9- Recombinant DNA and Cloning Vectors

Question 1

what is a vector?

Answer 1

a type of recombinant tool - used to transfer DNA into a biological system

Question 2

what is the Shine-Dalgarno sequence?

Answer 2

• short sequence 5-10bp upstream of the translation start codon
• ensures correct initiation of translation by aligning the small ribosomal subunit with the start codon

Question 3

list the 4 types of recombinant vectors used in molecular biology

Answer 3

plasmids
bacteriophages
viruses
artificial chromosomes

Question 4

describe the nature, characteristics and uses of plasmids as recombinant DNA vectors

Answer 4

nature: naturally occurring in many bacterial, sources of virulence factors and antimicrobial resistance

characteristics: restricted host range due to biological compatibility and restriction systems
- transferable by conjugation to other bacteria or natural transformation

uses: cloning, gene expression, producing recombinant proteins

Question 5

describe the nature, characteristics and uses of bacteriophages as recombinant DNA vectors

Answer 5

nature: naturally occurring from bacterial viruses

characteristics: transfer antimicrobial resistance and virulence through transduction
- have a restricted host range
- used as antibacterial agents = can restrict and lyse bacteria

uses: bacterial genetic engineering

Question 6

describe the nature and uses of viruses as recombinant DNA vectors

Answer 6

nature: different types, engineered as vectors to integrate DNA into mammalian cells or for recombinant expression in insect cells

uses: gene therapy, vaccine development, expression of foreign genes in eukaryotic cells

Question 7

describe the nature, characteristics and uses of artificial chromosomes as recombinant DNA vectors

Answer 7

(yeast) artificial chromosomes = YACs

nature: large DNA molecules designed to introduce large DNA segments, restricted to yeast
- similar to plasmids

uses:
- cloning, maintaining large genomic DNA fragments

Question 8

what are virulence factors?

Answer 8

molecule/structure possessed by a pathogen - enhances its ability to infect and cause disease to a host organism

allow pathogen to evade host’s defence mechanisms, establish infection and encourage disease progression

examples:
- toxins = can damage host cells
- adhesions = surface molecules, allow pathogens to adhere to host cells
- capsules = protective outer layers, shield bacteria from host immune system
- enzymes = aid in penetrating/ interfering with host defence mechanisms
- alter surface antigens to evade being detected by host immune system = antigen variation

Question 9

describe characteristics of plasmids

Answer 9

small, double stranded circular molecule - structure allows for easy replication with a high copy number, useful for recombinant DNA and cloning

naturally occurring, replicate independently, are sources of antimicrobial resistance and virulence factors

have a restricted host range due to limited biological compatibility

Question 10

describe the features of plasmids that make them useful for manipulating fragments of DNA and expression of recombinant proteins

Answer 10

1. (modified) origin of replication = replication at high multiplicity, independently in host cells, allows for high level of gene expression
2. selectable marker- e.g. antibiotic resistance gene = can select a specific recombinant cell by its resistance
3. multiple cloning site (MCS) = specific DNA sequence with multiple unique recognition sites for restriction enzymes
   - allow insertion of different compatible DNA fragments and to linearize & isolate plasmid
4. can be linearized = made linear with specific ends for DNA cloning and sequencing
5. promoters and enhancers
   - regulatory elements that control inserted gene expression and transcription
6. transcriptional terminator - signals end of transcription, controls gene expression
7. reporter gene - e.g. GFP (green fluorescent protein)
   - allows visualisation and identifying cells expressing the recombinant protein

Question 11

the use of inducible promoters in plasmids

Answer 11

inducible promoter = regulatory elements, can control gene expression by inducing/repressing it in response to certain signals

• involves introduction of inducible promoter upstream of plasmid = has a specific DNA sequence that interacts with repressive/inducive regulatory proteins

use: allows precise control over timing of gene expression
- temporal control = inducing gene expression at specific time points
- quantitative control = controlling level of gene expression and amount of protein produced

Question 12

describe how bacterial plasmids are used in making recombinant proteins

Answer 12

GOI or cDNA copy isolated - amplified using PCR. primers in PCR are designed to introduce restriction enzyme recognition sites at the ends of the PCR amplicon

bacterial plasmid is selected - has an origin of replication, selectable marker, multiple cloning site with unique recognition sites, and bacterial promoter and terminator for transcription

PCR amplicon and plasmid vector are digested by restriction enzyme - produces compatible ends that are ligated together by DNA ligase (phosphodiesters bonds form between compatible ends)

recombinant plasmid vector has been produced - transformed bacteria cells are placed on selective medium with antibiotics. only those that have taken up the plasmid vector with the selectable marker/ antibiotic resistance gene will survive

bacterial cells with recombinant plasmid survive, replicate and inserted gene is transcribed and translated

transcribed sequence must have proper Shine-Dalgarno sequence, start and stop codons before it undergoes translation

recombinant protein produced by bacterial culture - can be purified and studied

Question 13

importance of Shine-Dalgarno sequence?

Answer 13

Shine-Dalgarno = ribosomal binding site 5-10bps upstream of start codon
- often precedes start codon in prokaryotes - ensures correct positioning of ribosome for mRNA translation

start codon = AUG, site for initiating translation

stop codon = UAA, UAG or UGA = termination of translation, release of polypeptide chain by ribosome. prokaryotic release factors recognise stop codon and facilitate polypeptide chain release

Question 14

why use plasmids as recombinant tools?

Answer 14

can modify control elements - include different promoters, enhancers, regulatory elements and modulate gene expression
- inducible promoters allow temporal and quantitative control of gene expression

can alter properties of gene product
- add signal sequences for extracellular secretion of protein = easier purification
- add peptide tags to protein of interest = easier detection

can express recombinant genes in a living organism of choice - prokaryotic or eukaryotic

Question 15

clinical use of recombinant proteins?

Answer 15

biopharmaceutical production = recombinant plasmid vectors important in biopharmaceutical production

• produce human insulin for diabetes
• interferons for viral hepatitis
• erythropoietin for kidney disease and anaemia
• treat bleeding by producing factor XII and tissue plasminogen factor
• recombinant antibodies

Question 16

what are recombinant antibodies?

Answer 16

human antibodies derived from non-human sources and modified to be compatible for the human immune system

Question 17

describe relevant differences between prokaryotic and eukaryotic gene expression requirements

Answer 17

cellular differences:
- prokaryotes lack compartmentalisation - no nucleus or membrane bound organelles, transcription and translation occur in cytoplasm
= eukaryotic compartmentalisation is important in gene regulation - nucleus and may membrane-bound organelles, transcription in nucleus and translation in cytoplasm

• proks. have a smaller ribosome (70s - 30s and 50s), polysomes are common with multiple ribosomes translating one mRNA
  = euks. have larger ribosomes (80s - 40s and 60s), polysomes are common but transcription & translation occur in different compartments

gene regulation/ structure differences:
- prokaryotic genes lack introns, are continuous coding sequences
= eukaryotic genes have introns and exons, allow for alternate splicing and mRNA isoforms from one gene

• multiple prok. genes regulated by an operon, affected by binding of a repressor/activator to the operator region
  = complex euk. gene regulation with regulatory elements and transcriptional machinery, TFs
• prok. limited post-transcriptional processing and modifications
  = euk. capping, splicing and polyadenylation post-transcriptional modifications

transcription differences:
- in proks, occurs in cytoplasm, mRNA doesn’t undergo processing and modifications
= in euks, occurs in the nucleus, mRNA undergoes capping, splicing and polyadenylation afterwards

translation differences:
- in proks, occurs in cytoplasm, requires the Shine-Dalgarno sequence, start and stop codon, promoter and translation termination site
- smaller 70s ribosome, direct mRNA binding
= in euks, occurs in cytoplasm at RER, 5’cap ribosome needs to scan the mRNA to find the start codon. more complex process.

Question 18

what elements are required for the transcription and translation of a recombinant gene in a plasmid vector?

Answer 18

coding sequence that codes for the recombinant gene

Shine-Dalgarno sequence = 5-10bps upstream the promoter, ensures the small ribosomal subunit binds correctly to the start codon

bacterial promoter = initiates transcription at 5’ end of transcriptional unit

translational terminator = allows polymerase to end transcription, release polypeptide chain

Question 19

difference between constitutive and inducible promoter? what are their effects on recombinant gene expression?

Answer 19

constitutive promoter = continuously active promoter regardless of environmental conditions and signals - constant expression of downstream gene
= maintain a baseline gene expression level under normal conditions, don’t respond to stimuli
= e.g. promoter of housekeeping gene
= using it to drive recombinant gene expression means it’ll be expresses continuously at a constant level - useful if you need stable expression of a gene

inducible promoter = activity regulated in response to specific signals/conditions - allows control of gene expression
= inactive/ minimal activity in the absence of an inducer
= activity repressed/ induced by specific signals
= e.g. lac operon promoter in E.coli is activated in lactose presence and glucose absence - E.coli often used as bacterial plasmid vector
= using it to drive recombinant gene expression means gene expression level can be manipulated by controlling the presence of an inducer - fine-tune timing and level of recombinant gene production

Question 20

the use of gene fusions to improve purity of recombinant proteins

Answer 20

gene fusion = technique used to improve the purification and detection of recombinant proteins
- GOI is fused with affinity/ reporter tag, creating a chimeric protein = enhances purification and detection of gene
- choice of tag depends on available purification techniques and the effect of tag fusion with the protein (could cause structural/ functional/ activity changes)

affinity tag - e.g. FLAG tag, maltose-binding protein tag (MBP)
= facilitates purification and affinity chromatography
= FLAG tags useful in Western blot detection - can verify protein expression in cells

reporter tag - e.g. GFP (green) or YFP (yellow)
= facilitates purification, visualisation and monitoring gene expression
= allows real-time monitoring of protein expression and localisation in living cells

gene fusion purification methods:
1. affinity chromatography - affinity tags allow purification using chromatography columns with specific ligands for the tag = ligands bind to tag
2. tag cleavage - site-specific proteases cleave fusion tag-protein, leaves just the target protein

use of gene fusion:
- high-throughput proteomics = studying (recombinant) proteins and their activities
- fusing a target protein with a tag can aid in structural studies