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Biol 367

 


 

Molecular Biology

 


 

Midterm 2

 


 

November 2014

 


 

Name_______________________________ ID __________________

 

1- (5 points) The E. coli tryprophan operon is subject to regulation by attenuation (10-fold)

 

and repression (70-fold). The lacZ reporter gene shown in the figure below has been

 

constructed and cloned into an E. coli plasmid vector. When this plasmid is transformed

 

into an E. coli strain that has its lacZ gene deleted about 700 units of -galactosidase is

 

expressed in a medium without tryptophan.

 


 

How much -galactosidase activity will the combinations of E.coli strains and mutated reporter

 

genes (listed below) express under the growth conditions indicated?

 

-galactosidase activity

 

- tryptophan

 

+ tryptophan

 


 

Genotype of

 

E. coli strain

 


 

trp-lacZ reporter gene plasmid

 


 

LacZ-

 


 

plasmid as shown above

 


 

700

 


 

1

 


 

LacZ-

 


 

plasmid with region 1 and 2

 

deleted

 


 

___

 


 

___

 


 

LacZ- and

 

trpR-

 


 

plasmid with region 1 and 2

 

deleted

 


 

___

 


 

___

 


 

LacZ-

 


 

2 tryptophan codons deleted

 


 

___

 


 

___

 


 

LacZ- and

 

trpR-

 


 

2 tryptophan codons deleted

 


 

___

 


 

___

 


 

LacZ-

 


 

RBS and region 4 deleted

 


 

___

 


 

___

 


 

LacZ- and

 

trpR-

 


 

RBS and region 4 deleted

 


 

___

 


 

___

 


 

2- (4 points) Describe the fate of the following lambda phages after

 

they infect E. coli. Explain your rational. Please be brief.

 

a) PL- mutant

 

_________________________________________________________________________________________________________________________

 

_________________________________________________________________________________________________________________________

 

_________________________________________________________________________________________________________________________

 


 

b) Analysis of the lysis and lysogeny of E. coli by phage involved the

 

characterization of many mutations that disrupted the normal

 

capabilities of the virus. Predict the outcome of infection if a double

 

mutant phage bearing mutations in cro and OR1 is allowed to infect a

 

host bacterial cell that is already lysogenized by a wild-type phage

 

(Justify briefly your answer).

 

_________________________________________________________________________________________________________________________

 

_________________________________________________________________________________________________________________________

 

_________________________________________________________________________________________________________________________

 


 

3- (4 points) You have inserted into a lacI- strain of E. coli the following 2 genes.

 

Gene1 = Coding sequence of GFP and lambda CII under the control of the lac promoter

 

Gene 2 = Coding sequence of lacI under the control of the lambda PRE promoter

 

a) Are you going to see expression of GFP in a single cell grown in a medium that does not

 

contain IPTG? (justify your answer)

 

_________________________________________________________________________________________________________________________

 

_________________________________________________________________________________________________________________________

 

_________________________________________________________________________________________________________________________

 


 

b) Which of the following two media will give you the highest level of GFP expression?

 

(justify your answer)

 

medium 1 = glucose and IPTG

 

medium 2 = arabinose and IPTG

 

_________________________________________________________________________________________________________________________

 

_________________________________________________________________________________________________________________________

 

_________________________________________________________________________________________________________________________

 


 

4- (5 points) Consider both aspects of control (negative and positive) of the lac operon.

 

How much -galactosidase activity would you expect each of the following strains to

 

express under the growth conditions indicated.

 

Note: cap = cAMP binding protein; cbs = cap binding site

 

Strain genotype

 


 

glucose

 


 

glucose

 

and lactose

 


 

arabinose

 

and IPTG

 


 

cap+cbs+I+P+O+Z+Y+

 


 

0.5 units

 


 

10 units

 


 

500 units

 


 

cap+cbs+ I P+O+Z+Y/cap-cbs+ISP+O+Z-Y+

 


 

__________

 


 

__________

 


 

__________

 


 

cap-cbs- I P+O+Z+Y/cap+cbs-I P-O Z-Y+

 


 

__________

 


 

__________

 


 

__________

 


 

cap+cbs- I P+O+Z+Y+

 

/cap+cbs+I+P-O Z+Y+

 


 

__________

 


 

__________

 


 

__________

 


 

cap-cbs+ I P+O+Z+Y/cap+cbs-ISP-O Z-Y+

 


 

__________

 


 

__________

 


 

__________

 


 

-D

 


 

+

 


 

-D

 


 

C

 


 

+

 


 

+

 


 

+

 


 

C

 


 

Which strain(s) will be able to grow in presence of lactose only? _____________________

 

5) 3 points) The figure below illustrates a synthetic gene that has been introduced in the bacterium E.

 

coli.

 


 

A) Predict the pattern of expression of GFP over time in a single cell.

 

________________________________________________________________________

 

________________________________________________________________________

 

________________________________________________________________________

 

________________________________________________________________________

 

B) Four hours after the introduction of the construct in E. coli, GFP fluorescence was

 

monitored for few hours in a culture flask containing approximately 1 X 10 8 cells. Surprisingly,

 

the fluorescence was found to increase slowly over time. How can you explain the difference between

 

the predicted behavior and the one observed during the experimentation?

 


 

________________________________________________________________________

 

________________________________________________________________________

 

________________________________________________________________________

 

________________________________________________________________________

 

________________________________________________________________________

 

________________________________________________________________________

 


 

 


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