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This picture is showing the second part of the construction of our PACE device. The syringe pumps (green) as well as the valve control and the oxygen supply are shown. All necessary tubings and cables are inserted in the heating cabinet. | This picture is showing the second part of the construction of our PACE device. The syringe pumps (green) as well as the valve control and the oxygen supply are shown. All necessary tubings and cables are inserted in the heating cabinet. | ||
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Figure 2: Design of the Accessory Plasmids for the Evolution of Cas9| | Figure 2: Design of the Accessory Plasmids for the Evolution of Cas9| | ||
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Revision as of 20:59, 1 November 2017
PACE
Phage-assisted continous evolution
Introduction
Ave CaesarMaterials and Methods
Starting PACE
Before starting a PACE run, several prerequisites have to be fulfiled. Most of the preparations for the different PACE runs are the same in terms of tests for MP activity, F-Pilus plasmids and contamination. Additionally, several pre-tests are recommended to test the APs activity and the general functionality of the implemented genetic circuit.- Every part of the PACE device including all tubings and connectors have to be autoclaved. All open ends should be wrapped in aluminium foil. It is important to check all ends and tubings to be closed before start the dry autoclavation (Be aware of autoclaving only autoclavable parts of the PACE device).
- After autoclavation, the PACE device should be treated with highest carefulness to prevent phage contamination in the turbidostat. To make this possible, the use of 10% H2O2 or incidin as well as the usage of gloves is advised.
- Rebuild the PACE apparatus carefully using incidin to desinfect all for the autoclavation wrapped and thereby closed ends. Connect all necessary parts of the tubings.
- The medium should be prepared slightly different to the medium used in literature [Esvelt et al., 2011] by mixing 140g dikaliumhydrogenphosphate with 40g kaliumdihydrogenphosphate, 20g ammoniumsulfate and 20 ml tween-80 in 20l dH20. The medium should be autoclaved as well before using it.
- the autoclaved medium should be mixed with medium supplements, which should be prepared during autoclavation. 20g glucose, as well as 10g sodium citrate, 0.5g L-leucin, 0.5g and 100g casamino acids or trypton from casein have to be solved in at last 500 ml dH20. If the chemicals cannot be dissolved in this volume, water can be added until it can be solved. The resulting solution have to be steril filtrated.
- the appropiate volume of the prepared supplements can now be added to the autoclaved medium. This should be implemented in as steril conditions as possible, using incidin to sterilize the used pipette. In addition to the supplements, the appropiate antibiotics have to be added into the medium. Final concentrations should be choosen according the stock concentrations proposed by addgene. A blank for the OD600 measurements should be taken before connecting the medium to the tubings.
- After connecting the media line of the turbidostat to the medium container, the turbidostat should be filled with medium until a volume of 1.5l is reached, by starting the media pump.
- 50 ml bacterial culture resulting from the *MP testing* should be used for inoculation. Therefore draw up the culture into a syringe and inoculate the turbidostat using a cannula through the septa in the turbidostat. Reduce the flow rate to a minimum to ensure an efficient growth of the culture in the turbidostat.
- lagoon pump can be started when the turbidostat reaches an OD600 = 0.6 - 1.0. The lagoon volume can be adjusted at a range of 100 - 150 ml lagoon volume.
- induce mutagenesis by start adding 10% w/v arabinose to the lagoon. Arabinose should be added at last one hour before infection with bacteriophages to secure the induction of the MPs
- When the lagoon is ready, arabinose is added and the cells are on a constant optical density, the lagoon can be infected with bacteriophages. Add 1 ml of 10 10>sup> PFU/ml to the lagoon and start the existing PACE run
- during the PACE run, samples should be taken every four hours for the first 24 hours and every eight hours from the second day on until the run is finished. During a PACE run, phage detection PCRs and plaque assays should be implemented, proving the presence of the phage of interest and a contamination free turbidostat. Positive and negative control always have to be included into the detection PCR as well as the plaque assays. We recommend
- during the PACE run, samples should be taken every four hours for the first 24 hours and every eight hours from the second day on until the run is finished. During a PACE run, phage detection PCRs and plaque assays should be implemented, proving the presence of the phage of interest and a contamination free turbidostat. Positive and negative control always have to be included into the detection PCR as well as the plaque assays. We recommend
Results
Dicksinson-PACE
Based on the high-potential concept of Phage-assisted continous evolution the group of Bryan Dickinson evolved a T7 RNA polymerase in the "Evolution of a split RNA polymerase as a versatile biosensor platform"-paper, publicated in early 2017(- phage washout
- turbidostat contamination and working without contamination
- flow rates
- induction of mutagenesis
- evaluation of successfully performed PACE
-
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.
Random-mutagenesis PACE
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.PI-PACE
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.Table 1: Documentation of performed tasks with comments
TIme Date Operator Sample Action DONE ToDo: OD Turbidostat 2 Stock ID TO DOs: 12:00 24/10/2017 MP - Started filling the turbidostat (T2) with medium 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 13:30 24/10/2017 MP - Inoculation of turbidostat 2 with 50 ml Stock culture 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 14:00 24/10/2017 MP - Measured OD "0 18" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 15:00 24/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 16:00 24/10/2017 MP - Measured OD "0 511" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 17:00 24/10/2017 MP "Measured OD started filling the lagoon and the arabinose pump" "0 548" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 18:00 24/10/2017 MP 1 Inoculation with 1 ml phage supernatant "0 514" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 19:00 24/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 20:00 24/10/2017 MS 1 "Measured OD Took samples L1 T1 - 1" "0 637" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 21:00 24/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 22:00 24/10/2017 MP "Measured OD Flow rate for lagoon was to high from previous filling ( 7 ml/min in contrast to 1 666 ml/min) - potential washout " "0 791" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 23:00 24/10/2017 MP "Lagoon was infected with 0 8 ml 10^8 PFU/ml phages to counteract the potential washout " "0 818" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 00:00 25/10/2017 MP 2 "Measured OD Took samples L1 T1 - 2" "0 833" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) TO DO: Phage-PCR 01:00 25/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 02:00 25/10/2017 MS Measured OD "0 789" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 03:00 25/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 04:00 25/10/2017 MS 3 "Measured OD Took samples L1 T1 - 3" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 05:00 25/10/2017 "0 792" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 06:00 25/10/2017 MS Measured OD 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 07:00 25/10/2017 "0 817" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 08:00 25/10/2017 TB 4 "Measured OD Took samples L1 T1 - 4" Took samples 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 09:00 25/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 10:00 25/10/2017 TB Measured OD "0 816" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 11:00 25/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 12:00 25/10/2017 TB Measured OD Done PCR "0 81" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) TO DO: Phage-PCR 13:00 25/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 14:00 25/10/2017 PP Measured OD "0 843" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 15:00 25/10/2017 inoculated growth for plaque assay 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 16:00 25/10/2017 PP 5 "Measured OD Took samples L1 T1 - 5" "0 833" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 17:00 25/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 18:00 25/10/2017 PP Measured OD "0 836" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 19:00 25/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 20:00 25/10/2017 MP Measured OD "0 862" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) TO DO: Plaque Assay 21:00 25/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 22:00 25/10/2017 MP Measured OD "0 81" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 23:00 25/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 00:00 26/10/2017 LP 6 "Measured OD Took samples L1 T1 - 6" Done PCR "0 809" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) TO DO: Phage-PCR 01:00 26/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 02:00 26/10/2017 CG Measured OD "0 845" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 03:00 26/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 04:00 26/10/2017 CG Measured OD "0 864" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 05:00 26/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 06:00 26/10/2017 CG Measured OD "0 836" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 07:00 26/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 08:00 26/10/2017 TB 7 "Measured OD Took samples L1 T1 - 7" "0 831" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 09:00 26/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 10:00 26/10/2017 TB Measured OD "0 827" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 11:00 26/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 12:00 26/10/2017 TB Measured OD 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) TO DO: Phage-PCR 13:00 26/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 14:00 26/10/2017 TB Measured OD "0 738" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 15:00 26/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 16:00 26/10/2017 TB 8 "Measured OD Took samples L1 T1 - 8" "Took samples L1 T1" "0 652" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 17:00 26/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 18:00 26/10/2017 Measured OD 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 19:00 26/10/2017 MS "0 582" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 20:00 26/10/2017 Measured OD Plaque assay was performed successfully 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) TO DO: Plaque Assay 21:00 26/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 22:00 26/10/2017 MS Measured OD "0 633" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 23:00 26/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 00:00 27/10/2017 PP 9 "Measured OD Took samples L1 T1 - 9" "0 511" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) TO DO: Phage-PCR 01:00 27/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 02:00 27/10/2017 MS Measured OD "0 692" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 03:00 27/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 04:00 27/10/2017 MS Measured OD "0 937" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 05:00 27/10/2017 "0 945" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 06:00 27/10/2017 MP Measured OD "could not be measured as lagoon medium level was too high and therefore pump where set off for a short time " 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 07:00 27/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 08:00 27/10/2017 MS 10 "Measured OD Took samples L1 T1 - 10" "Phage detection PCR with samples 8 9 and 10 for Dickinson-Phage and PI-Phage" "0 912" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 09:00 27/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 10:00 27/10/2017 PP Measured OD "0 903" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 11:00 27/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 12:00 27/10/2017 LP Measured OD see above "0 924" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) TO DO: Phage-PCR 13:00 27/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 14:00 27/10/2017 MP Measured OD "0 942" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 15:00 27/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 16:00 27/10/2017 PP 11 "Measured OD Took samples L1 T1 - 11" took samples "0 942" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 17:00 27/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 18:00 27/10/2017 MP Measured OD "0 822" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 19:00 27/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 20:00 27/10/2017 MK Measured OD "0 59" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) TO DO: Plaque Assay 21:00 27/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 22:00 27/10/2017 CG Measured OD "0 61" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 23:00 27/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 00:00 28/10/2017 MP 12 "Measured OD Took samples L1 T1 - 12" Phage detection PCR with sample 11 "0 664" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) TO DO: Phage-PCR 01:00 28/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 02:00 28/10/2017 CG Measured OD "0 809" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 03:00 28/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 04:00 28/10/2017 MP Measured OD PCR: negative - phage washout "0 820" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 05:00 28/10/2017 "Flow rate was adjusted: 1 ml/min lagoon was infected by pooled samples 6 7 and 8 " 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 06:00 28/10/2017 MP Measured OD 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 07:00 28/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 08:00 28/10/2017 MP 13 "Measured OD Took samples L1 T1 - 13" "0 667" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 09:00 28/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 10:00 28/10/2017 PP Measured OD "0 62" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 11:00 28/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 12:00 28/10/2017 PP Measured OD Phage PCR was performed "0 768" 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) TO DO: Phage-PCR 13:00 28/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 14:00 28/10/2017 PP Measured OD 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 15:00 28/10/2017 199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4) 16:00 28/10/2017 PP 14 "Measured OD Took samples L1 T1 - 14" FINAL PROBES