Device

Part:BBa_K2082117

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


Six mutator genes under control of a modified Pbad

Genome wide mutator BBa_K2082117

This part is the assembly of six mutagenic genes with weak RBS (BBa_K2082111) under control of a modified PBAD (BBa_K2082112).
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
    Illegal BamHI site found at 2298
    Illegal XhoI site found at 3880
  • 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 BsaI site found at 4542
    Illegal SapI site found at 961


This expression device is our most potent genome wide mutator construct. It s overall structure is based on the mutagensis plasmid MP6 from Badran and coworkers. We rebuild and characterized this plasmid with coding sequences and regulatory elements from the iGEM parts registry.

The construct consists of six mutagenic proteins which impair the E. coli DNA fidelity mechanism on different ways (Figure 1) thus increasing the mutagenesis rate to (7.16±4.31)×10-6 bp-1×generation. The expression is controlled by a PBAD resulting in an 285x increase in mutagensis rate when induced.


Figure 1: Schematic view of the mutator genes of BBa_K2082117 and the affected DNA fidelity mechanism. (Badran, Liu 2015).

Characterization

This device expresses six mutagenesis genes from our construct BBa_K2082111 under control of the PBAD BBa_K2082112. We used BBa K2082117 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_K2082117 by using
As copy number we used 40 as determined in one of our high-throughput sequencing experiments.
We characterized BBa_K2082117 on pSB1C3 in reversion assays, measuring the reversion frequency of our stop beta lactamase reporter.

Figure 2: Mutation rate of BBa K2082117 determined by stop beta lactamase reversion assays.

The mutation rate of BBa_K2082117 is (7.16±4.31)×10-6 bp-1×generation induced and (2.51±3.18)×10-8 bp-1×generation-1 repressed.
Furthermore, we analyzed BBa_K2082117 by high-throughput sequencing to determine mutation rate (Figure 3) as well as mutation spectrum (Figure 4).

Figure 3: Mutation rate of BBa K2082117 (induced genome wide mutator) as determined by high-throughput sequencing.

Figure 4: Mutation spectrum of BBa K2082117 in comparison to MP6, a plasmid with a very similiar assembly of mutation genes (Badran and Liu, 2015)

BBa_K2082117 is fully functional and creates diverse mutations with high frequency. Therefore, it's a useful tool for creating libraries of proteins of interest for several applications.
When using BBa_K2082117 two things have to be kept in mind
  • BBa_K2082117 has basal mutagenic activity, therefore supplementing the growth medium with glucose to further repress promoter activity.
  • Induced BBa_K2082117 greatly decreases cell viability


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
  • NGS
    • Experimental setup as for reversion assays
    • Instead of plating isolate plasmids and use for Illumina MiSeq with the Illumina Nextera DNA Library Preparation Kit
    • Mapping of obtained read onto reference sequence, count coverage on non reference bases as number of mutations
    • Mutation rate: number of mutations/ number of sequenced bases
    • Divide mutations into groups based on refernce base and mutated base to obtain mutation spectrum


  • Badran, Ahmed H.; Liu, David R. (2015): Development of potent in vivo mutagenesis plasmids with broad mutational spectra. In: Nature communications 6, S. 8425. DOI: 10.1038/ncomms9425.


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