Difference between revisions of "Part:BBa K533003"

(→‎Usage and Biology)
(→‎Usage and Biology)
 
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|-
 
| mCherry
 
| mCherry
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| [[Image:Thuexp_release_p.png|350px]]
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| [[Image:Thuexp_release_p.png|250px]]
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| [[Image:Thuexp_release_n.png|350px]]
+
| [[Image:Thuexp_release_n.png|250px]]
 
|-
 
|-
 
| DAPI
 
| DAPI
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| [[Image:Thuexp_release_p_d.png|350px]]
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| [[Image:Thuexp_release_p_d.png|250px]]
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| [[Image:Thuexp_release_n_d.png|350px]]
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| [[Image:Thuexp_release_n_d.png|250px]]
 
|-
 
|-
 
| merge
 
| merge
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| [[Image:Thuexp_release_p_m.png|350px]]
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| [[Image:Thuexp_release_p_m.png|250px]]
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| [[Image:Thuexp_release_n_m.png|350px]]
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| [[Image:Thuexp_release_n_m.png|250px]]
 
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DAPI in the first set of data seems a bit diffusing, suggesting possible damage to the host cell.
 
DAPI in the first set of data seems a bit diffusing, suggesting possible damage to the host cell.
  
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<html>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K533003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K533003 SequenceAndFeatures</partinfo>

Latest revision as of 14:59, 5 October 2011

OmpA-HIV aspartyl protease

OmpA is used to present HIV aspartyl protease outside the E. Coli membrane so that it can cut other proteins with the specific sequence outside in the media.


Usage and Biology

We tested the function of this part. Our part BBa_K533002 contained an HIV-protease site. After BBa_K533002 was bound to mCherry, we cleaved it with bacteria expressing this part.

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

It shows that it works.

We further optimized the conditions for expression induction.

We induced OmpA-protease fusion protein with IPTG concentration of 0.1mM, 0.5mM and 1mM. After 12hours induction at 18 centigrade, protease activity were observed in all the samples.

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

Protease cleavage will extinguish the red fluorescence from mCherry.

As is shown, the cleavage activity is highest at 0.1mM induction, suggesting the too high expression level might hinder protein folding.

DAPI in the first set of data seems a bit diffusing, suggesting possible damage to the host cell.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 936
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]