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Molekularbiologie > Cloning strategies > Flashcards

Cloning strategies Flashcards

1
Q

What are clones?

A
  • Genetically identical offspring
  • Aggregate / cultures of genetically identical cells derived from a single cell
  • Identical genes as the (original) organism from which they are derived
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2
Q

What is molecular cloning?

A

Isolation of a nucleic acid sequence and its insertion into a vector (→ recombinant DNA), for replication into a host without sequence alteration

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3
Q

What are vectors?

A

Vectors are modified DNAs (more rarely RNAs) from bacteria, yeasts or viruses; often derived from natural bacterial plasmids.

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4
Q

What are vectors used for in molecular biology?

A

As a transport vehicle to bring a nucleic acid sequence in a recipient cell; to clone nucleic acids

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5
Q

What is GOI (gene of interest) and what are some examples?

A

Target / GOI (gene of interest) = nucleic acid sequence to clone
e.g.: a gene, genomic DNA, shRNA (small hair pin RNA) for knock-down, gRNA(guide RNA) for CRISPR-Cas-mediated knock-out

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6
Q

Name the steps of the general principle of cloning.

A
  1. Vector and insert preparation
  2. Recombination / Ligation
  3. Transformation
  4. Multiplication and Selection
  5. Plasmid purification
  6. Analysis
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7
Q

What are some possible applicatiosn of molecular cloning?

A
  1. Subcloning:
    * Exchanging vectors
    * Selection of sub fragments for experiments
  2. Sequence analysis
  3. Constitution of gene libraries:
    * Complete or partial genomic gene libraries
    * Complete cDNA libraries
  4. Protein expression / production
  5. Functional analysis
  6. Mutagenesis experiments
  7. Production of knock out, knock in or knock down constructs (shRNA, CRISPR-Cas9) in eukaryotic cells
  8. Generation of genetically modified plants or animals.
  9. Generation of vectors for gene therapy
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8
Q

What are the different vector types?

A
  1. Plasmids
  2. Phages
  3. Cosmids
  4. YAC
  5. BAC
  6. Mammalian Virus
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9
Q

What are plasmids?

A
  • Extra-chromosomal, circular DNA molecules
  • Naturally occurring plasmids confer, for example, bacteria resistance or enable genome exchange (F plasmid)
  • Carry (at least) one origin of replication (ORI)
  • Artificial “laboratory plasmids” carry MCSs (multiple cloning sites) and defined combinations of selection markers
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10
Q

Where do plasmids replicate?

A

Replicate autonomously in the cytoplasm of bacterial cell

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11
Q

What are some examples of single “cutter” / restriction enzymes?

A

EcoR I, Sst I, Kpn I, Sma I/ Xma I, BamH I, Xba I, Sa II/ Acc I /Hinc II, Pst I, Sph I, Hind III

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12
Q

What is the first plasmid vector found?

A

pBR322 (4361 bp)
* Has an ORI
* Selection marker: z.B. ampr - allows the selection of the transformed bacteria by Amp-resistance / tet
* Restriction enzyme: EcoR I

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13
Q

What is cloning capacity determined by?

A

Method of introducing the recombinant vector into the host cell

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14
Q

What is the laboratry favorite plasmid and why?

A

pUC19 (2686 bp)
* Large MCSs within a reporter gene, lac Z
* Selection of bacteria with recombinant vector relies on additional markers: e.g. second antibiotics resistance, X-Gal (blue/white screening), etc
* pUC19 is smaller than pBR322 → faster bacteria growth
* „High-copy”: between 500 and 700 plasmid copies per bacteria cell

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15
Q

What are some plasmid types and their applications?

A
  1. Plasmids for the (sub)cloning of DNA or cDNA:
    * pUC plasmid family
    * Plasmid vectors for special cloning procedures (e.g. pTOPO)
  2. Plasmids for the protein expression and purification
    * pET → Expression of fusion proteins containing an His6 tag; purification by immobilized metal ion chromatography (His6 tag: Nickel / Cobalt)
    * pGEX → Expression of fusion proteins containing a GST tag; purification by glutathione affinity chromatography GST-Tag (GST tag: glutathione), protein-protein interaction studies (=GST pull downs)
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16
Q

What length should the GOI have when transfecting recombinant plasmids?

A

< 5000 bps

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17
Q

Which phage vectors are most often used?

A

M13 (filamentous bacteriophages)-, Lambda- and P1-phages

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18
Q

What are phages?

A
  • Linear, dsDNA (becomes circular after infection of bacterial cell)
  • Have one ORI
  • Harbour „cos-sites“ (cohesive end sites) at the ends (through terminase and re-ligated through DNA ligase)
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19
Q

What happens during lysogenic cycle?

A

The phage DNA is integrated into the host genome (bacteria) and passed on to the offspring (= bacteria)
The lysogenic cycle is a dormant replication cycle where the phage genome integrates into the host’s chromosome as a prophage and replicates along with the host cell without killing it.
Example: λ phage can switch between lysogenic and lytic cycles

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20
Q

What are the steps of the lysogenic cycle?

A
  1. Attachment and Injection: The phage attaches to the bacterial cell and injects its DNA.
  2. Integration: The phage DNA integrates into the bacterial chromosome, forming a prophage.
  3. Replication: As the bacterium divides, the prophage DNA is copied and passed to daughter cells.
  4. Induction: Under certain conditions (e.g., stress, UV light), the prophage excises itself from the bacterial genome and enters the lytic cycle.
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21
Q

What happens during the lytic cycle?

A

The phage genome can reactivate (i.e. leave the bacterial genome) and produce phages; inducers for reactivation are e.g. environmental factors.
The lytic cycle is a destructive replication cycle in which the phage immediately hijacks the host cell’s machinery to produce new phages.
Example: T4 phages

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22
Q

What are the steps of the lytic cycle?

A
  1. Attachment: The phage binds to the surface of the bacterial cell using specific receptors.
  2. Injection: The phage injects its DNA into the bacterial cell.
  3. Replication: The phage DNA takes over the host’s machinery to replicate its genome and produce phage proteins.
  4. Assembly: New phage particles are assembled inside the host cell.
  5. Lysis: The bacterial cell bursts (lyses), releasing hundreds of new phages to infect other bacteria
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23
Q

How does DNA replication occur in phages and what does it yield?

A
  • Rolling circle principle of DNA replication
  • Replication of phage DNA yields in vivo concatemers (DNA multimers); concatemers are the substrate for packaging
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24
Q

What are concatemers?

A

Concatemers are linear or circular DNA molecules formed by multiple copies of the same DNA sequence arranged in tandem (DNA multimers)

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25
Q

How are concatemers in vitro formed?

A

In vitro, concatemers can be obtained by annealing cos-sites

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26
Q

Where do phages replicate?

A

Phages replicate inside bacterial host cells after infection

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27
Q

What are the two kinds of replication cycles used by bacteriophages (viruses that infect bacteria)?

A

Lytic and lysogenic cycles

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28
Q

What is rolling circle replication?

A
  • Rolling circle replication (RCR) is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids
  • Some eukaryotic viruses also replicate their DNA or RNA via the rolling circle mechanism.
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29
Q

What are the steps of in vivo packaging of Lambada phage DNA?

A

In vivo packaging of Lambda phage DNA:
1. Recognition of concatemeric viral genome
2. DNA cleavage and packaging motor (TerL & TerS rings) binding to procapsid (has portal ring)
3. ATP-dependent DNA packaging; packaging promoted by Nu1 and A
4. Nuclease stimulation after genome packaged
5. Mature, infectious virion

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30
Q

How is the phage DNA packaged?

A

The phage DNA is packaged from one cos-site to the next one; creating concatemers which are the substrate for packaging

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31
Q

What is the cloning capacity of phages as cloning vectors limited?

A

Limited by the size of the phage head

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32
Q

Which proteins promote packaging?

A

Nu1 and A

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33
Q

Name types of phages vectors

A
  1. Insertion phage vectors
  2. Replacement phage vectors = substitution vectors
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34
Q

What are insertion phage vectors and their applications?

A

Insertion phage vectors: the target DNA is incorporated at a specific site without removing any significant portion of the phage DNA –> Moderate cloning capacity, suitable for creating cDNA libraries

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35
Q

What are replacement phage vectors and their applications?

A

Replacement phage vectors = substitution vectors: a portion of the phage DNA is removed and replaced by the target DNA –> Larger cloning capacity, suitable for creating gene libraries

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36
Q

What are the steps of the process of creating replacement phage vectors to use for gene libraries?

A
  1. Phage genome modification; substitution of λ-phage genome by target DNA through DNA recombination
  2. In vitro packaging and assembly in λ-phages
  3. Infection of bacterial cells; replication through lytic and non-lytic cycles
  4. Phage gene libraries through plaques in bacterial lawn; each plaque corresponds to a single phage containing a unique DNA fragment from the library
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37
Q

What are cosmids?

A
  • Cosmids are hybrid DNA vectors that combine features of plasmids and phages
  • Are circular, extrachromosomal DNA with ORI and selection marker
  • Have COS-sites (from lambda phages) -> In vitro packaging in phage heads and infection as phages
  • Have MCSs
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38
Q

Where do cosmids replicate?

A

In the cytoplasm of bacterial cells, like plasmids

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39
Q

What can cosmids be used for?

A

Suitable for the generation of DNA libraries

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40
Q

What does the cloning capacity of cosmids depend on?

A
  • Depends on the size and stability of the phage head
  • But since cosmid vectors are small (a few kbp), they have a cloning capacity of 35-50 kbp → suitable for gene libraries
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41
Q

What are similarities between phage and cosmid as cloning vectors?

A
  • In both cases, the recombined DNA is in vitro packaged
  • This is followed by infection of the host cells (bacteria) by phages.
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42
Q

What are differences between phage and cosmid as cloning vectors?

A
  • Phage vector: The recombinant clones are phages, which
    lyse the bacterial lawn (plaques)
    Infection occurs as a phage, meaning the phage infects a bacterial host.
  • Cosmid vector: The recombinant clones are bacteria (appearing as bacterial colonies). No phage is produced (only cos-sites are present).
    Infection occurs via phage-like packaging, but after infection, they function as plasmids.
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43
Q

What are YACs?

A

YAC= artificial chromosome, linear DNA, with telomers and a centromere region, distributed to the daughter cells during mitosis
* Has an ARS (Autonomously Replicating Sequence), like ORI
* Has selection markers

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44
Q

What are yeasts?

A
  • Yeasts are unicellular fungi
  • 16 chromosomes, with telomers and a centromere region.
  • The chromosomes are distributed to the daughter cells during mitosis.
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45
Q

What elements of yeast chromosome do YAC vectors compromise of?

A

YAC vectors comprise essential elements of a yeast chromosome
- CEN: Centromer
- TEL: Telomer
- ARS: Autonomously replicating sequence
- Trp, ura: Marker genes → Selection
- Sup4: Additional marker for the selection of yeasts carrying recombinant vector (red/white)

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46
Q

What is important to happen to the circular YAC vector before cloing cloning can take place?

A

For YAC cloning, the circular YAC vector must be linearized through restriction enzymes (eg. EcoRI, BamHI) producing sticky ends (Marker gene)

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47
Q

How are YAC vectors amplified?

A

The circular YAC vectors are amplified in bacteria (E. coli K12 strains):
- ORI
- Prokaryotic resistance gene (ampr)

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48
Q

How are recombinant YACs recognized?

A

Recombinant YACs are recognized and segregated by the mitotic spindle -> Yeast-specific selection markers, e.g. specific amino acids or metabolites

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49
Q

What is the cloning capacity of YACs?

A

~ 500 kb (quite high)

50
Q

What happens to recombinant YACs with shorter insertions?

A

Recombinant YACs with shorter insertions dissociate from the mitotic spindle -> unstable

51
Q

What are BACs?

A
  • BAC (bacterial artificial chromosome) is circular DNA
  • Based on bacterial mini F plasmids
  • Has an ORI
    *Has diverse selection markers
  • Regulatory sequences
52
Q

What are some important elements that are included in BAC vectors?

A
  • ori2, repE, IncC – origin of replication (single copy); oriV – inducible origin of replication
  • par A, B, C – partition genes (from the F-plasmid): efficient partitioning as single-copy plasmid in the daughter cells during bacterial division
  • Cmr - chloramphenicol-resistance gene -> Selection
53
Q

What is the cloning capacity of BACs and application examples?

A

~ 500 -1000 kbp
Applications of BAC:
* Genomic libraries
* Sequencing large genomes (e.g., human genome projects)
* Studying large genomic regions, regulatory elements, or long-range gene interactions

54
Q

How is the recombinant BAC introduced into the E. coli cell?

A

Through electroporation

55
Q

How is the screening done to differentiate between recombinant and non-recombinant colonies when using BAC cloning?

A
  • Only cells with BACs (chloramphenicol-resistant) will grow.
  • lacZ – Blue/white screening of recombinant clones
  • White colonies (lacZ disrupted) indicate successful insertion of the DNA fragment, while blue colonies indicate non-recombinant BACs.
56
Q

What are application examples of mammalian viruses?

A

Applications:
* Introduction of vectors for gene expression (GOI)
* RNA interference (shRNAs)
* Generation of iPS cells, to introduce the components of the CRISPR-Cas 9 system, etc.

57
Q

What is the clonong capacity of mammalian viruses limited by?

A

Size of the viral particle

58
Q

What safety measure schould be considered when generating mammalian viruses?

A

Generation of replication-deficient viruses -> Biosafety

59
Q

How is the size of the cloning capacity of mammalian viruses?

A

Small

60
Q

How is it that the generated mammalian viruses cannot replicate?

A

A part of the virus genome is replaced by the target DNA → These vectors are therefore unable to replicate

61
Q

How can infectious mamamalian viruses be produced?

A

Infectious particles can only be produced, if the missing genomic information are available in the cells= Trans-complementation

62
Q

How are mammalian viruses produced in practice? (In which mammalian cells and how?)

A

In practice:
* Viral vectors are produced in HEK 293 T cells
* Co-transfection of the recombinant vector + packaging/ helper plasmids

63
Q

What are packaging / helper plasmids and what is their function?

A

Packaging/ helper plasmids: code for components of the viral capsid, of the replication machinery and the envelope glycoproteins -> allow the production of infectious recombinant virus particles but the corresponding DNAs are not incorporated in the virions!

64
Q

How can biosafety be increased when generating mammalian viruses?

A
  • „2-plasmid / 3-plasmid system “: Genetic information is split onto 2-3 different plasmids, which all have to be co-transfected with the vector plasmid to produce recombinant particles
  • Single-round infection (transduction): Replication-deficient viral particles can infect cells but lack the genetic information for the viral capsid, the envelope glycoprotein and the proteins responsible to replicate the viral genome
65
Q

What is the „2-plasmid / 3-plasmid system “ ?

A

To increase biosafety: genetic information is split onto 2-3 different plasmids, which all have to be co-transfected with the vector plasmid to produce recombinant particles

66
Q

What is single-round infection (transduction)?

A

Replication-deficient viral particles can infect cells but lack the genetic information for the viral capsid, the envelope glycoprotein and the proteins responsible to replicate the viral genome → single-round infection (transduction)

67
Q

Name commonly used mammalian viruses as vectors.

A
  1. γ-Retrovirus (e.g. Murine Leukemia Virus, MLV)
  2. Lentivirus (e.g. HIV)
  3. Adeno-Associated-Virus (AAV)
  4. Adenovirus
68
Q

What are γ-retrovirus? What type of genome does it contain? Does it integrate into host genome? Does it infect non-dividing cells?

A
  • Enveloped RNA viruses
  • ~ 100 nm diameter
  • Cargo capacity: ~ 8kb
  • Genome: ssRNA
  • Integrating: yes
  • Infection of non-dividing cells: no
69
Q

How can tropism in γ-retrovirus be manipulated?

A

By pseudotyping = replacement of the envelope proteins with those of a different virus, e.g. vesicular stomatitis virus envelope glycoprotein (VSV-G) to obtain a broad tropism

70
Q

How does the trans-complementation system for γ-retrovirus occur?

A

Trans-complementation system for γ-retrovirus uses the 3-plasmid system

71
Q

What elements are included in the retrovirus genome?

A

LTR, GAG, POL, ENV, LTR (Long Terminal Repeat)

72
Q

What is the GAG element of the retrovirus genome responsible for?

A

GAG = Packaging and assembly

73
Q

What is the POL element of the retrovirus genome responsible for?

A

POL = Reverse transcriptase + Integrase

74
Q

What is the ENV element of the retrovirus genome responsible for?

A

ENV -> often VSV-G (Pseudotyping)

75
Q

What are the retrovirus genome divided into using the 3-plasmid system?

A
  • Envelope plasmid (Promoter, ENV)
  • Packaging plasmid (Promoter, GAG, POL)
  • Transfer plasmid (LTR, Promoter, target DNA - [cDNA, sgRNA or shRNA], LTR) –> packaged in the viral particle
76
Q

How do γ-retrovirus integrate their genome in the host genome?

A

γ-retroviruses depend on the disassembly of the nuclear envelope during mitosis to integrate their genome in the host genome → only transduce dividing cells

77
Q

What are Lentivirus? What type of genome does it contain? Does it integrate into host genome? Does it infect non-dividing cells?

A
  • Enveloped RNA viruses
  • 8-10 nm diameter
  • Cargo capacity: ~ 9kb
  • Genome: ssRNA
  • Integrating: yes
  • Infection of non-dividing cells: yes
78
Q

How do lentivirus integrate their genome in the host genome?

A

Lentiviruses can cross the nuclear envelope using the nuclear pore complex → also suitable for non-dividing cells

79
Q

What are Adeno-Associated-Virus (AAV)? What type of genome does it contain? Does it integrate into host genome? Does it infect non-dividing cells?

A
  • Non-enveloped ssDNA virus
  • ~ 26nm diameter
  • Cargo capacity: ~ 4,7kb
  • Genome: ssDNA
  • Integrating: no / rare
  • Infection of non-dividing cells: yes
80
Q

How do Adeno-Associated-Virus replicate?

A

Episomal replication with the help of a helper virus (adenovirus), rare integration events

81
Q

What elements are included in the Adeno-Associated-Virus genome?

A

ITR, REP, CAP, ITR (Inverted Terminal Repeats)

82
Q

What is the REP element of the Adeno-Associated-Virus genome responsible for?

A

Replication

83
Q

What is the CAP element of the Adeno-Associated-Virus genome responsible for?

A

Capsid

84
Q

What are the Adeno-Associated-Virus genome divided into using the 3-plasmid system?

A

3-plasmid system: Helper plasmid (Promoter, E4, E2a, VA), Rep/Cap (Promoter, Rep, Cap), Transfer plasmid (ITR, Promoter, target DNA - [cDNA or shRNA], ITR) -> packaged in the viral particle

85
Q

What are adenovirus? What type of genome does it contain? Does it integrate into host genome? Does it infect non-dividing cells?

A
  • Linear dsDNA
  • Non-enveloped virus
  • Cold symptoms in human
  • 90 - 100nm diameter
  • Cargo capacity: up to 35kb
  • Genome: dsDNA
  • Integrating: no
  • Infection of non-dividing cells: yes
86
Q

What elements are included in the adenovirus genome?

A

ITR, Transcriptional units, ITR

87
Q

What are the adenovirus genome divided into using the 2-plasmid system?

A

2-plasmid system: Packaging plasmid (pAd Easy – Right arm, All components minus E1 and E3, Left arm), Transfer plasmid (Left arm, ITR, Promoter, target DNA - [cDNA], ITR, Right arm) -> packaged in the viral particle

88
Q

What are different cloning vectors, their host cells and their insert capacities?

A
  • Plasmid; E. coli; 0.1-10 kb
  • Bacteriophage Lambda; E. coli; 15-20 kb for replacement vector, 0,1-5 kb for insertion vector
  • Cosmid; E. coli; 35-45 kb
  • Bacteriophage P1; E. coli; 80-100 kb
  • BAC; E.coli; 50-300 kb
  • YAC; Yeast; 100-2000 kb
  • Human AC; Cultured human cells; >2000 kb
89
Q

Give two examples of of cloning procedures to fullfill a certain aim.

A
  1. Generation of a lentiviral expression construct for eukaryotic cells through overexpression of the GOI
  2. Generation of a knock down construct (shRNA) for eukaryotic cells
90
Q

What are the steps for the cloning of an expression construct in bacteria?

A

Cloning of an expression construct (protein expression in bacteria)
1. Vector restriction (e.g. pET or pGEX vectors)
2. Isolation and restriction of the target DNA, often obtained by PCR
3. Ligation (NB: must occur in frame!)
4. Transformation of competent bacteria and selection of the recombinant clones (GMOs)
5. Expression induction e.g. with IPTG, and bacteria collection
6. Cell lysis, verification of expression (SDS-PAGE, Coomassie staining), purification of the (fusion) protein using the His-(pET) or GST-(pGEX) tag

91
Q

Which type of restriction enzyme is usually used in cloning?

A

Type II restriction enzymes

92
Q

Where is the cut site of type II restriction enzymes?

A

The recognition site is the cut site

93
Q

What types of ends are generated through digestion in the cloning process?

A

Sticky ends (3´/ 5´overhangs) or blunt ends

94
Q

What does a combination of type II restriction enzymes allow?

A

Combination of type II restriction enzymes allow multiple cloning options

95
Q

Can identical sticky ends be produced through diffrenet enzymes?

A

Yes, Different enzymes can produce identical sticky ends

96
Q

Give examples of restriction enzymes that are 8-base cutters?

A

Not I

97
Q

Give examples of restriction enzymes that are 6-base cutters?

A

Bam HI, Bgl II, Stu I, Kpn I

98
Q

Give examples of restriction enzymes that are 4-base cutters?

A

Alu I, Sau 3a, Mbol

99
Q

What enzyme converts RNA to cDNA?

A

Reverse transcriptase, which is a retroviral enzyme

99
Q

What are the 3 functions of reverse transcriptase?

A
  • RNA-dependent DNA polymerase (reverse transcription)
  • DNA-dependent DNA polymerase
  • RNAse H
100
Q

What are the steps of typical cDNA cloning procedure?

A

Typical cDNA cloning procedure:
1. Restriction of the vector (plasmid or phage vector)
2. Isolation and purification of the mRNA of interest
3. Generation of the complementary DNA (cDNA) copy using a reverse transcriptase.
4. Ligation
5. Transformation in E. coli
6. Growth of the transformants under selection
7. Identification of the recombinants and expansion (selection)
8. Analysis (e.g. sequencing)

101
Q

What are some common protein tags?

A
  • His tag (6x Histidin)
  • GST tag (enzyme)
  • GFP tag (fluorescent protein)
  • FLAG tag or HA tag (epitope tags)
102
Q

What are protein tags used for?

A
  • Protein purification (affinity chromatography, pulldown)
  • Detection (Western blot, immunohistochemistry, fluorescence microscopy or live cell imaging)
103
Q

Where are protein tags inserted?

A
  • Tagging by in frame insertion of the tag in the cDNA of the GOI (N-terminal / C-terminal / internal insertion) using cloning techniques.
    *Respect the frame, preserve start and stop codons! Always verify the ORF.
104
Q

Which vectors and protein tags are used for the expression and purification of fusion (recombinant) proteins?

A

Expression and purification of fusion (recombinant) proteins:
* Using pET vector with His Tag
* Using pGEX vector with GST Tag

105
Q

What is GST pulldown used for?

A

To determine protein-protein interactions

106
Q

Which protein tags are used for the expression and visualisation of fusion (recombinant) proteins?

A

Expression and visualisation of fusion (recombinant) proteins:
* Using HA tag (peptide from Influenza A Virus hemagglutinin): YPYDVPDYA
- Recognized by well-characterized and commercially available antibodies
-> Investigate protein localization and colocalization with other proteins or organelles
* Using GFP-tagged ABHD5
- Fluorescent protein tags are compatible with live cell imaging

107
Q

What are examples of cloning shortcuts?

A
  • TA-cloning (e.g. TOPO TA-cloning)
  • Gateway technology
  • Gibson assembly
108
Q

Does TA-cloning / PCR cloning need restriction enzymes? How does it work?

A
  • No need for restriction enzymes
  • PCR product with A overhang (e.g. obtained with transferase activity of the Taq polymerase)
  • Vector containing a cloning site with a T overhang
109
Q

Does TOPO cloning need restriction enzymes? How does it work?

A
  • No need for restriction enzymes
  • TOPO vector with bound DNA topoisomerase I = restriction enzyme (5’…(C/T) CCTT…3’) + ligase
  • Quick cloning:
    1. Mix vector + GOI
    2. Incubate at room temperature
    3. Transform in competent bacteria
    4. Plate on agar
  • Can be combined with TA-cloning: TOPO-TA cloning
110
Q

Does gateway technology need restriction enzymes? How does it work?

A
  • No need for restriction enzymes or ligases
  • DNA recombination during phage integration / excision
  • Components: attP/B (recombination of this results in attL/R), BP reaction, LR reaction
  • Two-step process: entry clone creation, destination vector transfer
  • Many destination vectors possible, depending on application
  • Fast procedure, high cloning efficiency
  • Flexibility: simple transfer in different expression systems
111
Q

Does gibson assembly need restriction enzymes? How does it work?

A
  • No need for restriction enzymes
  • Exonuclease-based method
  • Seamless assembly of DNA fragments, in the correct order
  • Components: PCR fragments, or (commercial) DNA fragments synthetized chemically (15-20 bp overlapping ends), linear vector
  • Single-tube reaction: Gibson assembly master mix:
    1. 5’ exonuclease
    2. DNA polymerase
    3. DNA ligase
112
Q

What are some examples of selection strategies used when cloning?

A
  1. Auxotrophic markers in yeasts
  2. Blue-white screening
  3. CcdB: positive selection with a lethal gene
  4. Screening for recombinants by restriction
  5. PCR-based colony screening
  6. Limited dilution assay
113
Q

What is auxotrophy?

A

The inability of an organism to synthesize a compound required for its growth

114
Q

How do auxotrophic markers work?

A
  • Mutations in metabolic pathways lead to auxotrophic yeast strains (e.g. leu2Δ, his3Δ, ura3Δ strains auxotrophic for leucine, histidine or uracil) that cannot grow unless a specific nutrient is provided (e.g. amino acid)
  • Auxotrophic markers complement these deficiencies (e.g. URA3, LEU2, HIS3) -> transformants grow on medium lacking the required component
115
Q

How does blue-white screening work?

A
  • β-galactosidase cleaves X-Gal (chromogenic substrate) -> blue pigment
  • If insert within lacZ -> white colonies
  • If insert outside of lacZ or no insert present -> blue colonies
116
Q

How does selection through ccdB toxin work?

A

ccdB toxin (blocks the DNA gyrase, crucial for DNA replication and repair)
-> Bacteria death
-> Selection of recombinants (insertion in ccdB ORF) - positive selection
-> Reduces the need for extensive screening of clones

117
Q

How does screening for recombinants by restriction* work?

A
  • Recombinant clones can be isolated based on their restriction profile
  • Miniprep -> Restriction digests (with controls) -> Analysis by gel electrophoresis
118
Q

How does PCR-based colony screening work?

A
  1. Pick bacteria clones from the transformation plate with a toothpick
  2. Dip the toothpick in the PCR mix and then use it to inoculate a bacteria culture
  3. Run the PCR (to verify the presence of the insert, eventually also its orientation)
  4. Analysis by gel electrophoresis
  5. Extract the plasmid from the bacteria culture („miniprep“) and perform further analyses (e.g. sequencing)
119
Q

How does limited dilution assay work?

A

E.g.: isolation of a cell clone with a particular gene knockout (CRISPR-Cas9-edited knockout clone)

Molekularbiologie (9 decks)

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