Endy:Screening plasmid/v2.0/Testing

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There are a few tests we can get started on before the complete SP2.0 is constructed.

Induction system test

AHL-induction test

  • <bbpart>I13546</bbpart> (F2620 + GFP/RFP) on psb3K3
  • <bbpart>I13547</bbpart> (F2621 + GFP/RFP) on psb3K3

Results

Date?

  • <add images when finished processing data>
  • Looks like the GFP is responding well over a range of AHL conentrations. So the induction system itself looks pretty good. In fact the GFP expression was higher than we had seen with pBAD on a high copy (psb1A2) plasmid, and F262X were on 3k3.
  • However, the RFP is displaying low fluorecence intensity. Planning experiments to figure out why - see below.
  • Also, the data collection was set to linear scale, so will be redoing this experiment on 9/21/2006

9/21/2006

  • <bbpart>I13546</bbpart> (F2620 + GFP/RFP) on psb3K3
    • Induced 1nM, 10nM, 50nM, 100nM, 500nM, 1uM
  • <bbpart>I13547</bbpart> (F2621 + GFP/RFP) on psb3K3
    • Induced 1nM, 5nM, 10nM, 50nM, 100nM, 1uM
  • The variation in the induction levels was based on the results of the previous experiment, which demonstrated over which induction range the device was switching in response to AHL.

RFP expression tests

Date?

  • Will be comparing the expression of several costructs:
    1. <bbpart>I13530</bbpart> - pbad controlled RFP
    2. <bbpart>I13521</bbpart> - Ptet controlled RFP
    3. <bbpart>I13549</bbpart> - F2620 controlled RFP
    4. <bbpart>I13550</bbpart> - F2621 controlled RFP
    5. <bbpart>I13551</bbpart> - F2620 controlled RnaseE+hairpin+RFP
    6. <bbpart>I13552</bbpart> - F2621 controlled RnaseE+hairpin+RFP
  • All will be induced at the highest induction level (except tet which is constitive in this case because no tetR present).
  • <flojo plots here>
  • The RFP lacking the RnaseE site and the hairpin produced a weaker signal in response to equivalent induction. This is counter to some of our previous results using the pBAD induction system. Should consider revisiting those.

SP1.0 debugging

Understanding the issues with GFP/RFP relationship in SP1.0 will help us to better debug SP2.0 down the line.

Arabinose test

Will use a construct that has a constituitive promoter (R0074) driving expression of GFP/RFP. Will then grow in the presence of varying levels of arabinose and look at the effect on GFP/RFP levels.

  • <bbpart>I13553</bbpart>
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