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Operon Fusion

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Data & Analysis

Titer

Dilution of Lysate

Number Of Plaques

(pfu/mL in lysate)

 

Sue-Lynn

Joel & Isleen

Alex

Sue-Lynn

Joel & Isleen

Alex

10-5

0

tntc

TNTC

0

n/a

TNTC

10-6

400

273

69

4.0 x 109

2.73 x 109

6.9 x 108

10-7

0

30

10

0

3.0 x 109

1.0 x 109

 

Controls

 

Sue-Lynn

Joel & Isleen

Alex

Cell Control (# plaques)

0

37

0

Phage Control

(# colonies)

0

0

0

Sue-Lynn’s data provided by Mr.Sandoz

Sue-Lynn and Alex’s plaques that formed on these LBC plates were incubated at 30 degrees Celsius overnight and were all turbid. Joel & Isleen’s plaques were clear because they had been incubated at 42 degrees Celsius overnight.

If the plaques had been incubated ad 42 degrees Celcius overnight the plaques would be clear because a clear plaque shows that the bacteria was infected by the bacteriophage and then lysis of the cell occurred. Because MuCts is temperature sensitive, at 42 degrees the ability of the C gene in MuCts to repress its lytic genes is de-repressed. Lysis then occurs and a continuation of infection and lysis creates a number of clear plaques.

There should be no plaques on either of the control plates even though our data shows 37 plaques on one of the cell control plates.  A possible source of plaques on the cell control plate is a contamination of the lysate into the cell control tube. If there were colonies on the phage control plates then contamination is possible but the most likely source of colonies would occur because of accidental disturbance of the pellet, causing the presence of unlysed Mal 103.

Patched Arar Colonies

(Everyone’s Data)

 

Color of Patch

# of patches showing each pattern

Arabinose MacConkey

Lactose MacConkey

Arabinose + Lactose MacConkey

0   I.

white

white

white

0  II.

White

White

red

0 III.

White

Red

Red

All data provided by Mr.Sandoz

 

I               In case I the MudI phage inserted in either Ara A, Ara B, or Ara C with the lactose structural genes facing away from a promoter

II             In case II the MudI phage inserted in either Ara A, Ara B with its lactose structural genes facing towards an active promoter

III            In case III the MudI phage inserted in Ara C with lactose genes facing towards the e.coli’s promoter.

 

Selection of Mud1 Lysogens

 

Dilution of Lysate

 

Number of Colonies

White

 

Red

 

Total

 

% red colonies

SL

J&I

A

SL

J&I

A

SL

J&I

A

SL

J&I

A

10-1

0

45

TNTC

0

5

15

0

50

TNTC

0

10%

0

10-2

185

0

39

15

0

5

200

0

44

7.5%

0

11.3%

10-3

0

0

0

0

0

3

0

0

3

0

0

100%

Uninfected

0

0

0

0

0

0

0

0

0

0

0

0

Sue-Lynn, Joel &  Isleen’s  data provided by Mr.Sandoz

 

Joel  and Isleen did not get any colonies because the MC4100 was not infected by the lysate. This is why data was provided to them.

 

In order to select the Mud1 lysogens (above data) the plates used are Lactose MacConkey  with ampicillin. The MacConkey also has an acid base indicator and colonies that metabolize the sugar will produce acid and turn red. In our data specifically, some of the colonies are red because Mud1 inserted next to an active promoter in the MC4100 genome. This allows the lactose structural genes to be transcribed by the host promoter and continue to be translated and use these protein products to metabolize the lactose.

The white colonies are lysogenic for MuD1 and not MuCts because only MuD1 contains the bla genes which have the ability to make the cell resistant to ampicillin.

The average frequency of lysogeny to ampicillin resistance is 1.155 x 10-4 Ampr/pFu.

The frequency to Ampicillin resistance assuming the Viable Cell Count of the MC4100 is 2 X 109 cfu/ml is Less Than 5 x 10-9 mutations/vc.

Calculations:

Frequency of lysogeny to ampr:

Sue-Lynn’s Data

200 / .1 mL= 2000 lysogens/mL

4 x 109 pFu/mL x 10-2 x 1/3 = 1.33 x 107

2000/ 1.33 x 107= 1.5 x 10-4 Ampr/pFu

 

Joel & Isleens’s Data

50 / .1 mL= 500 lysogens/mL

2.73 x 109 pFu/ml x 10-1 x 1/3 = 9.1 x 107

500/9.1 x 107= 5.5 x 10-6 Ampr/pFu

 

Alex’s Data

44/0.1mL = 440

6.9x108 x 10-2 x 1/3 = 2.3x106

440/2.3x106 = 1.91 x 10-4 Ampr/pFu

 

Average Frequency of lysogeny to ampr: 

(1.5 x 10-4 Ampr/pFu + 5.5 x 10-6 Ampr/pFu + 1.91 x 10-4 Ampr/pFu)/3 = 1.155 x 10-4 Ampr/pFu

 

Mutation Frequency:

Everyone’s  Data

1/.1 mL= 10 cFu/mL

10/ 2 x 109= Less Than 5 x 10-9 mutations/vc

 

Selection of the Arar Lysogens

Dilution of lysate

Number and Description of the Colonies

Sue-Lynn

 

Joel & Isleen

 

Alex

Undiluted

10 W

1 R

12 W

4 R

0 W

0 R

10-1

0 W

0 R

0 W

0 R

5 W

20 R

10-2

0 W

0 R

0 W

0 R

0 W

0 R

Uninfected Mc4100

150 W

50 R

141 w

32 R

0 W

0 R

All data provided by Mr.Sandoz

Theoretically there should be colonies on the uninfected MC4100 plate but the probable source of no colonies here is human error. The lysate may have accidently been plated. For accurate calculation purposes we will only use Sue-Lynn, Joel and Isleen’s data to calculate the average concentration of Arar mutations and Ara+ mutations.

 The average frequency of lysogeny to Arabinose resistance is 7.92 x 10-7 Arar/pFu.

The ratio of Arar lysogens/pfu: Ampr lysogens/pfu is:

 

Sue-Lynn

Joel & Isleen

Alex

Ratio

1 : .0005

1 : .024

1 : .011

The average concentration of Arar mutations is (1500 + 1410)/2= 1455 cfu/mL

The average concentration of Ara+ mutations is (500 + 320)/2= 410 cfu/mL

 

 

The average mutation frequency for each of the above is:

Arar mutations: (7.5 x 10-7 + 7.05 x 10-7)/2= 7.28 x 10-7 cFu/mL

Ara+ mutations:  (2.5 x 10-7 + 1.6 x 10-7)/2= 2.05 x 10-7 cFu/mL

 

The most likely reason that these frequencies are high is because Arar was induced by the Mud1 insertion and was not a spontaneous mutation.  The Mud1 could insert anywhere in the arabinose operon, in which multiple genes are involved, which increases the odds of causing a change in phenotype. There are also multiple chances for mutation because there are multiple generations before ribulose-5-phosphate kills the cell.

Calculations:

                Sue-Lynn’s Data

  1. 10 W colonies/.1mL = 100 cFu/mL

100/ (4.0 x 109)(1)(1/3) = 7.5 x 10-8A Arar/pFu

 

  1. 7.5 x 10-8 Arar/pFu : 1.5 x 10-4 Ampr/pFu

1 : .0005

 

  1. 150 cFu/.1mL= 1500 cFu/mL

 

  1. 50 cFu/.1 ml= 500 cFu/mL

 

  1. 1500/2 x 109= 7.5 x 10-7 cFu/mL

500/2 x 109= 2.5 x 10-7 cFu/mL

 

Joel & Isleen Data

  1. 12 W colonies/.1mL = 120 cFu/mL

120/ (2.73 x 109)(1)(1/3) = 1.31 x 10-7A Arar/pFu

 

  1. 1.31 x 10-7A Arar/pFu : 5.5 x 10-6 Ampr/pFu

1 : .024

 

  1. 141 cFu/.1mL=  1410 cFu/mL

 

  1. 32 cFu/.1 ml= 320 cFu/mL

 

  1. 1410/2 x 109= 7.05 x 10-7 cFu/mL

320/2 x 109= 1.6 x 10-7 cFu/mL

 

Alex’s  Data

  1. 5 W colonies/.1mL = 50 cFu/mL

50/ (6.9 x 108)(10-1)(1/3) = 2.17 x 10-6 Arar/pFu

 

  1. 2.17 x 10-6 Arar/pFu : 1.91 x 10-4 Ampr/pFu

1 : .011

 

Average frequency of lysogeny to Arabinose resistance:

(7.5 x 10-8A Arar/pFu + 1.31 x 10-7A Arar/pFu + 2.17 x 10-6 Arar/pFu)/3= 7.92 x 10-7 Arar/pFu

 


Steric Hinderance - Sec 002 - BIOL 302L - FA09