General PCR methods and Sanger sequencing

A BioRad DNA Engine Thermal Cycler was typically used to perform Polymerase chain reaction (PCR). PCR tubes were strips of 8 thin-walled 0.2 ml tubes with domed-caps (Thermo Scientific). Most primers were designed with an annealing temperature of 55 to 60 °C, consequently the annealing temperature used for most applications was 56 °C.

The conditions typically used for different applications are listed below. The use of 4.8 % DMSO in PCR reactions facilitates the amplification of templates with a high GC content.

87 2.6.1 General analytical PCR

Amount/Concentration Volume (µl) Final Concentration

Template DNA 1-10 ng 1

Forward Primer 10 µM 1 0.4 µM

Reverse Primer 10 µM 1 0.4 µM

dNTPs 20 mM (5 mM each) 1 400 µM

(100 µM each)

Taq Buffer (Roche) 10 x 5 1x

DMSO 40 % 6 4.8 %

Taq (Roche) 5 U/µl 0.5 2.5 U

dH₂O 34.5

Total 50

95 °C 2 min 95 °C 45 s

55-60 °C 30 s x 27 cycles 72 °C 1-3 min

72 °C 7 min

2.6.2 High-fidelity amplification for cloning applications

Amount/

Concentration Volume (µl) Final Concentration

Template DNA 100 ng/µl 1

Forward Primer 10 µM 5 1 µM

Reverse Primer 10 µM 5 1 µM

dNTPs 20 mM (5 mM each) 1 400 µM (50 µM each)

Expand HiFi Buffer 2 (Roche)

10 x 2.5 1 x

DMSO 40 % 6 4.8 %

Expand HiFi Taq (Roche)

3.5 U/µl 0.71 2.48 U

dH2O 28.79

Total 50

88 94 °C 2 min

94 °C 45 s

55-60 °C 45 s x 27 cycles 72 °C 1-3 min

72 °C 7 min

2.6.3 Colony PCR in E. coli

The PCR reaction mix was as 2.6.1 but with an extra 1 µl dH2O. Colonies of E. coli were picked from agar plates using a sterile Gilson pipette tip (Starlabs) and were smeared onto the wall of a PCR tube before adding the appropriate PCR mix. The tubes were placed in the Thermo cycler using the conditions:

95 °C 5 min 95 °C 45 s

55-60 °C 45 s x 30 cycles 72 °C 1-3 min

72 °C 5 min

2.6.4 Colony PCR in Planomonospora

Single colonies of P. alba were patched on to plates of D/seed and incubated for three days at 30 °C. 50 µl 100 % DMSO were aliquoted into 1.5 ml microcentrifuge tubes for Planomonospora mycelium to be scraped into using a sterile toothpick. The tube was shaken vigorously for at least one hour and then briefly centrifuged to pellet cell debris.

2.5 µl of the supernatant were used as the template in the reaction as shown below.

Control DNA samples were diluted 1 µl into 1.5 µl DMSO and were used in the same way.

Amount/Concentration Volume (µl) Final Concentration

Forward Primer 10 µM 1 0.4 µM

Reverse Primer 10 µM 1 0.4 µM

dNTPs 20 mM (5 mM each) 1 400 µM (100 µM each)

Taq Buffer (Roche) 10 x 5 1 x

DMSO + template 100 % 2.5 5 %

Taq (Roche) 5 U/µl 0.5 2.5 U

dH2O 39

Total 50

89 95 °C 5 min

95 °C 45 s

56-60 °C 45 s x 30 cycles 72 °C 1 min

72 °C 5 min

2.6.5 Purification of PCR products

The QIAquick™ PCR purification kit (Qiagen) was used according to the manufacturer’s instructions to remove unincorporated primers, dNTPs and enzymes from PCR products

>100 bp. 5 µl of the PCR reaction mixture were used in agarose gel electrophoresis to check the correct product was amplified. The remaining PCR mixture was diluted 5 times in high salt buffer and applied to a silica-gel membrane mounted in a microcentrifuge tube.

The DNA fragment was washed then typically eluted in 2 x 25 µl elution buffer heated to 50 °C.

2.6.7 Sanger sequencing using Big Dye v3.1

The ABI BigDye® 3.1 dye-terminator reaction mix (Applied Biosystems) was used to label purified PCR products or vectors. The manufacturer’s instructions were consulted to find what amount of DNA was appropriate as a template, based on its length. Big Dye labeling reaction was performed as detailed below and subsequently submitted to The Genome Analysis Centre for ABI Sanger Sequencing. The resulting sequence chromatogram files were analysed using VectorNTI ContigExpress software.

Amount Volume (µl) Final Concentration

Template varies varies varies

Primer 10 µM 3.2 0.32 µM

ABI BigDye® 3.1 dye-terminator reaction mix

1

BigDye® 3.1 Reaction Buffer 5 x 2 1 x

dH2O varies

Total 10

95 °C 10 min 96 °C 10 s

50 °C 5 s x 25 cycles 60 °C 4 min

90 2.7 Growth conditions

2.7.1 Growth and storage of E. coli

E. coli strains were typically grown with the appropriate antibiotic selection either on L agar at 30 or 37 °C or in L broth with shaking at 250 rpm. For long-term storage strains were grown overnight in L broth and stored in a final concentration of 20 % glycerol at -20

°C or -80 °C.

2.7.2 Growth and storage of Micrococcus luteus

M. luteus ATCC 4698 was typically grown either on L agar at 30 or 37 °C or in L broth with shaking at 250 rpm. For long-term storage strains were grown overnight in L broth and stored in a final concentration of 20 % glycerol at -20 °C or at -80 °C.

2.7.3 Growth and storage of Streptomyces

Streptomyces was grown and manipulated as described in (Kieser et al. 2000). Growth media were as stated in Section 2.3.2 and 2.3.3. For manipulation of Streptomyces and growth of confluent lawns for long-term storage, SFM agar medium was used with the appropriate antibiotic selection at 30 °C. Spores were collected in approximately 2 ml 20

% glycerol using a sterile cotton pad through which spores were filtered by collecting with a 2 ml syringe and transferring to a 2 ml cryotube. Spore stocks were spun and 1 ml supernatant was removed before resuspension and storage at -20 °C. Spore stocks were titred by making serial dilutions in water and plating on SFM. The resulting colonies were counted from at least three dilutions and averaged. For liquid culture, ~10⁸ Streptomyces spores were inoculated into the appropriate media in a 250 ml flask containing a baffle and incubated at 240 rpm at 30 °C.

2.7.4 Growth and storage of Planomonospora

P. alba NRLL18924 and P. sphaerica NRLL18923 were obtained from the NRRL culture collection. Growth media were as stated in Section 2.3.2 and 2.3.3. For manipulation of Planomonospora and growth of confluent lawns for long-term storage, ISP4 agar medium was used with the appropriate antibiotic selection at 30 °C. Spores were resuspended in 10 ml H2O then filtered through non-absorbant cotton-wool in a glass collection tube as described in (Kieser et al. 2000). Spores were spun down and resuspended in 1 ml 20 %

91 glycerol then transferred to a 2 ml cryotube for storage at -20 °C. Spore stocks were titred

by making serial dilutions in water and plating on ISP4. The resulting colonies were counted from at least three dilutions and averaged. For liquid culture, ~10⁷ Planomonospora spores were inoculated into ISP4 medium in a 25-100 ml flask and incubated with shaking at 240 rpm at 30 °C. The use of 3-6 glass beads (2 mm diameter) instead of a baffle, prevents foaming and associated anaerobic growth.

2.7.5 Growth and storage of Nonomuraea

Nonomuraea ATCC 39727 was a gift from Prof Flavia Marinelli (University of Insubria, Italy). Nonomuraea ATCC 39727 was grown on V0.1 agar medium at 30 °C. For liquid culture, Nonomuraea ATCC 39727 was typically inoculated 1 in 10 into VSP medium in a 25-100 ml flask containing 3-6 glass beads (2 mm diameter) and incubated with shaking at 240 rpm at 30 °C. For long-term storage (master cell bank) culture broth containing mycelium was aliquoted into 2 ml cryotubes and stored at -80 °C.

2.7.6 P. alba growth curve

Cultures to assess the growth rate of P. alba were set up in the following way.

Approximately 10⁷ P. alba spores or 1 ml of broth containing mycelium were used to inoculate 10ml ISP4 in a 25 ml flask containing three glass beads. The culture was grown for 24-48 hours at 30 °C with shaking at 240 rpm. 100 µl of the resulting mycelium was diluted in 1 ml ISP4 and the optical density at OD450 assessed. Three 100 ml flasks containing 25 ml ISP4 and 10 glass beads were inoculated to a starting OD₄₅₀ of 0.1-0.2.

The flasks were incubated at 30 °C with shaking at 240 rpm. At appropriate time intervals samples were removed. The growth rate of P. alba was assessed in liquid ISP4 by two methods. First, optical density at OD450 (using a 1 in 10 dilution when OD450 > 1) with ISP4 as a blank. Alternatively a total protein assay was used. 1 ml of culture was sampled at the appropriate time intervals. The DC protein assay (Bio-Rad)was used according to the manufacturer’s instructions. Bovine serum albumin (BSA) standards of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 ng/ml were prepared in cuvettes in 0.5 ml volumes. The standard curve generated from the standards was used to calculate the protein concentrations in the experimental samples.

92 2.8 Microscopy

2.8.1 Phase-contrast microscopy

Samples were placed on 76x26 mm glass slides (VWR international) and covered with a glass cover slip (18x18 mm; VWR international). Slides were typically observed at 400 times magnification with a Photomicroscope II in phase-contrast mode (Zeiss).

2.8.2 Cryo-scanning electron microscopy

High-resolution SEM was carried out by Kim Findlay at the John Innes Centre using a Zeiss Supra 55 VP FEG SEM with a Gatan Alto 2500 cryo system at an accelerating voltage of 3 kV.