Difference between revisions of "Part:BBa K2871000"

Revision as of 11:55, 17 October 2018

Map20, T3SS export signal peptide from Map gene

Map20 is a signal sequence encoding a 20 amino acid long signal peptide derived from the map gene from the Enteropathogenic E. coli strain E22. It is used as an N-terminal tag for a protein to be translocated by the E. coli type-3-secretion system (T3SS). This T3SS signal tag is described by Charpentier & Oswald (2004) to be sufficient to target proteins to E. coli type III secretion pathway. Its short length seemed to be attractive to us because we wanted to minimize folding interference between the signal sequence and the intended protein fused to the signal sequence.

Sequence and Features

Experimental characterization

Map20-fusion protein expression plasmid construction

In order to be able to test the expression of N-terminal Map20-fusion protein, we constructed a plasmid consisting of a Map20-tagged reporter gene controlled by pBAD promoter, which can be activated by adding arabinose to the culture media. The Map20 T3SS signal is linked with the reporter protein by glycine-serine linker peptide to decrease folding interference. 6X-His tag has been added to the C-terminal to serve as an epitope tag for western blot. The plasmid map is shown in Figure 1.

Figure 1: Map20-mCherry expression plasmid.

To evaluate the function of CesF and CesT effector chaperone in the injectisome-dependent secretion of the fusion protein, the CesF-CesT chaperone cassette was inserted next to araC gene under a constitutive promoter pCAT as shown in Figure 2.

Figure 2: Map20-mCherry expression plasmid with CesF-CesT chaperone cassette.

Detection of signal peptide-dependent secretion without membrane

To create an E. coli strain with a different combination of injectisome, signal, reporter, and chaperone. The reporter plasmids with and without chaperone cassette were transformed into SIEC E. coli strain which express injectisome, and SIECΔP1 strain which doesn’t have injectisome. The E. coli strains with reporter plasmid were cultured in LB media overnight at 37 C. Reporter protein and injectisome expression was induced by adding 1% L-arabinose and 0.1mM IPTG respectively. The induced cultures were incubated at 16 C with 180 rpm shaking for 6 days. We collected 1 ml of sample from the induced culture every 24 hours and separate cell and supernatant by centrifugation. Cellular fraction were lysed by sonication and separated into soluble and insoluble parts by centrifugation. The supernatants, cellular soluble parts, and cellular insoluble parts from day 0, 3, and 6 were gel electrophoresis on 12% SDS-polyacrylamide gel (SDS-PAGE). The reporter protein was detected western blot with anti-his tag antibody. The constructs tested all contained the reporter protein, being mCherry or ꞵ-lactamase.

Result

Map20-mCherry fusion protein expressing E. coli lack visible mCherry colour characteristic

Visual observation of E. coli cell pellet on day 6 shows a difference in color between E. coli that express mCherry and Map20-mCherry fusion protein. The mCherry-expressing E. coli pellet (tube no. 4 and 6) has pink color while Map20-mCherry fusion protein-expressing E. coli pellet (tube no. 1, 2, 3 and 5) has pale white-yellowish color as shown in Figure 3. This observation suggests that Map20 signal might decrease mCherry reporter protein expression or interferes with its folding.

Figure 3 Visual observation of different E. coli strains. Tube number 1-7 contains:

1. I+, S+, C+ mCherry
2. I+, S+, C- mCherry
3. I-, S+, C+ mCherry
4. I-, S-, C- mCherry
5. I-, S+, C- mCherry
6. I+, S-, C+ mCherry
7. I+, S+, C+ beta-lactamase (TEM1)

Map20-mCherry fusion protein is expressed and present in both soluble and insoluble part of E. coli cell.