The metagenomic library, which consists of 18,432 clones, was constructed using DNA extracted from high-Andean forests soils from the National Natural Park “Los Nevados [17] . DNA was purified using the Ultra Clean Mega Soil DNA kit (MoBio) and fragments of approximately 30 kb were ligated to the pCC2FOS vector (Epicentre) and used to transform Escherichia coli EPI300™, following the manufacturer’s indications (Epicentre). The identification of positive clones for cellulose degradation was done as published [17] . Briefly, clones capable of growing on minimum salt medium (MM; 0.2% NaNO3, 0.1% K2HPO4, 0.05% MgSO4, 0.05% KCL, 0.02% Peptone, 12.5 μg/ml chloramphenicol) with pretreated OPEFB as only source of carbon for 35 days at 30 °C and 200 rpm, were presumed to be cellulases carriers. Then, these colonies were enriched in Luria-Bertani (LB) medium for 2 days and transferred to a solid MM containing carboxymethyl cellulose. To detect cellulose hydrolysis, Congo red staining was used and the presence of a hydrolysis halo surrounding a colony was taken as a positive clone for cellulose degradation.
Pcc2fos vector
Manufactured by Illumina
The PCC2FOS vector is a plasmid used in molecular biology research. It contains the FOS gene, which is a commonly used reporter gene for studying gene expression. The vector allows for the expression and detection of the FOS gene, which can be used as a marker for various cellular processes.
Automatically generated - may contain errors
4 protocols using pcc2fos vector
1
High-Andean Soil Metagenomic Library Screening
2
HMW DNA Fosmid Library Construction
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High-molecular-weight (HMW) DNA was isolated from 7-day cultured mycelia using the CTAB DNA isolation method. Genomic DNA was randomly sheared to approximately 40 kb in size. Then, the sheared molecules were size-selected using Pulse Field Gel Electrophoresis (Bio-Rad, Hercules, CA). The sheared DNA was ligated into the pCC2FOS vector (Epicentre, Madison, WI) after end-repair. The ligations were packaged using MaxPlax Lambda Packaging Extracts (Epicentre). The lambda phages carrying foreign DNA were used to infect EPI300-T1R E. coli cells (Epicentre). Positive fosmid clones were selected using lysogeny broth (LB)-chloramphenicol (12.5 μg/ml) plates. A total of 11,232 clones were selected and transferred to 96-well plates containing frozen LB media and stored at −80 °C.
3
Constructing Metagenomic Libraries for Biodiversity Exploration
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Two metagenomic libraries are used in this work. The first one is obtained from Dr. Wenjun Zhang's lab. Streptomyces sparsogenes (ATCC25498) genome (NZ_MAXF00000000.1) (18 (link)) fragments are inserted into the pCC2FOS™ vector (Epicentre) and transformed into Escherichia coli EPI300™ cells. A total of 1.5 × 104 clones with ∼40 kb inserts have been generated. Library cells are propagated in 2xYT media (Research Products International) supplemented with 25 μg/ml chloramphenicol at 37°C overnight, and then 1 ml of the culture further supplemented with 1× Fosmid Autoinduction Solution (Epicentre) is used for cell encapsulation in droplets. The second library is obtained from the Canadian MetaMicroBiome Library (19 (link)). The Arctic Tundra 2 (2ATN) metagenomic DNA sample is comprised of ∼6 × 104 unique clones, with each ∼31 kb random insert carried by the pJC8 within E. coli HB101 cells. Library cells are propagated in 2xYT media supplemented with 15 μg/ml Tetracycline at 37°C overnight, and 1 ml of the culture is used for cell encapsulation in droplets.
4
Cyanobacterial Fosmid Metagenomic Libraries
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Fosmid metagenomic libraries were constructed with the use of the CopyControl Fosmid Library Production Kit with pCC2FOS Vector (purchased from Epicentre Biotechnologies), according to manufacturer’s instructions. Cyanobacterial genomic DNA was isolated and purified as described previously [[13 (link)]], and 20 μg of DNA from each isolation experiment were used for library construction. The final product of each construction procedure was 1 ml of phage lysate which was then used to infect E. coli T1R host strain (provided in CopyControl Fosmid Library Production Kit). Number of obtained clones in each experiment is provided in Table 1 . Routinely, 0.1 ml of the lysate was used, and the rest (0.9 ml) is being stored for potential further use. For control experiments, a clone bearing the 41,285 bp fragment of E. coli genome, encompassing the region between abc2T and pdpN genes, was constructed and named 105–2.
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