Midterm 2 Flashcards
Coulter counters
A known volume of microbial suspension is forced through a small orifice
Movement of microbe through impacts the electric current (increases resistance) - these instances are counted
Flow cytometry
Similar to coulter counters but a light beam is disrupted rather than an electrical current.
Petrroff - Hausser Counting Chamber
Need to know the volume that was added (get a concentration)
Know the volume of the liquid based on the volume of the area under the coverslip.
What are viable counts?
Measurement of living, reproducing population of cells.
Ways to get viable counts?
Spread plate or pour plate method
- spread plate - can surface colonies
- pour plate - get surface and subsurface colonies
Most probable number technique
Similar to plate counts - but in LIQUID culture
Used to estimate the number of microorganisms in food, wastewater, and other samples.
Good for rapid determination of approximate populations
Indirect counting methods
Optical density, cell biomass, measuring of a cell component (like proteins), gene counting using quantitative PCR
Measure rate of production of a cell component or rate of consumption of a substrate
The most important indirect counting method in biol labs
Optical density! It is fast and has pretty good measurement of the actual cell count.
- shine light through culture to measure how much light gets scattered through
- the amount of scattered light is proportional to the amount of cells in the sample.
Too many cells –> get scattering effects however
what is a batch culture?
Growth in a closed system, medium NOT replenished
Growth conditions are constantly changing; it is impossible to control growth parameters
Continuous culture
An open system microbial culture of fixed volume.
Allows bacteria to always be in a state of growth
chemostat
the most common type of continuous culture
Both growth rate and population density can be controlled independently and simultaneously
The growth rate is controlled by the dilution rate
The growth yield (cell number/ml) is controlled by the concentration of the limiting nutrient
Both growth and population density of culture can be controlled independently and simultaneously
Dilution rate
Rate at which fresh medium is pumped in and spent medium is pumped out
What keeps the volume constant in a chemostat or continuous culture
Growth rate is controlled by the dilution rate
D = F/V where F is the flow rate and V is the culture volume, D is the dilution rate
What is controlled by the concentration of the limiting nutrient in a chemostat?
The growth yield (cell number/ml)
= density
What is controlled by the dilution rate in a chemostat?
The growth rate
Dilution Rate formula
D= F/V
where F is the flow rate and V is the culture volume, D is the dilution rate
What happens when a chemostat culture has too high of a dilution rate?
Organism is washed out
\What happens when a chemostat culture has too high of a dilution rate?
Organisms may die of starvation
Uses of a chemostat?
Fermentations (production of large amounts of cell mass)
Physiological studies (to determine max growth rate)
Useful tool because scientists can control growth rate and population density INDEPENDENTLY and obtain a steady cell supply
Tends to simulate natural conditions better than a batch culture
Ecological studies into competition, predation
What are the 4 basic -omic sciences?
DNA - RNA - Proteins - Metabolism
Genome, Transcriptome, Proteome, Metabolome
meta- meaning
Prefix implying beyond, more transcending - usually omics of a microbial community instead of a single strain
genome
Entire complement of genetic information in an organism
Includes genes, regulatory sequences, and noncoding DNA
Bacteria have small genomes relative to Eukaryotes but they have more efficient use of space (little garbage DNA relative to eukaryotes)
Genomics
Discipline of mapping, sequencing, analyzing, and comparing genomes
400 000 <
Genome size in ____bases correlates to the total number of thousands of ORFs in the genome
Mega
5 Megabases = 5000 ORFS
1 kilobase = 1 ORF?
ORF = gene that codes for a protein
Prokaryotes with larger genomes?
More versatile, can live in different environments
Like a swiss-army knife bacteria
Those with small genomes - endo symbionts/parasites - rely on their host for stuff as they don’t have the genes to produce it
As the genome increases in size, a larger proportion of the genes are dedicated to»>
Transcription, signal transduction
While a smaller proportion of genes are dedicated to translation and DNA replication (all organisms have roughly the same amount of genes dedicated to DNA replication)
sequencing
determining the precise order of nucleotides in a DNA or RNA molecule
genome assembly
taking short fragments of sequenced DNA and puzzling them together into larger configurations (ideally a complete chromosome)
genome annotation
Converting raw sequence (DNA) data into a list of genes and other functional sequences present in the genome.
- Deciding what the genes do
bioinformatics
analyzing sequences and structures of nucleic acids and proteins
Sanger sequencing
Copying of orginal single stranded DNA
small amounts of dideoxy nTPs are used with dNTPs. There is a diff fluorescent dye for each ddNTP base.
ddNTPs are CHAIN TERMINATORS preventing further elongation of DNA chain during PCR. They insert randomly, producing DNA chains of varying lengths
Capillary electrophoresis of fragments and detection of labels (sort by size of fragments) - automated sequencer reads the output.
Next generation sequencing
Much cheaper and allows much greater through put than Sanger sequencing.
Most of these techniques work by eaves dropping on a DNA polymerase as it copies a DNA strand
closed vs draft genome
A closed genome has an entirely circular chromosome with no missing pieces (rare)
More expensive, but provides us with more information
A draft genome - has gaps in it, is incomplete. Can be very close to being closed - Find pieces of overlap and build outward from here
ORF stand for______ and encodes for a_______
open reading frame and encodes a protein (functional ORF encodes a protein)
q
ORFs
Bacterial and Archael genomes are a series of open reading frames
The function of ORFs is predicted by searching for similarities in gene/protein data bases
Comparing ORFs found in an organism by using these data bases can help us determine the metabolic capabilities of the organism.
How long is the average gene?
1000 bps long
What is an indication that there is really an ORF present when the computer is scanning for them?
The presence of a ribosomal binding site at the beginning of the ORF
What are hypothetical proteins?
Uncharacterized ORFs - proteins that likely exist but whose function is currently unknown - about 30% of the ORFs
- lack of sufficient amino acid sequence homology with known proteins for identification
single cell genomics
Sequencing the genomes of single cells
Uses multiple displacement amplification MDA(modified PCR) for making multiple copies of the single cell’s genome
Dilute and distribute cells to microwells
lyse the cells
Amplify DNA by MDA
Sequence DNA
Sequence analysis
What is one discovery that comparative genomics/ gene sequencing has told us?
That horizontal gene flow is very common
What is one discovery that comparative genomics/ gene sequencing has told us?
That horizontal gene flow is very commongenomi
genomic islands
foreign DNA that is generally isolated to the same area within the cell
vertical gene transfer
genome replication and cell division (daughter cells with the same genomes)
horizontal gene transfer
Transformation, transduction, conjugation
codon bias
often species specific.
Although two species may have the same amino acid sequence, they may each have their own bias as to which codons are used to code for particular amino acids more often than others.
Codon bias (or lack of) may reveal that horizontal gene transfer likely occurred between two organisms.
What is the typical genome size range?
Between 5000 and 8000 genes or ORFs
Can range from 137 genes to 11559 genes.
Order of largest to smallest prokaryotic genome?
Free-living, parasite, endosymbiont
What kind of DNA do Bacteria and Archaea have?
Double stranded DNA
Folds into double helix
CIRCULAR CHROMOSOMES 99.9 percent of the time.
Proportion of guanine should be proportional to the amount of cytosine.
Same case for A and T
What is the method of DNA compaction in prokaryotes?
Compact DNA into supercoiled domains by DNA gyrase.
Eukaryotes use histones
can go from 350 micrometer long circular chromosome to less than 2 micrometer long when in super coiled domains
Chromosomes in Prokaryotes
~5 Mb
Have housekeeping/essential genes
One copy per cell
Replication is strongly regulated during the cell cycle
Housekeeping genes
Genes that are absolutely required for life. Can’t survive on its own if it doesnt have these genes
Plasmids
~100 kb
Bonus genes
One to many copies of multiple different plasmids
Where does DNA replication begin in a prokayote?
At the origin of replicate (oriC)
All the enzymes involved associate to form the replisome
Replisome
All of the enzymes required for DNA replication.
theta structure
Formed during DNA replication
Origin of replication at the top - replication forks spread either way and meet at the terminus of replication at the bottom of the chromosome
What sequences do sigma factors bind?
They bind consensus sequences. Theses are within the promotor, upstream of an initiation site . Include -35 and -10 (Pribnow) sequences
Sigma factors are absolutely required for all transcription to occur.
There are variations in the consensus sequences. The stronger the consensus, the stronger the sigma factor will bind (more transcription occurs)
Will genes that are transcribed a lot have stronger or weaker consensus sequences?
Stronger
Increasing the rate of transcription.
What does the RNA Pol core enzyme first need in order to bind to the DNA?
It needs a sigma factor to be bound as it recognizes the sigma factor and binds the aDNA the promoter.
RNA Pol core enzyme is a simplifies version of the RNA Pol ll enzyme
How does the RNA polymerase holoenzyme come into play?
The RNA polymerase holoenzyme unwinds the double helix, separates the strands, and transcribes the DNA in a 3’ to 5’ direction.
It continues transcription after the RNA polymerase core enzyme transcribes the first couple of nucleotides. It completes the transcription without sigma factor.
What do inverted repeats in the DNA sequence result in the formation of in the RNA transcript?
In the formation of a termination stem loop and a stretch of uracil’s
This stem loop causes RNA Pol to stall and fall off the DNA
This is the primary way that transcription ends
What is an operon?
Multiple genes related to one another, ordered sequentially under the control of a single promotor
the genes are cotranscribed to form a single transcript
What type of mRNA does the transcription of an operon produce?
polycistronic mRNA - contains multiple genes or ORFs
Can have multiple ribosomal binding sites so
What is a regulon?
multiple operons all controlled by the same molecule that operates one of the operons
When more than one operon is under the control of a single regulatory protein, these operons are collectively called a regulon
Open reading frame
portions of the mRNA THAT ACTUALLY ENCODE FOR AMINO ACIDS - genes
How is the termination stem loop form and what does it mean when it forms?
The transcription of two segments of inverted repeats caused the formation of a loop.
Causes the RNA Pol to stall and fall off the DNA, ending transcription
Enzyme repression
If the endproduct of the biochemical pathway is absent –> then the genes are expressed!
Product acids as an effector, called a corepressor that binds to a repressor protein
When bound to a corepressor the repressor will change conformation and go bind DNA in the OPerATOR region
- BLOCKS sigma and factor and RNA pol from binding
-product becomes ABSENT –> need more of it - want transcription to proceed –> so the product falls off the repressor, which falls off of the operator, and transcription proceeds
In enzyme repression: The _______ is used as a _________ and binds to ___________ and then both of them bind to the ___________
In enzyme repression: The product is used as a corepressor and binds to the repressor and then both of them bind to the operator site on the promoter –> this prevents trancription
- have the product dont need more of it
-dont have the product - product wont be binding to the repressor - repressor no longer stops transciption - transcription is allowed to promote formation of the product
repressors bind to ________ and block transcription by_____
Operator sites in the promotor region of DNA and prevent transcription by blocking Sigma factors and RNA Pol from binding
The binding of a corepressor promotes transcriptional repression while the binding of an inducer to a repressor removes the repressor from the operator site, allowing transcription
In enzyme induction the __________ of the biochemical pathway is an effector molecule called an _________ and binds to the _________, removing it from the ________ region
In enzyme induction the substrate of the biochemical pathway is an effector molecule called an inducer and binds to the repressor removing it from the operator region. So when substrate is present, transcription is promoted and when the subtrate is absentm, the repressor is bound, preventing sigma factor(s) and RNA from binding- prevetning transcription
What is the transcriptional regulation mechanism where the substrate of the pathways is an inducer?
Enzyme induction
In enzyme induction the substrate of the biochemical pathway is an effector molecule called an inducer and binds to the repressor removing it from the operator region. So when substrate is present, transcription is promoted and when the subtrate is absentm, the repressor is bound, preventing sigma factor(s) and RNA from binding- prevetning transcription
Which region of a DNA sequence do activators bind to?
activator binding site
In enzyme activation, the ________ of a biochemical pathway acts as an effector molecules called an ________ which will then bind to the __________ and together they will bind the ________ on the DNA molecule.
In enzyme activation, the substrate of a biochemical pathway acts as an effector molecules called an inducer will then bind to the activator and together they will bind the activator binding site on the DNA molecule.
This pathway is for rate substrates. The presence of the substrate helps recruit sigma factors and RNA pol tightly as it is a weak binding sequence - there is a weak binding
sigma factor doesnt bind as well to sequences of molecules not transcribed often
What are the differences between prokaryotic and eukaryotic translation?
- Prokaryotes have 70S ribosomes while eukaryotes have 80S
- first amino acid is formylmethionine instead of Met
- codon bias is different in different species
- Not all ORFs in polycistronic mRNA in the same reading frame**
- some species use “stop codons” for selenocysteine and pyrrolysine- instead of coding for stop, they code for rare amino acids.
What is a polysome?
An mRNA strand with ribosomes are attached all at different stages in translation. Polypeptides at different lengths
Occurs because transcription and translation can be couple in prokaryotes due to the fact that prokaryotes do not have a nucleus
attenuation
A transcriptional regulation mechanism that comes about due to the fact that transcription and translation can be coupled in prokaryotes.
Unique to prokaryotes
- During transcription, the 5’ end of the mRNA forms one of the two possible stem-loops.
region 3-4 stem loop
region 1-2 stem loop
Which stem loops forms depends on how fasst the mRNA leader sequence is translated.
region 3-4 stem loop vs
region 1-2 stem loop
Attenuation
Which one forms depends on how fast the mRNA leader sequence is translated.
LOTS of Trp /AA - ribosome reads the mRNA quickly – stemloop forms in region 3-4
-RNA Pol stalls when it reaches this stem loop. - trytophan structural genes are transcribed.
LAck of Trp - ribosome stalls as it waits for Trp - results in the formation of the stem loop in region 2-3 (closer to the 5’ end of the mRNA)
- not fed through fast
- Transcription of Trp structural genes transcribed.
What kind of regulation is sRNA?
Translational regulation
40-400 nt stretched of single stranded RNA
IT base pairs with mRNA altering the rates of translation
Translation inhibition with sRNA
if the correct sRNA is available, it can basepair with the ribosomal binding site on the mRNA transcript thus preventing ribosome binding and translation
Translation stimulation with sRNA
Sometimes the 5’ end of mRNA basepairs on itself thus occupying the RBS. sRNA can base pair with the mRNA covering the RBS, thereby allowing the RBS to be exposed
Stimulates translation
RNA degradation with sRNA
Translation can be prevented if:
sRNA binds to NEAR THE 3’ end of the mRNA transcript, recruits Ribonuclease. Ribonuclease degrades the RNA and translation doesnt occur
RNA protection with sRNA
Ribonuclease present initially and the mRNA would be degraded
BUT
if the right sRNA is available, it will bind RIGHT at the 3’ end of the mRNA transcript and prevent the ribonuclease from binding. so then the mRNA is not degraded and translation can occur
How do riboswitches work?
The aptamer region within the 5’ UTR of mRNA that can form alternate secondary structures in the presence/absence of a signal metabolite.
- signal metab present - binds to aptomer region - stem loop forms that occupys the RBs and NO TRANSLATION OCCURS
- no signal metabolite present - aptamer region remains bound to – RBS remains open - ribosome can bind and translation occurs
So signal metabolite signal = NO TRANSLATION
- ie if cell has enough amino acids for example
Horizontal Gene Transfer
movement of genes between cells (from donor to recipient) not directly descended from each other
What are two mechanisms which recombination can occur?
RecA-mediated homologous recombination
Site-specific (non-homologous) recombination
What do you call a cell that is capable of transformation?
competent
What is transformation?
The uptake of “naked” (free) DNA from the ENVIRONMENT
Naturally - uptake of single stranded DNA
Ways to induce competence in the lab?
-Negate charge differences
-Rupture membrane (cold/heat shock, electroporation)
- take up ds DNA this way
Conjugation
Transfer of plasmid DNA from a donor cell F+ to a recipient cell (F-)
Requires cell to cell contact
Genetically controlled by the plasmid
what is the tra region
The transfer region of the plasma. It encodes genes that form the pilus for conjugation . the pilus recognizes F- cells
- Type lV secretion system –allows DNA to transfer from one cell to another (it generates a conjugation bridge).
oriV
site of replication basically
No oriC in plasmids
oriT
This is where the plasmid gets nicked during conjugation so that the plasmid can get transported over
What does Hfr mean?
high frequency of recombination
Hfr cells have a plasmid that has been integrated into the chromosome of the cell. Genes on the plasmid are still expressed
Episome
The F plasmid is called an episome if it has the ability to integrate into the chromsome
HA
spike protein - hemagluttin
NA
spike protein - neuraminidase
Rotovirus
Class lll. dsRNA
Has multiple capsids, incl nucleocapsid
oral fecal transmission - causes diarrhea
DNA replication and transcription of the virus occur in the nucleocapsid while translation occurs in the viralplasm
HIV
Retrovirus –> Class Vl
ss (+) RNA enveloped, segmented
2 copies of the genome in each virion)
40 mill ppl currently infected, host = primates
its a provirus that can hide for years before initiating AIDS
Infects T cells and Antigen Presenting Cells (APC)
Spike proteins: gp120 and gp41 which bind to CCR5/CD4 on macrophages and T cells
Viral genome encodes: reverse transcriptase, integrase, viral tRNA, HIV protease
dsDNA is generated by RT so that the virus can integrate into the genome
HAART
Highly Active Anti-Retroviral Therapy
Includes:
Fusion inhibitors: block gp41
RT inhibitors: halt RT
Integrase inhibitors: prevent provirus from forming (preventing long term infection)
Protease inhibitors: prevent the post-translational modification of viral proteins
Parvovirus B19
Fifth Disease
ssDNA (+) genome Class ll
Causes a mild rash and swollen joints
naked icosohedral virion; the viral genome codes for 2 genes, one that aids with DNA replication and another that for capsid formation
Human Hepatitis B virus
dsDNA genome (Class Vll)
transmitted through blood, causes acute liver disease
vaccine - introduces spike protein genes to the recipient which makes them form antigens toward the spike protein
eveloped, spherical icosahedral virion
partially circular genome. all genes overlap
Where does DNA replication begin in the chromosome versus in the plasmid?
Chromosome = OriC
Plasmid = OriV
Plasmid conjugation transfer: OriT
What makes a cell competent?
It can do transformation
it has the rec2/com transporter than brings single stranded DNA into the cytosol
Tral
An enzyme encoded by the tra region of the plasmid. It nicks OriT site to begin rolling circle replication
