Supplemental Methods s6

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 pSMARTTM 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).