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Cell Bio Lab Test#2 > Lab4: Transformation > Flashcards

Lab4: Transformation Flashcards

1
Q

What is the process of transformation?

A

A bacterium is taking up the foreign DNA and incorporating it into its genome

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2
Q

What is a positive factor of transformation?

A

Increase the survival of the bacteria by acquiring new genetic material

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3
Q

What is a competent bacteria?

A

Bacteria with weakened cell walls capable to take up foreign DNA

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4
Q

What are 4 manners that a bacteria can become competent?

A

-Electro corporation (administering an electric shock to weaken the cell wall)
-Stressed by a low nutrients level
-Temperature shocking
-Presence of calcium chloride

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5
Q

Of what is compose a bacterial chromosome?

A

Single circular chromosome

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6
Q

What is a plasmid?

A

Small, circular pieces of DNA that exist outside the bacterial chromosome

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7
Q

What are to important functions of plasmid?

A

-Very stable
-Replicate independently

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8
Q

What is a vector in biology?

A

A vehicle used to carry foreign DNA into a cell

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9
Q

What is a pG plasmid?

A

Artificial plasmid to insert GFP gene into E.coli

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10
Q

What are the two genes of interest in the experiment?

A

-GFP gene
-AmpR gene

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11
Q

What is the selective marker in the experiment?

A

The AmpR gene

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12
Q

What is the function of the AmpR gene?

A

Confers ampicillin resistance to the bacterium, allowing it to grow on ampicillin-containing medium

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13
Q

What are the origin of the GFP gene? And what is its function?

A

Comes from a jellyfish and make bacteria glow green under UV light

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14
Q

What is the role of AmpR gene as a selective marker?

A

Allows only transformed bacteria (with the plasmid) to survive in the presence of ampicillin

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15
Q

What does PBP stands for?

A

Penicillin-binding protein

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16
Q

What is the role of PBP?

A

Helps the bacteria build their cell walls

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17
Q

What is the key role of Ampicillin?

A

Inhibits PBP, preventing cell wall synthesis, thus bacterial growth

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18
Q

What produces Beta-lactamase?

A

Becterai with AmpR gene

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19
Q

What is the function of Beta-lactamase?

A

Breaks down ampicillin, allowing the bacteria to survive and grow in its presence

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20
Q

Draw a scheme of GFP gene expression

A

GFP gene —- controlled by T7 promoter —- recognized by T7 RNA polymerase

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21
Q

What is the function of T7 RNA polymerase?

A

Binds strongly to the T7 promoter than transcribing viral genes

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22
Q

In engineered bacterium the T7 RNA polymerase is linked to what?

A

The lac Operon

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23
Q

What normally controls the lac operon?

A

Lactose

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24
Q

What is IPTG?

A

-An synthetic substitue for allolactose
-An inducer

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25
What is the function of IPTG?
-Can bind to the lac repressor -triggers gene expression
26
Why can IPTG trigger gene expression?
Because IPTG is not metabolized by the bacteria
27
What is a recombinant plasmid?
Plasmid that successfully incorporated the foreign gene (*In this experiment — GFP gene)
28
What is a non-recombinant plasmid?
A plasmid that has released without incorporating the foreign gene
29
True or false: During transformation, there is a mix of recombinant and non-recombinant plasmids is produced?
True
30
What is the function of the restriction site?
Cut the plasmid and gene of interest
31
Where do the plasmids can make many copies of themselves?
At the origin of replication
32
What is the reporter gene of the experiment?
GFP gene
33
What does the GFP gene can indicate?
Whether foreign gene is being expressed in the bacterium— bacterium will glow green when GFP gene is expressed
34
What is a avantage of using GFP gene?
Easy identification
35
Where is located the T7 RNA pol gene?
On bacterial chromosome
36
What has been attached to the lac promoter?
T7 RNA pol gene
37
What events does the presence of lactose triggers? (3 steps)
- Bacterium make T7 RNA pol — bind to the T7 RNA pol promoter on the plasmid — GFP is expressed in large quantities
38
What is the normal inducer for the lac operon?
Allolactose
39
Name the 3 steps of lac operon
-inducer binds to the repressor -falls of the operator -RNA pol transcribe the gene
40
What is the challenge with eukaryotic gene?
Bacteria cannot splice eukaryotic gene to remove introns
41
What is the solution to the challenge of eukaryotic gene?
Convert mature mRNA to cDNA — using reverse transcriptase
42
What is the specification of complementary DNA?
Lacks introns and can be inserted into bacterial plasmid for expression
43
What is the specification of retroviruses?
They have a -RNA (instead of a +RNA)
44
How retroviruses infect a cell?
Viral RNA is reverse-transcribed into cDNA then integrates the host genome
45
What is a colony?
It’s derived from a single bacterium
46
Is the genetical material of 1 colony is identical?
Yes
47
In presents of a recombinant gene, growing a colony represents what?
“Cloning” a gene
48
What is the objective of the lab?
To insert jellyfish into bacterium to make it glow green when exposed to UV light, in the presence of lactose
49
What is the dependent variable of the GFP lab?
Numbers of colonies
50
What is the independent variable of the GFP lab?
The presence of plasmid
51
What is the control variable of the GFP lab? And why?
IPTG because its concentration remains constant
52
What is the special treatment given to E.coli in this experiment?
Add of calcium chloride (CaCl2) so the bacteria can become competent
53
What is the function of PBP?
Normally helps the bacteria build their cell walls
54
What is the competitor for the active site of PBP?
Ampicillin
55
What is the normal inducer for the lac Oberon?
Allolactose
56
What is the normal function of the lac Operon?
Break downs the lactose in prokaryotic cells
57
What is the transformation effiency?
Numbers of cells transformed per microgram of plasmid DNA
58
What would be the equation to calculate the transformation effiency of colony calculations?
Number of transformed colonies/0.05 micrograms plasmids X total volume of bacteria at recovery/volume of bacteria plated onto agar
59
Why bacteria are induced?
To take up a plasmid through their weakened cell walls
60
Which plates contain transformed cells with the active GFP gene? How is IPTG activating the GFP gene?
*The plate IPTG + *IPTG is activating GFP gene by removing the repressor of the operator on the lac operon so transcription of T7 RNA pol can occur — will make T7 RNA pol — Allow GFP gene transcribes — GFP gene expressed
61
What is the point of comparing the plates: amp (+) and amp (+)IPTG?
Can prove that IPTG activates the GFP gene — plate containing IPTG was the only one to glow green *even though both plates have transformed bacteria
62
What is the point of comparing the plates: control and (-)Amp?
To determine the effiency of amipicillin — ampicillin prevents cell wall from growing — bacteria can’t grow since PBP is inactive *can clearly see that the (-)Amp plate had no bacteria growth while the control had many *ensure that the bacteria his healthy
63
Why would we compare plates (-)Amp and (+)Amp?
*(+)Amp have take up the plasmid so bacteria are capable of growing in the medium of ampicillin — because of AmpR gene *(-)Amp DON’T have the plasmid — incapable of producing enzyme capable of destroying the antibiotic — Can’t grow
64
What are non-glowing satellite?
Bacteria that did not take up the plasmid but are able to grow
65
How satellite non-glowing colonies are able to grow if they don’t have the plasmid?
They grow around colonies that have the plasmid containing AmpR gene — kills the presence of ampicillin around it — allowing satellite colonies that don’t have the plasmid to grow
66
What allow the bacteria to grow in the medium ampicillin?
The presence of the AmpR gene
67
How can we qualify that a bacteria has been transformed?
When it glows green — when GFP gene is expressed

Cell Bio Lab Test#2 (5 decks)

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