master deck Flashcards
purpose of this project
make a genomic library that makes E coli glow with lux
quantify gene expression changes in E Coli with GadA
A Fischeri
gram negative, contains lux operon
gram-negative bacteria
external LPS layer
thin peptidoglycan wall
name 2 gram negative bacteria
E coli. A. fischeri
operon
genetic regulatory system in when genes coding for functionally related proteins are clustered
ChDNA
large circular piece of DNA, where we got lux operon
restriction digestion
enzymatic rxns that cut DNA into smaller pieces, run by enzymes that cut DNA in specific places
what percent of deep sea marine organisms have a symbiotic relationship with bacteria
96%
what benefit do bacteria offer fish
attract prey, mate
communication
hide from predators
how do fish benefit bacteria
protection, reliable food source(glucose, amino acids)
how is lux operon gene expression regulated
quorum sensing
quorum sensing
regulation of gene expression in response to changes in cell-population density
what does production of light require
lots of energy and oxygen, no glow without it
how does bacteria sense growth
Auto-induction
Bacteria release small metabolic product
N-acyl homoserine lactone
how does AHL reach extracellular environment
free diffusion
as density of bacteria increases
more inducer is released into environment
at critical concentration
AHL diffuses back in to interact with lux operon
lux R
codes the transcriptional activator that binds to AHL
lux I
codes the AHL synthase
Lux C
codes the acyl reductase
lux D
codes the acyl transferase
lux A
codes the alpha subunit of luciferase
lux B
codes the beta subunit of luciferase
lux E
codes the acyl protein synthetase enzyme
what initiates gene expression of lux operon
AHL binding to lux R and lux I
what enzymes are needed to generate long chain fatty aldehyde
Lux C-removes FA’s from reg pathway to do run
Lux D-activates FA to form R-CO-AMP
Lux E-reduces activated fatty acid to form aldehyde
what is bioluminescence dependent on
NADPH
resuspension buffer
contains SDS to solubilize membrane proteins, disrupts lipid layer of gram negative cells, may be appearance of soap bubbles
proteinase K
responsible for freeing nucleic acids and disabling nucleases, stable in range of pH
Rnase A
an efficacious ribonuclease used to degrade RNA
how does RNase A cleave RNA
cleaves at 3’ side of phosphodiester bond after pyrimidines
why does DNA stay stable in presence of RNase A
does not have 2’ OH group
what does detergent do
disrupts lipid layer and brings proteins into lipid protein complexes
lysis buffer
contains chaotropic salt to disrupt regular hydrogen bonding with water, sets up conditions for DNA to bind to silica column
ethanol
enchance and influence binding of nucleic acids to silica by creating more hydrophobic solution
wash buffer
low amount of chaotropic salt that binds to and removes proteins and colored containments
wash buffer 2
contains ethanol to remove salts added from AW1
DNA grade water
water free of salts DNases and proteases which allows for rehydration
binding
SDS dissociates in the presence of chaotropic salts in lysis buffer and sodium ions form a cation bridge to bind DNA to the silica membrane
at what wavelength does DNA absorb light
260 nm
beer-lambert law equation
A = εcl
ε=extinction coefficient, constant
c= conc of substance
l=light path
beer-lambert law
light attenuation through a medium is proportional to the concentration of the light absorbers present in the substance the optical properties of the light absorber, and the optical pathlength traveled by the light beam
280 nm
absorbance maximum for proteins, due to Tyrosine, cysteine, tryptophan
230 nm
nucleic acids minimum absorbance, helps look for organic contaminants
A 260:280
chacks for protein/ RNA contamination
>2 = RNA contamination
<1.8= protein contamination, not great tho
A260:230
checks for organic contamination
outside of 2-2.2, presence of organic cmpds or salt
shotgun cloning
randomly digesting a large piece of DNA into smaller pieces that can be ligated into plasmids for transport to other organisms
once inside a vector
each vector contains separate frag of genome which is completely represented to refer back to
what do we get with shotgun cloning
frags with regulatory elements and coding sequences
lux operon is how long
8.5 kb
what makes a good plasmid vector
size
high copy number
ori
multiple cloning sites
selectable markers
size
large enough to hold foreign DNA, small enough to be retained by host and distinguished from host chDNA
high copy number
50-100 per cell
ORI
recognized by host machinery
multiple cloning sites
Region of DNA containing recognition sequences for many restriction enzymes
RNA polymerase promoter sequences
near mcs, mRNA can be made off inserted DNA
what is our vector of choice
pGEM3zf(+)
restriction endonuclease in vivo
cuts viral DNA to prevent infection, recognizes palindromic sequences
1 unit of enzyme is
the amount that catalyzes the conversion of 1 micro mole of substrate per minute
if conc of vector is .2µg/µL, volume needed to get us 1 µg is
1 µg ÷ 0.2 µg/µL = 5 µL of vector stock
agarose gel ranges between
.7 and 2%
.7% agarose
shows better separation of larger DNA fragments(5-10 kb)
a 2% agarose gel will
show good resolution for smaller DNA fragments (.2-1 kb)
GelRed
nucleic acid stain used to light up in gel electrophoresis, fluorophore
TAE buffer
component of electrophoresis to prevent inconsistent separation of restriction frags
loading dye
allows us to track DNA migration, as well as give weight to sample so it includes sugar or glycerol
blunt ends
no overhang
sticky ends
2-3 bp overhang
Sal I
has a high GC content, while A. fischeri has low, will cut DNA at few places
streaking of chDNA
good electrophoresis, completely digested chDNA
nicked DNA
damage in dsDNA, can be enzymatically induced or caused by physical damage during preparation
lambda control
well -studied bacteriophage of E. coli, known to have 2 restriction sites, making 3 frags, 48.5 kb
DNA ligation
reaction that forms recombinant DNA molecules by covalent bonding 2 restriction frags with compatible ends
T4 ligase
ATP
Mg2+ cofactor
free 3’OH and 5’ PO4 3- end
synthesizes ester linkage
4 outcomes of ligation reaction
Plasmid vector could ligate back on itself with no genomic DNA fragments
Multiple fragments could ligate to each other and become circularized
Plasmid could accept multiple fragments
Plasmid accepts one fragment
most likely outcome of ligation
Plasmid vector could ligate back on itself with no genomic DNA fragments
wanted ligation reaction outcome
plasmid accepts one fragment
best insert: vector
3:1
why do we set up mutliple ratios to improve overall chances of good ligation
to maximize chances of ligation
why is 3:1 optimal
It’s thought that this is simply due to a situation where there are more available free ends of chDNA to reduce the likelihood of plasmid self-ligation, while not being so much that many chDNA fragments ligate together
transformation
genetic alteration of organism by incorporation of foreign DNA into cells, DOES NOT HAVE TO INTEGRATE
induced competence
cold shock with CaCL2— heat – cold shock
attributes of a good host E. coli
no restriction endonucleases
no lacZ gene
no homologous recombination
limited resistance to ampicillin
interrupted lacZa
interrupted beta-gal will have glowing, white colony
preserved lacZa
preserved beta-gal,blue colonies
transformation efficiency equation
((# transformants/ µg pGEM )/(volume of transformant/volume plated)*dilution factor
week 1
isolate and purify chDNA
week 2
Digest chDNA and plasmid DNA for shotgun cloning of lux operon
week 3
ligate chDNA into plasmid DNA to create A. fischeri genomic library
week 4
transform E. Coli with plasmid library
week 5
screen E. coli for plasmids, containing lux operon
week 6
re-isolate plasmid
set up PCR for sequencing of lux I
rest of term
RNA isolation RT-PCR, qPCR
does E Coli have gene for auto-inducer
no
can E COli use quorum sensing
yes, by sensing AHL from other organisms
what is significantly upregulated when AHL is sensed
gadA
E Coli LuxR homolog
SdiA
ALKALINE lysis buffer
sugar, tris buffer, lysozyme, detergent, NaOH
neutralization buffer
glacial acetic acid, potassium acetate, RNase A , Chaotropic Salt
glacial acetic acid
neutralizes NaOH and allows plasmid to renature
potassium acetate
causes organic materials to precipitate out
project overview
First start by isolating the lux operon and ligating it into a vector (pGEM)
Transform E. coli with the recombinant vector
The glowing E. coli will be transcribing all genes in the lux operon, therefore the AHL encoded by luxI
From here we will collect RNA from the E. coli and use reverse transcription (RT) to turn it back to DNA (cDNA)
We will take the cDNA and run a real-time PCR (qPCR) to look at the transcripts of our target gene (GadA) and our reference gene 16S rRNA to quantify how much mRNA was made of each
We will compare our gene expression results with a sample of E. Coli without the lux operon.
wash buffer
wash buffer+ 90% EtOH
elution buffer
DNA grade water or TE buffer
PCR
polymerase chain reaction, amplifies a region of DNA to thousands of millions of copies
PCR requirements
DNA Template
Forward and Reverse Primers
Taq Polymerase
Deoxynucleoside triphosphates (dNTPs)
Buffer Solution
Bivalent Cation (Mg2+)
Monovalent Cation (K+)
PCR steps
denaturation
annealing
elongation
temps of each PCR step
denaturation 95
annealing 55
elongation 72
good primer attributes
20 bp long high GC content
negative control
pGEM from a blue colony
positive control
pGEM+lux+ from a commercially bought organism
experimental value
pGEM+lux+ from a glowing colony
furthest line on a PCR result
primer dimer
2nd to bottom line of PCR result
lux I PCR product
chomatogram
fluorophore on each ddNTP will be detected as it passes through the filter and recorded until an entire sequence read is detected. This will produce the nucleotide sequence of your results.
what is gadAX operon used for
acid tolerance of E coli
Culture A: E. coli + pGEMlux-
no lux operon to produce AHL, so no upreg of gadA in E coli
Culture B: E. coli + pGEMlux+
AHL is present E coli senses it with sdiA, upregs gadA
Why RNA
gives a screenshot of what is being produced at that very moment
most common method of extraction
acid guanidinium thiocyanate-phenol-chloroform extraction
RNA must be converted into DNA bc
DNA polymerase in PCR tubes will not work
cDNA
Using the poly-A tail a primer with a complimentary sequence is attached
Reverse Transcriptase is used to synthesize a single strand of DNA complementary to the original mRNA strand
The RNA template is then degraded
The newly synthesized DNA strand folds back on itself and acts as its own primer, allowing the DNA Polymerase to synthesize a double stranded piece of DNA
reverse transcriptase
compliments RNA to amplify only a transcript made at a certain point
what do we do with DNA after reverse transcription
qPCR instead of PCR
conventional PCR
- starts with DNA
- Amplicon is detected and analyzed at an end point
- Analyzes finished product
- usually to get a concentrated amount of DNA
qPCR
- starts with chDNA
- amplicon is based off of already existing concentration of nucleic acid
- analyzing product, in real time
- usually look at how much transcript was made
fluorescent dye
SYBR green
Cq
number of cycles of qPCR needed to detect fluorescence
what does a low Cq mean
not many cycles were needed for fluorescence, more cDNA of DNA in that region, with more mRNA being made
relative expression
2^(Ct(Ref)-Ct(Target))
two ratios of relative expression
Control Expression: control/control
Target Expression: target/control
