Bacterial Transformation

Question 1

how can foreign genes be introduced into organisms

Answer 1

• through bacterial transformation

Question 2

what is the purpose of a plasmid during transformation

Answer 2

a plasmid is a small, circular piece of double-stranded DNA

-plasmid DNA contains coding sequences (genes) which are expressed by the bacterium. Often, the gene carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance

Question 3

role of the beta-galactosidase (LacZ) (color marker gene)

Answer 3

• breaks down lactose into galactose and glucose
• can also cleave the synthetic analog of lactose, X-GAl
• creates a product that is blue when cleaved with XGal

Question 4

role of beta lactamase (ampicillin resistant gene)

Answer 4

• it destroys the activity of ampicillin by breaking down the beta lactic ring
• thus, bacteria with beta lactamase gene can resist ampicillin

Question 5

purpose of nutrient agar

Answer 5

to contain the bacteria

Question 6

purpose of ampicillin

Answer 6

ampicillin is a member of the penicillin family in antibiotics

• destroys the activity of bacteria by destroying its cell wall
• ampicillin contains a chemical group called BETA LACTAM RING which blocks the process by which bacteria link molecules to create a cell wall

Question 7

purpose of X-gal

Answer 7

• synthetic analog of lactose that when cleaved by the LacZ gene has a blue product
• allows for the identification of ampicillin-resistant bacteria

Question 8

purpose of pBlu

Answer 8

-a plasmid with all the necessary criteria to use in the bacterial transformation lab

Question 9

what is bacterial transformation

Answer 9

when a host organism takes in foreign DNA and expresses its gene
- the introduction of apiece of DNA, like a plasmid, into a bacterial cell

Question 10

what does a plasmid contain

Answer 10

• origin of replication
• gene for antibiotic resistance (encodes beta lactamase)
• color marker gene (beta galactosidase/lactZ)
• polylinker

Question 11

what is a recombinant plasmid

Answer 11

a genetically engineered plasmid (with a foreign gene inserted in it)

Question 12

why do we lower the temperature in the transformation experiment

Answer 12

to slow down the movement of the phospholipid in the membrane of the bacteria to efficiently neutralize the charges

Question 13

why do we use the heat shock

Answer 13

rapid change in temperature induces the formation of pores in the bacterial membrane so that the plasmid DNA can enter

Question 14

what are competent cells

Answer 14

when bacteria cells are in a state in which they are able to take up DNA, they are referred to as competent

-transformation rarely occurs naturally, so by subjecting bacteria to certain conditions, we can enable many of them to take up DNA

Question 15

• what is insulin +relation to bacterial transformation

- diabetes?

Answer 15

• a hormone that originates from the pancreas, regulates glucose in blood
• we can create insulin from bacteria by purifying it
• diabetes consists of type 1 and type 2. It is a disorder that deactivates the insulin

Question 16

how is a plasmid genetically engineered to contain a gene of choice

Answer 16

-a plasmid has sequences that restriction enzymes correspond to, so restriction enzymes can manipulate the plasmid to create a gene of choice by ‘cutting’ there

Question 17

why do we use CACl2 (calcium chloride) in the bacterial transformation lab

Answer 17

• Plasmid DNA is a hydrophilllic molecule which will normally pass through the plasma membrane of the bacterium in order to accomplish transformation, the bacteria cells must first become competent to make the plasmid DNA.
• this is done by neutralizing the negative charges of the membrane, the DNA molecule with calcium chloride

Question 18

origin of replication?

Answer 18

• a sequences of DNA is recognized by DNA replication enzymes
• when a bacterium divides all of the plasmids contained within the cell are replicated, thus each daughter cell receives a copy of each plasmid

Question 19

Results of bacterial transformation (with plasmid)

Answer 19

1. (LB) lawn of bacteria
2. (LB +AMP) bacterial growth ( white colonies)
3. (LP+AMP+X-GAL) Blue colonies (and white)

Question 20

Results of bacterial transformation (without plasmid)

Answer 20

1. (LB) lawn of bacteria
2. (LB +AMP) nothing
3. (LP+AMP+X-GAL)nothing

Question 21

where do transcription and translation take place

Answer 21

transcription: nucleus
translation: cytoplasm