Lab 1 - Gene regulation part 1

Question 1

Why do cells need to regulate the expression of their genes

Answer 1

external environmental conditions, developmental cues, in response to hormone signals, etc

Question 2

Most common type of gene regulation in bacteria

Answer 2

transcriptional regulation

Question 3

when does transcriptional regulation occur

Answer 3

during the first stage of gene expression, before significant amounts of mRNA are synthesized.

Question 4

what does the ara operon contain

Answer 4

genes that encode proteins required to metabolize a sugar - L arabinose

Question 5

what happens when L arabinose is present in bacterial growth mediums

Answer 5

the genes in the ara operon may be transcribed at high levels

Question 6

What happens when glucose is present in bacterial growth medium

Answer 6

there is little transcription of the ara operon genes because glucose is more easily metabolized

Question 7

pGLO plasmid

Answer 7

contains reporter gene that produces an easily visualized gene product

Question 8

what do arrows in the plasmid map represent

Answer 8

open reading frames or genes

Question 9

components in the plasmid map

Answer 9

araC, ori, bla, GFP, Pbad (not a gene but ara operon promoter)

Question 10

ori

Answer 10

origin of replication for plasmid

Question 11

bla gene

Answer 11

codes for a protein that makes the bacterial cell resistant to antibiotic ampicillin

Question 12

GFP gene

Answer 12

codes for a green fluroescent protein, when cells are exposed to UV light the GFP emits bright green fluorescence.

Question 13

Pbad

Answer 13

ara operon promoter

Question 14

what does Pbad do under normal conditions

Answer 14

regulates the transcription of genes in the ara operon (responsible for the metabolism or arabinose)

Question 15

in pGLO what relaces the regulatory genes (Pbad)

Answer 15

replaced by GFP gene

Question 16

what is GFP controlled by in this experiment and what is it normally controlled by

Answer 16

GFP ormally has its own eukaryotic promoter but in this case it is controlled bu the bacterial promoter Pbad.

Question 17

what happens when there is increased activity from the Pbad promoter

Answer 17

GFP will be produced

Question 18

ara C protein

Answer 18

allosteric regulatory protein with 2 bidning sites. When arabinose is present in the environment AraCi binds to the sugar. this makes AraCi to AraC that permits recognition and binding to aral.
it is hypothesized that araC facilitates RNA pol binding to the Pbad promoter.

Question 19

aral

Answer 19

activator sequence located just upstream of the Pbad promoter

Question 20

what happens in the absense of arabinose

Answer 20

AraC is not activated and thus cannot bind to aral sequence
RNAP will not bind efficiently to Pbad sequence
very little transcription will occur (little GFP made)

Question 21

How do we get the most efficient binding of RNAP to Pbad

Answer 21

two molecules ofAraC bind to aral sequence but CAP must bind to the cAMP-CAP binding site located just upstream of aral sequence.

Question 21

2 forms of CAP

Answer 21

cAMP bound to CAP - active
cAMP not bound - inactive and cannot bind to CBS

Question 22

what happens in the cell when theres high concentrations of glucose (in relation to CAP)

Answer 22

reduce synthesis of cAMP and thus CAP is less likely to be activated and bind to CBS sequence. Even in presence of arabinose, glucose will prevent formation of CAP.

Question 23

what happens when there is arabinose and low levels of glucose

Answer 23

2 molecules of AraC will bind to the aral sequence, CAP will bind to CBS sequence and binding of RNAP to Pbad will efficient. transcription of GF will be high

Question 24

catabolite repression

Answer 24

Since glucose is the preferred source of carbon in e coli, CAP ensures that other carbon utilization pathways are not expressed in the presence of glucose
(lack of repression due low cAMP-CAP)

Question 25

how are plasmids replicated

Answer 25

bacterial cell’s own DNA replication machinery and can be replicated independently of the host cell chromosomal DNA

Question 26

conjugation

Answer 26

when bacteria is in direct contact with one another, it can exchange genetic material naturallu

Question 27

competence

Answer 27

ability to take up foreign extracellular DNA from their surroundings

Question 28

how can plasmids be engineered in labs

Answer 28

bacteria can be rendered competent in the lab by pre treating with calcium ions, followed by a brief heat shock. this treatment introduces small pores into the bacterial plasma membrane , thereby producing bacteria that is transiently permeable to DNA.
it is hypothesized to involve the neutralization of electrostatic repulsion between DNA and the cell surface induced by interaction with divalent cations and increased fluidity and permeability of the membrane induced by increased temperature.

Question 29

heat shock

Answer 29

neutralization of electrostatic repulsion between DNA and the cell surface induced by interaction with divalent cations and increased fluidity and permeability of the membrane induced by increased temperature.

Question 30

how to differentiate transformed from non transformed bacterial cells

Answer 30

bla gene which encodes for beta lactamse, a bacterial enzyme that renders the bacterial cell resistant to ampicillin. ONLY cells that have been transformed with pGLO plasmid, nd hence express beta lactamase, will be able to grow on the medium,