One - step Phage Infection of Cyanobacteria ( large - scale )
Before T0 , use flow cytometry to measure cell concentrations of cyanobacterial cultures from Step 2 .
Use SYBR staining ( e . g . SYBR counts of viruses ) to determine concentration of your concentrated viral lysate from Step 1 .
Calculate volumes of cells , phage and media to add to each experimental bottle / tube .
We have an Excel template to do this calculation , based on the desired MOI , cell concentration , and final experimental culture volume .
For gene expression experiments , we use an MOI of 3 , final cell density of 5e7 / ml , and volume of 200ml for each replicate - - this scale allows us to sample for RNA , protein , and phage & cell concentration , over a 12 - 14 hour time course .
Working quickly , and with sterile technique , initialize experiment by mixing volumes ( calculated in Step 3 ) of media , cells , and phage .
First add appropriate volume of sterile medium to your experimental culture vessel ( e . g . sterile 250ml polycarbonate bottles for 200ml cultures ) .
Then add cells ( prepared in Step 2 ) .
Then , to uninfected control bottles , add lysate that has been filtered to remove phage particles ( e . g . the filtrate that goes through an Amicon centrifugal device ) .
[ This is important to control for small molecules that may be added with the phage particles in infected treatments ; however keep in mind that other large molecules will get retained , along with phage particles , in the Amicon reservoir . ]
Finally , just before T0 , add phage to their respective bottles .
This begins the one - step infection .
Mix bottles by gentle vortexing .
Sample immediately for time 0 .
Suggested sampling : FCM for cell counts , Fluorescence , extracellular phage , RNA - protein .
Place bottles in incubator until next time point .
Repeat sampling in Step 5 at each time point ( e . g . every 1 - 2 hours ) until lysis is complete .
Prepare cells for infection : Grow up sufficiently large volumes of cells to be infected .
Plan carefully so that cells are in mid - log phase on the day of the experiment .
If you are growing volumes of 1L or more , bubble your culture with an aquarium pump ( ambient air ) connected to a diffuser stone , to prevent CO2 limitation .
Prepare phage stockGrow : On day minus - 8 , add phage stock to 4L of mid - log cells to start the infection ( a low MOI is sufficient , e . g . 0.01 - 0.05 ) , and monitor for about a week until the culture clears .
Harvest : When the culture has cleared ( i . e . most cells have lysed ) , centrifuge the cleared culture ( max speed , 10 min ) to remove large cell debris and unlysed cells .
Pour supernatant ( containing phage ) into disposable sterile vacuum filtration units containing a 0.2µm PES filter ; discard the pellet containing cell debris .
Connect to vacuum source and filter the lysate ; save filtrate containing your phage particles .
Concentrate : Concentrate this clarified phage lysate using VivaFlow Concentration of Phage Lysate .
Further concentrate the phage stock , and exchange buffers / media if necessary ( e . g . to remove P , N , or other nutrients in the initial lysate ) using Amicon Concentration of Viruses .
