GRM3C shell proteins and design of the synthetic operon. All six genes encoding shell proteins in the GRM3C locus from R. palustris BisB18 are expressed in E. coli under the control of a T7 promoter. The Ribosome binding site (RBS) preceding BMC-H1 is derived from pET29b.[27] Native RBS sequences were used for BMC-T, BMC-H2, and BMC-H3 by including the intergenic sequences upstream of each gene in the R. palustris BisB18 genome. A synthetic RBS[28] and intergenic region from Synechocystis sp. PCC 6803 were used preceding BMC-H4 and BMC-Pstrep, respectively. Due to a 100 amino acid C-terminal extension, BMC-H2 is almost twice the length as BMC-H1-3. We modified the native BMC-P sequence to include a C-terminal Strep Tag II sequence for purification (BMC-Pstrep). The shell proteins can be further classified into clades relative to all identified BMC shell protein sequences: BMC-H1: H_robinEggBlue, BMC-H2: H_pumpkin, BMC-H3: H_tan, BMC-H4: Hp_GrpU_ickyGreen, BMC-T: Tsp_turquoiseBlue, BMC-P: P_stone according to the classification of Sutter et al.[25] and Melnicki et al.[29]
