Journals / Bitlis Eren Üniversitesi Fen Bilimleri Dergisi / 2020 / Cilt: 9 - Sayı: 1
P1 transduction method to construct Escherichia coli mutant strains lacking more than one gene
- Pages
- 143–156
- DOI
- —
Abstract
Assigning gene functions and performing phenotypic studies of the bacteria require the construction of specific mutants that at times should lack more than one gene due to the redundancy of particular gene activities. P1 phage is a generalized transducing phage that can be used in constructing double, triple or multiple mutants of Escherichia coli. This phage is able to mistakenly package random fragments of E. coli chromosome (~2 % of the genome into each particle) into the phage head during phage development. The phages with E. coli DNA can inject that DNA into recipient cells. Given homology between donor and recipient, and a selectable marker, transductants can be obtained. For transduction, a virulent derivative of phage P1 is preferably used because this form cannot integrate into the bacterial genome. The virulent phage can only develop using the lytic mode of development and produces phage progeny upon lysis of the host cell. This technique is based on recombination technology, allowing the DNA fragments of the E. coli genome to be transported among the strains. In this study, we optimized the P1 transduction methodology, which we aim to present to the Turkish scientific community in the native language with the experimental flow and sufficient details. For this reason, a number of four single gene mutants obtained from the KEIO collection were used. First, kanamycine (Kan) gene cassettes were removed from each mutant strain to create a recipient strain that is kan sensitive. The other four KEIO mutant strains with KanR cassettes were used as the donor strains and P1 phage were cultured with these strains and the lysates were obtained. These lysates and recipient strains were then transduced to integrate the DNA fragments of the lysates into the recipient strain genome. Upon selection with kanamycine, new mutant strains, double mutants are obtained. As a result, four double mutants namely DtsgAugpA, DyhdTompA, DugpAmdtG and DaroGmalF were obtained and the details pertaining to the protocols and results are presented and discussed.
Özet
In this study volume density of gray and white matter of cervical segments of spinal cords of rats were investigated using stereological method. Twelve male Wistar albino rats were used in the study as two different age groups 1 month and 5 months. All animals were fixed by perfusing 10% buffered formalin. Rats were dissected and spinal cords of rats were removed. In the study cervical segments of 1 and 5 months age rats were obtained. One of the first 15 section were selected randomly when the sections were taken. And following every 50th section was determined by systematic random sampling. Thus, 8-10 sections of 5µm thickness were obtained from cervical segments of each animal’s medulla spinalis. These sections were stained by hemotoxylin eosin and they were photographed at microscope. Densities of volumes of all tissue of cervical segments of whole spinal cord and white and gray matters were calculated using dotted area ruler by Cavalieri Principle. SHTEREOM 1.5 package programme was used for counting dotted area. In addition, the volume vales of total cervical segment, volume values of the white matter and the gray matter and the ratios of these volume values to each other were evaluated in the study.