Endy:Screening plasmid/v2.0/Construction

Screening plasmid 2.0 construction has two components:

1. Direct synthesis of the I/O reporter section
   • In progress - order placed 8/25/06
2. Construction of several induction system 'front ends'

Front End Construction

Construction steps:

1. Build induction system from BB parts, clone into 3K3, 1A3, 2K3?
2. Cap forward BB site

AHL-induced

1. <bbpart>F2620</bbpart> Ptet controls luxR expression
2. <bbpart>F2621</bbpart> lux Pl control luxR expression
3. Reversed <bbpart>I13212</bbpart> + R0062

Next Steps

1. Order primers to flip I13212
2. Move <bbpart>F2620</bbpart> & <bbpart>F2621</bbpart> into psb1a3, psb3k3, and psb4a3
3. Cap E/X for <bbpart>F2620</bbpart> & <bbpart>F2621</bbpart> in each plasmid.
4. Constructed reversed I13212, put it in plasmids, and cap E/X

IPTG-induced

1. Do we have (promoter)(rbs)(lacI)(Plac) already in registry?
   • Nope we don't even have lacI without the ssrA tag, <bbpart>C0013</bbpart> is a designed part w/o the tag, but it wasn't constructed. Though caitlin said she had ordered the primers for removing the LVA tag at one point.
     • "As for the primers, I'm not sure if they're still around. You can look in the "primers" binder above my old desk to find the name and sequence I used. It probably would have been "C0013_build_F" and "C0013_build_R". There are a few boxes of primers in the -20. (But that set might have gotten tossed...)"
2. Reverse lacI

Arabinose-induced

1. Do we have the right intermediate?
   • <bbpart>P1010</bbpart>
   • <bbpart>I13458</bbpart>+<bbpart>I13453</bbpart>
     • We could build this out just to have a split up version of P1010 (would be a useful registry part).