Team:Heidelberg/Pace
PACE
Phage-assisted continous evolution
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Figue 2: Random mutations in N-terminal domain of T7 RNAP
The figure is presenting the nucleotide sequence of the N-terminal domain of the T7 RNAP in light grey, a linker sequence in black and the leucine zipper in dark greyRN158 . At the top of the figure a consensus like sequence is shown, summarizing all mutations of the sequenced plaques, which are displayed as single bars under the summary. All mutations annotated are color coded. Recurrent mutations are divided into mutations leading to an amino acid exchange (red) and the mutations which do not have an amino acid exchange as a consequence (orange). In addition, mutations with amino acid exchanges, which are not recurrent are displayed in yellow, while the mutations without amino acid exchange are shown in blue. In total, only plaques showing mutations are shown. Sequences which did not contain mutations were omitted due to clarity reasons. Overall, sequencing was performed on 24 plaques.
By this, we demonstrated that the we can induce the mutagenesis plasmids successfully and sufficient manner. The evaluated conditions, hypothesized by our modeling, confirmed by our sequencing results from the real experiment should finally describe conditions in which a good induction of the MPs should be ensured.
PI-PACE<h1>
Coming to our final experiment we finally had the knowledge which is required to perform our own PACE run. This PACE run is just like Dickinson´s PACE approach based on protein-interaction of a split T7 RNAP. In contrast to the run before, both split sites are located on the selection phage. In principle, the split T7 RNAP is evolved on a better and faster reassembly of both fragments, yielding in a higher transcription of geneIII which is encoded under control of a T7 promotor. For further information on the principle of protein-interaction PACE, please visit our special site (hier Link einfügen).
Building up on the equivalent conditions to the random-mutagenesis PACE run, we used the same amount of glucose and arabinose for induction of the mutagenesis plasmids. In this case, we only used a strain with MP4 due to the observation of slightly more mutations in the random mutagenesis experiment. Since this PACE experiment was performed with selection pressure, we estimated for difficulties in the phage propagation during PACE, which is why the flow rate was decreased for enabling better phage propagation. Nevertheless, our plaque assays showed phage washout after only 38 hours. Regardless of these findings, plaque PCRs and sequencings were performed, using plaques from the last available time point. The sequencing results showed one mutation in each of the split sites, of which one mutation could have a functional input on the reassembly of both sites.
Figure 2: Agarose gel of phage detection PCR
This image shows the results of an phage detection PCR of samples from the final Dickinson-PACE run. NEB 2-log ladder was loaded on the first lane. All lanes are
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{Heidelberg/templateus/Imagesection|
https://static.igem.org/mediawiki/2017/0/08/T--Heidelberg--Team_Heidelberg_2017_MP_PI_PACE.png%7C
Figure 2: Design of the Accessory Plasmids for the Evolution of Cas9|
The AP consists of five subparts that are devided by homology regions for Gibson assembly (numbers). It carries an expression cassette for the transcription of a gRNA (between 1 and 5). GeneVI (2-3) is under control of a that can be activated by the Cas9-rpoZ in context with the respective gRNA. luxAB accounts as a reporter for fluorescent readout of geneIII activation (3-4). The whole plasmid can be produced with different origins of replication (4-5) to modulate the copy number and by exchanging the geneVI part with the RBS.}}
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References
Figure 2: Agarose gel of phage detection PCR
This image shows the results of an phage detection PCR of samples from the final Dickinson-PACE run. NEB 2-log ladder was loaded on the first lane. All lanes are
