Large Insert Environmental Genomic Library Production

Summary

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

Abstract

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE http://www.epibio.com/item.asp?ID=385). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Protocol

Fosmid library construction has been divided into four main steps and sub-divided into several parts (see Fig.1 for an overview).

Step I (see protocol "DNA extraction from 0.22 μM Sterivex filters" [3])

Part 1: Enzyme-catalyzed cell lysis

Part 2: Purification of environmental DNA by CsCl density gradient centrifugation and DNA recovery

Part 3: Quality control of extracted DNA by gel electrophoresis

Step II

Part 1: Enzymatic modification steps of recovered environmental DNA

Discussion

A procedure is described how to most efficiently generate a large-insert fosmid library with genomic DNA derived from a coastal water sample. Up-stream genomic DNA extraction is described in a separate protocol [3].

As the fosmid-library production is a multistep-process, plan at least two to three weeks in time for the whole procedure including all four presented steps. The extraction of genomic DNA is the most crucial step and all down-stream steps rely on the quality and quantity of extra.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Step II
CopyControl™ Fosmid Library Production Kit	Epicentre Biotechnologies	CCFOS110	sufficient to generate 9-10 fosmid libraries
SeaPlaque Agarose	Cambrex	50100
stirring hot plate	Corning	PC-420D
1.0X TAE running buffer
CHEF Mapper XA pulsed field electrophoresis system including cooling module and variable speed pump	Bio-Rad
λ DNA-HindIII Digest	New England Biolabs	N3012S
MidRange I PFG Marker	New England Biolabs	N3551S
10,000X SYBR Gold	Invitrogen	S11494
microwavable plastic wrap	Saran premium wrap
Safe Imager transilluminator	Invitrogen	S37102
GELase Agarose Gel-Digesting Preparation	Epicentre Biotechnologies	G09100
scalpel	Fisher Scientific	08-927-5D
Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane	EMD Millipore	UFC801024
Microcon YM-30 Centrifugal Filter Unit	EMD Millipore	42410
nuclease free water	Ambion	AM9932
Quant-iT PicoGreen dsDNA Assay Kit *2000 assays*#	Invitrogen	P7589
digital dry block heater	VWR international	12621-088
water bath	VWR international	14231-854
centrifuge	Beckman Coulter Inc.	Avanti J-E
centrifuge rotor	Beckman Coulter Inc.	JA 5.3
table top centrifuge	Eppendorf	5415D
microcentrifuge tubes, 1.7 mL, clear	Axygen Scientific	MCT-175-C
15 mL centrifuge tubes	Fisher Scientific	430052
Step III
LB broth	Fisher Scientific	BP 1426-2
MgSO4	Sigma-Aldrich	M2643
phage dilution buffer			10 mM Tris-HCl [pH 8.3], 100 mM NaCl, 10 mM MgCl2
cryogenic vials	Fisher Scientific	430488
Step IV
96 well plate with lid	Fisher Scientific	CS003370(3370)
384 well plate with lid	Fisher Scientific	07201157(3680)
Petri dishes 100 O.D. x 15mm H	Fisher Scientific	08-757-12
Petri dishes 150 Dia. x 15mmH	Fisher Scientific	08-757-14
BD Falcon BioDish XL 245 X 245 mm	VWR international	CABD351040
glycerol	Sigma-Aldrich	G5516
chloramphenicol	Sigma-Aldrich	C0378
LB broth	Fisher Scientific	BP 1426-2
Difco Agar	BD Biosciences	214530
glass beads	Fisher Scientific	10-310-1
QFill3	Genetix	X3050
Bleach	Javex
Ethanol	Sigma-Aldrich	E7023
15 mL centrifuge tubes	Fisher Scientific	430052
QPix colony picker	Genetix	QPix2
picking head (E. coli)	Genetix	X4006