Team:Goettingen/Experiments

Experiments

Overview of 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 Assay

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.

B₁₂ Assays

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.

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.

PCR methods

Entry 1

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ENtry 2

Entry 3

Entry 4

Plasmid Construction

Entry 1

ENtry 2

Entry 3

Entry 4

Analytic Tests

Agarose Gel Electrophoresis

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

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.

Team:Goettingen/Experiments

Experiments

Overview of Experiments

1. Production Strains

2. BioBricks

3. Transformation and Cultivation

4. Expression Test

5. Vitamin B12 Assay

B12 Assays

5.1. Microbial Assay

5.1. Photometric Assay

Protocols

Media

LB Medium

M9 Minimal Medium

RM Medium

Guttman Medium (B12 Assay)

S. blattae Medium Anaerobic

Cultivation and Transformation

Cultivation of Escherichia coli, Shimwellia blattae and Salmonella typhimurium TA 100

Cultivation of Raoultella planticola

Preparation of Electrocompetent Cells

Transformation Electroporation of Electrocompetent Cells

Heat Shock Transformation of Heat Competent Cells

PCR methods

Entry 1

ENtry 2

Entry 3

Entry 4

Plasmid Construction

Entry 1

ENtry 2

Entry 3

Entry 4

Analytic Tests

Agarose Gel Electrophoresis

SDS Polyacrylamid Gel Electrophoresis

Western Blot

Microbial B12 Plate Assay

Contact

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5. Vitamin B₁₂ Assay

B₁₂ Assays

Guttman Medium (B₁₂ Assay)

Microbial B₁₂ Plate Assay