BM214 Microbiology Test Revision

what is a biosensor

device using biological components to detect substances

what is the Gram stain

technique to classify bacteria based on cell wall

what is axenic culture

culture containing only one type of microorganism

what is the streak plate technique

method to isolate single bacterial colonies

what is the spread plate technique

technique to evenly distribute bacteria on agar

what is a plasmid

circular DNA molecule used in genetic engineering

what is a chromogenic protein

protein that produces colour for visualisation

what is MacConkey agar

selective medium for Gram-negative bacteria identification

what is Mannitol salt agar

selective medium for Gram-positive bacteria identification

what is crystal violet

primary stain used in Gram staining process

what is safranin

counterstain used in Gram staining process

what is iodine solution

used in Gram stain as a mordant

what is the alcohol rinse in the Gram stain used for

step in Gram staining to differentiate bacteria

what is the first step in constructing an antibiotic biosensor

isolate the plasmid containing the reporter gene

why should you streak for single colonies

single colonies represent genetically homogenous populations

steps of the Gram stain

crystal violet, iodine, alcohol, safranin

what colour do Gram-positive organisms stain

blue/violet

what colour do Gram-negative organisms stain

red/pink

what is the appropriate colony to plate range

30 - 300

what is a restriction digest

process of cutting DNA at specific sequences

what is agarose gel electrophoresis used for

separate DNA fragments by size

what is plasmid DNA used for

gene cloning

what is selective media

growth media favouring specific organisms growth

what is differential media

media allowing distinction between different organisms

what is alkaline lysis

method to isolate plasmid DNA from bacteria

what is GelRed

fluorescent dye for visualising DNA in gels

what is YMN medium

yeast medium with nutrients for yeast culture

what is mutagenesis

inducing mutations to improve microbial stains

what is pABS1

plasmid containing the shCP* gene

what is pLib

plasmid library containing a selection of different promoter regions

what is the purpose of Tris-EDTA in plasmids

EDTA chelates magnesium cations which are required for the activity of DNase

function of restriction enzymes

cleave specific DNA sequences, leaving sticky ends that can be annealed to homologous ends and ligated together

what charge does DNA have

negative - due to the phosphates and pH of the buffer

what percentage of agarose gel is in the electrophoresis

0.8%

what wavelength is used to irradiate the dye-DNA complex

300nm

what was the results of the first experiment

- pLib with HindIII formed one band

- pABS1 with HindIII + AflII forms three bands

what is MacConkey agar used for

isolation and/or enumeration of enteric bacteria

what is the selective and differential qualities of MacConkey agar

selective:

- bile salts

- crystal violet

differential:

- lactose

- neutral red

what is Mannitol salt agar used for

isolation and/or enumeration of staphylococci

what is the selective and differential qualities of Mannitol salt agar

selective:

- sodium chloride

differential:

- D-mannitol

- phenol red

what is transformation efficiency

ratio of transformed cells to total cells

what is PCR

technique to amplify specific DNA sequences

what is 16S rRNA gene

gene used for bacterial identification

what are dNTPs

building blocks for DNA synthesis in PCR

what is Taq DNA polymerase

heat-stable enzyme used in PCR

what is denaturation

step in PCR where DNA strands separate

what is annealing

step in PCR where primers bind to DNA

what is extension

step in PCR where new DNA strands are synthesised

what is optical density

measurement of cell density in cultures

what is light scattering

phenomenon used to estimate cell density

how to calculate transformation efficiency

(colonies/µg)/dilution

what should you observe when plates are incubated with MacConkey agar and Gram-negative bacteria

- pink/red colonies indicate lactose fermentation

- colourless colonies suggest non-fermentation

what should you observe when plates are incubated with Mannitol salt agar and Gram-positive bacteria

- agar will be red/orange when pH is neutral

- agar will be yellow when pH decreases after mannitol fermentation

what chemicals are in a PCR mastermix

- distilled H2O

- dNTPs

- Taq PCR buffer

- forward primer

- reverse primer

- MgCl2

steps of PCR

1. 96°C - denaturation

2. 55°C - annealing

3. 72°C - extension

what is Beer-Lambert law

A=lεc where A = absorbance, l = path length, c = concentration and ε = molar absorption coefficient

results from experiment 3 from lab 3

- optical density values for bacterial growth under different carbon sources

- S. cerevisiae prefers glucose as its carbon source but can still grow on lactose

what is DNA polymerase

enzyme-extending primers during DNA synthesis

what are primers

short sequences initiating DNA synthesis in PCR

what is Tm

melting temperature of DNA primers

what are primer dimers

self-annealed primers leading to non-specific amplification

what is Gibson assembly

method for joining DNA fragments without restriction enzymes

characteristics of successful primers

- GC content approximately 40-60%

- final nucleotide at 3' end must be G or C

- Tm is approximately 65-75°C

- Tm of the primers is within 5°C of each other

- delta G value of self-dimers, hairpins and heterodimers should be weaker than 9kcal/mol

additional applications and adaptations of PCR

- gene expression analysis

- genotyping and diagnostics

- cloning, mutagenesis, synthetic biology

how can you use PCR for gene expression analysis

by quantifying gene expression or detecting alternative splicing events

how can you use PCR for genotyping and diagnostics

- detect presence/absence of sequences in a given sample

- detect mutations

- detect large-scale inversions, expansions and contractions

how can you use PCR for cloning and mutagenesis

- restriction enzyme-based cloning

- site-directed mutagenesis

- Gibson assembly

what is hybridisation capture

involves designing DNA sequences that complement the gene you want to enrich, attaching probes to an array or a bead, introducing DNA fragments from a sample, and eluting DNA from the probes for targeted sequencing of specific genes

what is sequence searching

a computational hybridisation capture experiment where the DNA sample is the database being searched, and the probe is the query sequences

what is pairwise sequence alignment

in silico equivalent to observing whether two sequences hybridise

what is the scoring system in alignment

- matching identical letters score +1

- different letters align scores -1

- inserting a gap in the alignment scores -1

what is BLOSUM62

a common default scoring scheme for protein sequences

what is BLAST

Basic Local Alignment Search Tool

what are the three steps of BLAST algorithm

- seeding

- extension

- evaluation

what is a word hit

a match between a word from the query and a word in the database index

what does a diagonal in word hits mean

more word hits on a diagonal implies a more significant match

what is the score S in alignment

calculated after extension to determine the quality of alignment

what is E in alignment

E = kmne^-yS where k = minor constant, m = size of the query, n = size of the database, y = scaling factor, S = alignment score

what is local alignment

shows the best matching region between the two sequences without context of the rest of each sequence

is Needle global or local alignment

global

is Water global or local alignment

local

what is E in sequencing

the number of alignments with the same or larger S expected in a database of the same size and letter frequency if the sequences were completely random

what is BLASTn

nucleotide against nucleotide

what is BLASTp

protein against protein

what is BLASTx

translated nucleotide to protein

what is tBLASTn

protein to translated nucleotide

what is Silva

database for quality-checked rRNA sequences

what is the ACT service

search tool for matching rRNA sequences

what is turbidity

cloudiness in culture indicating cell density

what is ggplot2

R package for creating layered visualisations

what is geom_point

layer for adding points to ggplot

what is geom_smooth

layer for fitting models to data curves

what is scale_y_log10

function to set y-axis to logarithmic

what is annotation_logticks

adds ticks for logarithmic scale visualisation

what is scale_colour_grey

function to change plot colours to greyscale

what is theme_bw

applies a black and white theme to plots

what is panel.grid.major

controls major grid lines in ggplot

what is panel.grid.minor

control minor grid lines in ggplot

what is kanamycin

antibiotic used to test reporter gene response

what is facet_wrap

creates subplots for each sample in ggplot