This year our iGEM team has chosen the CDS of the PS2 S-layer protein for the basic part (BBa_K2641000). PS2 codes for a S-layer protein capable of assembly to a p2 symmetry and stems from Corynebacterium glutamicum ATCC 14067. This sequence was synthetically produced (many thanks to IDT for the free synthesis) to be BioBrick compatible.
The plasmid and the PS2 fragment were digested with EcoRI-HF and PstI-HF and ligated using T4 ligase. The plasmid was transformed in E. coli DH5α strain. Colony PCR was performed to proof successful ligation (Fig. 1). The isolated plasmid from clone #2 was shipped to iGEM as part.
Figure 1. Colony PCR of pSB1C3 PS2 clones.
Colony PCR was performed with single colonies after transformation and selection on chloramphenicol plates (lanes 4-11). The synthesized gBlocks fragment of PS2 from IDT was used as a positive control and the empty BioBrick vector (pSB1C3) was used as negative control for PS2. (Lanes 2 & 3 are from another experiment).
S-layer proteins are prokaryotic protein membranes that assemble into two dimensional symmetric lattices. They have two different domains: the first on attaches non-covalent to the cell wall of bacteria, the second one is for the self-assembly, which interacts hydrophobic as well as hydrophilic with other S-layer proteins. The PS2 gene encodes a protein with a molecular mass of 52 kDa (Fig. 2, left). The hydrophobic domain at the C-terminus is important for the interaction with the cell wall. The PS2 S-layer protein forms two-dimensional lattices with oblique symmetries (Fig. 2, right).
Figure 2. Coomassie stained SDS-PAGE of isolated PS2 from C. glutamicum (left). AFM picture of PS2 S-Layer membrane on C. glutamicum cell.
This part should be used as a base for S-Layer fusion proteins. If directly expressed in E. coli an additional affinity tag should be fused to the part as well, because the S-Layer will not attach to any other cell wall than the one of C. glutamicum. The sequence can be cloned into an C. glutamicum expression vector (e.g. pZ8-1) and expressed in stems without a functioning S-layer gene such as C. glutamicum ATCC 13032 for easy isolation without affinity tag.
Table of all parts
|BBa_K2641000||Coding||CDS of S-layer protein PS2||Selina Beck||1476|