Topic8 In Vivo Cloning Flashcards

1
Q

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

A

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)

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2
Q

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

A

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.

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3
Q

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

A

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

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4
Q

What is the purpose of Promotor Region and Terminator Region?

A

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.

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5
Q

After Modification what occurs?

A

Inset DNA into vector

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6
Q

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

A

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

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7
Q

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

A

Enzyme Lipase sticks DNA fragment and cut plasmid

Lipase catalyse the condensation reaction to form Phosphodiester bonds between nucleotides

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8
Q

How does Transform a host cell with the vector occur?

A

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

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9
Q

Why does identifying transformed cells happen?

A

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.

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10
Q

What are the three different marker genes?

What is the purpose of Marker genes?

A

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

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11
Q

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

A

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

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12
Q

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

A

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

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13
Q

If Bactria Survive Ampicillin Antibiotic agar what does it mean?

A

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

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14
Q

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

A

The Recombinant Plasmids is the DNA fragment inserted into plasmid.

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15
Q

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

A

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

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16
Q

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

A

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

17
Q

What is the last stage in identification of Fluorescent Markers?

A

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

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

18
Q

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

A

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

19
Q

What enzyme is used in Enzyme Markers?

A

Enzyme Lactase.

20
Q

What occurs in Enzyme Markers?

A

Enzyme Lactase turn a certain substance colourless to blue

21
Q

What fully occurs in Enzyme Markers?

A

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.

22
Q

How does Grow Host cell occur?

A

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.