Bottom-up genome assembly using the Bacillus subtilis genome vector

Transcription

1 Bottom-up genome assembly using the Bacillus subtilis genome vector Mitsuhiro Itaya, Kyoko Fujita, Azusa Kuroki & Kenji Tsuge Supplementary figures and text: Supplementary Table 1 Primers used to design dominos for mouse mtgenome. Supplementary Table 2 Primers used to design dominos for rice cpgenome. Supplementary Figure 1 Step-by-step integration of dominos in the BGM vector. Supplementary Figure 2 Nucleotide inheritance from dominos to mtg. Supplementary Figure 3 Cloned mtg separated by I-PpoI digestion. Supplementary Figure 4 Block elongation to completion of rice cpg. Supplementary Figure 5 Altered nucleotide sequences found in cloned cpg. Supplementary Methods 1

2 Supplementary Table 1. Primers used to design dominos for mouse mtgenome. Sequence data of strain BALB/c was downloaded from the web site: ( MMU txt). domino# domino name forward primer reverse primer 1 pc[ ] TTAGAATTCGCAGCCTGCGAAGCAGC ATACGGCCGGGACCAAACCTTTGTG pc[ ] TTAGAATTCGCAGCCTGCGAAGCAGC ATACGGCCGGGACCAAACCTTTGTG 2 pc[ ] CTAGTAAAGAATTCAAACACGTACGGAACAGATTAC pe[ ] ATACGGCCGGGACCAAACCTTTGTG GGACTGTAAGAATTCATCCTACAT GTACGGCTGTGGATCCGTTCG 3 pe[ ] GGCACCCAGAATTCTATATTCTTATCCTCCCAGG ATACGGCCGGGACCAAACCTTTGTG pc[ ]ci AAACTGCAGACCGGAGCAATCCAGGTC ATAAAGATCTGGGTGCCCAAAGAATC 4 pc[ ] ATTAATAAGCGGCCGCTACTCTCTACAAACACTTATTAC ATAAAGATCTGGGTGCCCAAAGAATC pe[ ] TTATTATCGCGGCCGCAGCAATCGTTCAC AAAAGATCTCGGATCCATAGGAATGTTG

3 Supplementary Table 2. Primers used to design dominos for rice cpgenome. Sequence data was provided by reference 15. domino# domino name forward primer reverse primer 1 pe[ ] TTTTTTTTCCTGCAGGTCTAGAGGGAAGTTGTGAGC TTTTTTTTGCGGCCGCAACGTGCGACTTGAAGGACAC 2 pc[ ] TTTTTTGCGGCCGCAAGGGATACTAGTGAGCCTCTC TTTTTTGCGGCCGCCCACCAAGCAAAACCGGTG 3 pe[ ] TTTTTTTTGCGGCCGCACACTCTTTGTTGCTACTATGG TTTTTTTTCCTGCAGGAGAGAATCGACTAAGCAGAGAC 4 pc[ ] TTTTTTTTGCGGCCGCATCGTTAGCTTGGAAGGC TTTTTTCCTGCAGGCTCACTCTTCATCAATCCCTAC 5 pe[ ] TTTTTTTTCCTGCAGGCGGCACCCAGATTTGAACTG TTTTTTTTGCGGCCGCACCTGCTCCTGTCGCAATTTC 6 pc[ ] TTTTTTTTCCTGCAGGAGACCCCTTCGAACAGCCT TTTTTTTTGCGGCCGCTCTTCCTCGAAGAGATCCTGTG 7 pe[ ] TTTTTTTTCCTGCAGGCCTCTCGATGGCTGATGATG TTTTTTTTGCGGCCGCAATACCTTCTACAGCTTGTCC 8 pc[ ] TTTTTTTTCCTGCAGGAGAATTAGCAGCGCCCACGA TTTTTTTTGCGGCCGCTTTCTTGATTGCCTCAAC 9 pe[ ] TTTTTTTTCCTGCAGGAACAACTCGAACGGTTTTCCC TTTTTTTTGCGGCCGCTCCCAAATTAGCCTGGAGTC 10 pc[ ] TTTTTTTTCCTGCAGGAGCTGTTAGCCTAGGCGCG TTTTTTTTGCGGCCGCCGTCTGGGAGCTTTACCAG 11 pe[ ] TTTTTTTTCCTGCAGGAGAGATGGCCGAGCGGTTC TTTTTTTTGCGGCCGCTCCGGTTCGTGAAGGACCAG 12 pc[ ] TTTTTTTTCCTGCAGGCCCAAAGATCTATGAACCACCTC TTTTTTTTGCGGCCGCAGCTACTGCAGCCCCTGCT 13 pe[ ] TTTTTTTTCCTGCAGGAACACTGAGAGGAGCTCCC TTTTTTTTGCGGCCGCGACGTTTTATTCCAGGGAATC 14 pc[ ] TTTTTTTTCCTGCAGGATAGAAAGGCCGCGAGATCG TTTTTTTTGCGGCCGCCAAGATCTTGAGCCCTATTCCG 15 pe[ ] TTTTTTTTCCTGCAGGCCATTTCCCCTACTTTCCTCC TTTTTTTTGCGGCCGCGCTTAAGGGATGTCCTCA 16 pc[ ] TTTTTTTTCCTGCAGGAGTTACCTCTCCGGGAATTCT TTTTTTTTGCGGCCGCACTGGGTTGGGTCGATTTGC 17 pe[ ] TTTTTTTTCCTGCAGGAGATGGCGACTAAAGTTGCTG TTTTTTTTGCGGCCGCGTTCGCCTAGAGAATGAC 18 pc[ ] TTTTTTTTCCTGCAGGTCGACACCCATTATGCGGC TTTTTTTTGCGGCCGCAATGGTTGGCCATACAATC 19 pe[ ] TTTTTTTTCCTGCAGGCACGCGCAATTTCTTTTCCCG TTTTTTTTGCGGCCGCTCTCCTCTATCTTCTGAGGT 20 pc[ ] TTTTTTTTCCTGCAGGATTGAACAACCGGGATCCGT TTTTTTTTGCGGCCGCATTATTCGAGGAGCCCTAGA

4 21 pe[ ] TTTTTTTTCCTGCAGGAAAGCCATATTTCGACCCGGAC TTTTTTTTGCGGCCGCTCGTAGTGACTTAGGGAGTC 22 pc[ ] TTTTTTTTCCTGCAGGTGCGGCTGGATCACCTC TTTTTTTTGCGGCCGCTGTCTCGGCTGTGCTAC 23 pe[ ] TTTTTTTTCCTGCAGGTCTCCGCAAAGTCGTAAGAC TTTTTTTTGCGGCCGCTTGGTGCGGTTGCGAAATCGG 24 pc[ ] TTTTTTTTCCTGCAGGCCCTCAGCACCCATCAATGC TTTTTTTTGCGGCCGCTATCCGTTGACAGGGTAGAG 25 pe[ ] TTTTTTTTCCTGCAGGTTCGAGTCCGAGTGGCGGCAG TTTTTTTTGCGGCCGCGTATGCCCTATAGATCTACC 26 pc[ ] TTTTTTTTCCTGCAGGCCGAGCCATAGTAATTGCACCTA TTTTTTTTGCGGCCGCATCGGAGGAGTTGCTGCC 27 pe[ ] TTTTTTTTCCTGCAGGAGCGGCAGCTCGTAGACCAC TTTTTTTTGCGGCCGCCACCTGGAGCTGTAGGTG 28 pc[ ] TTTTTTTTCCTGCAGGAACCTCCCTCTCCTCAGCC TTTTTTTTGCGGCCGCAGGGACCAGGAGGTTGG 29 pe[ ] TTTTTTTTCCTGCAGGGTCTATGGTCGGTCCGTGA TTTTTTTTGCGGCCGCGACAGCATCTAGGGCTCCTC 30 pc[ ] TTTTTTTTCCTGCAGGACTGGTGCCGACAGTTCATC TTTTTTTTGCGGCCGCTACTTATGGGCGTGCGCTC 31 pe[ ] TTTTTTTTCCTGCAGGAGGCCTTCCTCCACTAGCA TTTTTTTTGCGGCCGCCTAGCTGCTCTTGAAGTTCCATCTC

5 Supplementary Figure 1. Step-by-step integration of dominos in the BGM vector. Genomic pbr322 sequences, GpBR, indicated by two consecutive open arrows, equivalent to pbr322 sequences, was a cloning locus for the domino method. This was created at the prob locus of B. subtilis. Domino clones were prepared on E. coli pbr322-based plasmids, shown by arrow heads and arrow tails for pcisp401 or pcisp402 constructed in this study (Supplementary Methods online). The two plasmids are identical in structure, with the exception of the selection markers cat (closed circle) or erm (open circle) for B. subtilis. The homologous regions shared by two adjacent dominos are indicated by yellow horiaontal bars. Domino clones are presented as linear form, being opened up at the blasticidin S resistance gene (bsr; open rectangle). The 4.3-kb pbr322 sequence was separated into two halves, a 2.3-kb arrow head and a 2.1-kb arrow tail, which remained at

6 both ends of the cloned insert. During assembly, the first domino is integrated into the GpBR locus by homologous recombination (X) at the two halves of the pbr322 sequences. The tetracycline-resistance marker (closed rectangle indicated by 0) is replaced by the first domino possessing the cat marker. The subsequent domino integrations are characterized by the conversion of alternative makers: cat or erm. The regions that did not participate in the homologous recombination have been omitted from the intermediates. The GpBR sequence that always remained in the BGM clone provided a reusable sequence for subsequent domino integration. This approach, along with the alternative use of two antibiotic selection markers, allows limitless rounds of domino elongation. Two I-PpoI recognition sites ([I]) consisting of 26 bases, ATGACTCTCTTAAGGTAGCCAAA, provided the cloned segments resolved by contour-clamped homogeneous electric field (CHEF) gel electrophoresis shown in Figs. 2a and 2b and supplementary Fig. 3 online.

7 Supplementary Figure 2. Nucleotide inheritance from dominos to mtg. Only the three nucleotides that differed between the dominos. Other nucleotide changes incorporated during the domino preparation, probably due to PCR errors or differences between the mouse strains, were all inherited by the mtg, which means no unsuspected nucleotide alteration during domino and BReT in B. subtilis. These were accounted for by the location of the Holliday junctions indicated. The other 12 differences compared with those on MMU txt came from either PCR errors or strain differences. It was essential that the sequences of the dominos were rationally transmitted throughout the domino and BReT protocols in the BGM vector, and this was confirmed also in rice cpg (Supplementary Fig. 5 online).

8 Supplementary Figure 3. Cloned mtg separated by I-PpoI digestion. I-PpoI digestion from indicated BGM clones was resolved by CHEF gel electrophoresis. A step-by-step increase of the I-PpoI fragment is shown for all of the forms. The estimated size included the selection marker, cat (1.0 kb) or erm (1.2 kb). The running conditions for all cases were 1% agarose, 3 volts!cm 1, and a 6-s pulse time for 18 h.

9 Supplementary Figure 4. Block elongation to completion of rice cpg. Connection of block 3 to block 2 proceeded via homologous recombination (X) at the connecting domino 24 and the BGM region beyond the erm or cat gene. The genomic crosses produced several correct colonies. A representative BGM clone, BUSY2116, which was selected by erm followed by screening for the indicated antibiotic markers, possessed block 2+3. Connection of block 2+3 to block 1 of BUSY2048 similarly yielded BUSY2052, showing the indicated combination of antibiotic resistance. Details are described in Supplementary Methods.

10 Supplementary Figure 5. Altered nucleotide sequences found in cloned cpg. The numbered nucleotides that differed from the reported sequence (reference 9 in the main text) are shown along with the respective dominos. Four nucleotides in the coding sequence (indicated by grey and circled) were probably the result of mis-incorporation during the PCR reaction in the domino preparation. Some of the others might reflect differences in the rice strains (reference 9 in the main text). The descriptions of the numbered alterations were as follows: 1, replacement of C at 15,487 by T between the transfer RNA (trna) of domino 4; 2, insertion of A at 29,151 causing a frame-shift of ribosomal protein S2 of domino 7; 3, deletion of T at 36,542 causing a frame-shift of

11 ribosomal protein S14 of domino 9; 4, insertion of T at 44,412 before the trna of domino 10; 5, deletion of T at 49,278 causing a frame-shift of PSII G protein of domino 11; 6, replacement of C at 58,593 by T between ycf4-peta of domino 14; 7, replacement of G at 70,268 by A causing no amino-acid change of PSII B of domino 16; 8, replacement of C at 74,284 by T between rpoa and rps11 of domino 17; 9, deletion of T at 78,428 between rps3 and rpl16 of domino 18; 10, replacement of G at 80,018 by A at the end of ribosomal protein S2 of domino 18; and 11, replacement of A at 99,423 by G between the trna and open reading frame (ORF) 63 of domino 23.

12 Supplementary Methods Bacterial strains. E. coli JA221 and JM109 strains were routinely used as hosts for molecular cloning. Tetracycline (10!g/ml) and ampicillin (50!g/ml) were added for JA221 or JM109 selection. All of the B. subtilis strains including MI112(recA) were derived from a restriction-modification-deficient strain RM Competent B. subtilis cells were prepared as the protocol below. Chloramphenicol (5!g/ml), erythromycin (5!g/ml) and blasticidin S (250!g/ml) were added for B. subtilis selection. Luria-Bertani broth, unless specified, was used to grow B. subtilis and E. coli at 37 C. The preparation and transformation of competent E. coli cells were described 2. Competent B. subtilis and transformation. B. subtilis competent cell was developed as follows 4 : Stational stage of B. subtilis (50!l) in LB was inoculated in 950!l medium TFI. TFI medium was prepared by mixing of 20 ml of solution [2 grams (NH 4 ) 2 SO 4, 18.3 grams K 2 HPO 3 "3H 2 O, 6 grams KH 2 PO 4, 1 gram trisodium citrate 2H 2 O per liter], 0.2 ml 50 %(w/v) glucose, 0.3 ml 1.2 %(w/v) MgSO 4 "7H 2 O, 0.4 ml 10 %(w/v) Bacto yeast extract, and 0.5 ml 1 %(w/v) casamino acids. After shaking with vigorous aeration for 4-5 hours at 37 C to give an OD 600 reading about 0.6, 400!l culture was transferred in 3.6 ml medium TFII. TFII medium was prepared by mixing of 200 ml of solution of the square bracket as indicated for TFI medium, 2 ml 50 %(w/v) glucose, 14 ml 1.2 %(w/v) MgSO 4 " 7H 2 O, 2 ml 10 %(w/v) Bacto yeast extract, 2 ml 1 %(w/v) casamino acids, and 1 ml 0.1 M CaCl 2. After 90 min incubation at 37 C the cell was collected by centrifugation (8000g, 5 min) at 4 C. Cells gently suspended to the 250!l TFD medium containing 15 %(v/v) glycerol were for immediate use for transformation or saved at 70 C. TFD medium was prepared by mixing of 6.25 ml [2 grams (NH 4 ) 2 SO 4, 1 gram trisodium citrate 2H 2 O per 50 ml], ml [18.3 grams K 2 HPO 3 "3H 2 O, 6 grams KH 2 PO 4, per 50 ml], 1.25 ml 50 %(w/v) glucose, added up to 100 ml. Standard transformation was conducted in a 1.5 ml microtube. To the 50!l TFD medium, 1.25!l M MgCl 2, 1.25!l 2 %(w/v) MgSO 4, 5!l DNA solution, and 6.25!l competent cells were added this order and incubated at 37 C for 30 min. Successive incubation for 1 hour at 37 C by addition of 200!l LB including chloramphenicol at 200 ng/ml, or erythromycin at 10 ng/ml to induce respective antibiotic resistance gene. Cells were spread on plate containing selective media. The domino DNA, usually 200 ng in standard transformation, yielded more than dozens up to hundreds of colonies selected by chloramphenicol or erythromycin. In vitro preparation of domino clone. pcisp401(cat) or pcisp402(erm) was derived by the insertion of an EcoRI-NotI-Sse8387I-BamHI linker, 5!-AATTCGCGGCCGCCTGCAG/5!-GATCCTGCAGGCGGCCGCG, into the EcoRI or BamHI fragment of pcisp310b(cat) 3 or pcisp311b(erm) 3, respectively. PCR-mediated amplification was carried out by adopting a 4 6 kb size and polymerises ExTaq (Takara,

13 Shiga, Japan) for mtg or KOD-PLUS (Toyobo, Kyoto, Japan) for cpg. Different polymerises were adopted to obtain DNA with sufficient length but minimum spontaneous mis-incorporation. Primers for the dominos listed in Supplementary Tables 1 and 2 were synthesized by Nihon Gene Research Laboratories Inc. (Miyagi, Japan). PCR for 10 ng mouse DNA was programmed for one cycle at 95 C for 3 min, followed by 30 cycles at 95 C for 30 s, 60 C for 45 s and 72 C for 3 min, ending with a 10-min incubation at 72 C using ingredients included in the ExTaq-kit. Similarly for 100 ng rice genomic DNA, one cycle at 95 C for 3 min, followed by 30 cycles at 95 C for 15 s, 60 C for 30 s and 68 C for 5 min, ending with a 10-min incubation at 68 C using ingredients included in the KOD-PLUS-kit. PCR products for cpg after purification by ethanol precipitation were digested by NotI and Sse8387I. The digests were cloned in the same restriction enzyme site of pcisp401 or pcisp402. Plasmids were prepared by the alkali-sds method 2. Large-scale plasmid preparation was by the alkali-sds method, followed by ultracentrifugation in a CsCl-ethidium bromide gradient 2. Chromosomal DNA for B. subtilis transformation was prepared by a liquid isolation method 2. Type II restriction enzymes and DNA ligation kit were purchased from Toyobo (Tokyo, Japan) and Takara (Shiga, Japan) respectively, except for I-PpoI which was from Promega. The DNA sequence was determined using the ABI3100 sequencer (Applied Biosystems) and Big Dye terminators. Domino elongation by B. subtilis transformation. The BGM colonies selected by chloramphenicol or erythromycin were subjected for screening the newly elongated segment. For mtg, PCR analyses using the same primer as listed in supplementary table 1 scored colonies possessing correct elongation about %. For cpg, presence of the first domino-associated antibiotic resistance marker facilitated the screening and scored between %. I-PpoI digestion confirmed the insert DNA of BGM clones. The expected BGMs were constantly obtained in all the domino elongation experiments. Block elongation 5. Block 3 was first connected to block 2 (Supplementary Fig. 3 online). Genomic DNA isolated from BUSY2214 (block 3), when incorporated by BUSY2114 (block 2), elongated block 2 by double homologous recombination. BUSY2116, which was selected using the two antibiotic-resistance markers erm and spc located at its ends, lost the two internal antibiotic-resistance markers (underlined in Supplementary Fig. 3 online). BUSY2116 possessed the 63.8-kb continuous sequence of block 2+3 from numbers 17 to 31. The full-length kb cpg cloning was completed by the addition of block 2+3 to block 1 of BUSY2048. Once again, selection by a combination of antibiotic-resistance markers, erm and phl, resulted in the isolation of BUST2052. The structure of the IR sequences included in blocks 2 and 3 remained stable in all of the BGM clones. In block elongation, genome DNA solution isolated from the donor BGM strain was viscous due to their high molecular weight. On transformation, the TFD medium including the genomic DNA was gently shaken for at least 2 hours at room temperature prior to addition of competent cell. Approximately 5!g genome DNA was used to yield at least several colonies in all experiments.

14 Recombinational transfer in B. subtilis. DNA in the genomic pbr322 sequence (designated as GpBR) could be copied and transferred to an independent replicon via genetic process referred to as Bacillus Recombinational Transfer, BReT (reference 10 in the main text). Into the recipient plasmid possessing pbr sequences, the cloned DNA segment between the two GpBR halves transfer and bridges the gap. Indeed, copying a DNA segment from the genome and pasting it into the incomplete plasmid, causes the DNA transfer from the genome to plasmid in an apparent reverse direction as that of the domino cloning method. In this study, BReT plasmid, linearized by appropriate restriction enzymes prior to delivery, circularises only by retrieving the target organelle genomes and selected by the plasmid-associated marker. Two replicons for BReT, pgets109 and pgests113, were employed here. The copy number of both plasmids remained as low as one. In a typical BReT experiment, 1!g linearized pgets109 or pgets113 yielded several to dozens of candidate colonies by plasmid-associated antibiotic selection. At least three BReT isolates per retrieval were saved after confirmation by subsequent screening, including purification on ultracentrifugation in the presence of ethidium bromide 2. References 1. Itaya, M. Stability and asymmetric replication of the Bacillus subtilis 168 chromosome. J. Bacteriol. 175, (1993). 2. Maniatis, T., Fritsch, E. F. & Sambrook, J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (1982). 3. Itaya, M., Fujita, K., Ikeuchi, M., Koizumi, M. & Tsuge, K. Stable positional cloning of long continuous DNA in the Bacillus subtilis genome vector. J. Biochem. 134, (2003). 4. Spizizen, J. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl Acad. Sci. USA 44, (1958). 5. Itaya, M. Genetic transfer of large DNA inserts to designated loci of the Bacillus subtilis 168 genome. J. Bacteriol. 181, (1999).