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Evolution has selected for organisms that benefit from genetically encoded cell-cell communication. Engineers have begun to repurpose elements of natural communication systems to realize programmed pattern formation and coordinate other population-level behaviors. However, existing engineered systems rely on system-specific small molecules to send molecular messages among cells. Thus, the information transmission capacity of current engineered biological communication systems is physically limited by specific biomolecules that are capable of sending only a single message, typically "regulate transcription."
We have engineered a cell-cell communication platform using bacteriophage M13 gene products to autonomously package and deliver heterologous DNA messages of varying lengths and encoded functions. We demonstrate the decoupling of messages from a common communication channel via the autonomous transmission of various arbitrary genetic messages. Further, we increase the range of engineered DNA messaging across semisolid media by linking message transmission or receipt to active cellular chemotaxis.
We demonstrate decoupling of a communication channel from message transmission within engineered biological systems via the autonomous targeted transduction of user-specified heterologous DNA messages. We also demonstrate that bacteriophage M13 particle production and message transduction occurs among chemotactic bacteria. We use chemotaxis to improve the range of DNA messaging, increasing both transmission distance and communication bit rates relative to existing small molecule-based communication systems. We postulate that integration of different engineered cell-cell communication platforms will allow for more complex spatial programming of dynamic cellular consortia.
Keywords:Synthetic biology; Amorphous computing; Cell-cell signaling; Programmed pattern formation; Communication theory
The importance of cell-cell signaling throughout nature [1] suggests that better tools for engineering cell-cell communication will advance biotechnology. For context, other engineering disciplines have recognized the importance of understanding and programming systems across both space and time, and have even begun to map such work to biological substrates. As one example, Amorphous Computing (AC) was developed to help program system-wide patterns and other emergent behaviors within networks of independent elements [2]. Drawing inspiration from biology, AC-based systems are comprised of identical but autonomous agents that make use of local information to differentiate and act across space and time. The resulting space-time programming frameworks enable both the analysis and forward engineering of complex systems capable of programmed pattern formation and restoration (i.e., "healing") in the presence of noise or damage [3-5]. However, much improved cell-cell communication platforms are needed for scientists and engineers to practically benefit from and advance such research.
Autonomous engineered cell-cell communication was first demonstrated via the directed transmission of acyl-homoserine-lactones (AHLs) (Figure 1a) [6]. AHLs are freely-diffusing small molecules found in natural quorum sensing systems of various bacteria [7]. Subsequent pioneering examples have included Weiss et al.'s use of AHL concentration gradients to establish two-dimensional patterns [8], Tabor et al.'s engineering of an edge detector by integrating AHL-based signals to produce patterns in response to external stimuli [9], You et al.'s creation of synthetic predator–prey ecologies [10,11], and Danino et al.'s synchronization of genetic oscillators within microbial populations [12]. Most recently, Tasmir et al. demonstrated how cell-cell communication can be combined with serial plating of distinct cell types in order to approximate Boolean logic [13].
Figure 1. Existing cell-cell communication systems are limited by message-channel coupling. Four alternate schemes for engineering cell-cell communication. Gray circles represent cells. Colored arcs on left cells represent transmitters with different colors representing distinct transmitters. Stick-drawings on right cells depict receivers. Icons passing between cells represent information channels with colors representing distinct messages. (a) Single channel coupled to specific message (e.g., AHL-based systems), (b) Single channel coupled to specific message but with multiple receivers (e.g., AHL-based systems in which signaling molecule is recognized by distinct cognate response factors), (c) Multiple insulated channels each coupled to a specific message (e.g., a set of AHL-based systems with no crosstalk between signaling molecules and cognate response factors), (d) Single channel decoupled from any one message (e.g., M13-based systems, this work).
Most modern communication systems are based on the theoretical work of Shannon [14]. We thus mapped existing examples of engineered cell-cell communication into Shannon's framework in order to identify possible areas for fundamental improvement (Figure 2a). For example, of the engineered AHL-based systems noted above, an AHL signaling molecule is either directly added to cells or transmitted from sender cells to receiver cells. The "transmitter" in these systems is the source of signaling molecules, the researcher or encoded AHL synthase. The communicated "message" is, directly or indirectly, to transcribe genes under the control of a cognate signaling pathway or promoter. The message itself is encoded via the AHL concentration and the information "channel" is diffusion of AHL molecules. The "receiver" is the cognate receptor or transcription factor within receiver cells, which directly or indirectly initiate transcription in the presence of AHL.
Figure 2. Biological communication systems can be represented formally. (a) Schematic diagram of a general communication system, adapted from Shannon (19). (b) Representation of a bacteriophage M13-based cell-cell communication system within Shannon's framing.
Two limitations become immediately apparent in considering existing engineered AHL-based systems within Shannon's framing. First, such systems can transmit only a single message for a given information channel (Figure 1a). This "single message-single channel" limitation is due to the required molecular interaction between a given AHL and its cognate receptor or transcription factor. Attempting to encode a distinct message by changing the chemical structure of an AHL signaling molecule can adversely affect its interaction with its cognate receiver and disrupt the communication channel itself. In other words, the first limitation encountered in AHL-based systems is that the message and information channel are coupled such that the message is the molecule. Second, AHL-encoded messages are known to result in, directly or indirectly, regulation of transcription. Thus, AHL-based receivers can realize the receipt of just a single message type, "regulate transcription." Stated differently, the second limitation of AHL-based systems is their inability to transmit more than a single type of message.
One method for overcoming message-channel coupling is to increase the number of available independent channels (Figure 1c). However, members of the LuxR transcription factor family often respond to multiple AHL signaling molecules, leading to non-specific interactions and interference (Figure 1b) [15,16]. To explore and address molecular interference, researchers have employed directed evolution approaches to alter interactions among LuxR transcription factors and competing AHL molecules [17-19]. For example, the specificity of a LuxR variant for a specific AHL molecule was altered to allow recognition of straight-chained AHLs and to lack recognition of AHLs with a 3-oxo group. Unfortunately, so-evolved LuxR variants were not fully insensitive to AHL molecules with 3-oxo groups [18]. Moreover, while Balagaddé et al. was able to implement two independent AHL-based communication channels, receiver-specific genetic architectures were required as both AHL-responsive transcription factors recognized the same core promoter sequence [11,19]. Practically, only two AHL cell-cell communication channels, based on the LuxR and LasR transcription factors, have now been proven for independent use within any given engineered system.
Another approach for overcoming message-channel coupling would be to establish an information channel that is capable of transmitting arbitrary messages encoded via a common format. We immediately noted that many natural organisms can directly or indirectly exchange or uptake nucleic acids [20,21], a class of biomolecules whose underlying chemistry has itself been selected to support the encoding of information defining any genetic sequence [22,23]. We then specifically chose to focus on virus-mediated exchange of genetic material, as opposed to bacterial conjugation, since virus particles are typically physically released from infected cells whereas conjugation would require that sender and receiver cells interact directly.
Many viruses are capable of packaging and transmitting non-viral genetic material [24,25]. For example, "transvestite" bacteriophage lambda particles can package and transduce phage T7 genomes or other genetic material [26,27]. However, phage lambda and many viruses actively destroy the host cell in releasing virus particles. We thus instead considered viral systems that secrete progeny without destroying the infected host cell.
Bacteriophage M13 (M13) is a filamentous phage whose progeny particles are secreted from infected cells [28]. M13 is able to package single stranded DNA (ssDNA) of various lengths, a property that has allowed for the "hard-coding" of derivative M13 genomes that express various heterologous genes, including fusion proteins and transcription factors [29-31]. M13 is also able to package independent heterologous DNA sequences that contain the M13 packaging sequence. For example, "phagemids," plasmids encoding an phage DNA packaging sequence, have been used to generate M13 and other phage particles containing heterologous DNA that can then be applied to direct allelic replacement within E. coli[32-37]. However, such uses of phage particles to modify or exchange DNA typically require the physical purification or concentration of phage particles from infected cultures prior to their manual reapplication to transduce independent cultures.
Here, we repurpose the natural properties of M13 to engineer fully autonomous in situ cell-cell communication among bacterial cells. We show that an M13-based system can send DNA messages via a single information channel. Specifically, sender cells transmit an arbitrary DNA message that is encoded by a heterologous "messaging phagemid" and then packaged within M13 gene products. Receiver cells are transduced by so-produced M13 "message particles" containing user-defined DNA messages. We demonstrate transmission of various distinct DNA messages of varying lengths and encoded biochemical functions, including message-specific activation of genetic functions encoded within receiver cells, and long range transmission of DNA messages via active chemotaxis. Taken together, we have engineered a cell-cell communication platform that supports message-channel decoupling via phage particle-mediated transduction (Figure 1d).
We started by specifying three criteria for improving engineered cell-cell communication systems. First, a system should be "decoupled," which we define as the ability to transmit distinct messages via a reusable channel, such that the same system can be used in diverse applications without requiring changes to the system itself. Second, a system should be "flexible," which we define here as the ability to accommodate messages having different encoded lengths and biochemical functions, again to more readily enable applications. Finally, message transmission within the overall system should be "specific" such that certain cells can be made susceptible to message readout while others are not, so as to enable targeted cell-cell communication within mixed cultures.
We worked to meet our three criteria using M13. As noted above, we chose M13 because it can specifically package non-M13 genetic material and release so-produced particles without killing the host cell. In addition, binding of and infection by M13 particles requires cells to produce a pilus as encoded from the F-plasmid [38]. The resulting F+ strains are thus susceptible to transduction while F- strains are not.
Message transmission requires M13 packaging, particles, and F+ receiver cells
We first confirmed that message transmission occurs only if sender and receiver cells express M13 and F-plasmid gene products, respectively, and DNA molecules encode the M13 packaging sequence (Figure 2b). Stated differently, we considered if several imaginable modes of non-specific or alternate DNA exchange among cells might corrupt or bias M13-directed messaging. For such experiments we typically grew independent, well-mixed liquid cultures of sender and receiver cells to OD600 ≈ 0.7, combined equal numbers of sender and receiver cells to create a fresh co-culture, regrew a 1:100 dilution of the mixed population for five hours in the absence of antibiotic selection, and then regrew 1:1000 dilution split co-cultures with antibiotic selection for the messaging phagemid, receivers cells, or both (Methods).
To test if message transmission could occur in the absence of M13, we engineered F+ sender cells containing a messaging phagemid expressing green fluorescent protein (GFP) and ampicillin resistance, and F+ receiver cells expressing a red fluorescent protein (mKate2) and a chloramphenicol resistance marker (Methods). We co-cultured an equal number of sender and receiver cells in the absence of antibiotic selection. We assayed single cell fluorescence from chemically fixed samples taken at the start of the co-culture and following five hours of growth (Methods). We found no cells expressing both GFP and mKate2, suggesting that the messaging phagemid present in the sender cells was not transmitted to any receiver cells (Figure 3a, top row). We then regrew splits of the co-culture in the presence of ampicillin, chloramphenicol, or both antibiotics. No cells grew in the combined presence of ampicillin plus chloramphenicol (Additional file 1: Figures S3, S4).
Additional file 1. Supplemental Calculations.
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Figure 3. Targeted and autonomous communication of arbitrary DNA messages via a reusable cell-cell communication channel. (a) Bacteriophage M13-based cell-cell communication was used to send a DNA message, "GFP and ampicillin resistance", to receiver cells constitutively expressing mKate2. Schematic shows expected locations of cell populations after flow cytometry analysis. Data show fluorescence of GFP and mKate2 of co-cultures at the start of an experiment (t = 0) and after co-culture without antibiotic selection (t = 5 hr). Data are shown for experiments using cells with varying attributes, listed to the right of each pair of plots. (b) Schematic showing sender and receiver genotypes and expected message transmission of a lock-and-key system. Sender cells contain and transmit "T7 RNA polymerase autogene." Receivers contain a green fluorescent protein-lacZ alpha fragment fusion ("Gemini") under the control of a T7 promoter. Expression of Gemini is unlocked in Receivers given message transmission (34). (c) Communication of a lock-and-key message demonstrating decoupling and also the capacity for specificity of decoding received messages. Percentage of maximum events as a function of GFP fluorescence are shown for sender cells (XL1-Blue, Litmus-T7 RNA polymerase), receiver cells (XL1-Blue, pSB4C5-T7promoter-Gemini), co-cultures of receiver and sender cells, and a positive control (XL1-Blue, co-transformed with Litmus-T7 RNA polymerase and pSB4C5-T7promoter-Gemini). The curves for co-culture fluorescence are pooled data from three separate co-culture flasks. Data are shown for the start of the experiment (t = 0) and after selection with antibiotics (post-selection). Additional transmitted messages (Additional file 1: Figure S6).
We then transformed M13K07, a "helper phage" plasmid that encodes all M13 gene products along with a weak M13 packaging sequence, to our sender cells (Methods). We repeated the experiments as detailed above and found, following five hours of co-culture in the absence of antibiotic selection, that 92.4% of receiver cells (mKate2) also expressed GFP (Figure 3a, bottom row). We found that these cells were also able to grow in the combined presence of ampicillin plus chloramphenicol (Additional file 1: Figures S3, S4), suggesting that the messaging phagemid present in the sender cells was transmitted to receiver cells via M13 message particles. We note that the M13K07 helper phage also results in the production of a small fraction of secondary "M13K07" messages that convert receiver cells to potential rebroadcasting cells (below).
To cross-validate the above observations we used F- sender and receiver strains and repeated our messaging experiments in the presence of M13K07. We found no cells expressing both GFP and mKate2 (Additional file 1: Figure S2a). We also did not observe cell growth in the combined presence of ampicillin plus chloramphenicol (Additional file 1: Figures S3, S4). To confirm that M13 message particles were still being produced, we prepared a 0.2 micron filtrate from the supernatant of the initial co-culture and manually transduced separate F+ and F- receiver cell cultures (Additional file 1: Figure S2). We found that so-treated F+ receiver cells gained ampicillin resistance while F- receiver cells did not (Additional file 1: Figure S2b).
To confirm that the M13 DNA packaging sequence is required to package DNA into M13 particles we obtained a modified helper phage (HPdO) in which the packaging sequence is deleted [39]. HPdO sender cells should not produce any "HPdO" messages and therefore not convert any receiver cells to rebroadcasting cells. Both M13K07 and HPdO express a kanamycin resistance marker. We thus repeated our experiments using F- sender and receiver cells with HPdO and found no transmission of the messaging phagemid (Additional file 1: Figure S2a). We prepared a 0.2 micron filtrate from the co-culture and manually transduced independent F+ and F- receiver cell cultures. Like with M13K07, HPdO supernatants produced ampicillin plus chloramphenicol resistant cells only with F+ cultures, indicating successful messaging with either helper phage. However, only M13K07 supernatants produced kanamycin plus chloramphenicol resistant receivers, indicating that no M13 particles containing HPdO DNA were produced (Additional file 1: Figure S2). Stated differently, we did not observe any non-specific packaging of marker-encoding DNA into transducing M13 particles.
Message channel decoupling
To demonstrate that M13-based messaging can be used to transmit arbitrary genetic sequences we engineered a new messaging phagemid encoding a weak T7 RNA polymerase autogene [40] and ampicillin resistance, and receiver cells that regulate expression of a chimeric beta-galactosidase: GFP reporter [41] via a consensus T7 promoter (Methods). We termed this type of messaging "lock-and-key" since expression of T7 RNA polymerase from the sender cell message is required to activate the receiver cell reporter (Figure 3b). We co-cultured sender and receiver cells for five hours and then added antibiotics to select those cells that received the message. We used flow cytometry to assay GFP fluorescence in the co-culture, in independent sender and receiver cultures, and in a positive control in which we directly co-transformed receiver cells with the messaging phagemid. Only the co-culture of sender and receiver cells obtained GFP fluorescence levels equal to the positive control (Figure 3c).
Longer range messaging via chemotaxis
We then demonstrated that the range of M13-based DNA messaging can be increased using bacterial chemotaxis. For context, we confirmed by both experiment and theory that passive diffusion of M13 particles results in a detectable average run length of only ~2 mm after 24 hours on a semisolid low concentration agar plate (Additional file 1: Calculations & Figure S5). Moreover, we were initially unsure if M13 particle production, secretion, and transduction could be coupled to chemotaxis given imaginable issues arising from the combined metabolic burden of both systems, interference of partially secreted M13 filaments on flagella rotation, chemotactic shear forces destroying M13 particles during their secretion, or hindered transduction of receivers on swim plates in which chemotactic gradients are self-established via cell replication and maintenance.
The E. coli strain (RP437) used in our well-mixed liquid culture communication assays (Figure 3a) is chemotactic [42]. We thus obtained a non-motile strain (RP5838) for use as a control [42]. We reused the GFP plus ampicillin messaging phagemid for sender cells and the mKate2 plus chloramphenicol marker plasmid for receiver cells (Methods). In a typical chemotaxis experiment we first spotted equal amounts of sender and receiver cells on opposite sides of a 10 cm swim plate (Methods). We allowed cells to grow and move for 48 hours at 37C. We then assayed bright field plus green and red fluorescence using a tabletop imaging system (Methods). Lastly, we removed nine equally-spaced agar plugs spanning the region between the initial sender and receiver cells in order to directly observe cell phenotypes via microscopy or indirectly via antibiotic resistance in liquid culture (Methods).
We first spotted non-motile sender and receiver cells on opposite sides of swim plates (Figure 4a, left). As expected, we did not observe significant cell motility. GFP fluorescence encoded by the messaging phagemid was detected only with the sender cells. RFP fluorescence marking the receiver cells was found only on the opposite side of the plate. Liquid cultures inoculated from samples taken across the plate grew only in the presence of ampicillin (messaging phagemid within senders) or chloramphenicol (receivers), but not both antibiotics.
Figure 4. Active chemotaxis enables DNA messaging across centimeter lengths. (a) Bacterial chemotaxis using swim plates allows for interaction and messaging among sender and receiver cells. Three cases are presented: "non-motile" using non-motile sender and receiver cells; "pick-up" using non-motile sender cells but motile receiver cells and; and, "rendezvous" using motile sender and receiver cells. Stem plots give the measured less background absorbance (solid circles with stems) for well-mixed liquid cultures inoculated from plugs sampled in a equal spaced pattern across swim plates and cultivated with selection for sender cells (ampicillin), the messaging phagemid (chloramphenicol), or message transmission (both antibiotics). Distance from plate centers as noted; x-axes are consistent across each stem plot and cognate merged fluorescent / bright field plate photograph. (b) Active transmission of a DNA message across a 7 cm gap. Three representative photographs (upper row) merging RFP and GFP fluorescence from cells obtained directly from the merge region of a "rendezvous" swim plate; corresponding bright field images (lower row). Arrows indicate yellow cells expressing both reporters. (c) Merged multi-channel fluorescent micrographs of cells grown in replicate well-mixed liquid cultures inoculated with one sample taken from the merge zone of the rendezvous swim plate and cultivated under antibiotic selections, as noted.
We then spotted non-motile senders with motile receivers ("pick-up" messaging), and motile senders with motile receivers ("rendezvous" messaging). We observed receiver cell motility and plate-wide RFP levels and chloramphenicol resistance during pick-up messaging (Figure 4a, middle). We were able only to isolate cells resistant to ampicillin plus chloramphenicol from samples taken from where both sender and receiver cell populations merge. We observed sender and receiver motility and corresponding half-plate RFP and GFP levels plus antibiotic resistances during rendezvous messaging (Figure 4a, right). We again isolated cells resistant to ampicillin plus chloramphenicol only from samples taken from where both sender and receiver cell populations merged.
We also directly imaged single cells taken from the merge zone on the "rendezvous" plate for both GFP and RFP fluorescence prior to regrowth or selection for antibiotic resistance. We observed a mixed population of green (24.4 ± 5.3%), red (48.5 ± 5.3%), and yellow (27.1 ± 5.3%) cells (Figure 4b). When we cultured this mixed population in the presence of ampicillin, chloramphenicol, or both antibiotics, we recovered clonal sub-populations specific to the expected sender, receiver, and receiver plus message phenotypes (Figure 4c).
We observed that over 90% of individual receiver cells were transduced via M13-mediated messaging in a well-mixed liquid culture, as evidenced by message readout within receiver cells prior to selection for message receipt (Figure 3a). We also observed that over 33% of receiver cells were transduced via M13-mediated messaging within a semisolid medium across a ~7 centimeter gap bridged by chemotaxis of sender and receiver cells, again as observed by message readout within receivers prior to selection for message receipt (Figure 4a-b). In both cases 100% message receipt could be selected for using message-specific markers (Figure 4c, Additional file 1: Figures S3, S4). We expect that lower message receipt rates in semisolid media are due to a combination of factors, including the requirement for passive diffusion of M13 messaging particles within the agar microenvironment and, in our experiments here, chemotaxis driven by nutrient depletion due to cell growth, resulting in resource-limited cells within the messaging zone and thus a presumptive limited time window for messaging [43,44]. We note that a greater than 33% messaging rate compares favorably to pioneering work with AHL wherein limited fractions of single cells responded across a few millimeter gap [45], and expect that long range M13 messaging rates can be increased (below).
M13 phage particles are ~6 nm diameter rods whose length is defined by the packaged DNA molecule. For example, the wild-type M13 genome of 6,407 base pairs (bp) produces a ~930 nm rod; the longest reported DNA fragment packaged within a M13 particle, ~40,000 bp [46], would be expected to produce a ~5.8 micron rod. For comparison, the effective radius of an AHL molecule is ~0.5 nm. Thus, when the wild-type M13 particle is modeled as a cylindrical rod, the apparent spontaneous diffusion rate for an M13 particle in agarose (4.0E-13 m2/sec) is expected to be less than for AHL (7.1E-11 m2/sec), with a corresponding impact on expected average run lengths (<X > M13 = ~0.08 < X > AHL) (Additional file 2) [47,48]. We note that the AHL diffusion rate used here is ~4-fold greater than the widely-referenced value given in Basu et al. (1.7E-11 m2/sec, [8]) and that an increased rate seems better matched to reported results [49,50]. Meanwhile, most genetic messages of interest are likely to be encoded by DNA ranging from several hundred to many thousand base pairs. As a directly comparative example, LuxI, the enzyme that synthesizes 3OC6HSL (AHL) is encoded by a 579 bp open reading frame. If an expression cassette encoding for constitutive expression of LuxI were placed within a minimal messaging vector then a total of ~2,000 bp would be packaged, resulting in an M13 messaging particle with an expected diffusion rate of 1.0E-12 m2/sec, and thus an average daily run length ~8-fold less than AHL itself.
Additional file 2. Construct Sequences.
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We therefore coupled M13-mediated messaging to bacterial chemotaxis in order to increase the length across which DNA messages can be transmitted. The native E. coli chemotaxis system can actively translocate cells ~10-30 microns per second across short distances, generating apparent random diffusion coefficients in liquid of 1-3E-10 m2/sec (<X > = 6–18 mm/day), and biased directional movement over several centimeters per day in gradients of chemo-attractants or -repellents [51]. The coupling of cell movement to cell-cell communication is widespread in natural systems that establish complex dynamic patterns, from fruiting body formation in myxobacteria [52] to angiogenesis in mammals [53]. We expect that engineered small molecule signaling systems can also be coupled to cell motility, along with DNA-based messaging, in order to provide engineers with a full suite of tools for programming dynamic patterns that span the microscopic to observable length scales (e.g., scaffold-free tissue engineering).
We also expect that the M13 system established here can be improved. First, conditional control of M13 gene product synthesis would allow for M13-mediated messaging to be regulated by external or intracellular signals. Second, additional messaging vectors spanning a range of DNA copy numbers would allow for control of load effects within transmitter cells, and the matching of message-encoded signal levels to genetic devices pre-existing within receiver cells. Third, a suite of variable strength M13 packaging sequences might enable control over the production of "broadcasting" M13 particles that contain the "helper phage" plasmid, encoding M13 gene products, enabling engineers to program the rate at which receiver cells convert to rebroadcasting cells capable of transmitting or amplifying messages. Fourth, past work to develop M13 particles capable of transducing mammalian cells could be adapted to enable autonomous trans-kingdom communication of DNA messages [54,55]. More broadly, the ongoing development and improvement of all types of engineered cell-cell communication platforms is needed to enable future bioengineers to create systems that match the richness of biological patterns and programs found in nature.
We demonstrate the successful decoupling of a communication channel from message transmission within an engineered biological system via the autonomous targeted transduction of user-specified heterologous DNA messages. We also demonstrate that M13 particle production and transduction occurs among chemotactic bacteria. We use chemotaxis to increase the distance that DNA messages can travel, exceeding the transmission range and bit rate of existing small molecule-based communication systems. We postulate that integration of various engineered cell-cell communication platforms will allow for more complex spatial programming of dynamic cellular consortia.
Materials and methods
Strains and media
We conducted all co-culture experiments, except for lock-and-key messaging, in liquid culture using E. coli strain RP437 [42]. The lock-and-key co-culture experiment was conducted using E. coli strain XL1-Blue (Stratagene). We conducted experiments on agar plates using E. coli RP437, F+. RP437, F+ cells were created by mating RP437 with XL1-Blue via conjugation. We plated all transformations on LB agar supplemented with 10 μg/mL tetracycline, 30 μg/mL kanamycin, 50 μg/mL streptomycin, 25 μg/mL chloramphenicol, or 50 μg/mL carbenicillin, as necessary. We maintained clonal populations of transformants in glycerol stocks composed of 900 μL 60% sterile glycerol and 900 μL cell culture stored at -80C. We maintained liquid cultures of each cell type in 0.2 micron-filtered LB supplemented with appropriate antibiotics at 4C for no more than 5 days.
Messaging phagemid and plasmid construction
We constructed and cloned two BioBrick-based messages (noted below) into a Litmus28i backbone using standard molecular biology techniques [56][,57]. We created additional constructs in a similar fashion to identify specific cell types within our system. We purchased all enzymes from New England Biolabs unless otherwise stated. Linearized Litmus28i was prepared by restriction digest using the enzymes EcoRI and PstI.
We removed the BioBrick part I716104, which encodes the T7 RNA polymerase gene, from the BioBrick plasmid pSB1A2 by restriction digest using the enzymes EcoRI and PstI [57]. We then ligated the digested I716104 into linearized Litmus28i with T4 ligase and transformed the ligation into chemically-competent E. coli cells by heat shock.
We added the composite part J23115-B0032 to GFP, BBa_E0040, in the BioBrick plasmid pSB1A2 with a single PCR using primers (1) and (2). The resulting PCR product was digested with restriction enzymes EcoRI and PstI. We then ligated the construct to linearized Litmus28i with T4 ligase and transformed the ligation into chemically-competent E. coli cells by heat shock.
The original construction of Gemini, BBa_E0051, was described by Martin et al. [41]. We added a consensus T7 promoter (BioBrick part J64997) and ribosome binding site (BioBrick part B0034) in a single PCR reaction using primers (2) and (3). These primers also added the BioBrick prefix and suffix to the construct [57]. We digested the resulting construct with the restriction enzymes EcoRI and PstI. We ligated the digested construct into linearized BioBrick plasmid pSB4C5 (digested with EcoRI and PstI) with T4 ligase and transformed the ligation into chemically-competent E. coli cells by heat shock [57,58].
pSB4C5-J23119-RBS (C-dog)-mKate2
The mKate2 gene was synthesized by Blue Heron and cloned downstream of the constitutive BioBrick promoter J23119 and a strong RBS from the BioFAB C-dog pilot project onto a low copy BioBrick plasmid, pSB4C5 [58]. The resulting construct was transformed into chemically-competent E. coli cells by heat shock.
Primers used
Preparation of sender and receiver cells
Sender cells
For all experiments conducted using E. coli strain RP437 or RP5838, we prepared infected sender cells by co-transforming chemically-competent cells with M13K07 phagemid in addition to the messaging phagemid, Litmus28i_J23115-B0034-GFP.
We prepared sender cells for the lock-and-key messaging experiment by diluting overnight cultures containing the T7 RNA polymerase messaging phagemid ten-fold into fresh LB media containing carbenicillin and tetracycline. We then added M13K07 phage (NEB) at a multiplicity of 5–10. We incubated the diluted cultures at 37C with shaking to return them to early log phase (OD600 ≈ 0.2). We then diluted the early log phase cultures ten-fold further into fresh LB media containing tetracycline, ampicillin, and kanamycin, and incubated at 37C with shaking overnight to near saturation.
Receiver cells
For the co-culture experiments, we transformed chemically-competent RP437 cells with the pSB4C5-J23119-RBS (C-dog)-mKate2 plasmid via heat shock. For the lock-and-key experiments, receiver cells were similarly transformed with the pSB4C5-J64997-B0034-E0051 construct. We cultured receiver cells in LB media containing tetracycline and chloramphenicol overnight with shaking at 37C. For use in the 2D message transmission experiments, we also transformed RP5838, F+ cells with the pSB4C5-J23119-RBS (C-dog)-mKate2 plasmid via heat shock.
Liquid-based experiments
Dual-fluorescence experiments
Sender cells used in this portion of our study contained the Litmus28i-J23115-B0032-GFP message. Receiver cells contained the plasmid pSB4C5-J23119-RBS (C-dog)-mKate2. A positive control was constructed by co-transforming the pSB4C5-J23119-RBS (C-dog)-mKate2 and Litmus28i-J23115-B0032-GFP constructs into chemically-competent E. coli cells.
We diluted overnight cultures of sender, receiver, and positive control cells twenty-fold into fresh media with streptomycin. The diluted cultures were returned to log phase (OD600 ≈ 0.7) by incubation with shaking at 37C, after which we measured OD. From these log phase cultures we prepared four separate flasks of cells, each containing a total of 2×108 cells in 20 mL fresh media with streptomycin. The first flask contained an equal proportion of sender and receiver cells; the second flask contained only sender cells; the third flask contained only receiver cells. Immediately following addition of the appropriate cells we gently swirled the flasks and removed a 500 μL aliquot of diluted culture. After removing an aliquot, we incubated all flasks at 37C with shaking for 5 hours and then removed another 500 μl aliquot. We immediately fixed each removed aliquot by addition of paraformaldehyde (PFA) (Electron Microscopy Sciences #15714-S) to a final concentration of 1% followed by storage at 4C. Fixed cells were analyzed using a 488 nm blue laser and 532 green laser on a BD LSR II FACS (Stanford FACS Core Facility); for each sample, we collected 30,000 events triggered on side scatter.
Lock-and-key experiments
Sender cells used in this portion of our study contained the Litmus28i-I716103 message. Receiver cells contained the BioBrick-based construct pSB4C5-J64997-B0034-E0051. A positive control was constructed by co-transforming the pSB4C5-J64997-B0034-E0051 and I716103-Litmus28i constructs into chemically-competent E. coli cells.
We diluted near-saturated cultures of both sender cells, receiver cells, and the positive control ten-fold into fresh media containing tetracycline. The diluted cultures were returned to early log phase (OD600 ≈ 0.2) by incubation with shaking at 37C. From these early log cultures we prepared six separate flasks of cells. The first three flasks were replicates and contained a twenty-fold dilution of each sender and receiver culture in fresh media containing tetracycline; the fourth flask contained a ten-fold dilution of sender cells in fresh media containing tetracycline; the fifth flask contained a ten-fold dilution of receiver cells in fresh media containing tetracycline; the sixth flask contained a ten-fold dilution of the positive control in fresh media containing tetracycline. Immediately following addition of the appropriate cells we gently swirled the flasks and removed a 500μL aliquot of diluted culture. We immediately fixed each removed aliquot by addition of paraformaldehyde (PFA) (Electron Microscopy Sciences #15714-S) to a final concentration of 1% followed by storage at 4C. We incubated the culture flasks at 37C with shaking for for 5 hours after which we added chloramphenicol and ampicillin to flasks containing co-cultures or the positive control, ampicillin to the flask containing sender cells, and chloramphenicol to the flask containing receiver cells. Following incubation at 37C with shaking to near saturation, we removed and fixed a second 500μL aliquot. Fixed cells were analyzed using a 488 nm blue laser on a BD LSR II FACS (Stanford FACS Core Facility); for each sample, we collected 30,000 events triggered on side scatter.
2D message transmission experiments
We prepared swim plates with 23 mL tryptone broth supplemented with 0.2% agar, 50 μg/ml streptomycin, and 10 μg/ml tetracycline. Sender cells—either motile RP437, F+ or non-motile RP5838, F+ cells—used in this experiment contained the J23115-B0032-GFP-Litmus28i message and M13K07 phagemid. Receiver cells—either motile RP437, F+ or non-motile RP5838, F+ cells—contained the J23119-RBS (C-dog)-mKate2-pSB4C5 plasmid.
We inoculated swim plates with near-saturated cultures of the sender and receiver cells by spotting 1 μl of sender cell culture 1 cm from the left edge of the plate and 1 μl of receiver cell culture 1 cm from the right edge of the plate. We incubated all plates face up at 37C with humidity (provided by sponges soaking in DI water) for 48 hours. Plates were imaged within a black box for GFP fluorescence (excitation 490–510 nm; emission 520–540 nm) and RFP fluorescence (excitation 600–620 nm; emission 630–660 nm). Bright field images were also collected for each plate, and the fluorescence and bright field images were merged.
For the swim plate with both motile sender and receiver cells, we scraped the surface of the collision region and directly imaged these cells on an agarose pad via light and fluorescence microscopy. We also removed 9 equally spaced agar plugs from each plate along a horizontal line connecting the inoculation sites of the sender cells and receiver cells using 1.1 mm × 6.5 mm gel extraction tips (Phenix). We briefly resuspended each agar plug in 200 μl LB liquid media and then pipetted 10 μl of each resuspension into 1 mL LB liquid media containing ampicillin, chloramphenicol, or both ampicillin and chloramphenicol. These cultures were incubated with shaking at 30C for 24 hours.
After incubation, we transferred a 200 μl aliquot of each culture into a flat-bottomed 96-well plate (Nunc) and measured the OD600 of each culture on a Wallac Victor3 multi-well fluorimeter (Perkin Elmer). In addition, we imaged cells from these resulting cultures for GFP and RFP fluorescence (using 50 ms and 200 ms exposure times, respectively) and determined background fluorescence from monocultures of sender cells or receiver cells.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MEO and DE designed the experiments. MEO carried out the experiments. MEO and DE interpreted the data, and wrote and approved the manuscript. Both authors read and approved the final manuscript.
We thank M. Russel, J. Parkinson, A. Lam, F. St-Pierre, J. Bonnet, members of the Endy and Smolke labs, E. Cosgrove, S. Schneider, and the Stanford Shared FACS Facility. Support was provided by the NSF Synthetic Biological Engineering Research Center and Stanford University. MEO is a recipient of the Stanford Graduate Fellowship.
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Saint Clair Wairau Reserve Sauvignon Blanc
- Brand: Saint Clair, Marlborough
- Product Code: GWD-54519
- Availability: In Stock
Available Options
GOLD LIST - Sommelier Wine Awards 2012
GOLD MEDAL - Decanter World Wine Awards
This is Saint Clair's flagship variety and accounts for over 60% of sales. Saint Clair's aim is to be the best producer of this variety in New Zealand and results over the last two to three vintages would arguably indicate this aim is being achieved on a consistent basis.
Neal and Judy Ibbotson were pioneers in the Marlborough wine industry, first planting vineyards in the valley's unique climate and soils in 1978 and then establishing Saint Clair Estate in 1994.
Premium quality grapes, produced from vines that are now fully mature, combined with the proven experience of winemaker Matt Thomson, produce elegant, world class wines.
This is an outstanding alternative to Cloudy Bay, regarded as having a smoother, fruitier edge.
Grapes: 100% Sauvignon Blanc
Region: New Zealand / Marlborough / Lower Rapaura.
Maker: Matt Thompson.
Notes: This is a great pungent Sauvignon Blanc with powerful passionfruit, grapefruit, blackcurrant and an underlying mineral note. The palate is intensely concentrated and full with a long, lingering finish.
Serve: Ideally matched with oysters, scallops, salmon or pan-fried fish.
Suitable for Vegetarians

subset: commoncrawl


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Goodbye, Yasmin!UPDATED 8:41 AM HST Aug 17, 2012
From all of us here at KITV, your family and friends, we've enjoyed watching you grow from an aspiring high school drama student to one of the best traffic reporters in the State. Here's a look back at all the smiles you've given us along the way.
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- Filed Under
What do farmers do all summer? I work in town while they fish, hunt and mow grass. But don't threaten their million-dollar subsidies.
- What a strange upside-down world
Why are politicians afraid to cut welfare to farmers? Is it because of the well-funded lobbyists? Is it because some politicians own farms and are directly voting tax money into their pockets? How many businesses have a guaranteed income provided largely by taxpayers? Farmers do.
- Polk County Taxpayer
New license plates for all Iowans seem like a huge waste of money and metal when the "old" ones are just fine.
- 50266 Woman
Watch out for that word, "if." It's very dangerous and is a big trouble-maker. Lose your dreams, lose your mind. Isn't life so unkind?
- Iowa Mental Patient
"4 arrested, 33 pounds of meth seized in southeast Iowa." Small business in Iowa is alive and well.
- Barb Wire Dental Floss
The Do Not Call registry was created almost 10 years ago. I registered both my home and cellphone numbers on the Do Not Call list. I continue getting calls from telemarketers. I have yet to see any charges brought against these telemarketers that are ignoring the law. Why?
- Annoyed with the calls in Clive


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OneItem #: HL.48005027
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(from The Seven Vocal Fanfares). Conductor's Choice. Voicing/Format: 5 Part. 6.8x10.5 inches. 10 pages. Published by Boosey & Hawkes. (HL.48005027)
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The Seven Vocal Fanfares is an cappella work written especially for the renowned Girls' Choir of Christ Church Cathedral in Dublin, Ireland, tailored to their most unusual timbre and talents. The music is also designed to suit the acoustical attributes of the cathedral's famous crypt, the largest cathedral crypt in Europe.


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After quite the uproar over the revelation that Ralph Lauren is manufacturing the 2012 U.S. Olympic team's uniforms in China, the brand has released a double whammy statement not only announcing that it is the "Official Outfitter" of the 2014 Winter Olympic and Paralympic teams, but also promising that the uniforms will be 100% manufactured in the good ole' U.S. of A.
"Ralph Lauren promises to lead the conversation within our industry and our government addressing the issue of increasing manufacturing in the United States," the statement says. "…and has committed to producing the Opening and Closing ceremony Team USA uniforms in the United States that will be worn for the 2014 Olympic Games."
Nothing like a progressive public relations crisis management move. Perhaps our pals at Fox News will take a chill pill now, though it seems highly unlikely.
[image via Washington Post]


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From Fastiv.net isp ping 8-12
From Fastiv.net isp ping 8-12
City: Kyiv
Ping: ~15 ms
1. UkraInian Servers Online
2. Kyiv! Not Kiev!!!
Server down?
Seems that there are some problems with Ukranian servers.. I couldn't find any servers only and I was unable to create private match, QL said that there are no free servers or they are overloaded. Whats up?
no more ukrainian servers ?
what happend with ua servers?
Really! Why the server is not working?
online now
Can anybody tell, where are all ukrainian servers now?


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Last Friday's conference in Hyannis, put on by Community Action Committee of Cape Cod and Islands, Inc., offers human service agency administrators an inside look at preparation of the upcoming state budget and some advice on how to ask for what they need.
"We are far from out of the woods," said Murray, who remains Senate chairman of Ways and Means. She said tax revenues have grown 2.2 percent over five years while health care has gone up about 10 percent since 2001.
The senators criticized the federal pullback from funding hospitals' free care pool and other responsibilities, such as veterans' benefits, which will wind up on the state's chipped and overloaded plate.
"The federal government has got to step up to the health care issue," O'Leary said. "In the public realm, there's an appetite to take it on. (In Washington), they're pushing it under the rug."
"Thirty-three hundred jobs in Massachusetts were lost in December," Murray said. The drop came in state jobs and positions in education and tourism, as well as some high-paying tech jobs.
"The state is changing," O'Leary said. "Growth is elsewhere." But the chairman of the Senate's new higher education committee observed that Massachusetts has some strengths, including "one of the best health care systems in the country" and the state's public and private colleges. "There's lots of gloom and doom, but we've been through these periods," said O'Leary. "We've survived on our academic and intellectual skills and work ethic. Other states have big problems. The Southwest has growth, but it doesn't have the infrastructure and human talent we do."
Following the Money
One frustration for the Legislature, according to Murray, is the lack of accountability for the money going to school systems through Education Reform.
"We were giving money to systems and didn't know where it's going," she said. "We're going to ask for a money trail that's much more specific." When Murray criticized a series of superintendents in one Massachusetts city for spending state funds for their own benefit or on questionable programs, retiring Falmouth Superintendent Peter Clark said from the audience that "most districts are doing an outstanding job."
At the end of last year's legislative tive session, the Senate passed a bill that would revise the Education Reform Act to take into account factors beyond just property value in setting the funding formula, including median income. That would benefit the Cape, but Murray warned that relief was not likely before towns set their budgets for the fiscal year that begins July 1. Murray said she has asked her colleagues in the Senate to rank their funding priorities and get that information to her soon. Now's the time to lobby your representatives, she told the agency heads.
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Comment on: Patriots mock draft 7.0: What if the Patriots don't pick in the first round? - Extra Points - Boston.com
Posted 04/15/2014 02:08:02 EDT
This would be an unbelievable draft for the pats, but unfortunately it is a total pipe dream from start to finish. Nix at 39 is out of the question, along with some of the other picks. Good players, b more »
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Report on Loughner's mental condition due May 24
Web Producer: Ina Ronquillo
PHOENIX (AP) - The psychologist treating the Tucson shooting rampage suspect faces a May 24 deadline to hand in her latest report on efforts to make the suspect psychologically fit to stand trial.
The deadline falls two weeks before suspect Jared Lee Loughner's stay at a Missouri prison facility is set to end on June 7.
Loughner has been forcibly medicated with psychotropic drugs for about nine months in a bid to try to make him mentally fit for trial.
U.S. District Judge Larry Burns had ruled previously that Loughner wasn't ready for trial, but believed he can eventually be made ready to be tried.
Loughner has pleaded not guilty to 49 charges stemming from the shooting.
The report by Loughner's psychologist isn't expected to be publicly released.
(Copyright 2012 The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.)
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Gomez Comes To Gulfstream In Search Of Derby Victory
Gomez Comes To Gulfstream In Search Of Derby Victory
By Brian Skirka
HALLANDALE BEACH, FL - Two-time Eclipse Award winning jockey Garrett Gomez has never won the Derby...not at Churchill Downs or Gulfstream Park. But the 39-year-old rider hasn't given up trying and his quest for his first victory in the Run for the Roses will bring him to Gulfstream Park Sunday for the $1 million Florida Derby (G1).
Gomez will ship to South Florida from his California base to ride To Honor and Serve for Hall of Fame trainer Bill Mott in Gulfstream's most prestigious race.
"It's such a big boost of confidence to have a trainer like Bill Mott think of you to ride one of his best horses," said Gomez, who's ridden 2007 Woodbine Mile (G1) winner Shakespeare and 2009 Breeders' Cup Mile (G1) runner-up Courageous Cat for Mott among many others. "He's obviously a Hall of Famer and a great horseman, and it's always an honor to ride for him."
Although he's been based in southern California during recent winters, Gomez is no stranger to the Florida Derby. In 2009, Gomez guided Dunkirk to a second-place finish behind superstar Quality Road.
Despite having never experienced the thrill of victory, Gomez is no stranger to the Kentucky Derby. Just in the past two years, Gomez ran second in 2009 with Pioneerof the Nile before finishing a trouble-filled sixth last year aboard eventual 3-year-old champion Lookin at Lucky.
"That's why it's so hard to win the Derby," Gomez said. "No matter if you're on the best horse or not, you never know what is going to happen with a 20-horse field. Last year, the moment we drew the rail Bob [trainer Baffert] and I knew we were in trouble. As soon as the gates opened, I was already in a spot I didn't want to be."
His trip aboard Lookin at Lucky just reinforced to Gomez how hard it is to win the Kentucky Derby.
"As a trainer it's so hard to get a horse good so he can qualify for the Derby and then keep him good so he's competitive in the race," Gomez said. "They're 3-year-olds, so they're still growing and changing all the time. As a jockey, you just have to be on the right horse on the right day. There are a lot of variables, especially in a 20-horse field."
A few close calls and bad breaks haven't lessened Gomez's will to win the Derby.
"That's why it's exciting every year when spring comes around," Gomez said. "The top 3-year-olds are in top demand and the task of trying to find your Derby horse isn't easy. Two weeks ago I was at Fair Grounds, last week I was in Hot Springs for the Rebel, next week I'll be at Gulfstream, and then we have the Santa Anita Derby coming up."
Agent Ron Anderson, who no longer represents Gomez, secured the mount on To Honor and Serve, due in large part to his long-standing relationship with Mott.
"I've been dealing with Bill Mott going back to my days with Jerry Bailey," Anderson said. "In five years, Garrett has won 12 Breeders' Cup races – I've never been around anyone who has ever done that. I think [Mott] and Mrs. Weber [Charlotte, of Live Oak Plantation] just realized that Garrett is the best man for the job."
Gomez will be riding To Honor and Serve for the first time in the Florida Derby, but says he is somewhat familiar with the son of Bernardini.
"I know he can run," Gomez said. "He's certainly a talented horse. He ran a respectable race last time out in his first start of the year, and I'm expecting him to move forward off of that race."


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PAR56 Replacement Lamp - 120V, 300W MediumItem #: MC.300PAR56MFL
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Practically every young girl falls in love on or around her 16th birthday, but few of those affairs of the heart last a lifetime. Not so for Lori Tyler, though the object of her affection at that age wasn't the captain of the football team or a mysterious stranger from the wrong side of the tracks. No, Tyler fell in love with the food industry.
Stella a rising star in Oklahoma City's midtown
After studying psychology in college, working 16 years at other restaurants and spending seven years driving various nonprofit causes, Tyler has become what she's always dreamed of being: a restaurant owner.
Her dream came true April 22 when she opened Stella Modern Italian Cuisine. Stella is the new crown jewel of midtown, offering a sophisticated approach to rustic Italian food, chic interiors and attention to detail.
But Tyler is quick to point out the dream shared by her and her husband, Ty, could never have happened without help from the friends she made in her career working in other people's restaurants.
"This really has been a collective effort," Tyler said. "I've taken advice and been given help that has been invaluable in bringing this all together."
And it starts with the name, which came from an old friend who worked for her many years ago only to go on to his own success.
"The name came from Ryan Parrott," she said.
Parrott, the partner-chef of Iguana Mexican Grill, Season's Catering and Table One, said, "Lori told me she was opening an Italian restaurant and was looking for a name. I told her I'd always dreamed of opening an Italian place and calling it
"I asked him if I could use it," she said. "And he was gracious enough to say yes."
Tyler fell in love with the food business as a teenager working at Greenhouse Spa in Dallas, following up with a job at Bennigan's, which kept her funded through her degree work in psychology at the University of Dallas and set her up for the next big decision in life.
"My boyfriend lived in Oklahoma City, and I lived in Dallas," she said.
That boyfriend was Ty Tyler, long a local media mogul, and the two eventually married and settled in Oklahoma City. Lori went to work for the Val Gene restaurant group, helping open the original Pepperoni Grill in Penn Square Mall.
Pepperoni Grill brought the wood-oven concept to Oklahoma City for the first time, and Tyler has continued that tradition at Stella. As you walk in, the stainless brick oven greets you with a mellow, smoky hello. The aroma is a conduit to pleasant memories from bygone days by the fireplace.
But Tyler's business relationship with the wood oven was secondary to the one she had with her boss, Pete Holloway.
"Peter means the world to me," she said. "He's been a true mentor for me, and he's helped so much along the way in this process."
Holloway went on from Val Gene to open Boulevard Steakhouse and 501 Cafe in Edmond and to buy Classen Grill. Tyler worked in some capacity for all those concepts through the years. But after 16 years as general manager of Boulevard, she left the business altogether to concentrate on charity work.
Continue reading this story on the...
Stella Modern Italian Cuisine
• Where: 1201 N Walker Ave.
• Hours: 11 a.m. to 10 p.m. Mondays through Thursdays; 11 a.m. to 11 p.m. Fridays and Saturdays.
• Reservations: Call 235-2200 or go to www.
Read more on the Food Dude blog:
Watch the Food Dude's visit to Stella:


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It's day 3 in New York City and Jane is at it again, signing books and sharing success stories about species conservation. This time she's at the Barnes & Noble book store in Union Square. A great crowd of supporters and book fans gave Jane a warm welcome as she signed hundreds of books and spoke to the audience.
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Southfield, Mich. - A Memphis, Tenn.-based alumnus and entrepreneur has donated $2.5 million to name the Department of Mechanical Engineering at Lawrence Technological University.
The donor is A. Leon Linton, the founder and CEO of Southern Systems Inc. (SSI) of Memphis, which designs, builds and installs custom conveyor systems for manufacturing and distribution facilities. He received a bachelor's degree in mechanical engineering from Lawrence Tech in 1962.
"The education I received at Lawrence Tech has been the backbone of all the activities I have done throughout my working life," Linton said.
"We are delighted that our Department of Mechanical Engineering, one of the university's largest departments, will now be further distinguished by carrying the name of one of its most successful graduates," said Lawrence Tech President Lewis N. Walker.
Linton was born in a rural Mississippi town on the Tennessee River about 90 miles from Memphis. His family moved to Michigan when he was 12 after his father - a World War II veteran returned from Okinawa - took a construction job in Milford. After his father was transferred to another construction project, Linton finished high school in Ypsilanti.
While still in high school, Linton began a millwright craftsman trade apprenticeship and worked the night shift at a Lincoln-Mercury assembly plant. He expected to be called by the Army to serve in the Korean conflict following high school graduation, and in the interim worked in the millwright trade on the construction and installation of conveyor systems, machinery and automation equipment. When the Army call hadn't come after two years, Linton took the advice of his mentors and enrolled in the mechanical engineering program at Lawrence Tech.
Continuing to work full-time, he took Lawrence Tech classes at night and on some Saturdays. By the time he completed his bachelor's degree at the age of 28, he was a project engineer for Jervis B. Webb, now based in Farmington Hills, working on conveyor system installations in automotive plants.
"My evening courses at Lawrence Tech started paying real dividends very quickly," Linton recalled. "My ability to make physics calculations and solve basic problems expanded significantly to more complex issues. Other courses were teaching me about exciting materials and new methods that could be used in various equipment design applications."
After college graduation, Linton persuaded his employer to let him return to Tennessee and open a one-man office in Memphis. Five years later he went out on his own, and SSI quickly developed a niche market for the heavy-duty conveyor systems utilized in the manufacture and production of heavy trucks, construction equipment, military tanks, oil field pipe, aircraft, furniture, appliances and package handling.
The Bulk Handling Division of SSI operates as a general contractor in many states, from California to Florida, and is one of a very few companies in the United States capable of slipform, continuous-pour concrete construction of massive silos and related equipment associated with ethanol production, cement manufacturing, pet food processing and coal handling and storage.
Linton said he is confident Lawrence Tech will continue to thrive by maintaining its "theory and practice" approach to education, which he finds just as valuable in today's manufacturing environment as it was when he graduated.
"Lawrence Tech has always been mindful of and catered to serving the needs of industry by providing a top flight education for graduates in the meaningful disciplines needed for industry to stay competitive and move forward," he said.
A prior honor for Linton was receiving the Lawrence Tech Alumni Achievement Award in 1968.
Lawrence Technological University, ltu.edu, offers over 80 undergraduate, master's and doctoral degree programs in Colleges of Architecture and Design, Arts and Sciences, Engineering and Management. Founded in 1932, the 4,500-student, private university pioneered evening classes 75 years ago, and today has a growing number of weekend and online programs. Lawrence Tech's 102-acre campus is in Southfield, with education centers in Lansing, Livonia, Clinton Township, Traverse City and Petoskey. Lawrence Tech also offers programs with partner universities in Mexico, Europe, the Middle East and Asia.


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100 minutes
1-1/2 to 2 lbs. lean ground beef
1 large onion, finely chopped
Note: I like to add 1 green bell pepper that has been finely chopped but it's up to you.
1/2 tsp. oregano
2-1/2 to 3 cups Mozzarella cheese, shredded and divided
2 cups ricotta cheese
1 cup Parmesan/Romano cheese, grated and divided
1/4 cup chopped fresh parsley
1 egg, lightly beaten
1 jar (700 mL) spaghetti sauce (your favourite)
3-4 garlic cloves, minced
1-1/2 cups water
12 lasagna noodles, uncooked
Note: I sometimes add a layer of spinach. I use 1 package of frozen spinach that has been thawed and very well drained. Simple as that.
How to make it
Preheat oven to 350°F.
Cook meat, onion and oregano in a large skillet on medium heat.
Meanwhile, mix 1-1/4 cups of the mozzarella cheese, ricotta cheese, 1/4 cup of Parmesan or Romano cheese, parsley and egg until well blended and set aside.
Drain meat and return to skillet.
Stir in pasta sauce and minced garlic.
Pour water into empty sauce jar.
Replace lid and shake well.
Add to skillet and stir until well blended.
Spread approximately 1 cup of the meat sauce onto the bottom of a 13 x 9 inch baking dish.
Top with layers of 3 lasagna noodles, 1/3 of the ricotta cheese mixture and 1 cup of the meat sauce.
Repeat layers two more times.
Top with remaining 3 noodles and remaining meat sauce.
Sprinkle with remaining 1-1/4 cups or more of mozzarella cheese and 1/4 cup Parmesan/Romano cheese.
Cover tightly with PAM sprayed foil.
Bake for 1 hour.
Remove foil and continue baking for 15-20 minutes or until heated completely through.
Let stand 15 minutes before cutting to serve.
Substitute 2 pkg. (1 cup each) frozen, thawed simulated ground beef for the browned ground beef.
Adding water to the sauce helps cook traditional noodles during baking, so you don't have to cook them beforehand. This saves you 15 to 20 min. of prep time.
Print it!


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Today is October 13, 2015
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October 6, 2011
Carbon Sports
October 6, 2011
Carbon Sports
Carbon Volleyball drops league game to North Sanpete
The Carbon High Lady Dinos volleyball team is finding out Region 12 play will be very difficult to navigate through as they fell to North Sanpete in four sets.
Carbon High golf finishes season; golfer Etzel heads to state tourney
While the Carbon High golf teams have completed their seasons, much improvement was made and one golfer was on his way to the state tournament.
Leonard scores points with big elk
JD Leonard, Kyle Leonard and Zachery Rodriguez show of JD's prize Elk behind Miner's Trading Post in Sunnyside. Leonard was able to take the bull near Cold Spring on Sept. 22, after a full season of scouting and hunting. ...
Dinos football team looking to get back on winning track
The Carbon High Dinos football team attempt to put a two game losing streak to bed as they prepare to take on Payson on Friday night.
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Wheel of Fortune
Clever thief finds an inventive way to steal right under a guard's nose
[Collected via e-mail, 1998]
There was a man who had worked at a factory for twenty years. Every night when he left the plant, he would push a wheelbarrow full of straw to the guard at the gate.
The guard would look through the straw, and find nothing and pass the man through.
On the day of his retirement the man came to the guard as usual but without the wheelbarrow.
Having become friends over the years, the guard asked him, "Charlie, I've seen you walk out of here every night for twenty years. I know you've been stealing something. Now that you're retired, tell me what it is. It's driving me crazy."
Charlie simply smiled and replied, "Okay, wheelbarrows!"
The wheelbarrow legend has been set in a number of different countries, including Brazil, Mexico, South Africa, and the Soviet Union.
In a different form of this legend, a child wheels a bicycle bearing a sack of sand past Mexican or German border guards every day. The guards concentrate their energies on the sack of sand, completely failing to recognize the boy is smuggling
Another version of the "border guards" variant deals with a man who daily crosses a frontier leading a donkey bearing a sack of rice. The guards diligently search the bags of rice each day but find nothing. Of course, what's really being smuggled is rice (or, in some tellings, donkeys).
The concept of "hiding in plain sight" can often be an effective form of camouflage: sometimes the best form of concealment is not to try to hide something at all, but rather to make it such a familiar part of its surroundings that no one pays it any mind. That principle is at work in this tale of a clever thief who manages to steal from his employer for years while remaining completely undetected the whole time, because the manner and object of his pilfering are such thoroughly ordinary parts of his everyday environment.
This legend is actually an ancient story from Turkey (or Persia) about the famous trickster Nasradin (or the Hodja). The oldest print version of its modern form we've found so far comes from a 1952 joke book:
When Juan and Evita Peron were building a luxurious retreat for themselves some miles outside of Buenos Aires they established a rigid guard around the project
to prevent the stealth of valuable materials. Every day at noon, the story goes, the same workman began to appear at the exit gate with a wheelbarrow loaded with straw. The guard, convinced that there was dirty work afoot, searched the straw more carefully daily — even had it analyzed to see if it possessed special chemical values — but could find nothing to substantiate his suspicion, and had to let the workman pass.
A year later, the guard met the workman, evidently enjoying great prosperity. "Now that all is said and done," pleaded the guard, "just what were you stealing every day on that Peron project?" The workman whispered, "Wheelbarrows."
A version involving stolen trucks turned up in
Reader's Digest
in 1971:
A few years ago, the Army post where I was learning the ropes to be an M.P. was experiencing a rash of thefts. So when a
2 1/2-ton
truck came to the gate with a very nervous-looking civilian behind the wheel, my corporal decided to give me a lesson in how to search a departing vehicle. Springing into action, he looked in the back of the truck, under the canopy, in the tire well, under the hood, under the seat. Satisfied that the truck was "clean," the corporal waved the through, obviously proud of the lesson he had just given me. Later, we found out why the driver looked so nervous — he was stealing the truck.
No matter what form it takes — wheelbarrows, bicycles, trucks, or donkeys — the moral of the legend remains the same: Beware of red herrings; don't overlook the obvious.
The same message is also imparted in a slightly different form of the story:
Two guys purchase a park bench and are sure to get a receipt for it. They then carry the bench around Central Park, and are quickly stopped by the police. They show the cops the receipt and are allowed to go on their merry way. They do this for several weeks, until all the cops realize that the two running around the park with the bench actually OWN the bench. The guys then proceed to steal several dozen park benches right under the nose of the city's police.
Barbara "bench warranted" Mikkelson
This legend turns up in the 2001 film
Crocodile Dundee in Los Angeles
and the 1962 Benjamin Elkin book
Gillespie and the Guards
Last updated:
25 May 2011
Urban Legends Reference Pages © 1995-2015 by snopes.com.
This material may not be reproduced without permission.
snopes and the snopes.com logo are registered service marks of snopes.com.
Cerf, Bennett.
Good for a Laugh.
New York: Hanover House, 1952 (p. 64).
Hall, Jeffrey. "Humor in Uniform."
Reader's Digest.
August 1971 (p. 98).
Hershfield, Harry.
Laugh Louder Live Longer
New York: Gramercy Publishing Company, 1959. (p. 166).
Also told in:
Healey, Phil and Rick Glanvill.
Now! That's What I Call Urban Myths
London: Virgin Books, 1996. ISBN 0-86369-969-3 (pp. 34-35).
Spalding, Henry.
Encyclopedia of Jewish Humor.
New York: Jonathan David Publishers, 1969 (pp. 69-70).


text:
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One might imagine numbers like these would move the Obama team to pull the plug on the blame-fest, but one would be wrong. The Washington Post's E. J. Dionne rang out the old year by urging Democrats to double down, claiming that only by painting the Bush age as a "squandered decade" that ravaged the country could Obama's fortunes be revived. "Much of the contention surrounding Barack Obama's presidency is simply a continuation of our argument over the effects of George W. Bush's time in office," he said. It isn't: It's about the spending, the deficits, the enormous expansion of federal power, and the incredible corruption, deal-making, and squalor surrounding the health care reform bill. Dionne contends Obama was elected as a rebuke to the Bush foreign policy, but John McCain, who was for the surge before Bush was, was close to Obama through most of the year, and led in the two-week window between his convention and the financial collapse. Dionne sees Obama's election as a repudiation of Bush's governing policies, but Cheney won his debates with the president, and in the past few months conservative ideas have come back. Dionne says Obama is "both the anti-Bush and the leader of the post-Bush cleanup squad," but it's a strange kind of cleanup that leaves in place the Bush defense secretary (Robert Gates), the Bush generals in Iraq and Afghanistan, and the Bush prescription for the war in Afghanistan, not to mention Federal Reserve chief Ben Bernanke, the original architect in 2008 of Bush's response to the fiscal collapse. Dionne says "we have no choice but to reach a settlement about the meaning of the last 10 years"—i.e., to agree with him they have been a catastrophe—to give Obama a chance of succeeding. But Obama's ultimate success or failure will have very little to do with Bush's reputation. It will rest on his own ability to hold the line in the war against terror, restore some sense of fiscal stability, and keep the country safe from further attacks. These have very little to do with the Bush reputation. They rest in the hands of fate and Obama and have nothing to do with what anyone thinks about Bush.Dwight Eisenhower and John Kennedy ran to succeed a president of the opposite party whom they did not oppose in the election but whose administrations they felt obliged to critique. Once in office, however, they dropped the subject, and concentrated on governing, without further complaints about the messes they were handed: the Communists in Eastern Europe, the Communists with the H-Bomb, the conflict in Korea, the war in Vietnam and Laos, the potentially lethal flash point and mess in Berlin. Both Ike and Jack were judged in the end to be pretty good presidents, and their era—the early Cold War—as very important. They fought the enemy, not one another, at least not in public, and not once they had taken the oath of office. The crises they faced stand up to Obama's. And back then, the buck stopped with them. Noemie Emery is a contributing editor to THE WEEKLY STANDARD.
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