Application of an electro elution system for direct purification of linear covalently closed DNA fragments.

Gene therapy is a powerful treatment modality. Non-viral gene therapy vectors power one arm of this important approach, due to their enhanced safety profile compared to their viral counterparts. New non-viral approaches continue to be developed, but purification can bottleneck the scaleup and cost-effectiveness and quality of some of these advanced vectors. We require more advanced purification and separation techniques compared to conventional methods to maximize resolution in a scalable manner. The Prep Cell system is a continuous electro elution system that contains a circular gel casting tube where DNA mixtures can be run through and subsequently migrate into an elution chamber, to be eluted by a peristaltic pump. This DNA separation and purification process confers advantages over other conventional methods, including i) the elimination of multiple downstream purification process requirements; ii) its ability to be applied in mid-scale settings, and iii), its high-resolution power. In this study, we assessed the ability of this Prep Cell Model 491 system to purify a novel type of non-viral linear covalently closed (LCC) DNA minivector (ministring DNA) from its precursor parent plasmid DNA and process by-product DNA species by analyzing for effective separation via agarose gel electrophoresis, recovery yield, single enzyme digestion, and quality control assessments. Overall, effective separation and resolution of mini-DNA vectors was obtained using the Prep Cell system, conferring its potential to be applied towards mid-scale purification of DNA vectors for a variety of research, and eventually, clinical applications.

Treating cancer with infection: a review on bacterial cancer therapy.

There is an increasing need for new cancer therapies. The antitumour effect of bacterial infection has been well observed and practiced throughout history. Bacteria are well-suited to serve as anticancer agents due to their intrinsic mobility, cell toxicity, immunogenicity, and preferential accumulation within the anoxic tumour environment. Furthermore, advances in biotechnology and molecular techniques have made it easier than ever to engineer bacteria as both therapeutic agents themselves and as therapeutic vectors. Here, we review bacteriolytic therapy and immunotherapy strategies, and examine the development of bacteria as vehicles for cell- and tissue-targeted delivery of genetic cancer therapeutics.

Stationary phase-like properties of the bacteriophage lambda Rex exclusion phenotype.

The rex genes of bacteriophage lambda were found to protect lysogenic Escherichia coliK host cells against killing by phage T4 rII, when compared in parallel to isogenic Rex(-) lysogens and nonlysogens. This protective effect was abrogated upon mutation of the host stationary-phase sigma factor RpoS. Rex(+) lysogens infected by T4 rII contracted, formed aggregates and shed flagella, thus resembling cells entering stationary phase. These phenotypes were accentuated in nonlysogenic cells carrying multicopy plasmids expressing rexA-rexB: cells were about two-fold contracted in length, expressed membrane-bound and detached flagella, were insensitive to infection by a variety of phages and clumped extensively; in addition, cultures of these cells were odorous. Our observations support the hypothesis that the Rex system can cause a stationary-phase-like response that protects the host against infection by T4 rII.

Acquired mutations in phage lambda genes O or P that enable constitutive expression of a cryptic lambdaN+cI[Ts]cro- prophage in E. coli cells shifted from 30 degreesC to 42 degreesC, accompanied by loss of immlambda and Rex+ phenotypes and emergence of a non-immune exclusion-state.

The majority of bacteria, which carry the N+-cI857[Ts]-cro--O+-P+ fragment of lambda genome, are killed when derepressed by shifting from 30 degreesC to 42 degreesC. Among rare survivors, we observed a proportion of colony-forming units (cfu) that retained the typical immlambda-immunity phenotype when grown at 30 degreesC; however, when shifted from 30 degreesC to 42 degreesC, they lost lambda immunity and acquired a non-immune exclusion-state (Nie phenotype). We also found that the immlambda survivor cfu quickly lost their Rex+ exclusion phenotype (as measured by T4rII plating inhibition) when shifted from 30 degreesC to 42 degreesC, even though they produced CII, which stimulates pE-cI-rexA-rexB transcription. The Nie phenotype was characterized by an inhibition of plating of the homoimmune phage, lambdawt, and the heteroimmune phage, lambdaimm434. However, lambdavir and spontaneous mutants of lambdawt (lambdase mutations localized within oR) escaped the Nie exclusion-state and plated efficiently on lawns of Nie cfu at 42 degreesC. Thus, we examined the scope of the Nie exclusion-state toward lambda mutants blocked for lysogeny, and lambda hybrids substituted for immunity or replication genes. Phage like lambdawt, competent for lysogeny, were severely excluded compared to some mutants of lambda defective for lysogeny. Among this latter type, there was high variance in the Nie exclusion of various cI mutants; some of which were not excluded. The Nie exclusion-state was attributed to the constitutive expression of the defective lambda fragment in the survivor cfu, made possible by the acquired replication defect(s). We characterized, both genetically and physically, the mutations in the defective integrated lambda prophage that permitted growth of the survivor cfu at 42 degreesC. In five of seven survivor cfu, we identified IS2 insertions within lambda genes O and P that can block replication initiation from the lambda fragment. The remaining survivor cfu had multiple base substitutions within the C-terminal end of O and N-terminal half of P, the majority of which were silent. In some of these mutants, either an ochre nonsense mutation or a single-base frameshift deletion inactivated P.