Module 5

Insert

DNA sequence inserted into plasmid

Reproduced by bacterial replication

Vector

Plasmid in cloning

Cloning: Multiple Cloning Site (MCS)

Contain restriction enzyme recognition sites

Enzymatic connection to vector

Cloning: Origin of Replication (Ori)

Recognize by bacterial replication machinery

Determine plasmid copy number

Cloning: Selectable Marker

Selective culture bacteria

Antibiotic resistance genes

Cloning: Restriction Endonuclease

Enzyme

Cleave phosphodiester links in DNA

6 bp recognition site

Sticky Ends

Overhang from DNA cutting by restriction enzymes

Base pair interactions for ligation

Blunt Ends

No overhang from DNA cutting

No base-pair interactions for ligation

Cloning 1: Digestion

Cut circular vector DNA into linear DNA with 2 restriction enzymes

Cut insert with 2 restriction enzymes

MCS contain recognition sites

Purify products

Cloning 2: Ligation

DNA ligase join insert and vector sticky ends

DNA flanked by recognition sites

Vector with same recognition sites

ATP catalyzed

Cloning 3: Transformation and Selection

Competent E. coli transform with ligation reaction

Select cells with vector on antibiotic plates

Cloning 4: Confirmation

Grow liquid cultures (antibiotic and blue-white screening)

Isolate vector DNA

Confirm insert presence

Blunt-End Ligation

DNA ligase joins blunt ends

Unstable

Inefficient

Control Insert Direction

2 restriction enzymes (different sticky ends)

Only ligate sticky ends from same enzyme (1 direction)

E coli 1: Transformation

Produce artificial competent bacteria

Chemicals (heat-shock) or electric current

Take up DNA

E coli 2: Growth

On selective media with antibiotic

Select cells with cloning vector (antibiotic resistance)

E coli 3: Vector Purification

Inoculate liquid cultures

Add antibiotic (selective pressure)

Purify vector from cells

E coli 4: Evaluation

Characterize purified plasmid

Confirm vector identity and insert presence

Sanger sequencing or agarose gel electrophoresis

Blue-White Screening

Identify bacteria with self-ligated vector

Vectors with MCS in LacZ gene code for B-galactosidase (lactose digestion)

Convert colourless X-gal to blue

Use X-gal plate

Blue-White Screening: Blue Colonies

Vector with intact LacZ

Blue-White Screening: Colourless

Vector with disrupted LacZ

Promoter

Sequence signal transcription start

Upstream of start codon

Sigma Factors

Level 1 transcription control

Small proteins

Recognize by RNA polymerase

Initiate transcription

Operator

Level 2 transcription control

Between promoter and gene

Recognize by repressors (ligand control binding)

Prevent transcription

Operator: Inducible Operon

Ligand binding allows transcription

Conserve energy

Quick adaptation

Operator: Repressible Operon

Ligand binding prevents transcription

Conserve energy

Lac Operon

Inducible operon

Lactose metabolism

3 adjacent genes (LacZ, LacY, LacA)

LacI Protein

LacI gene upstream of Lac operon

Lac operon repressor

Block LacZ, LacY, LacA transcription

Allolactose

Inducible operon

LacI ligand

Prevent LacI binding

Allow Lac operon transcription

Trp Operon

Repressible operon

Tryptophan biosynthesis

5 adjacent genes

TrpR Protein

TrpR gene upstream from Trp operon

Trp operon repressor

Block Trp transcription

Tryptophan Levels

Repressible operon

TrpR ligand

Allow TrpR binding

Prevent Trp operon transcription

Heterologous Protein

Protein not naturally in bacteria

Recombinant Protein

Protein produced from recombinant expression vector

Expression Vector

Produce heterologous protein encoded by insert DNA MCS

Expression Vector: Promotor

Upstream from MCS

RNA polymerase and sigma binding factors transcribe downstream sequence

Expression Vector: Operator

Between promotor and gene sequence

Region in operon

Prevent overtranscription

Expression Vector: LacI Gene

Produce LacI protein repressor (bind lac operon)

Prevent transcription

Separate promotor regulation

Add Inducer: Trigger transcription

E Coli BL21(DE3)

E coli and bacteriophage derivative

Contain gene coding T7 RNA polymerase (lacUV5 promoter)

E Coli BL21(DE3): IPTG

Prevent LacI binding to operator

Induce T7 RNA polymerase transcription

E Coli BL21(DE3): T7 Promoter

Recognize by T7 RNA polymerase

Control insert and expression vector transcription

pET-28a Plasmid: MCS

Downstream from T7 promoter

pET-28a Plasmid: LacI Gene

Code LacI repressor

Control cloned gene transcription

pET-28a Plasmid: Ori Region

Allow plasmid copy production

pET-28a Plasmid: KanR Gene

Code antibiotic resistance protein

Selectable maker

MCS: Recognition Sites

Recognize by restriction enzymes

MCS: T7 Promoter

Recognize by T7 RNA polymerase

Transcription begin downstream

MCS: Lac Operator

Recognize by LacI repressor protein

IPTG prevent LacI binding Lac operator

Allow T7 RNA polyerase transcription

MCS: T7 Terminator

Signal T7 RNA polymerase stop

MCS: T7 Primer Arrows

Oligonucleotide primers

Confirm non-mutated insert in vector

MCS: Ribosome Binding Site

Nucleotide sequence in mRNA transcript

Recognize by ribosome

Direct to start codon

MCS: Start Codon

Begin translation

In frame with insert codons

MCS: Stop Codon

Stop translation

MCS: His-Tag

Short amino acid sequence

Add to protein end

Buffer

Weak acid and conjugate base mixture

Resist pH changes

Dialysis

Buffer exchange and desalting

Use tube with pores (selective molecule passage)

Dialysis Process

1. Protein solution with buffer 1 in tube
2. Tube in buffer 2
3. Buffer diffusion until equilibrium
4. Repeat

Diafiltration

Increase protein concentration

Use centrifugal filters

Diafiltration Process

1. Centrifuge solution in inner tube
2. Liquid forced through membrane filter
3. Protein concentration in tube increase

Lysis: Detergents

Amphipathic molecules dissolve bacterial membrane

Lysis: Sonication

High-power sound waves disrupt bacterial membrane

Lysis: Freeze/Thaw

Ice crystals around cell break bacterial membrane

Lysis: French Press

Force cells through narrow valve

High pressure and stress break bacterial membrane

Lysis Buffer

Stabilize protein during lysis

Protease Inhibitor Cocktail: Prevent protein degradation

Lysozyme

Degrade peptidoglycan

Weaken cell wall

In combination with lysis methods

Soluble Proteins from Lysis

Centrifuge

Remove insoluble components

Insoluble Proteins from Lysis

Use detergents

Chromatography

Separate components in mixture

Use resin trapping proteins

Chromatography Process

1. Load sample into column with resin
2. Add buffer to carry protein through resin (gravity, FPLC systems)
3. Protein separation
4. Protein elution at column bottom
5. Collect fractions monitored with spectrophotometry

Nickel Affinity Chromatography

Imidazole in his-tag bind Ni2+

Nickel resin bind and retain proteins

Nickel Affinity Chromatography Process

1. Load his-tagged proteins to nickel column
2. Wash with imidazole buffer to remove loose proteins
3. Wash with high imidazole buffer to elute his-tagged proteins

Size Exclusion Chromatography

Resin with pores

Separate proteins by size

Large pass quickly

Small pass slowly

Ion Exchange Chromatography

Charged exchange resin

Separate proteins by charge

Negative bind anion exchange resin

Positive bind cation exchange resin

Ion Exchange Buffer

Salts compete with protein to bind resin (depend on ionic strength)

Concentration changes in protein elution

Ion Exchange Buffer Gradient

Buffer ionic strength increase gradually

Multiple buffers

Ion Exchange Buffer Linear

Buffer percent composition increase gradually

1 buffer