Lecture 3: Flashcards
majority of life is aerobic / anaerobic
anaerobic
- photoautotrophs in light BUT when light not present chemoheterotrophs (dont fix CO2)
respiration & fermentation ___ energy from fixed carbon
liberate
different _____ to Oxygen allow respiration but result in lesser energy as the electrons dont drop as far
Terminal Electron Acceptors (TEAs) i.e. NO3, Fe3+
– fermentative bacteria (fermentate) for resp as well
how many options are there for respiration
lots !
- TEAs
- fermentation
sampling extreme environments
- is challenging
- potentially dangerous
- Don’t want to contaminate
- many microbes are difficult or impossible to culture
- direct study methods are the best way to examine bio-d
can many microbes be cultured
no, lots are difficult or impossible
Culturing :
- valuable approach
- e.g. Sulfolobus acidocaldarius can be cultured in lab, 75 DC, pH2, aerobic, yeast extract
- allows detailed analysis of growth, genetic analysis, manipulations of the environment
__ rRNA genes provide a useful measure of diversity
16S rRNA
- is a componenet of ribosomes
- some regions of the gene are highly conserved, others are highly variable
– advances in gene sequencing (sequence more genes, quicker)
16S rRNA sequencing
- short oligonucleotide primers are synthesised complementary to conserved regions of the 16S rRNA gene
- DNA is isolated from environmental samples or cultured organisms
- the 16S rRNA sequences are amplified by the PCR
- sequence diversity is assessed by sequencing or other methods
- metataxanomics (NOT metganomics as not doing whole genome)
sampling bias: culturing
- due to only some species being able to be cultured, certain species thrive when grown in large inoculums
- by using small inoculums species don’t have to compete
- culture in situ, agar in ground in field
- direct sequencing = no selection pressure, Multitude species, Many rRNA species, still some biases as some organisms difficult to get DNA from
only ___ % of organisms can be cultured
1-10%
example of sampling bias in culturing
- cyanobacteria Synechococcus suggested that cultures isolated from all over the world were identical
- BUT later direct amplification of 16 rRNA sequences (w/o culturing) showed there was as much diveristy in Synechococcus strains in Octopus Spring as was known in ALL cyanobacteria at that time
16S rRNA sequence can be used to study
biodiversity
e.g. cyanobacteria Synechococcus
denaturing gradient gel electrophoresis
- gel w increasing conc of denaturant
- one of the 16S rRNA primers is synthesised with a long GC ‘tail’
- high GC content of the primer means DNA will stick together very strongly
- denature occurs, gel stops
- at diff position from the gel it has a different sequence
- was used before sequencing became a thing
DGGE or sequencing?
DGGE: allows many samples to be processed cheaply, each lane may contain multiple bands, semi-quantitive data can be obtained
sequencing
- more precise
- resolve closely related species
- high throughout sequencing is revolutionising the field
- cheaper
