Gene transfer Flashcards
1
Q
What is horizontal gene transfer?
A
Movement of genes between cells that are not direct descendants of another to acquire new characteristics to fuel metabolic diversity and aid in evolution.
2
Q
What are the three mechanisms of genetic exchange in bacteria?
A
- transformation: free exogenous naked DNA released from one cell is taken up by another (natural or chemical)
- Transduction: DNA is packaged into a phage capsid and defective phage infects another cell, injecting bacterial DNA.
- Conjugation: DNA transfer from one cell to another via pilus
3
Q
What is a capsule?
A
Polysaccharide layer that allows bacteria to evade attack by immune system. Can have reversion between smooth and rough within the same strain (IIS-IIR, not IIS-IIIS)
4
Q
What were griffith’s contributions to the discovery of transformation?
A
- Griffith injected mice with safe streptococcus pneumonia IIR cells and lethal IS cells.
- He found that living S cells could be produced in a mixture of R and heat killed S cells, which showed that transforming material must be retained in dead S cells.
- He theorized and protein structure was responsible.
5
Q
What were McCloud and McCarty’s contributions to the discovery of transformation?
A
- reproduced griffith’s experiment with treatments of protease, DNAse and RNAse
- protease and RNAse with dead S cells killed the mouse, meaning that proteins and RNAs are not the material in the dead cells that allowed for transformation
- DNAse with dead S cells did not kill the mouse, meaning that DNA is the material in dead cells that allowed for transformation
6
Q
What are the requirements for natural transformation?
A
- DNA donor cells must be fragmented, linear and double stranded
- Recipient must be competent
7
Q
What makes a cell competent?
A
- High cell density at late log phase triggers excretion of competence factors
- competence factors bind to surface of cell to induce synthesis of other proteins involved in transformation
8
Q
How do pili make cells competent?
A
- Pilus binds to free double stranded DNA and actively retracts, brining DNA toward the cell surface.
- Nuclease near membrane degrades one of the two strands of DNA as the other enters the cell
- The ssDNA is paired with homologous region of host chromosome mediated by RecA
- ssDNA will be integrated into the bacterial genome by homologous recombination via RecBCD pathway
9
Q
What are the steps of the RecBCD pathway?
A
- Endonuclease nicks donor DNA
- RecBCD complex unwinds donor DNA w/ helicase activity, and the displaced ssDNA is coated with SSB proteins
- RecBCD loads RecA onto the ssDNA region, displacing SSB and searches for homologous sequences in the host
- RecA-bound ssDNA invades a homologous duplex in the host DNA, forming a cross-strand complex.
- Cross strand complex is cleaved on a horizontal or vertical plane by resolvase enzyme resulting in patches or splices
- Transformed now has donor DNA integrated and is successfully recombined, deetcted by change in phenotype.
10
Q
What’s the difference between a patch and a slice?
A
Patch: horizontal cut is made on two strands that did not exchange, resulting in one parental, and one recombinant strand (result of a horizontal cut of duplex)
Splice: vertical cut is made on two strands that did exchange, resulting in two recombinant strands (result of a horizontal cut of duplex)
11
Q
Are all bacteria transformable?
A
Not natrually but they may be induced by uptake of specifically designed plasmids following chemical treatment or exposure to electric current.
12
Q
What is an example of transformation mechanisms where host chromosome has sequences that recognize “self”
A
Haemophilus influenzae does not require competence factor, and will only take up linear dsDNA with an 11-bp DNA sequence recognized by cell surface receptors
13
Q
Why can’t dsDNA be used for artificial transformation?
A
It has exposed ends which are targets for an incompetent host endonucleases that protect against incorporation of foreign DNA.
14
Q
What are the two types of transduction?
A
Generalized: only bacterial DNA is carried in the transducing particle, any gene can be transferred
Specialized: some bacterial DNA and some phage DNA is carried, and only specific genes can be transferred from the donor
15
Q
What are the steps of generalized transduction?
A
- Transducing phage undergoes adsorption and attaches to cell
- Phage injects nucleic acid into cell
- DNA is recombined into host genome, lytic/lysogenic cycle does not occur due to lack of phage genes
16
Q
What are the elements involved in a generalized transduction?
A
Transducing phage: capable of transduction
Transducing particle: particle of phage that has picked up bacterial DNA (35-80 genes)
Donor strain: original bacterial strain that transducing particle picked DNA from
Transductant: cell that has received the donor DNA
17
Q
What are the conditions to be a generalized transducing phage?
A
- temperate or lytic
- must not completely degrade host DNA
- need to have general (non-specific) pac or cos sites for packaging of DNA
- broad host range of absorption of possibility on wide range of bacterial species
18
Q
What are the three possible fates for injected DNA?
A
- host restriction: host degrades DNA via restriction enzymes
- Abortive transduction: not degraded but fails to combine and is diluted out of population after many cell divisions
- Stable gene transfer: exogenous dsDNA paired with homologous region in chromosome and undergoes homologous reciprocal recombination.
19
Q
what are the steps of generalized transduction in E.coli?
A
- Donor strain (trp+) is infected with P1 linear dsDNA phage under conditions that will promote lysogeny
- Lysogenic culture is exposed to UV light to induce lytic cycle, allowing P1 to reproduce, lyse host cell and release lysate (progeny and transducing particles)
- Lysate is exposed to UV light to inactivate P1 phages by reducing ability to reproduce, but has little effect on the transducing particle
- Mix lysate with recipient cell culture (trp-) at MOI less than 1 (≤1 phage per cell)
- Select for transductants by plating infection mixture on selective medium to allow successful recombinant (trp+) cells to grow
20
Q
What is the frequency of transduction?
A
ratio of successful transductants/phage infected cells
= # successful transductants / phage infected cells
21
Q
What are pinpoint colonies?
A
Small colonies characterized by small size that grow on selective medium because of the expression of the donor gene WITHOUT being incorporated into the host chromosome
22
Q
What is co-transduction frequency?
A
transfer of two markers (traits) on the same fragment of transducing DNA, frequency depends on distance between two genes. The closer they are together, the more likely they will be transduced together.
= # cotransductants (express selected and unselected marker)/ number of single transductions (only express selected marker)
23
Q
How does co-transduction frequency relate to physical distance?
A
C = 1- t + t(lnt)
C = Cotransduction frequency
t = fraction of the length of complete phage genome
24
Q
What type of phage is used in specialized transduction?
A
Temperate phage with specific insertion sites in the host chromosome during lysogeny. Because of this, only the genes close to prophage insertion site are transduced after abberant (incorrect) excision of the prophage.
25
What are the steps of specialized transduction of lambda?
1. Lambda infects E.coli cell and enters lysogenic state, integrating between gal and bio operons
2. Lysogenized E. coli is exposed to UV, inducing lytic cycle
3. Aberrant excision is made and prophage takes nearby gal or bio genes, alongside lambda DNA, which leaves behind some head/tail genes.
4. faulty lambda DNA is packaged into a phage head which is released in the lysate to infect a new cell
5. Lambda phage injects DNA into the cell to incorporate into bacterial DNA
26
What are gene transfer agents?
tiny tailed bacteriophages created from bacterial genes that contain random small pieces of that host's DNA as a mechanism subpopulation of bacterial cells to sacrifice themselves to disperse genes in a protected manner
27
What is a conjugative plasmid?
Plasmid from a donor cell that can transfer copies of itself and the genes they encode to recipient host cells via cell to cell contact (mostly gram -ve)
28
How did lederberg and tatum contribute to the discovery of conjugation?
Mixed auxotrophic strains, incubated for several hours, plated on minimal medium. Growth of several prototrophic colonies due to Hfr strains
29
How did Davis contribute to the discovery of conjugation?
Determined that physical contact is necessary for genetic exchange by separating 2 auxotrophic strains by a fine filter, which prevented genetic exchange
30
What is the F factor?
F(ertility) plasmid: self replicating circular dsDNA with genes encoding F-pilus in order to self transfer F- cells via conjugation bridge
31
What are the features of the F plasmid?
tra region: 21 genes encoding F pilus and plasmid transfer protein
oriV: origin of replication
oriT: origin of transfer
IS2, IS3: insertion sequence elemnts
Tn1000: transposon
32
What are the steps of conjugation?
1. Pilus of F+ cell is assembled and establishes cell contact with F- cell
2. One strand of F plasmid is nicked at ori T, becoming linear with 5'-3' ends
3. 5' end is displaced and enters conjugation bridge establisehd by pilus and transferred to F- as ssDNA
4. 3' end is extended via rolling circle replication and once one round is complete, linear strand is cut.
5. Inside recipient cell, ssDNA is replicated to make dsDNA 5' to 3' via okazaki fragments
6. Double stranded ends join to make circular F-plasmid, converting F- cell into F+
33
what is an episome
plasmid that can integrate into host chromosome
34
What is the Hfr strain?
Bacterial strain that F+ cell is converted into after F factor is integrated into hosts chromosome rather than existing as a separate plasmid, therefore mediating transfer of chromosomal genes
35
What are the steps in the conjugation of Hfr genome?
1. Hfr cell meets F- recipient
2. Hfr produces pilus from tra genes on F factor, which attaches to the F- cell, forming a bridge
3. Enzyme nicks DNA at oriT (in middle of F factor) and one strand begins to unwind and transfer into the F- cell from the 5' end, the 3' end serving as a primer to replace the transferred strand.
4. Only about half of the F factor is transferred and rarely makes it through before the bridge snaps, so recipient stays F- but receives new bacterial genes from the donor after being recombined into the chromosome
35
How were Hfr strains used to map the E.coli chromosome?
1. Mix Hfr donor with F- recipient
2. Interrupt mating at various time points
3. place recipients on selective media
4. Record which genes show up at which times. the time it takes for a gene to show up reflects its distance from the origin.
5. Overlapping gene orders can form a composite map in units of time (for E. coli, 100 mins, 45 kbp/min)
36
What is the difference in the 4 Hfr's in ecoli?
Hfr 1: start with gene C and go clockwise
Hfr 2: start with gene L and go counterclockwise
Hfr 3: start with gene X and go clockwise
Hfr 4: start with gene G and go counterclockwise
37
What is the F' donor? What does conjugation with it result in?
bacterial cell with modified F plasmid that accidentally picked up chromosomal genes from the host when excised from the chromosome due to aberrant excision. It transfers the plasmid and any chromosomal genes into an F- cell, resulting in partial diploid or merodiploid that can be used for complementation tests.
38
how was complementation used in the lac study?
F' factors containing bits of lac operon were used to rescue the phenotype of lac mutants, allowing them to derive the functions from the F' DNA
39
What are transposable elements?
pieces of DNA that can move from place to place in the genome without an ori site, homology with destination, and are important in natural genome rearrangement and genetic analysis.
40
What are the two major types of transposable elements in bacteria?
insertion sequences (transposons): short, 10-50 base pairs, encode one transposase
composite transposons: two IS elements flanking antibiotic genes or other genes providing beneficial attributes, encode one transposase each but typically one is dysfunctional, allowing the other to move the unit as a whole
41
What are the two mechanisms of transposition?
conservative transposition: transposon is excised from the donor, resulting in transposon in target DNA and donor DNA with break (e.g. Tn5)
replicative: transposon replicates, resultin in transposon in new location and donor DNA undamaged (e.g. Tn3)
42
How does the conservative transposition work?
1. Transposon is moved from original DNA molecule to target site
2. Transposase recognizes inverted repeats on new location and cuts on either side (staggered) and inserts transposon
3. staggered ends gaps are filled by DNA pol I and DNA ligase, resulting in direct repeats flanking the transposon
43
How does replicative transposition work?
1. transposase cuts staggered nicks on either side of transposon, and staggered nicks on either side of the target site
2. Strands are exchanged and ligated with gaps corresponding to the target site and transposon (bottom strand of transposable element connects with bottom strand of target DNA)
3. Resolvase catalyzes recombination, resolving into two separate DNA molecules, each with a copy of the transposon
44
How many types of plasmids are there?
250 types of plasmids, fall into >50 incompatibility (Inc) groups that cannot exist in the same cell due to shared replication/partitioning control and thus makes a cell immune to superinfection by a similar type.
45
What are the uses of plasmids?
1. allow for mating
2. code for resistance genes
3. degrative: degradation of unusual substances
4. Col -plasmid: gene encoded colicins to kill other bacteria
5. virulence plasmids
46
What are R factors?
R plamids that confer resistance to one or more (1-8) antimicrobial agents and have mobility functions encoded (allows conjugation)
47
What are col plasmids?
Plasmids with genes that produce antibmicrobial proteins (colicins) that kills E. coli and makes the host immune to colicin (e.g. colE1)
48
What are Vir plasmids?
encode toxins and other virulence factors (e.g. agrobacterium tumefaciens pTi plasmid)
49
What are Conjugative plasmids?
F factors/F plasmids, carry out conjugation to help introduce genes into cells
50
How are plasmids named?
originally based on phenotype, now add P followed by capital letters
51
How is a plasmid host range determined?
narrow ori region: only replicates in particular bacterium and closely related species
braod ori region: replicates in wide range of hosts and must encode replication initiation proteins and can be recognized by host machinery
52
How do plasmids replicate?
- Plasmids are replicon: Replicate independently of chromosome.
- Can have one, or multiple ori sites
- plasmid encoded replication initiator protein while the replication proteins themselves are provided by host cell
- replicated via theta or rolling circle.
53
What is plasmid copy number?
number of copies of the plasmid in the daughter cell after host cell division.
54
What is the difference between low copy and high copy number of plasmids?
Low copy (stringent): - ≥1-20 copies per cell with tight regulation mechanisms, replicating one or a few times during host cell cycle
High copy (relaxed): 100-500 copies with inhibition on replication when threshold level is reached (typically used for lab cloning due to large amounts of plasmid DNA that can be harvested)
55
What does it mean when genes are altered in vitro?
Altered genes are reinserted into original source organism or into another host organism, and can be isolated into fragments and purified for further manipulation
56
What are restriction endonucleases?
Enzymes made by bacteria that cut DNA at palindromic sequences to make staggered or blunt ends. Viral DNA is attacked, host DNA is protected by methylation.
57
What are examples of restriction enzymes and how they cut?
HindIII: A-AGCT (cohesive)
BamHI: G-GATCG (cohesive)
AluI: AG-CT (blunt)
Sau3AI: -GATC (cohesive)
58
what is agarose gel electrophoresis?
DNA fragments are placed in a well and an electrical charge is applied. The fragments move through the gel towards the positive electrode due to negatively charged phosphate groups, and gel hinders progress of nucleic acid. Rate of migration is determined by charge of molecule, size, and shape.
59
What are the steps of an agarose gel electrophoresis nucleic acid analysis?
1. Digest DNA (cut) with endonuclease (37ºC, 2-4 hrs)
2. Prepare agarose gel (1% for average, 1%< for small pieces, <1% for big pieces)
3. Load gel with digested DNA and ladder size standard
4. Apply electrical current for a defined period of time
60
What was the first gene cloning experiment?
Stanley Cohen and Herbert Boyer cloned kanamycin cassette from one plasmid and insert it into another plasmid via cutting at same palindromic sites by EcoRi
61
What were the steps of Cohen and Boyers experiment?
1. Digest plasmid with EcoRI: pSC101 is cut at tetR site, pSC102 is cut three times into three pieces, one of which has the kanR site
2. Pieces are ligated together to form a pSC101 with piece A of pSC102, another with piece B, and another with the kanR site
3. make plasmid-less E. coli cells competent with CaCl2
4. mix competent cells with ligation reaction to allow transformation
5. Plate E coli cells on media containing ter and kan to see if the transformed bacteria have the altered plasmid with the kanR and tetR site.
6. Inoculate liquid media with one isolated colony and extract recombinant vector/plasmid
7. Do confirmatory restriction digest with EcoRI to confirm that the vector in the isolated colony has tetR and kanR sites (two pieces)
62
How does molecular cloning work?
Isolate desired gene from its original location and insert it into a small plasmid called a vector, resulting in recombinant DNA plasmid.
63
What are the features of a recombinant plasmid?
1. antibiotic marker
2. ori site (theta replication)
3. f1 ori site (rolling circle replication to make ssDNA to be packaged into phage particles)
4. multiple cloning site (many restriction sites)
5. lacZ (MCS within reading frame, disruption indicates presence of insert)
64
What is blue/white selection?
A way to screen for bacteria that successfully took up a recombinant plasmid via testing for insertional inactivation of lacZ reading frame.
65
What are the steps of blue/white selection?
1. Vector has lacZ fragement and MCS inside the gene.
2. Insert DNA into the MCS which disrupts the lac Z gene
3. plate transformed cells on media with antibiotic (since vector has resistance gene), X-gal (lactose analogue that turns blue when cleaved) and IPTG (allolactose analogue to induce expression of lacZ)
4. A) Colonies that are blue do not have an insert as lacZ is intact, making beta galactosidase to cleave X-gal.
4. B) Colonies that are white have an insert as lacZ is not intact and fails to cleave X-gal which cannot turn blue
66
What is polymerase chain reaction?
Method of DNA manipulation to amplify/copy DNA in substantial quantity without the use of vectors and host cells
67
What are the materials involved in PCR?
- template dsDNA
- pair of primers, one for each strand (18-25 nucleotides)
- thermostable DNA polymerase (usually Taq polymerase)
- Deoxyribonucleoside triphosphates
68
What are the steps of PCR?
1. DNA is heated ~90ºC to separate the two strands
2. DNA is cooled ~55ºC to allow primers to anneal to complementary sequences
3. DNA is heated to ~72º for optimal synthesis of DNA via Taq polymerase
4. Cycle is repeated and finished in 25-30 cycles, amplifying DNA to 2^(25-30)
69
What can you do with PCR products?
1. Clone PCR product directly into vector without the need for restriction digest.
2. Targeted insert PCR products into vectors by restriction digest
70
Why can you clone PCR product directly into vector?
Taq polymerase leaves adenine residues on 3' ends of fragments independent of template, which base pair with thymine residues on cloning vector.
71
How can PCR products be inserted into vectors by restriction digest?
Engineer restriction enzyme sites onto primers which after PCR will introduce restriction enzyme sites on both ends of PCR products. Digest the vector with the restriction enzymes that match the primers added onto the PCR, mix, ligate and allow transformation
72
How can you clone PCR product directly into vector with Pfu DNA polymerase?
If you use Pfu DNA polymerase instead of Taq polymerase, it will not add template independent nucleotides on the dsDNA (no T or A overhangs) and will have blunt ended PCR products. It will be ligated in a gap flanked by ccd (restriction endonucleasE) which targets DNA gyrase, allowing for insertional inactivation of ccd protein, meaning recombinants will have a working gyrase.
73
What are shuttle vectors?
Replicate and are stably maintained in two or more UNRELATED host organisms. Allows DNA to be cloned in a manipulatable organism, and create a vector that can replicate in a second host without any modifications. (e.g. E.coli to B. subtilis, yeast, mammalian cells)
74
What are the requirements for vector hosts?
1. nonpathogenic
2. easy to grow
3. genetically stable in culture
4. easily transformable
5. appropriate enzymes for vector replication
6. genomic/physiological info is available
7. tools for genetic manipulation is available
75
What is sequencing?
Determining the precise order of nucleotides on DNA and RNA (base sequence), first developed by Fred Sanger
76
How does sanger sequencing work?
1. ssDNA template undergoes PCR
2. ddNTPs are added with dNTPs in four reaction tubes (one per ddNTP base)
3. ddNTP insert randomly, and chains of varying lengths are produced in the synthesis reaction
4. A) Fragments are run under gel electrophoresis are lined up in order of size, then they are dried and exposed to film, and then are read from bottom upwards
4. B) Automated fluorescence sequencing labels each ddNTP base with a different colour which can be read at once
77
How does fluorescence labeled sequencing work?
Fluorescence labeled products are separated through capillary electorphoresis and laser detector records output sequence of copied strand. Reverse complementarity to determine original template sequence.
78
What is shotgun cloning/sequencing?
Method used to determine the seqeunce of a large piece of DNA by breaking it into smaller fragments, cloning them into vectors, and use MCS as primer sequences and overlaps to reassemble the full sequence.
79
80
What is the diference between first, second and third generation sanger based methods?
first: didieoxy method
second: pyrosequencing
third: nanopore
81
How does pyrosequencing work?
new dNTP inesrted in to s trand of DNA, pyrophosphtae is released and makes ATP from AMP via sulfurylase enzyme. ATP is consumed by luciferase which releases light. Unused dNTs are degraded by enzyme apyrase.
82
How does nanopore-MiniION sequencing work?
Palm sized device possesses 2000 nanopores. As ssDNA travels through, causes a change in electric current that are base specific. Individual nucleotides are identified in real time based on changes in electrical current.
83
What is genome annotation?
Annotating assembled genomic sequences to identify genes and other functional regions. Genomes typically consist of open reading frames (ORFs), short regulatory regions (binding sites), and transcriptional terminators.
84
How would you find probable ORFs when annotating a genome?
1. computer finds possible start codons
2. computer finds possible stop codons
3. computer counts codons between start and stop
4. computer finds possible ribosome binding site
5. computer calculates codon bias in ORF
6. computer decides if ORF is likely to be genuine
85
What is codon bias?
Some codons are used more frequently than others depending on the organism. If the codon bias in ORF differs greatly from the consensus for the organism containing it, the ORF may be non functional or functional but obtained by horizontal gene transfer.
86
How are ORF sequences ocmpared to other seqeunces?
What is the purpose ofSeqience of ORG can be compared to other sequences in genome sequence database (GenBank) using BLAST algorithm
BLAST (basic local alignment tool) algorithm uses DNA homology searches (hallmark domain features) in GenBank.
87
What fields did DNA sequencing give rise to?
bioinformatics: store and analyze sequences and structures of nucleic acids and proteins
functional genomics: studying one bacterial strain's genome
metagenomics: community genomic sampling
transcriptomics: RNA; gene expression
proteomics: protein expression
metabolomics: metabolic intermediates and small molecules
88
what is cDNA?
Complementary DNA synthesized from mRNA
89
How is cDNA made?
1. Processed total RNA
2. add primers to bind 3' end
3. Reverse transcriptase adds dNTPs
4. RNaseH degrades RNA template leaving ssDNA
5. add primers to bind 5' end, DNA pol elongates and ligase reseals the nicks.
90
What is cDNA used for?
- template for quantitative PCR (reflects quantity of mRNA transcripts per round)
- Microarray: fluorescently labeled and complimentarily bound to chips with gene DNA, flagging which genes were transcribed at the time of sampling
- RNA seq: sequencing cDNA assesses coverage to determine level of expression
- Structural gene organization: validate identification of operon structures by PCR of cDNA
91
How are nucleic acids hybridized in southern blot?
1. DNA embedded within agarose gel in electrophoresis are treated with chemicals to denature into ssDNA
2. ssDNA is transferred to solid matrix (nylon or nitrocellulose) called a membrane via capillary action or electric current
3. dsDNA fragment (PCR product) complementary to the DNA you're looking for is denatured to ssDNA and end labeled (coloured) to make a probe
4. probe is placed in buffer with membrane at certain temperature to allow complementary pairing with the membrane ssDNA
5. If the probe finds its match, it will stick.
92
What is the difference in high and low stringency in nucleic acid hybridization?
high: probe ssDNA will match exactly with target ssDNA
low: probe ssDNA will match with target ssDNA with some mismatches
93
what is the point of a southern blot?
Detect gene sequences in genomes to get an idea of level of identity or identify and confirm mutations
94
What is the purpose of the northern blot?
Asses gene expression levels using labeled DNA that complementarily detects mRNA transcript
95
What are protein expression vectors?
vectors that control the expression of a cloned gene via regulatory sequences that allow manipulation of gene expression to produce lots of mRNA
96
What is a features of a protein expression vector?
- Strong promoters to drive high level of transcription (e.g. Ptac)
- Strong repressor binding to operator sequence to block expression of cloned gene
- Inducer when approrpiate to bind to repressor to releave repression of cloned gene expression
- Strong ribosome binding site between promoter and cloned gene to allow for translation
97
Why do you engineer an protein experession vector system?
When you clone a gene from another organism into E. coli, its original promoter might not work well in E. coli as the sequence is specific to the transcription machinery of the original organism.
98
What is Ptac?
promoter that is synthetic hybrid of two naturally occuring promoters: -35 region from the trp promoter, and -10 region from the lac promoter. Even though it's part t promoter, it doesnt have a TrpR binding site so it cannot be controlled by Trp repressor, instead by lacI
99
How is Ptac controlled?
1. LacI from lac operon binds to lacO which is downstream of Ptac
2. This blocks transcription
3. Adding IPTG acts as allolactose analogue to bind to LacI releasing it from lacO and the promoter is activated
4. Promoter transcribes mRNA for protein translation and the resulting protein is fused with MalE to stabilize the protein and make purification easy by using a maltose resin column
100
How are elements of the T7 phage used to control gene expression?
1. lacI-encoded repressor binds lacO downstream of lac promoter
2. IPGT induction relieves repression to allow expression of T7 RNA polymerase
3. T7 RNA polymerase binds T7 promoter triggering expression of cloned gene on pET plasmid
101
What is western or Immuno blotting?
Detect proteins via use of primary antibodies making specific interactions with specific proteids, secondary antibodies recognizing primary antibody, and then separatee via protein gel, which is transferred to a membrane to be detected via colour
102
What is reverse genetics?
Transposons disrupt genes in order to determine the function, and can be selected for due to antibiotic resistance gene in transposon
103
What is the general method of transposons disrupting genes?
1. transform bacterium with plasmid that cannot replicate (incompatible ori) but carries transposon with antibiotic resistnace gene
2. Select for resistance marker
3. any colonies that grow will have transposon inserted into the chromosome and can be checked for altered phenotype
104
What are two ways that gene can be characterized?
1. mutagenize gene to figure out the function and verify phenotype of transposon mutant (via gene disruption and site directed mutagenesis)
2. Determine how gene is regulated (via reporter genes)
105
How does gene disruption allow for reverse genetics?
1. antibiotic resistance genes are engineered to be inserted within a the coding sequence of a gene they want to disrupt
2. The disrupted gene is put into a plasmid flanked by sequences that match the target gene
3. Plasmid enters the cell and uses homologous recombination to swap the real gene with the broken version resulting in knockout mutation
4. The plasmid cant replicate on its own so the cell gets rid of it, but the mutated gene stays behind int he chromosome
106
What does site directed mutagenesis allow?
amino acid seqeunce changes of a specific gene
107
What are the steps of site directed mutagenesis?
1. amplify gene of interest by PCR and inserted into plasmid vector
2. Put vector into E.coli and purify recombinant plasmid
3. Synthesize short primer (oligonucleotide) that matches a region on your gene with a SINGLE base difference (intentional mutation)
4. Recombineering: introduce mutated primer and PCR product into E.coli with defective prophage.
5. Red recombinase system (Bet, Gam, Exo) facilitate exchange generating plasmid featuring mutant gene.
108
How are reporter genes commonly used to determine how a gene is regulated?
encodes a protein that is easy to detect and assay, and can be used to fuse the promoter of other genes so that gene expression can be studied (e.g. beta-galactosidase LacZ, green fluorescent protein from jellyfish)
109
What is gene/operon fusion?
Transcriptional fusion of reporter gene to the promoter or regulatory region of the gene of interest to show how much the gene is being transcribed.
110
What is protein fusion?
Translational fusion of reporter gene's coding sequence to the coding region of gene of interest. This tracks protein localization.
