Organism used in this work

2.1.1.1 Bacteria strains and cultivation

Chemical competent E. coli strain Top10 was used during cloning. For heterologous protein expression in plants, electrocompetent A. tumefaciens strain UIA143, kindly supplied by Dr. Richard Strasser (DAGZ, BOKU), was used. This strain contains a

pMP90 Ti-helper plasmid containing gentamycin resistance and genes necessary for T- DNA transfer into the plant genome (Hamilton, 1997). Preparation of cells for transformation was self-made in both cases. Table 1 gives an overview about the two different bacteria strains.

strains genotype E. coli Top10F’

F’ [lacIq, Tn10(Tet^R)] mrA ∆(mrr-hscRMS-mcrBC)Φ80lacZ∆M15

∆lacX74 recA1 deoR araD139 ∆(ara-leu)7697 galU galK rpsL (Str^R) endA1 nupG)

A. tumefaciens UIA143 autC58 recA::Ery140(pAtC58)

Table 1: Overview about the different bacteria strains used in the experiments. 

Bacteria were cultivated either as liquid culture in common glass tubes containing LB liquid medium or on LB agar plates, both supplemented with antibiotics (see Table 2 and Table 3). Incubation of E. coli followed at 37 °C over night. A. tumefaciens cultures were incubated at 29 °C for 1 to 2 days. Liquid cultures were incubated on a shaker under agitation at 180 rpm. Plates of both bacterial species were then stored at + 4 °C for up to 1 month.

media compounds

LB bouillon, 1 L 10 g NaCl (Roth), 10 g pepton of casein (Merck), 5 g yeast extract (Merck), HQ-H2O up to 1 L

LB agar, 1 L 10 g NaCl (Roth), 10 g pepton of casein (Merck), 5 g yeast extract (Merck), 20 g agar-agar (Roth), HQ-H2O up to 1 L

SOB medium, 1 L

200 g tryptone (Merck), 5 g yeast extract (Merck), 0.5 g NaCl (Roth); after autoclaving added: 10 mL 1 M MgCl2 (Merck), 10 mL 1 M MgSO4 (Merck), filter sterilised

SOC medium, 100 mL 100 mL SOB medium, 1 mL 2 M filter-sterilised D-glucose (Merck) solution or 20 % (w/v) glucose, filter sterilisation

Table 2: Overview about the composition of media used for cultivation of bacteria.  

LB was the common medium for both E. coli and A. tumefaciens. SOC medium was used for transformation of  agrobacteria and was based on SOB medium. 

35  Antibiotics used during this work:

Antibiotic stock final concentration application Ampicilline 50 mg/mL 100 µg/mL (1:500) selection of E. coli

Kanamycine 50 mg/mL 50 µg/mL (1:1000)

selection of E. coli and A. tumefaciens; selection of Arabidopsis thaliana overexpression lines Gentamycine 50 mg/mL 25 µg/mL (1:2000) selection of A.

tumefaciens Tetracycline 10 mg/mL 10 µg/mL (1:1000)

preparation of competent cells for chemical transformation of E. coli Table 3: Overview about the used antibiotics in stock concentration and final concentration.  

Kanamycin was used at the same final concentration for screening of Arabidopsis overexpression plants. Ampicillin  sodium salt, Kanamycin sulfate from Streptomyces kanamyceticus and Gentamicin sulfate were purchased from  Sigma‐Aldrich, and Tetracylin‐Hydrochlorid from Roth. Ampicillin was used for E. coli transformed with cloning  vectors pGEM or pSTBlue‐1. Kanamycin was used for selection of plant expression vectors p20F eCaMV35S, p21GT  or ppT8 in E. coli. Kanamycin and Gentamycin was always used together for growth of Agrobacteria transformed  with plant expression vectors p20F eCaMV35S, p21GT or ppT8.  

2.1.1.2 Photometric measurement of the optical density (OD600) of bacteria cells

Photometric measurement of the optical density (OD) of bacteria cells - either E. coli or A. tumefaciens – has been performed at 600 nm (OD600) in half-micro plastic cuvettes from Sarstedt (Polystyrene, 10x4x45 cm) on a spectrophotometer (Genesys 10UV, Thermo Scientific).

2.1.1.3 Preparation of CaCl2 competent E. coli Top10 cells for transformation by heat shock

10 µL of a stock of CaCl2 competent E. coli strain Top10 cells (see Table 1: ) were transferred into 5 mL of LB liquid medium supplemented with 10 µg/mL tetracycline (see Table 3) and cultivated over night at 37 °C under agitation at 180 rpm. The following day, 2 mL of preculture were transferred into 200 mL of LB liquid medium supplemented with 10 µg/mL tetracycline and incubated at 37 °C until an OD600 of 0.5 was reached.

The cells were centrifuged at 4 000 g for 10 minutes at 4 °C and the cell pellet was resuspended in 20 mL (1/10 volume) of sterile 0.1 M CaCl2 (autoclaved). The cells were centrifuged a second time and the pellet was resuspended in 6 mL of 0.1 M CaCl2 and mixed with sterile glycerol (87 %). The competent cells were aliquoted into 100 µL

fractions in sterile 1.5 mL Eppendorf tubes and immediately frozen in liquid nitrogen.

CaCl2 competent cells were stored at – 80 °C until usage for transformation.

2.1.1.4 Preparation of electrocompetent A. tumefaciens UIA142 cells for transformation by electroporation

10 µL of a stock of electro-competent Agrobacteria strain UIA143 (see Table 1) were inoculated in 5 mL of LB liquid medium supplemented with 25 µg/mL gentamycin (see Table 3) and cultivated over night at 29 °C under agitation at 180 rpm. This pre-culture was transferred into 500 mL of LB liquid medium supplemented with 25 µg/mL gentamycin and cultivated at 29 °C until an OD600 of 0.5 – 1.0 was reached. After reaching this optical density, the cells were cooled on ice at 4 °C for at least 30 minutes (maximally 45 minutes). Cells were harvested by centrifugation at 5 000 rpm for 10 minutes at 4 °C. The cell pellet was re-suspended in 300 mL of 10 % glycerol and centrifuged a second time. The pellet was washed with 150 mL 10 % glycerol and centrifuged. This washing step was repeated with 75 mL of 10 % glycerol. The cell pellet was then resuspended in 3 mL concentrated glycerol and aliquoted into 100 µL and immediately frozen in liquid nitrogen. Electro-competent cells were stored at – 80 °C until transformation by electroporation.

2.1.1.5 Transformation of bacteria

Two transformation strategies have been applied during this work: Chemical transformation of E. coli TOP10 and electroporation of A. tumefaciens UIA143.

Chemical transformation: A frozen aliquot of -80 °C competent cells was thawed up on ice and mixed with the ligation mix. The reaction mix was incubated on ice for 30 minutes. Heat shock followed at 42 °C for 35 seconds in a water bath. The cell mix was incubation on ice for 2 minutes and then mixed with 250 µL of 37 °C prewarmed LB liquid medium. Incubation was performed at 37 °C for 1 hour on a shaker at 300 – 500 rpm. The cells were centrifuged for 3 minutes at 3 400 g (3 500 rpm). Most of the supernatant was removed and the pellet was resuspended in the residual medium. The cells were plated on LB plates supplemented with antibiotics and, in case of blue/white screening, with 14 µL of 1 M IPTG and 40 µL of 20 mg/mL X-Gal. Incubation of LB- plates followed over night at 37 °C in an incubator.

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Electroporation of A. tumefaciens UIA143: A frozen aliquot of -80 °C competent cells was thawed up on ice and mixed with the ligation mix. The cell mix was transferred into a cooled electroporation cuvette (Peqlab Electroporation Cuvette, 25 x 1mm electrode gap). Electroporation was performed on a BioRad MicroPulser with 1 pulse at 1,8 kV.

Subsequently, 900 µL of SOC medium prewarmed at 29 °C was transferred into the electroporation cuvette and carefully mixed by pipetting. The cell solution was transferred into a 1.5 mL Eppendorf tube and incubated at 29 °C for 2 – 3 hours. After incubation, 10 µL of cell mix were plated on LB supplemented with 50 mg/mL kanamycin diluted 1:1000 (final concentration 50 µg/mL) and 50 mg/mL gentamycin diluted 1:2000 (final concentration 25 µg/mL). Incubation followed at 29 °C over night for 1 – 2 days.

2.1.1.6 Cryostock preparation

In order to allow long-term storage of bacterial cells of both E. coli and A.

tumefaciens, cryostocks were prepared and stored at – 80 °C. A volume of 900 µL of a dense over night culture was mixed with 900 µL of sterile 87 % glycerol (Merck) and frozen immediately.