Table 1 List of strains and plasmids Strain or plasmid Relevant genotypea Reference or source Strains V. rotiferianus DAT722 DAT722 Wild-type [11] DAT722-Sm DAT722; Spontaneous SmR mutant. This study MD7 DAT722-Sm; Single recombination cross-over of pVSD2 into cassette 61, KmR This study d8-60a DAT722-Sm; Δcassettes 8-60, SmR, KmR This study d8-60b DAT722-Sm; Δcassettes 8-60, SmR, KmR This study d8-60b-S
DAT722-Sm; Δcassettes 8-60, SmR, KmR. Spontaneous mutant of d8-60b. This study d8-60c DAT722-Sm; Δcassettes 8-60, SmR, KmR This study d8-60c-S DAT722-Sm; Δcassettes 8-60, SmR, KmR. Spontaneous mutant of d8-60c. This study d16-60 DAT722-Sm; Δcassettes 16-60, SmR, KmR This study E. coli XL1-Blue F’ proAB lacI q ZΔM15 Selleck ARS-1620 Tn10/recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relAi, TcR EX 527 cost Stratagene SY327 λ pir Δ(lac pro) argE (Am) rif nalA recA56 [38] SM10 λ pir thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu, Tcr KmR [39] Plasmids pLOW2 Cloning vector, KmR [40] pGEM-T Easy
Cloning vector, ApR Promega pMAQ1080 pGEM-T Easy carrying a 1834-bp fragment. The fragment was created using fusion PCR and consists of, in order, a 448-bp of paralog group 1 sequence, a 964-bp fragment containing aphA1 and a 410-bp paralog group 2 sequence abutted by salI restriction sites. This study pCVD442 Mobilisable sacB counter-selectable suicide vector, ApR [41] RK600 pJAK16 pMAQ1081 pMAQ1082 ColE1 oriV; RP4tra + RP4 oriT; CmR; helper plasmid in triparental matings Low copy IPTG-inducible expression vector, CmR salI fragment from pMAQ1080 cloned into the unique salI site of pCVD442. pJAK16 containing cassette 11 [42] [43] This study This study aSmR, streptomycin resistance; KmR, kanamycin resistance; TcR, tetracycline resistance; ApR, Ampicillin resistance V. rotiferianus DAT722 was isolated Non-specific serine/threonine protein kinase from a mud crab aquaculture tank in Darwin (Northern Territory, Australia) [11]. It was typed by multi locus sequence analysis of the
recA, pyrH, rpoA, topA, ftsZ and mreB genes (data not shown). Transformation of E. coli XL1-Blue was performed as previously described [34]. Genomic DNA (gDNA) was extracted from overnight cultures using the Purelink genomic DNA mini kit (Invitrogen). Standard PCR was performed using high fidelity platinum Taq (Invitrogen) as per the manufacturer’s instructions. Primers (Table 2) were used at a final concentration of 0.5 μM each. Plasmid pMAQ1082 was created by amplifying the cassette 11 gene from V. rotiferianus DAT722 using primers B-VSD11-F and this website P-VSD11-R (Table 2). The resulting amplicon was directionally cloned in front of the lac promoter using BamHI and PstI. pMAQ1082 was conjugated into MD7 in a triparental conjugation using RK600 as the helper strain.