Difference between revisions of "Team:Heidelberg/Pace"
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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 grey<x-ref>RN158</x-ref>. 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. | 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 grey<x-ref>RN158</x-ref>. 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. | ||
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<h1 id="id">Starting PACE</h1> | <h1 id="id">Starting PACE</h1> | ||
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All parts, which were necessary for the assembly of Accessory Plasmids were generated by PCR with the respective homology regions in the extensions. Subsequently, they were assembled by Gibson assembly (Fig.:2). All APs carry a bicistronic operon for the expression of geneIII and luxAB as luminescent reporter downstream of the promoter, described above. An expression cassette with the required gRNA under the control of a constitutive promoter is located on the same plasmid. APs varying in the copy number of their origins of replication and the strength of the RBS upstream of geneVI were cloned. To evolve the PAM specificity, we generated PAM libraries with four randomized nucleotides next to the spacer sequence. In order to do so, the whole plasmid was PCR amplified with the four PAM nucleotides as primer extensions. Subsequently, the plasmid was reassembled by Golden Gate assembly. To avoid that nucleotides, that pair with the original PAM are preferred and overrepresented in the library, a BbsI site was inserted next to the PAM. Prior to the PCR, the plasmid was digested with the enzyme, resulting in a linear fragment. The four nucleotides were loceated in overhangs, in the strand, to which the primer cannot bind. Plasmids that were cloned for the evolution of PAM specificity, the plasmid names, and the functional parts they consist of are shown in (Tab.: 1). | All parts, which were necessary for the assembly of Accessory Plasmids were generated by PCR with the respective homology regions in the extensions. Subsequently, they were assembled by Gibson assembly (Fig.:2). All APs carry a bicistronic operon for the expression of geneIII and luxAB as luminescent reporter downstream of the promoter, described above. An expression cassette with the required gRNA under the control of a constitutive promoter is located on the same plasmid. APs varying in the copy number of their origins of replication and the strength of the RBS upstream of geneVI were cloned. To evolve the PAM specificity, we generated PAM libraries with four randomized nucleotides next to the spacer sequence. In order to do so, the whole plasmid was PCR amplified with the four PAM nucleotides as primer extensions. Subsequently, the plasmid was reassembled by Golden Gate assembly. To avoid that nucleotides, that pair with the original PAM are preferred and overrepresented in the library, a BbsI site was inserted next to the PAM. Prior to the PCR, the plasmid was digested with the enzyme, resulting in a linear fragment. The four nucleotides were loceated in overhangs, in the strand, to which the primer cannot bind. Plasmids that were cloned for the evolution of PAM specificity, the plasmid names, and the functional parts they consist of are shown in (Tab.: 1). | ||
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Taking this highly challenging experiment, we wanted build our own PACE device and investigate the general handling of the apparatus. We were first started with building up our PACE apparatus using the instructions of Kevin Esvelt back in 2011 (<x-ref>RN44´</x-ref>). For an accurate description of our PACE device and our procedure before starting PACE, please have a look at the Materials&Methods section. | Taking this highly challenging experiment, we wanted build our own PACE device and investigate the general handling of the apparatus. We were first started with building up our PACE apparatus using the instructions of Kevin Esvelt back in 2011 (<x-ref>RN44´</x-ref>). For an accurate description of our PACE device and our procedure before starting PACE, please have a look at the Materials&Methods section. | ||
Thankfully, Bryan Dickinson´s lab was very helpful, so that they were sending us their constructs, strains and the phages they used for their evolution. Based on this, we could design our own constructs. Beyond that, we could quickly test our turbidostat, which we used instead of a chemostat, Dickinson and colleagues used for there PACE device. First, we aimed at cultivating our bacterial strains at an nearly constant optical density. This turbidostat-test was also used for evaluating the media consumption of the apparatus during an estimated PACE run suitable for iGEM conditions. Since the original PACE run has to include 29 days of continous evolution as well as ten different strains with different positive and negative selection stringencies, we were targeting at a shorter variant of this PACE experiment. By analyzing most of the data for the PACE run, we detected 3 of 7 essential mutations of the final variant of the N-terminal domain to be present in phages after only seven days of evolution. Outlining a more realistic example of a PACE run during a project implemented in only five wet lab month, we aimed for reproducing some of the mutations detected in the first seven days. In this context, we especially wanted to learn to handle the most difficult aspects of PACE: | Thankfully, Bryan Dickinson´s lab was very helpful, so that they were sending us their constructs, strains and the phages they used for their evolution. Based on this, we could design our own constructs. Beyond that, we could quickly test our turbidostat, which we used instead of a chemostat, Dickinson and colleagues used for there PACE device. First, we aimed at cultivating our bacterial strains at an nearly constant optical density. This turbidostat-test was also used for evaluating the media consumption of the apparatus during an estimated PACE run suitable for iGEM conditions. Since the original PACE run has to include 29 days of continous evolution as well as ten different strains with different positive and negative selection stringencies, we were targeting at a shorter variant of this PACE experiment. By analyzing most of the data for the PACE run, we detected 3 of 7 essential mutations of the final variant of the N-terminal domain to be present in phages after only seven days of evolution. Outlining a more realistic example of a PACE run during a project implemented in only five wet lab month, we aimed for reproducing some of the mutations detected in the first seven days. In this context, we especially wanted to learn to handle the most difficult aspects of PACE: | ||
| + | <ul> | ||
| + | <li>phage washout | ||
| + | <li>turbidostat contamination and working without contamination | ||
| + | <li>flow rates | ||
| + | <li>induction of mutagenesis | ||
| + | <li>evaluation of successfully performed PACE | ||
| + | <ul> | ||
| + | |||
| + | During several short PACE runs we already faced two of these challenging problems. First we had to adjust the flow rates in comparison to the flow rates in Dickinson-PACE based on the fact that we (i) used a turbidostat instead of a chemostat and (ii) investigated a low growth rate of the bacterial culture and thereby a doubling time of more than the expected 30 minutes. Second, we faced problems with a phage contamination in the turbidostat due to an extendable design of our PACE device and the waste tubings. Based on this observations, we slightly adjusted the tubings on our apparatus, yielding the actually used design. | ||
| + | In the next steps we further tried to continously cultivate the N-terminal T7 RNAP phages with the first strain, used for PACE in the original paper. Since you cannot detect the phages simultanously during running PACE, we wanted to establish a quick detection method, which reveals a result in a shorter time than the usually used detection and quantification method - plaque assays. Several methods like the dot blot with M13 antibodies or the qPCR did not showed reliable and reproducible results. In contrast to these methods, a simple phage detection PCR using three characteristic PCR products in lengths of 200, 400 and 750 bp. The results of this PCR can be quickly analyzed on a agarose gel and yield an final result for the presence of your phages in the lagoon in only two hours. This detection method enables a more efficient and direct way of controlling the turbidostat. | ||
{{Heidelberg/templateus/Imagesection| | {{Heidelberg/templateus/Imagesection| | ||
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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 }} | 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 }} | ||
}} | }} | ||
| − | }} | + | |
| + | Following the phage propagation experiment further, we not only wanted to propagate the phages but trying to start our directed evolution in analogy to the first seven days of the original publicated PACE run. During this seven days, three different strains with different negative APs were used to integrate various selection stringencies. To implement this, the required APs were transformed with MP1 in S1030 cells following the *transformation protocol*. By performing seven days of PACE, it was necessary to change strains for the first time. This presents another challenging step, since a contamination during the build-up procedure pose a risk for contamination again. On this account, it should be avoided if possible. On this basis, first seven days of PACE were implemented. For efficient organisation, the expiry was documented before the run (siehe figure PI-PACE). This enables several team members to carry the experiment forward, without decrease in quality. | ||
| + | The PACE run was successfully performed for 137 hours, displaying an acceptable phage titer in comparison to the literature (Esvelt et al., Nature 2011; Pu et al., Nature 2017). Sadly, we were running into another problem, which was highly reported by Kevin Esvelt - phage washout. The results of our plaque assays showed no infective phages in our lagoons after the 23. timepoint, corresponding to 137 hours of PACE. In addition, we could not reveal any mutations in our sequencings in a number of eight plaques which were picked for detection of potential mutations in the N-terminal domain of the RNAP. | ||
| + | |||
| + | {{Heidelberg/templateus/Imagesection| | ||
| + | https://static.igem.org/mediawiki/2017/b/b8/T--Heidelberg--Team_Heidelberg_2017_MP_Dickinson-PACE_Phagetiter.png| | ||
| + | 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.}} | ||
| + | }} | ||
| + | |||
| + | Facing these problems, the general experimental design of our PACE device, as well as the bacterial strains and the inducer concentration were investigated for potential mistakes. Based on the observation, that we can detect phages even after almost six days, we focused on the induction of mutagenesis during our PACE run. As the mutagenesis is induced by arabinose and inhibted by glucose, we primarily concentrated on the glucose and arabinose concentrations in our PACE apparatus. Therefore, our modeling hypthesized a too high glucose concentration in our medium, as well as a low arabinose concentration, which could be a reason for neither mutations in our picked plaques. Together with this theoretical assumption, we developed an alternative PACE setup for testing of the induction of mutagenesis during PACE. | ||
| + | |||
| + | <h2 id="id">Random-mutagenesis PACE</h2> | ||
| + | Following our first PACE tests based on the the split T7 RNAP paper, we designed a new PACE test, using our modeling to estimate the glucose concentration in the lagoons as well as in the turbidostat. First tested in a pre-test using PREDCEL, we tried to transfer equivalent conditions yielding mutations in PREDCEL on the PACE apparatus. In order to do that, an alternative bacterial strain which contains the pJC175e plasmid, a plasmid usually used for plaque assays, providing geneIII under a psp-promotor for all phages was used. In addition to this propagation plasmid, MP1 and MP4 were transformed into one strain respectively. These strains were used for propagation of phages, used for the Dickinson-PACE testing too. Since the Dickinson-phage propagates in previously performed pre-experiments very well, the effect of the concurrent performed propagation and mutagenesis should be at its maximum. This is further underlined by the fact that in this case, geneIII is provided for free. Thus, there should be no selection pressure except from pressure on the best propagation regarding codon optimisation. Beyond that, a lot of random mutations should be observed when the MPs work reliable. To reach this point, three days of mutations using the MP1 and the MP4 strain simultanously in two turbidostats with one lagoon for each turbidostat were implemented. Sequencings were performed by picking eight plaques of each lagoon from the last sample included into the plaque assay. In this case, phage washout was not observed, displaying a phage titer which were settled between 10^4 and 10^7 in both lagoons. | ||
| + | |||
| + | {{Heidelberg/templateus/Imagesection| | ||
| + | https://static.igem.org/mediawiki/2017/8/88/T--Heidelberg--Team_Heidelberg_2017_MP_RM-PACE_-_Phage_titer.png| | ||
| + | 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.}} | ||
| + | }} | ||
| + | Sanger sequencing results from these plaques showed a high number of different mutations in the 16 picked plaques. Thereby, the number of mutations resulting as a consequence of MP4 usage was slightly higher than the number of mutations in phages cultivated with the MP1 strain. In total, the mutations were widely spread over the N-terminal domain of the RNAP. Certainly, one mutation was recurrent, even the fact that there were no selection pressure. Overall, 15 of 22 mutations acquired during this three day PACE run entailed an amino acid exchange in the domain, while one mutation which occured in four clones resulted in no amino acid exchange. | ||
| + | |||
| + | {{Heidelberg/templateus/Imagesection| | ||
| + | https://static.igem.org/mediawiki/2017/d/d2/T--Heidelberg--Team_Heidelberg_2017_MP_RM-PACE_mutations.png| | ||
| + | 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 grey<x-ref>RN158</x-ref>. 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. | ||
| + | |||
| + | <h1>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. | ||
| + | |||
{{Heidelberg/templateus/Imagesection| | {{Heidelberg/templateus/Imagesection| | ||
https://static.igem.org/mediawiki/2017/c/c8/T--Heidelberg--Team_Heidelberg_2017_MP_PI-PACE_phagedectionPCR.png| | https://static.igem.org/mediawiki/2017/c/c8/T--Heidelberg--Team_Heidelberg_2017_MP_PI-PACE_phagedectionPCR.png| | ||
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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 }} | 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 }} | ||
}} | }} | ||
| − | |||
| + | {Heidelberg/templateus/Imagesection| | ||
| + | https://static.igem.org/mediawiki/2017/0/08/T--Heidelberg--Team_Heidelberg_2017_MP_PI_PACE.png| | ||
| + | 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.}} | ||
| + | }} | ||
}} | }} | ||
}} | }} | ||
Revision as of 17:21, 1 November 2017
PACE
Phage-assisted continous evolution
{{{5}}}
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
}}
{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.}}
}}
}}
}}
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
