaCl | 10 g | |
| Yeast extract | 5 g | |
| optional: | ||
| Agar-agar | 15 g | |
- Add components to ddH2O and adjust volume to 1 L.
- Autoclave for 20 min at 121 °C.
M9 Minimal Medium
| Component | Amount |
|---|---|
| Na2HPO4 | 10 g |
| KH2PO4 | 10 g |
| NH4Cl | 5 g |
| NaCl | 15 g |
- Add components to ddH2O, adjust the pH to 7.0 and bring the volume to 987 mL.
- Autoclave for 20 min at 121 °C.
- Add the following
| Component | Stock solution | Amount |
|---|---|---|
| glucose | 20 % | 10 mL |
| MgSO4 · 7 H2O | 1 M | 1 mL |
| Thiamine · HCl | 30 mM | 1 mL |
| CaCl2 | 0.13 M | 1 mL |
RM Medium
The dehydrated medium is distributed by Laboratories Conda (Cat. 1542) and was prepared according to protocol.
Guttman Medium (B12 Assay)
| Component | Amount |
|---|---|
| K2HPO4 | 14 g |
| KH2PO4 | 6 g |
| (NH4)2SO4 | 2 g |
| MgSO4 · 7 H2O | 0.2 g |
| Na2citrate · 2 H2O | 1 g |
| L-Asparagine | 0.8 g |
| L-Arginine | 0.2 g |
| L-Glutamic acid | 0.2 g |
| Glycine | 0.2 g |
| L-Tryptophan | 0.2 g |
| L-Prolin | 0.2 g |
| L-Histidine | 0.2 g |
| Vitamin B12 | 40 µg |
- Add components to ddH2O, adjust the pH to 6.8-7.2 and bring the volume to 1 L.
- 50 mM glucose have to be added seperatly before the inoculation.
S. blattae Medium Anaerobic
Mineral medium with additon of yeast extract
| Component | Amount |
|---|---|
| K2HPO4 | 14 g |
| KH2PO4 | 6 g |
| (NH4)2SO4 | 3 g |
| MgSO4 · 7 H2O | 0.2 g |
| Yeast extract | 0.2 g |
| CoCl2 (50 mM) | 85 µL |
| SL-4 | 1 mL |
- Add components to ddH2O, adjust the pH to 7.5 and bring the volume to 1 L.
- 100 mM glycerine are added seperatly to the medium.
SL-4: Trace element solution (Pfenning and Lippert, 1966)
| Component | Amount |
|---|---|
| EDTA-Na2 | 5 g |
| FeSO4 · 7 H2O | 2 g |
| ZnSO4 · 7 H2O | 0.1 g |
| MnCl2 · 4 H2O | 0.03 g |
| H3BO4 | 0.3 g |
| CoCl2 · 6 H2O | 0.2 g |
| CuCl2 · 2 H2O | 0.01 g |
| NiCl2 · 6 H2O | 0.02 g |
| NaMoO4 · 2 H2O | 0.03 g |
- Add components to ddH2O, adjust the pH to 6.7 and bring the volume to 1 L.
- The solution is stored at 4 °C and protected from light.
Cultivation and Transformation
Cultivation of Escherichia coli, Shimwellia blattae and Salmonella typhimurium TA 100
S. blattae, S. typhimurium TA100, R. platicola and E. coli strains are grown in LB medium at 37 °C.
S. typhimurium TA100 has a natural ampicillin resistence. Therefore, 25 µg/mL ampicillin have to be added to the medium.
Cultivation of Raoultella planticola
R. platicola platicola is grown in LB medium at its temperature optimum of 30 °C.
Preperation of the Periplasm for Analysis
- Harvest 500 mL of cells from an induction experiment by centrifugation and wash three times with 0.9 % NaCl.
- Resuspend the pellet in 2 mL buffer P (pH 8.0) and incubate for 20-30 min on ice.
| Component | Concentration |
|---|---|
| Tris-HCl | 100 mM |
| Sucrose | 20 % (w/v) |
| EDTA | 100 mM |
- Centrifuge for 5 min (14000 rpm, 4 °C) and carefully remove the supernatant which contains the periplasm.
- Resuspend the pellet in 2 mL buffer P and incubate for 20-30 min on ice.
- After another centrifugation for 5 min (14000 rpm, 4 °C) the supernatant with the spheroplasts can be carfully removed.
- The samples can either be stored at -20 °C or directly used for analysis.
Preparation of Electrocompetent Cells
Material to prepare
- 2x 5 mL LB
- 250 mL LB in 1 L Erlenmeyer flask with chicane
- 500 mL sterile Millipore-H2O at 4 °C
- 50 mL sterile glycerol (10 % (w/v)) at 4 °C
- 30 sterile labeled Eppendorf cups at – 20°C or -80 °C
- 1x sterile GS3 jar at 4 °C
- 1x Aquatron at 30 °C
- 1x Sorvall RC6 centrifuge at 4 °C
- 1x Universal 320R centrifuge at 4 °C
- 1 L liquid nitrogen
Day 1
- Inoculate 2x 5 mL LB (if required with antibiotics) with your strain of interest, either from cryo culture or from agar plate.
- Incubate overnight at 37 °C and 150 rpm.
Day 2
- Inoculate 250 mL LB (without antibiotics) with 2 % (5 mL) preculture.
- Incubate in the Aquatron at 30 °C and 160 rpm until OD600 of 0.5-0.8.
- Check the culture via microscope for contaminations.
All liquids and containers must be cooled on ice. The major task of cell preparation is the removal of salts. In case of some strains like pLys-strains, the pellets must be resuspended very carefully.
- Let the cells (OD600 of 0.5-0.8) cool down in ice water for 10-20 min. All further steps are performed under cool conditions.
- Decant the cells in sterile GS3 jar and centrifuge at 4 °C and 5000 rpm (4230 g) for 5-10 min.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 250 mL sterile 4 °C ddH2O.
- Centrifuge the GS3 jar at 4 °C and 5000 rpm (4230 g)for 5-10 min.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 250 mL sterile 4 °C ddH2O.
- Centrifuge the GS3 jar at 4 °C and 5000 rpm (4230 g)for 5-10 min.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 10 mL sterile 4 °C glycerol (10 % (w/v)). Afterwards, transfer the culture into a 50 mL Falcon tube.
- Centrifuge the Falcon tube at 4 °C and 6200 rpm (4230 g) for 5-10 min.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 10 mL sterile 4 °C glycerol (10 % (w/v)).
- Centrifuge the Falcon tube at 4 °C and 6200 rpm (4230 g) for 5-10 min.
- Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 500 µL sterile 4 °C glycerol (10 % (w/v)).
- Aliquot the cells into the Eppendorf cups (40 µL per Eppendorf cup). While aliquoting, filled Eppendorf cups must be frozen directly in liquid nitrogen.
- Store the filled Eppendorf cups in a cryobox at -80 °C.
Transformation Electroporation of Electrocompetent Cells
- Mix 50-200 ng plasmid DNA with 40 µL of competent cells (always defrost on ice!). Transfer attempt into precooled electroporation cuvette. Incubate on ice for 10 min.
- Important: DNA must be salt free! Use either directly in ddH2O eluted plasmids, or (e.g. directly after ligation) desalt the DNA for 30 min on a Millipore filter (MF Membrane Filters).
- Electroporation: 2500 V for 2 mm cuvettes (1250 V for 1 mm cuvettes), 25 µF, 200 Ω, discharging time should be 3-5 msec. Contacts of the electroporation cuvette must be dry. Remove all air bubbles before electroporation.
- Immediately add 960 µL liquid LB (or, if available, SOC medium for a higher transformation efficiency) into the cuvette and transfer the content into a 2 ml Eppendorf cup.
- Incubate at 37 °C and 150 rpm for 1 h. Fix tube in horizontal position with tape.
- Plate 100 µL of 10-3 to 10-6 dilutions on plates with selection pressure
- Incubate at 37 °C overnight.
Heat Shock Transformation of Heat Competent Cells
One Shot®TOP10 Competent cells (ThermoFisher) were transformed following the respective protocol for chemical transformation:
- Add 3 µL of the TOPO®Cloning Reaction into a vial of competent E. coli cells and mix gently by inverting.
- Incubate on ice for 5-30 min.
- Heat-shock the cells for 30 s at 42 °C without shaking.
- Immediately transfer the tubes on ice.
- Add 250 µL of S.O.C. medium (RT), cap the tube tightly and shake it horizontally (200 rpm) at 37 °C for 1 h.
- Spread 100-200 µL of different volumes or dilutions from each transformation on a prewarmed selective plate and incubate overnight at 37 °C.
- Pick colonies for analysis.
Plasmid preparation
Small scale preparation of plasmid DNA was performed using the NucleoSpin® Plasmid kit distributed by Macherey-Nagel (REF 740588) according to protocol.
Plasmid Construction
Digestion of DNA with Restriction Enzymes
Plasmid DNA and PCR products were digested according to FastDigest protocol (Thermo Scientific).
| Component | Plasmid DNA | PCR product |
|---|---|---|
| Water, nuclease free | 15 µL | 17 µL |
| 10X FastDigest or 10X FastDigest Green buffer | 2 µL | 2 µL |
| DNA | 2 µL (up to 1 µg) | 10 µL (~0.2 µg) |
| FastDigest enzyme | 1 µL | 1 µL |
- Mix gently and spin down.
- Incubate at 37 °C in a heat block or water thermostat for X min.
- Inactivate the enzyme by heating for X min at X °C.
- If the FastDigest Green Buffer was used in the reaction, an aliquot of the reaction mixture can be loaded directly on an agarose gel.
Incubation time and inactivation time and temperature tepend on the FastDigest enzyme.
Phosphorylation of DNA
MutB2 DNA was phosphorylated with T4 Polynucleotide Kinase (T4 PNK) according to protocol (NEB, #M0201).
| Component | Amount |
|---|---|
| DNA | up to 300 pmol of 5' termini |
| T4 PNK Reaction Buffer (10X) | 5 µL |
| ATP (10 mM) | 5 µL |
| T4 PNK | 1 µL (10 units) |
| Nuclease-free water | up to 50 µL |
- Prepare reaction mixture on ice.
- Incubate at 37 °C for 30 min.
- Heat inactivate at 65 °C for 20 min.
Ligation
Text
Blunt End Ligation
Text
DNA Purification
DNA purification after PCR and restriction was performed using different kits
- SureClean Plus distributed by Bioline (BIO-37047)
- DNA Clean & Concentrator™-25 distributed by Zymo Research (D4033)
- NucleoSpin® Gel and PCR Clean-up distributed by Macherey-Nagel (REF740609)
The procedures were done according to the respective protocol.
Analytic Tests
Agarose Gel Electrophoresis
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Expression Test
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SDS Polyacrylamid Gel Electrophoresis
Acrylamid Stocksolution (AA)
- 40 % Acrylamide 4x cryst.- Mix 37.5:1 (Biomol 54535; stored at 4 °C)
Resolving gel Stocksolution (RG)
| Component | Amount |
|---|---|
| Tris-HCl | 18.2 g |
| SDS | 0.4 g |
- Add components to ddH2O, adjust the pH to 8.8 with HCl and bring the volume to 100 mL.
- Store at 4 °C.
Stacking gel Stocksolution (SG)
| Component | Amount |
|---|---|
| Tris-HCl | 6.1 g |
| SDS | 0.4 g |
- Add components to ddH2O, adjust the pH to 6.8 with HCl and bring the volume to 100 mL.
- Store at 4 °C.
10x running buffer
| Component | Amount |
|---|---|
| Tris-HCl | 30.3 g |
| Glycine | 144.1 g |
| SDS | 10 g |
- Add components to ddH2O and bring the volume to 1 L. The pH should bea around 8.4.
- Store at RT.
4x SDS Loading Dye
| Component | Amount |
|---|---|
| Tris-HCl | 0.4 g |
| SDS | 1.2 g |
| Glycerine (87 % w/v) | 7.5 mL |
| β-Mercaptoethanol | 2.5 mL |
| Bromphenol blue (2%) | 0.5 mL |
- Add Tris-HCl and SDS to ddH2O and adjust the pH to 6.8.
- Add the rest of the compnents and bring the volume to 50 mL
- Store at -20 °C.
Ammoniumpersulfat (APS)
- 10 % APS (w/v) in ddH2O
- Store at -20 °C.
Pipetting schemes for one gel
| Component | Resolving gel 10 % | Stacking gel 4 % |
|---|---|---|
| AA | 1 mL | 0.2 mL |
| RG | 1 mL | - |
| SG | - | 0.48 mL |
| ddH2O | 2 mL | 1.32 mL |
| TEMED | 3 µL | 2 µL |
| APS | 30 µL | 15 µL |
| Component | Resolving gel 15 % | Stacking gel 4 % |
|---|---|---|
| AA | 1.5 mL | 0.2 mL |
| RG | 1 mL | - |
| SG | - | 0.48 mL |
| ddH2O | 1.5 mL | 1.32 mL |
| TEMED | 3 µL | 2 µL |
| APS | 30 µL | 15 µL |
Preperation of SDS-Gels
- Glass plates, combs and spacers are cleaned with EtOH (70 %) and assembled.
- The resolving gel solution is prepared with TEMED and APS added last for polymerisation. The solution is poured between the assembled galss plates and covered with isopropanol.
- After polymerisation, the isopropanol is removed and the stacking gel is prepared and poured on top of the resolving gel. The combs need to be inserted immediatly.
- After polymerisation, the gels can either be put into the electrophoresis chamber with 1x running buffer or stored at 4 °C in a bag with wet tissue.
Preperation of samples
- Samples are mixed with SDS loading dye (4:1), denaturated at 95 °C for 5 min and breifly centrifuged.
- Up to 15 µL of a sample are loaded into one well.
SDS-PAGE
- After loading the samples and a protein marker onto the gel, it is run for 15 min with an electric current of 15 mA current (per gel). Afterwards, the gel is run with an electric current of 30 mA (per gel).
Western Blot
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Microbial B12 Plate Assay
The B12 Plate Assay allows the detection of corrinoids in cell extracts or supernatants. It is based on the fact, that E. coli DSM 4261 relies on its B12 dependent methionine synthase which is encoded by metH. This is caused by a point mutation in metE which encodes the B12 independent methionine synthase. In a medium without methionine, E. coli DSM 4261 therefore relies on the presence of B12.
Cultivation of E. coli DSM 4261
- 50 mL of Guttman medium are inoculated with a 5 mL preparatory overnight culture.
- The cells are harvested at an OD600 of 0.2, washed three times with 20 mL 0.9 % NaCl and afterwards resuspended in the same volume NaCl.
Preparation of samples
- After harvesting the cells from the induction experiments they are washed with 0.1 M KP-buffer:
| Component | Amount |
|---|---|
| K2HPO4 | 13.6 g |
| KH2PO4 · 3 H2O | 22.8 g |
- Add components to ddH2O, adjust the pH tp 7.0 and bring the volume to 1 L.
- Periplasm, cytoplasm and crude cell extracts from different expression studies are used as samples.
Assay
- 7 mL of the cell suspension, 50 mM and 0.2 g/L triphenyltetrazoliumchloride are added to 500 mL of the autoclaved mineral-agar (50 °C).
| Component | Amount |
|---|---|
| K2HPO4 | 7 g |
| KH2PO4 | 3 g |
| (NH4)2SO4 | 1 g |
| MgSO4 · 7 H2O | 0.1 g |
| Na2citrate · 2 H2O | 3 g |
| Agar Bacteriological | 15 g |
- Add components to ddH2O and bring the volume to 1 L.
- 4 sterile filter papers (Ø 0.9 cm, Fa. Schleicher & Schuell, Dassel) are laid onto the plates and loaded with 10-15 µL of a sample or a vitamin B12 calibration solution (0, 0.05, 0.25, 0.1, 5.0 μg/ml). Every sample or concentration is analyzed twice.
- The plates are incubated at 37 °C for 16-24 h.
If the samples contained vitamin B12, the cells grow in a red zone around the filter paper. The size is proportional to the vitamin B12 concentration and the reduction of triphenyltetrazoliumchloride causes the red staining.
- To analyze the B12 concentration in the samples, the diameter of the red zone is measured and compared to the calibrations.
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