For quiz 2

• Explain some of the underlying causes for our inability to culture the majority of Earths microbes?

  Specific Environmental Conditions,Complex Interactions,Unknown Growth Factors,Slow Growth Rates,Detection Limitations, Limits in tools
• An axenic culture is a pure culture that contains only one type of organism, without any contamination from other types of cells or microbes.

  1. Sample Collection: First, a sample is collected from the environment
  2. Isolation Techniques: streaking on agar plates, which helps separate individual organisms= single cells.
  3. Enrichment Culture: Sometimes, specific conditions (like temperature, pH, nutrients) are set up to encourage the growth
  4. Selection and Subculturing: Colonies selected and repeatedly subcultured
  5. Verification: confirm that the culture is axenic.
• Explain the concepts of “fast growing weeds” and dilution to extinction, as they relate to microbial isolations

  Fast growing weeds" rapidly multiplying microorganisms that dominate culture environments, often preventing the growth of slower-growing species.

  "Dilution to extinction" is a technique used to overcome this issue. It involves diluting a microbial sample so much that each final dilution likely contains only a single cell.
• Explain why culture-independent techniques are so critical and why, despite their availability, obtaining axenic cultures is still so essential

  Culture-independent techniques are crucial for studying diverse microorganisms in natural environments. They help identify unculturable species and reveal community structures. However, obtaining axenic cultures remains essential for in-depth studies, biotech applications, medical research, and genome sequencing. Axenic cultures allow detailed research on individual microbes.
• Describe the concept of microbial dark matter and provide a brief rationale for the relative paucity of human-associated dark matter vs the unfathomable extent of microbial dark

  Microbial dark matter = unidentified microbes. There's less human-associated dark matter because of extensive research. Environmental dark matter is vast due to diverse, challenging habitats and complex interactions.
• Briefly describe the challenge of cultivating pelagibacter ubique?an obligate oligotroph, meaning it can only grow in the extreme lack of nutrients. Needs light for its proteorhodopsin proton pump (export of H+) It has adapted to the oligotrophic (nutrient-poor) conditions of the open ocean, making it difficult to replicate those conditions in the lab. Cultivation: specialized cultivation medium that mimics the oligotrophic (nutrient-poor) conditions of the open ocean. They also used a technique called 'dilution to extinction' to isolate individual cells and obtain pure cultures.
• Briefly describe the challenge of cultivating Ca. P

  • This archaeon has a unique, tentacle-like morphology and relies on syntrophic relationships with other microorganisms, making it difficult to culture in isolation.
  • Cultivation: Scientists used co-culturing techniques, where they grew Ca. Prometheoarchaeum syntrophicum alongside its syntrophic partners to simulate its natural habitat and encourage its growth. This approach helped in obtaining pure cultures for study.
• Describe the various reasons why PCR amplification of part or all the SSU rRNA gene from metagenomic DNA would be useful

  PCR amplification of the SSU rRNA gene from metagenomic DNA is useful for identifying, classifying, and assessing the diversity of microorganisms in a sample. It aids in functional predictions, comparative analysis, biomarker discovery, community profiling, and experimental design while serving as a quality control step in metagenomic studies.
• Define the term FISH and explain the underlying principle

  FISH (Fluorescence In Situ Hybridization) is a technique that uses fluorescently labeled probes to bind to specific DNA or RNA sequences in cells or tissues. This allows researchers to visualize and locate these sequences within the sample using a fluorescence microscope.
• Explain how multiplex FISH can be used for taxonomic identification of multiple taxa at once

  Multiplex FISH simultaneously uses different fluorescently labeled probes that target specific microbial taxa. By observing the distinct colors emitted when these probes bind to their complementary targets in a sample, researchers can identify multiple taxa at once, providing a comprehensive view of microbial community composition.
• Distinguish between metagenomics & metagenomic DNA

  Metagenomics is the study of genetic material (usually DNA) collected from a complex mixture of microorganisms in an environmental sample.

  Metagenomic DNA refers to the total DNA extracted from a mixed microbial community within an environmental sample, allowing for the analysis of the collective genetic material of all organisms present.
• Define “diversity” as it relates to microbial communities and propose an experimental strategy for determining the taxonomic diversity of a sample

  Diversity means richness ( # of distinctive taxa/sequences) and evenness (relative abundance of each taxon)

  • Proposed experiment using PCR-based SSU variable region profiling AKA amplicon sequencing:

  o PCR amplification of SSU variable regions: product = amplicon

  NGS (next-gen sequencing) either illumina or nanopore

  Bioinformatic pipeline will trim off primers, filter out poor quality reads, and bin OTUs

  BLAST
• Define OTU and explain why that would be used during the characterization of a microbial community, instead of species?

  OTU = operational Taxonomic Unit

  • SSU variable regions aren’t specific enough to detect strains, or even species level. Mostly will only discriminate down to genus level.
• Define the term binning, its purpose and explain how that relates to microbial ecology studies?

  Binning: the process of grouping reads (nucleotide sequences generated via NGS, or Sanger) or contigs (continuous sequences) and assigning them to an individual genome

  The purpose of binning is to recover the microbial genomes from acquired metagenomic data

  • Sequences assigned to bins= share 97% or more homology with
• Discuss the basic principles of SYBR Green/Pico Green qPCR?

  Fluorescent dyes that have binding affinity for dsDNA – increases in fluorescence when bound

  High numbers of amplicons in solution corresponds to high levels of flurorescence

  SYBR green dye only fluoresces when it binds to the dsDNA amplicon

  • Constant light on mixture and monitoring for emissions
• SYBR Green is a fluorescent dye that binds to double-stranded DNA.
• In qPCR, SYBR Green is used to monitor the amplification of a specific DNA target during each PCR cycle.
• During PCR, as the DNA template is amplified, SYBR Green binds to the newly synthesized double-stranded DNA molecules.
• As the DNA strands separate during denaturation in each PCR cycle, the SYBR Green is released and emits fluorescence.
• The fluorescence signal increases proportionally to the amount of DNA amplified.
• More DNA means more SYBR Green binding and fluorescence.
• The amount of fluorescence is monitored in real-time after each PCR cycle.
• By comparing the fluorescence signal to a standard curve of known DNA concentrations, the initial amount of the target DNA in the sample can be quantified.
• Describe an experimental scenario in which qPCR would be used for absolute quantification

  In a medical scenario, qPCR is used for absolute quantification to determine the exact number of copies of a specific gene in a patient's DNA sample, aiding in the diagnosis and assessment of disease severity.qPCR Amplification: Data Analysis:
• Explain how & why qPCR was used in Imachi et al. and Daims et al.

  Used to determine the abundance (concentration) of the target gene (amoA in daims, SSU in imachi) at a certain point in time

  o In the case of

  daims, it wanted to see amoA levels when NH4+ levels decreased and NO3- levels increase
• Describe the underlying principles of stable isotope probing (SIP)

  Stable Isotope Probing (SIP) is a technique used to identify and characterize active microorganisms in a microbial community based on their ability to incorporate stable isotopes into biomolecules

  Isotope Labeling,Microbial Uptake,Density Gradient Centrifugation,Isolation of Labeled DNA,Characterization,Interpretation
• Briefly explain how Nano-SIP combined with FISH, can connect function with taxonomic identity?

  Isolate organisms and label them with a stable isotope, use NAno-SIP to observe the incorporation of the stable isotope into the cells ( what organism will degrade it) and then FISH to tag specific organisms. both will help identify what organism are taking up the stable isotope
• chain termination

  
  

  • Concept: This method is based on terminating DNA synthesis at specific points by incorporating chain-terminating dideoxynucleotides (ddNTPs) during DNA replication.
  • Technique: Sanger sequencing, also known as dideoxy sequencing, is the specific technique that employs chain-terminating ddNTPs to produce a set of DNA fragments of varying lengths, each ending with a ddNTP. These fragments are separated by size to determine the DNA sequence.
• sequencing by synthesis

  
  

  • Concept: Sequencing by synthesis involves the incorporation of labeled nucleotides (A, C, G, T) into a growing DNA strand during DNA synthesis.
  • Technique: Illumina sequencing, a widely used technique, is based on sequencing by synthesis. Fluorescently labeled nucleotides are added one at a time, and as they are incorporated into the DNA strand, the emitted fluorescence is detected, allowing for real-time sequencing.
• direct sequencing

  • Concept: Direct sequencing aims to determine the sequence of DNA without the need for amplification or cloning of the DNA region of interest.
  • Technique: Pyrosequencing, a specific technique, is an example of direct sequencing. It relies on the detection of light produced during the release of pyrophosphate (PPi) when a nucleotide is incorporated into the DNA strand. The light emission is proportional to the number of nucleotides incorporated, allowing for sequence determination without intermediate steps.
• Direct sequencing, also known as Pyrosequencing, is a method that eliminates the need for cloning and amplification steps, making it suitable for complex or unculturable samples.
• Direct sequencing allows for real-time sequencing and can provide results quickly.
• Direct sequencing is useful for studying complex microbial communities, a process known as metagenomics.
• The disadvantages of direct sequencing include limited read length and potentially higher error rates, particularly in homopolymeric regions.
• Direct sequencing requires specialized equipment and reagents.
• describe the steps involved in creating genome or metagenome- derived genomes (MAGs)Extract DNA from samples

  2. Sequence the DNA

  3. Bioinformatic pipeline: trim adaptor sequences and filter poor

  4. Bioinformatic pipeline: assemble the reads into contiguous (overlapping)

  5. Bioinformatic pipeline: identification of already known open reading frames and analyze them with sequence databases

  6. Bioinformatic pipeline: bin fragments into draft genomes to group them as possible species based on species-specific characteristics of their DNA

  7. generate a closed genome