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Agricultural biotechnology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Traditional plant-breeding techniques are very time-consuming and sometimes a lot of undesired genes are introduced into the genome of a plant. The undesired genes have to be “sorted out” by backcrossing. The use of restriction fragment length polymorphism (RFLP) greatly facilitates conventional plant breeding, because one can progress through a breeding program much faster, with smaller populations, and without relying entirely on testing for the desired phenotype. RFLP makes use of restriction endonucleases and these enzymes recognize and cut specific nucleotide sequences in DNA. For example, the sequence GAATTC is cut by the endonuclease EcoRl. After treatment of a plant genome with endo-nucleases, the plant DNA is cut into pieces of different lengths, depending on the number of recognition sites on the DNA. These fragments can be separated according to their size by using gel electrophoresis and are made visible as bands on the gel by hybridizing the plant DNA fragments with radiolabeled or fluorescent DNA probes. As two genomes are not identical even within a given species, due to mutations, the number of restriction sites and therefore the length and numbers of DNA fragments differ, resulting in a different banding pattern on the electrophoresis gel. This variability has been termed RFLP. The closer two organisms are related, the more the pattern of bands overlap. If a restriction site lies close to or even within an important gene, the existence of a particular band correlates with the particular trait of a plant, such as disease resistance. By looking at the banding pattern, breeders can identify individuals that have inherited resistance genes, and resistant plants can be selected for further breeding. The use of this technique not only accelerates progress in plant breeding considerably but also facilitates the identification of resistance genes, thereby opening new possibilities in plant breeding.
Plant Biotechnology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
Traditional plant-breeding techniques are very time-consuming and sometimes a lot of undesired genes are introduced into the genome of a plant. The undesired genes must be “sorted out” by backcrossing. The use of restriction fragment length polymorphism (RFLP) greatly facilitates conventional plant breeding, because one can progress through a breeding program much faster, with smaller populations, and without relying entirely on testing for the desired phenotype. RFLP makes “use of restriction endonucleases and these enzymes recognize and cut specific nucleotide sequences in DNA. For example, the sequence GAATTC is cut by the endonuclease EcoRI. After treatment of a plant genome with endonucleases, the plant DNA is cut into pieces of different lengths, depending on the number of recognition sites on the DNA. These fragments can be separated according to their size by using gel electrophoresis and are made visible as bands on the gel by hybridizing the plant DNA fragments with radiolabeled or fluorescent DNA probes. As two genomes are not identical even within a given species because of mutations, the number of restriction sites and therefore the length and numbers of DNA fragments differ, resulting in a different banding pattern on the electrophoresis gel. This variability has been termed RFLP. The closer two organisms are related, the more the pattern of bands overlap. If a restriction site lies close to or even within an important gene, the existence of a particular band correlates with the particular trait of a plant, such as disease resistance. By looking at the banding pattern, breeders can identify individuals that have inherited resistance genes, and resistant plants can be selected for further breeding. The use of this technique not only considerably accelerates progress in plant breeding, but also facilitates the identification of resistance genes, thereby opening new possibilities in plant breeding.
Approaches for increasing bioremediation capabilities of plants and microorganisms towards heavy metals and radionuclides
Published in Rym Salah-Tazdaït, Djaber Tazdaït, Phyto and Microbial Remediation of Heavy Metals and Radionuclides in the Environment, 2022
Rym Salah-Tazdaït, Djaber Tazdaït
A system based on the same principle has been developed by Torres, Jaenecke, Timmis, Garcia, et al. (2003) to limit the horizontal transfer of conjugative plasmids carrying cloned information. As in the previous case, and for the same reasons, it has two suicide functions. However, these were no longer induced in the absence of pollutants but when the plasmid which carries the suicide genes enters by conjugation in a bacterium not belonging to the constructed strain. The authors introduced into a conjugate plasmid of the HB101 strain of E. coli the colE3 gene coding for colicin E3, capable of killing many bacteria by destroying their 16S ribosomal RNA. In the chromosome of this strain, they introduced the gene immE3, which constitutively produces an antidote to colicin E3. Therefore, the strain is protected from the effects of colicin, but when the plasmid enters a receptor bacterium lacking the imm3 gene, colicin E3 exerts its effect and kills this bacterium. In addition, the plasmid carries a gene encoding the restriction enzyme EcoRI. This enzyme cuts all DNA when it recognizes a particular sequence of six base pairs. The strain of E. coli carries in its chromosome the gene coding for the corresponding modification enzyme, methylase, which protects the DNA sequences specifically recognized by EcoRI by methylating them. The DNA of the E. coli strain is therefore not cleaved by EcoRI. On the other hand, when the plasmid is introduced into a bacterium devoid of methylase, EcoRI destroys its DNA almost instantaneously. The frequency of transfer of the plasmid carrying this construct was reduced by a factor of 108 compared to the transfer of the same plasmid lacking the suicide system. This latest study describes the construction and evaluation of a laboratory strain. The use of this type of construction in natural conditions, however, raises two questions. The first concerns the expression of suicide genes in bacteria belonging to species other than E. coli. The promoters of the two genes are promoters of E. coli: their expression in other genetic contexts is not a certainty. The recipient bacteria which do not express them thus escape suicide. The second question relates to the dissemination of suicide genes by plasmids conjugative. The confinement of cloned genetic information would be drastic, but it would be done by the physical elimination of all bacteria capable of receiving these plasmids. The diversity of the environment concerned could be seriously affected (Torres, Jaenecke, Timmis, Garcia, et al. 2003, 3595–600).
Engineered Pseudomonas putida for biosynthesis of catechol from lignin-derived model compounds and biomass hydrolysate
Published in Preparative Biochemistry & Biotechnology, 2022
T4 DNA ligase, restriction enzymes (EcoRI, HindIII, kpnI, BamHI, PstI), shrimp alkaline phosphatase were purchased from New England Biolabs USA. PrimeSTAR Max DNA polymerase (2×) high fidelity PCR master-mix was purchased from Clontech (DSS TaKaRa Bio India Pvt Ltd.). Dream Taq Green PCR Master Mix (2×) was procured from ThermoFisher Scientific (India). NucleoSpin® Gel and PCR Clean-up kit was purchased from Macherey-Nagel (MN, India). Miniprep kit was purchased from GeneAll USA. Primers were procured from Eurofins Genomics, India.
Biotransformation of corn bran derived ferulic acid to vanillic acid using engineered Pseudomonas putida KT2440
Published in Preparative Biochemistry & Biotechnology, 2020
Priya Upadhyay, Nitesh K. Singh, Rasika Tupe, Annamma Odenath, Arvind Lali
T4 DNA ligase, restriction enzymes (EcoRI, HindIII), shrimp alkaline phosphatase were purchased from New England Biolabs, USA. PrimeSTAR Max DNA polymerase (2×) high fidelity PCR master-mix was purchased from Clontech (DSS TaKaRa Bio India Pvt Ltd.), and Dream Taq Green PCR Master Mix (2×) was procured from Thermo Fisher Scientific (India). NucleoSpin® Gel and PCR Clean-up kit was procured from Macherey-Nagel (MN, India) while Miniprep kit was from GeneAll, USA. Primers were procured from Eurofins Genomics, India.
Degradation of antibiotic resistance contaminants in wastewater by atmospheric cold plasma: kinetics and mechanisms
Published in Environmental Technology, 2021
Xinyu Liao, Donghong Liu, Shiguo Chen, Xingqian Ye, Tian Ding
DNA gel electrophoresis was used for the estimation of potential plasmid breakage after plasma treatment. The plasmid pBR322 was incubated with restriction enzyme EcoRI (Beyotime Biotechnology Inc., Shanghai, China) at 37°C for 1 h to obtain the linearized plasmid form. The gel electrophoresis was performed on 0.8% agarose gels at 3 V/cm for 50 min. The bands were visualized using a long wave UV light box and photographed.