Present proteins to the outer membrane

Molecular cloning

We fused GFP to the C-terminal of OmpA and inserted the his-tag in between them. The final plasmid is as follows.

Expression Test

Test for proper inducing conditions

Use 0.5mM IPTG, 18 centigrade, 12 hours induction in later experiments.

Fluorescence microscopy

Green fluorescence is bright.

In order to determine whether GFP is presented outside the membrane, cells were digested with Protease K at 50 centigrade for 30min.

Color Channel	Post-digest	Pre-digest
GFP
DAPI
Merge

The fluorescence disappeared completely after digestion, which seems too good to be true. Hence, we performed Western blotting to be sure of the result.

Western Blotting

The cell components were first fractionated before blotting.

1. Sonicate the bacteria culture.

2. Centrifuge 13000rpm, 30min to separate soluble protein and membrane components from the cell debris and the non-soluble protein.

3. 51000rpm 1h to separate soluble protein from membrane components.

The majority of the OmpA-GFP protein is in the pellet after the first centrifuge, indicating incomplete cell lysis or inefficient protein folding. Nonetheless, from the later result, we can see that it is exclusively in the membrane and not in the soluble proteins.

Expression of Substrate

Molecular cloning

We synthesized the multi-proline sequence and linked it to mCherry.

Test of Expression

After Pro-rich mCherry expression, significant color change can be observed (bacteria cells turned cherry red as a result of mCherry expression)

From left to right, IPTG concentration: 0, 0.1mM, 0.5mM, 1mM

SDS-PAGE

The bacteria culture was sonicated and then centrifuged at 13000rpm for 15min. Both the supernatant and the pellet were loaded onto SDS-PAGE.

Contrary to the sharp change in color, no obvious band can be seen in SDS-PAGE. Hence, further purification of protein is necessary.

Ni-column purification

The cell lysate was then purified by Ni-column. SDS-PAGE indicates that the protein is largely purified.

Western blotting

In order to be sure that the protein we got is multi-proline mCherry, Western blotting was performed.

As is shown, the protein eluted is indeed his-tagged mCherry.

Binding & Release Module

HIV Protease is fused with OmpA to be transported outside the membrane to facilitate substrate release.

SH3 domain is fused with OmpA as the binding module. In order to be readily released, an HIV protease site is settled in the middle.

Bind & Release

E. coli expressing OmpA-HIV protease site-SH3 was mixed with mCherry solution at room temperature for 30 minutes.

After washing with distilled water twice, some of the bacteria were fixed on a slip and stained with DAPI.

Color Channel	E. coli with IPTG induction	E. coli without induction
mCherry
DAPI
merge

The remaining were mixed with E. coli expressing OmpA-HIV protease at room temperature for 30 minutes to release the cargo.

After washing with distilled water twice, bacteria were fixed on a slip and stained with DAPI.

Color Channel	+ E. coli expressing protease	+ E. coli without protease
mCherry
DAPI
merge