Difference between revisions of "Part:BBa K533002:Experience"

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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===Applications of BBa_K533002===
 
===Applications of BBa_K533002===
 +
This part is used for specific binding to proteins with multi-proline peptide in the media and then releasing the cargo after interaction with HIV protease.
 +
 +
We first tested the expression under different concentration of IPTG induction.
 +
After 12 hours induction at 18 centigrade, we harvested the bacteria and mixed with multi-proline mCherry.
 +
 +
{| style="background: none; text-align:center;" align="center"
 +
| Color Channel
 +
| 0.1mM IPTG induction
 +
| 0.5mM IPTG induction
 +
| 1.0mM IPTG induction
 +
|-
 +
| mCherry
 +
| [[Image:Thuexp_bind_0.1.png|250px]]
 +
| [[Image:Thuexp_bind_0.5.png|250px]]
 +
| [[Image:Thuexp_bind_1.0.png|250px]]
 +
|-
 +
| DAPI
 +
| [[Image:Thuexp_bind_0.1_d.png|250px]]
 +
| [[Image:Thuexp_bind_0.5_d.png|250px]]
 +
| [[Image:Thuexp_bind_1.0_d.png|250px]]
 +
|-
 +
| merge
 +
| [[Image:Thuexp_bind_0.1_m.png|250px]]
 +
| [[Image:Thuexp_bind_0.5_m.png|250px]]
 +
| [[Image:Thuexp_bind_1.0_m.png|250px]]
 +
|}
 +
 +
As is shown, strongest binding is seen in 0.1mM induction, suggesting that high expression level might lead to inclusion bodies or inefficient transport onto the membrane.
 +
 +
E. coli with the plasmid for OmpA-HIV site-SH3 is induced with 0.1mM IPTG at 18 centigrade for 12 hours before harvesting by mild centrifuge.
 +
 +
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.
 +
 +
{| style="background: none; text-align:center;" align="center"
 +
! Color Channel
 +
! E. coli with IPTG induction
 +
! E. coli without induction
 +
|-
 +
| mCherry
 +
| [[Image:Thuexp_bind_p.png|350px]]
 +
| [[Image:Thuexp_bind_n.png|350px]]
 +
|-
 +
| DAPI
 +
| [[Image:Thuexp_bind_p_d.png|350px]]
 +
| [[Image:Thuexp_bind_n_d.png|350px]]
 +
|-
 +
| merge
 +
| [[Image:Thuexp_bind_p_m.png|350px]]
 +
| [[Image:Thuexp_bind_n_m.png|350px]]
 +
|}
 +
 +
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.
 +
 +
 +
{| style="background: none; text-align:center;" align="center"
 +
! Color Channel
 +
! + E. coli expressing protease
 +
! + E. coli without protease
 +
|-
 +
| mCherry
 +
| [[Image:Thuexp_release_p.png|350px]]
 +
| [[Image:Thuexp_release_n.png|350px]]
 +
|-
 +
| DAPI
 +
| [[Image:Thuexp_release_p_d.png|350px]]
 +
| [[Image:Thuexp_release_n_d.png|350px]]
 +
|-
 +
| merge
 +
| [[Image:Thuexp_release_p_m.png|350px]]
 +
| [[Image:Thuexp_release_n_m.png|350px]]
 +
|}
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 18:01, 5 October 2011

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K533002

This part is used for specific binding to proteins with multi-proline peptide in the media and then releasing the cargo after interaction with HIV protease.

We first tested the expression under different concentration of IPTG induction. After 12 hours induction at 18 centigrade, we harvested the bacteria and mixed with multi-proline mCherry.

Color Channel 0.1mM IPTG induction 0.5mM IPTG induction 1.0mM IPTG induction
mCherry Thuexp bind 0.1.png Thuexp bind 0.5.png Thuexp bind 1.0.png
DAPI Thuexp bind 0.1 d.png Thuexp bind 0.5 d.png Thuexp bind 1.0 d.png
merge Thuexp bind 0.1 m.png Thuexp bind 0.5 m.png Thuexp bind 1.0 m.png

As is shown, strongest binding is seen in 0.1mM induction, suggesting that high expression level might lead to inclusion bodies or inefficient transport onto the membrane.

E. coli with the plasmid for OmpA-HIV site-SH3 is induced with 0.1mM IPTG at 18 centigrade for 12 hours before harvesting by mild centrifuge.

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

User Reviews

UNIQ63466cd45a191a98-partinfo-00000000-QINU UNIQ63466cd45a191a98-partinfo-00000001-QINU