Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome

<p>Abstract</p> <p>Background</p> <p>A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into <it>Salmonella enteritidis </it>chromosome.</p> <p>Results</p> <p>The final product of this mutation strategy is the insertion of DNA encoding a foreign epitope into the <it>S. enteritidis </it>genome without the addition of any unwanted sequence. This experiment was performed by a two-step mutation process via PCR fragments, Red recombinase and counter-selection with the I-SceI enzyme site. First, the I-SceI site and kanamycin resistance gene were introduced into the genome of cells expressing Red recombinase enzymes. Next, this sequence was replaced by a chosen insertion sequence. DNA fragments used for recombination were linear PCR products which consisted of the foreign insertion sequence flanked by homologous sequences of the target gene. Described herein is the insertion of a section of the M2e epitope (LM2) of Influenza A virus, a domain of CD154 (CD154s) or a combination of both into the outer membrane protein LamB of <it>S. enteritidis</it>.</p> <p>Conclusion</p> <p>We have successfully used this method to produce multiple mutants with no antibiotic gene on the genome or extra sequence except those nucleotides required for expression of epitope regions. This method is advantageous over other protocols in that it does not require cloning or creating extra duplicate regions to facilitate homologous recombination, contains a universal construct in which an epitope of choice can be placed to check for cell surface expression, and shows high efficiency when screening for positive mutants. Other opportunities of this mutational strategy include creating attenuated mutants and site-specific, chromosomal deletion mutations. Furthermore, this method should be applicable in other gram-negative bacterial species where Red recombinase enzymes can be functionally expressed.</p

Compositions and methods of enhancing immune responses to Eimeria

Vaccines comprising TRAP polypeptides and Salmonella enteritidis vectors comprising TRAP polypeptides are provided. The vaccines may also include a CD154 polypeptide capable of binding to CD40. Also provided are methods of enhancing an immune response against Apicomplexan parasites and methods of reducing morbidity associated with infection with Apicomplexan parasites

Compositions and methods of enhancing immune responses to Eimeria

Vaccines comprising TRAP polypeptides and Salmonella enteritidis vectors comprising TRAP polypeptides are provided. The vaccines may also include a CD154 polypeptide capable of binding to CD40. Also provided are methods of enhancing an immune response against Apicomplexan parasites and methods of reducing morbidity associated with infection with Apicomplexan parasites

Using the Game of Mastermind to Teach, Practice, and Discuss Scientific Reasoning Skills

The code-breaking game Mastermind, which can be played in minutes at no cost, creates opportunities for students to discuss scientific reasoning, hypothesis-testing, effective experimental design, and sound interpretation of results

Exclusionary employment in Britain’s broken labour market

There is growing evidence of the problematic nature of the UK’s ‘flexible labour market’ with rising levels of in-work poverty and insecurity. Yet successive Governments have stressed that paid work is the route to inclusion, focussing attention on the divide between employed and unemployed. Past efforts to measure social exclusion have tended to make the same distinction. The aim of this paper is to apply Levitas et al’s (2007) framework to assess levels of exclusionary employment, i.e. exclusion arising directly from an individual’s labour market situation. Using data from the Poverty and Social Exclusion UK survey, results show that one in three adults in paid work is in poverty, or in insecure or poor quality employment. One third of this group have not seen any progression in their labour market situation in the last five years. The policy focus needs to shift from ‘Broken Britain’ to Britain’s broken labour market

Compositions and methods of enhancing immune responses to Eimeria

Vaccines comprising TRAP polypeptides and Salmonella enteritidis vectors comprising TRAP polypeptides are provided. The vaccines may also include a CD154 polypeptide capable of binding to CD40. Methods of enhancing an immune response against Apicomplexan parasites and methods of reducing morbidity associated with infection with Apicomplexan parasites are also provided

Social exclusion of older persons: a scoping review and conceptual framework

As a concept, social exclusion has considerable potential to explain and respond to disadvantage in later life. However, in the context of ageing populations, the construct remains ambiguous. A disjointed evidence-base, spread across disparate disciplines, compounds the challenge of developing a coherent understanding of exclusion in older age. This article addresses this research deficit by presenting the findings of a two-stage scoping review encompassing seven separate reviews of the international literature pertaining to old-age social exclusion. Stage one involved a review of conceptual frameworks on old-age exclusion, identifying conceptual understandings and key domains of later-life exclusion. Stage two involved scoping reviews on each domain (six in all). Stage one identified six conceptual frameworks on old-age exclusion and six common domains across these frameworks: neighbourhood and community; services, amenities and mobility; social relations; material and financial resources; socio-cultural aspects; and civic participation. International literature concentrated on the first four domains, but indicated a general lack of research knowledge and of theoretical development. Drawing on all seven scoping reviews and a knowledge synthesis, the article presents a new definition and conceptual framework relating to old-age exclusion