master deck Flashcards
(132 cards)
1
Q
purpose of this project
A
make a genomic library that makes E coli glow with lux
quantify gene expression changes in E Coli with GadA
2
Q
A Fischeri
A
gram negative, contains lux operon
3
Q
gram-negative bacteria
A
external LPS layer
thin peptidoglycan wall
4
Q
name 2 gram negative bacteria
A
E coli. A. fischeri
5
Q
operon
A
genetic regulatory system in when genes coding for functionally related proteins are clustered
6
Q
ChDNA
A
large circular piece of DNA, where we got lux operon
7
Q
restriction digestion
A
enzymatic rxns that cut DNA into smaller pieces, run by enzymes that cut DNA in specific places
8
Q
what percent of deep sea marine organisms have a symbiotic relationship with bacteria
A
96%
9
Q
what benefit do bacteria offer fish
A
attract prey, mate
communication
hide from predators
10
Q
how do fish benefit bacteria
A
protection, reliable food source(glucose, amino acids)
11
Q
how is lux operon gene expression regulated
A
quorum sensing
12
Q
quorum sensing
A
regulation of gene expression in response to changes in cell-population density
13
Q
what does production of light require
A
lots of energy and oxygen, no glow without it
14
Q
how does bacteria sense growth
A
Auto-induction
15
Q
Bacteria release small metabolic product
A
N-acyl homoserine lactone
16
Q
how does AHL reach extracellular environment
A
free diffusion
17
Q
as density of bacteria increases
A
more inducer is released into environment
18
Q
at critical concentration
A
AHL diffuses back in to interact with lux operon
19
Q
lux R
A
codes the transcriptional activator that binds to AHL
20
Q
lux I
A
codes the AHL synthase
21
Q
Lux C
A
codes the acyl reductase
22
Q
lux D
A
codes the acyl transferase
23
Q
lux A
A
codes the alpha subunit of luciferase
24
Q
lux B
A
codes the beta subunit of luciferase
25
lux E
codes the acyl protein synthetase enzyme
26
what initiates gene expression of lux operon
AHL binding to lux R and lux I
27
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
28
what is bioluminescence dependent on
NADPH
29
resuspension buffer
contains SDS to solubilize membrane proteins, disrupts lipid layer of gram negative cells, may be appearance of soap bubbles
30
proteinase K
responsible for freeing nucleic acids and disabling nucleases, stable in range of pH
31
Rnase A
an efficacious ribonuclease used to degrade RNA
32
how does RNase A cleave RNA
cleaves at 3' side of phosphodiester bond after pyrimidines
33
why does DNA stay stable in presence of RNase A
does not have 2' OH group
34
what does detergent do
disrupts lipid layer and brings proteins into lipid protein complexes
35
lysis buffer
contains chaotropic salt to disrupt regular hydrogen bonding with water, sets up conditions for DNA to bind to silica column
36
ethanol
enchance and influence binding of nucleic acids to silica by creating more hydrophobic solution
37
wash buffer
low amount of chaotropic salt that binds to and removes proteins and colored containments
38
wash buffer 2
contains ethanol to remove salts added from AW1
39
DNA grade water
water free of salts DNases and proteases which allows for rehydration
40
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
41
at what wavelength does DNA absorb light
260 nm
42
beer-lambert law equation
A = εcl
ε=extinction coefficient, constant
c= conc of substance
l=light path
43
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
44
280 nm
absorbance maximum for proteins, due to Tyrosine, cysteine, tryptophan
45
230 nm
nucleic acids minimum absorbance, helps look for organic contaminants
46
A 260:280
chacks for protein/ RNA contamination
>2 = RNA contamination
<1.8= protein contamination, not great tho
47
A260:230
checks for organic contamination
outside of 2-2.2, presence of organic cmpds or salt
48
shotgun cloning
randomly digesting a large piece of DNA into smaller pieces that can be ligated into plasmids for transport to other organisms
49
once inside a vector
each vector contains separate frag of genome which is completely represented to refer back to
50
what do we get with shotgun cloning
frags with regulatory elements and coding sequences
51
lux operon is how long
8.5 kb
52
what makes a good plasmid vector
size
high copy number
ori
multiple cloning sites
selectable markers
53
size
large enough to hold foreign DNA, small enough to be retained by host and distinguished from host chDNA
54
high copy number
50-100 per cell
55
ORI
recognized by host machinery
56
multiple cloning sites
Region of DNA containing recognition sequences for many restriction enzymes
57
RNA polymerase promoter sequences
near mcs, mRNA can be made off inserted DNA
58
what is our vector of choice
pGEM3zf(+)
59
restriction endonuclease in vivo
cuts viral DNA to prevent infection, recognizes palindromic sequences
60
1 unit of enzyme is
the amount that catalyzes the conversion of 1 micro mole of substrate per minute
61
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
62
agarose gel ranges between
.7 and 2%
63
.7% agarose
shows better separation of larger DNA fragments(5-10 kb)
64
a 2% agarose gel will
show good resolution for smaller DNA fragments (.2-1 kb)
65
GelRed
nucleic acid stain used to light up in gel electrophoresis, fluorophore
66
TAE buffer
component of electrophoresis to prevent inconsistent separation of restriction frags
67
loading dye
allows us to track DNA migration, as well as give weight to sample so it includes sugar or glycerol
68
blunt ends
no overhang
69
sticky ends
2-3 bp overhang
70
Sal I
has a high GC content, while A. fischeri has low, will cut DNA at few places
71
streaking of chDNA
good electrophoresis, completely digested chDNA
72
nicked DNA
damage in dsDNA, can be enzymatically induced or caused by physical damage during preparation
73
lambda control
well -studied bacteriophage of E. coli, known to have 2 restriction sites, making 3 frags, 48.5 kb
74
DNA ligation
reaction that forms recombinant DNA molecules by covalent bonding 2 restriction frags with compatible ends
75
T4 ligase
ATP
Mg2+ cofactor
free 3'OH and 5' PO4 3- end
synthesizes ester linkage
76
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
77
most likely outcome of ligation
Plasmid vector could ligate back on itself with no genomic DNA fragments
78
wanted ligation reaction outcome
plasmid accepts one fragment
79
best insert: vector
3:1
80
why do we set up mutliple ratios to improve overall chances of good ligation
to maximize chances of ligation
81
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
82
transformation
genetic alteration of organism by incorporation of foreign DNA into cells, DOES NOT HAVE TO INTEGRATE
83
induced competence
cold shock with CaCL2--- heat -- cold shock
84
attributes of a good host E. coli
no restriction endonucleases
no lacZ gene
no homologous recombination
limited resistance to ampicillin
85
interrupted lacZa
interrupted beta-gal will have glowing, white colony
86
preserved lacZa
preserved beta-gal,blue colonies
87
transformation efficiency equation
((# transformants/ µg pGEM )/(volume of transformant/volume plated)*dilution factor
88
week 1
isolate and purify chDNA
89
week 2
Digest chDNA and plasmid DNA for shotgun cloning of lux operon
90
week 3
ligate chDNA into plasmid DNA to create A. fischeri genomic library
91
week 4
transform E. Coli with plasmid library
92
week 5
screen E. coli for plasmids, containing lux operon
93
week 6
re-isolate plasmid
set up PCR for sequencing of lux I
94
rest of term
RNA isolation RT-PCR, qPCR
95
does E Coli have gene for auto-inducer
no
96
can E COli use quorum sensing
yes, by sensing AHL from other organisms
97
what is significantly upregulated when AHL is sensed
gadA
98
E Coli LuxR homolog
SdiA
99
ALKALINE lysis buffer
sugar, tris buffer, lysozyme, detergent, NaOH
100
neutralization buffer
glacial acetic acid, potassium acetate, RNase A , Chaotropic Salt
101
glacial acetic acid
neutralizes NaOH and allows plasmid to renature
102
potassium acetate
causes organic materials to precipitate out
103
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.
104
wash buffer
wash buffer+ 90% EtOH
105
elution buffer
DNA grade water or TE buffer
106
PCR
polymerase chain reaction, amplifies a region of DNA to thousands of millions of copies
107
PCR requirements
DNA Template
Forward and Reverse Primers
Taq Polymerase
Deoxynucleoside triphosphates (dNTPs)
Buffer Solution
Bivalent Cation (Mg2+)
Monovalent Cation (K+)
108
PCR steps
denaturation
annealing
elongation
109
temps of each PCR step
denaturation 95
annealing 55
elongation 72
110
good primer attributes
20 bp long high GC content
111
negative control
pGEM from a blue colony
112
positive control
pGEM+lux+ from a commercially bought organism
113
experimental value
pGEM+lux+ from a glowing colony
114
furthest line on a PCR result
primer dimer
115
2nd to bottom line of PCR result
lux I PCR product
116
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.
117
what is gadAX operon used for
acid tolerance of E coli
118
Culture A: E. coli + pGEMlux-
no lux operon to produce AHL, so no upreg of gadA in E coli
119
Culture B: E. coli + pGEMlux+
AHL is present E coli senses it with sdiA, upregs gadA
120
Why RNA
gives a screenshot of what is being produced at that very moment
121
most common method of extraction
acid guanidinium thiocyanate-phenol-chloroform extraction
122
RNA must be converted into DNA bc
DNA polymerase in PCR tubes will not work
123
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
124
reverse transcriptase
compliments RNA to amplify only a transcript made at a certain point
125
what do we do with DNA after reverse transcription
qPCR instead of PCR
126
conventional PCR
1. starts with DNA
2. Amplicon is detected and analyzed at an end point
3. Analyzes finished product
4. usually to get a concentrated amount of DNA
127
qPCR
1. starts with chDNA
2. amplicon is based off of already existing concentration of nucleic acid
3. analyzing product, in real time
4. usually look at how much transcript was made
128
fluorescent dye
SYBR green
129
Cq
number of cycles of qPCR needed to detect fluorescence
130
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
131
relative expression
2^(Ct(Ref)-Ct(Target))
132
two ratios of relative expression
Control Expression: control/control
Target Expression: target/control
