Difference between revisions of "Part:BBa J25500:Design"

 
(→‎Design Notes: added transcript from correspondance w/ natalie)
 
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===Design Notes===
 
===Design Notes===
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Question: in M30109, why is i15010 controlled by a separate promoter from i15008, i15009?  Could they all be contiguous and controlled by a single promoter?  
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Question: in M30109, why is i15010 controlled by a separate promoter from i15008, i15009?  Could they all be contiguous and controlled by a single promoter?
  
 +
I sent Natalie an email with the following:
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<blockquote>
 +
Your design places the two phycobilins (i15008 & i15009) under  the control of a pBad promoter and phytochrome Cph8 under the control  of a pTet promoter.  Did you incorporate additional compound parts to  generate araC and tetR proteins (the repressors of those two  promoters) in your final system, or if not, did your host cell do it  natively for you?
  
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<br><br>
 +
 +
If neither, then what was the activity of the two promoters?  Did  pBad basically stay shut off, lacking arabinose, regardless of the  lack of araC?  And did pTet basically stay activated, lacking tetR?  If so, then I'm missing something, because I thought the whole idea  was to express the genes controlled by pBad before Cph8.  Did you  initially pulse the system with arabinose or something?
 +
</blockquote>
 +
 +
She responded:
 +
<blockquote>
 +
Your questions make perfect sense. It's the science that doesn't. Just like in
 +
the Nature paper when they used no arabinose and no TetR protein, the three
 +
part light sensor uses basal (or readthrough) transcription of the promoters
 +
for gene expression. And it turns out the system is tough to calibrate, i.e.
 +
too much phycobilins and it fails, too little and it fails, too much light and
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the phycobilins are inactivated, etc. Robust it isn't but when it works it's
 +
very cool.
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</blockquote>
  
 
===Source===
 
===Source===

Latest revision as of 20:43, 9 August 2006


Light-repressed mCherry (uses photosensor)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1702
    Illegal BglII site found at 4190
    Illegal BamHI site found at 65
    Illegal XhoI site found at 2297
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1255
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Question: in M30109, why is i15010 controlled by a separate promoter from i15008, i15009? Could they all be contiguous and controlled by a single promoter?

I sent Natalie an email with the following:

Your design places the two phycobilins (i15008 & i15009) under the control of a pBad promoter and phytochrome Cph8 under the control of a pTet promoter. Did you incorporate additional compound parts to generate araC and tetR proteins (the repressors of those two promoters) in your final system, or if not, did your host cell do it natively for you?



If neither, then what was the activity of the two promoters? Did pBad basically stay shut off, lacking arabinose, regardless of the lack of araC? And did pTet basically stay activated, lacking tetR? If so, then I'm missing something, because I thought the whole idea was to express the genes controlled by pBad before Cph8. Did you initially pulse the system with arabinose or something?

She responded:

Your questions make perfect sense. It's the science that doesn't. Just like in the Nature paper when they used no arabinose and no TetR protein, the three part light sensor uses basal (or readthrough) transcription of the promoters for gene expression. And it turns out the system is tough to calibrate, i.e. too much phycobilins and it fails, too little and it fails, too much light and the phycobilins are inactivated, etc. Robust it isn't but when it works it's very cool.

Source

The photosensor (M30109) Natalie Kuldell used in her BE.109 course & mCherry+lva (J06505).

References