Our characterization is based on <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Reversion">reversion assays</a>, showing the mutation rate of dnaQ926.
Our characterization is based on <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Reversion">reversion assays</a>, showing the mutation rate of dnaQ926.
<b>Figure 1: Mutagensis rate of <i>dnaQ926</i> (<a href="http://parts.igem.org/Part:BBa_K2082116">BBa_K2082116</a>) and M6 (<a href="http://parts.igem.org/Part:BBa_K2082117">BBa_K2082117</a>) determined by reversion assays.</b> A plasmid carrying a stop beta lactamase was used as a reporter for the mutators. Revertants were counted, normalized on viable cell count and used for calculation of the mutation rate using mutation accumulation (Pope et al., 2008).
<b>Figure 1: Mutagensis rate of <i>dnaQ926</i> (<a href="http://parts.igem.org/Part:BBa_K2082116">BBa_K2082116</a>) and M6 (<a href="http://parts.igem.org/Part:BBa_K2082117">BBa_K2082117</a>) determined by reversion assays.</b> A plasmid carrying a stop beta lactamase was used as a reporter for the mutators. Revertants were counted, normalized on viable cell count and used for calculation of the mutation rate using mutation accumulation (Pope et al., 2008).
DnaQ is part of the DNA polymerase III and responsible for the proofreading activity of this complex. The dnaQ926 variant
loses this activity through mutation of two function essential amino acids inside the active site. The complete loss of proofreading as well as the resulting saturation of mismatch-repair makes dnaQ926 the single strongest mutator gene known (Fijalkowska und Schaaper 1996) (see her for more informations)
dnaQ926 is part of our genome wide mutator BBa_K2082117, but we also used it as a standalone mutator in BBa_K2082116. After initially
designing our genome wide mutator we looked for mutagensis gene already in the iGEM partsreg and found several candidates.
The most prominent one is iGEM SYSU-China 2014's dnaQ926 BBa_K1333108.
Furthermore, we use iGEM Uchicago IGSB2014'semrR and dam in our large genome wide mutator.
Characterization
We characterized BBa_K1333108 as part of the genome wide mutators
BBa_K2082116 and BBa_K2082117.
Our characterization is based on reversion assays, showing the mutation rate of dnaQ926.
Figure 1: Mutagensis rate of dnaQ926 (BBa_K2082116) and M6 (BBa_K2082117) determined by reversion assays. A plasmid carrying a stop beta lactamase was used as a reporter for the mutators. Revertants were counted, normalized on viable cell count and used for calculation of the mutation rate using mutation accumulation (Pope et al., 2008).
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).
In combination we added substentially to BBa_K1333108 characterization thus enabling future iGEM teams to use this part as an in vivo mutator.
