Chimeras are clones that possess two or more noncontiguous DNA inserts. DNA fingerprinting is a means of analyzing the similarity between several DNA samples based upon the presence or absence of specific restriction sites within their sequences. The digested DNA samples are run on a gel and blotted onto nitrocellulose Southern blotting. Those clones that have considerable overlap in their fingerprint patterns can be grouped together into contigs Marek and Shoemaker ; Marra et al.
False positive clones do not contain inserts from the experimental organism, yet exhibit the phenotype of recombinants based upon their growth pattern and colony color on selective media e. False positives generally do contain a vector molecule. However, the marker gene has been inactivated by an event other than insertion of a large DNA fragment into the polycloning site.
F factors are naturally occurring episomes i. In its free plasmid circular form, the typical F factor is approximately kb and is maintained at a level of one copy per bacterial genome. When inserted into the bacterial genome, F factors are replicated along with the bacterial genome. Integrated F factors can be present in more than one copy per cell. Bacteria that possess an F factor F-positive bacteria can conjugate with strains that do not contain an F factor F-negative bacteria.
During conjugation which is mediated by F factor gene products , an F-positive bacterium containing a free plasmid transfers a copy of the F plasmid to an F-negative bacterium.
If the F factor is integrated into the F-positive bacterium's genome, the F factor and part or all of the donor's chromosomal DNA may be transferred into the F-negative bacterium Willetts and Skurry ; Lewin Genes involved in conjugation and regions involved in insertion of the F factor into the bacterial genome have been eliminated.
Fluorescence in situ hybridization FISH is a technique in which hapten-labeled DNA probes are hybridized to chromosomes that have been spread on glass microscope slides. Antibodies or other affinity reagents conjugated to fluorochromes are used to detect directly or indirectly sites of hybridization Peterson et al. Genome coverage is the combined base pair length of all the inserts in a genomic library divided by the 1C genome content of the organism for which the library was made.
The level of genome coverage for a particular library can be determined using the following formula:. For example, suppose that a BAC library was constructed for soybean Glycine max.
The 1C DNA content for soybean is approximately 1. If the library contained , clones with an average insert size of , bp, the library coverage would be. To put it another way, the library would contain Naturally, increasing the genome coverage above 5X affords even higher confidence levels Paterson Insertional inactivation can be used to differentiate recombinant clones from non-recombinants. Most plasmid vectors including most BACs contain a reporter gene into which a polycloning site has been engineered.
Ligation of a piece of exogenous insert DNA into the polycloning site of a vector results in disruption of the reporter gene whereas a vector molecule that either was not cut by the restriction enzyme or that has had its termini ligated back together contains an intact reporter gene.
After transformation, bacteria are plated onto nutritive agar. In recombinant clones clones in which the reporter gene has been disrupted by an insert , a functional version of the reporter gene protein reporter protein will not be produced. In clones containing a plasmid without an insert non-recombinants , the reporter protein will be expressed. The reporter protein is typically an enzyme or part of an enzyme complex that catalyzes a colorimetric reaction using a component in the selective media as a substrate.
Consequently, recombinant and non-recombinant clones can be differentiated based on colony color see alpha-complementation. Biotechniques — Front Microbiol Antonie Van Leeuwenhoek — Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem — Springer-Verlag: New York, Chapter 1. Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature — BMC Biotechnol Int J Mol Sci Process Biochem — J Agric Food Chem — Biotechnol Lett — Biotechnol Prog — Electron J Biotechnol — DMMMH4 and its evaluation for anticancer activity. Download references. We would like to thank the Microbial chemistry Dep. You can also search for this author in PubMed Google Scholar.
AS made the screening test on the trans-conjugant strains and protein electrophoresis. AS, MK, and MA wrote the manuscript and participated in the data discussion, data analyses, and drafting of the manuscript. The authors have read and approved the final manuscript.
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Skip to main content. Search all SpringerOpen articles Search. Download PDF. Conclusion Trans-conjugant of E. Methods Strains, plasmids, and media Strains E. Media 1. Bacterial trans-conjugation The overnight cultures of recipient and donor strains were diluted fold in LB medium. Enzyme assay and protein estimation Enzyme activity was assayed by measuring the amount of o NP O -nitrophenolate liberated. Lactose bioconversion by E. Full size image. Table 5 Enzyme kinetics for the selected substrates Full size table.
Availability of data and materials The authors declare that all generated and analyzed data are included in the article. References 1. J Biotechnol —37 Article Google Scholar 3. Food Chem — Article Google Scholar 7. Food Chem — Article Google Scholar 9. Carbohydr Polym —83 Article Google Scholar Pharmacognosy Res Article Google Scholar Afr J Biotechnol — Google Scholar Biotechnol Adv — Article Google Scholar Food Chem — Article Google Scholar Res Microbiol —6 Article Google Scholar Gene — Article Google Scholar Bacteriol Rev Article Google Scholar Biotechniques — Article Google Scholar Front Microbiol Article Google Scholar Article Google Scholar The gene encoding this enzyme is called the LacZ or blue color gene.
The beta galactosidase enzyme will use galactose sugar as a substrate. If a special version of this sugar called X-gal is placed in the media, a neat thing happens inside the bacteria that have the blue color gene. These bacteria will be expressing the gene, making the beta-galactosidase enzyme and will cleave galactose sugar molecules.
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