Question

You are performing a DNA extraction of Prochlorococcus. Your goal is to extract 300 ng of DNA. You inoculated a flask with 1,000 cells. In the growth conditions you are using for your experiment, the lag phase of Prochlorococcus is 1.5 days and u = 0.6 days ^-1.

a) On what day should you perform the DNA extraction? (Assume 1 cell has 0.005 ng of DNA) Show your work for full credit.

b)Alas, 3 days into the incubation process all of your cells have died! You have done this experimental procedure before and it has gone well, but this time something got in the way. Discuss two possible reasons your phytoplankton cells could have died.

c) You perform the experiment again but now that you’ve lost 3 days you want to speed up the process. You decide to inoculate with more cells this time. How many cells of Prochlorococcus would use as an inoculum such that you can perform the DNA extraction on day 5?

Answer 1

a) DNA required = 300 ng

DNA in one cell = 0.005ng.

So minimun number of cells required for getting 300 ng DNA = 300 / 0.005 = 60,000 cells.

Now, we know that :

Nt = N0 x 2 ^ tμ (Exponential Growth kinetics)

Where Nt = No. of cells at time t.

No= Initial No. of cells

t = time

μ = Specific Growth Rate

Putting all values in this equation :

60,000 = 1000 x 2 ^ (t x 0.6)

60 = 2 ^ 0.6t

Taking logarithm on both sides :

log 60 = 0.6t x log 2

0.6t = log 60 / log 2

0.6t = 5.906

t = 9.843 days

So it means that after entering exponential phase, cells will take about 9.84 days to grow into 60,000 cells.

Therefore, DNA Extraction should be preferably done after :

9.84 days + 1.5 days (lag phase) = 11.34 day

So on 12th day, DNA extraction can be done.

b) The two possible reasons of cell death might be :

i) The nutrients in the culture/nutrient broth might have been exhausted, hence the cells may have died due to starvation. Lack of nutrients will stop the metabolism of cells, ultimately causing death.

ii) Due to lack of proper oxygen flow or insufficient sunlight, the cells may also die. Oxygen and sunlight are two most important factors in photosynthesis, and their insufficiency or absence is fatal for cells.

c) To perform DNA extraction on Day 5, the exponential/growth phase should last for 3.5 days (because lag phase lasts for 1.5 days, and 5 - 1.5 = 3.5).

So putting these values again in above equation -

Nt = N0 x 2 ^ tμ

60,000 = No x 2 ^ (3.5 x 0.6)

60,000 = No x 2 ^ 2.1

60,000 = No x 4.626

No = 12,970.16

So we need to inoculate about 12971 cells to perform DNA extraction on day 5.

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