Topic8 In Vivo Cloning

Question 1

What are the whole steps in DNA technology In Vivo Cloning?

Answer 1

1.Create DNA fragments for gene of interest.

2.Insert DNA fragments into a vector

3.Transform a host cell with the vector

4.Identity Transformed Cells

5.Grow then host cells (clones/make copies of the gene)

Question 2

What needs to happens in the first step of In Vivo Cloning?

Answer 2

Restriction EndoNuclease Enzymes cut gene of interest at recognition sites leaving sticky ends.

DNA fragments must be modified to ensure transcription of the genes can occur.

Question 3

What are the modification that occurs in the first step of In Vivo Cloning?

Answer 3

First Modification is a promoter region.
Promoter region added at start of DNA fragment.

Second Modification is a Terminator Region.
Terminator Region is added at the end of gene

Question 4

What is the purpose of Promotor Region and Terminator Region?

Answer 4

Promoter region- sequence of DNA which is binding site for RNA Polymerase to Enable transcription to occur.

Terminator Region- Causes RNA polymerase to detach and stop transcription so only one gene at a time is copied into mRNA so translated into one protein.

Question 5

After Modification what occurs?

Answer 5

Inset DNA into vector

Question 6

What occur in the Insert DNA into a Vector?
What is the first thing that occurs?

Answer 6

1.Plasmid curt open using same Restriction Endonuclease to create Same Sticky Ends
So DNA fragments sticky ends are complementary to sticky ends on the plasmid

Question 7

What is the last step in Insert DNA into a Vector?

Answer 7

Enzyme Lipase sticks DNA fragment and cut plasmid

Lipase catalyse the condensation reaction to form Phosphodiester bonds between nucleotides

Question 8

How does Transform a host cell with the vector occur?

Answer 8

Cell membrane of host cell must be more permeable

To increase permeability host cells are mixed with Ca2+ and heat shocked

Heat shocked is a sudden increase in temperature which enables vector to enter host’s cell cytoplasm

Question 9

Why does identifying transformed cells happen?

Answer 9

3 issues can occur:

1.Recombinant plasmid doesn’t Get inside the cell.

2.Plasmid re-joins before DNA fragments entered.

3.DNA fragments sticks to itself rather then inserting into plasmid.

Question 10

What are the three different marker genes?

What is the purpose of Marker genes?

Answer 10

The purpose of marker genes it to identify which bacteria successfully took up recombinant plasmid.

The three different marker genes used are:
1.Antibiotic Resistance genes

2.Genes coding for Fluorescent proteins

3.Genes coding for Enzymes

Question 11

What the first thing that occurs in Antibiotic-Resistance Marker genes?

Answer 11

Antibiotic Tetracycline and Antibiotic Ampicillin inserted Bacterial Plasmids

DNA fragments that we’re isolated that we want to clone inserted in the middle of Tetracycline Gene

So it gets disrupted so no longer be able to create protein that make bacteria resistant to Tetracycline

Question 12

What is the final step in identify Antibiotic-Resistance Marker Genes?

Answer 12

Grow the bacteria which Antibiotics on agar:

Transfer Bacterial colonies to plate with Ampicillin Antibiotics in agar

Transfer Bacterial colonies to plate with TetraCycline Antibiotic in the agar

Question 13

If Bactria Survive Ampicillin Antibiotic agar what does it mean?

Answer 13

Those that survive must have plasmids inside of it because it has gene resistant to Ampicillin

Question 14

What does it mean of Bacteria doesn’t grow in TetraCycline?

Answer 14

The Recombinant Plasmids is the DNA fragment inserted into plasmid.

Question 15

In Antibiotic-Resistance Marker genes what does it mean if bacterial colonies grew on Ampicillin and TetraCyline?

Answer 15

Must be original plasmid which doesn’t contain gene of interest.

Question 16

What is Fluorescent Marker comes from and what is the thing that occurs for identification?

Answer 16

Jellyfish contain gene which codes to create Green Fluorescent Protein (GFP)

GFP gene inserted into the Bacterial Plasmid

Insert DNA fragment into Bacterial Plasmid deliberately in the middle of GFP gene so any bacteria take up recombinant DNA plasmid not able to produce GFP protein because is disrupted

Question 17

What is the last stage in identification of Fluorescent Markers?

Answer 17

Grow Bacterial Plasmid with GFP gene (Bacteria) on Agar.

Expose colonies to U.V light Those that still have GFP gene intact glow.

Question 18

In Fluorescent Markers those that don’t glow what does it mean?

Answer 18

Those that are not glowing Contain the Recombinant plasmid because Gene have been disrupted so no longer produce green

Question 19

What enzyme is used in Enzyme Markers?

Answer 19

Enzyme Lactase.

Question 20

What occurs in Enzyme Markers?

Answer 20

Enzyme Lactase turn a certain substance colourless to blue

Question 21

What fully occurs in Enzyme Markers?

Answer 21

The gene for this Enzyme Lactase inserted into the plasmid (Bacterial Plasmid)

Put DNA fragment in the middle of the Bacterial Plasmid with Lactase gene to disrupt it

Grow bacteria on colourless substance
Colonies which cannot turn the colourless substance blue contain recombinant plasmid.

Question 22

How does Grow Host cell occur?

Answer 22

Fermenter used to grow multiple copies of the host cell which have been identified as containing+ recombinant plasmid

Large cloned population of host cell can produce protein.