TITLE: E_faecalis_conjugation_HR
AUTHORS: Elizabeth Fozo

[STEPS]
?. [+Enterococcus faecalis conjugation and homologous recombination]
Inoculate 5 to 10 mL BHI (add antibiotics if necessary) with donor and recipient strains and incubate overnight at 37°C
?. [+Enterococcus faecalis conjugation and homologous recombination]
In the morning, wash cultures 2x with BHI, then resuspend cells in the original volume of BHI.Dilute both cultures 1:10 in BHI and incubate at 37°C for 1 hour
?. [+Enterococcus faecalis conjugation and homologous recombination]
Dilute both cultures 1:10 in BHI and incubate at 37°C for 1 hour
?. [+Enterococcus faecalis conjugation and homologous recombination]
Mix donor and recipient (1 part donor to 9 parts recipient) to initiate conjugation
?. [+Enterococcus faecalis conjugation and homologous recombination]
Put 800µL of the conjugation mix into a 1.5 mL Eppendorf tube and centrifuge for 2 min at 13000 RPM at 4°C
?. [+Enterococcus faecalis conjugation and homologous recombination]
Resuspend in 150 µL BHI and spread on a BHI agar plate.
?. [+Enterococcus faecalis conjugation and homologous recombination]
Mate for 5 hours.
?. [+Enterococcus faecalis conjugation and homologous recombination]
Collect the cells from the surface by adding 2 mL of 1x PBS + 2 mM EDTA (20 mL 1x PBS + 80 µL 0.5M EDTA) and scraping the cells using a disposable hockey stick. Put the resuspended cells into a 1.5 mL Eppendorf tube.
?. [+Enterococcus faecalis conjugation and homologous recombination]
Prepare 10-fold serial dilutions using 1x PBS as diluent and plate the suspensions on selective media for donors and transconjugants. Typical dilutions for donors (-4 to –6) and transconjugants (-3 to –5). Incubate at 37°C. Blue colonies appear after 24-48 hours.
?. [+Enterococcus faecalis conjugation and homologous recombination]
Restreakblue colonies onto the same selective media 2x for single colony purification
?. [+Enterococcus faecalis conjugation and homologous recombination]
Inoculate 10 mL BHI with a colony in the absence of selection until the culture reaches stationary phase (grow as overnight)
?. [+Enterococcus faecalis conjugation and homologous recombination]
Prepare serial dilutions of the overnight: dilute the culture 1:100 three times, then 1:10 one time. Grow all as overnights.
Rachel suggests:  Do dilutions in 1.5 mL BHI; for final dilution, place 1 mL of diluent into 9 mL of BHI, leave 0.5 mL of higher concentrations in Eppendorf tubes; grow these in the incubator as well.Rachel suggests: There is no need to calculate CFUs of the original overnights before this step; just do the above dilutions – you will get growth!
?. [+Enterococcus faecalis conjugation and homologous recombination]
Prepare 10-fold serial dilutions using 1x PBS and plate on counterselection plates. Incubate at 37°C until colonies appear and are large enough to pick.
Rachel suggests: Incubating at 30°C may allow blue colonies to become more apparently blue, though growth will take longer. Rachel suggests: Placing plates with all apparently white colonies at 4°C may also help for blue color to developRachel suggests: hurting your eyes and squinting really hard at these plates will not necessarily help. Pick the whitest colonies of varying sizes if they all look white and screen these. Sometimes smaller slower growing colonies have had higher rates of successful recombination.
?. [+Enterococcus faecalis conjugation and homologous recombination]
Restreak white colonies on BHI. Screen with check primers for the wild-type or mutant allele using the E. faecalis colony PCR protocol.