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| − | Successful in vivo directed evolution by PREDCEL and PACE requires the thorough consideration of experimental parameters, e.g. phage propagation times, culture dilution rates and inducer/inhibitor concentrations. We employed extensive ODE-based and stochastic modeling to identify the most sensitive parameters and adapt our experiments accordingly. First, we calibrated our models using phage propagation experiments from our wet lab complemented with literature data. Simulations showed that the phage titer is highly sensitive to culture dilution rates. We simulated batch times and transfer volumes for PREDCEL and corresponding flow rates for PACE to determine optimized conditions for gene pool selection while avoiding phage washout. We also estimated phage titer monitoring intervals for cost/labor efficient QC as well as inducer/inhibitor concentrations required to express the required mutagenic polymerases. Finally, provide a web-based, fully interactive modeling platform, not only extensively employed by our wet lab, but highly informs future iGEM teams building on our work.
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| − | {{Heidelberg/templateus/Heading|Our Mutagenesis Plasmid Inter-Lab Study}}
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| − | In our directed evolution approaches PREDCEL and PACE, we select for beneficial mutations in a protein of interest encoded on a M13 phage genome. In order to generate a pool of protein mutants to select from in the first place, the efficient introduction of mutations during phage genome replication is essential. Mutagenesis-inducing plasmids (MPs) that enhance the mutation rate in <i>E. coli</i> by inhibiting DNA repair mechanisms have been described <x-ref>badran2015development</x-ref>. When transformed into <i>E. coli</i> hosts, these plasmids should cause mutation rates several orders of magnitude higher than usually expected under laboratory conditions.
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| − | The exact mutation rate induced by different mutagenesis plasmids could, however, vary between laboratories, as <i>E. coli</i> growth conditions and hence expression strength of the mutagenic proteins (e.g. error-prone polymerase subunits) could differ. However, robust induction of high mutation rates are critical for the success of PREDCEL and PACE and hence of major importance for other teams to reproduce our directed evolution methods. Therefore, we performed a small inter-lab study to evaluate the performance of different mutagenesis inducing plasmids and find the construct setup most robust and hence suitable for future iGEM teams to use for in vivo directed evolution. To enable an inter-lab comparison, we developed a standardized <a href="https://2017.igem.org/Team:Heidelberg/Collaborations/InterLab_HD"> mutagenesis plasmid test kit and assay
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| − | </a> and distributed it to iGEM team BOKU Vienna, ETH Zurich, Freiburg and Stuttgart. We are very grateful for their kind support and excited to share the <a href="https://2017.igem.org/Team:Heidelberg/Collaborations/InterLab_HD">results</a> from this small inter lab study with all iGEM teams
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| − | <b>Mutagenesis Assay Kit</b>
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| − | We shipped 3 mutagenesis plasmids (#1-3). All mutagenesis inducing plasmids contain an arabinose inducible promoter P<sub>BAD</sub>. Upstream of P<sub>BAD</sub> the araC protein is encoded in the opposite direction and regulates the activity of the P<sub>BAD</sub> promoter. The expression cassette downstream P<sub>BAD</sub> comprises multiple, different mutagenesis supporting elements, e.g. error-prone polymerase subunits. According to literature <x-ref>badran2015development</x-ref> the mutagenesis plasmids #1-3 were expected to cause increasingly high mutation rates.
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| − | <b>Mutagenesis Assay – Spontaneous Resistance Acquisition</b>
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| − | Due to exceptionally high mutagenesis levels of <i>E. coli</i> cells transformed with one of the mutagenesis plasmids, these cells can more quickly adopt to environmental changes. Hence, one way to measure the mutagenesis levels is simply to quantify the level of spontaneous antibiotic resistance acquisition. Therefore, <i>E. coli</i> were transformed with mutagenesis plasmids or remain untransformed (as control) followed by incubation in presence or absence of different antibiotics on agar plates. After 18-21 hours of incubation at 37 °C, colonies are counted. A higher number of colonies (i.e. clones with spontaneous resistance acquired) thereby indicates a higher mutation rate in the corresponding <i>E. coli</i> population due to the presence of the mutagenesis plasmid.
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| − | <b>Results from the Study</b>
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| − | To decide which mutagenesis plasmid to recommend to future iGEM teams for in vivo directed evolution approaches such as PREDCEL, it was essential to determine the mutagenesis plasmid setup most robustly functioning across different laboratories. The <a href="https://2017.igem.org/Team:Heidelberg/Collaborations/InterLab_HD">results</a> of iGEM team BOKU Vienna, ETH Zürich, Freiburg and Stuttgart indicate that MP #1 induces mutations most reliably .
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| − | Hence, we recommend to use mutagenesis plasmid #1 for PREDCEL experiments, which is also noted in our corresponding <a href="RFC https://2017.igem.org/Team:Heidelberg/RFC">RFC</a>. The results are shown and described on our <a href="https://2017.igem.org/Team:Heidelberg/Collaborations/InterLab_HD"> MP InterLab page</a>
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| − | As a first step to contribute to this year's iGEM competition we decided to participate in iGEM's fourth International Interlaboratory Study along with many other teams from around the world. This study is organized by iGEM's measurement committee in an effort to establish standardized, reliable and repeatable measurement tools for the iGEM community and the synthetic biology community as a whole. This year's iGEM InterLab study is about establishing a standardized protocol for the measurement of GFP using a plate reader. To start things off we needed a plate reader that is qualified to measure GFP fluorescence. Namely, Tecan Infinite M200 Pro plate reader. Additionally, we needed competent E.coli DH5. These were prepared from glycerol stocks. Together with the material iGEM had provided we were ready for work. Throughout our experiments, we tested 8 plasmids (2 controls and 6 test devices) by measuring the OD<sub>600</sub> and the fluorescence of the cells carrying the constructs.
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| − | The workflow can be separated into four segments. The first segment is the transformation of all plasmids into competent DH5 cells.
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| − | As a first step to contribute to this year's iGEM competition we decided to participate in iGEM's fourth International Interlaboratory Study along with many other teams from around the world. This study is organized by iGEM's measurement committee in an effort to establish standardized, reliable and repeatable measurement tools for the iGEM community and the synthetic biology community as a whole. This year's iGEM InterLab study is about establishing a standardized protocol for the measurement of GFP using a plate reader. To start things off we needed a plate reader that is qualified to measure GFP fluorescence. Namely, Tecan Infinite M200 Pro plate reader. Additionally, we needed competent E.coli DH5. These were prepared from glycerol stocks. Together with the material iGEM had provided we were ready for work. Throughout our experiments, we tested 8 plasmids (2 controls and 6 test devices) by measuring the OD<sub>600</sub> and the fluorescence of the cells carrying the constructs.
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| − | The workflow can be separated into four segments. The first segment is the transformation of all plasmids into competent DH5 cells.
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