2. Materials and Methods
2.2. Methods
2.2.6. Preparation and detection of DNA
Isolation of genomic DNA of B. subtilis
For the isolation of B. subtilis gDNA, the cells of a 4 ml LB overnight culture were harvested. The gDNA was extracted, using the peqGOLD Bacterial DNA Kit from PEQLAB. Deionized water was used for elution of the DNA from the columns (Blötz et al., 2017).
Isolation of plasmid DNA from E. coli
Plasmid DNA was extracted from E. coli cultures, using the NucleoSpin® Plasmid Kit from Machery-Nagel. Deionized water was used for the elution of the plasmids from the columns (Blötz et al., 2017).
Purification of DNA
The QIAquick PCR purification Kit was used for the purification of DNA fragments. For the elution of the DNA from the column, deionized water was used (Blötz et al., 2017).
Agarose gel electrophoresis
Agarose gel electrophoresis was used to separate DNA fragments by size and thereby analyse the respective size. 1% agarose gels (w/v) were prepared in TAE Buffer together with HDGreen™ DNA-Dye from Intas. The DNA samples were supplemented with 5x DNA loading dye and loaded onto the gel, together with an EcoRI/ HindIII digested λ-DNA marker to estimate the size of the DNA fragments. A voltage of 140 V was applied until the colour marker reached the last third of the gel.
For the detection of the DNA the GelDoc™ from Biorad was used by excitation with UV light (254 nm) (Blötz et al., 2017).
50x TAE Buffer 242 g Tris-base
57.1 ml Acetic acid (100%) 100 ml EDTA (0.5M, pH 8.0) Ad to 1000 ml with deionized water
5x DNA loading dye 5 ml Glycerol (100%)
200 µl 50x TAE
10 mg Bromphenol blue 10 mg Xylene cyanol 4.8 ml dH2O
25 Sequencing of DNA
Sequencing of plasmids and DNA fragments was performed by Microsynth AG with the chain termination method. Whole genome sequencing was carried out by the Göttingen Genomic Laboratory.
Cloning procedure
DNA restriction enzymes from ThermoFisher were used to cleave the PCR fragment and the respective plasmid. The manufacturer’s instructions for enzyme concentration, incubation conditions and heat inactivation after the reaction were followed. The vector was dephosphorylated by the addition of 1 µl alkaline phosphatase (ThermoFisher) and incubation at 37°C for 15 min. Afterwards the vector and the insert were ligated using T4-DNA ligase (ThermoFisher). The ligation reaction contained 150 ng of vector DNA and a 5-fold excess of insert DNA and was incubated 2 h at RT or overnight at 16°C (Blötz et al., 2017).
Polymerase chain reaction (PCR)
The PCR reaction was performed with genomic or plasmid DNA as template in a total volume of 50 µl with Phusion™ polymerase or DreamTaq polymerase.
Reaction mix for Phusion™ polymerase (50 µl):
10 µl 5x HF reaction buffer 2 µl dNTPs (12.5 µmol/ml) 2 µl forward primer (20 pmol) 2 µl reverse primer (20 pmol) 1 µl template DNA (1 ng/µl) 0.2 µl Phusion™ polymerase (2 U/µl) 32.8 µl dH2O
Reaction mix for DreamTaq (50 µl):
5 µl 10x DreamTaq reaction buffer 2 µl dNTPs (12.5 µmol/ml)
2 µl forward primer (20 pmol) 2 µl reverse primer (20 pmol) 1 µl template DNA (1 ng/µl)
0.25 µl DreamTaq polymerase (2 U/µl) 37.75 µl dH2O
26
The samples were briefly vortexed, centrifuged and the reaction was placed into the Themocycler with the following programs:
Standard program for the Phusion™ polymerase
Initial denaturation 98°C 5 min
Denaturation 98°C 30 s
Annealing 48-56°C 35 s 30 x
Elongation 72°C 30 s per 1 kb
Final elongation 72°C 10 min
Break 4°C ∞
Standard program for the DreamTaq polymerase
Initial denaturation 95°C 5 min
Denaturation 95°C 30 s
Annealing 48-56°C 35 s 30 x
Elongation 72°C 1 min per 1 kb
Final elongation 72°C 10 min
Break 4°C ∞
Long flanking homology PCR (LFH-PCR)
The long flanking homology PCR (LFH PCR) was used to generate a DNA fragment for the deletion of genes in the genome of B. subtilis. Therefore, the upstream and downstream regions of the target genes are amplified by PCR (1000 bp). The genes encoding for the resistance against chloramphenicol, kanamycin, erythromycin, spectinomycin, tetracyclin and zeocin are amplified from the plasmids pGEM-cat, pDG780, pDG646, pDG1726, pDG1513 and pDG148 respectively. The flanking regions and the resistance cassette were fused together in the LFH PCR, in which the first step was the joining of the three fragments without the oligonucleotides. In a second step the oligonucleotides are added to the reaction and the complete fragment was amplified.
Complementary sequences allow the joining of the fragments. Afterwards, B. subtilis competent cells were transformed with the LFH product and plated onto the respective selection plates.
27 Reaction mix for LFH PCR with Phusion™ polymerase (50µl):
10 µl 5x HF reaction buffer 2 µl dNTPs (12.5 µmol/ml) 4 µl forward primer (20 pmol) 4 µl reverse primer (20 pmol) 100 ng upstream flanking region 100 ng downstream flanking region 150 ng resistance gene
1 µl Phusion™ polymerase (2 U/µl) Ad to 50 µl with dH2O
Standard program for the LFH PCR with Phusion™ polymerase
Initial denaturation 98°C 3 min
Denaturation 98°C 30 s
Annealing 52°C 35 s 10 x
Elongation 72°C 2 min 15 s
Hold 15°C ∞
Addition of oligonucleotides
Denaturation 98°C 30 s
Annealing 52°C 35 s 30 x
Elongation 72°C 3 min 30 s + 5 s/ cycle
Final elongation 72°C 10 min
Break 4°C ∞
Marker-free deletion system
The marker-free deletion system is based on the mannose phosphotransferase system. In B. subtilis mannose uptake and phosphorylation is performed by the permease ManP. The resulting product mannose-6-phosphate is then further metabolized by the mannose-6-phosphate isomerase ManA.
Without the corresponding genes manP and manA, the uptake of mannose is not possible.
However, if only manP is present, mannose-6-phosphate accumulates in the cell to a toxic level.
The plasmids used in this method are pJOE6743 and pGP1022, which carry the manP gene as a counterselection marker and a spectinomycin resistance as a selection marker.
28
The upstream and downstream regions of the deletion target were amplified, fused together and cloned into the deletion plasmid. A strain without the manP and manA genes (ΔmanP-manA) was transformed with the plasmid and plated on LB agar with spectinomycin. A 4 ml LB culture was inoculated with a colony from the plates and after incubation at 37°C over the day, the cells were diluted 1:10-4 In LB medium supplemented with 0.5% mannose. The cultures were incubated overnight, then diluted 2x10-5 and plated onto LB agar plate with 0.5% mannose. The cells that could grow in the presence of mannose have lost the introduced manP gene and the uptake of mannose was not possible anymore. These colonies were again tested for their growth on LB medium with spectinomycin. Only the colonies which did not grow in the presence of spectinomycin were used for colony PCR to check for the correct deletion. This marker-free deletion method is illustrated in Figure 8 (Wenzel and Altenbuchner, 2015; Blötz et al., 2017).
Figure 8: Marker-free deletion system. A The upstream and downstream regions of the deletion region were amplified, fused in an LFH reaction and cloned into the vector pJOE6743 or pGP1022. B The strain was transformed with the plasmid, which was inserted into the genome via one of the homolog flanks. C Since the flanking region, which was not used for the integration is now present in two copies, a recombination occurs, and the deletion region is removed.
upstream downstream
down Deletion region
up
specR ampR
manP
A
B. subtilis ΔmanP-manA chromosome
specR ampR
manP
upstream downstream
B
manP specR ampR
recombination
Deletion region down up down
up down
C
29 Cre-lox deletion system
The Cre-lox system is used to generate clean deletions. In the first step, the gene of interest is exchanged with a resistance cassette from an LFH product. The LFH product contains the up- and downstream regions from the gene of interest, a resistance cassette and additionally lox sites between the flanking regions and the resistance cassette. These 34 bp lox sites, lox71and lox66, can be recognized by the Cre recombinase from the P1 bacteriophage, which cuts and recombines these specific sequences. The gene encoding this enzyme was previously introduced into the sacA locus of the B. subtilis chromosome and the expression can be induced by the addition of xylose. If the expression of the cre recombinase is induced, the resistance cassette is cut out and the lox72 site remains in the genome (Figure 9). (Yan et al., 2008; Kumpfmüller et al., 2013).
Figure 9: Cre-lox deletion. A The gene of interest was exchanged with an antibiotic resistance cassette via LFH. Additionally, lox sites were introduced. B The cre recombinase was induced by the addition of xylose and it cuts and recombines the lox71 and lox66 sites. Finally, the resistance cassette was removed and a lox72 site is left.