AB

Bioc192 Lecture 19 – Introduction to Recombinant DNA Technologies (Quick Reference)

What are recombinant DNA technologies?

  • Joining bits of DNA (sometimes from different species); inserted into an organism to produce (express) a useful protein.
  • Objective: use recombinant vectors to carry and express the gene of interest.

Major tools to manipulate and amplify DNA

  • Plasmids: circular double-stranded DNA; replicate independently; common in bacteria; can carry selectable markers.
  • Key components of plasmids:
    • Origin of replication (ORI): initiation of replication using host DNA polymerase.
    • Antibiotic resistance gene (selectable marker): provides survival advantage to cells carrying the plasmid.
    • Promoter: drives expression of the inserted gene in the host.
    • Screenable markers (e.g., GFP): enable visual identification of cells with the plasmid; used with fluorescence-based methods (e.g., FACS).
    • Restriction sites: allow ligation of the gene of interest into the cloning vector.
  • GFP as a reporter: emits green fluorescence under blue/UV light; used to identify cells expressing the recombinant protein; promoter determines cell-type expression.

The universal genetic code

  • All organisms read the same codons to produce the same amino acids.
  • Significance: a gene from one species can be expressed in another (e.g., human gene in bacteria).
  • Codon mapping examples:
    • \text{AUG} \rightarrow \text{Methionine}
    • \text{UGA} \rightarrow \text{Stop}

Gene expression and promoters

  • Promoter drives expression in cells with the appropriate transcription machinery.
  • Promoter specificity: different promoters work in different organisms (prokaryotic vs. eukaryotic) and cell types.

Introns and the use of cDNA

  • Prokaryotic genes typically lack introns; introns would disrupt expression in bacteria.
  • Use coding sequence only (cDNA) to ensure proper protein production.
  • Process: reverse transcription of mRNA to cDNA (introns removed).

Why use cDNA?

  • No introns allow successful translation in prokaryotes; reduces insert size; avoids alternative splicing.
  • Many vectors have insert size restrictions.

Construct synthesis (modern approach)

  • Chemically synthesize a DNA sequence (gene) from scratch and clone into a vector.
  • No biological material required; fully customizable (codon optimization, additions/removals).
  • Faster for complex constructs after design.

Cloning methods: traditional vs Gibson Assembly

  • Traditional cloning (restriction enzymes + ligase):
    • DNA cut at specific sites; insert and vector ligated; may leave restriction-site scars.
  • Gibson Assembly:
    • Joins multiple DNA fragments in a single, isothermal reaction; uses 5’ exonuclease, DNA polymerase, and DNA ligase; no need for restriction sites.

Comparative summary

  • Traditional cloning vs Gibson Assembly:
    • Restriction sites: required vs not required (Gibson).
    • Fragments: usually single vs multiple (Gibson can join 2–6+ fragments).
    • Speed: slower (multiple steps) vs faster (one-pot).
    • Precision: can leave scars vs seamless joins.
    • Flexibility: limited by restriction sites vs highly flexible design.
    • Cost: typically cheaper enzymes vs more expensive reagents.

Amplifying recombinant vectors: transformation

  • Transformation: transfer of vectors into bacteria.
  • Selection: antibiotic resistance on the vector allows survival of transformed cells.
  • Expression: vector gene expressed in bacteria (if bacterial promoter).
  • Amplification and purification for downstream uses (PCR, cloning, transfection).

What next after making the vector?

  • Expression of the gene in the host to produce protein; potential downstream applications.

The code that makes it all work: the genetic code recap

  • Universal code means the same codons map to the same amino acids across species.
  • Significance: cross-species gene expression is feasible.

Introns vs exons: practical design rule

  • Do not include introns in prokaryotic expression constructs; use cDNA.
  • Rationale: introns are not processed in bacteria; otherwise translation would fail.

Construct design: modern synthesis (recap)

  • DNA synthesis enables direct creation of gene sequences; overlaps and codon optimization can be designed in advance.
  • Synthesized genes are cloned into vectors for expression.

Key concepts (quick reference)

  • Recombinant DNA technologies combine DNA from different species.
  • Core elements: vector, promoter, ORI, selectable and screenable markers, restriction sites.
  • Traditional cloning vs Gibson Assembly: sites vs seamless overlaps; speed and flexibility differences.
  • Universal genetic code enables cross-species expression.
  • cDNA is used to express eukaryotic genes in prokaryotes due to lack of introns.
  • Modern synthesis allows direct construction of desired vectors.

Practice self-assessment (core questions)

  • 1. What are recombinant DNA technologies?
  • 2. What is the crucial element in recombinant DNA technologies?
  • 3. What are the key components of the element named in the question above?
  • 4. What are restriction enzymes?
  • 5. What are DNA ligases?
  • 6. How are restriction enzymes and DNA ligases useful in recombinant DNA technologies?
  • 7. Outline the basic method of Gibson Assembly.
  • 8. Briefly outline the process of transformation.
  • 9. How is recombinant DNA technology made possible via the genetic code?
    1. What issue may be encountered when cloning eukaryotic genes in prokaryotes and how is this issue overcome?