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On the northern edge of Gainesville, Devil's Millhopper Geologic State Park showcases a geologic formation known to generations of visitors to the area. You'd think you were in the Amazon, or Hawaii, when you hear and see the tumbling cascades behind a screen of dense vegetation. But the ephemeral waterfalls that occur at Devil's Millhopper are just one of the delights to be found in this small but intriguing state park.
It takes 232 steps to walk to the bottom of this 500-foot-wide, 120-foot-deep sinkhole. Each landing reveals a new layer of forest, each crack and crevice a bounty of botanical wonders. No matter what time of year you visit, the creek tumbles down into the abyss through a succession of rock outcroppings, but the best time to visit is after a heavy rain, when tall waterfalls gush down the sides of the sinkhole. A 0.9-mile trail rings the top of the sinkhole, leading you through habitats very different than what you'll find below.
Location: Gainesville
Length: 0.9 mile
Lat-Long: 29.705809, -82.394255
Type: loop
Fees / Permits: state park entrance fee
Difficulty: moderate to challenging
Bug factor: moderate to annoying
Restroom: yes
Open 8 AM to 5 PM. Closed Mondays and Tuesdays.
There is a visitor center with an orientation film and restrooms, plus picnic benches out at the parking area. Be cautious of poison ivy, which is prevalent along the sides of the footpath. Except in winter, mosquitoes are always intense here.
From I-75, take Gainesville exit 390 and head east into Gainesville on SR 222 for 3.4 miles, passing Santa Fe Community College. Turn left at the traffic light for NW 43rd Street. Drive 1 mile to NW 53rd Ave (Millhopper Road) and turn left. The park is on the right within 0.3 mile.
From the parking area, follow the paved path past the ranger station and around the interpretive center, where several exhibits provide interpretation of the geology and cultural history of the Devils Millhopper. A short film is offered in an open-air theater. The paved trail becomes a hard-packed sand path under the hickory and loblolly pine. When you reach the T intersection, turn right—my personal preference, so the plunge down the sinkhole is the last part of the hike.
If the water table is high, you'll hear the sound of running water, a good indicator that you'll see waterfalls later in this walk. The hardwood forest is heavy on oaks and hickories, so when the leaves are on the trees, you can't see the sinkhole for the forest. But you can certainly feel its presence, off to the left, where fencing discourages drawing close to the edge. The trail winds past a communications tower and into a scrub forest, where silk bay and rusty lyonia are among the larger shrubs in the understory. There are frequent benches along this topside nature trail, so it's not a challenge for folks with limited mobility.
After a half mile, you reach a bridge over a ravine that wouldn't look out of place in the Great Smoky Mountains. It carries a creek steeply downhill towards the sinkhole. A few moments later, you're at the top of the landing for the climb down into Devil's Millhopper. If you feel up to the 232-step challenge – and there are many landings along the way to rest – go for it! You'll be glad you did. The staircase begins by paralleling the creek, which tumbles down a series of rock outcroppings.
As you drop deeper into the sinkhole, the landings afford better panoramas of the inside of this unique world. Listen for the sound of water and look towards it to be rewarded with views of the various tall cascades that pour out of the water table when water is plentiful. They tumble down the rocky sides of the sinkhole behind the lush plants and trees. Look closely at plants on the ledges. Some of the more unusual species found down here are wakerobin, a variety of trillium that blooms in late winter, and needle palms, which are more frequently found along humid spring runs.
At the very bottom, the boardwalk jogs to the left and passes a cascade on the left. To the right, there's a sweeping view of the sinkhole's bottom, with its willow marsh. Water vanishes into a crack in the earth beyond. One final rise in the boardwalk provides access to a bench with a view.
There's no other way to go but up, so after a bit of exploring and enjoying, start your way back up the staircase. At the top, turn left to finish off the nature trail loop; you'll encounter the exit on your right.
For more information, visit the Devils's Millhopper website


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Having problems with your Electrolux Epic 6500 Vacuum ?
How do I take apart hand nozzle to repair hose?
\015 How do I take apart hand nozzle on my Electrolux 6500 sr to repair hose?\015
Answers :
Repair Help & Product Troubleshooting for Electrolux Epic 6500 Vacuum
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- Research
- Open Access
Evolutionary principles and synthetic biology: avoiding a molecular tragedy of the commons with an engineered phage
© Gladstone et al.; licensee BioMed Central Ltd. 2012
- Received: 24 May 2012
- Accepted: 22 July 2012
- Published: 1 December 2012
In prior work, adding a gene to phage T7 that degraded the host K1 capsule facilitated growth when plated on capsulated hosts. However, the transgenic protein (an endosialidase) is expressed as an exoenzyme, released from the cell at lysis but unattached to the phage particle. There is thus the possibility that the gene will be subject to a tragedy of the commons and be selected against, if the enzyme benefits other genomes.
This evolutionary perspective was supported in short term experiments. The genome carrying the endosialidase gene was favored on a capsulated host if grown in physical isolation of control genomes (lacking the gene) but was selected against otherwise.
These results challenge efforts to engineer phages with exoenzymes that degrade biofilm polymers. If biofilms do not facilitate spatially structured phage growth, the transgenic enzymes may be rapidly eliminated from the phage population after release in the environment, even if the transgene benefits overall phage growth on the biofilm.
- Capsid Protein
- Phage Particle
- Phage Type
- Major Capsid Protein
- Phage Suspension
Among many exciting prospects for synthetic biology, one is to engineer microbes that degrade environmental compounds – pollutants, bacterial biofilms, or foodstuff for fuel. For many applications, it would be ideal if the engineered microbial population persisted and purveyed its beneficial role indefinitely, generation after generation. Yet, the successful engineering of a genome that performs our desired function is not sufficient to confer transgenic immortality. The engineering must be evolutionarily stable, so that natural selection does not favor loss of the novel functions. For some purposes, engineered microbes must also not be inferior to wild competitors, although competitive inferiority may be desired as a way of limiting the environmental impact of the strain. The competition factor is ecological, however, and is not relevant if the engineered microbes are released into a closed environment from which outside competitors are excluded.
The focus of this paper is the first problem: evolutionary stability. A sufficient condition to ensure evolutionary stability of an engineered modification would seem to be benefit to the organism, such as a drug resistance gene in the presence of antibiotics, because beneficial traits are maintained by selection. However, there is a particular class of benefits that are not necessarily evolutionarily stable: although the gene products are beneficial, the genes are not. This paradox arises from the subtle matter of who benefits from the gene product, whether it is specifically the individual carrying the gene or others in the population. If the beneficiaries include unrelated individuals as well as the bearer of the gene, a 'tragedy of the commons' may result in which the gene is selected against because the non-producers benefit without paying the cost of production [1, 2]. In the parlance of evolutionary biology, the gene is producing a public good that benefits the whole.
These principles are broadly supported in the literature on evolutionary biology. It remains to be seen how relevant they are for engineering, and if they are relevant, how different engineering strategies may help solve them. We motivate our study with the specific example of bacteriophages engineered to degrade bacterial exopolymers. Lu and Collins [3] created a biofilm-degrading phage by endowing it with a gene for dispersin, an exoenzyme that degrades an essential biofilm component (β-1,6-N-acetyl-D-glucosamine). The inspiration for this engineering was that release of dispersin at lysis would provide high concentrations of enzyme where the enzyme was most useful for biofilm degradation, in the regions of highest cell density. Although the evolutionary advantage and evolutionary fate of the dispersin phage was not considered in that initial study, we might imagine that dispersin would be beneficial to the phage population by increasing the availability of hosts for the phage. However, even if dispersin provided such a benefit to the infecting phage population, the further question is whether the exoenzyme would be maintained over time or be lost in a tragedy of the commons. Here we test the basic principles behind this type of engineering. For practicality, we use an experimental system in which a phage produces an exoenzyme that degrades a capsular polysaccharide of its host [4].
A system with conflicting individual and group benefits
This type of system should obey a tragedy of the commons [5], a process in which unchecked selfish interests of the individual lead to overexploitation and eventual collapse of a common resource that benefits the entire group. In Hardin's original paper, the model was illustrated mostly with examples in which the public good was a pre-existing natural resource, but the paper also extended the process to public resources created by society (e.g., parking spaces, banks) as well as to evolutionary processes that favor the individual over the group. The metaphor has been so widely adopted in evolutionary biology that it has become virtually synonymous with a conflict between group and individual fitness, and indeed, our study can be viewed in this latter context without invoking a tragedy of the commons. In our study, the common resource benefitting the group is a phage-produced diffusible enzyme that degrades host capsule, allowing rapid infection of new hosts by any phage in the local group. The selfish behavior is the failure of an individual phage genome to produce enzyme, with a direct analogy to humans failing to support a public good by not paying taxes.
In a real system, these quantities will not be static. For phage growth in a confined environment such as a plaque or a small volume of liquid, the benefit B is likely to change with time since infection/inoculation, as phage density increases and protein concentrations build. If B varies with time, the overall growth of the transgenic phage depends on the average B over the interval. Thus the net effect of selection may depend on how long the competition lasts.
The model has thus far implicitly compared fitnesses of separate populations. Evolutionary inferences based on these comparisons thus assume that evolution is from competition between separate populations (groups) of each phage type, as when the two are grown in isolation. If the enzyme of the transgenic phage is produced as a free, diffusible molecule, that enzyme benefits not only growth of its own genome but also benefits the growth of any other (non-transgenic) phages in the local group – the enzyme pool is effectively a commons that can benefit all. In a population of fully mixed individuals of both types, the non-transgenic phage will always have the higher fitness because both types benefit equally from the enzyme – the group benefit – yet only the transgenic phage pays the cost of production. That is, both phages will experience H host encounters, but the difference in fecundity, F versus f , is unaffected. In a group with both types, fH applies to the non-transgenic phage and always exceeds FH for the transgenic phage. This asymmetry drives the tragedy of the commons. (The value of H in a mixed group would of course depend on the abundance of the transgenic phage, so a more formal argument would specify H as a function of phage density; the important point is that, whatever the value of H, it is shared between the two phage types.)
From these simple considerations, there are two ways to avoid a tragedy of the commons and maintain the transgenic individuals in the long term. One is to grow them in a spatially structured environment, so that the benefit of the enzyme (H) accrues only to those genotypes producing the enzyme - that even though the enzyme is produced as a free molecule, it cannot diffuse far enough to benefit other types. This strategy is one of group selection and works in the long term only if the populations are repeatedly dispersed into small, isolated subpopulations so that any enzyme-lacking mutants of the transgenic phage have little time to ascend before being required to grow on their own [6, 7]. The other solution is to abolish the potential for a commons, as by engineering the enzyme so that it is not a free molecule but is chemically coupled to the producing genome; then H remains associated with the transgene even in a mixed population. The purpose of the experimental design is to see if these principles can be successfully applied in a transgenic phage system.
Setup and general observations
Most experiments were done with two phages, T7E1 (carrying endosialidase) and T70 as a control. T70 carried a short insert in frame (an S-tag) considered to be of little consequence, whereas the engineered phage, T7E1, was cloned with the tailspike gene from phage K1-5 that encodes an endosialidase. The only strict requirements for the control phage are that its intrinsic growth be superior to that of T7E1 and that it benefit from the presence of exogenous endosialidase. It would have been feasible to use a wild-type T7 as the control, but use of the same genomic backbone for both phages ensured that fitness differences could be attributed largely to the engineered differences; the wild-type phage appears to have a substantially higher fitness than the vector (data not shown), an effect that would have complicated finding a suitable dynamic range for competing a wild-type control with an engineered vector.
The selective host was EV36, an E. coli K12 strain intrinsically permissive for T7 but expressing a K1 (sialic acid) capsule [11]. The capsule acts as a barrier between the phage and its bacterial receptors. Expression of the capsule thus protects the cell from the phage, and degradation of the capsule by the phage-encoded endosialidase augments infection. However, although the endosialidase protein made by T7E1 is the tailspike of phage K1-5 (see Methods), it does not attach to the T7 virion and thus is released as free protein. Consequently, the endosialidase produced by an individual T7E1 specifically helps that genome's progeny infect new EV36 only to the extent that the diffusing phage progeny remain spatially associated with the sialidase molecules after release at lysis.
Furthermore, there is a potential 'chicken and egg' problem with this phage. Given a suspension of phage particles, some host cells must be susceptible to T7 in the absence of enzyme so that the first generation of infections will occur to produce enzyme. If the capsule cannot be breached without enzyme, then phages alone cannot get the process started [4]. The success of T7E1 in our system thus depends on the presence of some cells that can be infected before capsule degradation.
Scholl [4] reported that T7 lacking the sialidase gene could not form plaques on EV36 grown in LB broth, whereas T7 with the sialidase gene formed plaques occasionally, at an efficiency of 10−3 to 10−4. In our hands, both T7E1 and T70 could form nearly normal plaques on EV36 grown in LB. With some effort, we found growth and plating conditions for EV36 that greatly impaired plaque formation by T70 (M9 glucose). A performance difference between alternative sources of LB media is not necessarily surprising, because LB is not a defined media and ingredients from different manufacturers are now known to vary in sRNAs that affect bacterial gene expression [12].
The transgenic phage T7E1 is at a disadvantage on a non-capsulated host
Separate competition on the capsulated host favors the endosialidase phage T7E1
The tragedy: mixed competition selects against the sialidase phage T7E1
A tragedy of the commons was avoided in the preceding experiment because the T7E1 phage type was grown separately and grown so briefly that mutants lacking the endosialidase could not arise and take over. As the endosialidase is produced as a free molecule, the system should be prone to a tragedy of commons when the two phages are grown together: T70 should outgrow T7E1 on a capsulated host when the two phages are mixed, because (1) T70 has an intrinsic growth advantage and (2) it can use the endosialidase produced by T7E1 when both phages are mixed. This prediction assumes that there is free mixing of endosialidase in the local environment infected by multiple phage types.
Alternative engineering: phage T7E2
From Figure 2, the level of gene expression may determine whether a gene capable of providing a benefit actually offers a benefit to the recipient genome. Although T7E1 was engineered to carry a gene beneficial for growth on a capsulated host, its level of expression was not optimized - the level of expression would have been high, similar to that of the major capsid protein, the gene immediately upstream (see above). Furthermore, a different method of cloning might be employed to avoid a tragedy of the commons altogether – as in coupling the endosialidase to the virion. It is thus expected that alternative designs for a transgenic phage could potentially yield better growth on the capsulated host.
A second transgenic design was implemented, designated T7E2, in which the endosialidase was cloned as a C-terminal fusion to the major capsid protein (Figure 3). If the cloning worked as designed, T7E2 would create phage heads in which all major capsid proteins were fusions with endosialidase. The isolates recovered all carried a base substitution that effected a stop codon at the end of 10A, terminating translations without any endosialidase residues. Although we might have hoped otherwise, this outcome is unsurprising because the 811 residue endosialidase protein is so large as to interfere with assembly if most capsid proteins carry it (the major capsid protein is 344 amino acids long). Although the vast majority of capsid proteins in the isolates of T7E2 recovered would have lacked residues of sialidase, fusions of capsid and sialidase would have resulted from low-level translational read through of the stop (maybe 1%, e.g., [13]), as a C-terminal domain on a small fraction of major capsid proteins. As pointed out by a reviewer, cryptic RBS activity immediately upstream of the endosialidase start (within the protein coding sequence of 10A) could also have allowed expression of endosialidase, and in this case, it would not have been fused to the capsid protein. Computational analysis of translational initiation of the 30 bases preceding the endosialidase start indicated a 15-fold lower rate than of the gene 10 rate, so a low level of unfused endosialidase expression is indeed a possibility. T7E2 exhibited endosialidase activity in phenotypic assays (data not shown), but we did not assess whether the protein with this activity was of the expected size for endosialidase versus endosialidase fused to capsid protein.
There are thus two possible differences between T7E2 and T7E1: the level of endosialidase production is perhaps two logs lower in T7E2, and the endosialidase may be fused with the capsid protein. As a consequence of the latter property, a third possible difference is that some of the endosialidase molecules might be attached to the phage head rather than be released as free proteins (albeit fused with capsid proteins). We did not determine whether any capsid-sialidase fusions were incorporated into the phage head.
The fate of a beneficial gene cloned into a bacteriophage genome obeyed evolutionary principles considered to underlie the natural selection of a group benefit. The transgene encoded an enzyme, an endosialidase, that degrades a sialic acid (K1) capsule found on some host strains. The protein thus improved phage infection rates. If the gene was cloned as a free protein, released into the environment at lysis, the engineered genome (T7E1) outgrew the control phage (T70, lacking the endosialidase) when two conditions were met: (1) The host expressed the K1 capsule. (2) The engineered phage was grown in physical isolation from the control phage. Mixing the two phages before growth or using a host lacking the capsule reversed the outcome. Our cloning the enzyme as a presumed fusion with the phage capsid protein resulted in superior growth over the control under these same conditions but only in a narrow range of high initial phage densities. However, it is also possible that the active enzyme in this latter case was in fact an endosialidase not fused with capsid, due to a low level of cryptic translation initiation, or that the fusion protein was not included in the capsid.
The ability of high concentrations of free endosialidase to strip K1 capsules and augment phage infection has precedent in previous work. Artificial infections of mice with K1 capsulated bacteria can be cured with high doses of exogenous endosialidase; this effect was interpreted as enzyme-mediated capsule degradation enhancing immune system clearance of the bacteria [14]. Also, in vitro growth of a wild siphovirid phage (not related to T7) on a K1 capsulated host was enhanced by addition of free endosialidase to the media [15]. These prior observations led to our design and led us to anticipate the outcome here. Nonetheless, a different outcome might well have been obtained here from the effects of several unmeasured variables (e.g., [7, 16]), and indeed, different ecological methods of creating spatial structure can lead to qualitatively different outcomes [6, 17]. Furthermore, there are ecological effects of depolymerase enzymes that can affect phage dynamics [18]. The main question both addressed and answered here is that a superficial understanding of basic evolutionary and engineering principles was robust enough to predict a qualitative outcome.
The results support theory and experimental work underlying a phenomenon generally known as a tragedy of the commons, which is more broadly interpreted as the evolution of cooperation or of group benefit. In a tragedy of the commons, individual selfish interests conflict with group benefit and prevent the evolution of traits that benefit the group. Experimental evidence supporting different aspects of the theory has been provided in the context of bacterial siderophores, experimental phage and plasmid evolution [1, 2, 6, 19, 20] and indeed is commonly observed as the presence of 'satellite' colonies when selecting ampicillin resistant plasmids in a transformation library. The biological context here differs in details from these precedents, but ours is also the first demonstration that the principles apply to the fate of a cloned gene outside of its natural context. Cloned genes may behave differently than native genes because cloned genes have not evolved to be integrated into the network of genomic interactions and thus may have fewer pleiotropic effects than native genes.
The experiments here have explored only a few of the many possible environmental sensitivities and quantitative difficulties in maintaining a group-beneficial transgene. The net benefit of a gene will generally depend on its level and timing of expression, the duration of the growth phase during which selection is operating, and on changes in the external environment that affect the momentary benefit [16]. In the present context, we attempted several variations in growth media before finding one that consistently allowed the engineered phage to outgrow the control phage. It is possible that this sensitivity in outcome reflected the thickness or other properties of the bacterial capsule that varied with growth conditions, but the situation may be considerably more complicated than suggested by our limited knowledge. Furthermore, two alternative methods of cloning the same gene led to quantitatively different evolutionary outcomes, which might have been due to how the cloning affected protein levels or to different molecular contexts for the protein (a free endosialidase versus an endosialidase fused to the capsid protein).
The second cloning method used here was motivated by one solution to avoid a tragedy of the commons: physically couple the beneficial gene product with the genome producing it. Attaching the endosialidase to the phage particle ensures that the engineered genome benefits from its transgene, although it is not clear if our attempted implementation of this method (endosialidase fused to capsid protein) indeed achieved this objective. Even so, coupling the transgenic protein to the viral particle does not ensure that the benefit goes entirely to the engineered genome: phage assembly is relatively inefficient, and lysis releases many unassembled components along with complete virions. Likewise, co-infection of a cell by multiple phages would allow one phage to capture the enzyme from another. Thus no cloning strategy is completely immune to a tragedy, but some are less prone than others.
The understanding achieved here may facilitate designing phages for controlling biofilms. One such original effort was the cloning of a dispersin gene into T7 [3]. Dispersin degrades an essential component of E. coli biofilms, so the engineered phage ought to be able to penetrate biofilms and deliver dispersin to successively deeper levels, ultimately destroying the biofilm. Short term assays of 24hr biofilms suggested that the transgene enhanced biofilm destruction. The question raised by the present study is whether the dispersin transgene would persist in the long term growth of phage in a biofilm. Produced as a 'public good,' the dispersin gene's fate would depend on phage dynamics and spatial structure of phage growth in the biofilm, which is presently unknown and may even vary from biofilm to biofilm; in particular, phage attacking a biofilm may persist by infecting the planktonic cells released from the biofilm rather than by penetrating it [21]. Whereas the spatial structure of a biofilm-infecting phage is unknown, we can invert the model and argue that long term fate of the dispersin-encoded transgenic phage in a biofilm can reveal the nature of phage population structure. However, spatial structure is a necessary condition for dispersin maintenance, not a sufficient one: failure of a transgenic dispersin to be maintained in T7 grown on a biofilm could stem from many causes even with spatial structure.
Even when a transgene is initially beneficial to its recipient genome, the engineering is unlikely to be without flaws. Directed evolution may then be employed to improve the engineered genome. For example, growth of our endosialidase phages under selective conditions might lead to improvements that broaden the spectrum of environmental conditions maintaining the transgene. Yet perpetual growth of a transgenic organism may favor improvements of some types but not others, and the tragedy of the commons perspective offers insight to which types of improvements may be expected. For example, mutations improving transgene expression, where the benefit of improved expression is manifest within the cell as increased burst or decreased lysis time, should not be subject to a tragedy of the commons, because any benefit is realized specifically by the mutant genome.
In contrast, mutations improving the extracellular performance of public good proteins (such as endosialidase activity) may suffer a tragedy of the commons even with spatially structured growth. A mutation creating a more efficient endosialidase would usually arise in a swarm of non-mutant enzymes produced by neighboring relatives such that its benefit is shared across a large population of phages. Only if the mutant phage was grown in isolation from others would it be able to benefit from its superior mutation. Thus, the tragedy of the commons guides both the maintenance of the engineered change as well as its improvement.
A tragedy of the commons does not preclude use of engineered genomes for the intended purpose. The rate of evolution away from the engineered state will dictate how long an infusion of transgenic organisms has its intended effect. As noted by a reviewer, decay of the engineered state can be temporarily reversed by re-introduction of engineered genomes. Where spread of the engineered gene to a natural fauna is to be avoided, an engineered senescence of the transgene may even be desirable.
The short term evolutionary fate of a gene cloned into a phage genome and expressed as a free protein obeyed principles underlying the evolution of cooperation. The gene encoded an endosialidase that degraded host capsular polysaccharides (K1) and was expected to expedite phage initiation of infection. The gene was highly disadvantageous when grown on a host lacking the capsule, but it was also disadvantageous on a capsulated host when the engineered phage was grown in a mix with the control phage lacking the endosialidase gene. The latter effect is a molecular tragedy of the commons, in which the free enzyme – the gene product – benefits all genomes in the local environment, even those that do not carry the gene and do not pay the cost of producing the enzyme.
There are two solutions to this tragedy of the commons. One is to employ 'group selection,' growing each phage type in isolation of the other. For phages released into the environment, the ecology will be beyond the control of the engineer and may not allow this solution. The second solution is to clone the transgene so that it is not expressed as a free protein, but is physically coupled to the genome producing it. Only the former approach was broadly successful here, but our attempted implementation of the second approach likely created problems with viral assembly and, even then, did not obviously achieve the objective of endosialidase coupled with the phage particle.
The evolutionary principles germane to this study apply broadly. Thus, a tragedy of the commons is possible with other enzymes cloned into phage genomes and even with secreted molecules of unicellular organisms. Efforts to engineer microbes with the capacity to release beneficial molecules into their local environment may thus need to account for – or modify – the organism's ecology, or find ways of tethering the extruded molecules to the producer.
E. coli K12 with K1 capsule
E. coli K12 fully permissive to T7 and lacking the K1 capsule
T7 Select 415 with 15 AA control insert and C74 left end
T7 Select 415 with sialidase insert expressed as free protein, C74 left end
cf. [4]
T7 Select 415 with sialidase insert expressed as stop codon readthrough fusion to 10A, C74 left end
Three phages were engineered (Table 1). All used the T7 Select 415 vector (Invitrogen) as a backbone [10]. The T7 capsid contains 415 copies of the capsid protein, and the '415' designation refers to the fact that all capsid proteins in the virion are derived from this vector. This phage is deleted for 2600 bases of the wild-type genome, mostly in the phage early region ('left' end), but our vector left end was exchanged with that of a phage carrying the C74 deletion [24] by a Bcl I fragment swap; this fragment carries an intact 0.3 gene, which enables the phage to grow on Type I RM hosts (which we used for work not reported here). The vector also includes a cloning site at the 3' end of the major capsid gene 10A, deleting the unique, non-essential portion of the minor capsid gene 10B.
Our control phage, T70, was modified to carry the short Invitrogen 15 amino acid control sequence in the cloning site between the Eco RI and Hind III sites, flanked by single amino acids; we consider this sequence to be of little consequence, and indeed, its nature is important only in that it does not degrade the K1 capsule. Phage T7E1 was modified to carry the full endosialidase gene and its ribosome binding site of phage K1-5 [25] between the Eco RI and Hind III sites of the vector; the fragment was amplified by PCR from phage K1-5 to have Eco RI and Hind III ends and to insert a stop codon in frame with 10A in addition to 30 bases of K1-5 sequence upstream of the endosialidase start. Phage T7E2 also carries the K1-5 endosialidase gene between Eco RI and Hind III sites, but was engineered so that its initial Met codon was in frame with 10A as the first unconstrained codon beyond the Eco RI site. The difference in these latter two designs is that T7E1 encodes sialidase as a free protein, released at lysis; T7E2 is expected to encode endosialidase as a protein fusion with 10A, but the only clones obtained had acquired a stop codon immediately in front of the endosialidase reading frame. The nature of the cloned endosialidase gene was verified by Sanger sequencing.
LB broth (10 g NaCl, 10 g Bacto tryptone, 5 g Bacto yeast extract per L) and M9 glucose (47.8 mM Na2HPO4, 22 mM KH2PO4, 8.5 mM NaCl, 1.87 mM NH4Cl, 1 mM MgS04, 0.1 mM CaCl2 with 0.2% glucose) were used. Plates used the indicated media with hard agar at 15g/L Bacto Agar, soft agar at 7 g/L Bacto Agar.
Phage competitions
Phage were competed in growth assays on plates. Assays on host EV36 were done on M9 glucose plates. EV36 was grown 24hr to high density (at least 109/mL) in liquid M9 glucose at 37°C. Fifty microliters of the suspension were added to 3 mL soft agar and poured on hard agar. After the top agar gelled, a small volume of phage suspension (2-8 μ L) was applied to the surface at a defined point, and the pipette tip depressed into the soft agar to enhance diffusion. Plates were incubated 16hr at 37°C. After growth, the zone of top agar surrounding the site of phage application was scraped into a known volume of media (with chloroform to kill cells), vortexed, allowed to sit 1hr, and plated to determine titer. Two types of assays were done in this fashion with M9 glucose media, differing only in whether the initial phage suspension was of a pure phage type ('separate' competitions) or a mix of phage types ('mixed' competitions).
Separate competition assays were also done on IJ1133 in LB. In these assays, phage were plated at low density and grown at 37°C for 24hr. Plaques were counted to determine initial titer, and the top agar was scraped into media (plus chloroform) to determine total phage numbers.
The growth rate of a phage in the separate competition treatment was measured as the number of phage recovered from the infected zone of a plate divided by the number of phage initially added to that zone - the total amplification. In mixed competitions, the proportion of each phage type was measured in the pool of phages used to initiate the competition and after the outgrowth of the competition.
Endosialidase status of phage isolates
In mixed competition assays, it was necessary to determine the proportion of phages carrying or lacking the endosialidase gene. We used a functional assay to determine this status. For both the initial phage mix and the final, post-competition mix, the phage suspension was plated on LB plates using IJ1133 (which does not discriminate against either phage type). Isolated plaques were transferred by sterile toothpick to a lawn of 98% EV36 (grown 24hr in M9 glucose) and 2% IJ1133 (grown 12-24 hr in LB). After 12-24 hr, plaques formed around the stabs exhibited a clear, broad halo if and only if the phage carried endosialidase.
where the bar represents the mean of (). For this test, independent measures of the initial phage counts can be performed on a sensitive host and the difference of the four means subjected to a t-test with the appropriately adjusted degrees of freedom [26].
We thank Eric Vimr for EV36 and several other K12 strains engineered to express the K1 capsule. Dean Scholl retested his published results to confirm inhibition against T7 in LB broth and sent us his strain of EV36, which was used for our assays (even though we used M9 medium). R. J. Bull did some early assays using EV36 grown in low salt LB. R. Springman confirmed the sequences of the 5' ends of the endosialidase genes.
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Relax with the girls on a fantastic Bath hen weekend!
You've chosen really well, without doubt Bath has to be one of the top picks in the whole of the UK for a stylish hen weekend. Its stunning Georgian streets and crescents, Roman history, cultural wealth, cafes, bars and restaurants make it worth a visit anyway but visiting for a pre-wedding send off with the girls... it doesn't get much better! We liked it so much we based ourselves here right in the city centre, so if anyone knows about getting the most out Bath - we do.
WHY: Reasons to choose a Bath hen do
It's known as one of the UK's most beautiful cities, home to an exceptional range of wining and dining that complements an enormous selection of quality, group pleasing activities and some gorgeous accommodation options. This unusual combination of range and quality puts Bath near the top of the list of Europe's very best hen do destinations. Click through to the Bath hen do planning page and you'll see dozens of exciting options for keeping the bride happy and the girls busy!
WHAT: Bath hen weekend adventures
Bath is a city where chocolate making can fill your morning, Grease dancing can exhaust your afternoon and cocktail mixing can spill into your early evening! You could easily follow that with dinner at a top restaurant like Carluccios or Jamie's, some fun at the comedy club and finish with guestlist treatment at the city's excellent nightclubs. What a day! Planning your Sunday? If you haven't already squeezed it in, a trip to Thermae Bath Spa is a must - bathe in the same natural waters as the Celts and Romans did over 2000 years ago, just a bit more polished. In between all of that there's top shopping, charismatic cafes, quaint pubs and plenty of bars to visit. Sit on one of the outdoor tables on popular George Street, explore the newly developed Southgate shopping centre or even catch Bath Rugby in action (it's popular with hens, especially if you can arrange to meet a couple of the players!). Whether you're looking to dive right into activities or spending a more relaxing weekend exploring the spa and sights, Bath's got more than enough hen weekend options for everyone!


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Finding Love After Tragedy
After being stabbed 32 times, survivor to marry paramedic who helped save her life
- Jun 10, 2015
- 1.8k have read
Fairy tales do come true—just ask Melissa Dohme. After a surprise proposal from her paramedic boyfriend at a Tampa Bay Rays baseball game in May, she's going to marry her knight in shining armor. But, as many fairy tales tend to go, this story began with tragedy.
In January 2012, paramedics from Clearwater, Fla., responded to a horrific scene: Melissa Dohme, 20, had been stabbed 32 times by her abusive ex-boyfriend. Firefighter-paramedic and first-responder Cameron Hill, 35 at the time, told the Tampa Bay Times, "It was so bad. You couldn't tell she was blond."
That evening, Dohme flat-lined four times and suffered a stroke in the hospital. Her injuries would leave her with permanent nerve damage to the left side of her face. She lost teeth and suffered a fractured skull and nose.
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But, she miraculously survived and, after recovering from that dreadful night, Dohme turned her energy to good and became a domestic violence advocate. She also met and thanked many of the people who cared for her after the attack.
Fate would have it that Hill and another first-responder surprised Dohme at a church where she was giving a talk, and romantic sparks flew. Hill and Dohme began dating and soon fell in love. In 2013, Hill was at Dohme's side when she watched her ex-boyfriend, Robert Burton, plead guilty to attempted murder. "He didn't win and I won," Dohme told the Tampa Bay Times. "He tried to murder me, and he failed."
On May 11 of this year, as recognition for her advocacy work, Dohme was asked to throw the first pitch at a Tampa Bay Rays baseball game. As she was about to throw out the first ball, Hill ran onto the field and gave her a baseball with the scrawled message, "WILL YOU MARRY ME?"
"He's the one I've been waiting for," Dohme told the Tampa Bay Times, "My fairy tale."
You can support Dohme by liking her Facebook page, Support Melissa but first, watch the video of the proposal here.
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Dark circles represent a problem and they rob us of brilliance. These areas of discolouration under the eyes appear for several reasons: fatigue, prolonged exposure to sunlight, lack of iron, vitamin K and vitamin B12, smoking, caffeine, dehydration, stress, liver or kidney disorders, etc. These are just some causes …Read More »
How to Clean your Lungs from Tobacco Naturally in a Week
Lungs detoxification prevents infections, reduces cancer risk and improves lung capacity. If you are a person who smokes constantly, or have to stay in environments with dust and smoke, it would be good to know that there are some ways and recipes you can help the body to cleanse …Read More »


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NPS Photo - Cookie Ballou
Buried Alive
The lowlands of Guadalupe Mountains National Park shimmer under the sun's rays. Mirages appear at the horizon and distant mountain ranges look like ships sailing on a desert sea. In this vast and timeless landscape the imagination is given free reign, but nothing prepares the mind for the reality that lies underfoot. Buried in earthen tanks created by ranchers and natural depressions are life forms ill-suited for the desert's extremes. Only after the drumming of summer's heavy downpours can they be summoned. For a brief span of time thousands of frogs become locally abundant, their nightly chorus audible up to a mile away. Amphibians are one of the desert's greatest surprises.
Nine species of amphibians can be found in Guadalupe Mountains National Park. Some of these amphibians, such as Rio Grande leopard frogs, are abundant in McKittrick Canyon where there is permanent water, but most of the park's amphibians spend most of their existence underground in a state of suspended animation.
Couch's spadefoot toads exemplify the ability of an animal vulnerable to the desert's heat to alter its lifestyle to meet the desert's demands. Like all amphibians, it must breathe through its skin and keep its skin moist. The toad can survive the dry spells (most of the year) by living underground. While underground a protective sheath of unshed skin forms around them. The toad's metabolism slows and it is able to tolerate a loss of 50% of its water.
For brief periods in summer water accumulates after monsoonal rains and provides the ideal breeding and feeding grounds for these water dependent creatures. These water sources often disappear quickly. For spadefoot toads timing is critical. Spadefoot toads will congregate at temporary pools to mate and lay their eggs. Once the eggs are laid the race is on. The eggs will hatch within 3 days and the tadpoles will reach maturity in as little as two weeks. After this frenzy of activity and growth, the frog chorus disappears with the rain. Once mature, the adult frogs will bury themselves alive and wait for next year's storms.
Last updated: February 24, 2015
400 Pine Canyon
Salt Flat, TX
(915) 828-3251


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Product Code:MMP-1050
Ex.Tax £5.00
Availability: 40
RasPiO InsPiRing is a system of programmable RGB LED boards which can connect together to make whatever 2D or 3D shape you want! All the boards are based on 1cm LED spacing and 10cm side. Through-holes enable use of standard 2.54mm headers, or you can wire directly to them if you prefer. Each shape has at least one set of inputs and outputs so they can be 'daisy-chained' together. The straights have 3 sets of input/output holes so they can be connected at 180°, 90° or 45°.
This purchase is for the Straight-8 Matrix, which consists of a single 8 LED run on a 10cm straight strip. Please note, you'll need a InsPiRing driver board to drive it! (Sold Separately).
The InsPiRing Driver Board is a pHAT-sized driver board for Raspberry Pi with a buffer chip to handle the 5V logic levels and a space for external power if needed. Pi GPIO ports are broken out and there is space for an optional ADC, which enables control of your LEDs with potentiometers or sensors. RasPiO InsPiRing uses SK9822 LEDs which are controlled using SPI.
With the RasPiO InsPiRing system, you could make a…
Or embed them in any project that requires really fine control over colour and intensity of light! RasPiO InsPiRing can be controlled by most popular electronics platforms, including…
You could even control it from your phone, if connected to wifi-enabled device like the Pi Zero W. Open a port on your router and you could switch on your 'TV simulator' if you're away from home (make it look like someone's home watching TV). But you can also hook up all sorts of other control mechanisms too, such as light, motion, sound or temperature sensors to trigger your LEDs.
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Kasie Pieri
Senior Account Manager
Behind the pearls, curls and sugary-sweet demeanor, you'll find one of Indy's most dynamic PR maestros, who is passionate about everything related to writing, media relations and social media marketing.
Fueled by the determination to see a client's name in the headlines, this Hoosier-bred gal has a tenacity to bring monumental levels of energy, sass and persistence to each client project, which has made her unstoppable in the PR realm. Her magical abilities to pull pie-in-the-sky coverage out of thin air has allowed her to snag national media coverage in TIME, FOX News, Entrepreneur, Business Insider, Inc., USA Today and more. Immersing herself in an assorted group of clients in a wheelhouse of industries, Kasie's strong PR persona as well as lighting speed "think-outside-the-box" mentality makes her a vibrant leader among the Dittoe PR office.
Kasie graduated from Ball State University (chirp, chirp) with a Bachelor of Science in public relations and a minor in communication studies. Outside of work, you'll find this one glued to her phone reading the latest sports news, watching re-runs of her favorite TV show or still waiting to receive her Hogwarts acceptance letter. This fashion-obsessed gal is also in love with her loveable pooch who's named after her favorite Parks & Recreation character, Ben Wyatt.
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- $40,000 price
- 5878215 listing id
- Neighborhood: White Mountain Vacation Village
- Status: Active
Developed & leveled HOME SITE available in beautiful White Mountain Vacation Village. This home-ready lot is located in the pine area in Show Low. It includes multiple pine trees, and is all set w/ electric, water & sewer connections. Move-in ready for your RV or a Park Model, this lot includes pavers and a sunken area w/picnic table. This popular vacation community is very quiet & peaceful, & offers plenty of activities & events throughout the year. Warm up by the fire in the clubhouse sitting room or relax w/a book in the library, enjoy a little competition at the pool tables, or host a gathering of family & friends in the clubhouse kitchen/ entertaining space, or the covered picnic area. Close to shopping & attractions, away from the valley heat, your perfect weekend retreat!
Courtesy of: Realty Executives
Office Phone: (623) 561-8800
Copyright Arizona Regional Multiple Listing Service, Inc. All rights reserved. Information is deemed reliable but not guaranteed.
Updated: 24th May, 2019 5:27 PM.
2310 E Broken Arrow Lane Show Low, AZ


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Maybe it is just coincidence that "aguila" means "eagle" in Spanish, but when a Steller's sea eagle from Woodland Park Zoo needed specialized surgery for a fractured bone, Dr. Alexander Aguila of the Animal Surgical Clinic of Seattle was ready!
On March 23rd, Dr. Darin Collins, Woodland Park Zoo Director of Animal Health, contacted the surgeons at Animal Surgical Clinic of Seattle regarding a potential fracture repair on the right wing of a Steller's sea eagle named Natasha. Exhibited in the award-winning Northern Trail exhibit at the zoo, Natasha is the female of a breeding pair acquired in 2010. Steller's sea eagles are among the largest and heaviest of the raptor family, and Natasha weighs in at 13 pounds with a wing-span of over 6 feet!
On the morning of March 22, Natasha (pictured in the background above) was found on the ground of her zoo enclosure. She appeared to be having difficulty maintaining her balance and was holding her wing lower than normal. Zookeepers quickly alerted the zoo's veterinary staff of their observations, and preparations were made for a physical examination.
Natasha had come to Woodland Park Zoo with an existing right wing abnormality from a previous injury sustained several years ago, and had been living quite successfully with her disability. She was navigating her zoo enclosure with minimal difficulty given her limited flight capability and mostly moved from branch to branch with short flights. Speculation was that this new injury could have been the result of Natasha falling from a higher branch.
After examining the eagle and taking X-ray films at the zoo's Animal Health Complex, Dr. Collins determined that she had fractured her humerus (equivalent to the human upper arm bone) and would require specialized surgery. Natasha was given fluids and pain medications and stabilized overnight while appropriate preparations were made to transport her to Animal Surgical Clinic of Seattle.
Upon receiving the news of the injury, ASCS board-certified surgeon Dr. Alexander Aguila swung into action. He and Dr. Collins collaborated with Dr. Ricardo de Matos, an avian specialist at the Cornell University College of Veterinary Medicine, to form a plan of action that involved placing an external fixator system to stabilize the fracture during the healing period.
Thanks to the knowledgeable zoo staff, Natasha's transport to ASCS and her subsequent anesthesia went very smoothly. Zoo and ASCS veterinary technicians worked side-by-side to place a breathing tube and IV catheter, as well as probes for monitoring vital signs. The surgical site was cleaned and Natasha was moved into the OR for her procedure.
Dr. Aguila worked alongside another ASCS board-certified surgeon, Dr. Russell Bennett, to place the external fixator, with the help of surgical assistant, Dr. Cristina Flamini. Anesthesia and vital signs were monitored constantly by Dr. Collins, and in just over an hour, the surgery was complete. Post-op X-ray films showed good positioning of the fixator and Natasha was removed from anesthesia. After placing a leather hood over her eyes and wrapping her securely, zoo staff transported her back to their Animal Health Complex, where her recovery continued uneventfully.
Natasha is doing very well now and her open wounds are beginning to close. Recheck X-rays are due at three weeks post-op to verify progress of healing, and again at six weeks to ensure completion of healing before removing the external fixator. The zoo vet and keeper staff members are keeping a close eye on her, and we expect an excellent outcome!
Thanks to the collaborative efforts between Woodland Park Zoo and Animal Surgical Clinic of Seattle, zoo-goers can continue to be inspired by this majestic conservation ambassador for the Steller's sea eagle species.
Photos: Sea eagles at Woodland Park Zoo by Ryan Hawk/Woodland Park Zoo, Surgery photos courtesy of Animal Surgical Clinic of Seattle.
This blog post originally appeared on the Animal Surgical Clinic of Seattle website.


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Living in California, I was never exactly oblivious to Dia de los Muertos celebrations.
But am I imagining it, or are sugar skulls becoming… well… bigger? Within the past few years, I've noticed more and more images of women (and some men) with faces painted like skulls. So I thought I'd do some research. What do these strangely seductive skulls mean?
1) November 1st is the Dia de Los Muertos: It's believed that the gates of heaven open at midnight on October 31st, and the spirits of departed children are allowed to return to their families on November 1st. On November 2nd, adult spirits can share in the festivities.
2) A 3,500 Year Old Tradition: The Aztecs had a month-long celebration honoring those who'd died, welcoming the spirits back to the earth for a visit. Skulls were often displayed as a part of the festivities. Today's Day of the Dead represents a mixture of Catholic and indigenous beliefs, and skulls are a colorful part of Day of the Dead symbolism.
3) Sugar skulls are made of… you guessed it: sugar. Italian missionaries brought sugar art to Mexico – a natural fit since Mexico is a sugar-producing country – in the 17th century. From the Italians, Mexicans learned the art of molding sugar into angels, skulls, and other religious imagery.
4) Sugar art was a part of All Saints Day (November 1st) in Europe dating back to the 12th century. But Mexico has certainly made the art its own.
5) Sugar skull face painting is somewhat newish (I wasn't imagining the uptick). Skull painting is derived from the custom of wearing skull masks during this holiday.
About the Author
Kirsten Weiss is the author of Steam and Sensibility, a steampunk novel of suspense, and the Riga Hayworth series of paranormal mysteries: the urban fantasy, The Metaphysical Detective, The Alchemical Detective, The Shamanic Detective, The Infernal Detective and The Elemental Detective. Watch for book six in the series, The Hoodoo Detective, available this Halloween.


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Lap - One Point Hoodie - White
Lap - One Point Hoodie - White
Product Code: AJ2CTS54
Available Colors: White, Black
Materials: Outshell: 100% Cotton
Size: FF, XL
Size Chart
Ships Internationally via EMS / DHL / Fedex
Return Policy
Harumio provides Korea proxy service.
Items are sourced from official retail channels from the respective manufacturers. Items sold non-returnable and non-refundable. Warranty is as provided by the manufacturer, however, buyer would need to ship the product back to Korea for any repairs.


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This is simply milk pudding covered with chocolates. Take a closer look at the chocolate layer.
Yes, it's indeed not smooth. I wanted to use up my leftover 40g of Swiss Mahony Chocolates from previous baking. It's been sitting in the fridge forever. I've mentioned before that this chocolate is really similar to Toblerone, hence the coarse texture from the pieces of nuts and nougat bits.
I made this to serve two only (or probably serve one if you're a big fans of dessert!)
It's called a candy spoon because when you dug into the pudding, the layer of chocolate will follow along and take its shape because of the canola oil used. I guess I didn't used enough because my chocolate layer was a little too hard for me to dug into the milk pudding layer when it was serving time. You could add more oil but I don't like the idea of it being unhealthy.
Here you go!
1 tbsp + 1/2 tsp cornflour
2 tbsp + 1/2 granulated sugar
Mix that two well in a pan heated over low heat.
Stir in 1 cup of cold full cream milk and stir till cornflour and sugar are dissolved. That will take about 2-3mins.
When the mixture is thick enough (you'll know when it can readily coat the back of a spoon evenly), turn the heat off and stir in 1/2 tsp of pure vanilla extract. Pour into two fine cups and chill well.
Microwave 40g of any chocolates with 1 tbsp of canola oil on high for 45 seconds (or more), stirring every 15 seconds interval. Pour on top of well chilled pudding and throw it back into the fridge to chill for another hour before serving.
He just dislikes milk so much and this is a very good way for me to get some calcium into his body!
Tuesday, May 13, 2008
Candy Spoon
Posted by Quinn at Tuesday, May 13, 2008
0 comments on "Candy Spoon"
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Mary dances in the street
QUESTION marks continue to surround the Mary Poppins mystery.
While Mary remains facing east, the public continue to scratch their heads about her mysterious rotation.
Project coordinator Paul McShane said theories about her mystical shift had been flooding in, but the mystery remained unresolved.
Mr McShane said one of the most interesting theories came from the grandchildren of Bowral resident Joan Smyth.
Six-year-old Gabrielle and four-year-old Rosana Smyth believed Mary had "come to life one night and danced around the playground having fun until the morning light appeared when she quickly jumped back onto the pedestal. Unfortunately she had too much fun during her adventures and she turned the wrong way when she became still again."
Mr McShane has deemed this as "one of the best theories yet".
The statue's sculptor Tanya Bartlett was also mystified when she heard of Mary's chim switch-aroo, but was not surprised.
"It's her magic, she's up to her tricks," Ms Bartlett said.
"It doesn't surprise me, but I hope she behaves herself a little," Ms Bartlett said.
Paul and Melissa McShane are still asking the community to send in their theories to firstname.lastname@example.com to help solve this head scratcher.
Discuss "Mary dances in the street"
Please note: All comments made or shown here are bound by the Online Discussion Terms & Conditions.


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Nancy Cardwell of IBMA reports:
An event producer friend of mine at Silver Dollar City, in Branson, Missouri, is exploring the idea of having an international bluegrass stage (where he would present bluegrass bands from outside the US) at a multi-day festival, the latter part of May 2009. If you know of any bands already planning to tour in the US in that time, please let me know and I'll pass along the info. This gig wouldn't pay enough to be the only reason for a band to fly over here, but if they're in the neighborhood anyway, it would be a great thing to pick up. Rhonda Vincent and I both played at this theme park in the 1970s - it's a nice place. (You work a lot, but the sound is good, you're treated well by management, the park is beautiful, the quality of bands is excellent, and it's definitely a chance to sell a lot of CDs - although a percentage of sales usually goes to SDC.)
Contact Nancy by e-mail if you're interested. The proposed date was originally, and wrongly, shown as May 2008 on this post.
Farm Hands – I Can't Go Home Anymore
4 days ago


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There can be no doubt that the Buddhas of Wat Chedi Luang look their best in the dark. When dusk turns to night and the lights come on around the temple, there's a quiet and relaxing ambiance that can't be found during the heat of the day in Chiang Mai.
Perhaps it was because there were far fewer people, or because I wasn't constantly on the look out for shade. Whatever the reason, this place was more magical once the sun had set, and so I spent an hour exploring the angles around the temple.
It took a while to get this shot all lined up. I was using a small mini-tripod which left me about 25cm from the ground setting up the camera. Unfortunately these things are never completely symmetrical, which I always find very disappointing!


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Helen Creighton collected songs and folklore in Nova Scotia and New Brunswick. Nova Scotia Archives and Records Management (NSARM) houses the Helen Creighton fonds, which include the bulk of her recordings, photographs, correspondence, manuscripts and notes etc. The Canadian Museum of Civilization and The Library of Congress (LOC) also have materials collected by Creighton while she worked under their sponsorship. In addition, Mount Allison University holds a collection of recordings. While NSARM and the LOC have separate searchable online catalogs, there does not yet exist any one master list of recordings, and other than by obtaining copies and listening, it is difficult to determine which entries are duplicates held by more than one archives.
Copies of selected recordings from the Nova Scotia Archives are available to the public in the non-circulating reference collection at the Alderney Gate branch of the Halifax Regional Library system. For information about using the other collections referenced here or obtaining copies of materials, contact the specific archives.
Canadian Museum of Civilization
Library of Congress Catalog Record: Helen Creighton collection of Nova Scotia recordings
Traditional Music and Spoken Word Catalog Search
Mount Allison University Archives
Nova Scotia Archives: Helen Creighton, Helen Creighton Fonds
Halifax Regional Library: Helen Creighton Collection
"From Acetate Disc to Digital Audio: Tracing the Copies of Helen Creighton's Sound Recordings."
Barrett, Creighton. 2009. Canadian Folk Music, Vol 43, No 2.


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Flashback Friday- Making A Christmas Card
Friday, November 19, 2010
Getting a late start, this Flashback Friday.
I thought it was Thursday.
Really though. I watched Greys Anatomy last night, I mentioned something about "so glad tomorrow is Friday, and hubby and I can have our on the couch with cable date night!", I even made a comment about Thanksgiving being in exactly one week.
...yet I still woke up, thinking that today was Thursday. Again.
So anyways. Since I've got Christmas card making on the mind (we're taking pictures for that this weekend, and I'm brainstorming), I thought I'd go back through my files and pick out some of the pictures from our photo shoot that we had at the Air Park (we live on an Air Force base, with miles of vintage parked planes on display) last year.
We went down there on Veterans Day, Santa hats in hand and briefly on heads, and played it by ear. It was just Charlie and Eleanore back then, Charlie not even walking yet. Eleanore mostly just wanted to run through the grass and pick flowers, rather than wear her holiday hat, but we managed to get at least one Christmas looking shot of her...
Yep, that second one there is the one that made the cut.
Charlie wasn't yet obsessed with planes, so he spent most of his time crawling around, in search of the most delicious looking rocks.
Yumm, must have found a good one.
Ok, but before you break the few teeth that you have...
There we go.
Oh, wait, whats that?
You're sitting right next to ant hill?!
Well of course you are! We're awesome parents, and totally saw it before we put you down.
Oh my God somebody please pick up the baby!
This was one of my favorite pictures of Eleanore. Sitting there all pretty in the grass, blowing the seeds off of flowers.
And this was another one, because she never sits still for pictures with me. Ever. This one didn't make it to the card, but it is in a collage frame in our living room.
And my boys.
Love these boys.
Me and Christopher.
Aaaaand the money shot. The centerpiece of our 3 blocked collage card.
I literally had to run, after setting the timer on the tripod. I think we did this shot 3 times before we got one where I had actually made it to the bench before the timer went off.
Oh wait wait wait! Theres one more!
As we were leaving, walking back to the car, a random shirtless jogger, an elderly man, stopped to take our picture. He came up, asked if he could get a shot of our "beautiful family" (he mentioned how beautiful we were at least 5 times, and kept thanking us?), suggested we take it "over there", and then proceeded to take my camera, stand us all in the sun, blind us, and wash us all out...
Ok but after playing with it a little, the photo turned out to be kind of perfect. Probably because of the half naked sweaty man who took it.
Flashback with us! Go through your polaroids, scrapbooks, old crappy cell phone pictures- whatever you've got. Scan them, upload them, get them onto the computer somehow, and then share them with the world! Or... with us at least. Post them onto your blogs, write a little bit to go with them- tell us your story. When you're all finished, grab one of our buttons from down below (wayyyy down there at the bottom of our page) and put it somewhere inside your post (or link back to us the old fashioned text way). Come back here to this post, and add your link to the photo list below!
And if you've got a minute, maybe go poke around some of the other flashbacks posted, and leave a comment on another blog or two?
This is a comment-free blog, but our email is at the bottom of the page, if you'd like to get in touch.


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|Mecha Kawasaki is on a destructive rampage! This crazy chef can fire all sorts of missiles!"
|— Description • Kirby's Return to Dream Land
Mecha Kawasaki is a boss in the Kirby series, debuting in Kirby's Return to Dream Land.
Physical Appearance
Mecha Kawasaki is a titanic robot designed in the likeness of Chef Kawasaki. It is egg-shaped with a brown face and hands. It has two orange eyes, a white jaw, and red feet. The robot wears mechanical chef's attire, complete with a star-adorned toque, a blue shirt, an orange tie, spiked collars on its arms, and a refrigerator-like apron adorned with a duck image. It carries a frying pan and a ladle.
Underneath Mecha Kawasaki's exterior is a steel skeleton. Beneath its face is a pink visor and yellow lips. The toque is revealed to be a smokestack when the exterior is stripped away.
Kirby's Return to Dream Land
Mecha Kawasaki is the boss of Mission 2 in the Scope Shot sub-game. It wanders back and forth across the sandy stage, waving its arms. It occasionally tries to protect its body from the player's shots by holding its frying in front of its chest. The robot also spins its ladle at blinding speeds, deflecting any shots that hit it. Destroying the robot's frying pan and right hand will disable these techniques.
Mecha Kawasaki can open its apron to reveal a missile, then fire it out of its toque. These missiles include a fish that swims back and forth toward the player, a meat bomb that bounces along the ground toward the player, and eggs that fire all around with one moving in the player's direction. All of these missiles stun the player for a short time if they hit him. If the missiles destroyed before it can be fired, Mecha Kawasaki will briefly falter, leaving it open to attack.
After Mecha Kawasaki has taken enough damage, the robot's weak point will appear inside its apron. One shot will be enough to destroy the machine, causing it to explode.
- The music that plays during the battle against this boss is an extended remix of "Dreaming of Food," the music that plays during the intro of Gourmet Race from Kirby Super Star.


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Good morning- Pinky here with a Happy Birthday Card
using the beautiful and fun Celebrate Life March Kit!
This kit is simply amazing, so pretty and fun to work with.
The colors are amazing.
For the flower I used the patterned paper and hand cut a flower,
then I added stickers from the 12X12 sheet.
The Liquid Pearls added the perfect touch.
What are you working on today?


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Congrats to "Good Day Philadelphia" anchor Karen Hepp who gave birth to her third child on Friday.
Kellen Alexander Sullivan was born at 2:48 a.m. at the Bryn Mawr Birth Center, weighing in at 8 pounds, 5 ounces and measuringi n at 21 inches long. Kellen is Hepp's third child with husband, Brian Sullivan, an insurance executive. Kellen joins big brothers Quinn, 6, and Macklin, 3.
Hepp, who used to work at NBC10 before making the switch, went on maternity leave June 28. She'll be back from maternity leave after Labor Day.
Check out a pic of the new big bros with new addition Kellen below:


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Status: Open (Call business to confirm holiday hours)
Know more about this business than we do? Cool! Please submit any corrections or missing details you may have.Help us make it right
American Institute Of Architects Award Of Excellence 1992 1995 1999
Our goal at Stephen Rhoads Architect is to provide clients with our highly skilled design services at an affordable price. We are a Princeton, NJ Architect who gives you personalized attention.
Be the first lehighvalleylive.com user to add photos or videos of Stephen Rhoads Architect Llc
Stephen Rhoads Architect LLC is located at 11 Alta Vista Dr, Princeton, NJ. This business specializes in Architecture and General Contractors.
Stephen Rhoads Architect LLC can be found at Alta Vista Dr 11. The following is offered: Preconstruction Consulting. The entry is present with us since Sep 9, 2010 and was last updated on Jun 4, 2015. In Princeton there are 1 other Preconstruction Consulting. An overview can be found here.


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I've been on twitter for about 6 years now, and it never ceases to amaze me how easy it is to find hilarious jokes for this list every week. You'd think I'd run out at some point, but nope. Twitter is a bottomless well of jokes (and lot's of other not so great stuff lately) but mostly jokes, and it is my absolute pleasure to bring you this list every week. I assure you it is as much fun for me to make as it is for you to read. So thank you for continuing to read this, because it means I get to continue putting it together. Please enjoy this week's roundup, and always remember: retweets mean everything.
in Funny


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Booz CEO: US fares better than Europe
Shumeet Banerji, chief executive of Booz & Co, tells the FT's Andrew Edgecliffe-Johnson why he thinks things are looking up for a US economic recovery but Europe's crisis may not yet be contained. He also weighs in on the FT's Capitalism in Crisis debate: The problem? Not enough of it.


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email: email@example.com
Alexandrite is a very rare gemstone which for a clear crystal cut 1 carat will sell well between $1500 -$3000 per/carat. The wonderful thing about Alexandrite is that it is a colour changeing gemstone, in natural light the colour is rich green and in incandscent light it changes to stunning redish purple . Alexandrite is one of the rarest gemstone in the world and some new pockets are been found but still it remains very rare. I have spent a lot of time chasing some Alexandrite and now have some for offer. This piece of Alexandrite has been certified through the HKD gemological laboratories Canada. This Alexandrite has strong colour change from good green to light pink to purple and is a stunning gemstone. This piece has some small inclusion but the clarity is fantastic very see through and clear.
Type: Alexandrite Weight: 0.89carat Size: 5.8 x 4.35 x 4.2 mm
Colour: Colour change (green- purple red) Shape: Oval faceted cut deep cut
This is from the chrysoberyl family and is 100% natural and certified.
Only have the one available in this list. This gemstone the Alexandrite is a very rare gemstone and are very hard to come by as gem grade cuttable stone so don't let this one get away!!


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Compiled by Brian Westcott
(Possibly a combination of the International, North Coast, and United States Titles)
1.Chris Hamrick
10/13/2001-Cleveland, OH
2.J-Rocc [2]
11/04/2001-Painesville, OH
3.Chris Hamrick [2]
05/11/2002-Painesville, OH
Stripped of title for failure to appear.
09/07/2002-Painesville, OH
4.Julio Dinero
09/07/2002-Painesville, OH
Awarded Title
5.Jerry Lynn
11/03/2002-Painesville, OH
6.Julio Dinero [2]
11/30/2002-Norwalk, OH
Defeats Crimson Dragon (Chris Hamrick). Jerry Lynn no-showed.
7.Chris Hamrick [3]
01/11/2003-Painesville, OH
02/08/2003-Painesville, OH
Defeated Al B. Damm and Chris Hamrick in a Three Way match.
Copyright 2002 - Jump City Poductions


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Preheat the oven to 325 degrees F. Line sheet pans with parchment paper.
In the bowl of a stand mixer with a paddle attachment, combine the butter and sugars on low speed. Mix until thoroughly combined and smooth. Add the maple syrup, vanilla and eggs. Mix on low until thoroughly combined, scraping down the sides as necessary. The mixture will look slightly broken. Add the flour, baking powder, baking soda and salt, and mix on low until the dough just comes together.
Place 1 tablespoon-size scoops on the prepared sheet pans. Bake 10 to 12 minutes, rotating the pan halfway through baking. Remove from the oven and cool to room temperature.
Place about a 4-ounce scoop of Sweet Potato Ice Cream between 2 cookies and sandwich.
Combine the cream and milk in a large saucepot over medium heat. Bring the liquid to an almost boil.
Whisk together the yolks, sugar and salt in a separate bowl. Gently add about 2 cups of the hot liquid, whisking until smooth. Add the egg mixture to the remaining cream/milk mixture, whisking until smooth. Remove from the heat.
In a separate container, combine the vanilla extract and the drained sweet potato. Using either a hand stick blender or a blender, puree until smooth.
Whisk the sweet potato puree into the egg mixture. Strain through a mesh strainer to remove any large chunks. Cool slightly over an ice bath, stirring constantly. You want it to be about room temperature.
Add the mixture to the ice cream machine, and make according to the machine's instructions.
Once the ice cream is complete, fold in swirls of the marshmallow creme, and freeze to set.
This recipe was provided by a chef, restaurant or culinary professional. It has not been tested for home use.
Recipe courtesy of Natasha Case and Freya Estrellar, Coolhaus Ice Cream Sandwiches


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Discussion in 'Suggestions/Comments' started by FRED_FLINTSTONE3, Oct 17, 2011.
sssssssssoooooooooo what award will I receive?
i'll make one for you..............lol
hahaha thanks bro.
Ok here's my list,
Outtlaw_Cassidy3: "Best spinner award"
kingvk3: "Best at close range no long range award"
xPrince_Mvpx: "Show your dot bro award"
Jandaro_CwC: "Good player award"
koolasice619: "1 on 1 award, 11-18 btw"
Ace-Pro_StAr: "Rapping award"
ndoor: "Front flip award"
and StomperCA187: "Am Just average award" LMFAO
Lmao wow you forgot some
Corvettezr1-13: "God of RDR"
BigDee: "No "tym" award" lmao
Born-2-Game: "Destined to be no.1"
ReallyGroovy: "Anti-Spin"
Lmao nah ReallyGroovy got the "I seen my brother whoop your ass award" Lol
How about the "Wrongfully Accused Award" for legit players
who are accused of cheating by shit cunts like Gvb & PunkBuster
lol @ awards - hahaha
farkin hell - what next?
Corvette = "Lying Award"
OuttLaw = "Quiet Award" or "Fake account Award"
Ndoor = "Jandaro Is a good player Award"
BORN-TO-GAME = "eyy Buddy Award"
xPrince_Mvpx = "No one wants to mess with me in real life Award"
Rara20grand1 = "Commmmme onnn Maynnne Award"
haha you and your little lying ass Corvette.
I never get a chance to talk with you two in the session lol
And we all know I only have this one account
King: "SoCalVal award"
Haha Wooow OuttLaw, I should have gave you the lying award haha Lmao
M1 has thought of an award for cassidy........well actually two!!!!
you have two choices, a bit like the blue or red pill in the matrix films,where neo has to choose....... haaaaa, lol.. so what you going choose?
award one: 'bad connection' award
award two: 'Lagged out' award
haaaa, only joking cassidy. hope you get everything sorted out soon dude... :)
I get the "Peace-Keeper award"
alot of people need the 'im not socalvalerie' award
Yep and we both know who should get the
I AM SoCalValerie Award don't we crome
what about most bias website award? site owner who should step up or step aside award? a blow out here as well.
the nobody gives a $h!T award
speaking of those awards, (notice this stays on topic) there should be an award for best thread (not really just keeping it on topic) i think we should all make a thread and go thru the threads on here and quote all the bias b.s by the admin's and owner. they're full of it, so why not show it? thatd probably def be longer than my old fb thread. its funny coming back on here and reading this stuff, makes me remember why i stopped coming here. i guess if we all take the owners advice tho and go to another site thats not bias where nothing gets deleted, the problem will be solved right? i think this logic deserves an award. (not really. but lets keep it on topic so the jabroni's out there cant taint it)
i second that shit bro
It was down at the end of jabroni drive, rdr.us ....lol if ya smell!!!!! what the stringz is cooking.....lol (i love the rock)
Separate names with a comma.


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Create relevant, fun yoga classes to engage your students and promote health, wellbeing, and academic success. In this workshop, you will learn the physical, mental, emotional, and social benefits of yoga for children ages 3-12 in order to develop age-appropriate lesson plans centered around central themes and concepts, as well as colorful yoga stories. You'll finish the course ready to sequence yoga poses to meet your students' needs , all the while adhering to National Standards for Physical Education.
Bonus! Download a free copy of the Yoga Ed. Lesson Planning Manual by enrolling in this course. This e-book details all the components of lesson planning covered in this workshop, comes complete with diagrams to facilitate learning.Recommended Background: No teaching experience necessary. Anyone interested in designing kids yoga lessons of their own is welcome to take this course.
Brynne Caleda, M.Ed., E-RYT is a leader in the field of yoga education. As CEO of Yoga Ed., Brynne champions the efficacy of innovative, evidence-based yoga programs for schools to establish lifelong foundations for student's fitness, wellness, and productivity. Over the past decade, Brynne has worked closely with educators, health professionals, and parents to enrich school communities with yoga tools to more authentically and effectively teach to their students and children. A pioneer of yoga education in Hawaii, Brynne is also the founder of Stretch Your Imagination, a non-profit that creates sustainable yoga programs for Hawaii's schools. When not at home in paradise with her two year old son, you can find Brynne leading trainings and empowering educators to integrate yoga into schools across Europe, Asia, and the United States.