4 Genetic Engineering Flashcards

Bibliography

• Brown, TA (2021) Gene Cloning and DNA Analysis: An Introduction. 8th edition. Ed. Wiley-Blackwell Chapter 2

• Nicholl, DST (2023) An Introduction to Genetic Engineering. 4th edition. Cambridge University Press.

• Real-García, MD., Rausell-Segarra, C., Latorre-Castillo A., (2017). Genetic engineering techniques. Ed. Síntesis. Chapters 4 and 6

• Prymrose & Twyman (2006) Principles of gene manipulation and genomics. 7th edition. Wiley-Blackwell Ed. Chapter 4 Molecular cloning: basic vectors

Molecular Cloning: Basic Vectors

• Basic Steps of Molecular Cloning:

  1. Construction of Recombinant DNA:

     • Vector + DNA Fragment = Recombinant DNA Molecule

  2. Transport into Host Cell

  3. Multiplication of Recombinant DNA

  4. Division of Host Cell

  5. Numerous cell divisions result in a clone

Molecular Cloning I

• Basic Steps of Gene Cloning:

  1. Fragment Insertion:

     • A DNA fragment containing the gene of interest is inserted into a circular DNA molecule called a vector, creating recombinant DNA.

  2. Vector Transport:

     • The vector carries the gene into a host cell, typically a bacterium, but other cell types can be used.

  3. Vector Multiplication:

     • Within the host cell, the vector replicates, producing numerous identical copies of itself and the carried gene.

  4. Recombinant DNA Inheritance:

     • As the host cell divides, copies of the recombinant DNA molecule are passed to the progeny, and further vector replication occurs.

  5. Clone Formation:

     • After many cell divisions, a colony (or clone) of identical host cells is produced.

     • Each cell contains one or more copies of the recombinant DNA.

     • The gene carried by the recombinant molecule is now considered cloned.

Basic Steps of Gene Cloning

1. Obtaining Fragments to be Cloned

   • PCR

   • Chemical synthesis

   • cDNA synthesis

2. Choice of Cloning Vector

3. Cloning Cell System

4. Construction of Recombinant DNA

5. Cell Transformation

6. Cloning Verification

7. Clone Purification

   • e.g., miniprep

Plasmids and Bacteriophages

1. PLASMIDS

   • Basic characteristics

   • Use as vectors

   • Examples: pBR322 and others

2. BACTERIOPHAGES

   • Bacteriophage λ (lambda)

   • Bacteriophage M13 and similar

Plasmids: Forms

• Plasmids exist in various forms:

  1. CCC DNA (Covalently Closed Circular DNA)

     • Supercoils or "supercoils" (SC DNA)

  2. OC DNA (Open Circular DNA)

  3. Linear DNA

Plasmid Migration in Electrophoresis

• Supercoiled (SC) plasmids migrate faster during electrophoresis.

Linear Plasmids

• Linear plasmids are found in:

  • Streptomyces sp.

  • Borrelia burgdorferi

Plasmids: Sizes

• Plasmids vary in size:

Plasmids: Origin

• Plasmids originate from prokaryotes, except for the 2 mm plasmid found in yeast.

Plasmids: Transfer

• Non-conjugative Plasmids

  • Cannot be transferred by conjugation.

  • Generally present in high copy numbers.

• Conjugative Plasmids

  • Contain tra genes, enabling transfer by conjugation between bacteria.

  • Typically present in low copy numbers.

Plasmids: Replication

• Autonomous (Non-integrative) Plasmids

  • Replicate independently.

• Episomes

  • Integrate into the host chromosome and replicate along with it.

Plasmids: Origin of Replication (ori)

• All plasmids contain an origin of replication (ori).

• Some replication proteins are encoded near the ori, while the main proteins (e.g., DNA polymerases) are encoded in the chromosome.

• The ori functions only in specific species, conferring bacterial host specificity.

Plasmids: Number of Copies

• Classification based on copy number:

  • High copy number: Relaxed plasmids

  • Low copy number: Stringent plasmids

Plasmids: Incompatibility

• Plasmids with similar characteristics can be incompatible within the same bacterium due to similar replication control mechanisms.

• Incompatibility groups include plasmids that are incompatible with each other. If one plasmid is present, the other is often lost.

Plasmids: Basic Features for Cloning

Basic features for use in cloning:

• Prepare DNA template using spin columns and centrifugation

• Grind plant material

• Purify DNA using silica spin column

• DNA extraction

• Elute DNA from spin column

• Add master mix to student samples and positive control

• Add master mix and nested PCR primers to student initial

• Amplify target

• Treat PCR

• product

• and positive

• control DNA

• sample with

• exonuclease I to

• remove primers

• Purity and blunt

• PCR products

• Two PCR steps and electrophoresis

• Purified

• blunted PCR

• products

• Jet ligase

• plasmid

• vector

• Grow liquid

• bacterial culture

• overnight at 37°C

• in shaking

• waterbath

• Ligation and transformation

• Amplify target

• sequences

• Analyze via DNA

• electrophoresis

• Combine PCR

• products, plet

• plasmid vector,

• and T4 ligase.

• Ligate 10 minutes

• Transform bacteria

• and incubate

• overnight at 37°C

• Purify plasmid

• DNA using alkaline

• lysis and miniprep

• spin column

• Restriction digest

• analysis via DNA

• electrophoresis

• and positive clones

• Combine purified

• plasmid containing

• cloned gene with

• sequencing primers

• and mail to

• sequencing facility

• Analyze sequences

• using Geospiza

• web portal

Plasmids: Cloning Features

• Small size and low molecular weight facilitate cloning, increase resistance to breakage, and result in a higher number of copies.

• Selectable markers are needed to identify bacteria containing the vector and insert.

Plasmids: Selection Genes

• Selection Gene: Used to identify colonies with the vector

  • To select colonies with the vector:

    • Antibiotic resistance genes

      • Ampicillin resistance (Amp^R) via Beta-lactamases

      • Kanamycin resistance (Kan^R)

      • Chloramphenicol resistance (Cm^R)

      • Tetracycline resistance (Tc^R)

      • Streptomycin resistance (Sm^R)

    • Reverse auxotrophy: Mutant bacteria that can only grow in minimal medium if they have incorporated the plasmid. For example, a hisB gene on a plasmid allows a hisB mutant bacterium to grow in a minimal environment without histidine.

  • To select colonies without the vector:

    • ccdB gene: Induces cleavage of bacterial DNA by DNA gyrase, lethal unless the bacterium has a gyrA mutation. This is used in the Gateway system.

Plasmids: Tracking Genes

• Tracking Gene (Screening or Reporter Genes): Used to identify colonies with both the vector and the insert.

  • Allows visual identification of colonies with the vector and insert.

  • These genes are inactivated upon insert integration into the vector.

  • Examples:

    • lacZ gene: Encodes β-galactosidase, resulting in blue colonies.

    • eco47IR, etc.

Plasmids: α-complementation with the lacZ gene

• The lacZ gene encodes the N-terminal region of β-galactosidase.

• This is introduced into a mutant E. coli strain that contains the C-terminal end of β-galactosidase.

• In vitro association of both subunits occurs.

• β-galactosidase catalyzes the conversion of X-gal to a blue substrate.

  • X-gal is a substrate but not an inducer of lacZ transcription.

  • Isopropyl-β-D-thiogalactoside (IPTG), an inducer, is added to the medium.

• The lacZ system requires adding X-gal + IPTG to the medium.

• IPTG is a lactose analog.

Plasmids: Bacterial Genotype Compatibility

• The genotype of the bacteria used must be compatible with the cloning method.

• Example: E. coli DH5α strain genotype:

  • fhuA2 lac(del)U169 phoA glnV44 Φ80' Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17

  • Transforms with high efficiency.

  • endA1 mutation inactivates an intracellular endonuclease that degrades plasmid DNA.

  • hsdR17 mutation eliminates the restriction endonuclease of the EcoKI restriction-modification system.

  • Δ(lacZ)M15 is the alpha acceptor allele needed for blue-white screening with lacZ-based vectors.

  • recA eliminates homologous recombination.

  • glnV44 is the systematic name for SupE44, an amber suppressor.

Plasmids: Unique Restriction Sites

• Unique restriction sites are very important for cloning.

  • The area of the vector containing the unique restriction sites is called the "Polylinker" or "Multiple Cloning Site" (MCS).

Plasmids: pBR322

• Initially, natural plasmids such as Col E1 were used. Later, in vitro designed vectors were developed.

• pBR322 was one of the first widely used vectors (late 1970s).

  • Small size

  • High copy number in E. coli

  • Two resistance genes:

    • A cloning marker

    • A marker for the presence of the plasmid

• pBR322 was designed using natural vectors (R1, R6-5, pMB1, etc.) and incorporating traits from each.

• PBR322 Vector Map:

  ECORI
  Apo I 4359
  Aat II 4284
  BsrB BciVI 4209

  SspI 4168
  Ear I 4155
  Eco57 1 4054-
  Xmn I 3961-
  Hinc II 3905
  ScaI 3844
  Pvu I 3733
  BsrD I 3608

  PstI 3607-
  Ase I 3537
  BsrD I 3420

  Ahd I 3361
  Eco57 1 3000
  AlwN I 2884-
  BciV I 2682
  Drd I 2575
  Afl III - BspLU111 2473-
  BsrB I 2408-
  Ear I 2351
  Sap I 2350

  Eag 1939
  Nru 1 972-
  BstAP I 1045
  BspM I 1063

  ROP

  Xmn I 2029
  Pvu II 2064
  BsmB I 2122
  Drd I 2162

  PflM I 1315
  Bsm I 1353
  Sty I 1369
  PflM I 1364
  Ava I - BsoB I 1425
  PpuM I 1438
  Msc I 1444

  Dsa I 1447
  PpuM I 1480
  Bsg I 1656
  BspE I 1664
  BsaB I 1668
  Nde I 2295
  BstAPI 2291-
  BstZ17I-Acc I 2244-
  BsaA I 2225
  PfiFI - Tth111 I 2217

  Clal - BspD I 23
  Hind III 29-
  ECOR V 185
  Nhe I 229
  BamHI 375
  SgrA I 409-

  Ban II 471

  TC

  Ban II 485

  *Dsa I 528
  *Sph 1 562
  EcoN I 622
  SalI 651
  Hinc II 651
  Acc 1 651
  PshA I 712
  BsaI 3433

Currently no longer in use

Plasmids: New Plasmids

• Many cloning plasmids have been generated from pBR322.

  • Introduction of a Multiple Cloning Site (MCS) or Polylinker:

    • A short DNA fragment with multiple restriction sites, allowing flexibility in cutting without disassembling the vector sequence.

  • Introduction of Visual Screening Genes, e.g., lacZ.

Plasmids: pUC8

• pUC8 is derived from pBR322 but retains only the ori and Amp^R (ampicillin resistance gene).

• Advantages:

  1. High copy number increases the chance of mutation during construction.

  2. Clustered restriction sites (MCS or polylinker).

  3. One-step clone selection due to the presence of the lacZ gene, with the MCS located within the lacZ sequence.

Plasmids: pUC8 and pUC18

• pUC18 is derived from pUC8.

• (a) pUC8
  amp (Ampicillin resistance gene)
  2750 bp
  lacZ' (lacZ alpha fragment)
  ori (Origin of replication)
  Cluster of sites

• (b) Restriction sites in pUC8
  --HindIII
  --Sphl
  --Pstl
  --Sall, Accl, HincII
  --Xbal
  --BamHI
  --Smal, Xmal
  --KpnI
  --Sacl
  EcoRI

• (c) Restriction sites in pUC18

Plasmids: pGEM3Z

• Characteristics:

  1. Amp^R (Ampicillin resistance gene)

  2. lacZ (lacZ alpha fragment)

  3. Polylinker (Multiple cloning site)

  4. Promoter sequences for RNA polymerases from phage T7 and SP6, allowing in vitro transcription.

• pGEM-T was designed from pGEM3Z and is widely used today.

Plasmids: pGEM-T Vector Map

• pGEM-T Vector Map and Sequence Reference Points

• Map:
  Xmnl 1994
  Scal 1875
  Nael 2692
  T7↓ (T7 promoter)
  f1 ori (Single-strand origin of replication from bacteriophage f1)
  1 start
  Apal 14
  Aatll 20
  Sphl 26
  BstZI 31
  Ncol 37
  Sacll 46
  Spel 55
  Notl 62
  BstZI 62
  Pstl 73
  Sall 75
  Ndel 82
  Sacl 94
  BstXI 103
  Nsil 112
  ↑ SP6 (SP6 promoter)
  126
  lacZ (beta-galactosidase alpha fragment)
  Amp (Ampicillin resistance gene) (Ampr gene)*
  ori (Origin of replication)
  T (Thymine over hang)*
  pGEM-T Vector (3000bp)

Plasmids: pKAN-R and pARA

• pKAN-R Plasmid:

  • kan^R (Kanamycin resistance)

  • 5,512 bp

  • HindIII

  • BamHI

  • ori

• pARA Plasmid:

  • amp^R (Ampicillin resistance)

  • pBAD

  • araC

  • PBAD-rfp (RFP under PBAD promoter)

  • 4,872 bp

  • PARA (Arabinose-inducible promoter)

  • BamHI

  • HindIII

Plasmids: pKAN-R Exercise

1. Which of the following functions can the pKAN-R plasmid carry out when present in bacterial cells?

   • A) Initiate replication of the pKAN-R plasmid.

   • B) Initiate transcription of the rfp gene.

   • C) Produce a protein that allows bacteria to survive in the presence of Kanamycin.

   • D) Produce a protein that allows bacteria to survive in the presence of Ampicillin.

   • E) Regulate expression of the rfp protein.

Plasmids: pARA Exercise

2. Which of the following functions can the pARA plasmid carry out when present in bacterial cells?

   • A) Initiate replication of the pARA plasmid.

   • B) Initiate transcription of the rfp gene.

   • C) Produce a protein that allows bacteria to survive in the presence of the antibiotic Kanamycin.

   • D) Produce a protein that allows bacteria to survive in the presence of the antibiotic Ampicillin.

   • E) Regulate expression of the rfp protein.

Plasmids: gfp Expression

3. You have a piece of DNA containing the gene for green fluorescent protein (gfp) and the pBAD promoter. This DNA fragment has been cleaved with BamHI and HindIII and can be inserted into either the pKAN-R plasmid or the pARA plasmid.

   Which plasmid would you choose to use as the plasmid vector to express gfp protein in bacterial cells?

   • A) pKAN-R plasmid

   • B) pARA plasmid

   • C) Neither plasmid

pARA and pKAN-R Plasmids: gfp Explanation

4. Select the response(s) below that best explain your answer.

   • A) Once the gfp DNA fragment is inserted, the pKAN-R plasmid could express the gfp protein.

   • B) The pKAN-R recombinant plasmid would not be maintained in bacterial cells as they replicate.

   • C) Once the gfp DNA fragment is inserted, the pARA plasmid could express the gfp protein.

   • D) The pARA recombinant plasmid would be maintained in bacterial cells as they replicate.

   • E) Neither plasmid contains the necessary components to express the gfp protein in bacterial cells.

Phage Structure

• Head-and-tail Phages:

  • Head (contains DNA)

  • Tail

  • Example: T4, lambda

• Filamentous Phages:

  • Protein molecules (capsid)

  • DNA molecule

  • Example: M13

Phages: Lytic vs. Lysogenic

• Lytic phages:

  • Endogenous gene replication of the phage.

  • Example: Phage T4, T3, T7 (T = tail).

• Lysogenic phages:

• Incorporation into the bacterial genome

• Lytic phase destroys host cells

  • Example: Phage λ (lambda).

Phages: Phage Lambda

• Lysogenic phage: phage lambda

  • Over 50 kb

  • Genes are grouped in functional clusters.

  • dsDNA linear

  • ends: 12nt overhangs: cos sites

    • allow recirculation when injecting the DNA: only circular DNA can be integrate into the genome bact

    • circle replication rolling

• endonuclease A recognizes the sites cos

• new ones are packaged virus

Phages: Limitations of Phage Lambda

• Initial Limitations:

  • Only 5% size could be inserted of the phage genome

  • Large genome: very many sites of restriction

• Now are capable of cloning 37-52kb (much more than plasmids and M13)

Phages: Cloning Strategies with λ

• Cloning Strategies with λ:

  • Circular:

    • Cutting and ligation

    • Transfection in E. coli (not very efficient)

  • Linear:

    • Recombinant viruses: they are concatenated

    • E. coli infection

    • In vitro packaging

    • Larger number of neighborhoods

Phages: Types of Vectors Based on Phage λ

• Types of vectors based on phage λ:

  • a) INSERTIONAL

    • Example: λ gt 10

    • Insertion into the phage genome

  • b) REPLACEMENT

    • Example: λEMBL3 and λEMBL 4

    • Replacement of a fragment of the genome by the foreign DNA: the fragment is called "STUFFER".

    • The most widely used (and improved)

    • e.g. EMBL3 and EMBL 4: capable of host 9-23 kb

Phages: M13

• Phage M13:

  • ssDNA circular 6.5 kb

  • step to dsDNA in bacteria

  • M13 is NOT integrated in the genome

  • No lysis occurs cell: bacteria releases viruses continuously

• Phage M13: it is not a lytic phage (it does not destroy the bacterium) but neither is it incorporated into the bacterial genome.

Phages: M13 replication and Vector Use

• Phage M13:

  • step ssDNA à dsDNA (RF)

  • RF: replicative form

  • Rolling circle replication

• It was useful as a vector: < 10kb single stranded DNA genes are obtained à very useful for e.g. mutagenesis in vitro

Phages: M13 Vectors

• Phage vectors need to maintain all genes necessary for replication

• M13 genome:

  • ->10 genes (I-X) needed for replication

  • ->only the intergenic sequence "IS" allows for enter DNA

Phages: M13 Modifications summary

• Modifications:

  • 1st) lacZ'

  • 2°) EcoRI

  • 3rd) polylinker
    a long primer with 4 restriction sites was introduced

Phages: M13 Vectors - Example M13mp18

• REP: genes for DNA replication

• CAP: genes for capsid formation

• MOR: genes for morphogenesis

Commercially available bacteriophage vectors:

• CAP: genes for capsid formation

• MOR: genes for morphogenesis