Efficient DNA Transfer Research Articles

Enhancing In Vivo Electroporation Efficiency through Hyaluronidase: Insights into Plasmid Distribution and Optimization Strategies.

Electroporation (EP) stands out as a promising non-viral plasmid delivery strategy, although achieving optimal transfection efficiency in vivo remains a challenge. A noteworthy advancement in the field of in vivo EP is the application of hyaluronidase, an enzyme with the capacity to degrade hyaluronic acid in the extracellular matrix, which thereby enhances DNA transfer efficiency by 2- to 3-fold. This paper focuses on elucidating the mechanism of hyaluronidase's impact on transfection efficiency. We demonstrate that hyaluronidase promotes a more uniform distribution of plasmid DNA (pDNA) within skeletal muscle. Additionally, our study investigates the effect of the timing of hyaluronidase pretreatment on EP efficiency by including time intervals of 0, 5, and 30 min between hyaluronidase treatment and the application of pulses. Serum levels of the pDNA-encoded transgene reveal a minimal influence of the hyaluronidase pretreatment time on the final serum protein levels following delivery in both mice and rabbit models. Leveraging bioimpedance measurements, we capture morphological changes in muscle induced by hyaluronidase treatment, which result in a varied pDNA distribution. Subsequently, these findings are employed to optimize EP electrical parameters following hyaluronidase treatment in animal models. This paper offers novel insights into the potential of hyaluronidase in enhancing the effectiveness of in vivo EP, as well as guides optimized electroporation strategies following hyaluronidase use.

A toolbox for genetic manipulation in intestinal Clostridium symbiosum

Gut microbes are closely related with human health, but remain much to learn. Clostridium symbiosum is a conditionally pathogenic human gut bacterium and regarded as a potential biomarker for early diagnosis of intestinal tumors. However, the absence of an efficient toolbox that allows diverse genetic manipulations of this bacterium limits its in-depth studies. Here, we obtained the complete genome sequence of C. symbiosum ATCC 14940, a representative strain of C. symbiosum. On this basis, we further developed a series of genetic manipulation methods for this bacterium. Firstly, following the identification of a functional replicon pBP1 in C. symbiosum ATCC 14940, a highly efficient conjugative DNA transfer method was established, enabling the rapid introduction of exogenous plasmids into cells. Next, we constructed a dual-plasmid CRISPR/Cas12a system for genome editing in this bacterium, reaching over 60 % repression for most of the chosen genes as well as efficient deletion (>90 %) of three target genes. Finally, this toolbox was used for the identification of crucial functional genes, involving growth, synthesis of important metabolites, and virulence of C. symbiosum ATCC 14940. Our work has effectively established and optimized genome editing methods in intestinal C. symbiosum, thereby providing strong support for further basic and application research in this bacterium.

Identification of the lipodepsipeptide selethramide encoded in a giant nonribosomal peptide synthetase from a Burkholderia bacterium

Bacterial natural products have found many important industrial applications. Yet traditional discovery pipelines often prioritize individual natural product families despite the presence of multiple natural product biosynthetic gene clusters in each bacterial genome. Systematic characterization of talented strains is a means to expand the known natural product space. Here, we report genomics, epigenomics, and metabolomics studies of Burkholderia sp. FERM BP-3421, a soil isolate and known producer of antitumor spliceostatins. Its genome is composed of two chromosomes and two plasmids encoding at least 29 natural product families. Metabolomics studies showed that FERM BP-3421 also produces antifungal aminopyrrolnitrin and approved anticancer romidepsin. From the orphan metabolome features, we connected a lipopeptide of 1,928 Da to an 18-module nonribosomal peptide synthetase encoded as a single gene in chromosome 1. Isolation and structure elucidation led to the identification of selethramide which contains a repeating pattern of serine and leucine and is cyclized at the side chain oxygen of the one threonine residue at position 13. A (R)-3-hydroxybutyric acid moiety decorates the N-terminal serine. Initial attempts to obtain deletion mutants to probe the role of selethramide failed. After acquiring epigenome (methylome) data for FERM BP-3421, we employed a mimicry by methylation strategy that improved DNA transfer efficiency. Mutants defective in selethramide biosynthesis showed reduced surfactant activity and impaired swarming motility that could be chemically complemented with selethramide. This work unveils a lipopeptide that promotes surface motility, establishes improved DNA transfer efficiency, and sets the stage for continued natural product identification from a prolific strain.

The Interaction of the F-Like Plasmid-Encoded TraN Isoforms with Their Cognate Outer Membrane Receptors.

Horizontal gene transfer via conjugation plays a major role in bacterial evolution. In F-like plasmids, efficient DNA transfer is mediated by close association between donor and recipient bacteria. This process, known as mating pair stabilization (MPS), is mediated by interactions between the plasmid-encoded outer membrane (OM) protein TraN in the donor and chromosomally-encoded OM proteins in the recipient. We have recently reported the existence of 7 TraN sequence types, which are grouped into 4 structural types, that we named TraNα, TraNβ, TraNγ, and TraNδ. Moreover, we have shown specific pairing between TraNα and OmpW, TraNβ and OmpK36 of Klebsiella pneumoniae, TraNγ and OmpA, and TraNδ and OmpF. In this study, we found that, although structurally similar, TraNα encoded by the Salmonella enterica pSLT plasmid (TraNα2) binds OmpW in both Escherichia coli and Citrobacter rodentium, while TraNα encoded by the R100-1 plasmid (TraNα1) only binds OmpW in E. coli. AlphaFold2 predictions suggested that this specificity is mediated by a single amino acid difference in loop 3 of OmpW, which we confirmed experimentally. Moreover, we show that single amino acids insertions into loop 3 of OmpK36 affect TraNβ-mediated conjugation efficiency of the K. pneumoniae resistance plasmid pKpQIL. Lastly, we report that TraNβ can also mediate MPS by binding OmpK35, making it the first TraN variant that can bind more than one OM protein in the recipient. Together, these data show that subtle sequence differences in the OM receptors can impact TraN-mediated conjugation efficiency. IMPORTANCE Conjugation plays a central role in the spread of antimicrobial resistance genes among bacterial pathogens. Efficient conjugation is mediated by formation of mating pairs via a pilus, followed by mating pair stabilization (MPS), mediated by tight interactions between the plasmid-encoded outer membrane protein (OMP) TraN in the donor (of which there are 7 sequence types grouped into the 4 structural isoforms α, β, γ, and δ), and an OMP receptor in the recipient (OmpW, OmpK36, OmpA, and OmpF, respectively). In this study, we found that subtle differences in OmpW and OmpK36 have significant consequences on conjugation efficiency and specificity, highlighting the existence of selective pressure affecting plasmid-host compatibility and the flow of horizontal gene transfer in bacteria.

Biological role of the major AP (abasic site) endonuclease of an archaeon from geothermal environments.

Archaea and bacteria in geothermal environments are predicted to suffer DNA depurination in vivo at high rates, which raises questions regarding the biological roles of their abasic-site-repair enzymes. Gene deletion and enzymatic assay demonstrated that the saci_0015 gene of Sulfolobus acidocaldarius encodes an AP endonuclease (Apn) accounting for as much as 95% of the assayable activity in cell extracts and is not essential for viability. To identify genetic functions of this enzyme, deletion (ΔApn) strains were examined with respect to growth, spontaneous mutation, transformation by ssDNA containing an abasic site, and conjugation. Relative to its isogenic control, the ΔApn strain did not exhibit any change in growth rate or final cell density, rate or spectrum of spontaneous mutation, transformation by DNA containing an abasic site, or efficiency of DNA transfer and recombination. The apparent lack of genetic impact of removing the major AP endonuclease was unexpected and indicated that abasic sites are rarely bypassed directly by DNA polymerases in S. acidocaldarius. AP endonuclease deficiency had no obvious effect on survival of S. acidocaldarius under several test conditions, but it accelerated the death of cells at 4º C under illumination. Our results suggest that the normal level of AP endonuclease in S. acidocaldarius is well above the minimum required for growth and cell division but not for recovery from prolonged exposure to certain low-temperature conditions. This situation illustrates a biological challenge that has not been emphasized in experimental studies of extremophiles, i.e., the problem of long-term survival under "non-extreme" conditions.

Engineering Agrobacterium tumefaciens Adhesion to Target Cells.

Agrobacterium tumefaciens is a plant pathogen commonly repurposed for genetic modification of crops. Despite its versatility, it remains inefficient at transferring DNA to many hosts, including to animal cells. Like many pathogens, physical contact between A. tumefaciens and host cells promotes infection efficacy. Thus, improving the strength and specificity of A. tumefaciens to target cells has the potential for enhancing DNA transfer for biotechnological and therapeutic purposes. Here, we demonstrate a methodology for engineering genetically encoded exogeneous adhesins at the surface of A. tumefaciens. We identified an autotransporter gene we named Aat that is predicted to show canonical β-barrel and passenger domains. We engineered the β-barrel scaffold and linker (Aatβ) to display synthetic adhesins susceptible to rewire A. tumefaciens to alternative host targets. As a proof of concept, we leveraged the versatility of a VHH domain to rewire A. tumefaciens adhesion to yeast and mammalian hosts displaying a GFP target receptor. Finally, to demonstrate how synthetic A. tumefaciens adhesion can improve transfer to host cells, we showed improved protein translocation into HeLa cells using a sensitive split luciferase reporter system. Engineering A. tumefaciens adhesion has therefore a strong potential in generating complex heterogeneous cellular assemblies and in improving DNA transfer efficiency against non-natural hosts.

Mating pair stabilization mediates bacterial conjugation species specificity

Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane (OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNA transfer. We show that TraN from the plasmid pKpQIL (Klebsiella pneumoniae) interacts with OmpK36, plasmids from R100-1 (Shigella flexneri) and pSLT (Salmonella Typhimurium) interact with OmpW, and the prototypical F plasmid (Escherichia coli) interacts with OmpA. Cryo-EM analysis revealed that TraNpKpQIL interacts with OmpK36 through the insertion of a β-hairpin in the tip of TraN into a monomer of the OmpK36 porin trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified a fourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classification scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNβ (OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect the distribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pair stabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emerging resistance plasmids in high-risk bacterial pathogens.

Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum.

ABSTRACTThe majority of the genes present in bacterial genomes remain poorly characterized, with up to one-third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to those species in which high rates of DNA transfer can be achieved. Here, we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum by using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as the sole carbon and energy source.IMPORTANCE Although microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterized, leaving the function of many as either hypothetical or entirely unknown. High-throughput transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here, we developed approaches to facilitate transposon insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases CO and CO2 into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth, providing insights into the utilization of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon insertion sequencing to other microbes where DNA transfer has until now represented a barrier to progress.

An Electrochemical Protocol for CRISPR-Mediated Gene-Editing of Sheep Embryonic Fibroblast Cells

Genetic engineering of farm animals is commonly carried out via cell-mediated transfection followed by somatic cell nuclear transfer. However, efficient transfer of exogenous DNA into ovine embryonic fibroblast (EF) cells without compromising cell viability has remained a challenging issue. Here, we aimed to develop a protocol for electrotransfection of sheep EF cells. First, we optimized the pulsing condition using an OptiMEM-GlutaMAX medium as the electroporation buffer and found 2 pulses of 270 V, each for 10 ms and 10 s interval, is the most efficient condition to have a high rate of transfection and cell survival. Moreover, supplementing 3% dimethyl sulfoxide (DMSO) into the electroporation medium considerably improved the cell viability after the electroporation process. The electroporation procedure resulted in >98% transfection efficiency and >97% cell survival rate using reporter plasmids. Finally, using CRISPR/Cas9-encoding vectors, we targeted BMP15 and GDF9 genes in sheep EF cells. The electroporated cells are associated with a 52% indels rate using single gRNAs as well as a highly efficient target deletion using 2 gRNAs. In conclusion, we have developed an electrotransfection protocol using the OptiMEM-GlutaMAX medium supplemented with 3% DMSO for sheep EF cells. The electroporation method can be used for cell-mediated gene-editing in sheep.

Molecular Mechanisms Influencing Bacterial Conjugation in the Intestinal Microbiota.

Bacterial conjugation is a widespread and particularly efficient strategy to horizontally disseminate genes in microbial populations. With a rich and dense population of microorganisms, the intestinal microbiota is often considered a fertile environment for conjugative transfer and a major reservoir of antibiotic resistance genes. In this mini-review, we summarize recent findings suggesting that few conjugative plasmid families present in Enterobacteriaceae transfer at high rates in the gut microbiota. We discuss the importance of mating pair stabilization as well as additional factors influencing DNA transfer efficiency and conjugative host range in this environment. Finally, we examine the potential repurposing of bacterial conjugation for microbiome editing.

A molecular view on the escape of lipoplexed DNA from the endosome.

The use of non-viral vectors for in vivo gene therapy could drastically increase safety, whilst reducing the cost of preparing the vectors. A promising approach to non-viral vectors makes use of DNA/cationic liposome complexes (lipoplexes) to deliver the genetic material. Here we use coarse-grained molecular dynamics simulations to investigate the molecular mechanism underlying efficient DNA transfer from lipoplexes. Our computational fusion experiments of lipoplexes with endosomal membrane models show two distinct modes of transfection: parallel and perpendicular. In the parallel fusion pathway, DNA aligns with the membrane surface, showing very quick release of genetic material shortly after the initial fusion pore is formed. The perpendicular pathway also leads to transfection, but release is slower. We further show that the composition and size of the lipoplex, as well as the lipid composition of the endosomal membrane, have a significant impact on fusion efficiency in our models.

Two pKM101-encoded proteins, the pilus-tip protein TraC and Pep, assemble on the Escherichia coli cell surface as adhesins required for efficient conjugative DNA transfer.

Mobile genetic elements (MGEs) encode type IV secretion systems (T4SSs) known as conjugation machines for their transmission between bacterial cells. Conjugation machines are composed of an envelope-spanning translocation channel, and those functioning in Gram-negative species additionally elaborate an extracellular pilus to initiate donor-recipient cell contacts. We report that pKM101, a self-transmissible MGE functioning in the Enterobacteriaceae, has evolved a second target cell attachment mechanism. Two pKM101-encoded proteins, the pilus-tip adhesin TraC and a protein termed Pep, are exported to the cell surface where they interact and also form higher order complexes appearing as distinct foci or patches around the cell envelope. Surface-displayed TraC and Pep are required for an efficient conjugative transfer, 'extracellular complementation' potentially involving intercellular protein transfer, and activation of a Pseudomonas aeruginosa type VI secretion system. Both proteins are also required for bacteriophage PRD1 infection. TraC and Pep are exported across the outer membrane by a mechanism potentially involving the β-barrel assembly machinery. The pKM101 T4SS, thus, deploys alternative routing pathways for the delivery of TraC to the pilus tip or both TraC and Pep to the cell surface. We propose that T4SS-encoded, pilus-independent attachment mechanisms maximize the probability of MGE propagation and might be widespread among this translocation superfamily.

Crystal structure of TcpK in complex with oriT DNA of the antibiotic resistance plasmid pCW3

Conjugation is fundamental for the acquisition of new genetic traits and the development of antibiotic resistance in pathogenic organisms. Here, we show that a hypothetical Clostridium perfringens protein, TcpK, which is encoded by the tetracycline resistance plasmid pCW3, is essential for efficient conjugative DNA transfer. Our studies reveal that TcpK is a member of the winged helix-turn-helix (wHTH) transcription factor superfamily and that it forms a dimer in solution. Furthermore, TcpK specifically binds to a nine-nucleotide sequence that is present as tandem repeats within the pCW3 origin of transfer (oriT). The X-ray crystal structure of the TcpK–TcpK box complex reveals a binding mode centered on and around the β-wing, which is different from what has been previously shown for other wHTH proteins. Structure-guided mutagenesis experiments validate the specific interaction between TcpK and the DNA molecule. Additional studies highlight that the TcpK dimer is important for specific DNA binding.

Engineered integrative and conjugative elements for efficient and inducible DNA transfer to undomesticated bacteria.

Engineering microorganisms to promote human or plant health will require manipulation of robust bacteria that are capable of surviving in harsh, competitive environments. Genetic engineering of undomesticated bacteria can be limited by an inability to transfer DNA into the cell. Here we developed an approach based on the integrative and conjugative element from Bacillus subtilis (ICEBs1) to overcome this problem. A donor strain (XPORT) was built to transfer miniaturized integrative and conjugative elements (mini-ICEBs1) to undomesticated bacteria. The strain was engineered to enable inducible control over conjugation, to integrate delivered DNA into the chromosome of the recipient, to restrict spread of heterologous DNA through separation of the type IV secretion system from the transferred DNA, and to enable simple isolation of engineered bacteria through a D-alanine auxotrophy. Efficient DNA transfer (10-1 to 10-7 conjugation events per donor) is demonstrated using 35 Gram-positive strains isolated from humans (skin and gut) and soil. Mini-ICEBs1 was used to rapidly characterize the performance of an isopropyl-β-D-thiogalactoside (IPTG)-inducible reporter across dozens of strains and to transfer nitrogen fixation to four Bacillus species. Finally, XPORT was introduced to soil to demonstrate DNA transfer under non-ideal conditions.

Peyer's Patches as a Portal for DNA Delivery by Lactococcus lactis in Vivo.

Application of food-grade Lactococcus lactis (L. lactis) as a safe delivery tool for DNA vaccines and therapeutic proteins has been well investigated. Although some studies showed that eukaryotic expression plasmids were transferred from L. lactis to enterocytes, the precise mechanism of the DNA transfer remains unknown. In this study, we generated an invasive L. lactis strain that expresses "murinized" Internalin A, an invasin of intracellular bacteria Listeria monocytogenes with two amino acid alterations for invasion into murine cells, and confirmed that this L. lactis strain delivered DNA in an invasin-dependent manner into a monolayer of epithelial cells polarized to mimic the gastrointestinal tract environment. Although invasive L. lactis inoculated orally can deliver DNA into enterocytes in the gastrointestinal tract of mice, the efficiency of DNA transfer was similar to that of non-invasive L. lactis strain, suggesting that the in vivo DNA transfer from L. lactis occurs invasin-independently. A ligated-intestinal loop assay, a method for a short-term culturing of the whole intestine filled with materials to evaluate the interaction of the materials with intestinal cells, demonstrated that both non-invasive and invasive L. lactis strains were present in the Peyer's patches of the small intestine. On the other hand, few L. lactis was detected in the non-Peyer's patch epithelial region. Thus, our observations lead us to speculate that DNA transfer from L. lactis occurs predominantly in the Peyer's patches in an invasin-independent manner.

Development of an efficient conjugal DNA transfer system between Escherichia coli and a non-sporulating Streptomyces strain

Bacterial conjugation is a powerful tool used for DNA transfer from Escherichia coli into various bacteria including streptomycetes. In this methodology, spores are usually employed as recipient cells of the genetic information. However, some industrially important Streptomyces do not produce spores making difficult their genetic manipulation. In these strains, the use of mechanically fragmented mycelia allows DNA transfer with low efficiency. Streptomyces peucetius var. caesius is a non-sporulating bacteria which produces the antitumor compound doxorubicin. The use of aerial mycelia of this microorganism, failed to get intergeneric conjugation with E. coli. In the present work, by using young aerial mycelia of this microorganism and an excess of E. coli cells (~7×108cellsmL−1) in soybean-mannitol medium (MS) supplemented with 20mMMgCl2 resulted in a high number of exconjugant colonies (5×10−4) when compared to other reports from this genus (1.1×10−5 to 2.5×10−8). The effectiveness of these conditions was confirmed by isolating null mutants of two different glucokinases from S. peucetius var. caesius. The novelty in using young aerial mycelia as receptor cells, allowed an efficient conjugative process and opened the way for genetic manipulation of additional non-spore forming actinobacteria exhibiting natural resistance to be genetically manipulated.

Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from Escherichia coli to Bacillus subtilis.

The ability to efficiently and reliably transfer genetic circuits between the key synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the major hurdles of the rational genome engineering. Using lambda Red recombineering we integrated the thermosensitive lambda repressor and the lysis genes of several bacteriophages into the E. coli chromosome. The lysis of the engineered autolytic cells is inducible by a simple temperature shift. We improved the lysis efficiency by introducing different combinations of lysis genes from bacteriophages lambda, ΦX174 and MS2 under the control of the thermosensitive lambda repressor into the E. coli chromosome. We tested the engineered autolytic cells by transferring plasmid and bacterial artificial chromosome (BAC)-borne genetic circuits from E. coli to B. subtilis. Our engineered system combines benefits of the two main synthetic biology chassis, E. coli and B. subtilis, and allows reliable and efficient transfer of DNA edited in E. coli into B. subtilis.

Lessons from genome skimming of arthropod-preserving ethanol.

Field-collected specimens of invertebrates are regularly killed and preserved in ethanol, prior to DNA extraction from the specimens, while the ethanol fraction is usually discarded. However, DNA may be released from the specimens into the ethanol, which can potentially be exploited to study species diversity in the sample without the need for DNA extraction from tissue. We used shallow shotgun sequencing of the total DNA to characterize the preservative ethanol from two pools of insects (from a freshwater habitat and terrestrial habitat) to evaluate the efficiency of DNA transfer from the specimens to the ethanol. In parallel, the specimens themselves were subjected to bulk DNA extraction and shotgun sequencing, followed by assembly of mitochondrial genomes for 39 of 40 species in the two pools. Shotgun sequencing from the ethanol fraction and read-matching to the mitogenomes detected ~40% of the arthropod species in the ethanol, confirming the transfer of DNA whose quantity was correlated to the biomass of specimens. The comparison of diversity profiles of microbiota in specimen and ethanol samples showed that 'closed association' (internal tissue) bacterial species tend to be more abundant in DNA extracted from the specimens, while 'open association' symbionts were enriched in the preservative fluid. The vomiting reflex of many insects also ensures that gut content is released into the ethanol, which provides easy access to DNA from prey items. Shotgun sequencing of DNA from preservative ethanol provides novel opportunities for characterizing the functional or ecological components of an ecosystem and their trophic interactions.

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae.

Shuttle vectors allow for an efficient transfer of recombinant DNA into yeast cells and are widely used in fundamental research and biotechnology. While available shuttle vectors are applicable in many experimental settings, their use in quantitative biology is hampered by insufficient copy number control. Moreover, they often have practical constraints, such as limited modularity and few unique restriction sites. We constructed the pRG shuttle vector series, consisting of single- and multi-copy integrative, centromeric and episomal plasmids with marker genes for the selection in all commonly used auxotrophic yeast strains. The vectors feature a modular design and a large number of unique restriction sites, enabling an efficient exchange of every vector part and expansion of the series. Integration into the host genome is achieved using a double-crossover recombination mechanism, resulting in stable single- and multi-copy modifications. As centromeric and episomal plasmids give rise to a heterogeneous cell population, an analysis of their copy number distribution and loss behaviour was performed. Overall, the shuttle vector series supports the efficient cloning of genes and their maintenance in yeast cells with improved copy number control.

Structures of malonic acid diamide/phospholipid composites and their lipoplexes.

As a continuation of previous work, the self-assembly process of cationic lipid formulations in the presence and absence of DNA was investigated with respect to binary lipid mixtures suitable as polynucleotide carrier systems. The lipid blends consist of one malonic-acid-based cationic lipid with a varying alkyl chain pattern, either N-{6-amino-1-[N-(9Z)-octadec-9-enylamino]-1-oxohexan-(2S)-2-yl}-N'-{2-[N,N-bis(2-aminoethyl)amino]ethyl}-2-hexadecylpropandiamide () or N-[6-amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-N'-{2-[N,N-bis(2-aminoethyl)amino]ethyl}-2-hexadecylpropandiamide (), and one neutral co-lipid, either 1,2-di-[(9Z)-octadec-9-enoyl]-sn-glycero-3-phosphocholine (DOPE) or 1,2-di-(hexadecanoyl)-sn-glycero-3-phosphocholine (DPPC). Although the cationic lipids exhibit only slight differences in their structure, the DNA transfer efficiency varies drastically. Therefore, self-assembly was studied in 3D systems by small- and wide-angle X-ray scattering (SAXS and WAXS) and transmission electron microscopy (TEM) as well as in 2D systems by infrared reflection-absorption spectroscopy (IRRAS) on Langmuir films. The investigated lipid mixtures show quite different self-assembly in the absence of DNA, with varying structures from vesicles (/DOPE; /DOPE) and tubes (/DOPE) to discoid structures (/DPPC; /DPPC). Twisted ribbons and sheets, which were stabilized due to hydrogen-bond networks, were found in all investigated lipid mixtures in the absence of DNA. The addition of DNA leads to the formation of lamellar lipoplexes for all the investigated lipid compositions. The lipoplexes differ in crucial parameters, such as the lamellar repeat distance and the spacing between the DNA strands, indicating differences in the binding strength between DNA and the lipid composition. The formation of associates with an ideal charge density might emerge as a key parameter for efficient DNA transfer. Furthermore, the structures observed for the different lipid compositions in the absence of DNA prepare the way for other applications besides gene therapy.