Device

Part:BBa_K2082116

Designed by: Carsten Hain   Group: iGEM16_Bielefeld-CeBiTec   (2016-10-14)


dnaQ926 under control of a modified Pbad

Genome wide mutator BBa_K2082116

This part is the function expression device of BBa_K2082110 under control of BBa_K2082112. Its effective the smaller version of BBa_K2082117.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal BamHI site found at 1411
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
    Illegal AgeI site found at 1283
    Illegal AgeI site found at 1772
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961


This device expresses dnaQ926 (BBa_K1333108) from our construct BBa_K2082110 under control of the PBAD BBa_K2082112. We used BBa K2082116 as a mutator on pSB1C3 in E. coli Top10. We measured the performance in reversion assays and quantifiy the revertant frequency at two timepoints of a cultivation. The reversion frequencies f and the total cell count N enables us to calculate induced and basal mutation rate of BBa_K2082116 by using
As copy number we used 40 as determined in one of our high-throughput sequencing experiment.
Our results show a increased mutation rate by induction with 20 mM arabinose. The basal mutation rate is still considerable even when the promoter is repressed with 20 mM glucose.

Figure 1: Mutation rate of BBa K2082116 determined by stop beta lactamase reversion assays.
The mutation rate of BBa_K2082116 is (2.3±1.12)×10-6 bp-1×generation-1 induced and (6.67±4.09)×10-8 bp-1×generation-1 repressed.

Application Protocol

  • Create competent cell of E. coli with arabinose expression genotype (e.g. araD139 Δ(ara-leu)7697).
  • For library generation
    • Transform mutator plasmid and plasmid with the gene of interest
    • Grow cells in LB mit appropiate anitbiotics
    • Induced mutagensis by addition of 20 mM arabinose upon reaching mid-log phase
    • Grow until saturation
  • Coupling with selection of improved proteins can be useful to screen more variants

Characterization protocols

  • Reversion assay
    • Transform reporter plasmid and pSB1C3::K2082117 into E. coli Top10
    • After regeneration inoculate prewarmed 10 mL LB with appropriate antibiotic and 20 mM glucose with 10 μL
    • Grow until OD600 ~0.3
    • Add 20 mM arabinose
    • Grow until saturation
    • Plate serial dilutions on LB agar plates with and without ampicillin
    • Determine total cell count and revertant count
    • Reversion frequency: number of Revertants/ number of viable cell count

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