L14: Culture Independant Microbiology Flashcards
What are the benefits of environmental microbiology
- in lab
- bypass limitations of culture based by extracting nucleic acids directly
- manipulate + chartacterise individual DNA + RNA molecules from complex microbial communities
- helps moniter populations
- helps with biodegredative pathways/ biogeochemcal cycling
What are the 4 types of molecular biology techniques
- nucleic acid analysis
- liquid profile analyeses
- fluorescent in situ hybridiation
- linking phylogentics to functions
What are the steps in nuclec acid analsys
- extraction of total community nucleic acids from environmental samples
- PCR dependant methodologies
- methodologies to characterise PCR amplification products (cloning + sequencing of 16S rRNA fragments)
What makes good nucleic acid analyses
- reliability
*yield - purity
- shearing degree
Compare extraction of DNA, proteins, enzyme, mRNA
- can extract DNA cus stable + proteins enzymes, mRNA is harder and rapidly degraded
Step 1 of nucleic acid analyses (aim, main component, main success, success depends on)
aim: high quality/quantitiy extract to provide snapshop of indigenous microbial community
main component: successful extraction + purification of a good amount of NA
main success: soil DNA isolation good in reliability, yield, purity, shearing degree
what does success depend on: source of sample, co-extracted interfering substances like heavy metals, pollutants)
What are 2 general methods in nucleic acid extraction
a. direct lysis of microbial cells by chemical treatments, freeze-thaw, sonication or beatng => DNA shearing
b. extraction of bacterial cerls from the samples then cell lysis
What is often extracted among soil, what happens
- humic + fulvic acids are co-extracted from soil with NA
- must be removed cus it interferes with next moleculare reaction
- DNA polymerae amplification
- DNA DNA hydrbirdation
- DNA labelling
- Restriction nuclease digestion
What are the problems in nucleic acid extraction, + fix
- physical, chemical, biological step in prep is source of bias
- was there suffucient/preferential disruption of microbial cells
- rigorious conidtion for gram+ lyse could lead to excessive shearing of gram= cells
- reported diversity of sample becomes bias
- creates artifcants + chimeric PCR products
- fix: checking soil via argose gel electro indicate quality and extent of sheeing
How can nucleic acids be purified
- chromatography
- silicia gel columnPVPP spin filter
- magnetic bands
How does quantification take place + problems
- quality + quantity by argose gel electro
- spectrometry ⇒ nanodrop
- fluoremeter ⇒ qubit
- problem: spectrometry is inaccurte in comparison
Why is it PCR dependant called that, and what is the main objective
- name: cus reliatn on PCR amplification
- obj: amplify target gene sequences from community so they can be characterised
What are the steps in the PCR procedure
- denaturation: dsDNA seperarted to ss at high temps (denatures)
- annealing: two oligonucleotide primers anneal at lower temps to complementary regions that flank the target sequence of ssDNA
- Extension: heat stable DNA polymerase sytheises new strand be extending the primer using complementary strand as a template ⇒ makes duplicate copy of the target sequence
- repeated 20-30 times = exponentioal amplifcation of target
What is amplified, with examples
- functional genes of interest
- contaminated soil: catabolic genotypes to see biodegradative genotypes to see functional microbial populations to see the appropiate strategies
- phylogentic/taxonomic genes of interest
- two types or rRNA in ribosomes(pro: 16s, 23s, 5s // euk: 18s,28s,5.8s)
Describe ribosomal components
- 30S: small subunit
- 16s + 21 proteins
- 50s: large subunit
- 23S + 5S + 30 proteins
What makes rRNA good chronometer + phylogenetic markers
- 16s highly conserevd and is essential, low mutation
- also has signature sequence motifs that is hypervariable and differentiates phylogentic distances
List other phylogenetic markers
- chaperonin 60
- rpoB
- gyrB coding for topoisomerase II
How are PCR primers selected
- based on superkingdom, family, genus, subspecies
- cover many conservaed + variable regions
What is the routine procedure in 16S rRNA sequencing
- amplification of gene encoding 16S rRNA = PCR
- sequencing amplified genes = illuminia miseq
- analyzing of sequence in ref to other sequenences ⇒ Bioindormatics
- Identify microorganisms bases on 16S rRNA sequences, determine community composition
- Based on identities, extrapolate function / metabolisms of the microbial community inhabiting / detected in a given sample
- Use bioinformatics / ecological statistical methods to determine diversity, compare similarity between samples
What happens in 16S sequence comparison
- when samples are sequenced, you submit to blast and fasta for comparison
- they approximate Smith Waterman but needs a lot of computing power
- want similar hits, top e-value otherwise study conserved regions, variable regions, chimeras
FASTA
fast approximation
How does tree analysis work
- distances are relative, distance is relative to all others in the set
- nodes spin to analyse relationships
- bootstrap to determine phylogenetic accuracy by resampling dataset + % of times group occurs
What is the bootstrap method of tree analysis
dataset resampled and % of times group occurs is calculated
What is the caveat of rRNA
rrn operon: 16S, 23S, 5S, internal spacer + 1 tRNA
How many rRNA operons can baccterium posses
- rrn copy numbers:
- bacterial genomes have max 15 operons
- 7 is most common
- 40% organisms have 1-2
- divergence among operons is small, <1% nucleotide difference
- very few (5) with higher level nucleotide divergence is detected
- in themophilic bacteria meaning HGT probably happended in this type
What is the role of rRNA in relationships
Genome sequences currently confirm that the rRNAs provide a relatively solid framework for the estimation of phylogenetic relationships
Go deep into step 3 of nucleic acids analyses
Step 3 = methodologies to characterise PCR amplification products: cloning and sequencing of 16S rRNA fragments
What are the steps in cloning + sequencing 16S rRNA fragments
- 16S rRNa is PCR amplified then cloned to PCR fragment vector to make clone library
- unique clones are screened by ARDRA which is a RFLP
- the 16S rRNA fragments are sequenced and origin/identification determented by BLAST or FASTA
What is the general approach for cloning and sequencing 16s fragments
- compare SSU rRNA genes (16 for pro, 18 for euks)
- SSU rRNA gene similarity isued to define OTU of working species and diveristy is measured by rarefaction curve
What is OTU and how is species diversity measures
- operational taxonomic units to measure gene similarity
- rarefraction curve for species diversity
- plotting # of OTUs as function of increasing sample size
List the phlyogentic composition of bacteria 16S rRNA clone library
- high GC gram positive
- delta-proetbacteria
- alpha proteobacteria
- CFB
What are examples of new technologies and what do they help us do
- 454 pyroseqeuncing, illuminia, oxfoed nanpore
- generates larger 16s rRNa database
What is the problem with PCR-Dependant Techniques
- very dependent of PCR primers and can be too specific
- only looks at what we already know
- PCR amplifies errors
- hard to quantify genotypes for environmental samples BUT qPCR is making it better
- only amplifies limited regions ⇒ limits the amount of useful sequencing information
What are other molecular techniques in env microbio
- nucleic acid analyses
- lipid profile analyses
- FISH
What are examples of lipid profile analyses
- FAME analysis by GC (gas chromatrography)
- based on extraction of membrane lipid components
- then you seperate, identify and quantificaton of membrane fatty asids
- good for new species, using fatty acids as biomarkers
What is FISH
- fluorescent in sity hybridisation
- microscopic tech using biofilrms
- put sample on slides and cells are fixed
- specific cells are hydrbised with specific fluorescently labeled probe targeting 16S
- singal detected + quantified by fluroecent microsopy/CSLM
- determine composition + spatial distribution in situ
- enchamces signal CARD-FISH
What methods are used to linking phylogenetics to function
- stable isotope probing (SIP) technique
- MAR-microautoradiography-FISH
What is SIP tehcnique
- identifies specific groups resposible for in situ biogeochemical tranformation process
- identify active members in communities (methanogens…) by linking metabolic activity to diveristy using isotopes
- microorganisms metabolisim a stable isotope incorportate it into DNA
What is MAR Fish
- use radioisotopes to measure microbial activity
- follows in vivo uptake of radiolabeled substraces with speicifc organism idenitified by fish
- linking specific genes and function to specific organisms
- combines phylogenty activity of cells
