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Methods

Molecular Biology Methods

Isolation of Plasmid DNA from E.coli (miniprep)

Plasmid DNA from E. coli was isolated from overnight cultures using the DNA extraction mini-prep kit (Qiagen). The principle of this method is alkaline lysis of bacterial cells followed by a selective immobilization of the plasmid DNA on a column, subsequent washing steps to remove impurities and the elution of plasmid DNA.

Determination of DNA Concentration

DNA concentration was measured using a NanoDrop Spectrophotometer by Thermo Scientific. The concentration was calculated after determination of DNA specific absorbance at 260 nm. Furthermore, the ratio of sample absorbance at 260 and 280 nm as well as at 260 and 230 nm were measured to specify the purity of the samples. A ratio of 260/280 of ~1.8 is generally accepted as “pure” for DNA. If the ratio is appreciably lower, it may indicate contamination with proteins. The 260/230 nm ratio indicates contamination with thiocyanates and phenolate ions since these absorb at 230 nm. The value is expected to be in the range of 1.8-2.2 in case DNA is relatively pure.

Agarose Gel-Electrophoresis

Agarose gel-electrophoresis was used to separate double-stranded DNA fragments by length. Ethidium bromide was applied as a nucleic acid stain (Sambrook et al., 1989). This method was used for the restriction analysis of plasmids (analytical gel-electrophoresis) as well as for the isolation of DNA fragments (preparative gel-electrophoresis). After preparative gel-electrophoresis, the bands were cut out and purified using a Qiagen Gel extraction kit.

TAE buffer (pH 8.0):

Tris	40mM
acetic acid	20mM
EDTA	1mM

Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) was used for the selective amplification of desired DNA fragments (for example from a plasmid). Primers were designed for the desired target sequences. The PCR reaction was divided in to three steps which were repeated up to 30 times. Firstly, the DNA template strand was heat-denatured at 95 °C to produce single-stranded DNA. Secondly, the temperature of the reaction batch was lowered to 55 – 60 °C to allow the primers to bind. Thirdly, the temperature was raised to 72 °C. This enabled the DNA polymerase to synthesize the other DNA strand. Special PCR methods that were used include colony and genomic PCR.

Colony PCR

A method to allow for higher throughput of clone screenings. Colonies were picked with a sterile toothpick or pipet tip. Some of the cells were smeared onto the wall of the PCR tube. Subsequently the toothpick was put into a cell culture tube with LB-medium and suitable antibiotic. Colony PCR was performed using OneTaq Hot Start DNA Polymerase (Qiagen) following this temperature scheme:

Initial denaturation	94 °C	10 min
30 cycles	95 °C	30 s
	59 °C	30 s
	68 °C	1 min 55 sec
Final extension	68 °C	5 min
Hold	4 °C

Purification of PCR products

PCR products were purified using the PCR purification kit by Qiagen.

Dephosphorylation of DNA

Dephosphorylation of digested PCR products via Shrimp Alkaline Phosphatase (Fermaentas) was done to avoid religation of the insert and enhance ligation rate. Before dephosphorylation was performed, digest solution of restriction enzymes and buffer were purified with PCR Purification Kit (Qiagen). Afterwards 1 µg sample DNA was mixed with 10 % of 10x SAP Buffer and 1 unit SAP. The mixture was incubated at 37 °C for 30 min. Inactivation occurred at 65 °C for 15 min.

DNA Restriction Enzyme Digest

For the preparation of DNA fragments and the restriction analysis of plasmid DNA, DNA was cut using restriction endonucleases. Buffers and DNA concentrations were used according to the manufacturer's suggestions.

Ligation / Cycled Ligation

After digestion with an restriction enzyme, plasmid fragments were inserted into vectors (which were cut with matching restriction enzymes) by ligation. The enzyme T4 ligase connected complementary overhangs of fragments by catalyzing the formation of the bond between the 5'phosphoryl group and the 3' hydroxyl group.

Oligohybridization of Single-Stranded DNA

Oligohybridization of oligo-nucleotides was used to create a mini MCS for cloning RFC10 compatible parts between promoter and terminator. This was achieved by using complementary oligo-nucleotides that contained the desired sequence with specific overhangs for cloning. For oligohybridization, 25 ml of 100 mM of forward and reverse oligos were put together in one tube and heated to 90 °C for 5 min. The samples were slowly cooled to room temperature in a styrofoam box overnight.

Site-Directed Mutagenesis

Site-Directed Mutagenesis was used to mutate specific bases of DNA sequences. Therefore, specific primers, which bind at the same site and contain a mismatch at the specific base, were required. The original base pair that had to be replaced was replaced by the mismatch. The method works just as PCR by amplifying the desired product that contains the mismatch. Afterwards, the product was digested with the restriction enzyme DpnI to destroy the plasmids strands which do not contain the desired base pair exchange. The QuikChange Site-Directed Mutagenesis Kit by Agilent Technologies was used.

Sequencing of Plasmid DNA

DNA constructs were sequenced by [http://eurofinsdna.com/ Eurofins mwg operon] using our own sequencing primers.

Protein Biochemical Methods

Protein production in E. coli

Cell disruption

Streptavidin-Affinity chromatography (SAC)

concentrating proteins using a centrifugal filter unit

Size-exclusion chromatography (SEC)

SDS Polyacrylamide Gelelectrophoresis (SDS-PAGE)

Firstly, glass plates were wiped with 70 % ethanol and then assembled onto a setting rig. No rubber spacers were added as they were already fixed to the glass plates. A 15 % resolving gel was made as follows:

• 5 ml protogel
• 2.5 ml 4 x Lower Tris (pH 8.8)
• 2,5 ml H2O
• 50 μl ammonium persulphate (APS) (10 %)
• 2,5 μl N,N,N’,N’-tetramethylethylenediamine (TEMED)

The tube was mixed thoroughly and added to the setting rig between the glass plates, covered with water and left to set for about 30 minutes. Then the water was poured off and a stacking gel was prepared as follows:

• 1 ml protogel
• 1.5 ml 4 x Upper Tris
• 3,5 ml H2O
• 36 μl ammonium persulphate (APS) (10 %)
• 3 μl N,N,N’,N’-tetramethylethylenediamine (TEMED)

All substances were mixed by inversion. 1 ml was applied on top of the already set resolving gel and a comb was put in. Once the gel was set, the comb was taken out and the wells were cleaned out with sterile water. The set gels were removed from the setting rig and placed in the running rig. 1x running buffer was poured into the rig ensuring the plates were covered. For protein preparation, 30 µg protein in 10 µl H20 and 2,5 µl 5 x Laemmli buffer were denatured for 5 minutes at 95 °C. Then 6 µl marker (unstained marker for coomassie-staining or prestained marker for Western Blot) was put into a well. The remaining wells were filled with 12,5 µl of the protein that was to be analyzed. The SDS-PAGE was performed at 120 V for about 1,5 h. For coomassie staining, the gel was incubated in coomassie staining dye for 20 minutes, then put into the first decolorizing solution for 20 minutes. Finally. the gel was put into a second decolorizing solution until the background color was gone. Another method that was used for protein staining was silver staining.

Western Blot

After SDS-PAGE, gels were transferred to a nitrocellulose membrane in transfer buffer (20 ml 5x SDS buffer, 20 ml methanol, 60 ml H2O) at 500 mA for 1h. Membranes were washed 3x 15 min in PBS-T0.1 (PBS + 0,1 % v/v Tween 20) and subsequently blocked with 3 % BSA for one hour. For antibody detection, membranes were washed 3x 15 min in PBS-T0.1 and then incubated in detection solution containing the antibody straptavidin-AP (1:4000 in PBS-T0.1) for one hour. The Western blot was washed 2x 10 min in PBS-T0.1 and then 2x 10 min in PBS. Afterwards, the developing solution (15 ml alkaline phosphatase buffer, 45 µl BCIP (50 mg/ml in DMF) and 7,5 µl NBT (75 mg/ml in 70 % DMF) was added. After the appearance of bands, the blot was washed and stored in water.

Microbiological Methods

Cultivation of E.coli

E.coli strain XL1-Blue was cultivated in LB-medium (lysogeny broth) and LB plates. for the preparation of 1 liter of LB, dissolve the following and autoclave:

• 10 g tryptone
• 5 g yeast extract
• 10 g NaCl

For making plates, add 15 g bacto-agar before autoclaving.

Heat Shock Transformation of E.coli with Plasmid DNA

Before transformation, CaCl₂ competent cells were produced after Cohen et al., 1972. For the production of competent cells, 50 ml LB medium were inoculated with an overnight culture of the used ‘’E.coli’’ strain and incubated at 37 °C, 180 rpm. After an OD ₅₅₀ of 0,5 was reached, the culture was centrifuged for 4 minutes at 5000 g for 10 minutes. The pellet was then resuspended in 40 ml pre-chilled in 0,1 M MgCl₂ solution, centrifuged again and resuspended in 20 ml of pre-chilled 0,05 M CaCl₂ solution. After 30 minutes of incubation on ice, the cells were centrifuged and resuspended in 2 ml 0,05 M CaCl₂ solution, 15 % v/v glycerol. The competent cells were aliquoted and stored an – 80 °C. For the transformation, 100 µl competent cells and 1 ng plasmid or 5 µg of a ligation product were mixed and incubated for 30 minutes on ice. Afterwards, the cells were heat shocked at 37 °C for 5 minutes, then mixed with 2 ml LB medium and incubated at 180 rpm and 37 °C for 30-45 minutes. The transformed cells were then plated on LB medium containing an antibiotic.

Physcomitrella Methods

The techniques used for the cultivation and manipulation of Physcomitrella patens are based on the knowledge of the chair for Plantbiotechnology of Prof. Dr. Reski at Freiburg University which are availible at [http://www.plant-biotech.net Plant-Biotech.net].

Preparation of Knob-Media

According to Reski and Abel, 1985:

Stock solutions 100x:

25 g/l	KH2PO4
25 g/l	KCl
25 g/l	MgSO4 x 7 H2O
100 g/l	Ca(NO3)2

Knop media:

	stock solutions
12.5 mg/l	FeSO4x 7 H2O

The pH is adjusted to pH 5.8 with KOH or HCl, then the medium is sterilized by autoclaving.

Knop media agar plates:

	Knop medium
1.2% (w/v)	agar (Oxoid Ltd.)

The pH is again adjusted to pH 5.8 with KOH or HCl, the agar medium is sterilized by autoclaving and poured into dishes while still warm.

Knop media:

stock solutions
12.5 mg/l	FeSO4x 7 H2O
50 g/l	glucose
30 g/l	mannitol

The pH is adjusted with KOH to pH 5.8 and the medium is sterile filtered.

Cultivation of P. patens

The standard conditions for P. patensare 25°C ±1°C under a 16/8 h light/dark photoperiod with a lightintensity of 55 μmol m-2s-1.

In our lab, we cultivated P. patens in Erlenmeyer flasks eqipped with a sterile filter in the lid made of aluminum foil. To provide CO2, we used compressed air which went through the sterile filter and through an attached Pasteur pipette into the liquid medium. The resulting bubbles helped to continously stir the culture. The liquid cultures were regularly homogenized and provided with fresh medium. We also experimented with liquid cultures in the incubator shaker as well as in unstirred medium and with cultivation on solid medium and on medium soaked felt, as well as some other surfaces. We tried to keep the temperature constant by air conditioning, but it was of course subject to many fluctuations. The appropriate light/dark photoperiods were produced by hooking striplights up to a time switch.

After Transformation, the regenerating protoplasts were incubated in regeneration medium in 6-well-plates sealed with parafilm for 10 days and then cultivated on a layer of autoclaved cellophane (seperated with Whatman paper during autoclavation, so they dont stick together), on top of Knop medium agar plates for three days under standard conditions. Then we transferred the cellophane layers with the regenerated moss onto Knop medium agar plates containing 25 µg/ml G418 antibiotic for two weeks. The official protocol schedules a two week release period followed by another selection period to ensure stabile transormation, but we didn´t have that much time and went with a single round of selection. We plated only half of our transformed protoplasts. The other half was left in the 6-well-plates where 2 ml of selection medium were added to the 2 ml of regeneration medium from the transormation, with G418 diluted 1:8000.

Homogenization

The moss in liquid culture in Erlenmeyer flasks has to be homogenized weekly to disintegrate the protonema filaments. The process is carried out on a laminar flow bench to prevent contamination. Before homogenization, we let the moss settle and decanted the supernatant medium. We used an Ultra Turrax at 19.000 rpm. The duration depends on the density of the culture. The homgenized moss is distributed into several previously autoclaved flasks to achieve a lower density. Fresh medium is added to the new flasks and autoclaved air-filters are integrated into their lids.

Bioinformatical Methods

Generation of Annimated GIFs

• A homology search was performed for the amino acid sequece using [http://toolkit.tuebingen.mpg.de/hhpred HHpred]
• Entry for the best matching structure(> 50%)in PDB-database was opened
• PDB-file was was saved
• The structure was opened with Pymol
• The approach was adapted from a PyMol-Tutorial http://ihome.cuhk.edu.hk/~b102142/pymol/pymol_tutorial.html Wong
• The following commands were entered into the command line:

PyMOL> orient
PyMOL> hide everything, all
PyMOL> show cartoon, all
PyMol> bg_color white
click> Settings>Rendering>Shadows>Occlusion
PyMOL> color purple, ss h; color yellow, ss s; color green, ss ""
PyMOL> show surface, all
PyMOL> set transparency, 0.75
PyMOL> set surface_quality, 1 (command will prolongue the process significantly, you may skip it)
PyMOL> set ray_trace_frames=1 (command will prolongue the process significantly, you may skip it)
PyMOL> mset 1 x180
PyMOL> util.mroll 1,180
PyMOL> mpng frame


• Afterward the *.png files were automatically converted to *.tif files using xxx
• The program xxx was used to generate the annimated GIFs
  • A new working folder was generated and the *.gif files were imported
  • The background was set to white
  • The range of colors was set to 256
  • Finally the annimated Gif was computed

The result is the following:

References:

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Emil-Erlenmeyer-Forum 5
85354 Freising, Germany

Email: igem@wzw.tum.de
Phone: +49 8161 71-4351