Week 11 Flashcards

Question

What is the method for High throughput sequencing of rRNA gene PCR products?

Answer 1

Take sample Prep gDNA Carry out PCR on gDNA using 16S or 18S primers and purify PCR product Amplicon sequence the product: Prepare a sequencing library (normally Illumina) –a process that adds short DNA adaptors enabling sequencing Sequenced en masse (typically Illumina MiSeq) Given all sequence reads ~30 million rRNA gene sequences Bioinformatics analysis to classify different microbes in the community

Answer 2

Can say % of pretty much any bacterium present in a sample Much more information than from DGGE

Answer 3

Gives a clue into which bugs are undergoing the process of interest eg catabolishing DMS Same method as in previosu flashcard it promotes growth of the bacterial species that undergo a large amount of bacterial growth

Answer 4

When combined these techniques allow you to identify microorganisms important in your process of interest These techniques are universal

Answer 5

This is the process of applying high throughput sequencing on metagenomic DNA. Will give taxonomic information on the community (based on all genes in data)

Answer 6

Gives info on the metabolic potential of the community via indicating the abundance of diagnostic gene that drive processes, e.g. ddd genes for DMSP lysis Potential to identify the key microbes driving your process, e.g. the most abundant microbe that has your process gene

Answer 7

The percent composition of an organism of a particular kind relative to the total number of organisms in the area. A relative abundance of 80% means that Piscirickettsiaceae are predicted to comprise 80% of the bacteria in the sample. This can also be applied to genes, but in this case you gene of interest is normalised to genes that are known to be single copy in genomes, e.g. recA in bacteria

Answer 8

Part of the basic metabolic processes of the cell or its physical structure they are essential for survival

Answer 9

Organic acids and sugars Aminoacids Nucleosides Fatty acids and other lipids Vitamins Their intermediates in biosynthesis/degradation pathways

Answer 10

Fermentation of alcohol Shikimic acid pathway, present in all organisms except animals involved in the generation of precursors of aromatic amino acids, vitamin cofactors and other metabolites Vitamin B12 or cobalamin, is biosynthesised by some bacteria and archaea. We are not able to make it and have to incorporated it through the diet

Answer 11

Primary metabolites are essential for the normal growth, development, and reproduction of the organism They are also the building blocks for secondary metabolism and other non-essential processes

Answer 12

Also called specialised metabolites or natural products Small organic molecules not directly involved in the normal growth, development or reproduction of the organism

Answer 13

They mainly mediate environmental interactions both with other bacteria and fungi, as well as with higher organisms (chemical ecology) Technically non-essential, but can confer selective advantages for the producer organism

Answer 14

Competition Communication Toxicity/virulence Nutrient scavenging Protection against toxic chemicals/ UV

Answer 15

Natural products are derived from primary metabolites, but have their own specialised biosynthetic pathways

Answer 16

1 trillion bacteria, or 95% of all the bacteria in our bodies All use secondary metabolites for competition and cooperation between other bacteria living in the gut

Answer 17

Differential distribution in the different areas of the soil and the plant, and also inside the insects, both as symbiotic and beneficial partners or as pathogens. Plants produce nutrients in form of exhudates and other compounds that repel or attract specific microbes. Produce different molecules, including plant hormones, stimulate immune system, volatile compounds Microbes also communicate among themselves both in antagonistic (e.g. antibiotics) and synergistic ways (e.g. quorum sensing molecules)

Answer 18

Bacterial chromosome the genes involved in biosynthesis or secondary metabolites are located contiguously forming BIOSYNTHETIC GENE CLUSTERS (BGCs)

Answer 19

Composed of one or several operons Expression of the genes in the cluster is coordinated by specific regulation responding to environmental factors Genes encode enzymes that create the backbone of the molecules and introduce tailoring modifications BGCs can span from 5 kb bp to well over 100kb

Answer 20

Diversity and distribution of BGCs is highly enhanced by spontaneous mutations and horizontal gene transfer. BGCs are also great targets for gene editing and engineering techniques.

Answer 21

Polyketides Non ribosomal peptides Ribosomal peptides (RiPPs) Terpenes Alkaloids

Answer 22

Produced by the condensation of multiple simple ketone precursors (acetyl CoA, malonyl CoA…).

Answer 23

Biosynthesised by modular multienzyme complexes called polyketide synthases (PKSs) function in an assembly line-like fashion – each module incorporates one extender unit

Answer 24

After release from the PKS, the products are often cyclised and further modified by other enzymes in the pathway.

Answer 25

Made from proteinogenic and non-proteinogenic amino acids

Answer 26

Biosynthesised by modular multienzyme complexes called non ribosomal peptide synthetases (NRPSs) that function in an assembly line-like fashion – each module incorporates one extender unit, like in PKSs

Answer 27

The peptide backbone can also suffer multiple tailoring modifications

Answer 28

Bleomycin - have other molecules like sugars attatched

Answer 29

Ribosomally synthesised and posttranslationally modified peptides (RiPPs)

Answer 30

The peptide backbone of the molecule is encoded within a short gene present in the BGC After translation, this short peptide (precursor peptide) is modified by other tailoring enzymes included in the cluster, including cleavage of leader, follower sequence(s) Originally made of proteinogenic amino acids, the tailoring modifications can hugely modify the final product

Answer 31

Nisin: produced by Lactococcus lactis and used as a food preservative. It has 34 amino acid residues (allows for lager peptides than NRPSs) Thiostrepton: produced by Streptomyces laurentii. Core has 17 aminoacids out of 54 the full precursor. Antibiotic used in veterinary medicine Thioalbamide: produced by Amicolatopsis alba antitumor compound

Answer 32

Natural products built from polymers of isoprene, often volatile

Answer 33

The enzymes involved in the biosynthesis of these molecules are called terpene synthases. They take activated forms of isoprene units and condense them to make longer carbon chains that can be cyclised and further modified by other enzymes in the cluster.

Answer 34

The main producers of terpenes are plants, but fungi and bacteria also produce them. Brasilicardin, first discovered as a product of Nocardia

Answer 35

Gibberellin A4 plant hormone, but also found to be biosynthesised by fungi and then multiple Gram- bacteria from the soil

Answer 36

Diverse and loosely defined family of natural products. Basic molecules containing at least one nitrogen, usually as part of an heterocycle. Plant and fungi are the most prevalent producers, but there are also hundreds of bacterial alkaloids.

Answer 37

Alkaloids have varied biosynthetic origins, but are mainly derived from amino acids. Alkaloids can come from different biosynthetic origins (aminoacids, terpenes, poliamines) and even the boundaries of what are considered alkaloids is very blurry.

Answer 38

Many alkaloids have relevant pharmacological applications. The best known are analgesic, stimulants, psycotropic molecules or toxins

Answer 39

Ergotamine, vasoconstrictor used alongside caffeine to prevent migraines. Fugu, or Japanese puffer fish bioaccumulates tetrodotoxin of likely bacterial origin

Answer 40

Specialised metabolites mediate the communication between microorganisms and the response to changes in the environment (nutrient depletion, presence of competitors, unfavourable abiotic factors)

Answer 41

Bacteria can monitor their own cell density and display coordinated population behaviours through the secretion of signalling molecules in their environment

Answer 42

Motility Biofilm formation Onset of virulence Production of other secondary metabolites

Answer 43

N-acyl homoserine lactones are quorum sensing molecules in gram- bacteria Gram+ this is mediated by autoinducer peptides, There is a wide range of signalling molecules and only a part of them are involved in quorum sensing mechanisms.

Answer 44

Aliivibrio fischerii (formerly known as Vibrio fischerii) are symbionts of the bobtail squid Euprymna scolopes The squid provides a colonisation niche for the bacteria, whereas the bacterial luminescence provides protection against predators for the squid

Answer 45

Broad definition can include all molecules that inhibit the growth or kill other organisms

Answer 46

Other bacteria (antibacterial molecules) Fungi (antifungals) Higher organisms (insecticides, nematicides, cytotoxic molecules, herbicides)

Answer 47

Production is usually triggered by nutrient depletion in the environment or when competing organisms are detected

Answer 48

Gram-positive, high GC content bacteria, often filamentous, with huge morphological diversity Environmental bacteria, traditionally associated with the soil, but present pretty much in any habitat

Answer 49

It is estimated that around two-thirds of all antibiotics in use today are natural products from actinomycetes or semi-synthetic derivatives of them

Answer 50

Within Actinobacteria, the genus Streptomyces is particularly talented: just this genus is responsible for 30% of all known bioactive natural products

Answer 51

Streptomyces dedicate large parts of their genome to biosynthetic gene clusters and each strain has the potential to produce dozens of secondary metabolites

Answer 52

Molecules with high affinity for iron (and other essential metal ions). Produced in response to low levels of iron, they are secreted to the environment to scavenge it and retrieve it for the cell. Diverse biosynthetic origins, but many of them are non ribosomal peptides or hybrid PKS-NPRS products

Answer 53

Enterobactin - a catecholate Pyochelin - phenolate Alcaligin - Hydroxamate Rhizoferrin - a carboxylate

Answer 54

Uptake among clonal cells - both benefit Cheater - cheater benefits and actor loses Locking away - actor benefits and other loses Competition with siderophores - both small benefit

Answer 55

Pseudomonas aeruginosa, an opportunistic pathogen responsible for multiple hospital infections, uses iron acquisition as an additional virulence factor

Answer 56

Use potent siderophores to get iron from environment Use siderophores to strip iron away host cells

Answer 57

Natural products have many diverse functions, but often end up affecting gene expression and help microorganisms adapt to changes in their environment

Answer 58

Antibiotics targeting transcription or translation. Signalling molecules that activate or repress expression of specific genes

Answer 59

Antibiotics - huge discovery from 30s to 70s then barely any after, and no antibiotics for gram negative Food biosecurity - natural products to help increase productivity through pesticides

Answer 60

Sanger sequencing was the original DNA sequencing method to identify and determine the sequence (nucleotide) of DNA. Enzymatic method of sequencing developed by Fred Sanger in 1977

Answer 61

A typical electropherogram each peak represents a single nucleotide in the DNA sequence, andeach nucleotide has a different colour: A is green T is red C is blue G is black

Answer 62

Template DNA DNA polymerase Nucleotides Primers Fluorescently labelled dideoxynucleotides (lacks OH group to prevent extension of DNA seqeunce)

Answer 63

A laser beam excites the fluorescently labelled dideoyxnucleotide after gel electrophoresis to shape the size of the strand. The colour produced form excitement shows the base

Answer 64

The Human Genome Project (HGP) was an international scientific research project with the goal of determining the base pairs that make up human DNA, and of identifying, mapping and sequencing all of the genes of the human genome. Completed in 2003 (92% of the genome, $3B), then in 2021 (99.97%), but fully finished in 2022

Answer 65

454-pyrosequencing Illumina sequencing

Answer 66

DNA polymerase ATP sulfurylase Luciferase Apirase

Answer 67

When dNTP is added in the DNA during the PCR (DNA polymerase), pyrophosphate is released. ATP sulfurylase converts pyrophosphate to ATP. ATP powers oxidation of luciferin by luciferase which generates a light signal recorded as a pyrogram pick. Apirase removes any unincorporated nucleotide remaining in the reaction

Answer 68

Ligation of adaptors to DNA fragments Emulsion of PCR Break emulsions and deposit beads into picotiter plate Pyrosequencing occurs, using enzymes

Answer 69

Library prepared - DNA flanked by adaptors Cluster formation - DNA amplified on a slide Sequencing - Fluorescent terminator base anneals are added to DNA stran is completed then Laser removes fluorescent terminator Sequence identification using sequencing both 3' and 5' end

Answer 70

Sequencing of 16S rRNA V3-V4 sequenced by Illumina For rRNA sequences by PacBio

Answer 71

1 - A fluorescently-labelled nucleotide associates with the template in the active site of the polymerase. 2 - The fluorescence output of the colour corresponding to the incorporated base is elevated. 3 - The dye-linker-pyrophosphate product is cleaved from nucleotide and diffuses out, ending the fluorescence pulse. 4 - The polymerase translocates to the next position. 5 - The next nucleotide associates with template in the active site of the polymerase, initiating the next fluorescence pulse

Answer 72

Pass a DNA molecule through a nanoscale pore in a membrane from head to tail and read off each base when it is located at the narrowest constriction of the pore, using the ion current passing through the pore to probe the identity of the base

Answer 73

Genomes may be analyzed and ORFs assigned to COG categories Assignments are based on homology Demonstrates the relative importance of processes to each organism Reflects the ecology of that organism

Answer 74

Metagenomics is the study of the collective genome of microorganisms from an environmental sample to provide information on the microbial diversity and ecology of a specific environment

Answer 75

Metagenomics provides a relatively unbiased view not only of the community structure but also of the functional (metabolic) potential of a community.

Answer 76

Sample Extract DNA Sequence Assembly Binning (MAGs) Functional annotation Metabolic reconstruction

Answer 77

Construct as many and as much of the sequenced genomes as possible Assign taxonomy and function

Answer 78

k-mer refers to all of a sequence's subsequences of length ie 3-mers use 3 base sequence Slowly build up the size of the Kmers

Answer 79

Identify ORFs (Operational Reading Frames) and assign function to assembled sequences

Answer 80

Phred quality score allows for data control which assings each base reading a symbol which can be used to generatir a Phred Quality score

Answer 81

10 - 1in 10 - 90% accuracy 20 - 1in 100 - 99% accuracy 30 - 1in 1000 - 99.9% accuracy 40 - 1in 10000 - 99.99% accuracy

Answer 82

HPC provides access to a variety of hardware that can support intensive computation, including high memory, GPU, parallel and standard compute nodes

Answer 83

A scheduler (queueing system) that takes user’s jobs and submits them to the best-fit node in the requested queue taking into account the job’s requirements.

Answer 84

Pools of compute nodes (specialist computer hardware) which provide lots of CPU and RAM for cluster users to use. Storage which is available throughout the cluster, giving each cluster user a home directory (250GB, backed up) and a scratch directory (500GB, not backed up).

Week 11 Flashcards

(109 cards)