Bacterial strains and growth conditions
E. coli JM109 [32]
was used as a host for the cloning experiments, the measurement of
fluorescence intensity, plasmid stability experiments and microscopy.
Additionally, E. coli W3110 [33], the rhaB- derivative BW3110, BL21 [34] and the rhaB- derivative BL21 Rha- were used for microscopy. LB liquid and LB agar plates were used as complete medium [35], supplemented with 100 μg ml-1 ampicillin. For induction of the rhaPBAD promoter, sterile filtered L-rhamnose was added as indicated.
General methods
Restriction enzymes and DNA modifying enzymes were purchased from
Roche Applied Science (Mannheim, Germany). For restriction enzyme
analysis and cloning experiments, standard methods were used [36]. Plasmid DNA was isolated according to a published protocol [37]. E. coli was transformed with plasmid DNA as described by Chung et al.[38]. All changes made in the expression vectors during this study were confirmed by DNA sequencing of the corresponding regions.
Construction of plasmids
Table 1 lists the plasmids used in this study, Figure 1 gives a schematical overview.
Plasmid pJOE4056.2 is a fully sequenced expression vector derived from pJOE3075 [39] containing eGFP as a reporter gene, which is positively controlled by the inducible rhaBAD promoter (rhaPBAD) together with the CRP-cAMP binding site, a ribosomal binding site and a transcription terminator from the rrnB operon. The plasmid pJOE5058.1 was made by restriction endonuclease digestion of pJOE4056.2 with EcoRI and AflII,
and the 5297-bp fragment was then ligated to the oligonucleotides S3977
(5'-AAT TCA GGC GCT TTT TAG ACT GGT CGT AGG GAG ACC ACA ACG GTT TCC CTC
TAG AAA TAA TTT TC-3') and S3978 (5'-TTA AGA AAA TTA TTT CTA GAG GGA
AAC CGT TGT GGT CTC CCT ACG ACC AGT CTA AAA AGC GCC TG-3'), in order to
replace the original transcription initiation region by that of the
gene 10 from bacteriophage T7 (rhaPBAD-T7sl).
The ccd locus of plasmid F consisting of the two genes ccdA and ccdB was
amplified by PCR using the primers S3991 (5'-GGC GCG CTG ATT TGT GCG
GCA TAA-3') and S3992 (5'-GGC TGC CCG GCA GAA TAC ACT GCC-3'). A lysate
of E. coli JM109 obtained by boiling and subsequent
centrifugation was used as a template. The 667-bp fragment was treated
with Klenow enzyme to remove overhanging nucleotides at the 3'-ends and
inserted into the positive selection vector pJOE4780.1, which was cut
with the restriction endonuclease NaeI, to get pWA17.2. The
DNA sequence was verified by sequencing with the primers S3767 (5'-TAA
TAC GAC TCA CTA TAG GG-3') and S3768 (5'-ATT TAG GTG ACA CTA TAG-3').
The plasmid pWA19 was received by cleaving pWA17 with NaeI and ligating the ccdAB containing fragment into pJOE5058.1, which was cut with the same restriction enzyme.
In order to remove one of the two cer regions, the plasmids pJOE5058.1 and pWA19 were cut with MluI,
a 264 bp-fragment was excised, the vectors were religated to obtain the
plasmids pWA21 and pWA22, respectively. For the construction of the
plasmids pWA23 and pWA28 the rop locus was eliminated by restriction endonuclease digestion with PvuII and NaeI and religation of pJOE5058.1 and pWA21, respectively. Similarly, the rop locus of pWA19 and pWA22 was excised by cutting with PvuII and KpnI,
removing the protruding 3'-overhang with Klenow enzyme, and religating
the DNA to get the plasmids pWA24 and pWA29, respectively. The plasmids
pWA124.1 and pWA125.1 were made by restriction endonuclease digestion
of pJOE4056.2 and pWA21, respectively, with EcoRI and AflII, a subsequent Klenow fill-in of the recessed 3'-ends and religation.
To study the influence of the CRP-binding site, the promoter region of pWA21 was cut out with the restriction endonucleases MluI and XbaI
and the fragment was ligated into pIC20HE, which was cut with the same
restriction enzymes, to obtain the plasmid pWA58.1. A site-directed
mutagenesis was performed by replicating both strands in a PCR reaction
with Pfu DNA polymerase using the primers S4113 (5'-CAG CAA
ATT GTG ATC ATG ATC ACA TTC ATC TTT CCC TGG TTG CC-3') and S4114
(5'-GGC AAC CAG GGA AAG ATG AAT GTG ATC ATG ATC ACA ATT TGC TG-3'),
which do not only change the nucleotide sequence in the CRP-binding
site but additionally introduce a new restriction site for BspHI. The template was degraded by a digest with DpnI, an enzyme which cuts only dam methylated
DNA and leaves the newly synthesized untouched. The nicked vector DNA
bearing the desired mutation then was transformed into E. coli JM109 via electroporation. Colonies were picked, the DNA was isolated and tested in a BspHI
digest, then the CRP-binding region of the plasmids pWA61.12 and
pWA70.7 was sequenced with the oligonucleotide S4083 (5'-GGC TCG TAT
GTT GTG TGG-3') which binds in lacZ. The modified CRP-binding
regions were reintroduced into the pWA21 context by means of cloning,
resulting in the plasmids pWA64.1 and pWA73.1. The sequence in pWA73.1
is adjusted to the consensus sequence, whereas pWA64.1 differs from
this sequence as one nucleotide is missing.
Table 2 lists the sequences of CRP-binding sites in the described plasmids, comparing the consensus sequence with the original rhaPBAD sequence and the modified ones.
Measurement of fluorescence intensity
20 ml LB in a sterile 100 ml flask were inoculated with 200 μl
of an overnight culture and incubated at 37°C for 2 h shaking with
approximately 200 rpm. Then the cultures were induced with 0.2% (w/v)
L-rhamnose and shifted to 30°C. Samples were taken every 60 min, and
the OD600 was measured. The samples were diluted to an OD600 = 0.1 with LB. For the measurement of fluorescence, three aliquots of 100 μl
of each cell suspension were added to 96 well flat-bottom polystyrene
microplates (Greiner, Germany), and the fluorescence was measured with
a GENios fluorometer (Tecan, Austria), at an excitation wavelength of
485 nm and an emission wavelength of 535 nm. The background
fluorescence was determined by using E. coli JM109 cultures without plasmid.
Determination of plasmid stability
20 ml LB with 0.2% (w/v) L-rhamnose in a sterile 100 ml flask were
inoculated with approximately 1000 cells derived from an overnight
culture grown in LBamp and incubated at the indicated
temperature for 24 h shaking with roughly 200 rpm. 1000 cells of this
induced culture were used to inoculate the following one, again 20 ml
LB with 0.2% (w/v) L-rhamnose, and incubated for another 24 h. The OD600 of
the culture was measured, the samples were diluted in LB and plated on
LB agar. The plates were incubated for 12 h at 37°C, then 100 colonies
of each sample were stroked on LBamp agar with 0.2% (w/v) L-rhamnose and again incubated for 12 h at 37°C.
The estimation of 1000 cells is based upon the assumption that one ml culture of an OD600 = 1 matches 109 cells.
Microscopy
An overnight culture was diluted 1:100 and the cells were grown in
20 ml LB for 24 h at 30°C with 0.2% (w/v) L-rhamnose, L-arabinose or
D-glucose where indicated. A drop of the living culture was placed
between a microscope slide and a cover glass. The cells were viewed
immediately with a Plan-APOCHROMAT 100×/1,4 oil dic objective on a
Zeiss Axioplan 2 with Immersol™ 518F (Zeiss, Germany), using a
combination of UV and visible (phase-contrast) light. Photographs were
captured with a monochrome cooled CCD camera (AxioCam MRm, Zeiss,
Germany) controlled by AxioVision 3.1. For each culture the lengths of
100 cells were determined.
Answers to all your Biology Questions
