Team:Goettingen/Notebook/May

As seen in this figure, both strains grow well on CS-glucose without glyphosate. Strain BP233 also forms a lawn on plates with 10mM glyphosate, while the wildtype does not form a lawn, but single colonies, which could be suppressor mutants. This leads to the hypothesis that GltT, which is a glutamate transporter, transports glyphosate into the cell. The strain BP235 was not stable, so it was constructed again!

Identification of upregulated proteins in presence of glyphosate in Bacillus subtilis

07.05.18

The two B. subtilis strains 168 and the tryptophan prototroph strain SP1 were grown at different glyphosate concentrations. To identify upregulated protein expression in the presence of glyphosate.

For cultivation, the two strains were inoculated in 4 mL LB medium and incubated overnight at 37°C (200 rpm) The overnight cultures were transferred into 4 mL fresh LB medium and grown for 2 h at 28°C (200 rpm).

CS-Glucose medium was prepared with and without tryptophan

Components	Volume (mL)
5 × C salts	10
Iron Ferric Ammonium Citrate (CAF; 2.2 mg/mL)	0.5
III´ salts	0.5
Sodium Succinate (30%)	1
50% glucose	0.5
(Tryptophan	0.5)
Ad to 50 mL with sterile water	100

B. subtilis 168 and B. subtilis SP1 were transferred into CS-Glucose medium containing increasing glyphosate concentrations to an OD₆₀₀ of 0.1. OD measurement was carried out every 2 hours until the cells reached an OD₆₀₀ of ∼2. The cells were harvested by centrifugation for 1 min at 13000 rpm and stored at -20°C.

         <img src="/Notebook/images/notebook_sp1_070518.png">

         <img src="/Notebook/images/notebook_168_070518.png">

Eins

Zwei

Growth curves of the wild type SP1 and the 168 mutant, auxotrophic for tryptophan

14.05.18

Proteins were isolated from cultures by lysing the cells with 50 µL lysis buffer and a Bradford assay was carried out to determine the protein concentration.

Materials for the Bradford assay:

Bradford solution, 5-fold concentration
buffer from protein elution

Procedure:

Prespare for each portein mixture 3 samples with different concentrations.

Sample	Reference sample
800 µL H₂O	800 µL H₂O
0.5, 1.0 & 2.0 µL crude extract	no protein but equal volume of buffer
200 µL Bradford Solution	200 µL Bradford Solution
Sodium Succinate (30%)	1
50% glucose	0.5
(Tryptophan	0.5)
Ad to 50 mL with sterile water	100

After mixing the samples with the protein solutions, the mixtures have to be incubated for 5 minutes at room temperature. Transfer the mixtures and the reference sample to 1.5 mL cuvettes and measure the absorption at a wavelength of 595 nm.

Calculate the amount of protein in your sample using the following formula:

         <img class="article_formula"
              src="/Notebook/images/notebook_formula_140518.png">

Protein concentration:

Sample	Protein concentration (µg/mL)
168 in 0 mM Glyphosate	0.375
168 in 0.5 mM Glyphosate	1.049
168 in 1.5 mM Glyphosate	1.049
SP1 in 0 mM	0.159
SP1 in 0.5 mM	0.496
SP1 in 1.5 mM	1.4045

The protein expression was then further analysed by SDS-PAGE and subsequent silver staining.

The Material

6× SDS loading dye (98 C)
6× SDS loading dye (98 C)
10× Running (PAGE) buffer (98 S)
1.5 M Tris-HCl, pH 8.8 (95 B)
1.56 M Tris-HCl, pH 6.8 (95 B)
TEMED/TMEDA (Tetramethylethylenediamine)
Acrylamide:Bis-Acrylamide (37.5:1, TOXIC!!!)
APS (Ammonium persulfate, 10 %)
SDS (Sodium dodecyl sulfate, 10 %)
Power supply & SDS PAGE devices
Iso-propanol (100 %)

The procedure

Clean the glass plates, combs and mats with ethanol and assemble the glass plate sandwich. Be sure that the plates are well aligned. Place the plates in the casting frame and tighten them. Place the casting frames into the casting stands.
Wear gloves for making the gels because acrylamide is a neurotoxin in its unpolymerized form!!! Running gel for denaturing SDS PAGE (enough for 2 gels).
Components for gel 15 %, 12 %, 10 %
- H₂O 2.3 mL 3.3 mL 4.0 mL
- Acryl-Bisacrylamide 30 % 5.0 mL 4.0 mL 3.3 mL
- 1.5 M Tris-HCl, pH 8.8 2.5 mL 2.5 mL 2.5 mL
- SDS solution 10 % 0.1 mL 0.1 mL 0.1 mL
- APS 10 % 0.1 mL 0.1 mL 0.1 mL
- TEMED 8 µl 8 µl 8 µl
Stacking gel for denaturing SDS PAGE (5 %; enough for two gels)
- H₂O 6.83 mL
- Acryl-Bisacrylamide 30 % 1.3 mL
- 1.5 M Tris-HCl, pH 6.8 0.87 mL
- SDS solution 10 % 0.1 mL
- APS 10 % 0.1 mL
- TEMED 10 µl
Mix the ingredients of the running gel and pour the solution quickly (gel starts to polymerize once TEMED has been added) into the gap between the glass plates. Leave enough room for the stacking gel – appr. 1 cm below the bottom of the comb´s teeth. After a minute you can overlay the solution with iso-propanol or ethanol. This excludes the access of oxygen and plane the surface
After polymerization, pour off the iso-propanol and rinse with distilled water. Mix the reagents for the stacking gel and pour it on top of the running gel till the space is completely full. Then insert the comb. No alcohol overlay is needed due to the comb.
After polymerization clamp the gel into the electrophoresis apparatus and fill it with running buffer. Remove the comb carefully and use a pipette or syringe to wash the wells with running buffer.
The protein samples (up to 15 µl) are mixed with SDS loading buffer (2.5 µl) and incubated for 10 min at 95°C.
After heating briefly centrifuge the samples to spin down the condensed water.
Then you can apply the samples on a SDS PAGE with a pipette or Hamilton microliter syringe. Do not forget to add molecular weight marker in one lane.
Run the gel at 80 V until the front has moved into the running gel. Then you can increase the voltage to 120 V. Run the gel until the running front has reached the bottom of the running gel.
Pry the plates apart by using a gel spacer. The gel can then be stained (Coomassie staining, method 56 or Silver staining, method 57) to visualize the separated proteins or processed further (Western Blotting, method 58).