Difference between revisions of "Part:BBa K3187008"

Profile

Name	GGGG-mCherry
Base pairs	1028
Molecular weight	28.5 kDa
Origin	synthetic, derived from Discosoma sp.
Parts	mCherry, GGGG-sequence, T7 Promoter, lac Operator, GASPAG Linker, Strep-Tag II, T7 Terminator
Properties	Red fluorescent, Ex λ: 587nm, Em λ: 610 nm

Usage and Biology

mCherry (BBa_K3187026) is a red fluorescent protein. Which is a synthetic protein derived from Discosoma sp. by directed evolution. The N-terminal GGGG-sequence (BBa_K3187018) can be fused to a protein with a C-terminal LPETGG-Sortase A link (BBa_K3187019) by Sortase A. We use mCherry as an easily imaged reporter for checking if the coupling worked.
The coding sequence was cloned in pET24 vector, containing the sequence of mCherry, a GGGG-sequence, a GASPAG Linker (BBa_K3187038), a Strep-Tag II (BBa_K3187025) for Purification, a T7 Promoter with lac Operator and an RBS (BBa_K3187029), a T7 Terminator (BBa_K3187032), a Start-Codon (BBa_J70593) and a Stop-Codon (BBa_K2868029). The coding sequence consists of 851 bp which are translated to 260 amino acids.^[1]

Methods

Purification

The GGGG-mCherry was heterologously expressed in E. coli BL21 and purified with GE Healthcare ÄKTA Pure machine which is a machine for FPLC. The used affinity tag was Strep-tagII.

SDS-Page and Western blot

To verify that the CP-LPETGG was produced, a SDS-Page SDS-Page followed by a Western blot was performed.

Results

Cloning and Expression

The successful cloning was proven with sanger sequencing and production with a Western blot.

Fig. 1 shows that the band of the GGGG-mCherry is approximatley found by the 28.5 kDa band. Consequently, the successful protein production was proven.CP-LPETGG is detected with Strep-Tactin-HRP.

References

1. ↑ Nathan Shaner, Robert Campbell, Paul Steinbach, Ben Giepmans, Amy Palmer and Roger Tsien, Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein, Nature Biotechnology, 2004, 22: 1567-1572 [1]