DsRED, mKate2, mCherry, YFP & CFP and fluorescent reporters in plasmid pCM29

DsRed.T3(DNT), mKate2, mCherry, YFP (venus) and CFP (Cerulean) coding genes were amplified by PCR from several templates (templates and primers are presented in Table 3). For amplification of mCherry and mKate2 the forward primer was designed to include XmaI, KpnI and Bsu36I restriction sites, a RBS, and spacing to start of the gene encoding the reporter protein identical to the one present in pCM29 (Table 3). For mKate2 genomic DNA of S. pneumoniae strain MK119⁹ was used as a template. This strain contains a S. pneumoniae codon optimized gene encoding mKate2 C-terminally fused to a histone protein. To create a single gene also the ATG start codon was added to the primer sequence (Table 3).

To create the mAmetrine, CFP, YFP, and DsRED expressing vectors, the pCM29 plasmid and PCR amplified reporter DNA were digested with KpnI and EcoRI. To create the mKate2 and mCherry constructs, pCM29 and PCR amplified reporter DNA were digested XmaI-EcoRI. After digestion, pCM29 was dephosphorylated, separated on an agarose gel, and the plasmid backbone was isolated from gel. The digested reporters were ligated into pCM29-backbone. The resulting plasmids were named pTH1 (DsRED), pTH2 (CFP), pTH3 (YFP), pRN10 (mKate2), pRN11 (mCherry), pRN12 (mAmetrine). All plasmids were transformed to competent E. coli DC10B and, after initial selection based on expected fluorescence spectrum of the colonies using the ImageQuant LAS4010 (GE Healthcare), checked for correct ligation and absence of mutations by restriction analysis and sequencing.