Bioluminescent Imaging of Bacterial Biofilm Infections In Vivo - Bacterial Pathogenesis: Methods and Protocols - page 214

3.2. Transformation Procedure for Gram-negative Bacteria
(General Methodology)
Gram-negative bacteria are made bioluminescent (e.g., P. aeruginosa Xen-
5 and P. mirabilis Xen-44) through the stable integration of an unaltered P.
luminescense luxCDABE cassette onto their chromosome as first described by
Winson et al. (16) .
1. Mate the Gram-negative strain to be transformed (the recipient) with the E. coli
strain S17-1 pir pUT Amp R mini-Tn5 luxCDABE Tc R (the donor) ( see Note 1 ).
2. To allow the recipient strain to be more readily distinguished from the donor
strain, initially transform the recipient with a blank vector carrying a unique
antibiotic resistance gene (primary resistance marker) using standard electropo-
ration procedures.
3. Use a single resistant transformant (carrying the primary resistance marker) to
inoculate 10 mL of LB containing the primary selective antibiotic and incubate
overnight at 37 °C. Similarly, use a single colony of E. coli S17-1 pir pUT mini-
Tn5 luxCDABE Tc R to inoculate 10 mL of LB containing 100 μg/mL ampicillin.
4. Individually, pellet and resuspend both bacterial cultures in 10 mL of LB.
5. Inoculate a fresh 10-mL volume of LB containing 10 m M MgSO 4 with 100 μL
volumes of each of the suspensions and incubate this mixture overnight at 37 °C
to allow mating to occur.
6. Plate 100 μL volumes of the mating onto LB containing both the primary selective
antibiotic and 20 μg/mL tetracycline.
7. After an overnight incubation at 37 °C, screen the plates for bioluminescent
transposants using a highly sensitive optical imaging system, such as an IVIS®
Imaging System.
8. Select a single highly bioluminescent colony and characterize to ensure that it
is stable [e.g., bioluminescent photon counts correspond to colony-forming units
(CFUs)] and behaves similarly to the parental strain, both in vitro and in vivo
(e.g., not attenuated).
3.3. Transformation Procedure for Gram-positive Bacteria
(Staphylococcus aureus Methodology)
Gram-positive bacteria are made bioluminescent (e.g., S. aureus Xen-29)
through the stable integration of an optimized P. luminescense luxABCDE
cassette onto their chromosome as first described by Francis et al. (3) . This
broad host range plasmid can be used to transform a variety of Gram-
positive bacteria to a stable bioluminescent phenotype. However, the procedure
described below is optimized for the transformation of S. aureus .
1. Grow-up an overnight culture of S. aureus in 10 mL of BHI.
2. Dilute the overnight culture 1:100 in 50 mL of fresh BHI broth in a 500-mL
flask and incubate at 37 °C in an orbital shaker at 200 rpm until the OD 600
reached 0.8 (exponential growth phase).