Team:Tsinghua/experiment

From 2011.igem.org

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Revision as of 16:03, 4 October 2011

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Present proteins to the outer membrane

   We used OmpA-His tag-GFP to test the conditions for presenting protein to the outer membrane.
   The design allows double check whether the C-terminal moeity can be presented outside. One is by green fluorescence, the other is by Western blotting with antibody against his tag.

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℃

Ni-column purification

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

Thuexp cherrysds2.png1. Sonicated 2. Supernatant 3. Pellet 4. Flowthrough 5. Buffer wash 6. Resin 7. 5mM Elution 8. 20mM Elution 9. Resin 2 10. 200mM Elution 

 1    2    3    4    5      6  Marker  7   8    9    10

Western blotting

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

Thuexp cherrywb.png


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