Lecture 4: Plasmids and Vectors

Question 1

What are plasmids?

Answer 1

Plasmids are double-stranded extra chromosomal DNA found naturally in bacteria. Genes carried on plasmids often confer advantages to the bacteria, such as antibiotic resistance,

Question 2

What are vectors?

Answer 2

Vectors are artificially made molecules that can facilitate the incorporation of foreign DNA and manipulation.

Question 3

What are clones?

Answer 3

Clones are populations of cells or organisms that are derived from a single progenitor and are, therefore, genetically identical.

Question 4

What is sub-cloning?

Answer 4

Involves the isolation of clones of bacteria expressing a particular DNA sequence of interest inserted, usually, into a plasmid.

Question 5

What are restriction endonucleases/ enzymes?

Answer 5

Proteins that cleave DNA molecules at specific sites, producing discrete fragments of DNA. “Molecular scissors” that can cut DNA. Can destroy the DNA without destroying the whole plasmid.

Question 6

What is needed for the restriction endonucleases used in plasmid engineering?

Answer 6

The same restriction endonuclease used to cleave a particular gene is needed to cut the plasmid for its insertion, in plasmid engineering processes.

Question 7

What is transformation?

Answer 7

Transformation is the process by which exogenous DNA is transferred into the host cell and replicated.

Question 8

What does exogenous mean?

Answer 8

Exogenous refers to any material that is outside the cell.

Question 9

What is biosynthesis?

Answer 9

Biosynthesis is the ability of an organism to produce a substance from substrate(s).

Question 10

What is biotransformation?

Answer 10

Biotransformation is the ability of an organism to convert one substance into another, usually through an enzymatic process.

Question 11

What are transgenic organisms?

Answer 11

Organisms that possess DNA from another organism, which it is able to transcribe into functional proteins.

Question 12

Where are cloning and vectors used?

Answer 12

1. Production of DNA vaccines
2. Gene Therapy
3. Production of pharmaceuticals like insulin

Question 13

What is super-coiled DNA?

Answer 13

DNA with a high number of helical turns. Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling.

Question 14

What is open circular DNA?

Answer 14

Double-stranded circular DNA molecule that has been nicked in one of the strands to allow the release of any superhelical turns present in the molecule.

Question 15

What is linear DNA?

Answer 15

Single linear strand of DNA with 2 ends. Linear chromosomes make it easier for transcription and replication of large genomes

Question 16

What type of DNA is best for use in PCR?

Answer 16

Linear DNA as it can be run on Agarose gel to help identify size and other characteristics of the DNA.

Question 17

Describe the first generation vector PSC101

Answer 17

One of the first vectors discovered. It contains only a single restriction site for E.coli and tetracycline is used for screening.

Question 18

Describe the first generation vector pBR322

Answer 18

Used to store genes, of which can be taken out and sub-cloned. Would need a promoter region in order to transcribe the genes in this plasmid.

Question 19

What are multiple cloning regions (MCRs)?

Answer 19

A region of DNA on the plasmid that has multiple singular restriction sites for many restriction endonucleases/enzymes.

Question 20

What are multiple cloning regions useful?

Answer 20

Multiple cloning regions reduce the number of cut sites on the plasmid and contains cutting to a certain region on the plasmid.

Question 21

What happens when a plasmid is cut in its multiple cloning region?

Answer 21

A single linear strand of DNA is formed due to the presence of only singular restriction sites in the region. If the DNA was cut outside the multiple cloning region, it is likely to form 2+ fragments s there is often more than 1 copy of a particular restriction site for a particular restriction enzyme.

Question 22

What are ‘sticky ends’?

Answer 22

Overhangs, following the cleavage of DNA by restriction endonucleases

Question 23

What are ‘sticky ends’ preferred over blunt ends?

Answer 23

Sticky ends tend to be easier to ligate back into new plasmids, using T4 DNA ligase, during plasmid engineering/ manipulation.

Question 24

What is star activity in restriction endonucleases?

Answer 24

Star activity in restriction enzymes limits the duration of their functionality.

Question 25

What is recombinant DNA?

Answer 25

DNA that has been formed artificially by combining constituents from different organisms.

Question 26

Why is star activity in restriction enzymes bad for investigations and plasmid engineering?

Answer 26

Endonuclease activity tends to be rogue after an hour, for those endonucleases with star activity, and they can target many different sites, and not just their specific restriction site.

Question 27

How does DNA ligase work?

Answer 27

Hydroxyl group added to 3’ end and a adenyl group is added to a 5’ group. Responsible for catalysing the formation of a phosphodiester bond between 3' hydroxyl ends of one nucleotide ("acceptor"), with the 5' phosphate end of another ("donor").

Question 28

What is used to re-join DNA fragments following cleavage?

Answer 28

T4 DNA ligase is used to re-join DNA fragments

Question 29

What is needed for DNA ligase to work?

Answer 29

DNA ligase requires the use of ATP in order to function; it is an active process so often not reversible unless the DNA is cut again.

Question 30

What is amplification?

Answer 30

Amplification in the process of inducing replication, by binary fission (VGT), of the recombinant DNA to generate multiple copies of the new DNA. Produces large amounts of plasmid DNA using using a plasmid with recombinant DNA introduced into a complement (bacterial) host cell.

Question 31

What features can be added to plasmids to aid in their identification?

Answer 31

1. Antibiotic resistance 2. Colorimetric ‘markers’ 3. Strong or weak promoters for driving expression of a protein.

Question 32

What is gene splicing?

Answer 32

Gene splicing is a post-transcriptional modification in which a single gene can code for multiple proteins. Gene Splicing is done in eukaryotes, prior to mRNA translation, by the differential inclusion or exclusion of regions of pre-mRNA.

Question 33

Why is gene-splicing important?

Answer 33

Important source of protein diversity.

Question 34

How can antibiotic resistance be used to aid identification?

Answer 34

Antibiotic resistance can help to identify the successful insertion of a gene into a plasmid. When plated on a medium containing a particular antibiotic, a zone of clearance will be visible for all colonies of bacteria that contain the plasmid with the antibiotic resistance genes.

Question 35

How can colorimetric markers be used to aid identification?

Answer 35

Insertion of a gene into a lacZ gene within a multiple cloning region (MCR) aids in colorimetric identification of successful insertion of a gene into a plasmid. Insertion of gene into a lacZ gene within a MCR causes a change in colour which is visible in the colonies; change from blue to white, no successful insertion or insertion outside the lacZ results in blue colonies.

Question 36

What cells are capable of taking up plasmid DNA?

Answer 36

Only competent host cells are capable of taking up plasmid DNA

Question 37

How can competent cells be formed?

Answer 37

Bacterial cells can be treated to increase their competency and increase the likelihood of DNA being taken up through treating them with an electrical current, heat shock, or divalent cations.

Question 38

What 2 methods are used to transform bacterial host cells?

Answer 38

1. Heat Shock | 2. Electroporation

Question 39

Describe the heat shock process

Answer 39

1. Chemically competent bacteria and DNA are combined in a medium 2. Short incubation on ice 3. Heat shock at 42 degrees Celsius for 45 seconds 4. Back to ice 5. Growth media is added 6. Transformed cells are incubated at 37 degrees Celsius for 30 mins with agitation

Question 40

What is electroporation?

Answer 40

Electroporation is the application of an electrical current across a bacterial cell that creates temporary ‘pores’ in the cell membrane, increasing the permeability of cell membranes to the DNA, and forces negatively charged DNA into the cells.

Question 41

What conditions are electroporation carried out under?

Answer 41

Electroporation is carried out at 0 degrees Celsius to minimise heat damage to the cells, and in the presence of calcium chloride. DNA is forced into the cells by applying a short 42 degrees Celsius heat shock; the thermal current sweeps DNA into the cells.

Question 42

What is a restriction digest?

Answer 42

A procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation. Uses restriction endonucleases/enzymes that are able to cleave DNA molecules at the positions at which particular short sequences of bases are present.

Question 43

What environmental factors need to be considered when selecting a restriction enzyme?

Answer 43

1. Temperature 2. Salt Concentration 3. Purity of the DNA

Question 44

How would you determine the orientation of DNA fragments?

Answer 44

The usual PCR technique for fragment orientation involves one primer specific of the vector (upstream or downstream of the cloning site) and one primer within the cloning sequence. If the primer set is correctly oriented, PCR will give a unique band with a specific size according to the distance between primers. If not properly oriented, there will be no amplification.

Question 45

What reasons may lead to the lack of a functional protein being produced from a gene inserted into a plasmid?

Answer 45

1. Gene may not be intact. 2. Mutations introduced may have disrupted gene. 3. Protein encoded by gene may require post-transcriptional modifications in order to be expressed. 4. Some enzymes are also a complex of different peptides expressed from separate genes, so other genes may be necessary for its expression.

Question 46

Describe the process of plasmid engineering for the production of human insulin

Answer 46

1. Isolate target gene from host organism. 2. Prepare target DNA using restriction endonucleases 3. Insert DNA into the plasmid via ligation; human insulin gene is now recombined with the bacterial DNA plasmid. 4. Insert plasmid back into host cell, where human insulin gene can be transcribed. 5. Induce plasmid multiplication within the host cell 6. Target cells reproduce and generate the insulin protein 7. Human insulin protein molecules are then concentrated and purified for later use in diabetes treatment.