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The Role of Calcium and Phospholypase C Signaling in Flagellum Regeneration in Chlamydomonas

Autor:   •  February 12, 2019  •  1,617 Words (7 Pages)  •  92 Views

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In experiment 2, the effects of neomycin were measured. We had 4 test tubes in total. The first two test tubes received acid shock treatment, neutralization, and cell washings. Test tube 3 didn’t receive any acid shock treatment, while tube 4 received acid shock treatment but no neutralization. Centrifugation and cell washings were done similarly as in experiment 1. However, test tube 3 and 4 were washed by adding 1mL of neomycin (not 1mL of distilled water). After three washings, the supernatant was removed and each tube received their designated solutions: Tube 1: 1mL distilled water; tube 2: 1mL colchicine mixture; tubes 3 and 4: 1mL TAP and neomycin solution. As before, the tubes were placed under the light and the number of non-motile cells were calculated.

Results

During the 10-minute intervals, data was collected for both experiments. The number of non-motile cells were counted using a hemocytometer; this number was recorded. The data was plotted.

Table 1: Effect of Calcium on Chlamydomonas Reflagellation

Mins

Test tube 1 - Acid Shock treatment

Test tube 2 - Acid shock treatment + colchicine treatment

Test tube 3 – Acid shock treatment + TAP solution with Calcium

Test tube 4 – TAP solution with EGTA

10

20

42

36

11

20

34

25

17

23

30

43

2

27

18

40

42

27

39

11

50

29

37

26

8

60

19

28

23

6

[pic 1]

Graph 1: Visual representation of the effect of calcium on Chlamydomonas reflagellation based on the number of non-motile cells.

Table 2: Effect of Neomycin on Chlamydomonas Reflagellation

Mins

Test tube 1 - Acid Shock treatment

Test tube 2 - Acid shock treatment + colchicine treatment

Test tube 3 – No acid shock treatment + washing with neomycin + TAP solution with neomycin

Test tube 4 – Acid shock treatment + washing with neomycin + TAP solution with neomycin

10

6

12

1

82

20

15

29

1

67

30

7

18

8

49

40

6

28

10

101

50

9

60

9

81

[pic 2]

Graph 2: Visual representation of the effect of neomycin on Chlamydomonas reflagellation based on the number of non-motile cells.

Discussion

The purpose of this lab was to explore this the effects of colchicine, calcium, and neomycin on the reflagellation of Chlamydomonas. Based on the data collected, the effects we should have observed were observed for the most part. It also can be concluded that colchicine inhibits flagella regeneration and Chlamydomonas requires Ca2+ to swim around and regenerate flagella. Overall, colchicine, no calcium ions, and adding neomycin all contribute to inhibiting the regeneration of the flagella. While our data is a little off, general trends can be observed

In the first experiment, it was expected that cultures treated with an acid shock, tubes 1, 2 and 3, should all be non-motile at time zero. EGTA chelated with the calcium ions and reduced the amount of free calcium ions available to promote regeneration. Therefore, those cells remained largely immotile since they could not regenerate their flagella. However, this tube shows a slight decreasing trend, concluding that the flagella regenerated and were able to swim due to the calcium in the TAP medium. In the second experiment, it was expected that cultures treated with an acid shock, tubes 1, 2 and 4, should all be non-motile at time zero. Tube 4, treated with neomycin, which should have inhibited phospholipase activity, would demonstrate a high number of immotile cells. However, the number of non-motile cells decreased, not following the expected results. This could be due to human error since these cells may have not been properly acid-shocked.

References

CCNY Biology 22900 Lab Manual.

Quarmby LM, Yueh YG, Cheshire

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