Module 7 - Microbial Genomics Flashcards
1
Q
What is genomics?
A
Methods to study the entire genome of a microbe
2
Q
Why is genomics important?
A
It provides insight into evolutionary relationships and detection of unknown organisms
3
Q
What does introduction of DNA into microbes often lead to?
A
Gene disruption, allowing for studying loss-of-function on phenotype
4
Q
What is a genome?
A
An organism’s complete set of DNA (including all genes)
5
Q
What is genomics?
A
The collective characterization and quantification of genes (entire genome)
6
Q
What is genomics?
A
The collective characterization and quantification of genes (entire genome)
7
Q
What does genomics focus on?
A
The structure, function, evolutionary mapping, and editing of genomes
8
Q
What needs (3) were created based on genomics?
A
- Improved DNA sequencing techniques
- Formats for storage of very large data sets
- Tools for analysis of large data sets
9
Q
What needs (3) were created based on genomics?
A
- Improved DNA sequencing techniques
- Formats for storage of very large data sets
- Tools for analysis of large data sets
10
Q
When was DNA sequencing first developed?
A
1970
11
Q
What is sequencing?
A
The process of determining nucleic acid sequence
12
Q
What did Walter Gilbert do?
A
Developed a chemical degradation method of DNA sequencing
13
Q
When was Sanger sequencing developed?
A
Around 1970 (same time as Gilbert sequencing)
14
Q
When was Sanger sequencing developed?
A
Around 1970 (same time as Gilbert sequencing)
15
Q
What did Fredrick Sanger do?
A
Developed an enzymatic method using DNA polymerase
16
Q
What is another name for Sanger sequencing?
A
Dideoxy sequencing, or chain termination sequencing
17
Q
What are the three steps of Sanger sequencing?
A
- Clone a gene of fragment of DNA of interest
- Synthesize DNA with DNA polymerase
- Use electrophoresis to separate fragments of DNA at different lengths
18
Q
What are the three steps of Sanger sequencing?
A
- Clone a gene of fragment of DNA of interest
- Synthesize DNA with DNA polymerase
- Use electrophoresis to separate fragments of DNA at different lengths
19
Q
What is Sanger sequencing based on?
A
The fact that DNA polymerase requires a free 3’ OH group to continue DNA synthesis
20
Q
What holds the two strands of DNA together?
A
Hydrogen bonds
21
Q
What holds two consecutive nucleotides together?
A
Phosphodiester bonds (between 5’ phosphate group and 3’ hydroxyl group)
22
Q
What is the significance of the 3’ OH group?
A
It is essential for chain elongation
23
Q
What is the significance of the 3’ OH group?
A
It is essential for chain elongation
24
Q
What is a nucleotide with a 3’ H called?
A
A dideoxyribonucleoside triphosphate (ddNTP)
25
What is the principle of dideoxy sequencing?
ddNTPs will stop chain elongation
26
What is the principle of dideoxy sequencing?
ddNTPs will stop chain elongation
27
How is the DNA sequence determined in Sanger sequencing?
By detecting labeled nucleotide at the end of each fragment separated by gel electrophoresis
28
How is the DNA sequence determined in Sanger sequencing?
By detecting labeled nucleotide at the end of each fragment separated by gel electrophoresis
29
What are the components (4) of the reaction mixture for Sanger sequencing?
1. A template DNA to be sequenced
2. A short oligonucleotide primer
3. DNA polymerase enzyme
4. All 4 dNTPs
30
How is the primer designed in Sanger sequencing?
To be complementary to the vector sequence
31
Why is the primer labeled in Sanger sequencing?
To allow for ease of detection of DNA pieces
32
Why is the primer labeled in Sanger sequencing?
To allow for ease of detection of DNA pieces
33
How is the Sanger sequencing reaction carried out?
In 4 tubes
34
What is the different with each tube in Sanger sequencing?
The specific ddNTP used (ddATP, ddTTP, ddCTP, and ddGTP)
35
What happens when a ddNTP is incorporated in a reaction in Sanger sequencing?
DNA synthesis is terminated
36
What happens when a ddNTP is incorporated in a reaction?
DNA synthesis is terminated
37
What happens when a dNTP is incorporated in a reaction in Sanger sequencing?
The DNA chain elongation will continue
38
What determines how many products will be in each reaction tube in Sanger sequencing?
Based on the number of times a specific nucleotide appears in the template DNA sequence (number of incorporation points)
39
How does a gel separate DNA fragments?
Larger fragments are at the top, while smaller fragments are at the bottom
40
How does a gel separate DNA fragments?
Larger fragments are at the top, while smaller fragments are at the bottom
41
What types of labels can be used in Sanger sequencing?
Radioactive labels, or fluorescent labels
42
How come fluorescent labels are used more frequently than radioactive labels?
Fluorescent labels are safer, cheaper, and easier than radioactive labels
43
How come fluorescent labels are used more frequently than radioactive labels?
Fluorescent labels are safer, cheaper, and easier than radioactive labels
44
How many bases can be sequenced by Sanger sequencing in a day?
700-1000 bases
45
How many bases can be sequenced by Sanger sequencing in a day?
700-1000 bases
46
How is a gel read in Sanger sequencing?
From bottom to top (Guitar Hero)
47
If a gel from Sanger sequencing reads 5'-AGTCT-3', what is the DNA sequence from 5' to 3'?
5'-AGACT-3'
48
If a gel from Sanger sequencing reads 5'-AGTCT-3', what is the DNA sequence?
5'-AGACT-3'
49
What is primer walking?
Designing primers such that the 5' end complements the end of the last DNA segment sequenced
50
What is primer walking?
Designing primers such that the 5' end complements the end of the last DNA segment sequenced
51
What is pyrophosphate?
Two phosphates bonded together
52
What is pyrophosphate?
Two phosphates bonded together
53
How is pyrosequencing similar to Sanger sequencing?
It uses DNA polymerase
54
What happens if a dNTP is incorporated in pyrosequencing?
A pyrophosphate is released, which can be detected
55
What happens if a dNTP is incorporated in pyrosequencing?
A pyrophosphate is released, which can be detected
56
What reaction does pyrophosphate undergo in pyrosequencing?
APS + PP --> ATP
57
What does APS stand for?
Adenosine phosphosulfate
58
What enzyme catalyzes the reaction of pyrophosphate in pyrosequencing?
ATP-sulfurylase
59
What does ATP-sulfurylase do?
Converts APS and pyrophosphate into ATP
60
What does ATP do in pyrosequencing?
It can be used in a luciferase reaction to produce detectable light
61
What does CCD stand for?
Charged-coupled device
62
What does CCD stand for?
Charged-coupled device
63
What happens in pyrosequencing after a wash?
The reaction is repeated with a different dNTP base
64
When is light detected in pyrosequencing?
Only when the dNTP is complementary to the template base
65
How many bases can be sequenced by pyrosequencing in a day?
300-500
66
What is pyrosequencing used for?
Resequencing or sequencing genomes for which a close relative is already available
67
What is pyrosequencing used for?
Resequencing or sequencing genomes for which a close relative is already available
68
What are the steps of whole-genome shotgun sequencing?
1. Shear the DNA into short pieces
2. Sequence the fragments
3. Use a computer algorithm to reconstruct
69
What are the steps of whole-genome shotgun sequencing?
1. Shear the DNA into short pieces
2. Sequence the fragments (Sanger sequencing)
3. Use a computer algorithm to reconstruct
70
How can the fragments of DNA be sequenced in whole-genome shotgun sequencing?
1. Cloning fragments, then Sanger sequencing
| 2. Directly on fragments using high-throughput sequencing
71
What do computer programs do in whole-genome shotgun sequencing?
Identify regions of sequence overlap from the fragments
72
What do computer programs do in whole-genome shotgun sequencing?
Identify regions of sequence overlap from the fragments
73
How come ~10x genome size is needed for whole-genome shotgun sequencing?
There is a random distribution of fragments generated
74
How come ~10x genome size is needed for whole-genome shotgun sequencing?
There is a random distribution of fragments generated
75
How many microorganisms have currently been sequenced?
3,000
76
How many microorganisms have currently been sequenced?
3,000
77
What is another name for high-throughput sequencing?
Next-generation sequencing
78
How many base pairs can high-throughput sequencing sequence at a time?
25-500
79
How many reads can high-throughput sequencing generated?
Hundreds, thousands, or millions of reads
80
What is the consequence of high-throughput sequencing having many reads?
High coverage, but a more computationally intensive assembly process
81
What are some examples of high-throughput sequencing?
Illumina (Solexa), Nanopore DNA sequencing, single molecule real time (SMRT), DNA nanoball, and SOLiD sequencing
82
What are some examples of high-throughput sequencing?
Illumina (Solexa), Nanopore DNA sequencing, single molecule real time (SMRT), DNA nanoball, and SOLiD sequencing
83
What DNA sequencing is currently under active development?
Third-generation sequencing
84
How does third-generation sequencing work?
By reading nucleotide sequences at the single molecule level
85
What is bioinformatics?
An interdisciplinary field that develops software and methods to understand large and complex biological data
86
What is bioinformatics?
An interdisciplinary field that develops software and methods to understand large and complex biological data
87
What is the purpose of annotation?
It allows for researchers to predict ORFs
88
What does ORF stand for?
Open reading frames
89
What is the importance of ORFs?
They allow researchers to better determine the start and stop points for a given gene
90
How can functions for newly discovered proteins be suggested?
Based on observed similarities
91
What can researchers speculate about a gene by looking at its sequence?
Whether it is a transcriptional factor, transport protein, or some enzyme
92
True or false: many genes have known proteins associated with them
False: many genes predicted by sequencing data encode gene products whose functions remain unknown
93
What is functional genomics?
The study of finding out the biological role of unknown genes
94
What is functional genomics?
The study of finding out the biological role of unknown genes
95
What types of experiments are part of functional genomics?
Using metagenesis to study phenotypes
96
How is gene expression generally regulated?
At the transcriptional level
97
What is a genomic library?
A collection of all the genes in a genome (cloned DNA fragments)
98
What can a genomic library be used for?
Whole genome sequencing
99
What does the method for obtaining a genomic library depend on?
The desired outcome
100
How can an mRNA library be formed?
By using cDNA generated from reverse transcriptase
101
What does reverse transcriptase do?
Convert RNA into DNA
102
What does cDNA stand for?
Complementary DNA
103
What does cDNA do?
It is the complement to the expressed mRNA
104
How can a true genomic library be formed?
By shearing the genome and cloning the fragments
105
How are DNA fragments prepared for a genomic library?
Through restriction analysis
106
How are clones generated for a genomic library?
By ligating the DNA fragments into plasmids, and transforming the cells
107
What determines how many clones are needed to represent the whole genome in a genomic library?
The size of the genome, and the average size of each cloned fragment
108
What equation can be used to calculate how many clones are needed for a genomic library?
N = ln(1-p)/ln(1-f)
109
In the equation for a genomic library, what is N?
The number of cloned fragments
110
In the equation for a genomic library, what is p?
The probability of generating a complete library
111
In the equation for a genomic library, what is f?
The average size of a fragment divided by the total genome size
112
What is the difference between a cDNA library and a true genomic library?
A true genomic library has all the genes, while the cDNA library only has genes that encode proteins
113
What is the difference between a cDNA library and a true genomic library?
A true genomic library has all the genes, while the cDNA library only has genes that encode proteins
114
What understanding do expression patterns allow for?
How organisms function under different conditions
115
What is a transcriptome?
A set of transcribed mRNA molecules in a cell
116
How can a transcriptome be measured?
Through northern blots
117
What is the procedure for a northern blot?
1. Total RNA is isolated from cells of interest, and separated with gel electrophoresis
2. RNA is transferred to a membrane
3. RNA is probed with labeled DNA fragments
118
What is the procedure for a northern blot?
1. Total RNA is isolated from cells of interest, and separated with gel electrophoresis
2. RNA is transferred to a membrane
3. RNA is probed with labeled DNA fragments
119
What happens after gel electrophoresis in a northern blot?
RNA is transferred to a membrane made of nitrocellulose or nylon
120
How is RNA linked to the nitrocellulose membrane?
Through exposure to UV light
121
What probe is used in a northern blot?
A labelled DNA probe which is complementary to the mRNA being examined
122
How can the location of the signal on the membrane be measured in a northern blot?
Through autoradiography or photography
123
What is the disadvantage of a northern blot?
It is cumbersome to track the expression of multiple genes at once
124
What is the disadvantage of a northern blot?
It is cumbersome to track the expression of multiple genes at once
125
What is a DNA microarray?
A collection of microscopic DNA spots attached to a solid surface
126
How does the procedure of a microarray compare to northern blotting?
A microarray is essentially the reverse procedure
127
How does the procedure of a microarray compare to northern blotting?
A microarray is essentially the reverse procedure
128
What are the steps of the microarray procedure?
1. A glass slide is spotted with synthesized oligonucleotides
2. Total mRNA is extracted from the cell
3. mRNA is converted into cDNA, incorporating a fluorescent label
4. The cDNA is passed over the chip
129
In the microarray procedure, what happens if more mRNA copies are present in a sample?
More fluorescently labeled cDNA corresponding to that mRNA is produced
130
How are the fluorescent labels used in a microarray?
They are captured by a scanner and analyzed by a software to quantify the amount of binding to each individual spot
131
How are the fluorescent labels used in a microarray?
They are captured by a scanner and analyzed by a software to quantify the amount of binding to each individual spot
132
How can a microarray be used with two different samples?
The cDNA from two different samples can be labeled differently, and hybridize to a single microarray
133
How is the relative amount of cDNA in a microarray seen?
Through color output
134
How is the relative amount of cDNA in a microarray seen?
Through color output
135
What does Yersinia pestis do?
Infects a variety of rodents, and can replicate within fleas
136
What is the primary transmission of Yersinia pestis between rodents?
Flea bites
137
How can Yersinia pestis be transmitted to humans?
Via fleas or an infected animal
138
What is special about the hosts for Yersinia pestis?
They differ greatly in internal body temperature
139
What is the internal body temperature of a human
37 C
140
What is the internal body temperature of a flea?
26 C
141
What conditions were used in the microarrays studying Yersinia pestis?
Two different temperatures, corresponding to the flea and human
142
What can be done to the genes identified from the Yersinia pestis microarray?
They can be studied further to identify function, role in pathogenesis, or potential as targets for therapeutic applications
143
What can be done to the genes identified from the Yersinia pestis microarray?
They can be studied further to identify function, role in pathogenesis, or potential as targets for therapeutic applications
144
What is the most recent technology in studying transcriptomes?
RNA-sequencing
145
What is another name for RNA-sequencing?
RNA-seq
146
What are the steps for RNA-seq?
1. RNA is isolated from cells, and the rRNA is removed
2. Remaining RNA is converted to cDNA by reverse transcriptase
3. The cDNA can be sequenced through high throughput sequencing
147
In RNA-seq, before cDNA is sequenced, what is done?
Sequencing linkers are attached to cDNA fragments
148
What does the cDNA represent in RNA-seq?
The transcripts within a cell
149
What happens after the cDNA is sequenced in RNA-seq?
The resulting sequences are compared to known RNA sequences (bioinformatics)
150
How can the abundance of RNA transcripts be confirmed in RNA-seq?
Through qPCR
151
What was RNA-seq first used for?
Used to examine the transcriptome of Saccharomyces cerevisiae
152
Besides microorganisms, what can RNA-seq be used on?
The genome of RNA viruses
153
Which is used more frequently: RNA-seq or microarrays?
RNA-seq
154
What will cause RNA-seq to evolve?
Advancements in DNA sequencing and analysis methods
155
What is a proteome?
A collection of all the proteins present in a cell under specific conditions
156
What do differences in protein types and abundance reflect?
Changes in gene expression and/or protein stability
157
Why is the study of proteomes important?
Proteins can vary due to stability or post-translational modifications, which cannot be detected by analyzing gene expression or mRNA
158
What are some methods to study proteomics?
2D-PAGE, mass spectrometry, X-ray crystallography, and NMR
159
What does NMR stand for?
Nuclear magnetic resonance
160
What does 2D-PAGE stand for?
2D-polyacrylamide gel electrophoresis
161
What is 2D-PAGE?
A 2D separation method to separate proteins based on two properties
162
What properties are proteins separated by in a 2D-PAGE?
Isoelectric point and mass
163
What does pI stand for?
Isoelectric point
164
What is an isoelectric point?
The pH where a protein has no net charge
165
What determines the isoelectric point of a protein?
The amino acid sequence
166
What will a protein do in a pH gradient?
Migrate to the pH that matches pI
167
What do proteins in a 2D-PAGE look like?
Spots on a 2D matrix
168
What does the pattern of spots represent on a 2D-PAGE?
The protein composition of a cell
169
What does the different pattern of spots in a 2D-PAGE suggest?
A measure of gene expression in different conditions
170
How are many protein in a cell expressed?
Under conditions where they function or are needed by the cell
171
How can the identities of proteins in an individual spot in a 2D-PAGE be determined?
By using mass spectrometry
172
What makes mass spectrometry possible for identifying proteins?
Being able to compare amino acid sequences with corresponding genome sequence databases
173
What are the steps to identify a protein from a 2D-PAGE?
1. Spots are extracted from the gel
2. The protein is digested into smaller fragments
3. The fragments are analyzed by mass spectrometry to determine amino acid sequence
4. These sequences are compared to a sequence database
174
How is a protein digested into smaller fragments?
Through proteases
175
What do proteases do?
Break down proteins into smaller fragments
176
How is the amino acid sequence determined from mass spectrometry?
By considering the individual mass/charge ratio
177
What is comparative genomics?
The study of evolutionary processes using the tools of genomics
178
What is the goal of comparative genomics?
To determine relationships between species
179
What can comparative genomics identify (in terms of genes)?
Genes associated with virulence and pathogenicity
180
Where does genetic variability come from?
Mutations in the DNA sequence
181
What are homologs?
Genes in a given genome that belong to related gene families that share a common ancestral DNA sequence
182
What are the two types of homologs?
Paralogs and orthologs
183
How can homologs arise?
From a gene duplication event
184
What happens (in terms of evolution) what a gene is duplicated?
One copy of the gene can evolve novel functions, while the other copy performs the original function
185
What are paralogs?
Homologous genes that arise from a duplication event
186
What is an example of a paralog?
Malate dehydrogenase and lactate dehydrogenase
187
What is an example of a family of paralogs?
ABC transporters
188
True or false: paralog families are tiny
False: they can be quite large
189
What is important in the evolution of genomes?
The duplications of genes
190
What are orthologs?
Homologs that evolved from the same ancestor with the same function in different organisms
191
What is an example of an ortholog?
Malate dehydrogenase in two different genomes
192
What does HGT stand for?
Horizontal gene transfer
193
What is horizontal gene transfer?
The movement of DNA between organisms other than transmission from parent to offspring
194
What is another name for HGT?
Lateral gene transfer
195
True or false: the genome of every organism contains foreign genes
True: HGT is important in all organisms
196
True or false: plasmids are the only way HGT can occur
False: the genome of every organism contains foreign genes
197
What is an indication that a gene is foreign?
If the base pair composition differs significantly from the rest of the chromosome
198
What is a characteristic of a specific genome (specific to a particular organism)?
The GC content
199
What is the GC content of E. coli?
50%
200
What is the GC content of Streptomyces coelicolor?
72%
201
What is the GC content of S. cerevisiae?
38%
202
What is some evidence for HGT?
Gene phylogeny and differences in nucleotide pair patterns
203
What was found when comparing genome sequences of related microbes?
Large segments of DNA exist in one genome, but not in a closely related genome
204
What do large segments of DNA that exist in one genome, but not a closely related one, suggest?
Introduction or removal of large stretches of DNA occur through one or more gene transfer mechanisms with the assistance of transposable elements
205
What are genomic islands?
Introduced DNA segments greater than 10kb up to 200 kb
206
How are genomic islands detected?
By comparison of genomic sequences and analysis of nucleotide composition
207
What is a characteristic of genomic islands?
They often contain GC content different from the majority of the genome
208
What are genomic islands usually associated with?
tRNA genes, transposable elements, plasmids, or bacteriophages
209
What is metagenomics?
A process where DNA is extracted directly from microbial communities and analyzed as a composite mixture
210
What happens to extracted DNA in metagenomics?
They can be sequenced directly or cloned into vectors to make libraries for further analysis
211
What is another name for metagenomics?
Environmental genomics
212
What is the consequence of having many organisms in microbial communities?
It is difficult to complete DNA sequences of each individual genome
213
How come only a subset of microbial communities are targeted for metagenomics?
There are many organisms in microbial communities
214
In what environments is metagenomics used?
Freshwater, wastewater treatment systems, acid mine drainages, and deep-sea thermal vents
215
What can metagenomics lead to?
The discovery of new genes encoding novel enzymes and antibiotics, or evidence of new organisms
216
What are the steps for metagenomics?
1. Obtain DNA from a sample
2. Sequence using rapid next-gen sequencing
3. Analyze sequences using a computer
217
What must be done to analyze sequences in metagenomics?
Sequences from already known organisms must be eliminated
218
In Sanger sequencing, which dNTP is radiolabeled?
dCTP (32P-dCTP)
219
What is the importance of radiolabeling dCTP?
It is important for collecting data
220
True or false: radiolabeled dCTP can be added to the DNA strand
True: it still has the 3' OH group
221
What helped increase the number of nucleotides that can be read from Sanger sequencing?
Improvements in gel electrophoresis technology
222
What were some improvements of Sanger sequencing over the years?
Thermostable polymerases, fluorescent labels for each ddNTP, and base-calling softwares
223
How are thermostable polymerases advantageous for Sanger sequencing?
It allows multiple rounds of synthesis from a single template strand
224
How are fluorescent labels for each ddNTP advantageous for Sanger sequencing?
The reaction can be done in one tube instead of four
225
How are base-calling softwares advantageous for Sanger sequencing?
They can interpret the raw data automatically and provide a direct sequence output
226
What is automated sequencing?
Using longer electrophoresis in the Sanger reaction to lead to more sequencing data
227
What is another name for automated sequencing?
Cycle-sequencing
228
When was high-throughput sequencing developed?
2004
229
What does apyrase do?
Removes unincorporated dNTPs and ATP from the system
230
Where is apyrase used?
In pyrosequencing
231
What is 454-pyrosequencing?
An adaptation of pyrosequencing that dramatically increased throughput
232
Why is it called 454-pyrosequencing?
"454" is the code name of the sequencing technology project
233
What are the steps of 454-pyrosequencing?
1. DNA is fragmented into short pieces, then ligated onto beads
2. Each bead can be separated into its own PCR reaction
3. The pyrosequencing reagents are added to each well
234
What is the advantage of 454-pyrosequencing?
It increases sequencing throughput
235
What is emulsion PCR?
PCR carried out on the surface of a bead in an oil emulsion
236
Where is emulsion PCR used?
In 454-pyrosequencing
237
What is Ion Torrent?
A high-throughput sequencing method
238
What is Ion Torrent related to?
454-pyrosequencing
239
How does Ion Torrent work?
Like 454-pyrosequencing, but it measures protons instead of pyrophosphate each time a nucleotide is incorporated
240
What is another name for Ion Torrent?
Ion-semiconductor sequencing
241
How does Illumina sequencing work?
1. DNA polymerase adds fluorescently labeled nucleotides
2. An image is taken, and the fluorescent label is cleaved
3. Paired-end reads can be analyzed separately
242
What is PacBio?
A system for genome sequencing
243
What is the advantage of more coverage in shotgun sequencing?
It helps reduce gaps in the final assembly that would need to be filled in with time consuming methods like primer walking
244
How can gaps be reduced in shotgun sequencing?
By combining a long, error-prone sequencing with short sequences
245
In shotgun sequencing, which method produces long, error-prone sequences?
PacBio
246
In shotgun sequencing, which method produces short sequences?
Ion Torrent or Illumina
247
What does the long error-prone sequence do in shotgun sequencing?
Gives a backbone (scaffold) for the overall genome
248
What do the short sequences do in shotgun sequencing?
Ensure that the final genome does not contain errors
249
What is a pan-genome?
The full complement of all genes within a species of bacteria (in all different strains)
250
What fundamental technique is used to study the transcriptome?
Nucleic acid hybridization
251
What does a Southern blot do?
Detects specific DNA sequences
252
What are the steps for a Southern blot?
1. Generate DNA fragments through restriction enzymes
2. Separate the fragments through gel electrophoresis
3. Transfer the DNA fragments to a membrane for further analysis
253
What advances led to DNA microarrays?
Advances in photolithography and the availability of complete genome sequences
254
What does photolithography refer to?
The technique of creating computer chips
255
In the Y. pestis experiment, which genes could be important in pathogenicity?
The green and red genes (not the yellow genes)
256
How come the green and red genes may be important in Y. pestis pathogenicity?
They respond differently according to the different host temperatures
257
What does SDS-PAGE stand for?
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
258
What does an SDS-PAGE do?
Separates denatured proteins based on mass
259
How does an SDS-PAGE work?
It mitigates charge, so proteins can be separated based on mass alone
260
How come most of the proteome would not resolve on an SDS-PAGE?
Many polypeptides have similar mass
261
What are the steps of a 2D gel?
1. Apply a protein sample to a pH gradient on a polyacrylamide strip
2. Use a current to separate by pI
3. Use this strip in electrophoresis to separate by mass
262
What does LC-MS stand for?
Liquid chromatography-mass spectrometry
263
What is the purpose of LC-MS?
To separate proteins by LC before analyzing them with MS
264
What methods can be used to study the structures of proteins?
X-ray crystallography and NMR
265
How does X-ray crystallography work?
Crystallized proteins are subjected to X-rays. The scattering patterns can be detected and analyzed
266
How easy is protein crystallization?
It is a delicate and often difficult process
267
What are the disadvantages of protein crystallization?
Not all proteins crystalize well
268
Which proteins are especially difficult to crystallize?
Hydrophobic proteins that normally associate with cytoplasmic membranes
269
What is the advantage of NMR?
It is able to determine the structure of proteins while in solution
270
How does NMR work?
It measures the distances between atomic nuclei
271
What is the disadvantage of NMR?
It has a maximal size limit of about 30 kDa (the size of an average bacterial protein)
272
How are the different proteins in a paralog family related?
They probably carry out similar functions, but on different substrates
273
How are the different proteins in an ortholog family realted?
They probably carry out the same function (and same substrate) in different organisms
274
What does the evolutionary relationship of orthologs mirror?
The evolutionary history of their respective genomes
275
What do dehydrogenases do?
They carry out NAD or NADP dependent reactions
276
What does malate dehydrogenase do?
Converts malate into oxaloacetate
277
What does lactate dehydrogenase do?
Converts lactate into pyruvate
278
Why can genomes be considered as mosaics?
They have arisen from evolutionary changes and horizontal gene transfer
279
How come scientists first believed that HGT had no evolutionary advantage to microbes?
1. There were different gene regulatory elements involved
2. Expression was needed for the new gene
3. The new gene could be detrimental
280
Besides GC content, what are some clues that HGT has taken place?
Differences in patterns of nucleotide base pairs, codon usage patterns, and presence of repetitive sequences
281
How can gene phylogeny imply HGT?
If evolutionary relationships from the gene do not match the core genome, this is evidence that HGT has taken place
282
When is HGT most successful?
When two organisms have similar DNA and GC content
283
What are some examples of advancements due to metagenomics?
1. The discovery of proteorhodopsins
2. The discovery of ammonia-oxidizing archaea
3. The discovery of vitamin B12 producers in the ocean
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What do proteorhodopsins do?
Harness light energy for metabolism in marine environments
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Who pioneered functional metagenomics?
Jo Handelsman
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How can sequences from metagenomics be linked to specific microbes?
Through databases or SSU rRNA genes
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What are some limitations of metagenomics?
Researchers cannot make firm predictions based solely on available sequence information
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What does FACS stand for?
Fluorescently associated cell sorting
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How does FACS work?
A DNA probe for a SSU rRNA is used in a permeabilized cell to recover individualized uncultivated cells
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What is single-cell genomics?
Understanding the genome of one cell through FACS
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What are some challenges of metagenomics?
Co-extracted contaminants and community complexity
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What is metatranscriptomics?
The study of RNA transcripts directly extracted from a commuinty
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What is metaproteomics?
The study of environmental proteins
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How do metatranscriptomics and metaproteomics work?
Through bioinformatics (similar to individual organisms)
