Lab 4

Question 1

Purpose of streak plate method

Answer 1

siolate pure colonies

Question 2

Whats a mixed culture

Answer 2

a microbial culture that consists of 2 or more species -> mixed culture

Question 3

Whats a pure culture

Answer 3

containing organism from 1 spieces

Question 4

What media is used

Answer 4

NYZ, containing maltose and magnesium sulfate

Question 5

Whats the system used for bacterial transformation

Answer 5

the pGLO system

Question 6

Whats essential in bacterial transformation

Answer 6

The bacteria used does not have nucleases to break down foregin DNA

Question 7

Whats bacterial transfomration

Answer 7

Making cells competent so they can take up foreign DNA

Question 8

Whats bioluminiscnece

Answer 8

protein that naturally fluoresnces

Question 9

How does the arabinose operon work

Answer 9

Arabinose -> bind to the regulator section -> conformational seuqnece -> ranscription of genes

Question 10

Whats the GFP protein

Answer 10

Green Fluorescent protien ioslated and engineered to be a part of the arabinose

Question 11

So in arabinose, the GFP will

Answer 11

fluosce

Question 12

What does beta lactamase do

Answer 12

Break down the beta lactam ring in ampicillin

Question 13

What is part of the pGLO plasmid

Answer 13

1. Origin of replication
2. gene for AraC
3. arabinose promotor
4. gene for GFP
5. Ampicillin resistnace gene - “bla” gene

Question 14

purpose of recovery at room temperature

Answer 14

plasmid replication and expression of ampicillin
resistance.

Question 15

Steps of transformation

Answer 15

1. Make cells competent: Addition of CaCl2 on ice
2. Brief heat shock
3. Recovery at room temeprature

Question 16

Purpose of CaCl2

Answer 16

Ca complexes with the negatively charged phosphates on the lipids. The low temepratures congeal the cell membrnae.

Once DNA is added, the Ca++ ions bind to the negative charges on phosphate backbone helping DNA stick to the membrnae.

Question 17

Purpose of heat shock

Answer 17

thermal imbalance -> causes changes in the membrane composition and allows for the uptake to DNA -> increasing transformation efficineciny

Question 18

How is the success of transformation determined
and how is the expression of GFP manifuluated

Answer 18

By plating of LB media alone

LB media containing ampilicin (2 plates)

LB media containi ampicclilin and arobinose (manipulate expression of the GFP)

Question 19

Procedure for transformation

Answer 19

1. obtain 2 tubes (one with and one without)
2. put cacl2 on ice
3. then add E. colicells with inoculating loop and disperese the cells
4. After 10 min, transfer to heat for 50s
5. Put back on ice again
6. remove and place on bench.
   ADD LB BORTH. MIX
7. Allow 10 minutes for recovery

Question 20

Bacterial conjugation requires

Answer 20

Direct contact between host and recipeint

Question 21

The donor strain is

Answer 21

HT-99

resistant to chloramphenicol

encoded on conjugative plasmid

Question 22

The recipient strain is

Answer 22

J-53R

Rifampicin resistant

Bacterial chromosome

Question 23

What type of strain is J-53R

Answer 23

Auxotrophic strain

Question 24

Whats an auxotrophic strain

Answer 24

A mutant strain that has specific growth requiremenst

Question 25

How is the J-53R an auxotrophic strian

Answer 25

it requires the amino acids methoionie and proline be added in the media to grow -> ensures that J-53R cannot grow in nature

Question 26

What happens if conjugation takes place between donor HT-99 and recipeint J-53R?

Answer 26

the J-53R will be both resistant to chloramphenicol and rifampicin

Question 27

Whats the ratio of donor to recipient

Answer 27

Having more recipient than donor. Ensures each donor will come into contact with at least 1 recipient

Question 28

Results of the HT-99 ctontrol

Answer 28

It is Chloraphenicol resistant so it will grow on Chloraphenicol plate but not rifampicin

Question 29

Results of the J-53R ctontrol

Answer 29

It is rifampicin resistant. So it will grow on the rifampicin plate and not the chloramphenicol plate

Question 30

What will you observe on the double antibiotic plate

Answer 30

If genetic exchange took place, you will observe individual colonies on the double antibiotic plate. Each colony represents a J-53R cell that underwent conjugation with HT-99 and obtained a plasmid