Difference between revisions of "Part:BBa K1604010"
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===Usage and Biology===
 
===Usage and Biology===
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PR is a light-powered proton pump that belongs to the rhodopsin family.
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This protein has the property to use light energy to generate an outward proton flux that can subsequently power cellular processes, such as ATP synthesis, chemiosmotic reactions and rotary flagellar motor.[1] It was demonstrated that engineered <i>E. coli</i> with PR, whose cellular respiration is inhibited and undergoes anaerobic conditions, become light-powered in presence of light. [2]
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<div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/d/d6/Unitn_pics_PRmechanism.jpg"></img></div></html>
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<p style="width:600px; margin-left:150px; margin-bottom:60px;
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text-align:justify "><b>FIGURE 1. Proteorhodopsin can drive ATP synthesis.</b> Proposed mechanism of PR associated to ATP-synthase complex. Light-activated proteorhodopsin pumps protons outwards increasing the proton motive force, protons can reenter the cells through ATP-synthase complex powering ATP production.</p>
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<div style="text-align:center"><html><img src="https://static.igem.org/mediawiki/parts/1/1e/Unitn_pics_PRinduction.png"style="width:75%;"></img></div></html>
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<p style="width:600px; margin-left:170px; margin-bottom:60px;
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text-align:justify "><b> Figure 2. Optimal conditions for a proper folding.</b> NEB10&beta; cells transformed with BBa_K1604010 and grown in LB and induced in LB or M9 with 5 mM arabinose and 10 uM of retinal at 30C or 37C. Negative controls were cells transformed with BBa_K731201 (i.e. araC-pBAD). By the screening of several parameters (media, temperature, time of induction) we discovered that the optimal expression conditions were in LB, at 37 °C overnight in the presence of 10 μM of all-trans retinal. It is a membrane protein that needs time to fold properly into the membrane and requires retinal for the correct folding.</p>
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Revision as of 14:08, 6 September 2015

araC-pBAD + Proteorhodopsin

AAA

Usage and Biology

PR is a light-powered proton pump that belongs to the rhodopsin family.

This protein has the property to use light energy to generate an outward proton flux that can subsequently power cellular processes, such as ATP synthesis, chemiosmotic reactions and rotary flagellar motor.[1] It was demonstrated that engineered E. coli with PR, whose cellular respiration is inhibited and undergoes anaerobic conditions, become light-powered in presence of light. [2]

FIGURE 1. Proteorhodopsin can drive ATP synthesis. Proposed mechanism of PR associated to ATP-synthase complex. Light-activated proteorhodopsin pumps protons outwards increasing the proton motive force, protons can reenter the cells through ATP-synthase complex powering ATP production.


Figure 2. Optimal conditions for a proper folding. NEB10β cells transformed with BBa_K1604010 and grown in LB and induced in LB or M9 with 5 mM arabinose and 10 uM of retinal at 30C or 37C. Negative controls were cells transformed with BBa_K731201 (i.e. araC-pBAD). By the screening of several parameters (media, temperature, time of induction) we discovered that the optimal expression conditions were in LB, at 37 °C overnight in the presence of 10 μM of all-trans retinal. It is a membrane protein that needs time to fold properly into the membrane and requires retinal for the correct folding.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1740
    Illegal BamHI site found at 1144
    Illegal XhoI site found at 1313
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
    Illegal AgeI site found at 1502
    Illegal AgeI site found at 1877
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961