Methods

1. Cloning

1.1 Restriction and phosphatase treatment of plasmid DNA

Originally found in bacteria, endonucleases belong to a class of enzymes that have the ability to cleave the phosphodiester bond of DNA molecules by hydrolysis. Depending on the type they recognize site specific base sequences of 4, 6 or 8 bases that are often palindromic. Treatment of different DNA molecules with the same endonucleases allows the combination of the resulting DNA fragments in a following ligation reaction.

The treatment of DNA fragments, in particular plasmid DNA with phosphatase is beneficial if the restriction was performed with only one restriction enzyme or if the restriction with two different restriction enzymes was unbalanced and single cut plasmid DNA is left. As the nomenclature indicates the phosphatase cleaves the phosphate group at the 5´-terminus of DNA fragments and thereby minimizes the background of mono ligated plasmid DNA. Consequently the amount of transformants with insert DNA in the plasmid construct is considerably increased.

The following components were used for a restriction batch by default:

The batch was incubated for 10 min at 37 °C. The success of the restriction was checked in a following agarose gel electrophoresis.

1.2 Restriction of PCR products:

Corresponding restriction sites were added in a PCR reaction at the 5´and 3´-termini of Insert DNA via modified primers. After gelextraction the following components were used for a restriction batch by default

The batch was incubated for 10 min at 37 °C followed by purification via QIAquick Gel Extraction Kit (Qiagen).

1.3 Polymerase chain reaction (PCR):

The PCR is a standard procedure in molecular biology that enables the amplification of distinct DNA sequences in vitro. Short flanking DNA oligomers define the DNA sequence that is to be amplified. The amplification process itself is performed by a polymerizing enzyme also referred as DNA polymerase. Since its invention in 1983 numerous variations have been established for different applications.

1.3.1 Amplification of genomic DNA:

The following components were used for the amplification of genomic DNA of Bacillus megaterium by default:

The following speed cycle program was used:

Presence, absence and size of amplification products were determined by agarose gel electrophoresis.

1.3.2 Splicing by overlapping extension (SOE)-PCR:

The SOE-PCR is a common variation of the standard PCR reaction and primarily used for the generation of fusion genes. The name refers to the special set of primers. Their overhangs are complementarily overlapping with the DNA sequences of the DNA fragments to be fused. Figure 1 shows the flowchart of the SOE-PCR for the generation of a hyaluronan synthase (has2) - green fluorescent protein (gfp) fusion gene.

Figure 1: Scheme of a SOE-PCR for the generation of a hyaluronan synthase (has2) - green fluorescent Protein(gfp) fusion gene. First step of the SOE-PCR comprises the amplification of the individual gene sequences of has2 and gfp with primer combinations a/b and c/d. Complementary sequences of their counterparts derived from primer b and c allow the mutual annealing of the amplification products. A second amplification with the flanking primers a and d results in the generation of the respective has2-gfp fusion gene.

The following components were used for the first PCR reactions by default:

The following components were used for the second PCR reaction by default:

The following speed cycle program was used:

Presence, absence and size of amplification products were determined by agarose gel electrophoresis.

1.3.3 Colony PCR:

After transformation of competent E. coli cells the colony PCR is a popular high-throughput technique in molecular biology for the quick identification of the presence, absence or orientation of insert DNA in plasmid constructs. An extended heating step at the beginning of the PCR reaction results in cell lysis and release of plasmid DNA which serves as template for the following amplification with insert specific primers. Time consuming DNA isolations and restrictions of multiple colonies are superfluous.

The following components were used for the second PCR reaction by default:

The following speed cycle program was used:

Presence, absence and size of amplification products were determined by agarose gel electrophoresis.

1.3.4 Quick Change (QC)-PCR:

The QC-PCR is a common variation of the standard PCR and useful for site-directed mutagenesis of plasmid DNA. The special design of mutagenic primers allows the insertion or deletion of whole sequences as well as the substitution of single nucleotides for applications such as restriction site mutagenesis or amino acid exchange. Figure 2 shows the experimental setup for a QC-PCR.

The following components were used for the PCR reactions by default:

The following speed cycle program was used:

The following components were used for digestion and removal of the template plasmid DNA :

Presence, absence and size of amplification products were determined by agarose gel electrophoresis after digestion with DpnI.

1.4 Isolation of plasmid DNA

The isolation of plasmid DNA was performed with a QIAprep® Spin Miniprep Kit (Qiagen). The provided instructions were followed precisely.

1.5 Isolation of genomic DNA of Bacillus megaterium:

The isolation of genomic DNA of Bacillus megaterium was performed with a NexttecTM 1-Step DNA Isolation Kit for Bacteria. The provided instructions were followed precisely.

1.6 Measurement of DNA concentration:

The measurement of DNA concentration after plasmid DNA isolation was performed with the UV2100 UV/Vis Recording Spectrophotometer (Shimadzu). Spectrum was analyzed between wavelengths of 300 nm and 200 nm. Level of DNA purity was assessed with the q-value of A260 /A280.

1.7 Agarose gel electrophoresis (AGE)

The agarose gel electrophoresis is a standard method in molecular biology primarily used for separation of DNA or RNA molecules of varying lengths. The separation effect is due to the sieving properties of the three dimensional agarose gel matrix that enables short negatively charged DNA molecules to travel faster towards the positive anode than large negatively charged DNA molecules after applying an electric field. As a consequence the distribution pattern of DNA molecules is inversely proportional to the log of their molecular weights. DNA fragments with identical lengths form bands inside the gel. After separation and staining with an appropriate dye (e.g. ethidium bromide) the DNA bands can be visualized under ultra violet light.

The agarose gel electrophoresis was performed with prestained (peqGREEN) 1 % agarose gels (w/v) in 0.5 x TBE buffer. The 2-Log DNA Ladder (NEB) was used as indicator for DNA size. 120 V were applied for 50 min for appropriate separation.

1.8 Gel extraction and purification of DNA fragments

Both procedures were performed with the QIAquick Gel Extraction Kit (Qiagen). The provided instructions were followed precisely with the exception that the elution step was performed with 25 µl preheated ddH2O.

1.9 Ligation of DNA fragments

During ligation process selected DNA fragments such as insert DNA and plasmid constructs are enzymatically linked to form recombinant DNA molecules. The phosphodiester bond is formed between 3`-hydroxyl groups and 5`- phosphoryl groups of terminal nucleotides. The energy for the reaction is provided by addition of the co-factor ATP.

The following components were used for the ligation batch by default:

The ligation reaction was incubated for 10 min at 22 °C. Heat inactivation was performed for 10 min at 65°C. Success of the ligation reaction was checked via agarose gel electrophoresis with 5 µl of the ligation batch.

1.10 Dialysis of the ligation batch

Dialysis of the ligation batch was performed on MF-Membrane Filter Type VSWP02500 (Millipore) in 10 % glycerol for 1h at RT. Transformation of competent E. coli cells was subsequently performed with 5 µl of the dialyzed ligation batch.

2. Transformation and cultivation of E. coli and B. megaterium

2.1 Measurement of cell density

Measurement of cell density was regularly performed after inoculation of cultures with 1 ml samples. Fresh LB medium or TB medium was used as reference for measurement of E. coli or B. megaterium density. The measurement itself was performed with a Novaspec visible spectrophotometer at a wavelength of 600 nm.

2.2 Preparation of electro competent E. coli cells

E. coli cells belong to the class of Gram-negative bacteria and are therefore naturally not competent for absorption of DNA molecules. For a successful transformation with plasmid DNA, E. coli cells have to be artificially made competent. The following protocol was used for preparation of electro competent E. coli cells with high transformation efficiency of 2 x 108 cfu/µg pUC19 DNA:

2x 400 ml LB medium in 2000 ml Erlenmeyer flasks were inoculated with one hundredth of a fresh overnight culture of the E. coli cloning strain TOP10F´. The main cultures were incubated at 37 °C and 180 rpm until a cell density of A600=0.4. The measurement of the cell density was regularly performed over a period of time of approximately 3 hours. The flasks were subsequently chilled on ice for 30 minutes. Afterwards each culture was centrifuged at 4000 g and 4 °C for 10 min. The supernatant was discarded and each pellet resuspended in 400 ml of ice cold, sterile water. Centrifugation step was repeated. The supernatant was discarded and each pellet resuspended in 200 ml of ice cold, sterile water. Centrifugation step was repeated. The supernatant was discarded and each pellet resuspended in 100 ml of ice cold, sterile water. Centrifugation step was repeated. The supernatant was discarded and each pellet resuspended in 50 ml of ice cold, sterile water. Centrifugation step was repeated. The supernatant was discarded and each pellet resuspended in 20 ml of ice cold, sterile 10 % glycerol. Centrifugation step was repeated. The supernatant was discarded and each pellet resuspended in 10 ml of ice cold, sterile 10 % glycerol. Centrifugation step was repeated. The supernatant was discarded and each pellet resuspended in 2 ml of ice cold, sterile 10 % glycerol. The electro competent E. coli cells were immediately frozen in aliquots of 50 µl and kept at -70 °C until reconstitution.

2.3 Transformation of electro competent E.coli cells:

During the electroporation process, the membrane of E. coli cells is made permeable by applying a high voltage over a short period of time. Frozen electro competent E.coli cells were slowly thawed on ice and mixed with up to 5 µl of prechilled, dialyzed DNA. After careful resuspension the transformation batch was incubated for 5 minutes on ice and subsequently filled into a prechilled electroporation cuvette (gap width 0.1 cm). The transformation was performed via electroporation with the Easyject Prima (Equibio) at 1800 V. The transformation batch was immediately mixed with 500 µl of preheated SOC medium and incubated at 37 °C and 700 rpm for 1 h. Different dilutions of the transformation batch were grown on LB agar plates with an appropriate selection medium at 37 °C over night. Presence and size of insert DNA in plasmid constructs of transformants were checked by colony PCR.

2.5 Cultivation of E. coli cells

A 300 ml Erlenmeyer flask was filled with 50 ml of fresh LB medium and 50 µl of an ampicillin stock solution (100 mg/ml). The LB medium is inoculated with an E. coli colony that has been positively tested for insert DNA by colony PCR. The resulting culture was incubated at 37 °C and 180 rpm over night.

2.6 Preparation of B. megaterium protoplasts

B. megaterium belongs to the class of Gram-positive bacteria. Thus it is naturally competent for absorption of DNA. The thick surrounding peptidoglycane layer however prevents the absorption of DNA and has to be degraded. The following protocol was used for preparation of protoplasts.

50 ml LB medium in a 300 ml baffeled flask was inoculated with one hundredth of a fresh overnight culture of the B. megaterium strain MS941. The main culture was incubated at 37 °C and 180 rpm until a cell density of A600=1.0. The measurement of the cell density was regularly performed over a period of time of approximately 4 hours. The culture was transferred to a 50 ml Falcon tube and centrifuged at 1280 g and 4 °C for 15 min. The supernatant was discarded, the pellet was resuspended in 5 ml of fresh SMMP and transferred to a new 15 ml Falcon tube. The protoplast formation was initiated by addition of 100 µl of lysozyme solution (10 mg/ml in SMMP). The batch was incubated at 37 °C without shaking until 80-100 % of the cells showed the typical globular morphology of protoplasts. The progress of the reaction was checked every 10 min under a microscope. If the formation of protoplasts didn`t initiate, another 100 µl of the lysozyme solution should be added. After successful protoplast formation, the batch was centrifuged at 1280 g and RT for 10 min. The supernatant was carefully decanted and the pellet was washed with 5 ml of SMMP. Centrifugation and washing step were repeated. Subsequently 750 µl of sterile 87 % glycerol were added to protoplast suspension and carefully mixed by tilting the tube. Finally the protoplasts were frozen in 500 µl aliquots and kept at-70 °C until reconstitution

2.7 Transformation of B. megaterium protoplasts

A 15 ml Falcon tube was prepared and filled with 1.5 ml of PEG-P which serves as a carrier matrix for the DNA. The protoplast suspension was slowly thawed on ice and carefully mixed with 5-10 µg of DNA. The protoplast-DNA-solution was subsequently pulled vertically through the PEG-P in circling motions with a micropipette. The resulting batch was mixed by tilting the tube and was incubated for 2 min at RT. Afterwards the batch was resuspended with 5 ml of SMMP and centrifuged at 1280 g and RT for 10 min. The supernatant was immediately decanted, the pellet resuspended in 500 µl SMMP and transferred into a sterile 1.5 ml reaction vessel. The regeneration of the cell wall was triggered in two steps. First incubation step was performed at 30 °C without rotations, while the second one was performed at 30°C and 300 rpm. During these 90 min incubation time, 3.5 ml of CR5 overlay agar were prepared in a 15 ml Falcon tube and kept warm at a temperature of 42 °C in a water bath. After incubation the transformation batch was added to the overlay agar and carefully mixed by tilting the 15 ml Falcon tube. The agar is poured onto LB plates with an appropriate selection medium. The plates were incubated over night at 37 °C. The next day single colonies were transferred to fresh LB plates with selection medium and incubated again over night at 37 °C. To avoid contamination, single colonies were identified the following day via microscopy representatives of B. megaterium.

2.8 Cultivation of B. megaterium cells

A 300 ml Erlenmeyer flask was filled with 50 ml of LB medium and 50 µl of a tetracycline stem solution (10 mg/ml). Transformed B. megaterium strains were taken from the corresponding glycerol stock and put into the Erlenmeyer flask. This preculture was incubated at 37 °C and 180 rpm overnight. The next day a mix of 45 ml fresh TB medium, 5 ml 10 x KPP buffer and 50 µl tetracycline stem solution was inoculated in a 300 ml baffled flask with one hundredth of the fresh overnight culture of the B. magisterium and incubated at 30 °C and 180 rpm until a cell density density of A600 = 0,4. Protein expression was induced with addition of 1 ml of a xylose solution (250 mg/ml).