SUPPLEMENTAL TABLE S1. Additional descriptions of plasmid constructions and the oligonucleotides used Plasmid or Oligonucleotide

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1 SUPPLEMENTAL TABLE S1. Additional descriptions of plasmid constructions and the oligonucleotides used Plasmid or Oligonucleotide former/ working Description a designation Plasmids pes213a b pes213-tn5 KnR: Tn5-<NotI-Kan-3>-insert in native pes213 (upstream of bp 1 in GenBank AY465897) pes213c b pes213-tn5- CmR, KnR: pevs118 b SpeI-XbaI fragment in SpeI site of pes213a 118 pes213kn b pblinker KnR: pes213c digested with BamHI and self-ligated yielding pes213d b (formerly p Bam). Linker inserted into pes213d between BsrGI and NotI sites, replacing 5 - CAT CTT-3 with 5 -CTG CAG ATA TCG ATC CCC GGG CTA GC-3 and yielding pes213kn pes213tp b pblinkertp TpR: NotI-BamHI TpR fragment from pjlb1 b replaced KnR in pes213kn pvsv2 pmsb6 KnR, gfp: pqbi63 (Quantum Biotechnologies, Montreal) SphI-BamHI (gfp) fragment into same sites in pevs122 b yielded pmsb5. pmsb5 NotI (gfp) fragment cloned into pes213kn NotI site yielded pvsv2 pvsv3 pdma30 KnR, lacz: pkv43 c BamHI (lacz) fragment in BamHI site of pes213kn pvsv4 pmsb8 KnR, laczα-(sphi, AvrII, HpaI, SalI, NcoI, KpnI, SacI) d : pevs122 b NotI laczα fragment (originally in pgem3z, Promega, Madison Wisc.) into NotI pes213kn yielding pmsb7. Linkers Lac1 and Lac2 self-annealed and cloned into KpnI to SphI to add more sites to laczα, yielding pvsv4 pvsv33 pjlb33 KnR, oriv R6Kγ, promoterless CmR-gfp: BamHI-ApaI fragments of pqbi63 (Quantum Biotechnologies) and pbc(sk) (Stratagene) were fused, and the resulting vector used as template for PCR with Pfu and primers EVS88 and EVS89. This PCR product was digested with HindIII and self- 1

2 pvsv33 (cont.) ligated to generate a plasmid where the stop codon of CmR and the start codon of gfp are separated by TAA AAG CTT AAG GAG ATA TAC AT ATG-3. The AseI site in this plasmid was destroyed by AseI digestion, Klenow fill in, and self-ligation, yielding pevs121. The streptomycin/spectinomycin resistance omega cassette in putntn5s/s was cloned as a BamHI fragment into pevs121, yielding pevs123. The orit in pevs94n e was deleted by BglII-BamHI digestion and self-ligation, generating pevs124. pevs123 and pevs124 were linearized with AseI and NheI, respectively, made blunt with Klenow fragment, and ligated together to generate pevs127. EcoRV-digested pevs127 was ligated to BstZ17I-digested pevs126 b generating pevs130, which was used as template for PCR with primers EVS95 and EVS96, the product was digested with BclI and self-ligated to generate pevs132, in which the CmR promoter is replaced with a transcriptional terminator (adjacent to another terminator in the plasmid) and a multiple cloning site. At this stage sequencing pevs132 revealed deviations from the predicted sequence, notably resulting in an EcoRI site within the CmR gene. Oligonucleotides TT1 and TT2 were annealed and cloned into the BglII site of pevs132 to add a third transcriptional terminator upstream of the CmR gene, yielding pevs141. The V. fischeri ndh promoter was cloned between the SalI and SphI sites of pevs141, yielding CmR expression, and this promoter-reporter and flanking transcriptional terminators were PCR amplified using primers 141F and 141R. This PCR product was digested with NotI and SmaI and cloned into NotI- and SmaIdigested pes213kn, selecting for CmR, generating pblinkerndh141. This plasmid was digested with AvrII, the overhangs filled with Klenow and self-ligated to destroy the AvrII site, and the SalI to SphI ndh promoter fragment was then replaced by a linker composed of Newlink1 and Newlink2, yielding pblinkerndh141-avriilink. The oriv R6Kγ in pevs94 e was PCR amplified using primers JBR6K1 and JBR6K2, and ligated into the XmaI site of pblinkerndh141-avriilink yielding pvsv33. 2

3 pvsv102 pmsb6+r6k KnR, gfp, oriv R6Kγ : The oriv R6Kγ in pvsv105 was PCR amplified using primers JBR6K1 and JBR6K2, and ligated into the XmaI site of pvsv2 yielding pvsv102. pvsv103 pdma30+ R6K KnR, lacz, oriv R6Kγ : The oriv R6Kγ in pvsv105 was PCR amplified using primers JBR6K1 and JBR6K2, and ligated into the XmaI site of pvsv3 yielding pvsv103. pvsv104 pjlb40 KnR, laczα-(sphi, AvrII, HpaI, SalI, NcoI, KpnI, SacI) d, oriv R6Kγ : pvsv107 (see below) NheI- BsrGI (oriv R6Kγ ) fragment into NheI-BsrGI digested pvsv4 yielded pvsv104 pvsv104h pjlb105 Increased copy-number variant of pvsv104: When attempting to isolate promoter-up mutants in pjlb38 (see construction of pvsv209) by selecting for increased resistance to chloramphenicol, copy-up derivatives of the plasmid origin were isolated. The BamHI fragment containing this origin was used to replace the BamHI origin-containing fragment in pvsv104 generating pvsv104h. pvsv105 pes213-tn5-118sx-lac CmR, laczα-(sphi, SalI/HincII, XbaI, SmaI/XmaI, KpnI, SacI) d, oriv R6Kγ : pes213c digested with XhoI and SpeI, Klenow filled and self-ligated to form pes213-tn5-118sx. Then pevs122 b laczα NotI fragment cloned in the NotI site of pes213-tn5-118sx forming pvsv105. pvsv106 pdma41.1sa TcR, laczα-(apai, XhoI, SalI/HincII, SmaI/XmaI, XbaI) d, oriv R6Kγ : PCR amplified TcR gene from pkv139 c using primers dma27 and dma28 and cloned into pcr-bluntii-topo (Invitrogen) yielding pdma39. This contained regions of the tetm gene corresponding to Genbank U09422, bp to SpeI-NcoI TcR pdma39 fragment cloned into SpeI-NcoI digested pes213-tn5-118sx (removing CmR) yielding pdma40. PCR amplified (using Pfu) laczα fragment from pbluescript SK+ (Stratagene) with primers EVS81 and EVS82 and cloned the product into EcoRV-digested pevs77 e yielding pbc1 (contains multiple inserts and vectors combined). AvrII (laczα) fragment from pbc1 in NheI site of pdma40 yielding pvsv106. 3

4 pvsv107 pdma38 TpR, laczα-(kpni, ApaI, XhoI, SalI, SpeI, XbaI, NotI, SacI) d, oriv R6Kγ : pvsv33 XmaI (oriv R6Kγ ) fragment in pes213tp yielding pjlb35. pjlb35 NotI-digested, Klenow filled, self-ligated yielding pdma37. AvrII (laczα) fragment from pbc1 (see construction of pvsv106) in SpeI site of pdma37 yielding pvsv107 pvsv209 pakd209 KnR, oriv R6Kγ, rfp, promoterless CmR-gfp: AvrII-digested pvsv210 self-ligated ( lux promoter) pvsv210 pakd210 KnR, oriv R6Kγ, rfp (DsRed.T3[DNT]), P lux -cat-gfp: The lux promoter from V. fischeri ES114 was PCR amplified with primers EVS109 and EVS110, cloned into pcr-bluntii-topo, and subcloned as an AvrII fragment into the AvrII site of pvsv33 yielding pjlb38. The DsRed.T3[DNT] allele was PCR amplified using primers RedliftF and RedliftR, digested with EagI, and ligated into the NotI site in pjlb38 to generate pvsv210. The rfp-bearing PCR product contained four EagI sites, two on either end, and sequencing revealed that both EagI sites upstream of rfp were present in pvsv210, although only the EagI site proxminal to rfp was retained downstream. Oligonucleotides f 141F 141R dma27 dma28 EVS81 EVS82 EVS88 EVS89 ATT AGC GGC CGC AGG ACA AGT TTT GGT GAC ATT ACC CGG GGC CAA CAT AGT AAG CCA G CGC GGT CTA CAG GAG GGC TTA GTT TTT TGT ACC C CGG GCC ATG GCG GGC TAG CCG TAA AGC CTG TTA TCT CCC CC CCT AGG ATG CAT AGC TCA CTC ATT AGG CAC CCC AGG CCT AGG ATG CAT GCT AGC ACG TGA ACC ATC ACC CTA ATC AAG CCC CAA GCT TAC GCC CCG CCC TGC CAC TCA TCG C CCC CAA GCT TAA GGA GAT ATA CAT ATG GCT AGC AAA GG 4

5 EVS95 EVS96 EVS109 EVS110 JBR6K1 JBR6K2 Lac1 Lac2 Newlink1 Newlink2 RedLiftF RedLiftR TT1 TT2 CCC CTG ATC AAT GCG CCT TTT TTA TAA GAT CTG TCG ACA GGC CTA GGA ATT CAG GAG CTA AGG AAG CTA AAA TGG CCC CTG ATC AAT GCG CCT TTT TAT ATT CAC TCC GCT AGA AAT ATT TTA TCT G CCG CCC TAG GTT ATT CAG ATA AGC ATT GAT TAA TAT C CCG CCC TAG GGC ATG CTT AAC CTC TAT ACT CCT CCG ATG GAA TCC CCC CGG GAA TTC CCA TGT CAG CCG TTA A TCC CCC CGG GAT CCG GCC ACG ATG CGT C CCC TAG GGT TAA CGT CGA CCA TGG TAC CAT GGT CGA CGT TAA CCC TAG GGC ATG TCG ACG CTA GCT CTA GAG GTA CCT AGG GCA TG CCC TAG GTA CCT CTA GAG CTA GCG TTA TCG GCC GTT AAG CTG TCT CTT GTA CAC ATC TAT ACG GCC GAT CCT CTA GAG TCA AAA GGA TC GAT CAT AAA AGA CCC TTC ATT TAT GAA GGG TCT TTT TGC ATG CGG CGC CT GAT CAG GCG CCG CAT GCA AAA AGA CCC TTC ATA AAT GAA GGG TCT TTT AT a All pvsv plasmids contain orit RP4, oriv pes213. KnR, kanamycin resistance; CmR, chloramphenicol resistance; TcR, tetracycline resistance; TpR, trimethoprim resistance b Described in: Dunn, A.K., M.O. Martin, and E.V. Stabb Characterization of pes213, a small mobilizable plasmid from Vibrio fischeri. Plasmid In press. c Provided by Karen Visick d unique restriction sites present in the laczα multiple cloning site e Described in: Stabb, E.V. and E.G. Ruby RP4-based plasmids for conjugation between Escherichia coli and members of the Vibrionaceae. Methods in Enzymology 358: f Oligonucleotides are shown 5 to 3 5