Lecture 5

Host Organisms & Bacterial Genotypes

• E. coli K12 Derivatives: Primary bacterial host for cloning and propagation.

• Biological Containment: Carries mutations that prevent survival outside laboratory conditions.

• Essential Modifications: Altered restriction systems and lac operon machinery allow plasmid stability and regulated expression.

• Eukaryotic Hosts: Yeast (S. cerevisiae, Pichia) for basic eukaryotic protein folding; insect and mammalian cells for complex post-translational modifications (e.g., glycosylation).

Bacterial Genotype Nomenclature Rules

• Mutated Genes Only: Strains list only mutated alleles; normal/wild-type genes are omitted.

• 3-Letter Designation: Indicates the specific pathway or phenotype (e.g., rec = recombination, end = endonuclease).

• Delta (Δ): Indicates a complete gene deletion.

• F' Episome [in brackets]: Lists genes carried on the F fertility plasmid; these represent functional wild-type alleles unless noted.

• Identification Markers: Nutritional mutations (e.g., requiring thiamine) allow strain verification using selective minimal media like M9.

Restriction & Modification (R-M) Systems in Bacteria

Natural Role:

- Serves as a bacterial immune defence against bacteriophage infection.

- Host DNA is protected by methylation (-CH3 groups added to specific bases).

- Unmethylated foreign phage DNA is cleaved and destroyed by host restriction endonucleases.

Application in Molecular Biology Hosts:

- Restriction mutants (r-): Allow uptake and survival of foreign/unmodified DNA.

- Modification mutants (m-): Prevent the host from methylating DNA, ensuring downstream restriction enzymes can cut target sites.

Methylation in PCR & Cloning (DPNI Digest)

1. Methylation Status Differences:

- Parental plasmid DNA isolated from E. coli = Methylated.

- Newly synthesized PCR product generated in vitro = Unmethylated.

• This happens because in PCR, no enzyme methylates the newly synthesized DNA, compared to within a bacterium; thus, we can get rid of the methylation.

• Also, Taq polymerase can synthesize methylated regions, which is how PCR can still be performed on methylated DNA.

2. DpnI Selection Strategy:

- DpnI is an endonuclease that ONLY cuts methylated GATC sites.

- Adding DpnI digests and eliminates the background parental template DNA.

- The desired, unmethylated PCR amplicon is left untouched for downstream transformation.

Overview of the lac Operon

Biological Purpose: An inducible bacterial operon in E. coli that coordinates lactose transport and breakdown.

• Structural Genes (Transcribed together):

• lacZ: Encodes beta-galactosidase (cleaves lactose into glucose and galactose).

• lacY: Encodes lactose permease (membrane transporter for lactose entry).

• lacA: Encodes beta-galactoside transacetylase (transfers acetyl groups for detoxification).

• Regulatory Elements:

• lacI: Regulatory gene that continuously produces the Lac repressor protein.

• Promoter (P): Binding site for RNA polymerase.

• Operator (O): DNA regulatory sequence between promoter and structural genes where the repressor binds.

• CAP site (c): Binding region for CAP-cAMP activation under low-glucose conditions.

Mechanisms of Regulation (Lac operon)

Repressed State (Inducer Absent):

• Lac repressor binds to the Operator site.

• RNA polymerase is sterically blocked from moving forward.

• Transcription of lacZYA structural genes is shut off.

Induced State (Inducer Present: Allolactose / IPTG):

• Inducer molecule binds to the allosteric site of the Lac repressor.

• Repressor changes shape and detaches from the Operator.

• RNA polymerase transcribes the structural genes (lacZYA) into a polycistronic mRNA.

Molecular Biology Application (IPTG):

• IPTG functions as a non-metabolizable ("gratuitous") inducer. It’s often used in labs instead of allolactose.

• Because it is not degraded by beta-galactosidase, IPTG maintains steady, long-lasting transcriptional induction in recombinant protein expression systems. Meaning this system will always remain turned on.

Lac Operon Mutation & Alpha Complementation

1. Engineered Host Mutations:

- lacI^q: Overexpresses Lac repressor (~10x); ensures tight transcriptional shutdown of multi-copy plasmid promoters until IPTG induction.

- lacZ ΔM15: Deletes amino acids 11-41 from beta-galactosidase; produces an inactive C-terminal beta-galactosidase (omega fragment).

2. Principles of Alpha-Complementation:

- Host Cell: Supplies the inactive omega fragment (from F' episome).

- Cloning Vector: Supplies the functional N-terminal alpha peptide (lacZ 5' region).

- Reconstitution: The alpha and omega pieces associate non-covalently into an active enzyme complex (beta-galactosidase).

3. X-Gal Selection Breakdown:

- Non-recombinant / Empty Vector: Alpha peptide produced -> Active beta-galactosidase -> Cleaves X-Gal -> BLUE COLONIES.

- Recombinant / Inserted Gene: Insert disrupts alpha peptide coding frame -> Inactive enzyme -> Cannot cleave X-Gal -> WHITE COLONIES.

- White colonies are the ones that took up the plasmid WITH the insert in it, thus its the colonies we are concerned with.

Common features of vectors

• They are able to replicate in a host cell.

• They can accept foreign DNA.

• They are able to be transformed into host cells.

Plasmid Replication & Copy Number Control

• Host Enzyme Utilization: Plasmids rely on stable host cellular machinery (e.g., host DNA polymerases) to initiate and execute DNA replication.

• Replication Control Modes:

• Stringent Replication: Tightly coupled to the host chromosome cycle; results in low copy numbers (1–5 copies/cell).

• Relaxed Replication: Decoupled from host chromosomal division, can replicate whenever; produces medium-to-high copy numbers (e.g., ColE1, pMB1 give 15–20+ copies/cell).

• Independence from Protein Synthesis: Relaxed replicons utilize long-lived host enzymes and regulatory RNAs, allowing replication to proceed even when bacterial protein synthesis is inhibited.

• Biosafety Disarming (tra Deletion): Molecular biology vectors lack tra (transfer) genes to eliminate conjugative transfer and prevent horizontal gene spread outside the lab.

Replicons & Plasmid Incompatibility

• Replicon Definition: The functional genetic unit containing the origin of replication (ORF) and its control elements (e.g., pMB1, ColE1, pBR322, pUC series).

• Plasmid Incompatibility: The failure of two different plasmids to stably coexist within the same host cell in the absence of continuous selection pressure.

• Molecular Basis of Incompatibility:

• Plasmids sharing identical or closely related replication control systems cross-regulate each other.

• Competition for shared regulatory factors and unequal partitioning during division leads to the rapid loss of one plasmid type.

• Co-Transformation Requirement: To stably propagate two distinct plasmids simultaneously, each plasmid must possess an origin from a distinct, mutually compatible incompatibility group (e.g., ColE1 paired with p15A).

Plasmid Development

1. Key Improvements in Modern Vectors:

• Compact size: Accommodates larger inserts, increases transformation yield, simplifies gel mapping.

• Multi-Cloning Sites (MCS) / Polylinker: Cluster of unique restriction enzyme sites for flexible cloning.

• Efficient markers: Antibiotic resistance (selection) + lacZ alpha-peptide (blue/white screening).

2. pUC18 (Plasmid) Vector Features:

• Size: 2686 bp.

• Replicon: High-copy pMB1 origin.

• Screening: lacZ alpha-peptide gene interrupted by the MCS.

3. pBluescript (Phagemid) Specialized Functions:

• ColE1 Origin: Drives normal high-copy double-stranded plasmid replication.

• f1 Phage Origin: Produces single-stranded DNA (ssDNA) upon helper phage infection.

• T3 & T7 Promoters: Flank the MCS to allow in vitro synthesis of sense and antisense RNA transcripts.

pUC18 Vector

Gene Cloning Workflow & Blue-White Screening

1. Standard Cloning Protocol:

- Digestion: Vector and DNA insert are cut with the same restriction enzyme(s) to create matching sticky ends.

- Ligation: DNA ligase covalently seals the vector and insert into circular recombinant plasmids.

- Transformation: Plasmids are introduced into competent host bacteria.

- Plating: Grown on LB agar containing Ampicillin + X-Gal + IPTG.

2. Colony Phenotype Interpretation:

- Antibiotic Selection: Only cells containing a plasmid (AmpR) can grow into colonies.

- Blue Colonies = Non-recombinant (Empty Vector): Intact lacZ alpha-peptide -> Active beta-galactosidase -> Cleaves X-Gal -> Blue color.

- White Colonies = Recombinant Clones (Target Insert Present): Insert disrupts lacZ gene (insertional inactivation) -> No beta-galactosidase activity -> Cannot cleave X-Gal -> White colour. - Action: White colonies are selected and cultured for plasmid DNA extraction and verification.

Bacteriophage Lambda Vector

1. Biological Basis:

- Linear dsDNA genome (~48.5 kb).

- Middle 1/3 of the genome is dispensable for lytic growth (contains non-essential lysogeny genes).

- Essential lytic/structural genes reside on the left and right terminal arms.

2. Major Advantages:

- High Efficiency: in vitro packaging into viral capsids allows near 100% infection efficiency (1:1 plaque formation), compared to <1/1000 for standard plasmid transformation.

- High Capacity: Can clone fragments up to 20-25 kb.

3. Packaging Rules:

- Capsid size limit: Only DNA lengths between 38 kb and 52 kb (~78% - 105% of WT) can be successfully packaged into infectious phages. Anyting larger or smaller will lead to no replication.Anything

4. Types of Lambda Vectors:

- Insertion Vectors: Single cloning site; accommodates small inserts (0 - 10 kb); commonly used for cDNA libraries.

- Replacement Vectors: Contains a removable "stuffer fragment" between arms. Arms alone (~30-36 kb) are too small to package. Foreign insert (10 - 20 kb) restores total length into the 38-52 kb packaging range. * Serves as automatic positive selection for recombinants (genomic libraries).

Insert Capacity:

• Insertion Vectors: Carry small inserts (0–10 kb).

• Replacement Vectors: Accommodate large fragments (10–20 kb).

Cloning Mechanism:

• Insertion Vectors: Single-restriction-site cleavage; target DNA is inserted directly into the vector backbone.

• Replacement Vectors: Dual-restriction-site cleavage; a disposable stuffer fragment is excised and replaced with the target DNA.

Selection Strategy:

• Insertion Vectors: Requires screening methods (e.g., plaque phenotype or insertional inactivation of cI / lacZ) to distinguish recombinants from empty vectors.

• Replacement Vectors: Relies on physical size constraints for automatic selection; vector arms alone (38 kb) are too small to package, ensuring only recombinants form viable phage particles.

Primary Use Cases:

• Insertion: cDNA library construction.

• Replacement: Genomic DNA library construction.

Gateway Cloning System

• Recombination-Based: Replaces traditional restriction endonuclease and DNA ligase reactions with site-specific recombination machinery derived from bacteriophage $\lambda$.

• Recombination Sites (att): Target genes flanked by specific att sequences are transferred directionally and in-frame.

• Universal Entry Clone: Once a gene is captured inside an Entry Vector, it can be recombined simultaneously into various specialized Destination Vectors (e.g., His-tag, GST-tag, mammalian CMV, or Baculovirus).

Cosmid Vectors

• Hybrid Architecture: Standard plasmid backbone (plasmid ORF + antibiotic marker) engineered with phage lambda cos sites.

• High Cloning Capacity: Accommodates large inserts up to ~42 kb.

• Delivery and Maintenance:

• Packaged in vitro into phage lambda capsids for high-efficiency infection into E. coli.

• Circularizes inside the host via cos overhangs and replicates as a large extra-chromosomal plasmid (does not cause lytic plaque formation).

Large-Scale Vectors: BACs vs. YACs

Bacterial Artificial Chromosomes (BACs):

• Insert Capacity: ~300 kb; based on the E. coli F-factor plasmid.

• Replication Control: repE Controls low-copy plasmid replication complex formation.

• Partitioning Stability: sopA, sopB, and sopC ensure accurate segregation to daughter cells.

• Markers: Cm^R (chloramphenicol selection) and lacZ (blue/white screening).

Yeast Artificial Chromosomes (YACs):

• Insert Capacity: ~1,000 kb (1 Mb); maintained as linear eukaryotic chromosomes in S. cerevisiae.

• Essential Functional Units:

  • ARS: Autonomously Replicating Sequence (yeast origin of replication).

  • CEN: Centromere sequence for spindle attachment and equal division.

  • TEL: Telomeric repeats protecting terminal ends from exonucleases.

  • TRP1 / URA3: Auxotrophic selection markers ensuring retention of both arms.