SUPPLEMENTAL METHODS
Genomic Library Construction & Cloning
Overnight cultures of the E. coli strain K12 were cultivated in 150ml of LB
at 37°C to an optical density of 1.0 measured by absorbance at 600nm. The
culture was pelleted by centrifugation. The pellet was then washed in 50 ml of
TES buffer: 10mM Tris HCl, 1mM EDTA 1.5% w/v NaCl, pH = 8.0 and again
centrifuged. The pellet was again resuspended in 50ml of TES buffer. 300 µl of
20mg/ml proteinase K (Fisher Scientific, Pittsburgh, PA). and 3 ml of 10% w/v
SDS were added to the cell suspension which was then incubated at 55°C for 16
hours. The genomic DNA was then extracted twice with equal volumes of TE
(10mM Tris HCl, 1mM EDTA, pH=8.0) saturated phenol followed by two
extractions with TE saturated phenol/chloroform/isoamyl alcohol (25:24:1).
Genomic DNA was then precipitated with 1/10 volume 3M sodium acetate pH=
5.5 and 0.6 volumes of isopropanol. DNA pellets were washed with 70% ethanol
and resuspended in TE buffer pH= 8.0.
Six samples of 50 µg of purified genomic DNA were digested with two
blunt cutters AluI and RsaI (Invitrogen Carlsbad, CA) both having four base pair
long recognition sequences. 50 µl reactions with four units of each enzyme plus
50 mM Tris-HCl (pH 8.0), and 10 mM MgCl2 were carried out for 10, 20 30, 40,
50, and 60 minutes respectively, at 37°C. The reactions were heat inactivated at
70°C for 15 minutes. Restriction digestions were mixed and the fragmented DNA
was separated based on size using agarose gel electrophoresis. DNA fragments
of 0.5, 1, 2, 4, and greater than 8 kb were excised from the gel and purified with a
Gel Extraction Kit (Qiagen, Valencia, CA), according to manufacturer’s
instructions.
The purity of the DNA fragments was quantified using U.V.
absorbance each with an A260/A280 absorbance ratio of >1.7.
Ligation of the purified, fragmented DNA with the pSMART TM LC-Kan
vector was performed according to manufaturer’s instructions (Lucigen). The
ligation product was then electroporated into E. Cloni 10 G Supreme
Electrocompetent Cells (Lucigen) and plated on LB plus kanamycin.
1/1000
volume of the original transformations was plated on LB plus Kanamycin in
triplicate in order to determine transformation efficiency and transformant
numbers. Three of these dilutions were plated, in order to get average
transformant counts. Plates were incubated at 37°C for 24 hours.
Colonies were harvested by gently scraping the plates into TB media and
incubating at 37°C for 1 hour. The low copy plasmids were then amplified by
adding chloramphenicol to the culture and incubating at 37°C for 30 minutes
before
centrifugation.
The
plasmid
DNA
was
extracted
according
to
manufacturer’s instructions using a HiSpeed Plasmid Midi Kit (Qiagen).
In order to confirm insert sizes and transformant numbers, plasmids were
isolated from random clones for each library using a Qiaprep Spin MiniPrep Kit
from Qiagen. Plasmids were then analyzed by either PCR or restriction digestion.
PCR using the SL1: 5’-CAG TCC AGT TAC GCT GGA GTC-3’ and SR2: 5’-GGT
CAG GTA TGA TTT AAA TGG TCA GT-3’ primers was performed on ten clones
from the 0.5, 1, and 2 kbp insert libraries. Restriction digestions with the enzyme
SwaI (New England Biolabs, Beverly, MA) were carried out for ten clones from
the 4 and 8kbp insert libraries. Inspection by electrophoresis showed that greater
than 80% of the colonies contained an insert of the expected size and that
chimeras were not present. All DNA sequencing was performed by Macrogen
(Seoul, Korea).