3.1 Preparation
for Conjugation
1. One to two weeks prior to conjugation, aliquot L. biflexa into
25 mL of fresh EMJH liquid media in a 50 mL conical, and
incubate at 30
C with or without agitation until bacteria reach
mid-late exponential growth (2–3 Â 10
8 cells/mL) (see Note
13).
2. One day prior to conjugation, aliquot E. coli β2163 harboring
pSW29T-TKS2 from freezer culture into 5 mL of fresh LB
liquid medium adulterated with 0.3 mM DAP and 50 μg/mL
of kanamycin. Incubate overnight at 30
C (see Note 14).
3. The morning of conjugation, transfer 0.5 mL of the E. coli
incubated overnight into 4.5 mL of fresh EMJH liquid
medium adulterated with 0.3 mM DAP (without kanamycin)
(see Notes 15–17).
Table 1
Primers used for Tn insertion site determination
Primer name Sequence (5
0
! 3
0 )
Purpose
Expected
size
VTK1-TKS2
GATCATGATATCGATTACAAGG
Transposon-specific
sequence
258 bp
VTK2-TKS2
GTTAATATTTTGTTAAAATTCGCG
LEPBIa0024F AATATAAAACTCTCGATTCGCTTAACA Amplification of
LepBIa0024 in L. biflexa
500 bp
LEPBIa0024R TCACAATTGCTTGTGTCAACC
Deg1
GGCCACGCGTCGACTAG
TACNNNNNNNNNNGATAT
Semi-random degenerate
primer for first round of
nested PCR with 5’ Tag
N/A
Deg2
GGCCACGCGTCGACTAG
TACNNNNNNNNNNTCTT
N/A
TnK1
CTTGTCATCGTCATCCTTG
Transposon-specific primer
for first round of nested
PCR
N/A
TnK2
GTGGCTTTATTGATCTTGGG
Transposon-specific primer
for first round of nested
PCR
N/A
TnKN1
CGTCATGGTCTTTGTAGTCTATGG
Transposon-specific primer
for second round of
nested PCR
Varies
TnKN2
TGGGGATCAAGCCTGATTGGG
Transposon-specific primer
for second round of
nested PCR
Varies
Tag
GGCCACGCGTCGACTAGTAC
Tag sequence of semirandom degenerate
primers for second round
of nested PCR
Varies
Random Transposon Mutant Library
81