Chapter 7 – Complementary Experimental Tools  311

◾Microfluidics has transformed our ability to monitor the same biological sample

under different fluid environments.

◾Bulk tissue measurements can provide useful ensemble average information and have

led to several developments in biomedical techniques.

QUESTIONS

7.1

A polyclonal IgG antibody that had binding specificity against a monomeric variant

of GFP was bound to a glass coverslip surface of a microscope flow cell by incuba­

tion, and then GFP was flowed through the flow cell and incubated, and any unbound

GFP was washed out. The coverslip was then imaged using TIRF, which results in

bright distinct spots visible that were sparsely separated on the camera image much

greater than their own point spread function width. Roughly 60% of these spots had a

total brightness of ~5000 counts on the camera used, while the remaining 40% had a

brightness of more like ~10,000 counts. Explain with reasoning what this could indi­

cate in light of the antibody structure. (For more quantitative discussion of this type

of in vitro surface-​immobilization assay, see Chapter 8.)

7.2

What are the ideal properties of a model organism used for light microscopy

investigations? Give examples. Why might a biofilm be a better model for investi­

gating some bacteria than a single cell? What problems does this present for light

microscopy, and how might these be overcome?

7.3

Why does it matter whether a genetically encoded tag is introduced on the C-​terminus

or N-​terminus of a protein? Why are linkers important? What are the problems

associated with nonterminus tagging?

7.4

Outline the genetic methods available for both increasing and decreasing the concen­

tration of specific proteins in cells.

7.5

Image analysis was performed on distinct fluorescent spots observed in Slimfield

images of 200 different cells in which DNA replication was studied. In bacteria, DNA

replication is brought about by a structure of ~50 nm diameter called the replisome

that consists of at least 11 different proteins, several of which are used in an enzyme

called the DNA polymerase. One protein subunit of the DNA polymerase called ε was

fused to the yellow fluorescent protein YPet. Stepwise photobleaching of the fluores­

cent spots (see Chapter 8) indicated three e-​YPet molecules per replication fork. In

this cell strain, the native gene that encoded for the ε protein was deleted and replaced

entirely with ε fused to YPet. It was found that there was a 1/​4 probability for any ran­

domly sampled cell to contain ~80 e-​YPet molecules not associated with a distinct

replisome spot and the same probability that a cell contained ~400 e-​YPet molecules

per cell.

a

Estimate the mean and standard error of the number of e-​YPet molecules per cell.

In another experiment, a modified cell strain was used in which the native gene

was not deleted, but the e-​YPet gene was instead placed on a plasmid under con­

trol of the lac operon. If no IPTG was added, the mean estimated number of

e-​YPet molecules per cell was ~50, and using stepwise photobleaching of the

fluorescent replisome spots, this suggested only ~1–​2 ε-​YPet molecules per

spot. When excess IPTG was added, the stepwise photobleaching indicated ~3

molecules per spot and the mean number of nonspot e-​YPet molecules per cell

was ~850.

b

Suggest explanations for these observations.

7.6

Live bacteria were immobilized to a glass microscope coverslip in a water-​based

medium expressing a fluorescently labeled cytoplasmic protein at a low rate of gene

expression resulted in a mean molar concentration in the cytoplasm of C. The protein

was found to assemble into one or two distinct cytoplasmic complexes in the cell of

mean number of monomer protein subunits per complex given by P.

a

If complexes are half a cell’s width of 0.5 μm from the coverslip surface and the

depth of field of the objective lens used to image the fluorescence and generate a