scholarly journals inPOSE: a flexible toolbox for chromosomal cloning and amplification of bacterial transgenes

2021 ◽  
Author(s):  
Ranti Dev Shukla ◽  
Agnes Zvara ◽  
Akos Avramucz ◽  
Alyona Biketova ◽  
Akos Nyerges ◽  
...  
Keyword(s):  
Transposable Elements ◽  
Bacterial Species ◽  
Single Copy ◽  
Single Step ◽  
Copy Numbers ◽  
Proper Design ◽  
The Stability ◽  
Genetic Constructs ◽  
Copy Number Amplification ◽  
Double Digit

Cloning genes and operons encoding heterologous functions in bacterial hosts is almost exclusively carried out today using plasmid vectors. This has multiple drawbacks, including the need for constant selection and the variation in copy numbers. Chromosomal integration of transgenes has always offered a viable alternative, however, to date it has been of limited use due to its tedious nature and to being limited often to a single copy. We introduce here a strategy that uses bacterial insertion sequences, the simplest autonomous transposable elements to insert and amplify genetic cargo into a bacterial chromosome. Transgene insertion can take place either as transposition or homologous recombination, and copy-number amplification is achieved using controlled copy-paste transposition. We display successful use of IS1 and IS3 for this purpose in Escherichia coli cells, using various selection markers. We demonstrate the insertion of selectable genes, an unselectable gene, and a five-gene operon in up to two copies in a single step. We continue with the amplification of the inserted cassette to double-digit copy numbers within two rounds of transposase induction and selection. Finally, we analyze the stability of the cloned genetic constructs in the lack of selection, and find it to be superior to all investigated plasmid-based systems. Due to the ubiquitous nature of transposable elements we believe that with proper design, this strategy can be adapted to numerous further bacterial species.

scholarly journals New Vector System for Random, Single-Step Integration of Multiple Copies of DNA into the Rhodococcus Genome

2010 ◽  
Vol 76 (8) ◽  
pp. 2531-2539 ◽  
Author(s):  
Khalid Ibrahim Sallam ◽  
Noriko Tamura ◽  
Noriko Imoto ◽  
Tomohiro Tamura
Keyword(s):  
Cultured Cells ◽  
Single Copy ◽  
Inducible Promoter ◽  
Single Step ◽  
Biologically Active ◽  
Host Cells ◽  
Vector System ◽  
Copy Numbers ◽  
Multiple Copies ◽  
Cell Genome

ABSTRACT We designed a new vector system for creating a random mutant library with multiple integrations of DNA fragments into the Rhodococcus genome in a single step. For this, we cotransformed two vectors into Rhodococcus by electroporation: pTip-istAB-sacB regulates the expression of the transposase (IstA) and its helper protein (IstB) under the influence of a thiostrepton-inducible promoter, and pRTSK-sacB provides the transposable-marker DNA. Both are multicopy vectors that are stable in the host cells; transposition of the transposable-marker DNA occurs only after the induction of IstA/IstB expression. With the addition of thiostrepton, all cultured cells harboring the two vectors, irrespective of the volume, can be mutated by random insertion of the transposable-marker DNA into their genome. Among the generated mutants examined, 30% showed multiple (two to five) insertion copies. The multiple integrated DNA copies were stable in the genome for more than 80 generations of serial growth without the addition of any selective antibiotics. This system can also be used for integrating various copy numbers of stably maintained protein expression cassettes in the host cell genome to modulate the expression level of biologically active recombinant proteins. We successfully applied this system to integrate multiple copies of expression cassettes for proline iminopeptidase and vitamin D3 hydroxylase into the Rhodococcus genome and verified that the clones containing double or multiple copies of the integrated cassettes produced higher levels and showed higher enzymatic activities of the target protein than clones with only a single copy of integration.

scholarly journals Polyhydroxyalkanoate Nanoparticles for Pulmonary Drug Delivery: Interaction with Lung Surfactant

Nanomaterials ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1482
Author(s):  
Olga Cañadas ◽  
Andrea García-García ◽  
M. Auxiliadora Prieto ◽  
Jesús Pérez-Gil
Keyword(s):  
Drug Delivery ◽  
Drug Delivery Systems ◽  
Bacterial Species ◽  
High Surface ◽  
Lung Surfactant ◽  
Delivery Systems ◽  
Pulmonary Drug Delivery ◽  
Epifluorescence Microscopy ◽  
Energy Storage Materials ◽  
The Stability

Polyhydroxyalkanoates (PHA) are polyesters produced intracellularly by many bacterial species as energy storage materials, which are used in biomedical applications, including drug delivery systems, due to their biocompatibility and biodegradability. In this study, we evaluated the potential application of this nanomaterial as a basis of inhaled drug delivery systems. To that end, we assessed the possible interaction between PHA nanoparticles (NPs) and pulmonary surfactant using dynamic light scattering, Langmuir balances, and epifluorescence microscopy. Our results demonstrate that NPs deposited onto preformed monolayers of DPPC or DPPC/POPG bind these surfactant lipids. This interaction facilitated the translocation of the nanomaterial towards the aqueous subphase, with the subsequent loss of lipid from the interface. NPs that remained at the interface associated with liquid expanded (LE)/tilted condensed (TC) phase boundaries, decreasing the size of condensed domains and promoting the intermixing of TC and LE phases at submicroscopic scale. This provided the stability necessary for attaining high surface pressures upon compression, countering the destabilization induced by lipid loss. These effects were observed only for high NP loads, suggesting a limit for the use of these NPs in pulmonary drug delivery.

scholarly journals Temporal Stability and Biodiversity of Two Complex Antilisterial Cheese-Ripening Microbial Consortia

2003 ◽  
Vol 69 (7) ◽  
pp. 4012-4018 ◽  
Author(s):  
Ariel Maoz ◽  
Ralf Mayr ◽  
Siegfried Scherer
Keyword(s):  
Species Composition ◽  
Bacterial Species ◽  
Temporal Stability ◽  
Individual Characteristics ◽  
Microbial Consortia ◽  
Cheese Ripening ◽  
Safe Food ◽  
The Stability ◽  
The Individual ◽  
First Time

ABSTRACT The temporal stability and diversity of bacterial species composition as well as the antilisterial potential of two different, complex, and undefined microbial consortia from red-smear soft cheeses were investigated. Samples were collected twice, at 6-month intervals, from each of two food producers, and a total of 400 bacterial isolates were identified by Fourier-transform infrared spectroscopy and 16S ribosomal DNA sequence analysis. Coryneform bacteria represented the majority of the isolates, with certain species being predominant. In addition, Marinolactobacillus psychrotolerans, Halomonas venusta, Halomonas variabilis, Halomonas sp. (106 to 107 CFU per g of smear), and an unknown, gram-positive bacterium (107 to 108 CFU per g of smear) are described for the first time in such a consortium. The species composition of one consortium was quite stable over 6 months, but the other consortium revealed less diversity of coryneform species as well as less stability. While the first consortium had a stable, extraordinarily high antilisterial potential in situ, the antilisterial activity of the second consortium was lower and decreased with time. The cause for the antilisterial activity of the two consortia remained unknown but is not due to the secretion of soluble, inhibitory substances by the individual components of the consortium. Our data indicate that the stability over time and a potential antilisterial activity are individual characteristics of the ripening consortia which can be monitored and used for safe food production without artificial preservatives.

Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits

1993 ◽  
Vol 13 (5) ◽  
pp. 2835-2845
Author(s):  
M Deshmukh ◽  
Y F Tsay ◽  
A G Paulovich ◽  
J L Woolford
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Subunit ◽  
Single Copy ◽  
5S Rrna ◽  
Gene Encoding ◽  
Ribosomal Subunits ◽  
Ribosomal Protein L1 ◽  
The Stability ◽  
And Function

Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another ribosomal protein, L16. Preliminary results suggest that transcription of RPL1 is not autogenously regulated by L1.

Coordinate expression of amplified metallothionein I and II genes in cadmium-resistant Chinese hamster cells

1985 ◽  
Vol 5 (12) ◽  
pp. 3525-3531
Author(s):  
J K Griffith
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Recombinant Dna ◽  
Chinese Hamster ◽  
Single Copy ◽  
Ovary Cell ◽  
Gene Copy ◽  
Mrna Levels ◽  
Protein Levels ◽  
Copy Numbers

Recombinant DNA probes complementary to Chinese hamster metallothionein (MT)-1 and MT-2 mRNAs were used to compare MT gene copy numbers, zinc-induced MT mRNA levels, and uninduced MT mRNA levels in cadmium-resistant (Cdr) Chinese hamster ovary cell lines. Quantitative hybridization analyses determined that the MT-1 and MT-2 genes are each present at approximately single-copy levels in the genome of cell line Cdr2C10 and are coordinately amplified approximately 7, 3, and 12 times over the Cdr2C10 value in the genomes of cell lines Cdr20F4, Cdr30F9, and Cdr200T1, respectively. The maximum zinc-induced MT-1 mRNA concentrations in cell lines Cdr20F4, Cdr30F9, and Cdr200T1 were equal to 1, 3, and 15 times that measured in Cdr2C10, respectively. Similarly, the maximum zinc-induced MT-2 mRNA concentrations were equal to 1, 3, and 14 times that measured in Cdr2C10, respectively, and in each instance they were 90 to 150 times greater than their respective concentrations in uninduced cells. Thus, relative MT gene numbers are closely correlated with both zinc-induced and uninduced MT mRNA levels in Cdr2C10, Cdr30F9, and Cdr200T1, but not in Cdr20F4. Each of the latter two lines possesses structurally altered chromosomes whose breakpoints are near the MT locus. Nonetheless, the ratio of the levels of MT-1 to MT-2 mRNAs was constant in each of the four cell lines, including Cdr20F4. These results demonstrate that MT-1 and MT-2 mRNAs are induced coordinately in each Cdr cell line. Therefore, the coordination of the induction of MT-1 and MT-2 mRNA is independent of MT gene amplification, MT gene rearrangement, and the relative inducibilities of amplified MT genes. However, MT mRNA and protein levels each indicate that MT-1 and MT-2 expression is non-coordinate in uninduced cells. Thus, regulation of MT expression may involve two different mechanisms which are differentially operative in induced and uninduced cells.

scholarly journals SEVA 3.0: an update of the Standard European Vector Architecture for enabling portability of genetic constructs among diverse bacterial hosts

2019 ◽  
Vol 48 (D1) ◽  
pp. D1164-D1170 ◽  
Author(s):  
Esteban Martínez-García ◽  
Angel Goñi-Moreno ◽  
Bryan Bartley ◽  
James McLaughlin ◽  
Lucas Sánchez-Sampedro ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Bacterial Species ◽  
Web Based ◽  
Genomic Engineering ◽  
Bioinformatic Tools ◽  
Synthetic Biology Open Language ◽  
User Friendly ◽  
Machine Readable ◽  
Genetic Constructs

Abstract The Standard European Vector Architecture 3.0 database (SEVA-DB 3.0, http://seva.cnb.csic.es) is the update of the platform launched in 2013 both as a web-based resource and as a material repository of formatted genetic tools (mostly plasmids) for analysis, construction and deployment of complex bacterial phenotypes. The period between the first version of SEVA-DB and the present time has witnessed several technical, computational and conceptual advances in genetic/genomic engineering of prokaryotes that have enabled upgrading of the utilities of the updated database. Novelties include not only a more user-friendly web interface and many more plasmid vectors, but also new links of the plasmids to advanced bioinformatic tools. These provide an intuitive visualization of the constructs at stake and a range of virtual manipulations of DNA segments that were not possible before. Finally, the list of canonical SEVA plasmids is available in machine-readable SBOL (Synthetic Biology Open Language) format. This ensures interoperability with other platforms and affords simulations of their behaviour under different in vivo conditions. We argue that the SEVA-DB will remain a useful resource for extending Synthetic Biology approaches towards non-standard bacterial species as well as genetically programming new prokaryotic chassis for a suite of fundamental and biotechnological endeavours.

APOBEC3 proteins: major players in intracellular defence against LINE-1-mediated retrotransposition

2007 ◽  
Vol 35 (3) ◽  
pp. 637-642 ◽  
Author(s):  
G.G. Schumann
Keyword(s):  
Transposable Elements ◽  
Tandem Repeats ◽  
Genetic Disorders ◽  
Single Copy ◽  
Long Terminal Repeat Retrotransposons ◽  
Variable Number ◽  
Ltr Retrotransposons ◽  
Negative Effects ◽  
Processed Pseudogenes ◽  
Retrotransposon Activity

Mammalian genomes are littered with enormous numbers of transposable elements interspersed within and between single-copy endogenous genes. The only presently spreading class of human transposable elements comprises non-LTR (long terminal repeat) retrotransposons, which cover approx. 34% of the human genome. Non-LTR retrotransposons include the widespread autonomous LINEs (long interspersed nuclear elements) and non-autonomous elements such as processed pseudogenes, SVAs [named after SINE (short interspersed nuclear element), VNTR (variable number of tandem repeats) and Alu] and SINEs. Mobilization of these elements affects the host genome, can be deleterious to the host cell, and cause genetic disorders and cancer. In order to limit negative effects of retrotransposition, host genomes have adopted several strategies to curb the proliferation of transposable elements. Recent studies have demonstrated that members of the human APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide 3) protein family inhibit the mobilization of the non-LTR retrotransposons LINE-1 and Alu significantly and participate in the intracellular defence against retrotransposition by mechanisms unknown to date. The striking coincidence between the expansion of the APOBEC3 gene cluster and the abrupt decline in retrotransposon activity in primates raises the possibility that these genes may have been expanded to prevent genomic instability caused by endogenous retroelements.

scholarly journals Specific Detection of Clavibacter michiganensis subsp. sepedonicus by Amplification of Three Unique DNA Sequences Isolated by Subtraction Hybridization

1997 ◽  
Vol 87 (8) ◽  
pp. 853-861 ◽  
Author(s):  
Dallice Mills ◽  
Brian W. Russell ◽  
Janet Williams Hanus
Keyword(s):  
Dna Sequences ◽  
Bacterial Species ◽  
Single Copy ◽  
Cross Reactivity ◽  
Clavibacter Michiganensis ◽  
Subtraction Hybridization ◽  
Detection Systems ◽  
Clavibacter Michiganensis Subsp ◽  
Polymerase Chain ◽  
Primer Sets

Three single-copy, unique DNA fragments, designated Cms50, Cms72, and Cms85, were isolated from strain CS3 of Clavibacter michiganensis subsp. sepedonicus by subtraction hybridization using driver DNA from C. michiganensis subsp. insidiosus, C. michiganensis subsp. michiganensis, and Rhodococcus facians. Radio-labeled probes made of these fragments and used in Southern blot analysis revealed each to be absolutely specific to all North American C. michiganensis subsp. sepedonicus strains tested, including plasmidless and nonmucoid strains. The probes have no homology with genomic DNA from related C. michiganensis subspecies insidiosus, michiganensis, and tessellarius, nor with DNA from 11 additional bacterial species and three unidentified strains, some of which have been previously reported to display cross-reactivity with C. michiganensis subsp. sepedonicus-specific antisera. The three fragments shared no homology, and they appeared to be separated from each other by at least 20 kbp in the CS3 genome. Internal primer sets permitted amplification of each fragment by the polymerase chain reaction (PCR) only from C. michiganensis subsp. sepedonicus DNA. In a PCR-based sensitivity assay using a primer set that amplifies Cms85, the lowest level of detection of C. michiganensis subsp. sepedonicus was 100 CFU per milliliter when cells were added to potato core fluid. Erroneous results that may arise from PCR artifacts and mutational events are, therefore, minimized by the redundancy of the primer sets, and the products should be verifiable with unique capture probes in sequence-based detection systems.

scholarly journals Origin and evolution of Mytilus mussel satellite DNAs

Genome ◽  
2005 ◽  
Vol 48 (2) ◽  
pp. 247-256 ◽  
Author(s):  
A Martínez-Lage ◽  
F Rodríguez-Fariña ◽  
A González-Tizón ◽  
J Méndez
Keyword(s):  
Phylogenetic Reconstruction ◽  
Single Copy ◽  
High Similarity ◽  
Dot Blot ◽  
Promoter Regions ◽  
Satellite Dnas ◽  
Crustacean Species ◽  
Origin And Evolution ◽  
Copy Numbers ◽  
Parsimony Trees

A phylogenetic reconstruction based on the amplification of 3 satellite DNAs (stDNAs) was carried out in 1 crustacean species and 15 bivalve species of the subclass Pteriomorphia (10, subfamily Mytilinae; 1, subfamily Litophaginae; 1, subfamily Modiolinae, all belonging to family Mytilidae; 1, family Arcidae; and 2, family Pectinidae). The sequences obtained showed motifs with high similarity to those of A and B boxes of tRNA promoter regions. Dot-blot hybridizations revealed that the 3 stDNAs are present mainly in high copy numbers for each species of the genus Mytilus, whereas for the other species they appear in low copy numbers. Maximum-parsimony trees evidenced a tendency to group Mytilus clones together, and species containing these sequences as a single copy were distributed among the different mytilids. Finally, the possible origin and evolution of these stDNAs is discussed.Key words: bivalves, Pteriomorphia, satellite DNA, phylogeny, dot-blot.

scholarly journals The distribution of transposable elements within and between chromosomes in a population of Drosophila melanogaster. I. Element frequencies and distribution

1992 ◽  
Vol 60 (2) ◽  
pp. 103-114 ◽  
Author(s):  
Brian Charlesworth ◽  
Angela Lapid ◽  
Darlene Canada
Keyword(s):  
Population Dynamics ◽  
Drosophila Melanogaster ◽  
Linkage Disequilibrium ◽  
Transposable Elements ◽  
Copy Number ◽  
Polytene Chromosomes ◽  
Chromosome Band ◽  
High Frequencies ◽  
Copy Numbers

SummaryData were collected on the distribution of nine families of transposable elements among second and third chromosomes isolated from a natural population of Drosophila melanogaster, by means of in situ hybridization of element probes to polytene chromosomes. It was found that the copy numbers per chromosome in the distal sections of the chromosome arms followed a Poisson distribution. Elements appeared to be distributed randomly along the distal sections of the chromosome arms. There was no evidence for linkage disequilibrium in the distal sections of the chromosomes, but some significant disequilibrium was detected in proximal regions. There were many significant correlations between different element families with respect to the identity of the sites that were occupied in the sample. There were also significant correlations between families with respect to sites at which elements achieved relatively high frequencies. Element frequencies per chromosome band were generally low in the distal sections, but were higher proximally. These results are discussed in the light of models of the population dynamics of transposable elements. It is concluded that they provide strong evidence for the operation of a force or forces opposing transpositional increase in copy number. The data suggest that the rate of transposition perelement per generation is of the order of 10−4, for the elements included in this study.

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