plasmids 1

Question 1

What is Recombinant DNA Technology?

Answer 1

Genetic engineering in vitro techniques for isolation, manipulation, recombination & expression of DNA (& proteins)

Dependent on and developed from naturally-occurring mobile genetic elements and activities of naturally-occurring enzymes.

Question 2

What are the main types of cloning vectors?

Answer 2

• Plasmids
• Bacteriophages
• Viruses
• Hybrids (cosmids & phagemids)
• BAC/YAC

Cloning vectors vary in suitability for different purposes.

Question 3

What are key features of cloning vectors?

Answer 3

• Modifiable
• Selectable (e.g. antibiotic resistance)
• Transferable
• Detectable

Derived from natural mobile genetic elements.

Question 4

What is a plasmid?

Answer 4

DNA molecules that you can insert other DNA sequences into & that replicates in one or more host organisms

Naturally found in bacteria & some eukaryotes.

Question 5

How many plasmid sequences are available in GenBank?

Answer 5

49499 plasmid sequences

Example: Natural bacterial plasmid pNY2385-KPC from Citrobacter freundii strain NY2385.

Question 6

What is the role of the ‘origin of replication’ in plasmids?

Answer 6

Allows plasmids to replicate autonomously in bacterial cells independent of the chromosome

It ensures synchrony with chromosome replication & cell division.

Question 7

What are the requirements for plasmid maintenance within cells?

Answer 7

• Incompatibility type
• Specific stability & partition proteins
• Copy number
• Selection based on a phenotype conferred by genes (e.g. antibiotic resistance)

Maintenance depends on combination of these factors.

Question 8

What is the significance of plasmid incompatibility groups?

Answer 8

Determined by ‘origin of replication’ sequence; distinct plasmids with the same ori type cannot coexist in a single cell

This has implications for the use of plasmids in recombinant DNA technology.

Question 9

What is Conjugation in the context of plasmid transfer?

Answer 9

A method where some plasmids are ‘conjugative’ and can transfer DNA between donor and recipient cells

Significant means of horizontal gene transfer.

Question 10

What is required for Transformation to occur?

Answer 10

Bacteria must be in a ‘competent’ physiological state to take up ‘naked’ DNA

Some bacteria are naturally competent (e.g. Bacillus subtilis).

Question 11

Which bacterium is primarily used as a cloning host?

Answer 11

Escherichia coli (E. coli)

E. coli is a model organism with known culture and molecular biology characteristics.

Question 12

What are the characteristics of E. coli cloning host strains?

Answer 12

• Antibiotic sensitive (or have known resistances)
• Auxotrophy (specific gene mutations affecting metabolic capabilities)

These strains require specific growth additives and cannot grow outside laboratory conditions.

Question 13

What is the importance of ‘selectable markers’ in plasmids?

Answer 13

Allows for the selection of plasmids in host cells

Essential for identifying plasmids with inserted DNA and verifying the insert.

Question 14

What is the function of the ‘par’ locus in plasmids?

Answer 14

Ensures each daughter cell receives one plasmid during cell division

Not all plasmids have such sequences for segregation.

Question 15

What does the term ‘competent’ refer to in bacteria?

Answer 15

A physiological state that allows bacteria to uptake DNA

This state involves specialized mechanisms for DNA uptake.

Question 16

What is the role of the F-factor in plasmids?

Answer 16

A conjugative plasmid that carries tra (transfer) genes

It enables horizontal gene transfer and can transfer DNA other than solely the plasmid.

Question 17

What type of plasmid is pTiAch5?

Answer 17

Crown gall tumour plasmid from Agrobacterium tumefaciens

Size: 213 kbp.

Question 18

What is the significance of ‘oriT’ in plasmids?

Answer 18

An origin of transfer that allows plasmids to be mobilized

These plasmids carry functions needed for transfer.

Question 19

What is the difference between ‘compatible’ and ‘incompatible’ plasmids?

Answer 19

Compatible plasmids can coexist in a single cell, while incompatible plasmids cannot

This is based on their origin of replication type.

Question 20

Define ‘transduction’ in genetic engineering.

Answer 20

A method of introducing DNA into host cells using phages & viruses

It is one of the main methods of genetic transfer.

Question 21

What is a common characteristic of E. coli strains used for cloning?

Answer 21

They can be antibiotic sensitive or have known resistances

This characteristic is crucial for selecting transformed cells.

Question 22

Define auxotrophy in the context of E. coli strains.

Answer 22

Auxotrophy refers to specific gene mutations or alleles that affect metabolic capabilities, requiring specific growth additives

These strains cannot grow outside laboratory conditions.

Question 23

What are some specific chromosomal mutations or alleles in E. coli for?

Answer 23

They allow detection of plasmids with inserts and prevent incoming plasmid DNA from being destroyed

This is achieved through restriction deficient/modification proficient mutations.

Question 24

List three examples of commonly used E. coli cloning host strains.

Answer 24

• BL21
• DH5α
• JM109

Other strains include BW25113, DH10B, HB101, MG1655, TOP10, and XL1-Blue.

Question 25

What is the approximate number of genes in a typical E. coli genome?

Answer 25

Approximately 4500 genes ## Footnote Typical cloning strains have around 4200 genes.

Question 26

What does the term 'wild-type' refer to in the context of E. coli genotypes?

Answer 26

'Wild-type' refers to unaltered genes that are not listed in a stated genotype ## Footnote Only mutations or relevant alleles are shown.

Question 27

What is the genotype of E. coli XL-1 Blue?

Answer 27

endA1, gyrA96(nalR), thi-1, recA1, relA1, lac, glnV44, e14-Δ(mcrCB-hsdSMR-mrr)171, recB, recJ, sbcC, umuC::Tn5, uvrC, F'[::Tn10 proAB+ lacIq Δ(lacZ)M15 Amy CmR] ## Footnote This genotype provides insights into its genetic manipulation capabilities.

Question 28

What are the two main laboratory methods to make E. coli competent?

Answer 28

* Calcium chloride treatment * Electroporation ## Footnote Calcium chloride involves chilling cells and heat shocking, while electroporation uses an electrical charge to disrupt the cell wall.

Question 29

Fill in the blank: The introduction of exogenous DNA into a recipient cell is known as _______.

Answer 29

Transformation ## Footnote This process is essential for genetic engineering.

Question 30

What does a selectable marker do in transformation?

Answer 30

Enables selective isolation of cells containing the plasmid ## Footnote Selection is typically done by plating onto medium containing antibiotics.

Question 31

True or False: In a typical transformation, most cells are transformed.

Answer 31

False ## Footnote Most cells are not transformed, which is why selectable markers are necessary.

Question 32

What are some examples of plasmids used in E. coli transformation?

Answer 32

* pBR322 * pUC8 * pBluescript ## Footnote These plasmids often carry antibiotic resistance genes for selection.