Lecture 3-4 - Plasmid

Question 1

Plasmid

Answer 1

A plasmid is a small circular DNA molecule found in bacteria, archaea and yeast.

Plasmids are physically separate from chromosomal DNA and replicate independently.

They typically have a small number of genes — notably, some associated with antibiotic resistance — and can be passed from one cell to another.

Question 2

Similarities between plasmid and genomic DNA

Answer 2

• Types of DNA present in living organisms.

*Contain genes.

• Present in bacteria and archaea.
• The building blocks of both are deoxyribonucleotides.
• Double-stranded.

Question 3

Origin of replication

Answer 3

DNA sequence which allows initiation of replication within a plasmid by recruiting transcriptional machinery proteins.

Question 4

Antibiotic Resistance Gene

Answer 4

Allows for selection of plasmid-containing bacteria.

Question 5

Multiple Cloning Site (MCS)

Answer 5

Short segment of DNA which contains several restriction sites allowing for the easy insertion of DNA. In expression plasmids, the MCS is often downstream from a promoter.

Question 6

Insert

Answer 6

Gene, promoter, or other DNA fragment cloned into the MCS for further study.

Question 7

Promoter Region

Answer 7

Drives transcription of the target gene. Vital component for expression vectors: determines which cell types the gene is expressed in and amount of recombinant protein obtained.

Question 8

Selectable Marker

Answer 8

The antibiotic resistance gene allows for selection in bacteria. However, many plasmids also have selectable markers for use in other cell types.

Question 9

Primer Binding Site

Answer 9

A short single-stranded DNA sequence used as an initiation point for PCR amplification or sequencing. Primers can be exploited for sequence verification of plasmids.

Question 10

Promoters for gene expression

Answer 10

Promoters for gene expression are sequence of DNA to which proteins bind to initiate transcription of a single RNA from the DNA downstream of it.

They are located near the transcription start sites of genes, upstream on the DNA (towards the 5’ region of the sense strand).

Question 11

Ideal features for a promoter

Answer 11

• It is strong enough to allow product accumulation up to 50% of the total cellular protein.

*It exhibits a minimal level of basal transcription (a highly repressible promoter is especially important, when the protein of interest is toxic or detrimental to the growth of the host cell).

*It is easy to induce

Question 12

Operators

Answer 12

Additional sequences controlling the strength of a promoter

Question 13

What affects strength of gene expression

Answer 13

Promoter type,
Strength of a promoter depends on the -10, -35 and ~17 bp nucleotide sequence between the consensus sequences

Question 14

Activators and repressors

Answer 14

A repressor is a protein that binds to a promoter or regulatory region to block gene transcription

An activator is a protein that binds to a promoter to enhance or initiate gene transcription.

Question 15

Types of promoters

Answer 15

Promoters can be constitutive (regulated instead by RNA polymerase levels or sigma factors),

Positively regulated (increased promoter activity based on some transcription factor, also known as an activator, increases in cellular concentration)

Negatively regulated (decreased activity as a repressor protein is present)

Question 16

Dynamically regulated promoters

Answer 16

These promoters respond to changing conditions in the cell and are likely regulated by multiple factors, including both positive and negative regulatory mechanisms.

Question 17

Repressed promoters

Answer 17

Repressed promoters are inhibited by a repressor protein, preventing transcription. When the repressor is removed or inactivated, transcription is allowed.

Question 18

Multiple regulated promoters

Answer 18

These promoters are regulated by multiple factors, both activators and repressors, which allow for fine-tuned control of gene expression.

Question 19

Inducible promoter

Answer 19

positively regulated, requiring an activator or removal of a repressor to turn on gene expression.

Question 20

Transcription terminator and roles

Answer 20

The transcription terminator is placed at the 3’ end of the target gene.

It enhances plasmid stability by preventing transcription through the replication region and other promoters on the plasmid.

Transcription from strong promoters into the replication region can destabilize plasmids by causing overproduction of ROP protein, which controls plasmid copy number.

Terminators prevent the formation of antisense RNAs from downstream promoters operating in reverse orientation relative to the target gene.

The transcription terminator also increases mRNA stability through the formation of a stem-loop structure at the 3’ end.