PAGE 3 OF 7 sequence conﬁrmed, and the sequence has been deposited in GenBank (accession no. AY916793). Library construction The SU66E20 bacterial artiﬁcial chromosome (BAC) from the sea urchin S.purpuratus was prepared using the Large Construct Plasmid Isolation kit (Qiagen). The template DNA was sonicated and end repaired with T4 DNA polymerase (Fermentas), Klenow fragment (Fermentas) and T4 polynuc- leotide kinase (Fermentas). Fragments of approximately 1.5–3 or 2–4 kb were isolated by gel puriﬁcation. The pGRFP plas- mid was cut with EcoRV (NEB), 50 dephosphorylated with CIAP, and gel puriﬁed. The linearized vector was blunt-end ligated to the SU66E20 BAC library inserts using T4 DNA ligase. Preparation of cells Ligation products were electrotransformed into Electromax DH10B cells and incubated in 1 ml SOC for 1 h. An aliquot of 5 ml of fresh LB–carbenicillin (50 mg/ml) was inoculated with 100 ml of the SOC mixture. Cells were grown at 30 C with shaking for 24 h. Approximately 10–50 ml of culture was added to 4 ml of 0.9% NaCl (saline) for ﬂow-cytometric analysis. Fluorescence microscopy A culture transformed with pGRFP harboring ligated sea urchin DNA was harvested for cells that were ﬁxed on a coverslip with 10% formaldehyde. Cells were photographed using a Zeiss Axiophot ﬂuorescence microscope with attached Nikon digital camera. Illumination was by arc lamp light ﬁltered to the blue range (450–490 nm). Flow cytometry Flow cytometry was performed using an inFlux ﬂow cyto- meter (Cytopeia). Samples were injected into a sterile saline ﬂow stream. The sample cells were analyzed for green and red ﬂuorescence according to the set-up shown in Figure 1B. Excitation was by 5 W Innova argon laser (Coherent) at 488 nm emission with 400 mW power. The ﬂow cytometer was conﬁgured to trigger on the forward scatter channel. Emis- sion ﬂuorescence was passed through a 488 nm rejection band ﬁlter and split using a 550 nm dichroic long pass beam splitter. The green ﬂuorescence was ﬁltered through a 560 nm short pass ﬁlter before measurement by using a Hamamatsu PMT. The red ﬂuorescence was passed through a 590 nm long pass ﬁlter before measurement. Red signal intensity was plotted versus green for each cell on linear bivariate dot plots. Cells displaying low red-to-green ﬂuorescence ratio were selected for sorting. The ﬂow cytometer was programmed to deposit a single cell into each well of a 96-well plate containing 200 ml LB–carbenicillin (50 mg/ml). To establish enrichment of insert-bearing clones after cell- sorting, individual cells were sorted onto LB–carbenicillin plates in a grid pattern. Cells were either selected GFP+/ DsRed cells or randomly selected cells from the original culture. After 24 h growth at 37 C, plates were counted to determine the number of DsRed+ and DsRed colonies. PCR amplification Shotgun library inserts from SU66E20 in the 1.5–3 kb size range were cloned into pGRFP. From the single sorted cells 46 cultures Nucleic Acids Research, 2005, Vol. 33, No. 5 e49 were grown. An aliquot of 5 ml from each culture was diluted into 50 ml ddH2O and heated to 95 C for 5 min. As a template for subsequent PCRs 1 ml of lysate was used. Taq polymerase (Promega) was used according to the manufacturer’s recom- mended protocol with forward (TGTAAAACGACGGC- CAGT) and reverse M13 primers (CAGGAAACAGCTA- TGACC) ﬂanking the linker region of pGRFP. Thermocycle conditions were as follows: 95 C for 5 min; and 35 cycles of 95 C for 45 s, 51 C for 45 s, 72 C for 3 min 30 s; and a ﬁnal extension step of 72 C for 5 min. PCR products were photo- graphed after gel electrophoresis. BAC sequencing Shotgun library inserts from SU66E20 in the 2–4 kb size range were cloned into pGRFP. Single cells were sorted into 96-well plates as described above. After overnight growth, 1 ml of cultures were used as templates for rolling circle ampliﬁcation (RCA) using TempliPhi DNA Sequencing Template Ampli- ﬁcation kits (Amersham) according to the manufacturer’s recommended protocol. RCA reactions were performed in 10 ml volume at 30 C for 18 h and then stopped by heating to 65 C for 10 min. Products were dephosphorylated using 1 U shrimp alkaline phosphatase at 37 C for 45 min with heat inactivation at 80 C for 10 min. An aliquot of 1 ml of the product was used as template for Big Dye Terminator v3.0 (Perkin-Elmer) sequence reactions with 4 pmol of either for- ward or reverse M13 primers. Sequences were analyzed and assembled using Phred/Phrap (18) and the assembly was viewed using Consed (19). RESULTS Single reporter Initial experiments using single ﬂuorescent proteins were limited by large inter-bacterial variations in ﬂuorescence intensity. Even when picked from a single colony on a plate, individual bacteria showed large variations in GFP con- centration and occasionally bacterial cell size. Figure 3A shows a typical ﬂow cytometry dot plot of E.coli expressing GFP from a constitutive promoter. The spread of ﬂuorescence intensities reﬂects stochastic cell-to-cell variations, despite the cells’ clonal nature. When using only a single ﬂuorescent protein, large ambiguities exist owing to the large degree of inherent variation in expression. These observations are con- sistent with earlier studies in clonal E.coli populations (11). Identification of insert bearing clones using dual-fluorescence reporting system The problems associated with single ﬂuorochrome markers can be circumvented with a dual-reporter system. We con- structed the pGRFP vector that expresses two translationally fused ﬂuorescent proteins (Figure 1A). The linker region between the two proteins contains several cloning sites. With- out an insert, the uninterrupted protein linker will lead to a hybrid protein consisting of GFP and DsRed ﬂuorochrome groups. On the other hand, insertion of a DNA fragment into the linker region will introduce stop codons so that these clones will express only GFP. Excitation then produces an intense green ﬂuorescence signal.