Plasmids

Question 1

what is genetic engineering?

Answer 1

the process of manually transferring genetic information from one organism to another

Question 2

what is the difference between the structure of an animal cell and bacterial cell?

Answer 2

bacterial cells do not contain a nucleus, their DNA is found in a ring called a plasmid

Question 3

what is a plasmid?

Answer 3

circular piece of DNA that replicate independently from the host chromosomal DNA

Question 4

what are plasmids created in a lab called?

Answer 4

constructs or vectors

Question 5

list the parts of a plasmid

Answer 5

• origin of replication
• antibiotic resistance gene
• selectable marker
• promoter
• restriction sites
• inserted gene

Question 6

what is the role of a restriction site?

Answer 6

recognise the insertion site and allow for genes to be cut an new genes to be inserted

Question 7

what is the role of a promoter?

Answer 7

allows for gene transcription

Question 8

Origin of Replication

Answer 8

• DNA sequence which directs initiation of plasmid replication (by bacteria) by recruiting DNA replication machinery.
• The ori is critical for the ability of the plasmid to be copied (amplifies) by bacteria, which is an important characteristic of why plasmids are convenient and easy to use.

Question 9

Insert

Answer 9

• The insert is the gene, promoter, or other DNA fragment cloned into the MCS.
• The insert is typically the genetic element one wishes to study using a particular plasmid

Question 10

Promoter

Answer 10

• Drives transcription of the insert
• the promoter is designed to recruit transcriptional machinery from a particular organism or group of organisms
• meaning, if a plasmid is intended for use in human cells, the promoter can also direct cell-specific expression, which can be achieved by a tissue-specific (e.g. a liver-specific promoter)
• the strength of the promoter is also important for controlling the level of insertion expression
• i.e., a strong promoter directs high expression, whereas weaker promoters can direct low/endogenous expression levels

Question 11

Selectable Marker

Answer 11

• The selectable marker is used to select for cells that have successfully taken up the plasmid for the purpose of expressing the insert
• This is different than selecting for bacterial cells that have taken up the plasmid for the purpose of replication
• The selectable marker enables selection of a population of cells that have taken up the plasmid and that can be used to study the insert.
• The selectable marker is typically in the form of another antibiotic resistance gene (this time under the control of a non-bacterial promoter) or a florescent protein (that can be used to select or sort the cells by visualization or FACS)

Question 12

What are the Stages of Genetic Modification?

Answer 12

1) The required gene is identified and extracted from the chromosome using enzymes
2) The plasmid is extracted from the bacterial cell
3) An enzyme is used to cut open the plasmid
4) The required gene is inserted into the plasmid and sealed by an enzyme
5) The modified plasmid is inserted into the bacterial cell, producing a genetically modified organism. The bacteria reproduce rapidly, producing new bacteria with the required with the required characteristic