Plasmid Amplification Research Articles

Amplified DNA heterogeneity assessment with Oxford Nanopore sequencing applied to cell free expression templates.

In this work, Oxford Nanopore sequencing is tested as an accessible method for quantifying heterogeneity of amplified DNA. This method enables rapid quantification of deletions, insertions, and substitutions, the probability of each mutation error, and their locations in the replicated sequences. Amplification techniques tested were conventional polymerase chain reaction (PCR) with varying levels of polymerase fidelity (OneTaq, Phusion, and Q5) as well as rolling circle amplification (RCA) with Phi29 polymerase. Plasmid amplification using bacteria was also assessed. By analyzing the distribution of errors in a large set of sequences for each sample, we examined the heterogeneity and mode of errors in each sample. This analysis revealed that Q5 and Phusion polymerases exhibited the lowest error rates observed in the amplified DNA. As a secondary validation, we analyzed the emission spectra of sfGFP fluorescent proteins synthesized with amplified DNA using cell free expression. Error-prone polymerase chain reactions confirmed the dependency of reporter protein emission spectra peak broadness to DNA error rates. The presented nanopore sequencing methods serve as a roadmap to quantify the accuracy of other gene amplification techniques, as they are discovered, enabling more homogenous cell-free expression of desired proteins.

Optimizing CAR-NK Cell Transduction and Expansion: Leveraging Cytokine Modulation for Enhanced Performance.

Cellular immunotherapy has emerged as one of the most potent approaches to treating cancer patients. Adoptive transfer of chimeric antigen receptor (CAR) T cells as well as the use of haploidentical natural killer (NK) cells can induce remission in patients with lymphoma and leukemia. Although the use of CAR T cells has been established, this approach is currently limited for wider use by the risk of severe adverse events, including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. Moreover, the risk of triggering graft vs host reactions in settings of allogeneic T cell infusion limits the use to autologous CAR T cells if advanced CRISPR engineering is not applied. In contrast, NK cell-based cancer immunotherapy has emerged as a safe approach even in allogeneic settings. However, efficient transduction of primary blood NK cells with vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentivirus commonly used for T cell modification remains challenging. This article presents a detailed method that significantly enhances the transduction efficiency of NK cells by utilizing a short-term culture in cytokine-supplemented medium. It also encompasses the preparation of high-titer and high-quality lentiviral particles for optimal NK cell transduction. Overall, this protocol details the step-by-step culture of NK cells in cytokine-supplemented medium, their transduction with VSV-G lentiviral vectors, and subsequent expansion for functional assays. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of NK cells from human peripheral blood mononuclear cells (PBMCs) Basic Protocol 2: NK cell expansion and transduction with lentivirus for generating CAR-NK cells Support Protocol 1: Plasmid amplification Support Protocol 2: Lentivirus preparation Support Protocol 3: Lentivirus titration.

Amplified DNA Heterogeneity Assessment with Oxford Nanopore Sequencing Applied to Cell Free Expression Templates.

In this work, Oxford Nanopore sequencing is tested as an accessible method for quantifying heterogeneity of amplified DNA. This method enables rapid quantification of deletions, insertions, and substitutions, the probability of each mutation error, and their locations in the replicated sequences. Amplification techniques tested were conventional polymerase chain reaction (PCR) with varying levels of polymerase fidelity (OneTaq, Phusion, and Q5) as well as rolling circle amplification (RCA) with Phi29 polymerase. Plasmid amplification using bacteria was also assessed. By analyzing the distribution of errors in a large set of sequences for each sample, we examined the heterogeneity and mode of errors in each sample. This analysis revealed that Q5 and Phusion polymerases exhibited the lowest error rates observed in the amplified DNA. As a secondary validation, we analyzed the emission spectra of sfGFP fluorescent proteins synthesized with amplified DNA using cell free expression. Error-prone polymerase chain reactions confirmed the dependency of reporter protein emission spectra peak broadness to DNA error rates. The presented nanopore sequencing methods serve as a roadmap to quantify the accuracy of other gene amplification techniques, as they are discovered, enabling more homogenous cell-free expression of desired proteins.

Large T antigen mediated target gene replication improves site-specific recombination efficiency.

With advantages of high-fidelity, monoclonality and large cargo capacity, site-specific recombination (SSR) holds great promises for precise genomic modifications. However, broad applications of SSR have been hurdled by low integration efficiency, and the amount of donor DNA available in nucleus for SSR presents as a limiting factor. Inspired by the DNA replication mechanisms observed in double-stranded DNA virus SV40, we hypothesized that expression of SV40 large T antigen (TAg) can increase the copy number of the donor plasmid bearing an SV40 origin, and in consequence promote recombination events. This hypothesis was tested with dual recombinase-mediated cassette exchange (RMCE) in suspension 293F cells. Results showed that TAg co-transfection significantly enhanced SSR in polyclonal cells. In the monoclonal cell line carrying a single landing pad at an identified genomic locus, 12% RMCE efficiency was achieved, and such improvement was indeed correlated with donor plasmid amplification. The developed TAg facilitated RMCE (T-RMCE) was exploited for the construction of large libraries of >107 diversity, from which GFP variants with enhanced fluorescence were isolated. We expect the underlying principle of target gene amplification can be applicable to other SSR processes and gene editing approaches in general for directed evolution and large-scale genomic screening in mammalian cells.

A Comprehensive Study of the Effects by Sequence Truncation within Inverted Terminal Repeats (ITRs) on the Productivity, Genome Packaging, and Potency of AAV Vectors.

One of the primary challenges in working with adeno-associated virus (AAV) lies in the inherent instability of its inverted terminal repeats (ITRs), which play vital roles in AAV replication, encapsidation, and genome integration. ITRs contain a high GC content and palindromic structure, which occasionally results in truncations and mutations during plasmid amplification in bacterial cells. However, there is no thorough study on how these alterations in ITRs impact the ultimate AAV vector characteristics. To close this gap, we designed ITRs with common variations, including a single B, C, or D region deletion at one end, and dual deletions at both ends of the vector genome. These engineered ITR-carrying plasmids were utilized to generate AAV vectors in HEK293 cells. The crude and purified AAV samples were collected and analyzed for yield, capsid DNA-filled percentage, potency, and ITR integrity. The results show that a single deletion had minor impact on AAV productivity, packaging efficiency, and in vivo potency. However, deletions on both ends, except A, showed significant negative effects on the above characteristics. Our work revealed the role of ITR regions, A, B, C, and D for AAV production and DNA replication, and proposes a new strategy for the quality control of ITR-bearing plasmids and final AAV products.

Multi-enzymatic systems synergize new RCA technique amplified super-long dsDNA from DNA circle

BackgroundIn the field of DNA amplification, there are great challenges in the effectively amplify of long-chain amplification, especially amplification up to several hundred kb level. ResultsA novel technique for the unbiased whole genome amplification from a thimbleful of DNA circles, such as low as 10 ng/ 10 μL of the circular cpDNA or low as 5 ng/ 10 μL of the plasmid, is developed, which can amplify an abundance of the whole genome sequences. Specifically, the new technique that combines rolling-amplification and triple-enzyme system presents a tightly controlled process of a series of buffers/reactions and optimized procedures, that applies from the primer-template duplexes to the Elution step. The result of this technique provides a new approach for extending RCA capacity, where it can reach 200 kb from the circular cpDNA amplification and 150 kb from the plasmid DNA amplification, that demonstrates superior breadth and evenness of genome coverage, high reproducibility, small amplification bias with the amplification efficiency. Significance and noveltyThis new technique will develop into one of the powerful tools for isothermal DNA amplification in vitro, genome sequencing/analysis, phylogenetic analysis, physical mapping, and other molecular biology applications.

Determining the prevalence and genetic diversity of plasmid-mediated sulfonamide resistance in Escherichia coli from commercial broiler samples

Sulfonamides are commonly used antibacterials in commercial poultry, contributing toward the development of multidrug-resistant (MDR) phenotypes among Escherichia coli and that has emerged as global concern. The current study aimed to assess the sulfonamide resistance among isolated E. coli strains among commercial broilers. The bacterial strains were identified from fecal samples (n = 100) using selective media, followed by initial identification based on biochemical profiles. The susceptibility was determined by measuring the minimum inhibitory concentration (MIC) against sulfamethoxazole. The study also evaluated mobile genetic elements (MGEs), the mediators of antibiotic resistance, by amplification of plasmid DNA using specific primer PCR. Additionally, the isolates were subjected to multilocus sequence typing (MLST) analysis to investigate the genetic diversity among E. coli carrying sulfonamide resistance genes. The results revealed that 58% (58/100) E. coli strains were resistant to sulfonamides, with 36.20% (21/58) of the strains exhibiting an MIC breakpoint ≥512 µg/mL. PCR analysis showed that 42.85% (9/21) of the strains harbored the sul-1 gene, while 38.09% (8/21) carried the sul-2 gene, and 19.04% (4/21) had both genes. No isolate showed the presence of the sul-3 gene. Furthermore, class 1 and class 2 integrons were identified among 80.95% (17/21) and 19.04% (4/21) of the strains, respectively. MLST analysis confirmed that the strains belonged to sequence types (STs) including ST1638, ST155, ST48, ST350, ST23, ST156, and ST746. These findings underscore the diversity among E. coli strains in commercial poultry, which poses a significant risk.

Association of Blaoxa-1 Gene with Multidrug Resistance in K. pneumoniae Clinical Isolates

OBJECTIVES This study aimed to isolate K. pneumoniae from patients samples and find an association of the plasmid-mediated bla-OXA-1 gene with multidrug-resistant K. pneumoniae. METHODOLOGY This cross-sectional study was conducted at Mardan Medical Complex and Khyber Medical University Peshawar. K. pneumoniae was isolated from pus, urine and blood samples by culture and confirmed by biochemical techniques. Antibiotic susceptibility was done by disc diffusion according to the CLSI 2022 guidelines. A polymerase chain reaction was done for the gene after extraction and amplification of plasmid DNA. Furthermore, an association of antibiotic resistance was confirmed with blaOXA-1. RESULTSA total of 160 K. pneumoniae isolates were cultured from the patient’s samples, including pus (135, 84.37%), urine (15, 9.37%) and blood (10, 6.26%). There were 154 (96.3%) isolates resistant to Penicillin-G, followed by Ceftriaxone 151 (94.4%), Cefepime 143 (89.4%), Amoxicillin 125 (78.1%), Tigecycline 110 (68.8%), Imipenem 92 (57.6%) and Ertapenem 75(49.9%). However, Tetracycline had 1.9% resistance. The blaOXA-1 gene was positive in 41(25.62%) isolates with a different pattern of antibiotics resistance to Penicillin-G, Ceftriaxone, Cefepime, Amoxicillin, Tigecycline, Imipenem and Ertapenem as compared to the negative isolates. Among the blaOXA-1 gene-positive K. pneumoniae isolates, resistance to Penicillin-G was 100%, followed by Ceftriaxone (92.7%), Cefepime and Amoxicillin (80.5%), respectively. However, resistance to Imipenem and Ertapenem was 46.3% and 41.5%, respectively, and Tetracycline was not resistant. CONCLUSION Our data suggest that the presence of plasmid associated blaOXA-1 gene in K. pneumoniae isolates may contribute to multidrug resistance in beta lactamase-containing antibiotics along with other internal mechanisms of resistance present in these bacteria.

Colicin production, antibacterial effect and molecular sequencing of Escherichia coli isolated form different sources

Bacteria especially Escherichia coli (E. coli) possess several mechanisms enabling them to respond to changing environment and to out compete other bacteria. E. coli strains are known to produce bacteriocins, one of which is colicin. Colicins are effective against closely related enterobacteriaceae and are considered viable alternatives to antibiotics. In this study Twelve (12) E. coli strains were isolated from food, water soil and anal swab. They were characterized, confirmed and identified as E. coli. The isolates were subjected to stress condition to stimulate colicin production. The colicin produced was tested against three indicator organisms namely Salmonella Sp, Klebsiella Sp and Staphylococcus aureus. Comparative antibiotic susceptibility test against standard antibiotics and the colicin produced was carried out to ascertain the efficacy of the colicin. E. coli isolates were also subjected to antibiotic sensitivity test to determine the resistance profile of the colicin producing strains of E. coli. Plasmid amplification to identify colicin plasmid was also carried out to determine the relationship between colicin production and resistance to antibiotics of colicinogenic E. coli. Result obtained showed that six out of the twelve strains of E. coli isolated from water produced colicin that showed high antibacterial activity against the indicator organisms. The comparative antibiotic resistance profile revealed that the colicin had better antibacterial activities than some of the antibiotics used. Furthermore the colicinogenic E. coli also showed resistance to some of antibiotics used. The plasmid amplification showed the colicinogenic E. coli possess high molecular weight (4kb) colicin plasmid while the gene sequencing revealed the presence of ColF and ColR plasmids. The result of this work underscores the fact that the use of colicin as an alternative to standard antibiotics can be further explored, especially in the search for alternatives to antibiotics considering the high level of bacteria resistance to standard antibiotics.

Inhibition of the PCR by genomic DNA.

qPCR, is widely used for quantifying minimal residual disease (MRD) and is conventionally performed according to guidelines proposed by the EuroMRD consortium. However it often fails when quantifying MRD levels below 10-4. By contrast, HAT-PCR, a recent modification designed to minimise false-positive results, can quantify MRD down to 10-6. The factors leading to failure of conventional qPCR to quantify low levels of MRD were studied by analysing PCR reagents, protocol and primers and by testing for inhibition by adding primers to a plasmid amplification system. Complementary primers, ending in either G/C or A/T, were used to determine the effect of the 3' end of a primer. Inhibition of conventional PCR resulted from interaction of primers with genomic DNA leading to exponential amplification of nonspecific amplicons. It was observed with approximately half of the EuroMRD J primers tested. Inhibition by a primer was significantly related to primer Tm and G/C content and was absent when extension at the 3' end was blocked. Nonspecificity and inhibition were decreased or abolished by increasing the annealing temperature and inhibition was decreased by increasing the concentration of polymerase. Primers terminating with G/C produced significantly more nonspecificity and inhibition than primers terminating with A/T. HAT-PCR produced minimal nonspecificity and no inhibition. Inhibition of the PCR may result from the presence of genomic DNA and resultant exponential amplification of nonspecific amplicons. Factors contributing to the phenomenon include suboptimal annealing temperature, suboptimal primer design, and suboptimal polymerase concentration. Optimisation of these factors, as in HAT-PCR, enables sensitive quantification of MRD. PCR assays are increasingly used for sensitive detection of other rare targets against a background of genomic DNA and such assays may benefit from similar improvement in PCR design.

Profiling and antibiotic resistance of some human intestinal isolates

The cases of antibiotic resistance in Enterobacteriaceae are of great global concern. This study aimed at contributing towards the fight against antibiotic resistance and ameliorate the management/treatment of Enterobacteriaceae-linked diseases. Ten (10) rectal swab samples were collected from five (5) male and five (5) females and two isolates were identified from the samples collected. The isolates were identified through colonial, morphological and biochemical tests carried out following standard procedures. Isolates were investigated for their antibiotic resistance profile, Multiple Antibiotic Resistance indices (MARi), pathogenicity and their resistance genes were identified through molecular means using plasmid amplification and primers. Primers used include. ermB, BlaTem, qnrB genes Result obtained showed the isolates to be Escherichia coli and Klebsiella oxytoca. E. coli showed Alpha (α) heamolysis, while Klebsiella oxytoca showed gamma (γ) heamolysis . E. coli was resistant to 75% of the antibiotics used, Klebsiella oxytoca was sensitive to 42% of all the antibiotics. All the test organisms were resistant to all classes of Cephalosporins. The plasmid profiling revealed that all isolates have low molecular weight plasmids. The molecular fingerprinting of the isolates using gene primers viz -a viz ermB, BlaTem, qnrB genes showed E. coli to have resistance genes for macrolides (ermB gene) while none of the isolates had resistance factor against quinolones (qnrB gene). This study showed a high carriage of Enterobacteriaceae having phenotypic resistance with corresponding plasmid-borne resistance genes. The need to understand how bacteria adapt to the antibiotic environment will lead to new therapeutic strategies for antibiotic-resistant infections. Interventions measures to minimize the abundance of antibiotic-resistant commensals and opportunistic pathogens may include faecal microbiota transplantation and the use of live biotherapeutics.

Characterization of a novel theta-type replicon of indigenous plasmid pTE15 from Lactobacillus reuteri N16

BackgroundpTE15 is a ~ 15-kb narrow-host-range indigenous plasmid from Lactobacillus reuteri N16 that does not replicate in selected Bacillus spp., Staphylococcus spp., and other Lactobacillus spp.MethodsCombined deletion analysis the minireplicon essential of pTE15 with replicon-probe vector pUE80 (−) to confirmed sufficient for replication and from the ssDNA intermediate detection, plasmid amplification tested by chloramphenicol treatment, and replication origin sequence analysis to delineated the novel theta-type replication of pTE15.ResultsSingle-stranded intermediate of pTE15 DNA was not detected in L. reuteri, indicating that this plasmid does not replicate via a rolling circle mechanism. The replicon of pTE15 did not display the structural organization typical of rolling-circle plasmids, nor were they similar to known rolling-circle plasmids. We further provided evidence that this plasmid applied a new mode of theta-type replication mechanism: (1) the size of this plasmid was > 10-kb; (2) the minireplicon consisted of AT-rich (directed repeat, iteron) and DnaA sequences; (3) the minireplicon did not contain double-strand origin (DSO) and essential rep genes, and it also showed no single-strand origin (SSO) structure; (4) the intermediate single-stranded DNA products were not observed for pTE15 replication; (5) the minireplicon did not contain a typical essential replication protein, Rep, (6) its copy number was decreased by chloramphenicol treatment, and (7) genes in pTE15 replication region encoded truncated RepA (TRepA), RepB and RepC, which were replication-associated proteins, but they were not essential for pTE15 replication.ConclusionsCollectively, our results strongly suggested that the indigenous plasmid pTE15 of L. reuteri N16 belongs to a new class of theta replicons.

Construction of an adenovirus vector expressing engineered splicing factor for regulating alternative splicing of YAP1 in neonatal rat cardiomyocytes

To construct an adenovirus vector expressing artificial splicing factor capable of regulating alternative splicing of Yap1 in cardiomyocytes. The splicing factors with different sequences were constructed against Exon6 of YAP1 based on the sequence specificity of Pumilio1. The PCR fragment of the artificially synthesized PUF-SR or wild-type PUFSR was cloned into pAd-Track plasmid, and the recombinant plasmids were transformed into E. coli DH5α for plasmid amplification. The amplified plasmids were digested with Pac I and transfected into 293A cells for packaging to obtain the adenovirus vectors. Cultured neonatal rat cardiomyocytes were transfected with the adenoviral vectors, and alternative splicing of YAP1 was detected using quantitative and semi-quantitative PCR; Western blotting was performed to detect the signal of the fusion protein Flag. The transfection efficiency of the adenovirus vectors was close to 100% in rat cardiomyocytes, and no fluorescent protein was detected in the cells with plasmid transfection. The results of Western blotting showed that both the negative control and Flag-SR-NLS-PUF targeting the YAPExon6XULIE sequence were capable of detecting the expression of the protein fused to Flag. The results of reverse transcription-PCR and PCR demonstrated that the artificial splicing factor constructed based on the 4th target sequence of YAP1 effectively regulated the splicing of YAP1 Exon6 in the cardiomyocytes (P < 0.05). We successfully constructed adenovirus vectors capable of regulating YAP1 alternative splicing rat cardiomyocytes.

Development of a defined medium for Corynebacterium glutamicum using urea as nitrogen source.

The online version contains supplementary material available at 10.1007/s13205-021-02959-6.

Antibiotics Interfere with the Evolution of Plasmid Stability

Extra-chromosomal genetic elements are important drivers of bacterial evolution, and their evolutionary success depends on positive selection for the genes they encode. Examples are plasmids encoding antibiotic resistance genes that are maintained in the presence of antibiotics (e.g., [1-3]). Plasmid maintenance is considered a metabolic burden to the host [4]; hence, when the cost of plasmid carriage outweighs its benefit, plasmid-free segregants are expected to outcompete plasmid-carrying cells, eventually leading to plasmid loss [5-7]. Thus, in the absence of positive selection, plasmid survival hinges upon stable persistence in the population. The ubiquity of plasmids in nature suggests that plasmids having a negligible effect on host fitness may evolve stable inheritance and thus gain a long-term persistence in the population, also in the absence of positive selection [8]. Nonetheless, the transition of plasmids into stably inherited genetic elements remains understudied. Here, we show that positive selection for a plasmid-encoded gene interferes with the evolution of plasmid stability. Evolving plasmids under different selection regimes in Escherichia coli, we find that antibiotics led to plasmid amplification, resulting in plasmid instability. Thus, under positive selection, suboptimal solutions for plasmid stability were maintained in the population hindering long-term plasmid persistence. Indeed, a survey of Escherichia plasmids confirms that antibiotic resistance genes are rarely found on small plasmids. Our results show that a plasmid-mediated advantage for the host may manifest in reduced plasmid evolutionary success. Considering plasmids as autonomously evolving entities holds promise for understanding the factors that govern their evolution.

The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System.

Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host Escherichia coli presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts are made to establish plasmid-free expression, ensuring more stable process conditions. Another strategy to stabilize production processes is lactose induction, leading to increased soluble product formation and cell fitness, as shown in several studies performed with plasmid-based expression systems. Within this study we wanted to investigate lactose induction for a strain with a genome-integrated gene of interest for the first time. We found unusually high specific lactose uptake rates, which we could attribute to the low levels of lac-repressor protein that is usually encoded not only on the genome but additionally on pET plasmids. We further show that these unusually high lactose uptake rates are toxic to the cells, leading to increased cell leakiness and lysis. Finally, we demonstrate that in contrast to plasmid-based T7 expression systems, IPTG induction is beneficial for genome-integrated T7 expression systems concerning cell fitness and productivity.

Optimization of medium composition for propagation of recombinant Escherichia coli

AbstractRecombinant protein production in heterologous hosts often seems a simpler and more effective way than its production by natural producer. The secretion of recombinant protein inEscherichia colihas many advantages comparing to than in insect or mammalian cells. The important factor for high-level recombinant protein production is the sufficient amount ofE. colibiomass. Therefore, the aim of this study was to optimize the composition of propagation medium resulting in the maximum biomass yield of recombinantE. colias the part of fermentation strategy for neuraminidase (NA) production. Three independent variables including glucose, asparagine and phosphate concentrations, and four dependent variables, such as biomass yield, residual concentrations of glucose or asparagine and pH of the propagation medium after fermentation, were chosen to the optimization by Response Surface Methodology (RSM). The optimal conditions for the maximum biomass yield expressed as dry cell weight (DCW) (16.57±0.55 g DCW.L−1) were as follows: glucose concentration of 39.37 mM, asparagine concentration of 62.68 mM and phosphate concentration of 14.80 mM. For this model, the predicted values for the responses are close to the experimental values. The yield of desired pET15b-neu plasmid fromE. colicells cultivated in optimized propagation medium was almost 23 % higher than in commonly used Luria-Bertani (LB) medium suggesting that asparagine may be involved in the induction of plasmid amplification.

The role of amino acids in the amplification and quality of DNA vectors for industrial applications.

In this study, we have demonstrated that the type and feeding regimen of amino acids have a significant impact on the quality as well as the quantity of DNA vectors produced. Nutrient pool and factorial design experiments were carried out in order to identify the amino acids involved in increased biomass and induction of plasmid amplification. Leucine, glycine, and histidine were responsible for increased biomass and leucine starvation in the presence of histidine was implicated in plasmid amplification. Supercoiling of the plasmid was optimized using a dual feeding strategy. As a result of this, a fed-batch fermentation strategy for the production of a 6.9 kb plasmid, pSVß, in Escherichia coli DH5α was developed. In batch fermentation, a maximum plasmid yield of 39.4 mg/L equivalent to 11.3 mg/g dry cell weight (DCW) was achieved with casein hydrolysate limitation. About 90% of plasmid was in the supercoiled (SC) form after 31 hr of fermentation but only remained so for a short period, leading to a very brief window for harvesting cells at scale. Subsequently, a fed-batch fermentation using a dual feeding strategy was employed. A mean maximum plasmid yield of 44 mg/L equivalent to 9.1 mg plasmid/g DCW was achieved. After 25 hr, 90% of plasmid was in the SC form and remained at this level for the remaining 10 hr of the fermentation, allowing adequate time for the harvesting of cells without the loss of supercoiling of product. This study emphasized that optimizing fermentation strategy and identifying the essential nutrients are beneficial for bioprocessing of plasmid DNA for therapeutic applications.

Insights into the DNA sequence elements required for partitioning and copy number control of the yeast 2-micron plasmid.

The yeast 2-micron plasmid is an almost perfect selfish DNA. The entire coding capacity of the plasmid is dedicated to ensuring its own inheritance, with no benefit to its host. Despite high copy number, the plasmid confers no phenotype. It manages this feat by possessing mechanisms for plasmid copy-number control and for partitioning. The former increases plasmid numbers when they fall, but is repressed at high copy number, while the latter ensures 2-micron copies are equally partitioned during host cell division. Although the plasmid amplification mechanism is well established, the partitioning system and the means by which the 2-micron plasmid partitioning proteins, Rep1 and Rep2, regulate plasmid copy number remain incompletely understood. This review focuses on recent efforts to determine the nature of Rep protein complexes formed at the plasmid stability locus (STB) and at plasmid gene promoters, the identity of DNA sequence elements required for Rep protein association, and the mechanism by which the Rep proteins manage their dual roles of plasmid partitioning and plasmid gene repression.

Recombinant multicopy plasmids in yeast - interactions with the endogenous 2 μm.

Flp-mediated site specific intramolecular recombination in Saccharomyces cerevisiae is considered responsible for amplification of the endogenous 2 μm plasmid. For YEp-type vectors, a similar mechanism can be imagined by which such plasmids achieve high copy numbers, a trait desired for many research applications and necessary for industrial production. We have cultivated yeast carrying one of six isomeric YEp-type model expression plasmids under two different conditions and back transformed the shuttle vectors into Escherichia coli. Our analysis of 586 ampR clones represents a high-resolution snapshot of plasmid forms present in the transformed yeast cells with a detection limit of structural changes of <2%. Altered forms summed up to about 11%, constituting likely a lower limit. We have observed two categories of recombination events. One is Flp based, with products of intermolecular recombination with the 2 μm, likely intermediates that are prerequisites for YEp-type plasmid amplification. The other type is based on Flp-independent homologous recombination leading to oligomerization of such plasmids also in a 2μm-free [cir°] strain, i.e. in the absence of Flp. Beyond the general maintenance and its functional sequences, only the gene of interest and its expression might have an impact on the physiology of the host.