Segmentación de los Consumidores de Detergentes en Trujillo

2.2.2.1 DNA extraction and purification

For plasmid DNA extraction from E. coli, commercial kits based on alkaline lysis methods were used according to the manufacturers’ protocols. An AxyPrep Plasmid Miniprep Kit (Corning, USA) was used for small-scale plasmid isolation. A PureLink™ HiPure Plasmid Filter Midiprep Kit (Life Technologies, USA) was used if larger quantities of plasmid were needed. The E.Z.N.A.® M13 DNA Mini Kit (Omega Bio-Tek, USA) was used to purify ssDNA from phage particles.

For purification of DNA fragments from agarose gels, AxyPrep DNA Gel Extraction (Corning, USA) and QIAquick Gel Extraction (Qiagen, The Netherlands) kits were used as per the manufacturers’ protocols.

Metagenomic DNA was extracted from bovine rumen plant-adherent microbiota as described in section 2.2.4.3. Purified DNA was stored at –20°C until required.

2.2.2.2 Agarose gel electrophoresis

The gels contained 0.8 – 1% (w/v) agarose and the 1× final concentration of SYBR® Safe DNA Gel Stain (Life Technologies, USA) in 1× TAE buffer. DNA samples were mixed with OrangeG DNA Loading Dye (1× final) prior to loading, except for the DNA size standard

(1 Kb Plus DNA ladder; Life Technologies, USA). Gels were run in 1× TAE buffer at 5 V cm-1

in a Wide mini-Sub® Cell GT horizontal electrophoresis system (Bio-Rad Laboratories, Inc., USA) for 90 min or in an Owl Horizontal A2 Large Gel System (Thermo Fisher Scientific, USA) for 30 min. DNA was visualised using a UV transilluminator and digitally photographed using a Nikon D700 digital camera with Kodak Gel Logic 200 Imaging System software (Eastman Kodak, USA). A Safe Imager™ 2.0 Blue Light Transilluminator (Life Technologies, USA) was used to visualise DNA for gel extraction.

2.2.2.3 DNA quantification

Fluorometric and spectrophotometric methods were used to measure the concentration, and estimate the purity of DNA samples. Fluorescence-based quantification assays were performed using a Qubit® dsDNA BR Assay Kit and Qubit® ssDNA Assay Kit with the Qubit® 2.0 Fluorometer (Life Technologies, USA) according to the manufacturer’s instructions. A NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, USA) was used for spectrophotometric quantification, where the DNA concentration was calculated from absorbance at 260 nm wavelength.

2.2.2.4 Preparation and transformation of electro-competent E. coli cells

Electro-competent E. coli cells used in this work were prepared using a modified

protocol by Jacobsson et al. [329], with the exception of the TG1 electro-competent cells used

to generate the large shotgun library, that were purchased from Stratagene (USA).

Electro-competent cells were transformed by electroporation using 0.1 cm gap electroporation cuvettes and a Gene Pulser® II Electroporation System (Bio-Rad), under the following conditions: 1.8 kV, 25 μF, 200 Ω, unless stated otherwise. Transformed cells were transferred to a tube containing 950 μL of freshly prepared SOC medium and incubated for 1 h at 37°C with rotary agitation. Ten-fold serial dilutions of the transformed cells were plated on 2×YTCm25 agar and incubated overnight at 37°C to allow colonies to form before counting.

89

2.2.2.5 Polymerase chain reaction (PCR) amplification

2.2.2.5.1 Bacterial colony PCR

Recombinant plasmids were analysed by PCR using DNA released from resuspended bacterial colony cells as a template. Primers pspF03 and pspR03 (Table 2.3) used in these reactions were complementary to the vector sequences flanking the multiple cloning site (MCS). The reverse primer pspR03 anneals 137 nt downstream of the cloning site, resulting in amplification of the vector sequence encoding the c-myc peptide tag and the first 30 amino acid residues of the C-terminal domain of pIII.

To prepare the cell suspension for PCR, individual colonies were picked from the transformation plates using sterile toothpicks and resuspended in 100 µL 1×TBS (25 mM Tris, 137 mM NaCl, 3mM KCl, pH 7.4) and this suspension was used at 1/10 volume of the colony PCR reactions (e.g. 5 µL in a 50 µL reaction, see Table 2.5). Colony PCR amplifications were carried out using Platinum® Taq DNA Polymerase High Fidelity (Life Technologies, USA) in the Mastercycler®pro system (Eppendorf, Germany). The components and the thermal profile of this PCR reaction are listed in Tables 2.5 and 2.6, respectively.

Table 2.5 Components of a PCR reaction mixture for colony PCR.

Component Volume per reaction

(50 μL)

Final concentration

10× High Fidelity PCR Buffer 5 μL 1×

10 mM dNTP mixture 1 μL 0.2 mM each

50 mM MgSO4 2 μL 2 mM

200 pM sense primer pspF03 (section 2.1.4) 0.125 μL 0.5 pM 200 pM antisense primer pspR03 (section 2.1.4) 0.125 μL 0.5 pM

Cell suspension in 1×TBS 5 μL N/A

Platinum® Taq HiFi polymerase (5U μL-1) 0.2 μL 0.02 U μL-1 DNA-free PCR water (section 2.1.2.2) 36.55 μL N/A

Table 2.6 Thermal profile of the colony PCR reaction.

PCR step Temperature Time

1 Initial denaturation 94ºC 5 min

2 Denature 94ºC 30 s

3 Anneal 55°C 30 s

PCR step Temperature Time

5 Repeat steps 2 – 4 total 30×

6 Final extension 68ºC 7 min

* Extension time recommended by manufacturer: 1 min per Kb of DNA template

The amplicons obtained after completion of the bacterial colony PCR were analysed by agarose gel electrophoresis and/or used as templates for sequencing. The PCR products that

served as sequencing templates were further purified using the NucleoSpin® Gel and PCR

Clean-up (Macherey-Nagel, Germany) and USB® ExoSAP-IT® PCR product clean-up (Affimetryx, USA) kits according to the manufacturers’ protocols.

The cell suspensions that were used in the PCR reactions were further clonally purified by streaking onto the selective plates (2×YTCm25). These purified transformants were used as a source of recombinant plasmids (section 2.2.2.1) for further analyses.

2.2.2.5.2 PCR amplification of ssDNA for pyrosequencing

PCR amplification of the recombinant phagemid ssDNA pool, derived from the large- scale shotgun metagenomic library after metasecretome selection, was performed in order to amplify the secretome-selected inserts as a template for pyrosequencing. The hot-start

PrimeSTAR® Max DNA Polymerase (Takara Bio Inc., Japan) was used for amplification in the

Mastercycler®pro system (Eppendorf, Germany). Primers PCRF2 and PCRR2 (see section 2.1.4) were designed to anneal to the phagemid vector sequences 361 bp upstream and 367 bp downstream of the library insert, respectively.

The components and the thermal profile of this PCR reaction are listed in Tables 2.7 and 2.8, respectively.

Table 2.7 Components of a PCR reaction mixture for ssDNA amplification.

Component Volume per reaction

(50 μL)

Final concentration

PrimeSTAR Max Premix (2×) a 25 μL 1× 20 pM sense primer PCRF2 (section 2.1.4) 1.25 μL 0.5 pM 20 pM antisense primer PCRR2 (section 2.1.4) 1.25 μL 0.5 pM

ssDNA template 2.5 μL 5 – 50 pg μL-1

DNA-free PCR water (section 2.1.2.2) 20 μL N/A

a

2× premix contains the PrimeSTAR® Max DNA Polymerase, reaction buffer, 2 mM Mg2+ and

91

Table 2.8 Thermal profile of the PCR reaction (rapid amplification protocol).

PCR step Temperature Time

1 Denature 98ºC 10 s

2 Anneal 55°C 5 s

3 Extend 72ºC 25 s a

4 Repeat steps 1 – 3 total 35×

a

Extension time recommended by manufacturer: 5 s per Kb of DNA template