Rao:CRIM

Overview

The CRIM (Conditional-replication, integration, and modular) plasmid system is a series of plasmid vectors which use the R6K replicon for maintenance as an autonomous DNA element in pir+ hosts and contain an attP site for site-specific integration into the chromosome of non-pir+ hosts. Integration into non-pir+ hosts requires the expression of the corresponding lambdoid phage Int protein (ie integration into attBλ requires attPλ and Intλ). The procedure outlined here is directly adapted from the 2001 Haldimann and Wanner paper.

Materials

• Desired host containing the Int expressing helper plasmid at 30°C. All of these helper plasmid have the Ts SC101 replicon and the int gene under λP_RM regulated by the Ts λcI857 repressor. The idea is to cure the plasmid while expressing the corresponding Int.
  • pINTts for Int(λ)
  • pAH69 for Int(HK022)
  • pAH123 for Int(Φ80)
  • pAH121 for Int(P21)
  • pAH130 for Int(P22) - Use of this att site isn't recommended in e14+ strains (ex. MG1655)
• Purified CRIM plasmid.
  • A useful note. The pir allele allows for low copy replication of your CRIM plasmid and the pir-116 allele allows for high copy replication of your CRIM plasmid.
  • Rao lab pir strains: BW25141, DH5αλpir+, REB114λpir+.
  • Rab lab pir-116 strains: BW25142, Ec1000D::pir-116
• Some LB, or whatever rich media suits your fancy.

Method

• Grow an overnight of your Int expressing strain in LB(Amp 100 μg/mL) at 30°C.
• The following morning, subculture your Int expressing strain 1:100 in fresh LB(Amp 100 μg/mL).
  • I usually concentrate cells for electroporation 100X. So 5 mL of culture will make 50 μL of electrocompetent cells, etc.
• Grow with vigorous shaking at 30°C, until your culture has reached an OD600 of around 0.6.
  • Do not let the cells enter late-log/stationary phase. You need them to continue growing during recovery so they can express Int effectively.
• Prepare the cells for electroporation at 100X concentration.
• In an 1.8 mL tube mix 50 μL of 100X cells and 2 to 5 μL of your purified CRIM plasmid.
• Electroporate, and recover in 1 mL LB for 1 hr at 37°C.
• After one hour, move the cultures to 42°C for 30 minutes before plating.
  • This step helps cure the plasmid and also allows for high level expression of the Int protein. We've found this step increases the efficiency of integration and should not be omitted. A high level of false positives have been observed when recovery is only done at 37°C.
• Plate on selective agar for your CRIM's marker.
  • For kanamycin we've found 40 μg/mL is better than 10 μg/mL suggested in the Haldimann paper. All other antibiotics are typically used at concentrations suggested.
  • It is also advisable to plate 1:10 and 1:100 dilutions as the integration process is usually efficient and this helps reduce false positives.
• The next day, restreak some colonies on LB w/ or w/o antibiotic (user preference here) and grow at 42°C to clear the helper plasmid.
• The third day (or later the second day depending on how late you stay), PCR check the integration using the primers suggested in the paper.

Notes

• It is advisable to restreak a few times to make sure your integration is stably maintained, especially if you're using the TetR CRIM derivatives or you've integrated into the P22 att site.
• Also, P1 transduction of this insertion back into your parent strain is sometimes useful if you are worried about illegitimate recombination events. We've not observed this to occur very often, but it has happened.