Biology Reference
In-Depth Information
oligo ligation. Four oligos were synthesized, which when annealed together left
a sticky end corresponding to the desired restriction site. The oligos were diluted
to 20
µ
l, and complementary sets were annealed together by mixing, heating to
95°C for 10 minutes, then cooling to room temp gradually. The annealed oligos
were kinased, and finally mixed with the vector in a ligation reaction, producing
pTSH117, with 10 bp within the fim and hin mirrored inverted repeats, and
pTSH118, with 30 bp within the fim mirrored inverted repeats and 10 bp within
the hin mirrored repeat.
The strong hairpins formed by the mirrored pairs prevented sequencing
through them, so their successful insertion was verified by checking the insert
length via PCR and by the presence of an un-sequenceable hairpin. A construct
containing hixC in place of the hix mirrored pairs was also made (pTSH89, 10-
bp gap fim mirrored repeat, pTSH90, 30-bp gap fim mirrored repeat). The com-
pleted switch, which includes hixC, the mirrored fim repeats, the terminator, gfp
and rfp, were named pTSH97 (10 bp gap fim mirrored pair) and pTSH98 (30 bp
gap fim mirrored pair), respectively.
testing
The testing of the double inversion switch was performed in vivo, in E. coli
DH10B. The vector containing the switch, along with any reporter fluorescent
genes, was co-transformed with another vector containing the recombinases fimB
and hin. The expression of FimB and Hin was performed through induced ex-
pression via P
BAD
(fimB) and P
Te t
(hin). The ribosome binding sites (RBS) of the
genes were mutated to adjust the expression level. This was done via addition of
overhanging sequences during PCR.
Induction experiments were performed as follows. The strain was grown at
30°C overnight in LB medium containing kanamycin (50 µg/ml), chlorampheni-
col (30 µg/ml), and dextrose (5% w/v). Dextrose was added to prevent spurious
PBAD expression. The overnight culture (50 µl) was inoculated into 2 ml LB
medium containing kanamycin and chloramphenicol and incubated at 37°C with
shaking. After reaching an OD600 of 0.2 to 0.5, the inducer (10 mM arabinose
for fimB induction and/or 100 nM anhydrous tetracycline (aTc) for hin) was
added. The culture was incubated at 37°C with shaking for 6 hours or overnight.
Control cultures were inoculated into non-inducing medium. To test for states 3
and 4, the cultures in states 1 and 2 were inoculated into fresh medium (50 µl of
culture into 2 ml of LB) without an inducer, grown overnight, and induced with
the complement inducers.
After the prescribed induction time, a 1 ml sample of the culture was centri-
fuged (15,000×g, 1 minute), and the pelleted culture was used as the template for
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