scholarly journals Natural Transformation of Plasmid DNA in Escherichia coli in Sterilized Soil Column

1970 ◽  
Vol 25 (1) ◽  
pp. 49-52
Author(s):  
M Mahabub-Uz-Zaman ◽  
Zia Uddin Ahmed
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Natural Transformation ◽  
Soil Column ◽  
Limiting Factor ◽  
Broad Host Range ◽  
E Coli ◽  
Competent Cells ◽  
Broad Host Range Plasmid ◽  
Plasmid Puc18

The present study was carried out to assess transformability of natural and laboratory strains of Escherichia coli by plasmid DNA under different transformation conditions in sterilized soil column. Transformation experiments were carried out in laboratory conditions and in sterile soil columns with CaCl2-treated competent cells and non-competent cells at log phase and stationary phase of growth using the broad host range plasmid pUC18. In soil column experiments, transformants were obtained after CaCl2 induced competence in both E. coli K12 DH5α and strain BM09 in the frequency of 10-8 to 10-9. In natural transformation assays, transformants appeared only in log phase cells of strain DH5α at a lower frequency (5.0 x 10-9), and in CaCl2-competent BM09 cells, but not in fresh cells. Thus the major limiting factor for natural transformation in environmental E. coli in soil column is probably the absence of a competent state. The significance of this finding has been discussed with respect to generally observed lower antibiotic resistance in environmental E. coli isolates from aquatic sources. Keywords: Natural transformation; Plasmid DNA; Escherichia coli; Competent stateDOI: http://dx.doi.org/10.3329/bjm.v25i1.4856 Bangladesh J Microbiol, Volume 25, Number 1, June 2008, pp 49-52

scholarly journals Expanding the toolbox of broad host-range transcriptional terminators for Proteobacteria through metagenomics

2018 ◽  
Author(s):  
Vanesa Amarelle ◽  
Ananda Sanches-Medeiros ◽  
Rafael Silva-Rocha ◽  
María-Eugenia Guazzaroni
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Pseudomonas Putida ◽  
Host Range ◽  
Broad Host Range ◽  
Functional Metagenomics ◽  
Alternative Hosts ◽  
E Coli ◽  
Broad Host Range Plasmid

AbstractAs the field of synthetic biology moves towards the utilization of novel bacterial chassis, there is a growing need for biological parts with enhanced performance in a wide number of hosts. Is not unusual that biological parts (such as promoters and terminators), initially characterized in the model bacteria Escherichia coli, do not perform well when implemented in alternative hosts, such as Pseudomonas, therefore limiting the construction of synthetic circuits in industrially relevant bacteria. In order to address this limitation, we present here the mining of transcriptional terminators through functional metagenomics to identify novel parts with broad host-range activity. Using a GFP-based terminator trap strategy and a broad host-range plasmid, we identified 20 clones with potential terminator activity in Pseudomonas putida. Further characterization allowed the identification of 4 unique sequences between 58 bp and 181 bp long that efficiently terminates transcription in P. putida, E. coli, Burkholderia phymatum and two Pseudomonas strains isolated from Antarctica. Therefore, this work presents a new set of biological parts useful for the engineering of synthetic circuits in Proteobacteria.

One-Step Preparation of Competent E. coli: Transformation and Storage of Bacterial Cells in the Same Solution

2021 ◽  
Vol 2021 (11) ◽  
pp. pdb.prot101212 ◽  
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Escherichia Coli ◽  
Convenient Method ◽  
Plasmid Dna ◽  
Transformation Efficiency ◽  
Bacterial Cells ◽  
E Coli ◽  
One Step ◽  
And Storage

This protocol describes a convenient method for the preparation, use, and storage of competent Escherichia coli. The reported transformation efficiency of this method is ∼5 × 107 transformants/µg of plasmid DNA.

scholarly journals In VivoTransmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration, and Acquisition of the Plasmid Results in a Variable Cost of Fitness

2015 ◽  
Vol 81 (10) ◽  
pp. 3561-3570 ◽  
Author(s):  
Timothy J. Johnson ◽  
Randall S. Singer ◽  
Richard E. Isaacson ◽  
Jessica L. Danzeisen ◽  
Kevin Lang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Dose ◽  
Broad Host Range ◽  
Antibiotic Administration ◽  
Dose Administration ◽  
Content Type ◽  
E Coli ◽  
The Impact ◽  
Selection Of

ABSTRACTIncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. Although antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types of antibiotic administration on the selection of plasmid-containing multidrug resistant isolates. In this study, chlortetracycline treatment at different concentrations in pig feed was examined for its impact on selection and dissemination of an IncA/C plasmid introduced orally via a commensalEscherichia colihost. Continuous low-dose administration of chlortetracycline at 50 g per ton had no observable impact on the proportions of IncA/C plasmid-containingE. colifrom pig feces over the course of 35 days. In contrast, high-dose administration of chlortetracycline at 350 g per ton significantly increased IncA/C plasmid-containingE. coliin pig feces (P< 0.001) and increased movement of the IncA/C plasmid to other indigenousE. colihosts. There was no evidence of conjugal transfer of the IncA/C plasmid to bacterial species other thanE. coli.In vitrocompetition assays demonstrated that bacterial host background substantially impacted the cost of IncA/C plasmid carriage inE. coliandSalmonella.In vitrotransfer and selection experiments demonstrated that tetracycline at 32 μg/ml was necessary to enhance IncA/C plasmid conjugative transfer, while subinhibitory concentrations of tetracyclinein vitrostrongly selected for IncA/C plasmid-containingE. coli. Together, these experiments improve our knowledge on the impact of differing concentrations of tetracycline on the selection of IncA/C-type plasmids.

Multiple mechanisms for expression of incompatibility by broad-host-range plasmid RK2

1982 ◽  
Vol 152 (3) ◽  
pp. 1078-1090
Author(s):  
R Meyer ◽  
M Hinds
Keyword(s):  
Dna Replication ◽  
Host Range ◽  
Plasmid Dna ◽  
Plasmid Vector ◽  
Broad Host Range ◽  
Plasmid Maintenance ◽  
Broad Host Range Plasmid ◽  
Expression Of Genes ◽  
Plasmid Partitioning ◽  
Plasmid Rk2

By cloning fragments of plasmid DNA, we have shown that RK2 expresses incompatibility by more than one mechanism. One previously identified (R. J. Meyer, Mol. Gen, Genet. 177:155--161, 1979; Thomas et al., Mol. Gen. Genet. 181:1--7, 1981) determinant for incompatibility is linked to the origin of plasmid DNA replication. When cloned into a plasmid vector, this determinant prevents the stable inheritance of a coresident RK2. However, susceptibility to this mechanism of incompatibility requires an active RK2 replicon and is abolished if another replicator is provided. We have also cloned a second incompatibility determinant, encoded within the 54.1- to 56.4-kilobase region of RK2 DNA, which we call IncP-1(II). An RK2 derivative remains sensitive to IncP-1(II), even when it is not replicating by means of the RK2 replicon. The 54.1- to 56.4-kilobase DNA does not confer susceptibility to the IncP-1(II) mechanism, nor does it encode a detectable system for efficient plasmid partitioning. The incompatibility may be related to the expression of genes mapping in the 54.1- to 56.4-kilobase region, which are required for plasmid maintenance and suppression of plasmid-encoded killing functions.

scholarly journals Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Dongchang Sun ◽  
Xudan Mao ◽  
Mingyue Fei ◽  
Ziyan Chen ◽  
Tingheng Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Natural Transformation ◽  
Inducible Promoter ◽  
Regulation Mechanism ◽  
Type I ◽  
Content Type ◽  
E Coli ◽  
Double Stranded Dna ◽  
Dna Binding Site

ABSTRACT Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (Pcas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli. Although the H-NS paralogue StpA also binds Pcas, its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of Pcas. Third, by comparing transcriptional activities of the intact Pcas and the Pcas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in Pcas. Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of Pcas. Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. IMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli. However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli. In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.

Core-Shell Structured Fe3O4/PPy Microspheres with High Magnetization for Purification of Plasmid DNA

2013 ◽  
Vol 716 ◽  
pp. 314-319 ◽  
Author(s):  
Hai Hui Jiang ◽  
Yan Zhou ◽  
Xiao Yun Han ◽  
Xin Cheng Chen ◽  
Yun Hua Hou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Drug Delivery ◽  
Plasmid Dna ◽  
Magnetic Particles ◽  
Interfacial Polymerization ◽  
Core Shell ◽  
Magnetic Composite ◽  
Amino Group ◽  
High Quality ◽  
E Coli

Amino group-functionalized magnetic particles have wide applications in enzyme immobilization, DNA extraction, drug delivery, water purification, catalysis, and sensor. In this paper, Fe3O4/PPy microspheres with a well-defined coreshell structure have been prepared through an interfacial polymerization approach without surfactant. The magnetic composite spheres were characterized with XRD, FTIR, SEM, TEM, and magnetometry techniques, and further tested as the adsorbent to isolate plasmid DNA from Escherichia coli (E. coli) DH5α cells. The magnetic separation yields high-quality plasmid DNA in satisfying productivity as compared to the conventional phenolchloroform extraction.

Physiological effects of plasmid DNA transformation on Azotobacter vinelandii

1986 ◽  
Vol 32 (2) ◽  
pp. 145-148 ◽  
Author(s):  
Baernard R. Glick ◽  
Heather E. Brooks ◽  
J. J. Pasternak
Keyword(s):  
Plasmid Dna ◽  
Azotobacter Vinelandii ◽  
Genetic Manipulation ◽  
A Priori ◽  
Broad Host Range ◽  
Physiological Effects ◽  
Dna Transformation ◽  
Physiological Changes ◽  
Physiological Processes ◽  
Broad Host Range Plasmid

Genetic transformation of Azotobacter vinelandii by the introduction of broad-host-range plasmid DNA (i.e., pRK2501, RSF1010, or pGSS15) causes a number of physiological changes. As shown here, the capacity for nitrogen fixation, mean cell size, and synthesis of siderophores are decreased, whereas the production of capsular slime is enhanced. These findings suggest that the presence of plasmid DNA imposes a "metabolic load" on Azotobacter vinelandii. Therefore, it cannot be assumed a priori that the introduction of plasmid DNA into Azotobacter vinelandii will not disrupt some normal physiological processes. The implications of these findings are discussed, specifically in the context of developing Azotobacter vinelandii as an effective bacterial fertilizer by genetic manipulation.

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