Lecture 3: Expression vectors, host cells and gene expression

Recap: The Gene Cloning Process 🔄

In the previous lectures, we discussed the fundamental tools and initial steps of gene cloning. We learned how to prepare a DNA fragment of interest (the INSERT) and ligate it into a digested plasmid (the VECTOR). The next crucial steps involve introducing this recombinant DNA into host cells, selecting for cells that have successfully taken up the plasmid, and then screening to identify those clones that contain the specific insert.

A Note on Preventing Vector Recircularization: When digesting a plasmid vector with a single restriction enzyme, there's a risk the vector might simply re-ligate to itself (recircularize) instead of incorporating the insert. To minimize this:

• Use a high molar ratio of insert to vector during ligation.

• Treat the cut vector with phosphatase to remove the 5' phosphate groups. This prevents self-ligation of the vector, as DNA ligase requires a 5' phosphate to form a phosphodiester bond. The insert, which retains its 5' phosphates, can still be ligated into the dephosphorylated vector.

• Use two different restriction enzymes to cut the vector and prepare the insert, creating incompatible ends on the vector that cannot re-ligate to each other.

Introducing Recombinant DNA into Host Cells 🦠

Methods for E. coli

There are three common methods to introduce recombinant DNA into E. coli:

The general process for plasmid transformation involves mixing plasmid DNA with host cells, inducing DNA uptake (via heat shock or electroporation), selecting for cells that contain plasmid, and then screening these to find clones with the desired insert.

Methods for Eukaryotic Cells

Introducing cloned DNA into eukaryotic cells often uses different techniques:

Selection and Screening of Recombinant Clones 🎯

After attempting to introduce the recombinant DNA, we need to identify which cells successfully incorporated the plasmid and, furthermore, which of those plasmids actually contain our gene of interest.

Selection for Transformed Cells (Containing a Plasmid)

Most plasmid vectors contain a selectable marker, typically an antibiotic resistance gene (e.g., the amp<sup>R</sup> gene in the pUC19 plasmid confers resistance to ampicillin). This marker gene is part of the plasmid itself and has its own promoter for expression in the host cell. When transformed cells are plated on growth medium containing the specific antibiotic, only cells that have successfully taken up a plasmid (and can therefore express the resistance gene) will be able to survive and form colonies. This process is called selection. Other common antibiotic resistance markers include Tet<sup>R</sup> (tetracycline), Kan<sup>R</sup>/nptII (kanamycin), CAT/Cm<sup>R</sup> (chloramphenicol), and Strep<sup>R</sup>/Spec<sup>R</sup> (streptomycin/spectinomycin).

Screening for Recombinant Plasmids (Containing the Insert)

Selection ensures cells have a plasmid, but not necessarily the recombinant plasmid (i.e., one with the DNA insert ligated in). The vector might have re-ligated to itself without an insert. Screening methods help distinguish between cells carrying recombinant plasmids and those with non-recombinant (empty) vectors.

Essential Features of Plasmid Vectors for Cloning:

• An origin of replication (ori) that allows the plasmid to replicate in the host cell and controls its copy number (high or low).

• One or more unique restriction enzyme sites, often clustered together in a Multiple Cloning Site (MCS) or polylinker, into which foreign DNA can be ligated.

• A selectable marker (usually an antibiotic resistance gene).

• Ideally, a means of screening for recombinant plasmids.

Screening Methods:

1. Insertional Inactivation using pBR322 (Older Method):

   • pBR322 contains two antibiotic resistance genes, amp<sup>R</sup> and tet<sup>R</sup>. If the DNA insert is cloned into a restriction site within, for example, the tet<sup>R</sup> gene (e.g., SalI site), the tetracycline resistance is lost (insertional inactivation).

   • Cells transformed with a non-recombinant pBR322 will be resistant to both ampicillin and tetracycline. Cells with a recombinant plasmid (insert in tet<sup>R</sup>) will be ampicillin-resistant but tetracycline-sensitive (Amp<sup>R</sup> Tet<sup>S</sup>).

   • This requires replica plating: colonies are first grown on ampicillin plates, then transferred (replicated) to tetracycline plates. Colonies that grow on ampicillin but not on tetracycline are the desired recombinants.

2. Blue-White Screening using pUC Plasmids (e.g., pUC18/19):

   • pUC vectors (e.g., pUC18) are high copy-number plasmids containing an amp<sup>R</sup> gene and the lacZα gene fragment, which has an MCS within it. The lacZα gene encodes the α-peptide of β-galactosidase.

   • Principle (α-complementation): The E. coli host strain used often carries a mutation in its own lacZ gene (e.g., lacZΔM15), producing an inactive β-galactosidase. If the plasmid expresses the LacZα peptide, it can complement the host enzyme fragment, restoring β-galactosidase activity.

   • Screening Process: Transformed cells are plated on media containing ampicillin, X-gal (a chromogenic substrate that turns blue when cleaved by β-galactosidase), and often IPTG (an inducer of the lac operon, sometimes needed to ensure lacZα expression).

     • Non-recombinant plasmid (empty vector): The lacZα gene is intact, functional β-galactosidase is produced, X-gal is cleaved → blue colonies.

     • Recombinant plasmid (insert in MCS): The lacZα gene is disrupted by the inserted DNA, no functional β-galactosidase is made, X-gal is not cleaved → white colonies.

     • White colonies are selected as likely containing the recombinant plasmid. Agar plate showing blue and white bacterial colonies. White colonies typically represent cells transformed with a recombinant plasmid where the lacZα gene has been disrupted by an insert.

3. Insertional Inactivation using the ccdB Gene:

   • The E. coli ccdB gene encodes a toxin that inhibits DNA gyrase, which is lethal to most lab strains of E. coli that lack the CcdA anti-toxin (normally found on the F plasmid).

   • Cloning vectors can be designed with the ccdB gene containing an MCS. If a DNA fragment is successfully cloned into the MCS, the ccdB gene is disrupted, and the cell survives.

   • If the vector re-ligates without an insert, the ccdB gene remains functional, and its expression kills the host cell. Thus, only cells transformed with recombinant plasmids survive.

4. Screening by Plasmid Analysis or PCR:

   • If no insertional inactivation system is available, individual colonies can be picked, grown, and their plasmid DNA extracted.

   • The plasmid can then be digested with restriction enzymes expected to release the insert, and the products analyzed by gel electrophoresis to check for an insert of the correct size.

   • Alternatively, Colony PCR can be performed: a small amount of a bacterial colony is used directly as the template in a PCR reaction with primers specific for the insert. A successful amplification product indicates the presence of the recombinant plasmid.

Preparing and Expressing Cloned DNA 📜➡Protein

Plasmid DNA Extraction from Recombinant Colonies

Once recombinant colonies are identified, they are grown in liquid culture to produce more cells from which plasmid DNA can be purified in larger quantities. A common method is the alkaline lysis method:

1. Cells are harvested and resuspended. The cell wall is disrupted (e.g., with glucose/EDTA).

2. Cells are lysed with a solution of NaOH/SDS, which denatures cell membranes, chromosomal DNA, and proteins.

3. An acidic solution (e.g., potassium acetate/acetic acid) is added to neutralize the lysate. The small, supercoiled plasmid DNA rapidly renatures (re-anneals), while the larger, more complex genomic DNA and denatured proteins precipitate.

4. Cellular debris, genomic DNA, and proteins are removed (e.g., by centrifugation or phenol/chloroform extraction).

5. Plasmid DNA is precipitated from the cleared supernatant using isopropanol or ethanol.

6. The plasmid DNA pellet is washed with 70% ethanol to remove residual salts and impurities, then dried and dissolved in water, often containing RNase to degrade any co-purified RNA.

7. The purified plasmid DNA can be checked for integrity and conformation by gel electrophoresis.

Expression Vectors and Gene Expression

Standard cloning vectors are mainly for DNA propagation. To produce protein from a cloned gene, an expression vector is required.

• Promoters: Expression vectors contain specific DNA sequences called promoters that are recognized by the host cell's transcription machinery to drive the expression (transcription and translation) of the inserted gene. The promoter must be functional in the chosen host organism (e.g., a plant promoter for expression in plants, a bacterial promoter for E. coli, etc.).

• cDNA for Eukaryotic Genes in Prokaryotes: When expressing a eukaryotic gene in a prokaryotic host like E. coli, it is essential to use the cDNA version of the gene. cDNA is made from mRNA and therefore lacks introns. Prokaryotes do not have the machinery to splice introns from eukaryotic pre-mRNA.

The pET System for Protein Expression in E. coli: The pET system is a widely used and powerful set of expression vectors for producing high levels of recombinant protein in E. coli.

• Host Strain: It requires a specific E. coli host strain, such as BL21(DE3), which carries the gene for T7 RNA polymerase integrated into its chromosome. The expression of this T7 RNA polymerase is under the control of an IPTG-inducible lac promoter.

• Vector Features: The gene of interest is cloned into the pET vector downstream of a very strong T7 promoter and a lac operator sequence. The lacI gene, encoding the Lac repressor protein, is often present on both the host chromosome and the pET plasmid to ensure tight repression of both the T7 RNA polymerase gene and the target gene in the absence of an inducer.

• Induction of Expression:

  • Addition of IPTG (a lactose analog) binds to the Lac repressor, causing it to dissociate from the lac operators.

  • This de-repression allows the host E. coli RNA polymerase to transcribe the T7 RNA polymerase gene.

  • The newly synthesized T7 RNA polymerase then specifically recognizes the T7 promoter on the pET plasmid and drives high-level transcription of the cloned gene of interest. IPTG also de-represses the lac operator controlling the target gene on the plasmid.

• This system offers very tight control over expression (low basal levels before induction) and allows for very high levels of protein production upon induction. pET vectors typically do not use lacZ for blue-white screening; the lac regulatory elements are for controlling target gene expression. Diagram of the pET expression system in an E. coli host cell, showing IPTG induction leading to T7 RNA polymerase production, which then drives high-level expression of the target gene from the pET plasmid.

• Protein Purification (His-tag): Many pET vectors facilitate protein purification by allowing the expression of the target protein as a fusion with a 6xHis tag (a sequence encoding six consecutive histidine residues). This tag has a high affinity for nickel ions, so the His-tagged recombinant protein can be purified from cell lysates using nickel affinity chromatography. The purified protein can then be used for biochemical studies, structural analysis, antibody production, etc..

  • Note on Fusion Proteins: When creating C-terminal fusions (e.g., gene-of-interest-His-tag or gene-of-interest-GFP), the stop codon of the gene of interest must be removed, and the coding sequences of both parts must be ligated in-frame to ensure correct translation of the fusion protein.

Other Types of Cloning Vectors 🚀

Besides standard plasmids, other types of vectors are used for specific purposes, particularly for cloning larger DNA fragments or for efficient library construction:

• Bacteriophage Vectors (e.g., based on M13 or λ phage): Insert DNA is incorporated into the phage genome. E. coli cells are infected, and recombinant phages produce plaques (clearings) in a bacterial lawn. Ideal for constructing large-scale libraries. Plasmids containing a phage origin of single-stranded DNA replication are called phagemids.

• Cosmids: Hybrid vectors containing plasmid components and the cos sites from bacteriophage λ. They can accommodate larger inserts than plasmids (up to ~100 kb), replicate as plasmids in E. coli, but can also be packaged into phage particles for efficient delivery into cells.

• BAC (Bacterial Artificial Chromosome): Based on the E. coli F (fertility) plasmid, BACs are used for cloning very large DNA fragments (up to ~300 kb).

• YAC (Yeast Artificial Chromosome): Used for cloning extremely large DNA fragments (up to ~1 Mb or more). YACs contain yeast centromeric and telomeric sequences, as well as a yeast chromosomal origin of replication, allowing them to be maintained as linear artificial chromosomes in yeast host cells. (Students are advised to be aware of these other vector types, but detailed knowledge is not required for assessment on this specific lecture).

Learning Outcomes & Recommended Reading 📚

By the end of this lecture, you should understand that:

• DNA uptake into bacterial host cells (transformation) is followed by selection for cells containing a plasmid (usually via antibiotic resistance) and screening to identify recombinant clones (those with the desired insert).

• Plasmid vectors possess essential features: an origin of replication (controlling copy number), a multiple cloning site (MCS) for inserting DNA, and a selectable marker.

• Recombinant plasmids can be screened using methods like insertional inactivation of a marker gene (e.g., lacZ for blue-white selection or ccdB for toxic selection).

• Plasmid DNA is commonly isolated from bacterial cultures using the alkaline lysis method.

• Expression vectors are used to produce protein from a cloned gene insert, requiring appropriate promoters for the chosen host organism.

• Various types of vectors exist (plasmids, bacteriophages, cosmids, BACs, YACs), each suited for different insert sizes and applications.