8. Send round 2 nested PCR product for Sanger sequencing
analysis using oligo 3 as the sequencing primer (i.e., either
TnKN1 or TnKN2) (see Note 46).
9. Use sequencing results to run a BLAST analysis to determine
transposon insertion site (see Notes 47 and 48).
10. Confirmation of transposon insertion site is conducted by
running standard PCR with primers which flank approximately
100 bp proximal and distal to insertion site, which should
increase PCR product corresponding to size of transposon
insertion.
4 Notes
1. Ensure EMJH base has cooled to at least 50
C before adding
BSA supplement or rabbit serum.
2. Can also add 0.0001% superoxide dismutase (SOD), 1% lactalbumin hydrolysate, and 0.004% sodium pyruvate to further
enrich media and improve growth of more sensitive Tn
mutants.
3. Saprophytic Leptospira which may be present in municipal
water are thin enough to fit through a 0.22 μm filter membrane. Therefore, we autoclave our ultrapure water before
addition of BSA and other growth factors to ensure no microorganisms contaminate the medium.
4. Please bear in mind BSA enrichment supplement should not be
warmed above 50–55
C as BSA can denature.
5. A low passage, verified, or sequenced strain should be used for
generation of the transposon mutant library. This helps to
avoid a secondary phenotype as a result of SNPs that may be
present in the parental strain [43].
6. Preliminary studies in our lab of transformation efficiency of
Himar1 via conjugation with the saprophytic strains L. meyeri
and L. yanagawae did not result in a high yield of transconjugants. Therefore, these strains are not recommended until
expertise of this protocol is completed first with the highly
transformable strain L. biflexa.
7. pSW29T-TKS2 contains the Himar1 random transposon system which confers kanamycin resistance for transconjugant
selection along with a c9 hyperactive transposase, Ori R6K
and OriT RP4. OriT and c9 are located outside of the transposable element. Other plasmids carrying the Himar1 system are
also useful for random transposon mutagenesis, such as
pAL-614 [44], which confers spectinomycin resistance. However, kanamycin resistance-conferring transposons are
88
Christopher J. Pappas et al.
analysis using oligo 3 as the sequencing primer (i.e., either
TnKN1 or TnKN2) (see Note 46).
9. Use sequencing results to run a BLAST analysis to determine
transposon insertion site (see Notes 47 and 48).
10. Confirmation of transposon insertion site is conducted by
running standard PCR with primers which flank approximately
100 bp proximal and distal to insertion site, which should
increase PCR product corresponding to size of transposon
insertion.
4 Notes
1. Ensure EMJH base has cooled to at least 50
C before adding
BSA supplement or rabbit serum.
2. Can also add 0.0001% superoxide dismutase (SOD), 1% lactalbumin hydrolysate, and 0.004% sodium pyruvate to further
enrich media and improve growth of more sensitive Tn
mutants.
3. Saprophytic Leptospira which may be present in municipal
water are thin enough to fit through a 0.22 μm filter membrane. Therefore, we autoclave our ultrapure water before
addition of BSA and other growth factors to ensure no microorganisms contaminate the medium.
4. Please bear in mind BSA enrichment supplement should not be
warmed above 50–55
C as BSA can denature.
5. A low passage, verified, or sequenced strain should be used for
generation of the transposon mutant library. This helps to
avoid a secondary phenotype as a result of SNPs that may be
present in the parental strain [43].
6. Preliminary studies in our lab of transformation efficiency of
Himar1 via conjugation with the saprophytic strains L. meyeri
and L. yanagawae did not result in a high yield of transconjugants. Therefore, these strains are not recommended until
expertise of this protocol is completed first with the highly
transformable strain L. biflexa.
7. pSW29T-TKS2 contains the Himar1 random transposon system which confers kanamycin resistance for transconjugant
selection along with a c9 hyperactive transposase, Ori R6K
and OriT RP4. OriT and c9 are located outside of the transposable element. Other plasmids carrying the Himar1 system are
also useful for random transposon mutagenesis, such as
pAL-614 [44], which confers spectinomycin resistance. However, kanamycin resistance-conferring transposons are
88
Christopher J. Pappas et al.