Plasmids and Conjugation Notes
Plasmids
Plasmids are modified from naturally occurring plasmids to allow scientists to insert genes of interest for study.
The first use of plasmid technology was in 1973, where a frog gene was inserted into a plasmid.
Amplifying Genes of Interest
Plasmids allows for artificial amplification of genes to study their phenotypic effects on organisms.
Cloning a gene (not a protein) onto a plasmid allows for easier manipulation of the gene sequence to study its function.
Protein Purification
Plasmids can include a DNA sequence encoding of six histidines (His-tag).
The gene of interest is cloned in frame with the His-tag, the protein expressed from this gene will include His-Tag.
His-tag binds to nickel, facilitating protein purification using a nickel column.
After washing the column the protein of interest can be eluted.
Purified proteins facilitates various biochemical assays (e.g., Km, kcat values).
Cloning and Restriction Enzymes
Cloning involves restriction enzymes that generate sticky ends or blunt ends.
Sticky ends are produced by staggered cuts, they have overhangs.
Blunt ends are produced by straight cuts, they have no overhangs.
Ligation efficiency: sticky ends are more efficient than blunt ends because of their tendency to base pair and come together.
Ligase Function
Ligase joins a 5' phosphate to a 3' hydroxyl group, forming a phosphodiester bond. Ligase does not add nucleotides.
Directional Cloning
Directional Cloning: Uses two different restriction enzyme sites, ensuring the insert goes in the desired orientation.
Non-Directional Cloning: Uses the same restriction enzyme on both ends of the insert, allowing it to insert in either orientation.
In non-directional cloning, the insert has an equal chance of inserting in either direction, but in practice, one orientation may be preferred.
Restriction Enzyme Considerations
When using restriction enzymes, it's important to have approximately six nucleotides on either end of the cut site for efficient cutting.
Some restriction enzymes, like NDE one, have larger recognition sites and require more space.
NEB.com provides information on restriction sites and optimal conditions.
Cloning Workflow
After restriction digest of a PCR product and plasmid, purification is performed to remove small DNA fragments and the enzyme.
Always assume that not 100% of the molecules are cut, even if the gel suggests otherwise but always include plasmid backbone vector.
Select for successfully transformed plasmids using antibiotic resistance (e.g., ampicillin).
Antibiotic Resistance
The BLA gene encodes beta-lactamase, which confers ampicillin resistance.
Ampicillin blocks peptidoglycan synthesis, leading to cell lysis.
Transformation
Transformation involves introducing the ligation mix into E. coli cells so these cells can uptake the plasmid.
E. coli is made artificially competent to take up DNA, often through electroporation.
The plasmid must be circular to be maintained in E. coli; linear DNA will be degraded.
Screening Colonies
Colonies are screened to confirm the presence of the insert.
Phosphatase Treatment
To prevent self-ligation of the cut plasmid, treat with a phosphatase to remove phosphates.
Gibson Assembly
Gibson assembly is a restriction enzyme-free cloning method using PCR, exonucleases, and ligase.
Gibson Assembly Steps:
Drawing what you want in the end.
Primers are designed with overhangs that match the plasmid sequence.
The PCR product and linearized plasmid have complementary overhangs.
An exonuclease chews back the 5' ends of the DNA fragments, creating single-stranded overhangs.
The complementary overhangs anneal, and a polymerase extends the DNA.
Ligase seals the nicks, creating a complete plasmid.
Primer Consideration:
Overhangs should be about 18 nucleotides long.
Advantages of Gibson Assembly:
No need for restriction enzymes.
Higher efficiency compared to restriction enzyme cloning.
Allows for the assembly of multiple DNA fragments.
Disadvantages of Gibson Assembly:
Requires longer primers, increasing primer costs.
Gibson Assembly Master Mix is expensive.
Transduction and Conjugation
Transduction is the transfer of genes by a phage.
Conjugation is plasmid-mediated direct cytoplasmic transfer.
Regulation of Downstream Sequences
A riboswitch is a region of RNA that binds a ligand, affecting transcription or translation.
Transformation
Transformation involves making cells competent to take up external DNA.
Mating Pair Formation and DNA Transfer
MPF (mating pair formation) genes encode proteins involved in forming the conjugation complex and pilus.
DTR (DNA transfer) genes encode proteins involved in preparing and transferring the DNA from the donor to the recipient.
Coupling proteins facilitate communication between the MPF and DTR, activating relaxase to nick the DNA.
Coupling proteins recognize relaxases and other proteins at the channel, acting as gatekeepers.
They interact with ATPases to generate energy for DNA transfer.
DTR and Relaxase
Relaxase makes a single-stranded break at the NIC site within the origin of transfer (ORI-T).
Relaxase forms a covalent bond with the DNA, transferring the 5' phosphate to a tyrosine residue.
The reaction does not require any energy, it is a transesterification activity.
A plasmid-encoded helicase unwinds the DNA to facilitate transfer but the helicase can transfer to the recipient as well.
After transfer, both the donor and recipient cells need to replicate the complementary strands.
Primases
Conjugal plasmids often encode their own primase to synthesize the primer for replication in the recipient cell.
Coupling Proteins Role
All proteins crossing into the pilus must interact with the coupling protein.
Relaxase must be transported in every system studied to date.
Regulation of Conjugation
Transfer is heavily regulated because mating pili can be co-opted by phages as receptors.
Pili are often only made right after entering a new cell and then quickly turned off.
F Plasmids
F Plasmids are an example of regulated conjugation. They are often studied to teach how conjugation works.
DNA in Recipient Cells
The transferred DNA is single-stranded but once transferred will replicate to become double stranded again.
FinOP System
FinO is a protein that stabilizes FinP, an antisense RNA.
FinP controls the translation of TraJ, a transcriptional activator for conjugation.
FinP base pairs to the TraJ mRNA, preventing its translation.
The system relies on the single-stranded nature of the transferred DNA.
Mobilizable Plasmids
Mobilizable plasmids have an origin of transfer (ORI-T) and are dependent on a self-transmissible plasmid for transfer.
Many encode their own genes for DNA preparation (DTR functions), including relaxase and helicase.
They do not encode their own coupling protein which is part of the MPF.
Mobilizable plasmids encode a relaxase that must be recognized by the coupling protein of the helper plasmid.
HFR Strains
HFR (high-frequency recombination) strains have a plasmid integrated into their chromosome.
HFR bacteria can transfer part of their chromosome during conjugation.
Plasmids often integrate into IS (insertion sequence) elements, which are sequence repeats in the chromosome.
The integrated plasmid can excise itself and reform as a closed circular plasmid, potentially bringing chromosomal DNA with it
Plasmids bringing chromosomal DNA are called prime factors and have are desiginated as F'.
Mapping E. Coli Chromosome
HFR strains can be used to create a circular chromosome map by disrupting conjugation at different time points which maps to the E.Coli clock system.
Gram-Positive Conjugation
Gram-positive bacteria also undergo conjugation with self transmissible plasmids.
The recipient cell releases a pheromone signal to attract donor cells.
Donor cells recognize the pheromone and activate the transfer process.
Once a plasmid is acquired, the recipient cell synthesizes an inhibitory protein to bind up the pheromones. This inhibits the synthesis of more pheromenones.
ICE Elements
ICE (integrating conjugative elements) integrate into the chromosome, have tra functions, and can transfer themselves.
ICE elements excise from the chromosome, transfer as single-stranded DNA to the recipient, and then integrate into the recipient's chromosome.
ICE elements often encode antibiotic resistance genes, such as tetracycline or vancomycin resistance.
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