Persister

Question 1

How to identify a persister cell

Live dead stain

Answer 1

• Kill off the susceptible population, antibiotics
• Isolate the persister population with microscopy, a live dead stain
• The intact cells will take up a dye. Live= green, Red= dead

Question 2

How to identify a persister cell

FACS

Answer 2

• If a bacterial cell is healthy it will be expressing allof the components of a ribosome
• Put GFP downstream of the ribosome promoter, you will have fluorescent bacteria
• Use FACS to separate cells on size and colour
• Fluorescent= not persister cells
• Persister cells are not growing

Question 3

How to identify a persister cell

Microfluidics

Answer 3

• Visualise cells that don’t grow
• After using antibiotics to kill cells that are growing
• we can regrow them

Question 4

How to identify a persister cell

Fluorescence dilution

Answer 4

• Put a reporter gene in
• GFP has a short half life
• Use an inducer
• The cells that are growing would dilute the fluorescence

Question 5

Persister cell mechanism

Answer 5

• They have an upregulation of genes, toxin/ antitoxins

Question 6

Toxin/ Antitoxin

Answer 6

• They are co located
• When they are expressed together they negate the other
• Bacterial cellular proteases (clpAP and lon) that are under the control of environmental factors
• If the levels of clipAP and lon are elevated, they will degrade the antitoxin and release the toxin which will go and mediate growth arrest

Question 7

Direct ELISA

Start with an antibody to test for an antigen

Answer 7

• a specific antibody is adsorbed onto the walls of microwells
• A suspension of serum is added to the wells to test for the presence of a complementary antigen
• If there is a complementary antigen it will bind to the antibodies
• It is strong enough to withstand rinsing, which removes the fluid and unbound antigens
• After rinsing another aliquot of the monoclonal antibody is added to the well
• The antibodies are modified so that they carry a reporter enzyme
• A reporter enzyme is designed to change colour when the enzyme reacts with its substrate
• The sample is rinsed to remove unbound antibodies
• If the antigen is present a complex forms that includes the antibody, antigen and antibody with enzyme
• The enzyme substrate is added
  A colour change indicates the antigen was present in the substrate

Question 8

Indirect ELISA

Start with antigen to test for an antibody

Answer 8

• It determines whether a specific antibody is present in a sample
• The antigen is absorbed onto a strip of microwell
• Serum that could contain the antibody is injected into the microwell
• If the specific antibodies are present they will bind to the antigens
• Rinsing removes antibodies not attached to the enzymes
• If antibodies attach to the antigen they can be detected by the addition of an antibody enzyme conjugate
• The reporter antibodies bind to the antibodies used in the previous stage
• As the specimen uses human antibodies, we use enzyme conjugated anti-human IgG antibodies for the reporter
• If the antibody was present in the sample it will be bound to the adsorbed antigen and the antibody reporter will be bound to the complex
• It is rinsed
• Substrate is added
• A colour change indicates a positive result