41 | Overview over plasmids used in this study

All plasmids used in this study are listed in table 2.2. Gene inactivation in Bacillus strains was performed by plasmid integration and homologous recombination using recombinant derivatives of the thermo-sensitive shuttle vector pMAD (Arnaud et al., 2004; figure 2.1). Recombinant vectors that contain up to 500 bp of the N- and C-terminal sequences of target genes were introduced into the corresponding restrictions sites of the mcs. Resulting vectors were introduced in E. coli SCS110 as intermediate cloning host and selected on LB agar plates containing ampicillin at 37°C. Finally, these vectors were transformed into Bacillus strains and selected on TSA agar plates containing the selection marker erythromycin (5 mg/l) at 30°C. Homologous recombination and insertion of the vectors into target genes was performed by overnight cultivation at the non- permissive temperature (45°C) in TSB (25 mg/l EM) and, subsequent, plating onto TSA agar plates (25 mg/l EM).

Figure 2.1: Physical map of pMAD (Arnaud et al., 2004). The shuttle vector pMAD contains a thermo-sensitive origin of replication and confers resistance to ampicillin (bla) and erythromycin (ermC). Blue-white screening of recombinants is facilitated by a constitutively expressed β- galactosidase (bgaB).

Vectors of the pET expression system (Merck-Novagen, Darmstadt, Germany) were employed for heterologous expression of recombinant, HIS-tagged proteins in E. coli. In this system, proteins of interest are expressed under control of a strong bacteriophage T7lac promoter and are repressed by the LacI repressor protein. Expression hosts are lysogenic for

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bacteriophage DE3 and, therefore, carry a chromosomal copy of the T7 RNA polymerase gene under control of a lacUV5 promoter, which is also repressed by LacI. By addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to bacterial cultures, LacI is inactivated which results in the expression of T7 polymerases and, consequently, in expression of target genes. In pET22b, the coding sequence of six histidine residues is located downstream of the multiple cloning site (mcs) and enables a C-terminal fusion of the HIS-TAG to target proteins (figure 2.2). pET28b harbors two putative HIS-TAG sequences for expression of C- or N- terminally HIS-tagged proteins (figure 2.2).

Figure 2.2: Physical map of expression vectors pET22b and pET28a (Novagen/Merck chemicals). Plasmids confer resistance against ampicillin (Ap) or kanamycin (Kan). HIS-TAG coding sequences are located next to the mcs and allow cloning of N- or C-terminal HIS-tagged proteins.

Photometric determination of nucleic acid concentration and purity

UV-light based photometric determination of nucleic acid concentrations was performed using a Nanodrop spectrometer (Nanodrop Technologies, Wilmington, USA) at 260 nm. Purity was determined by the quotient of extinction at 260 nm over 280 nm. A quotient below 1.8 indicated contamination with proteins.

| 43 | Table 2.2: Plasmids used in this study.

Plasmid Information Aim Source

pMAD shuttle vector harboring a temperature sensitive ORI, AMPr_,

EMr

homologous recombi-

nation in Bacillus Arnaud et al., 2006

pMADLicM1AC pMAD derivative carrying an approx. 500 bp C- and N-terminal fragment of bli4126

knock out of LicM1 this study pMADLicM1AC pMAD derivative carrying an approx.

500 bp C- and N-terminal fragment of bli4128

knock out of LicM2 this study pET22b HIS-TAG expression vector, AMPr _{heterologous expres-}

sion of C-terminally HIS-tagged proteins in

E. coli

Novagen

pET22PunM pET22 derivative harboring the C-

terminally HIS-tagged punM heterologous expres-sion of PunM this study pET22AnaM pET22 derivative harboring the C-

terminally HIS-tagged anaM heterologous expres-sion of AnaM this study pET28b HIS-TAG expression vector, KANAr _{heterologous expres-}

sion of N-terminally HIS-tagged proteins in

E. coli

Novagen

pET28PunA1 pET22 derivative harboring the N-

terminally HIS-tagged punA1 heterologous expres-sion of the PunA1 pre- peptide

this study pET28PunA1_XA pET22 derivative harboring the N-

terminally HIS-tagged punA1 including a XA-factor cleavage site

heterologous expres- sion of the PunA1 pre- peptide

this study pET28AnaA pET22 derivative harboring the N-

terminally HIS-tagged gene anaA heterologous expres-sion of the AnaA pre- peptide

this study pET28AnaA_XA pET22 derivative harboring the N-

terminally HIS-tagged besciin including XA-factor cleavage site

heterologous expres- sion of the AnaA pre- peptide

this study pUC19 shuttle vector; AMPr_{, CM}r _{intermediate cloning}

vector for lantibiotic precursors

Yanisch- Perron et

al., 1985

pUC19AnaA pUC19 derivative harboring the

structural gene anaA templates for site directed mutagenesis of protease cleavage sites

this study pUC19PunA1 pUC19 derivative harboring the

structural gene punA1 this study

pOPAR1 pCU1 derivative harboring mrsR1 and

mrsA including its operator structure expression of the lanti-_{biotic mersacidin in} B. amyloliquefaciens

Schmitz et

al., 2006

pPAR1/1 pCU1 derivative harboring mrsR1 and

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DNA sequencing

DNA sequencing was performed by Sequiserve (Vaterstetten, Germany) or Seqlab (Göttingen, Germany) employing the Sanger chain determination method (Sanger et al., 1977). For sequencing, recombinant plasmids and PCR products were dissolved in sterile MilliQ water. Integrity of recombinant plasmids was confirmed by sequencing of inserts with vector-specific primers supplied by Sequiserve, if available.

Agarose gel electrophoresis

Standard agarose gel electrophoresis was used to analyze and separate DNA fragments employing 0.8-2% agarose gels (Top VisionTM LE GQ agarose, Fermentas) in a horizontal electrophoresis apparatus (Peqlab, Erlangen, Germany) according to Sambrook et al., 1989. DNA was stained with ethidium-bromide and visualized using ImageMaster VDS (GE Healthcare/Pharmacia).

Polymerase chain reaction (PCR)

Amplification of DNA was performed by the polymerase chain reaction in accordance with Mullis et al., 1986. DreamTaq Polymerase (Fermentas) was used for standard PCR batches. For cloning or subsequent sequencing DNA was amplified using PhusionTM_{HF DNA}

Polymerase (NEB, Frankfurt/Main, Germany) as detailed in the manual. Desoxynucleotides were ordered as premixed solutions (25 mM each; Fermentas). PCRs were performed in 50 µl batches on the PCR express Thermal Cycler (Hybaid, ThermoLifeScience, Engelsbach, Germany) or the SensoQuest Labcycler (SensoQuest, Göttingen, Germany).

All primers were synthesized by Metabion (Martinsried, Germany) or Microsynth (Balgach, Switzerland) and are listed in table 2.3. Sequencing and standard cloning primers were designed using the Primer3 tool: http://biotools.umassmed.edu/bioapps/primer3_www.cgi.

Enzymatic modification of DNA

Plasmids and PCR products were digested using restriction enzymes (FastDigestTM) from Fermentas. Digestion was performed as detailed in the manual. For cloning of large inserts (> 2000 bp), vector backbones were dephosphorylated using alkaline dephosphatase (Fermentas) during digestion. After heat inactivation (10 min, 65°C) digested nucleic acids were stored at -20°C until further use.

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