Team:Stuttgart/Experiments

Methods

Media and antibiotic

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LB Agar for E.coli

1. 10 g/L tryptone.

2. 10 g/L NaCl.

3. 5 g/L yeast extract.

4. 20 g/L agar.

5. Autoclave the bottle with medium.

6. Add 1ml of 150 mg/mL of ampicillin solution into 1 liter medium.

LB Medium for E.coli

1. 10 g/L tryptone.

2. 10 g/L NaCl.

3. 5 g/L yeast extract.

4. Autoclave the bottle with medium.

5. Add 1ml of 150 mg/mL of ampicillin solution into 1 liter medium.

TB-Medium

1. 12 g to 900 ml tryptone.

2. 5 g to 900 ml glycine.

3. 24 g to 900 ml yeast extract.

4. Autoclave bottle with medium.

5. Add 100 ml 10x TB-Salt

TB salt

1. 23,1 g/L KH2PO4.

2. 125,4 g/L K2HPO4.

Ampicillin solution

1. Prepare a solution containing a mass concentration of 150 mg/mL of ampicillin.

2. Dissolve ampicillin in dH20.

3. Filter the ampicillin solution through a syringe with a suitable filtration tip.

4. Aliquot the solution and store at -20 °C.

5. 1ml Ampicillin was added to 1 L medium.

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Cloning

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Processing of the ordered primers and fragments

1. Centrifuge the fragments and primers with Short Spin for approx. 10 sec.

2. Dilute the fragments and primers with ddH2O (final concentrations 50 nM for fragments and 100 µM for primers).

3. Vortex thoroughly

4. Short spin for approx. 10 sec.

Primer and fragment sequences

Sequence of wildtype dsup fragment, optimized for E.coli : here is the sequence

Sequence of N-terminal dsup fragment, optimized for E.coli : here is the sequence

Sequence of C-terminal dsup fragment, optimized for E.coli : here is the sequence

Sequence of C-terminal dsup fragment, optimized for HeLa : here is the sequence

Sequence of wildtype dsup forward primer, optimized for E.coli : here is the sequence

Sequence of wildtype dsup reverse primer, optimized for E.coli : here is the sequence

Sequence of N-terminal dsup forward primer, optimized for E.coli : here is the sequence

Sequence of N-terminal dsup reverse primer, optimized for E.coli : here is the sequence

Sequence of C-terminal dsup forward primer, optimized for E.coli : here is the sequence

Sequence of C-terminal dsup reverse primer, optimized for E.coli : here is the sequence

Sequence of N-terminal dsup forward primer, optimized for HeLa : here is the sequence

Sequence of N-terminal dsup reverse primer, optimized for HeLa : here is the sequence

PCR for amplification

 

1.    pBAD18 and the dsup Fragments for E. coli

Method for 50 µl approach:

·       The components listed in Table 1 were mixed in a PCR tube and run threw the cycles listed in Table 2.

Table 1: Reagents and corresponding concentrations used for a single PCR amplification

Components	Volume	Concentration
Q5 2x Master Mix	25 µl	-
10 µM Primer fw	2,5 µl	0,5 µM
10 µM Primer rv	2,5 µl	0,5 µM
ddH2O	19 µl	-
DNA Template	1 µl	-

 

Table 2: General heat cycler program for PCR amplification. The cycle steps were repeated for 30 times for the fragments and 40 times for the vector

Step	Temp for fragment	Time for fragment	Temp for vector	Time for vector
Initial Denaturation	98°C	30 s	98°C	30 s
Cycle: Denaturation	98°C	10 s	98°C	10 s
Cycle: Annealing	70°C	30 s	70°C	30 s
Cycle: Elongation	72°C	90 s	72°C	180 s
Final Extension	70°C	150 s	70°C	210 s
Hold	8°C	-	8°C	-

 

2.    pcDNA3.1 and dsup fragments for HeLa

Method for 50 µl approach:

·       The components listed in Table 3 were mixed in a PCR tube and run threw the cycles listed in Table 4.

Table 3: Reagents and corresponding concentrations used for a single PCR amplification

Components	Volume	Concentration
Q5 2x Master Mix	25 µl	-
10 µM Primer fw	2,5 µl	0,5 µM
10 µM Primer rv	2,5 µl	0,5 µM
ddH2O	19 µl	-
DNA Template	1 µl	-

 

Table 4: General heat cycler program for PCR amplification. The cycle steps were repeated for 30 times for the fragments and 40 times for the vector

Step	Temp for fragment	Time for fragment	Temp for vector	Time for vector
Initial Denaturation	98°C	30 s	98°C	30 s
Cycle: Denaturation	98°C	10 s	98°C	10 s
Cycle: Annealing	71.5°C	30 s	72°C	30 s
Cycle: Elongation	74°C	90 s	74°C	210 s
Final Extension	71.5°C	150 s	72°C	210 s
Hold	8°C	-	8°C	-

 

Preparation of the agarose gel

1. Briefly boil 0,8% w/v Agarose I n1x TAE Buffer until it´s solved.

2. Add 5 µl of Midori Green or 1 µl of Green Pack.

3. Pour into the mold and wait until the gel is fully polymerized.

4. Remove air bubbles or place in the bottom corner/side of the mold.

Gelectrophoresis

1. Preparation of a 0,8% w/v agarose gel.

2. Add 5 µl or 10 µl loading dye (6x) to 25 µl or 50 µl of the PCR product, respectively.

3. Mix the mixture well using a pipette and load the whole volume in the gel pocket.

4. Run the gel on 100 V for 50 minutes.

5. Visualize your DNA fragments with UV light.

DNA Gel Extraction

The DNA gel extraction was carried out with the Monarch® DNA Gel Extraction Kit from NEB. The gel extraction was performed as described in the protocol of this kit (https://international.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020).

All centrifugation steps will be performed at 13,000 rpm.

1. In the end the DNA was eluted with 10 µl DNA Elution Buffer.

2. DNA concentration is determined by using a Nanodrop.

PCR purification

The PCR purification was carried out with the Monarch® PCR and DNA Cleanup Kit from NEB. The PCR purification was performed as described in the protocol of this kit (https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol).

All centrifugation steps will be performed at 13,000 rpm.

Dilute sample with DNA Cleanup Binding Buffer (from NEB Monarch® PCR and DNA Cleanup Kit), for dsDNA > 2 kb ratio Binding Buffer:Sample 2:1, for dsDNA > 2 kb ratio Binding Buffer:Sample 5:1.

Gibson Assembly

1. In approach A: 3 µl fragment and 2 µl vector is added to 15 µl of Gibson Master Mix.

2. In approach B: 2,5 µl fragment and 2,5 µl vector is added to 15 µl of Gibson Master Mix.

3. In approach C: 2 fragment/1 vector mass ratio is added to 10 µl NEBuilder®.

4. For approach A and B the sample was incubated for 1 hour at 50 degrees.

5. For approach C the sample was incubated for 15 minutes at 50 degrees.

DNA cleanup and concentration

The DNA cleanup and concentration was carried out with the Monarch® PCR and DNA Cleanup Kit from NEB. The DNA cleanup and concentration was performed as described in the protocol of this ki (https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol).

All centrifugation steps will be performed at 13,000 rpm.

Dilute sample with DNA Cleanup Binding Buffer (from NEB Monarch® PCR and DNA Cleanup Kit), for dsDNA > 2 kb ratio Binding Buffer:Sample 2:1, for dsDNA > 2 kb ratio Binding Buffer:Sample 5:1.

Preparation of E.coli Overnight Cultures

1. Add 5mL of LB or TB medium, depending on the purpose, to a culture tube.

2. Add 5 µL of the ampicilin.

3. Use a pipette tip to pick a single colony from the plate.

4. Immerse pipette tip in the medium-antibiotic mixture and incubate at 37°C.

Competent cell production

The evening before, inoculate pre-culture in LB-medium with single colonies and incubate at 30°C. The next morning over-inoculate in 20 ml LB medium and incubate at 37°C until the OD600 value is about 0.4.

1. Centrifuge the culture at 13000 rpm for 6 min at 4°C.

2. Discard the supernatant and resuspend the pellet in 1 ml 0.1 M CaCl2 (ice cold).

3. Centrifuge the cells at 13000 rpm for 1 min 4°C.

4. resuspend the pellet in 0.2 ml 1 ml 0.1 M CaCl2.

Transformation into E. coli cells

1. Thaw 25 µL of competent cells on ice.

2. Incubate for 30 min on ice.

3. Place the tube with the cells and DNA in thermocycler at 42 degrees for 45 sec.

4. Place the cells in ice for 1 min.

5. Add 450 µL of LB medium to the cells.

6. Plate out 100 μl of the cell suspension directly onto LB agar culture plates with the appropriate antibiotic.

7. Incubate the culture plates overnight at 37 °C.

For transformation of the empty vector, 1 µl of plasmid DNA was added to 25 µl of competent cells and for transformation of the Gibson plasmid, 3 µl was added. The Gibson plasmid was centrifuged at 13000 rpm for 1 minute before plating out in the agar plates, 350 µl of the supernatant was discarded, resuspended and the remainder was plated out onto the agar plates.

Plasmid Isolation

The plasmid isolation was carried out with the innuPREP Plasmid Mini Kit 2.0from Jena Analytik. The plasmid isolation was performed as described in the protocol of this kit. (https://www.analytik-jena.de/fileadmin/content/products/02_Kits/innuPREP_Plasmid_Mini_Kit_2_0/Manual_innuPREP_Plasmid_Mini_Kit_2.0.pdf).

All centrifugation steps will be performed at 13,000 rpm.

Protein expression

1. (Transform plasmid into BW25113. Plate on antibiotic selection plates and incubate overnight at 37°C.

2. Add a single colony to 5 ml TB medium still containing TB salt and ampilicin and incubate overnight at 37°C.

3. Inoculate the whole overnight culture into 500 ml of TB medium.

4. Incubate at 37°C with shaking.

5. Continuously measure OD at 600 nm and record.

6. When the OD value is between 0.4 and 0.6, induce with 5 ml of 2% arabinose to total volume.

7. Express protein at 37°C, at 30°C or at room temperature overnight.

Protein purification: Nickel affinity chromatography

1. Equilibrate the HisTrap FF column with 5 column volume (CV) of binding buffer.

2. Apply the lysed cell extract.

a) C-term was purified on a 10 mL column.

b) N-term was purified on a 30 mL column.

3. Wash with 5 CV binding buffer.

4. Elute with 5 CV 500 mM imidazole in a one-step elution (used with C-term lysate) or a gradient (used with N-term lysate).

5. Collect the peak in a Falcon tube.

6. Pool the samples of the same variant.

7. Desalinate the samples on a HiTrap FF column, with Tris(Hydroxymethyl)Methylamin-Hydrochlorid (Tris-HCl) at pH 7.6.

Bradford Assay

1. Prepare 1 mg/mL BSA standard solution by dissolving 1 mg Bovine Serum Albumine (BSA) in 1 mL ddH2O

2. Pipet the triplicate standard samples directly into cuvettes according to following scheme:

BSA [µg]	BSA standard [µL]	H2O [µL]
0	0	100
2	2	98
5	5	95
7	7	93
10	10	90
15	15	85
20	20	80

3. Before adding the Bradford reagent make sure the photometer is set to 595 nm

4. Add the Bradford reagent and measure after exact 5 min (invert the samples by using Parafilm during the 5 min)

5. Note the absorbance at 595 nm.

Preparing samples:

1. Always use 100 µL of the diluted sample and add 900 µL of Bradford

2. Example: Use 2 µL of a 1:10 diluted sample with 98 ddH2O

3. Measure as in steps 3.-5. of the preparation of the calibration line

4. Adjust the used sample volume as needed to stay in the range of the standard samples.

Linear range: 2-25 µg/mL

SDS-PAGE (qualitative)

1. Apply approximately 10 µg protein per sample, if needed dilute the sample.

2. Mix 7,5 µL of the samples with 7,5 µL 2x SDS loading buffer.

3. Boil at 95 °C for 10 min.

4. Apply 5 µL of marker and 15 µL of each sample mixed with loading buffer.

5. Run SDS-PAGE for 1,5 h at 100 V and 200 mA.

6. Stain with Coomassie for 40 min.

7. Destain with 20 % acetic acid for 60 min and subsequently with ddH2O overnight

HeLa cultivation

Medium: Gibco Roswell Park Memorial Institute (RPMI) 1640 Medium + 10% FBS + 1% PenStrep.

Washing buffer: phosphate-buffered saline (PBS).

Detaching Reagent: Trypsin-EDTA.

Staining solution: Trypanblue.

All cell cultivation work was performed on a safety cabinet under aseptic conditions. Culture medium is stored in the fridge at 8 ° C, while trypsin has to be stored in the freezer at -20° C. The PBS was stored at RT. The culture media as well as the enzymes used were warmed to RT before use. The cultivation was carried out at 37 °C and 5% CO2. To cultivate the cells, cell culture flasks in 2 sizes were used (T75 and T25).

Splitting

HeLa cells are adherent, as a result, the given space is limited. To ensure that the cells don’t overgrow each other, they have to split into several flasks to give them enough space.

1. Remove the medium.

2. Wash with PBS.

3. Add 2 ml Trypsin-EDTA (1x).

4. Incubate at 37 °C for 5 min (until cells are detached).

5. Transfer cells into a 10 ml falcon.

6. Centriuge at 2000 rpm for 5 min.

7. The pellet is resuspended in medium and then divided into new T75 flasks. If necessary the cells can be counted.

Counting

For the live cell imaging experiments, as well as for the correct cultivation, the determination of the cell number is necessary. To count the cells, a Neubauer chamber and Trypanblue is used.

1. Detach the cells as described before.

2. Resuspend the cell pellet in 1 ml medium.

3. Take 20 μl of the cell suspension and add 80 μl Trypanblue.

4. Transfer 20 μl to the Neubauer chamber.

5. The number of cells is counted under a microscope.

6. The total number of cells (zz) is calculated by multiplication of the dilution factor, cell number, the voulum of the sample and the factor 10^4:

zz = counted cell number * 5 * 10^4 * 1

Experiments

First irradiation Experiment with intracellular Dsup in E. coli

1. Apply approximately 10 µg protein per sample, if needed dilute the sample.

2. Mix 7,5 µL of the samples with 7,5 µL 2x SDS loading buffer.

3. Boil at 95 °C for 10 min.

4. Apply 5 µL of marker and 15 µL of each sample mixed with loading buffer.

5. Run SDS-PAGE for 1,5 h at 100 V and 200 mA.

6. Stain with Coomassie for 40 min.

7. Destain with 20 % acetic acid for 60 min and subsequently with ddH2O overnight

Second irradiation experiment with E.coli

To eliminate the potential of a negative effect by the antibiotic on the cells, no ampicillin was used in the second experiment. There weren’t enough materials for a complete second experiment with triplicates for each genotype. Therefore, the same genotype with C-terminal His-Tagged Dsup as in the extracellular experiments was used, for better comparison. To minimize the variability introduced by the many dilution steps, the cells were only grown to a OD600 of 0,1. Please read the according paragraph in the troubleshooting section for further information about the difficulties of this experiment.

1. Inoculate 5 mL LB Medium with cells.

2. Add 50 µL 200 g/L Arabinose, except to the negative control.

3. A OD600 0,1 value was reached after approximately 3 h.

4. Dilute the suspension directly in 1:10 dilution steps to a total dilution of 10-5.

5. Plate 100 µL of the 10-5 diluted suspension on agar-LB-plates.

6. Incubate a 30 °C for 30 min.

7. Irradiate the cells at 100 µJ/cm2 without the cover of the agar plate.

8. Cover the plates again and place them back into the incubator.

9. Let the cells grow overnight at 30 °C.

10. Count the colonies on the next day.

Live Cell Imaging

The Zeiss Axio Observer system with an integrated incubator was used for the live cell imaging experiments. The cells were incubated under standard conditions. In most live cell imaging experiments, the structures of the cell are stained. We would like to exclude the influence of a fluorophore on the protein, therefore the experiments were carried out without fluorophores. To achieve a realistic experimental setup, the cells were irradiated with a wavelength of 380 nm. Although there are only a few dyes that are excited at this wavelength (1-anilinonaphthalene-8-sulfonic acid), this range already belongs to UVA radiation. UVA is the lowest-energy form of UV radiation, but it can pass through the earth's atmosphere unhindered. [1]

For the live cell imaging experiments Hela cells were used. First, a setting had to be found which shows a phototoxic effect, but which does not immediately kill the cells. Such a setting would not correspond to a real experiment and no differences would occur between positive and negative control. Under these conditions, the DNA protection potential of intracellularly Dsup should be verified. Unfortionally, the Gibson assembly for the Hela plasmid did not work. Therefore, this part could not be performed. The other part is to test the extracellular Dsup protection potential. For this part, the purified Dsup is added to the media of the Hela cells.

[1] https://www.bfs.de/DE/themen/opt/uv/einfuehrung/einfuehrung_node.html (20.10.2021)

Experimental setup

1. Two times: Seed 30 000 HeLa cells to a confocal dish (20 mm)
   (one positive control without any treatment)
2. Transfer the dish into the incubator Axio Observer (37 °C and 5% CO2)
3. Choose settings by ZEN 3.1 (blue edition)
   Two blocks can be set up. The first block is identical for each test. The cells were only exposed to brightfield every 15 min for 20 h. They can recover from the transport to the microscope. The second block (approximate 52 h) determines phototoxicity. The setups for block two are shown in the following table.

For the extracellular experiment, the settings of test 3 were chosen. A quadripartite confocal dish was used to test different Dsup concentrations:

1. Seed 18 000 HeLa cells per quarter
2. Add different amounts of purified Dsup to the media (0 ng, 450 ng, 4500 ng, 0,1125 mg)
3. Choose settings of Test 3 by ZEN 3.1
4. Transfer the dish into the incubator Axio Observer (37 °C and 5% CO2)