Rao:CRIM

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(New page: =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 eleme...)
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=Materials=
=Materials=
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* 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 λ P<sub>R</sub> regulated by the Ts λcI857 repressor. The idea is to cure the plasmid while expressing the corresponding Int.
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** pINTts for Int(λ)
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** pAH69 for Int(HK022)
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** 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.
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** 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.
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** Rao lab pir strains: BW25141, DH5αλpir+, REB114λpir+.
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** Rab lab pir-116 strains: BW25142, Ec1000D::pir-116
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* Some LB, or whatever rich media suits your fancy.
=Method=
=Method=
 +
* Grow an overnight of your

Revision as of 18:48, 12 January 2008

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 λ PR 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
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