Endy:Library-based construction/Inverter library protocol

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

Contents

Generate inverter library via mutagenic PCR

  1. 50 ul PCR reaction using Stratagene's GeneMorph® II Random Mutagenesis kit
    • Amplify VF2-VR
    • Generally I have been using the "high" mutation frequency protocol, with ~20 ng initial target DNA
  2. Perform a PCR cleanup and elute with 30 ul

Prepare cut SP1.0

  1. Midi or maxiprep SP1.0 with P1010 insert
  2. Digest 10 ug of vector XP (or ES) in 50 ul using 1 ul of each enzyme
    • 1 hr at 37 C? --> should find out optimal/minimum time required for amt of DNA we're using, 1 hr is probably enough incubation time for BioBricks enzymes, which are listed by NEB as time-saver qualified restriction enzymes
  3. Heat inactivate the reaction and perform a PCR cleanup with elution volume of 30 ul
    • This won't get rid of P1010, but the ccdB gene should kill any transformants that get this insert

Digest the library and ligate into SP1.0

  1. Perform a 50 ul restriction digest using all of the PCR reaction
    • 1 hr incubation time? -->should find out optimal/minimum time required for amt of DNA we're using
  2. Heat-inactivate the digest for 20 min
    • 65 C for EcoRI, XbaI, SpeI
    • 80 C for PstI
  3. Perform a PCR cleanup on the digest and elute with 30 ul EB
    • Note: This doesn't get rid of the cut ends from VF2-VR amplification, which are each about ~140 bp from the BioBricks prefix and suffix sites. This probably reduces the subsequent ligation efficiency, but from qualitative experience, the DNA lost from performing a gel extraction reduces the resulting library size by about the same or more. Could possibly improve this by amplifying directly from the BB prefix and suffix, have ordered primers for this but haven't determined if they work yet.
  4. Perform a 20-ul ligation:
    • 2 ul 10X T4 DNA ligase buffer
    • 8.5 ul cut SP1.0
    • 8.5 ul library insert
    • 1 ul (400 units) T4 DNA ligase
    • Note: NEB recommends a DNA concentration of 1-10 ug/ml (2-20 ng in a 20-ul reaction) for efficient circularization and subsequent transformation. In general, try to max out the DNA concentration in the ligation, since it allows you to electroporate more DNA without arcing later on.
  5. Heat inactivate ligation reaction 10 min at 65 C unless you are using Quick Ligation Kit buffer, which contains PEG. If the ligation contains PEG, purify before using for transformation.
  6. Dialyze the ligation product for 30 mins

Transform into electrocompetent CW2553/pJat8

Controls

Procedure

  1. Keep 2mm cuvettes on ice
  2. Thaw electrocompetent cells (40 ul per tube) on ice
  3. Add 1 ml of SOC to 12x75mm culture tube(s)
  4. 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?)
  5. Add cell/DNA mix to the cuvettes in the center channel
  6. 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
  7. 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
  8. 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
  9. Quickly add the 1 ml of SOC from the culture tube and pipette up and down to get cells out of the chamber, then pipette everything back into the culture tube
  10. Put the cells on ice for a couple minutes to allow them to recover
  11. Grow for one hour at 37 C without antibiotic
  12. Plate to determine library size and transformation efficiency
    • plate a portion on selective media in order to determine the library size (50 ul = 1/20 of total library, could plate less depending on how big you think your library is)
    • plate a portion on non-selective media in order to determine the number of surviving cells following electroporation (generally a serial dilution to 1/1e6 works fine)
  13. Grow the rest of the culture at 37 C overnight in 25 ml of M9+Amp/Gent

Rest of procedure

  1. Store X glycerols of the libary from the overnight culture
  2. Spin down cells, remove SOC/M9 media (catabolite repression from glucose in SOC affects arabinose induction system), and resuspend in ~10 ml M9
  3. Measure OD to determine how many cells to add to your experimental cultures
    • To be safe, take at least 10 times (more?) the library size (calculated from plates)
  4. Start experimental cultures with low (0%-1e-6%) induction and high (1e-4%-1e-3%) induction
    • Be sure to make a high enough dilution to allow for at least 9-10 doublings without going into stationary (OD600 < 1)
  5. Grow overnight (rough doubling time is ~1.2-2 hrs)
  6. Sort cells with LOW in/HIGH out and HIGH in/LOW out into sub-libraries
    • Have 1 ml M9+AG in 12x75mm tubes ready for collecting cells
    • Be sure to also run analytical samples of the populations, so that we have a record of what the libraries looked like (data from the actual sorting can't be saved)
  7. Grow up the sub-libraries in the 12x75mm tubes until they are dense enough to transfer to 5 ml cultures (let them double a number of times so you won't lose clones)
  8. Grow up the 5 ml culture and store X glycerols
  9. Measure OD's of the sub-library cultures and innoculate experimental cultures 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)
    • To be safe, take at least 10 times (more?) the number of cells we sorted
    • Be sure to make a high enough dilution to allow for at least 9-10 doublings without going into stationary (OD600 < 1)
  10. Grow overnight (rough doubling time is ~1.2-2 hrs)
  11. Conduct a second round of sorting to create sub-libraries that we've tested under both HIGH input and LOW input conditions
  12. Grow up the sub-libraries and store X glycerols, as well as plate to get individual mutants
  13. Test individual mutants, and if necessary, repeat the rounds of screening to tighten up the library
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