Lecture 51: Techniques 2- cloning Flashcards
Tuesday 25th February 2025
What are reasons for wanting to clone or express a gene?
To Determine its nucleotide sequence
To Identify and analyse its control sequences (promoters, translational signals, etc)
To Identify mutations in the gene e.g., gene defects related to specific diseases
To Investigate the structure and function of the encoded protein
To Produce large amounts of the encoded protein
To Make ‘tagged’ versions of the product for ease of purification
To investigate the intracellular targeting of the gene product (where does the protein work in the cell?)
What steps need to be considered when you want to clone a gene?
- Obtaining the gene of interest (insert)
-Constructing a vector/genetic construct to express it in E. coli
- Introducing the vector into E. coli (transformation)
How is the insert made in a prokaryote?
- The insert is the DNA fragment that contains the gene we want to clone. The method of obtaining the gene depends on whether it is from a prokaryote or a eukaryote.
- If the sequence of the gene is known, then the ends of the gene are modified to have restriction enzyme recognition sequences.
- PCR is then performed with these restriction sites to amplify the gene. They will all have the same incorporated restriction site at both ends of the gene
How is a eukaryotic gene expressed in e coli?
- Solution: Use cDNA (complementary DNA) instead of genomic DNA.
- mRNA will be made and the introns will be taken out.
- Reverse transcription is performed using reverse transcriptase. This generates a single stranded cDNA from the mRNA strand.
- The mRNA strand is degraded under alkaline conditions to leave just the cDNA.
- The second DNA strand is synthesized using terminal transferase and DNA polymerase.
- The DNA can then be amplified and modified for cloning.
- This results in double-stranded cDNA ready for cloning.
- Primers for PCR can be designed to include restriction enzyme recognition sites.
- After amplification, the insert is digested with restriction enzymes to allow ligation into a plasmid vector.
Why can’t e coli initially express a eukaryotic gene?
because eukaryotic genes contain introns, and bacteria cannot process these.
How do we make the construct?
- Restriction enzymes digest where you want to insert your DNA, then ligates the gaps together.
What are the essential features of a plasmid vector?
Origin of replication (ori): Allows the plasmid to replicate in bacteria.
Selectable marker (e.g., antibiotic resistance gene, bla for ampicillin resistance): Ensures that only transformed bacteria survive.
Multiple Cloning Site (MCS): A sequence with several restriction sites for inserting the gene.
Disruptable marker (e.g., lacZ’ gene in pUC vectors): Helps distinguish recombinant plasmids that have the marker inside of them.
No conjugate ability: The plasmid vector cannot easily be spread from cell to cell – it can only be transmitted by cell division, forming a clone.
What are the 4 major classes of restriction enzymes/
- Type I
- Type II
- Type III
- Type IV
Which class of restriction enzyme is extensively used for DNA manipulations?
Type II enzymes are used extensively for DNA manipulations
Describe type II restriction enzymes
- Type II enzymes typically recognise a 4, 6 or 8 bp sequence known as a restriction site and hydrolyse a phosphodiester bond in each strand of the DNA.
- Most Type II restriction sites are palindromic (read the same in either direction) with the cleavage sites symmetrically arranged.
What is an example of a type II restriction enzyme?
EcoRI
Give an example of a pUC vector
- pUC8 plasmid
- contains:
- lacZ’ gene: Used for blue-white screening
- Ampicillin resistance gene (bla): Allows selection of bacteria that have taken up the plasmid.
- Multiple cloning site (MCS): Contains restriction sites for inserting the gene.
How is the plasmid introduced to the vector?
- Once the gene insert is ligated into the plasmid, it must be introduced into bacteria via transformation.
- Calcium chloride (CaCl₂) treatment neutralizes the negative charges on DNA and bacterial cell membranes.
- A heat shock (42°C for ~30–60 sec) increases membrane permeability, allowing plasmid uptake.
- After transformation, only cells that contain the plasmid should grow. This is achieved using antibiotic selection and blue-white screening.
- Antibiotic selection: Only bacteria with the ampicillin-resistant plasmid survive on ampicillin plates.
- Blue-white screening:
If the plasmid has no insert, the lacZ’ gene remains functional → Blue colonies (X-gal is cleaved).
If the plasmid has an insert, the lacZ’ gene is disrupted → White colonies (no X-gal cleavage).
- White colonies are selected for further analysis.
What is bla?
An ampicillin resistant gene that is a selectable marker
After construct is made, cells are transformed to accept the plasmid
After construct is made, cells are transformed to accept the plasmid
once transformed, e coli can….
Once transformed, E. coli can produce large amounts of protein encoded by the inserted gene.
What are the key components for protein expression?
- Bacterial promoter (e.g., lac promoter): Controls transcription.
- Shine-Dalgarno (SD) sequence: Helps ribosomes recognize the start codon.
- Selectable marker: Ensures plasmid maintenance.
Describe the recombinant protein human insulin
Human insulin was one of the first recombinant proteins produced in bacteria.
The mRNA from pancreatic cells was used to generate cDNA.
The cDNA was inserted into a bacterial expression vector.
E. coli synthesized human insulin, which was then purified.
To facilitate protein purifcation, what do researchers add to recombinant proteins/
They add tags
Describe his-tag purification
- Histidine tag (His₆): A short sequence of six histidines added to the protein.
- His-tagged protein binds to a nickel-coated column.
- Impurities are washed away.
- Imidazole elutes the His-tagged protein.
- This method allows highly pure protein extraction.
How do we create a fusion protein
- Fusion proteins combine two proteins into one polypeptide chain.
- PCR is used to fuse two genes together.
- Used for tracking proteins, studying protein interactions, and creating research tools.
Give some examples of fusion proteins
- GFP (Green Fluorescent Protein) Fusion Proteins
- Single-Chain Variable Fragment (scFv) Antibody
- Cyclin-CDK Fusion Proteins
Why is CaCl2 added before the plasmid is taken up by the cell?
becayse the LPS of e coli is negatively charged. the calcium is positively charged and so it decreases repulsion.
Apart from adding CaCl2, what other methods allows the plasmid to be taken up by the cell?
- Heat shock will allow E.coli to temporarily move thjrough holes in the membrane
- A breif electric shick creates pores that are big enough for E.coli to be taken in
Making a recombinant protein in e.coli
- Bacterial promotors control gene expression
- Promoter can be induced to produce the protein
- Central dogma
- polarity needs to be taken into account -> make a sense strand
- Ribosomes will bind to the shine dalagarno sequence, resulting in translocation
- Don;t need a selectable marker
What can fusion proteins be used in?
Antibody engineering -> immunotherapy as a treatment for cancer
His-tag
6 proteins
Key Takeaways
Basic Cloning Steps: Insert preparation, vector construction, transformation.
Selection Methods: Antibiotic resistance and blue-white screening.
Gene Expression: Requires a bacterial promoter and proper regulatory sequences.
Recombinant Proteins: Used in medicine, research, and biotechnology.
Fusion Proteins: Allow visualization, purification, and functional studies.
