Fig 1.
SbcCD/palindrome-induced replication-dependent site-specific DNA double-strand break system in E. coli.
A) A schematic representation of E. coli circular chromosome showing bidirectional replication originating from oriC and the location of the palindrome within the lacZ region in the right replichore. B) An illustration showing SbcCD-mediated cleavage of the 246bp interrupted palindrome that results in a two-ended DSB. The palindrome is highlighted by orange arrows.
Fig 2.
Optimisation of psoralen crosslinking using different concentrations of psoralen.
A) NdeI digestion map of the region surrounding the DSB. NdeI restriction sites and the distance between them are marked with blue vertical arrows and numbers (in kb), respectively. The palindrome and the radiolabelled probe that was used to detect the fragment are marked by an orange triangle and a red line, respectively. Ori and Ter mark the orientation of the DNA fragment with respect to the origin and terminus of DNA replication. B) Detection of DNA fragments by Southern blotting and hybridization of samples treated with different concentrations of psoralen (0–50 μg/ml). ‘+’ indicates that the sample was boiled and immediately cooled and ‘-’ indicates that the sample was not boiled. 1 kb NEB ladder was used as a marker. The control DNA samples were neither treated with TMP nor irradiated.
Fig 3.
Separating DNA fragments of interest from the rest of the chromosome.
A) I-SceI digestion of the chromosome will produce two DNA fragments: an extremely big fragment and a much smaller segment containing the DSB region (palindrome shown by an orange triangle). B) Using a synthetic region devoid of 4-base cutting sites of REs, when digested by the frequent cutters, the rest of the chromosome will be cleaved into small fragments, leaving the 40 kb DSB region (palindrome) intact. C) Design of the 40 kb synthetic DNA region. The palindrome, Chi arrays, and kanamycin resistance gene are indicated by the orange triangle, orange angle bracket and a blue box, respectively. Different restriction sites are marked with black vertical lines and different coloured vertical arrows. The 4 base cutters (BstUI and HhaI) that have been removed from the synthetic region and been used in this study are shown in the homology arms. The selective restriction sites that are kept in the synthetic DNA are shown below the sequence. The 5 kb homology arms to E. coli chromosome are indicated by green boxes.
Fig 4.
Gel electrophoresis analysis of I-SceI digested DNA fragments.
A): I-SceI, NdeI and SalI digestion maps of the E. coli chromosomal region surrounding the DSB site. The restriction sites and the distance between them are marked with blue vertical arrows and numbers (in kb), respectively. The DSB site is marked by an orange triangle. Ori and Ter mean origin and terminus, respectively. The radiolabelled probe that was used to detect the fragment is marked by a red line. B): Southern blot of gel electrophoresis of a DSB+ and a DSB- strain where a radioactive probe is used to detect a 26 kb, 14 kb and 16 kb DNA fragments surrounding the DSB site, resulting from I-SceI digestion, a 8 kb DNA fragment resulting from NdeI digestion and a 23 kb DNA fragment resulting from SalI digestion. The position of the well and the linear fragments are shown by horizontal black arrows. 1 kb NEB ladder was used as a marker. C): Quantification of branched DNA molecules represented as the proportion of well DNA content out of the sum of well DNA band and gel DNA band. The values are shown on the top of each bar. Strains used were DL5743 (ΔruvAB, lacZ::246, DSB+, I-SceIcs across 26 kb),DL5744 (ΔruvAB, lacZ+, DSB-, I-SceIcs across 26 kb), DL5745 (ΔruvAB, lacZ::246, DSB+, I-SceIcs across 14 kb), DL5746 (ΔruvAB, lacZ+, DSB-, I-SceIcs across 14 kb), DL5747 (ΔruvAB, lacZ::246, DSB+, I-SceIcs across 16 kb), DL5748 (ΔruvAB, lacZ+, DSB-, I-SceIcs across 16 kb). Error bars represent the standard error of the mean where n = 3.
Table 1.
Conventional gel electrophoresis conditions that were tested.
Fig 5.
Gel electrophoresis analysis of DNA isolated from the wells of an electrophoresis gel upon β-agarase treatment.
A) The steps of the entire protocol are outlined. B) Southern blot of the first electrophoresis gel of the 26 kb DSB region resulting from I-SceI digestion. C) Southern blot of the second electrophoresis gel of the β-agarase treated DNA that was recovered from the well of the first gel. The solution resulting from β-agarase treatment of one agarose plug from the first gel was loaded into three lanes in the second gel. Both DSB+ and DSB- strains were used. Strains used were DL5743 (ΔruvAB, lacZ::246, DSB+), and DL5744 (ΔruvAB, lacZ+, DSB-). The marker used was 1 kb DNA ladder from NEB.
Fig 6.
Effects of β-agarase treatment on DNA degradation.
Detection of β-agarase treated DNA fragment that was trapped in the agarose plugs after the 1st gel electrophoresis analysis. The first lane represents non-crosslinked DNA fragment containing agarose plug that was washed with only TAE buffer and afterwards boiled without β-agarase treatment, and then run again in this gel. Lanes 2–4 represent non-crosslinked DNA that was washed with only TAE buffer and then treated with β-agarase. The samples in lanes 5–7 are non-crosslinked DNA that was washed with both TAE and β-agarase buffer and then treated with β-agarase. The samples in lanes 8–10 and 11–13 are crosslinked DNA that was equilibrated with only TAE buffer, and TAE followed by β-agarase buffer, respectively, before a β-agarase digestion reaction. The sets of three lanes, containing the DNAs treated similarly, are technical replicates of the same β-agarase treated samples.
Fig 7.
Second gel electrophoresis for further purification of branched DNA molecules.
A) I-SceI digestion map of the E. coli chromosomal region surrounding the DSB site. The restriction sites and the distance between them are marked with blue vertical arrows and numbers (in kb), respectively. The DSB site is marked by an orange triangle. The radiolabeled probe that was used to detect the fragment is marked by a red line. Ori and Ter mean origin and terminus, respectively. B) Southern blot of a gel electrophoresis where two types of DNA samples are represented including DNA sample undergoing 1st gel electrophoresis and DNA sample recovered from the 1st gel well and undergoing 2nd gel electrophoresis. C) Quantification of the proportion of well DNA content out of the sum of well and gel DNA band after 1st and 2nd gel electrophoreses. The values are shown on the top of each bar. Strains used were DL5743 (ΔruvAB, lacZ::246, DSB+), and DL5744 (ΔruvAB, lacZ, DSB-). NEB 1 kb DNA ladder was used. Error bars represent the standard error of the mean where n = 3.
Fig 8.
Gel electrophoresis analysis of the synthetic DNA.
A) Ethidium bromide staining of a 0.5% agarose gel containing genomic DNA digested with either only HhaI or a combination of HhaI and BstUI. B) Detection of the synthetic DSB region following Southern hybridisation of the gel in A. Both DSB+ and DSB- cultures of DL7672 were used. A radiolabelled probe was used to detect the synthetic region. The probe did not detect the marker. C) Quantification of the well DNA content out of the total lane is shown.
Fig 9.
Quality of final DNA sample as depicted by agarose gel electrophoresis and staining following two rounds of gel electrophoresis.
Ethidium bromide staining of the gel electrophoresis of β-agarase treated DNA that had undergone DSB and recovered from the wells of a 2nd gel.
Fig 10.
Optimized protocol of sample preparation for studying branched DNA molecules under TEM.