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BIO219 > master deck > Flashcards

master deck Flashcards

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

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2
Q

A Fischeri

A

gram negative, contains lux operon

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3
Q

gram-negative bacteria

A

external LPS layer
thin peptidoglycan wall

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4
Q

name 2 gram negative bacteria

A

E coli. A. fischeri

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5
Q

operon

A

genetic regulatory system in when genes coding for functionally related proteins are clustered

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6
Q

ChDNA

A

large circular piece of DNA, where we got lux operon

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7
Q

restriction digestion

A

enzymatic rxns that cut DNA into smaller pieces, run by enzymes that cut DNA in specific places

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8
Q

what percent of deep sea marine organisms have a symbiotic relationship with bacteria

A

96%

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9
Q

what benefit do bacteria offer fish

A

attract prey, mate
communication
hide from predators

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10
Q

how do fish benefit bacteria

A

protection, reliable food source(glucose, amino acids)

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11
Q

how is lux operon gene expression regulated

A

quorum sensing

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12
Q

quorum sensing

A

regulation of gene expression in response to changes in cell-population density

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13
Q

what does production of light require

A

lots of energy and oxygen, no glow without it

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14
Q

how does bacteria sense growth

A

Auto-induction

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15
Q

Bacteria release small metabolic product

A

N-acyl homoserine lactone

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16
Q

how does AHL reach extracellular environment

A

free diffusion

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17
Q

as density of bacteria increases

A

more inducer is released into environment

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18
Q

at critical concentration

A

AHL diffuses back in to interact with lux operon

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19
Q

lux R

A

codes the transcriptional activator that binds to AHL

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20
Q

lux I

A

codes the AHL synthase

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21
Q

Lux C

A

codes the acyl reductase

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22
Q

lux D

A

codes the acyl transferase

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23
Q

lux A

A

codes the alpha subunit of luciferase

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24
Q

lux B

A

codes the beta subunit of luciferase

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25
Q

lux E

A

codes the acyl protein synthetase enzyme

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26
Q

what initiates gene expression of lux operon

A

AHL binding to lux R and lux I

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27
Q

what enzymes are needed to generate long chain fatty aldehyde

A

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

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28
Q

what is bioluminescence dependent on

A

NADPH

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29
Q

resuspension buffer

A

contains SDS to solubilize membrane proteins, disrupts lipid layer of gram negative cells, may be appearance of soap bubbles

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30
Q

proteinase K

A

responsible for freeing nucleic acids and disabling nucleases, stable in range of pH

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31
Q

Rnase A

A

an efficacious ribonuclease used to degrade RNA

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32
Q

how does RNase A cleave RNA

A

cleaves at 3’ side of phosphodiester bond after pyrimidines

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33
Q

why does DNA stay stable in presence of RNase A

A

does not have 2’ OH group

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34
Q

what does detergent do

A

disrupts lipid layer and brings proteins into lipid protein complexes

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35
Q

lysis buffer

A

contains chaotropic salt to disrupt regular hydrogen bonding with water, sets up conditions for DNA to bind to silica column

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36
Q

ethanol

A

enchance and influence binding of nucleic acids to silica by creating more hydrophobic solution

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37
Q

wash buffer

A

low amount of chaotropic salt that binds to and removes proteins and colored containments

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38
Q

wash buffer 2

A

contains ethanol to remove salts added from AW1

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39
Q

DNA grade water

A

water free of salts DNases and proteases which allows for rehydration

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40
Q

binding

A

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

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41
Q

at what wavelength does DNA absorb light

A

260 nm

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42
Q

beer-lambert law equation

A

A = εcl

ε=extinction coefficient, constant
c= conc of substance
l=light path

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43
Q

beer-lambert law

A

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

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44
Q

280 nm

A

absorbance maximum for proteins, due to Tyrosine, cysteine, tryptophan

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45
Q

230 nm

A

nucleic acids minimum absorbance, helps look for organic contaminants

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46
Q

A 260:280

A

chacks for protein/ RNA contamination
>2 = RNA contamination
<1.8= protein contamination, not great tho

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47
Q

A260:230

A

checks for organic contamination
outside of 2-2.2, presence of organic cmpds or salt

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48
Q

shotgun cloning

A

randomly digesting a large piece of DNA into smaller pieces that can be ligated into plasmids for transport to other organisms

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49
Q

once inside a vector

A

each vector contains separate frag of genome which is completely represented to refer back to

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50
Q

what do we get with shotgun cloning

A

frags with regulatory elements and coding sequences

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51
Q

lux operon is how long

A

8.5 kb

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52
Q

what makes a good plasmid vector

A

size
high copy number
ori
multiple cloning sites
selectable markers

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53
Q

size

A

large enough to hold foreign DNA, small enough to be retained by host and distinguished from host chDNA

54
Q

high copy number

A

50-100 per cell

55
Q

ORI

A

recognized by host machinery

56
Q

multiple cloning sites

A

Region of DNA containing recognition sequences for many restriction enzymes

57
Q

RNA polymerase promoter sequences

A

near mcs, mRNA can be made off inserted DNA

58
Q

what is our vector of choice

A

pGEM3zf(+)

59
Q

restriction endonuclease in vivo

A

cuts viral DNA to prevent infection, recognizes palindromic sequences

60
Q

1 unit of enzyme is

A

the amount that catalyzes the conversion of 1 micro mole of substrate per minute

61
Q

if conc of vector is .2µg/µL, volume needed to get us 1 µg is

A

1 µg ÷ 0.2 µg/µL = 5 µL of vector stock

62
Q

agarose gel ranges between

A

.7 and 2%

63
Q

.7% agarose

A

shows better separation of larger DNA fragments(5-10 kb)

64
Q

a 2% agarose gel will

A

show good resolution for smaller DNA fragments (.2-1 kb)

65
Q

GelRed

A

nucleic acid stain used to light up in gel electrophoresis, fluorophore

66
Q

TAE buffer

A

component of electrophoresis to prevent inconsistent separation of restriction frags

67
Q

loading dye

A

allows us to track DNA migration, as well as give weight to sample so it includes sugar or glycerol

68
Q

blunt ends

A

no overhang

69
Q

sticky ends

A

2-3 bp overhang

70
Q

Sal I

A

has a high GC content, while A. fischeri has low, will cut DNA at few places

71
Q

streaking of chDNA

A

good electrophoresis, completely digested chDNA

72
Q

nicked DNA

A

damage in dsDNA, can be enzymatically induced or caused by physical damage during preparation

73
Q

lambda control

A

well -studied bacteriophage of E. coli, known to have 2 restriction sites, making 3 frags, 48.5 kb

74
Q

DNA ligation

A

reaction that forms recombinant DNA molecules by covalent bonding 2 restriction frags with compatible ends

75
Q

T4 ligase

A

ATP
Mg2+ cofactor
free 3’OH and 5’ PO4 3- end
synthesizes ester linkage

76
Q

4 outcomes of ligation reaction

A

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
Q

most likely outcome of ligation

A

Plasmid vector could ligate back on itself with no genomic DNA fragments

78
Q

wanted ligation reaction outcome

A

plasmid accepts one fragment

79
Q

best insert: vector

A

3:1

80
Q

why do we set up mutliple ratios to improve overall chances of good ligation

A

to maximize chances of ligation

81
Q

why is 3:1 optimal

A

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
Q

transformation

A

genetic alteration of organism by incorporation of foreign DNA into cells, DOES NOT HAVE TO INTEGRATE

83
Q

induced competence

A

cold shock with CaCL2— heat – cold shock

84
Q

attributes of a good host E. coli

A

no restriction endonucleases
no lacZ gene
no homologous recombination
limited resistance to ampicillin

85
Q

interrupted lacZa

A

interrupted beta-gal will have glowing, white colony

86
Q

preserved lacZa

A

preserved beta-gal,blue colonies

87
Q

transformation efficiency equation

A

((# transformants/ µg pGEM )/(volume of transformant/volume plated)*dilution factor

88
Q

week 1

A

isolate and purify chDNA

89
Q

week 2

A

Digest chDNA and plasmid DNA for shotgun cloning of lux operon

90
Q

week 3

A

ligate chDNA into plasmid DNA to create A. fischeri genomic library

91
Q

week 4

A

transform E. Coli with plasmid library

92
Q

week 5

A

screen E. coli for plasmids, containing lux operon

93
Q

week 6

A

re-isolate plasmid
set up PCR for sequencing of lux I

94
Q

rest of term

A

RNA isolation RT-PCR, qPCR

95
Q

does E Coli have gene for auto-inducer

A

no

96
Q

can E COli use quorum sensing

A

yes, by sensing AHL from other organisms

97
Q

what is significantly upregulated when AHL is sensed

A

gadA

98
Q

E Coli LuxR homolog

A

SdiA

99
Q

ALKALINE lysis buffer

A

sugar, tris buffer, lysozyme, detergent, NaOH

100
Q

neutralization buffer

A

glacial acetic acid, potassium acetate, RNase A , Chaotropic Salt

101
Q

glacial acetic acid

A

neutralizes NaOH and allows plasmid to renature

102
Q

potassium acetate

A

causes organic materials to precipitate out

103
Q

project overview

A

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
Q

wash buffer

A

wash buffer+ 90% EtOH

105
Q

elution buffer

A

DNA grade water or TE buffer

106
Q

PCR

A

polymerase chain reaction, amplifies a region of DNA to thousands of millions of copies

107
Q

PCR requirements

A

DNA Template
Forward and Reverse Primers
Taq Polymerase
Deoxynucleoside triphosphates (dNTPs)
Buffer Solution
Bivalent Cation (Mg2+)
Monovalent Cation (K+)

108
Q

PCR steps

A

denaturation
annealing
elongation

109
Q

temps of each PCR step

A

denaturation 95
annealing 55
elongation 72

110
Q

good primer attributes

A

20 bp long high GC content

111
Q

negative control

A

pGEM from a blue colony

112
Q

positive control

A

pGEM+lux+ from a commercially bought organism

113
Q

experimental value

A

pGEM+lux+ from a glowing colony

114
Q

furthest line on a PCR result

A

primer dimer

115
Q

2nd to bottom line of PCR result

A

lux I PCR product

116
Q

chomatogram

A

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
Q

what is gadAX operon used for

A

acid tolerance of E coli

118
Q

Culture A: E. coli + pGEMlux-

A

no lux operon to produce AHL, so no upreg of gadA in E coli

119
Q

Culture B: E. coli + pGEMlux+

A

AHL is present E coli senses it with sdiA, upregs gadA

120
Q

Why RNA

A

gives a screenshot of what is being produced at that very moment

121
Q

most common method of extraction

A

acid guanidinium thiocyanate-phenol-chloroform extraction

122
Q

RNA must be converted into DNA bc

A

DNA polymerase in PCR tubes will not work

123
Q

cDNA

A

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
Q

reverse transcriptase

A

compliments RNA to amplify only a transcript made at a certain point

125
Q

what do we do with DNA after reverse transcription

A

qPCR instead of PCR

126
Q

conventional PCR

A
  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
Q

qPCR

A
  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
Q

fluorescent dye

A

SYBR green

129
Q

Cq

A

number of cycles of qPCR needed to detect fluorescence

130
Q

what does a low Cq mean

A

not many cycles were needed for fluorescence, more cDNA of DNA in that region, with more mRNA being made

131
Q

relative expression

A

2^(Ct(Ref)-Ct(Target))

132
Q

two ratios of relative expression

A

Control Expression: control/control
Target Expression: target/control

BIO219 (1 decks)

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