METAGENOMICS & BIOREMEDIATION

METAGENOMICS & BIOREMEDIATION

1. METAGENOMICS & BIOREMEDIATION

2. BIOREMEDIATION Bioremediation is the process of cleaning up the environment with the help of biological entities. Microorganism-based bioremediation is now regarded as a cost-effective and environmentally benign method for environmental management. They alter their existing metabolic pathways with the help of genetic transformation to breakdown or conversion of contaminants. Micro - organisms like fungus, yeast, or bacteria have long been thought to be superior organisms for pollution detoxification. These are versatile in terms of nutrition and have the flexibility to cope to harsh environmental conditions. They also have a variety of extracellular and intracellular enzymes that break down complicated contaminants into simpler molecules including carbon, water, methane, and sources of energy.

3. METAGENOMICS: Culture-Independent . Insight Metagenomics is a technique for analyzing genetic material extracted directly from environment. Only around 1% of the microbes can be grown using standard microbiological procedures i.e traditional culture-based techniques ( Handelsman 2004). “Metagenomics” is increasingly used for determining the genetic composition of both culturable and non-culturable microorganisms from any source. Jo Handelsman et al. introduced the term metagenomics in 1998 .

4. APPLICATION OF METAGENOMIC IN BIOREMEDIATION A pool of genomes taken from a polluted sample is used in the metagenomic method to identify the genes involved in bioremediation. With advancements in vector selection and construction research, it is now possible to operate effectively on huge genomic segments and then screen vast clone libraries with functioning metagenomes Metagenomics seeks to recognize microbe-related genes in order to better understand the real diversity of microorganisms, their activities, structures, dynamics, cooperation, relationships, and evolution in a range of environments, and so improve bioremediation processes. Stable Isotope Probing (SIP) can be used to increase the RNA, DNA, or phospholipids of dynamic microbial populations. DNA fragments obtained from environment are cloned in a suitable vector [phage, plasmid, bacterial artificial chromosome (BAC)] and then rebuilt into a host bacterium to create metagenomic reference libraries.

5. Continue… Pre- and post-contamination, metagenomic data can reveal taxonomic and enzymatic diversity , allowing for the identification of potentially active genes and species. It will be able to correlate changes in contaminant composition and concentration to individual genes and taxa by accumulating metagenomes from a range of contaminated and uncontaminated similar environment. This will provide answers to concerns regarding the polluted system's microbial ecology , especially how microorganisms respond to the contaminant's perturbation. metagenomic investigations of bioremediation will also offer information on how microbial populations respond to changes in a range of environments.

6. Approaches to Metagenomic Analysis Sequence-Based Analysis Relies on sequence analysis to get a foundation for function prediction. Sequence-based screening consists mostly of two steps: identifying metagenomic reads with desirable sequences ( gene prediction ) and connecting the desirable sequences to a database ( gene annotation ). Gene identification, genome assemblages, elucidating entire metabolic pathways, and comparing organisms from various communities Function-Based Analysis Functional metagenomics is a strong and effective approach for researching the functions of genes . Its purpose is to isolate DNA from environment in order to investigate the functionalities of the encoded protein . DNA fragments are cloned, expressed in a laboratory host, and tested for enzyme activity in functional-based study. This method is dependent on the expression profiles of the clones of the metagenomic library. This method has a lot of potential for detecting new gene segments that code for already-identified or unknown functions. Phenotype-based screening is part of the functional screening method.

7. Workflow of metagenomics research.

8. Metagenomic strategies and tools for bioremediation First generation sequencing (complete genome shotgun sequencing) The first-generation DNA sequencing technologies were Frederick Sanger's and Allen Maxam's—Walter Gilbert's approaches. Sanger sequencing generates DNA fragments of varied lengths using a denatured DNA template, radioactively tagged primer, DNA polymerase, and chemically modified nucleotides termed di-deoxynucleotides. The integrated dNTPs determine the length of the DNA fragment. On gel electrophoresis, the DNA fragments are separated depending on their size and may be seen using an X-ray or UV-light imaging equipment. Since it employs chemicals to break nucleotides, Maxam-Gilbert sequencing is known as the chemical degradation technique. Chemical treatment causes nucleotide base breakage, resulting in a collection of marked fragments that may be separated by gel electrophoresis based on their size.

9. Next generation sequencing (high throughput sequencing) ( i ) Pyrosequencing technique It is a synthesis-based sequencing method that detects the release of pyrophosphate when a nucleotide is added to a freshly produced DNA strand. It is ideal for sequencing small DNA fragments. (ii) Illumina/ Solexa sequencing the DNA sequence is examined base-by-base, hence very accurate. Cells are not required, the throughput is maximum, the reads are relatively short (up to 300 bp), the cost per base is lowest, and the output is suitable with most applications. Sequencing by synthesis with reversible terminators is used to determine the nucleotide in the sequences, with four modified nucleotides, sequencing primers, and DNA polymerases included such that the primers are hybridised to the sequence.

10. (iii) Sequencing by ligation on beads It is made up of several rounds of sequencing. The location of the nucleotide is revealed during sequencing by ligating universal primer to a fluorescently tagged DNA octamer. The procedure is repeated until every base has been sequenced twice, increasing the platform's accuracy. (iv) Ion torrent sequencing It works in a similar way as pyrosequencing technology. This method depends on the discharge of a hydrogen when a dNTP is introduced to DNA polymer instead of fluorescently tagged nucleotides. The incorporation of nucleotides into DNA strands by polymerase produces hydrogen ions as a by-product, which lowers the pH, which is detected by a pH sensor at the microwell's base and converted into a voltage proportionate to the amount of nucleotides integrated.

11. Third generation sequencing (single molecule long-read sequencing) It does not require PCR amplification for sample preparation. ( i ) Pacific Biosciences Fluorescent labelling, like other sequencing methods, is used in this method. In real time, it identifies nucleotide signals. Each base is enzymatically incorporated, resulting in a flash of light that identifies the base and is analysed repeatedly to form the DNA sequence

12. (ii) Oxford nanopore technology The DNA/RNA molecule is passed through a nanopore using electrophoresis. It makes use of electrolyte solution as well as a constant electric field . A termination repair stage shears double-stranded DNA and forms blunt-ended DNA molecules with this technique. The DNA is then modified with two adaptors (a Y adapter and a hairpin adaptor ) coupled with a unique motor protein that aids in unzipping the double-stranded DNA at the Y adapter and moving the DNA as a single strand via the nanopore. The activity of the motor protein as the nucleic acid travels through the nanopore creates a change in ionic current due to mobile nucleotides filling the pore. The ionic current variation is graphically shown and then clarified for sequence identification . (iii) HeliScope It is another technological platform for single DNA molecule sequencing that uses an exceptionally sensitive fluorescence detection device. Restriction enzymes fragment DNA strands, which are identified by the insertion of a poly-A tail . The DNA molecules are hybridized to the flow cell plate, which has billions of oligo(dT) chains attached to its surface, resulting in an array of primer-annealed single DNA templates . Labelling is done in " quads ," which are made up of four cycles for each of the four nucleotide bases . A template-dependent extension is created by adding fluorescently labelled bases one at a time. The label is illuminated by a laser light , which reads the strands that have taken up a specially designated base, which is then recognized and recorded by a camera. These signals are translated into a nucleotide sequence by a variety of computer systems. The label is then cleaved , and a fresh base is used in the following cycle.

13. Bioinformatic tools for metagenomic bioremediation Bioinformatics performs a variety of functions in the field of metagenomic bioremediation, most notably during the analysis of metagenomic data.

14. A comparative overview of functions and suitability of mostly used tools for metagenomic analysis +Yes, -No