Team:Tsinghua/experiment

From 2011.igem.org

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After washing with distilled water twice, some of the bacteria were fixed on a slip and stained with DAPI.
After washing with distilled water twice, some of the bacteria were fixed on a slip and stained with DAPI.
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Revision as of 16:52, 4 October 2011

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Present proteins to the outer membrane

Molecular cloning

OmpA, GFP His tag 1507bp confirmed by sequencing

Thuexp og.png

The final plasmid is as follows.

Thuexp ogp.png

Expression Test

Test for proper inducing conditions

Group 1 2
Volume 3ml
IPTG(mM) 0.1
0.5
1.0
Temperature 30oC 18oC
Duration 12h 12h
Result Media remained yellowish.
E. coli white
Media turned greenish.
E. coli green

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 Thuexp gn.png Thuexp gp.png
DAPI Thuexp gn d.png Thuexp gp d.png
Merge Thuexp gn m.png Thuexp gp m.png

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.

Thuexp west.png

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.

Thuexp omp.png


Test of Expression

Group 1 2
Volume 3ml
IPTG(mM) 0.1
0.5
1.0
Temperature 30oC 18oC
Duration 12h 12h
Result Media remained yellowish.
E. coli white
Media turned purple.
E. coli cherry pink

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

Thuexp cherryexp.png

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.

Thuexp cherrysds1.png

1 2 3 4 5 6 7 8 Marker 9 10 11 12 13 14
1. Pellet, 1mM 18℃ 2. Supernatant 1mM 18℃ 3. P 0.5mM 18℃
4. S 0.5mM 18℃ 5. P 0.1mM 18℃ 6. S 0.1mM 18℃
7. P NC 18℃ 8. S NC 18℃ 9. S 1mM 30℃
10. P 1mM 30℃ 11. S 0.5mM 30℃ 12. P 0.5mM 30℃
13. S 0.1mM 30℃ 14. P 0.1mM 30℃


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.

Thuexp cherrysds2.png

1 2 3 4 5 6 Marker 7 8 9 10 Marker
1. Sonicated 2. Supernatant 3. Pellet
4. Flowthrough 5. Buffer wash 6. Resin
7. 5mM Elution 8. 20mM Elution 9. Resin 2
10. 200mM Elution

Western blotting

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

Thuexp cherrywb.png

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.

Thuexp op.png

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.

Thuexp ohs.png

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 Thuexp bind p.png Thuexp bind n.png
DAPI Thuexp bind p d.png Thuexp bind n d.png
merge Thuexp bind p m.png Thuexp bind n m.png

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 Thuexp release p.png Thuexp release n.png
DAPI Thuexp release p d.png Thuexp release n d.png
merge Thuexp release p m.png Thuexp release n m.png