I. Transposon Mutagenesis and Colony Selection
i. TnPhoA Matings
Saturated cultures of PA14 and E. coli carrying pRT733 were mixed in a 1:1 ratio,
dropped on King's B media plates (2% w/v peptone, 6.57 mM K2HP04x3H2O, 6.08 mM MgSO4X7H2O, 1% v/v glycerol)
[Jones DL, 1954] and incubated for 6 hours at 37°C. Multiple matings were collected, resuspended in 100
mM MgSO4, and plated onto Luria Broth (LB) agar containing 200µg/mL neomycin and 100µg/mL Irgasan. Colonies
were picked with a Qbot into 250µL LB containing 200µg/mL kanamycin and 50µg/mL irgasan and grown at 37°C statically
approximately 40 hours. 70µL of each culture was transferred robotically to plates for arbitrary PCR and sequencing.
60µL 60% glycerol was added to the remaining culture in the working plates before transfer to 384-well plates for long-term storage.
ii. MAR2xT7 Matings
Tripartite matings were performed using saturated cultures of PA14, MC4100 carrying pMAR2xT7 and E.
coli carrying pRK2013. 1:2:2 mixtures of the cultures respectively, were dropped on Kings' B media plates and incubated at
37°C for 2 hours before collection and plating on Luria broth (LB) agar containing 15µg/mL gentamycin and 1µg/mL irgasan.
Colonies were picked with a Qbot (Genetix, Hampshire, United Kingdom) and grown statically in 96-well "working" plates
containing 280µL LB and 15µg/mL gentamycin for approximately 40 hours at 37°C. 70µL of each culture was transferred
robotically to plates for arbitrary PCR and sequencing. 70µL 60% glycerol was added to the remaining culture in the
working plates (for a final glycerol concentration of 15%) before transfer to 384-well plates for long-term storage.
II. Arbitrary PCR and Sequencing
i. First-Round Arbitrary PCR
70 μL culture transfered from original working culture plates to polypropylene plates were stored at -20°C.
Plates were thawe, incubated at 99°C for 10 minutes to lyse the cells and spun at 3500 rpm for 5 minutes to pellet cell debris.
3 μL of lysate was used as template for the first round of arbitrary PCR (ARB 1). ARB1 PCR reaction mix included 1x Taq Buffer (Roche),
10% DMSO, 2.5 μM dNTPs, 1.25 U of Taq polymerase (Roche) and 1.0 ng/μL of each of primer. For MAR2xT7 mutants, the transponson-specific
primer, PMFLGM.GB-3a, (5'-TACAGTTTACGAACCGAACAGGC-3') was used. For TnPhoA mutants, Tn5Ext (5'-GAACGTTACCATGTTAGGAGGTC-3') was
used as the transposon-specific primer. We found that different arbitrary (ARB 1) primers were more effective at different times
during the project. The ARB 1 primer with the highest success rate was ARB1D (5'-GGCCAGGCCTGCAGATGATGNNNNNNNNNNGTAT-3'). Plates
were sealed with adhesive foil (Diversified Biotech Alum-1000). After initial denaturation at 95°C for 5 minutes, plates
(Corning Incorporated Costar #6511) were cycled 30 times at 95°C for 30 seconds 47°C for 45 seconds and 72°C for 1 minute.
Plates were incubated 5 minutes at 72°C to allow for extension of PCR products. PCR products were stored at 4°C.
ii. Second-Round Arbitrary PCR
5 μL ARB1 reaction was used as template for the ARB 2 PCR reaction. ARB 2 reaction mix included 1x Taq Buffer
(Roche), 10% DMSO, 2.5 μM dNTPs, and 1 ng/μL of each the transposon specific primer and ARB2 primer. For MAR2xT7 mutants, the
PMFLGM.GB-2a primer (5'-TGTCAACTGGGTTCGTGCCTTCATCCG-3') was used. For TnPhoA mutants,
Tn5Int2 (5'-GGAGGTCACATGGAAGTCAGATCCTGG-3') was used as the transposon-specific primer. The ARB 2 primer,
ARB2A (5'-GGCCAGGCCTGCAGATGATG-3') was used. Plates were sealed with adhesive foil. Reactions were cycled 40 times:
95°C for 30 seconds, 45°C for 30 seconds and 72°C for 1 minute with a 5 minute extension at 72°C.
Plates were stored at 4°C.
iii. PCR Cleanup
5 μL ARB2 reaction was mixed with 2 μL EXOSAP-IT enzyme mix (USB #78205) in a new reaction plate (Abgene #AB-0800).
Plates were sealed with adhesive foil and incubated according to the manufacturer's instructions.
iv. Sequencing Preparation
Sequencing primer was added directly to each PCR cleanup reaction for a final concentration of 5 ng/μL.
For MAR2xT7 mutants, PMFLGM.GB-4a (5'-GACCGAGATAGGGTTGAGTG-3') was used as the sequencing primer. For TnPhoA mutants, the
Tn5Int primer (CGGGAAAGGTTCCGTTCAGGACGC-3') was used.
III. PA14NR Set
i. Mutant Selection Criteria
Selection of mutants for the non-redundant set was automated by creating a set of priorities. Mutants
were prioritized as follows.
- The mutants of each individual gene were divided into two groups: those with BLAST bit
scores >=80, and those with scores <80. Priority was given to those with blast scores above 80.
- Both of these two groups
were further subdivided by background and transposon, with priority given to PA14/MAR2xT7 over PA14/PhoA and PA14/PhoA over
exoU/MAR2xT7 or exoU/spcU/MAR2xT7.
- Each of these six groups were ordered by distance of the transposon insertion site
from the start of the gene, with priority given to more 5' insertions.
- Lastly, the mutants were ordered by BLAST bits score such that,
if all other criteria are equal, the mutant with the higher BLAST score had priority. In cases where there was a mutant
available in the PA14 background, but a more 5' mutant was available in either the exoU or exoU/spcU background, both mutants
were included in the set.
ii. Colony Purification
Mutants were picked manually from frozen 96-well working plates on dry ice and streaked onto LB agar
containing 15µg/mL gentamycin. Plates were incubated at 37°C for 14-16 hours, cooled to room temperature and stored
at 4°C for no more than 8 days before picking. Colonies were examined under a dissecting scope. A single colony with
a morphology representative of the majority of colonies for a given mutant was used to inoculate 600µL LB, 15µg/mL gentamycin
in a 96-deep well Master block (USA Scientific). Small colony variants were avoided except in cases where non-variants were not
iii. Culture Conditions
Master deep well blocks were incubated in a HiGro (Genomic Solutions, Ann Arbor, MI) set to 37°C with O2
injection for 2 seconds every 5 minutes of incubation for 14-16 hours to select against RSCV. Plates were set at room temperature for 0-6 hours
before transferring culture to Storage plates.
iv. Biomek Transfer to Master plates
Culture transfer methods were performed using a Biomek FX (Beckman Coulter, Inc., Fullerton, CA). 200µL
sterile 60% glycerol was transferred from a reservoir to each deep well Master block and mixed 3 times by pipeting up and
down 200µL at a time. Tips were touched to the side of the wells to remove liquid clinging to the end of the tips and
discarded. Fresh tips were used to transfer 40µL of the mixed cultures to each of 10 96-well Storage plates containing 160µL
LB with 15µg/mL gentamycin and 15% glycerol. The Biomek FX was set to pipet up culture 11 mm from the top of the culture for
each transfer to minimize the surface area of the transfer tip coated with culture. Master blocks and Storage plates were sealed
(Alumina Seals, Diversified Biotech, Boston, MA) and stored and -80°C immediately after transfer.