Experiment details.
Strain: Escherichia coli strain MG1655 (F-
- ilvG rfb50 rph1) (Jensen, 1993) was used.
Transposome formation and transposon mutagenesis. Plasmid pMOD<MCS> containing
artificial transposon EZ::TN<KAN-2> (Epicentre Technologies, Madison, WI) was isolated from the
MG1655 strain to avoid restriction/modification problems. Transposon DNA was released by PvuII
digestion, as recommended by the manufacturer and gel purified using QIAquick Gel Extraction
columns (Qiagen, Valencia, CA). Transposomes were pre-formed by incubating 7 ng/µl transposon
DNA with 0.1 U/µl hyperactive Tn5 EZ::TN transposase (Epicentre Technologies and generous gift
from W. Reznikoff) in 40 mM Tris-acetate, pH 7.5, 100 mM potassium glutamate, 0.1 mM EDTA, 1
mM dithiothreitol, and 0.1 mg/ml tRNA. Samples were incubated for 30 minutes at 37oC and
dialyzed against 10 mM Tris-acetate, pH 7.5, 1 mM EDTA on 0.025 micron filters (Millipore,
Bedford, MA) for 1 hour. Dialyzed samples were mixed with electrocompetent E. coli in 1:2 ratio
(v/v) and transformed by electroporation. Cultures were immediately diluted with an LB-based rich
media (see below) without kanamycin and incubated at 37oC for 40 minutes with gentle agitation.
The efficiency of electroporation for E. coli strains MG1655 and DH10B was 5x104 and 2x106 Km-
resistant colonies per 1 µg of transposon DNA respectively.
Outgrowth of mutagenized population. Half of the mutagenized population was immediately
frozen and stored as the time zero sample. The rest of the culture was used to inoculate a BIOFLO
2000 fermentor (New-Brunswick Scientific, Edison, NJ) containing 950 ml of the following media:
tryptone 10 g/L, yeast extract 5 g/L,
NaCl 50 mM,
NH4Cl 9.5 mM,
MgCl2 0.528 mM,
K2SO4 0.276 mM,
FeSO4 0.01 mM,
CaCl2 5x10-4 mM,
and K2HPO4 1.32 mM; supplemented with micronutrients:
(NH4)6(MoO7)24 3x10-6 mM,
H3BO3 4x10-4 mM,
CoCl2 3x10-5 mM,
CuSO4 10-5 mM,
MnCl2 8x10-5 mM,
and ZnSO4 10-5 mM
(Neidhard et al., 1974). The following vitamins were added (mg/L): biotin
0.12, riboflavin 0.8, pantothenic acid 10.8, niacinamide 12.0, pyridoxine 2.8, thiamine 4.0, lipoic
acid 2.0, folic acid 0.08, and p-aminobenzoic acid 1.37. Kanamycin was added to 10 µg/ml.
Throughout the fermentation temperature was held at 37o C, dissolved oxygen at 30-50% of
saturation, and the pH at 6.95 (via titration with 5% H3PO4). Media and growth conditions were
designed to minimize the number of genes required for cell survival. Cells were grown in batch
culture for 23 population doublings (12 hrs) to a cell density of 1.4x109. Genomic DNA was isolated
and used to generate genetic footprints.
Detection of transposon insertions by nested PCR. Two pairs of primers were used
consecutively, with the second pair of primers nested within the first as illustrated in Figure 1. Each
primer pair contained one transposon-specific primer and one chromosome-specific primer.
Chromosome-specific landmark primers were designed as an ordered set of unidirectional nested
primer pairs covering the entire E. coli genome using custom software. Pairs were separated on
average by 3500 bp, while primers within each pair were separated by the shortest possible distance
in the range: 3 to 900 bp. Average primer length was 27 bp (sequences available upon request).
Transposon-specific primers were chosen to avoid any significant similarity with the E. coli
chromosome, using PrimerSelect software (DNASTAR, Inc., Madison, WI). Two pairs of nested,
outwardly directed transposon-specific primers (one at each end) were used to detect transposons
inserted in both orientations. The forward primer pair includes
an external primer 5'-
GTTCCGTGGCAAAGCAAAAGTTCAA-3' and
an internal primer 5'-
GGTCCACCTACAACAAAGCTCTCATCA-3'. The reverse primer pair includes
an external
primer 5'-CCGACATTATCGCGAGCCCATTTAT-3' and
an internal primer 5'-
GCAAGACGTTTCCCGTTGAATATGGC-3'.
The first of two consecutive PCR amplifications (the external PCR reaction) was performed
under the following conditions: 95oC for 1 min; 94oC for 12 s, 70oC for 6 min (2 cycles); 94oC for 12
s, 69oC for 6 min (2 cycles); 94oC for 12 s, 68oC for 6 min (36 cycles); 68oC for 6 min.
Amplification reactions contained: 0.3 µg of template DNA (equivalent of 6x107 E. coli genomes),
0.2 mM each dNTP, 0.4 µM each primer, PCR buffer (40 mM Tricine-KOH pH 9.2, 15 mM
potassium acetate, 3.5 mM magnesium acetate, 3.75 µg/ml BSA), and 0.4 µl of Advantage cDNA
Polymerase Mix (CLONTECH Laboratories, Palo Alto, CA) in 20 µl. The second internal PCR was
performed in the same reaction mix, except the DNA templates consisted of the products of the first
PCR diluted 103-fold. Amplification conditions for internal PCR were: 95oC for 1 min; 94oC for 12
s, 69oC for 6 min (2 cycles); 94oC for 12 s, 68oC for 6 min (9 cycles); 68oC for 6 min. The products
of the internal PCR reactions (3 µl aliquots) were size-separated on 0.65% agarose gels. All insert
detection and analysis procedures were performed in 96-well format.
Image capture and analysis were performed with 1D Image Analysis Software (Eastman Kodak
Company, Rochester, NY). Mapping of the detected Tn5 insertions was done using custom software,
which calculates insert positions within a genome sequence using the addresses of the internal
landmark primers and the size of the corresponding PCR products. Visualization of insert locations
was done using custom software Chromosomal Viewer integrated into the ERGO database
(Overbeek et al., 2003).
References
Jensen, K. F. (1993). The Escherichia coli K-12 wild types W3110 and MG1655 have rph
frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels.
Journal of Bacteriology 175, 3401-3407.
Neidhard, F. C., Bloch, P. L., and Smith, D. F. (1974). Culture Medium for Enterobacteria.
Journal of Bacteriology 119, 736-747.
Overbeek, R., Larsen, N., Walunas, T., D'Souza, M., Pusch, G., Selkov, E. J., Liolios, K.,
Joukov, V., Kaznadzey, D., Anderson, I., et al. (2003). The ERGO(TM) genome analysis and
discovery system. Nucleic Acids Res 31, 164-171.
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