Transforming Caulobacter Introduction B5 BAC Plasmid Preparation Positive Control 20 Once you have confirmed that your gene of interest is cloned in frame with the CX leader and the truncated RsaA ORF, you are ready to express your protein of interest in Caulobacter. To express your recombinant fusion product, you will transform your pCX- TOPO ® construct into B5 BAC Caulobacter cells included with the kit. Guidelines and instructions to transform your construct into One Shot ® B5 BAC cells are provided in this section. You will use One Shot ® B5 BAC Electrocomp Caulobacter cells to express your recombinant fusion protein from pCX-TOPO ® . The B5 BAC strain has been specifically designed to allow high-level expression of recombinant proteins from plasmids containing the RSF1010 origin of replication (oriV). In addition, the B5 BAC strain contains a mutation which eliminates its ability to express native RsaA protein. Do not use this strain for propagation and maintenance of your plasmid. We have found that expression of recombinant proteins from pCX-TOPO ® can decrease with successive subcultures when the expression construct is maintained in B5 BAC cells. We recommend that you store expression strains as frozen stocks or perform a fresh transformation of your pCX-TOPO ® plasmid into B5 BAC cells before each expression experiment. For propagation and maintenance of your plasmid, use TOP10F′. It is possible to use other Caulobacter host strains for expression of your recombinant fusion protein from pCX-TOPO ® , however, these strains must contain the repBAC genes. If you wish to prepare electrocompetent Caulobacter cells, a protocol is provided in the Appendix, pages 37-38 for your convenience. Alternatively, One Shot ® B5 BAC electrocompetent Caulobacter cells are available separately from Invitrogen (see page x for ordering information). Purified plasmid DNA may be isolated using your method of choice. We recommend isolating plasmid DNA using the S.N.A.P. MiniPrep Kit (10-15 µg DNA, Catalog no. K1900-01), the S.N.A.P. MidiPrep Kit (10-200 µg DNA, Catalog no. K1910-01), or CsCl gradient centrifugation. Note: You will need approximately 10 ng of plasmid DNA per transformation. The pCX vector provided with the kit produces a fusion protein consisting of the CX leader and the truncated RsaA protein and may be used as a positive control for transformation, expression, and purification. The pCX vector will produce a recombinant RsaA fusion protein that is 33 kDa in size. For a detailed map and a description of the features of the vector, see page 41. continued on next page Transforming Caulobacter, continued Suggested Controls Important Before Starting We recommend including the following controls in your expression experiments. • No DNA (mock) • pCX vector (positive control) Caulobacter requires a different type of medium and temperature than E. coli for optimal growth. For general growth purposes, culture Caulobacter cells in PYE medium and at 30°C. To prepare PYE medium, see the recipe in the Appendix, page 43. Note that because of the osmolarity (salt content), Caulobacter cells cannot grow in LB medium. For the same reason, Caulobacter cells grows extremely poorly or not at all in tissue culture media. This is an important point to note for those users who maintain multiple gene expression systems. Be sure you have the following reagents on hand. • PYE medium (see page 43 for a recipe) • PYE plates containing 2 µg/ml chloramphenicol (3 per transformation) • Electroporator • 0.1 cm cuvettes, on ice • Microcentrifuge tubes • 30°C incubator • 30°C shaking incubator in a 30°C warm room continued on next page 21 Source.