Part:BBa_K562019:Experience

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••••• iGEM Dundee 2011

This part was seen work in practice. When expressed in E. coli, fragments of broken-down GFP was detected by Western immunoblotting (Figure 1), indicating the ssrA tag was directing proteolysis of Pdu40-GFP. When Pdu40-GFPssrA was co-expressed with the microcompartment from BBa_K562009, then the GFP was stable and protected inside the compartment (Figure 1).

Results

E. coli was transformed either with BBa_K562009 encoding a synthetic microcompartment, BBa_K562019 encoding the Pdu40-GFPssrA fusion protein, or both together. Cells were grown aerobically overnight in LB medium. 1 ml samples of whole cells were collected, washed in buffer, and taken up on Laemmli disaggregation buffer. Proteins were separated by SDS-PAGE, blotted onto nitrocellulose and challenged with anti-His or anti-GFP antobodies.

Thsi small scale experiment allows the detection of the BBa_K562009 microcompartment, because several of the proteins are His-tagged. In addition the presence of stable GFP is only readily detectible when PduD40-GFPssrA is co-expressed with the microcompartment. This is strong evidence that the PduD40 tag is targeting GFP inside the microcompartment, which then protects the ssrA tag from degradation by cytoplasmic proteases.

Figure 1: The BBa_K562009 microcompartment shields ssrA-tagged proteins from degradation. Lane 1: Pdu40-GFP-ssrA expressed alone in MG1655 E. coli cells. Lane 2: Synthetic bacterial microcompartment genes (BBa_K562009) expressed alone in MG1655. Lane 3: Pdu40-GFP-ssrA expressed together with the synthetic bacterial microcompartment genes (BBa_K562009) in MG1655 E. coli cells.

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