Designed by: Zhang Fangyingnan Group: iGEM14_SYSU-China (2014-10-17)
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how you used this part and how it worked out.
dnaQ926 is part of our genome wide mutator BBa_K2082117, but we also used it as a standalone mutator in BBa_K2082116.
Characterization
We characterized BBa_K1333108 as part of the genome wide mutators
BBa_K2082116 and BBa_K2082117in 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. Figure 1: Mutagensis rate of dnaQ926 (BBa_K2082116) and M6 (BBa_K2082117). The mutation rate of standalone dnaQ926 is (2.3±1.12)×10-6 bp-1×generation-1 induced and (6.67±4.09)×10-8 bp-1×generation-1 repressed. The mutation rate of M6 (assembly of six mutator genes amongst other dnaQ926) is (7.16±4.31)×10-6 bp-1×generation-1 induced and (2.51±3.18)×10-8 bp-1×generation-1 when repressed.
Furthermore, we determined precise dnaQ926 mutational spectrum via high-throughput sequencing.
Figure 2: Mutagenic spectrum of the genome wide mutators dnaQ926 (BBa_K2082116) and M6 (BBa_K2082117). Teh mutagenic spectrum was determined by distribution all oberserved mutations to the different mutations (reference base &arr; mutated base). The tables show the mutation distribution in percent. n is the number of counted mutation events.
Other important information for everyone using dnaQ926 are that expression of dnaQ926 decreases cell viability. Furthermore, even very low amounts of dnaQ926 create a strong mutator phenotype thus use of a very tightly controlled promoter is recommended. For this application iGEM Bielefeld 2016 used a modified PBAD, further repressed by addition of 20 mM glucose.
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
