Endy:Library-based construction/Inverter library protocol

We should be fleshing this out as we work through it a few times

Generate inverter library via mutagenic PCR

• 50 ul PCR reaction using Stratagene's GeneMorph® II Random Mutagenesis kit
• PCR cleanup and elute with 30 ul

Digest the library and ligate into SP1.0

1. Perform a 50 ul restriction digest using all of the PCR reaction
2. Separate the insert out on a gel and perform gel extraction
   • Elute the entire reaction into a single 30 ul volume (reuse on multiple columns if necessary)
3. Perform a 40-ul ligation:
   • 4 ul 10X T4 DNA ligase buffer
   • All of the digest
     • Can also save some of the ligation and use less, probably don't need this much
   • 1 ul of the cut SP1.0 vector
   • 2 ul T4 DNA ligase
   • H₂0 to 40 ul

Transform into electrocompetent CW2553/pJat8

• controls:
  • (+): pUC18 (Amp resistance)
  • (-): cells only
• Procedure:
  1. Keep 2mm cuvettes on ice
  2. Dialyze ligation product ~20 min
  3. Thaw electrocompotent cells (40 ul per tube) on ice
  4. Add 1 ml of SOB (from media room) to 12x75mm culture tube(s)
  5. Quickly spin down the thawed cells and add 3 ul (more, less?) of ligation product to the cells
     • Keep on ice as much as possible
     • Swirl with pipette tip to mix, pipetting is bad (heat, shear?)
  6. Add cell/DNA mix to the cuvettes in the center channel
  7. On the electroporation machine's menu, go to 4) pre-set protocols; 1) bacterial; 2) E.Coli-2mm, 2.5kV
     • Should end up on a screen that lists the voltage and other parameters
     • Should have a blinking 'P' in the corner of the screen
  8. Tap the cuvette on the table to settle cells to the bottom of the chamber, wipe the metal with a kim wipe and place in electroporation machine
  9. Press pulse button, wait, and check for a exponential decay graph with ~1800 volts applied and a time of ~4s
     • If the screen goes back to the main menu, it worked
     • If you hear a cracking noise/loud pop, sample arced, cells died, try again
  10. Quickly add the 1 ml of SOB from the culture tube and pipette up and down to get cells out of the chamber, then pipette everything back into the culture tube
  11. Grow for one hour without antibiotic (1ml)
      • plate a portion (50 ul? more, less?) on selective media in order to determine the library size
      • plate a portion (1/1e6? 1e7?) on non-selective media in order to determine the number of surviving cells following electroporation
  12. Move the rest of the culture into a higher volume (10ml?) of selective media

Rest of procedure

1. Store 5 glycerols of the libary from the overnight culture
2. Spin down cells and switch to M9 media
3. Measure OD to determine how many cells to add to your experimental cultures
   • X times larger than library size calculated from plates?
4. Start experimental cultures (in 25 ml?) with no induction and high induction
5. Sort cells with LOW in/HIGH out and HIGH in/LOW out into sub-libraries
   • Be sure to also collect analytical samples of the populations after sorting, so that we have data of what the libraries looked like.
6. Grow up the sub-libraries under the reverse condition (and the same condition)
   • The reason for growing under the same conditions is to see how effective the sorting was (control, basically).
7. Conduct second round of sorting to create sub-libraries that we're tested under both HIGH input and LOW input conditions
8. Grow up library and store 5 glycerols, as well as plate to get individual mutants
9. Test individual mutants, if necesary can repeat the rounds of screening to tighten up the library