Team:Heidelberg/Pace


PACE
Phage-assisted continous evolution

Introduction

Phage-assisted continuous evolution (PACE) is a powerful in vivo directed evolution method invented by Kevin Esvelt (now at the MIT media lab) and David Liu (Harvard University) (Esvelt et al, Nature, 2011RN44'). It can in principle be applied for directed evolution of any gene or protein of interest (GOI and POI, respectively), provided that the function to be evolved can be coupled to gene expression in E. coli (Esvelt et al, Nature, 2011RN44'). Conceptually, PACE mimics a closed evolution circle composed of (i) variant replication, (ii) mutation and (iii) selection in a bioreactor. The basic principle of PACE is shown in Figure 1. In brief, PACE employs M13 phages that encode protein of interest (POI) to be evolved. These phages have a knockout for gene III, an essential M13 gene essential for phage replication.
Figure 1: Basic principle of phage-assisted, continuous evolution. (see main text for details).
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During PACE, the POI-encoding phages are propagated of E. coli host cells carrying two plasmids. One it the mutagenesis plasmid (MP), which encodes highly mutagenic genes strongly reducing phage replication fidelity and error repair. As these genes are toxic to the E. coli host, they are set under control of an arabinose-inducible pBAD promoter. Different MP variants have been reported, which cause mutation rates up to ~2.3 substitutions per kb during phage replication when fully induced (Badran et al, Nature Communications, 2015RN159), thereby highly accelerating the evolution.

The second plasmid is called accessory plasmid (AP), and is the PACE component inducing the selection pressure directing the evolution towards the desired goal. The AP thereby encodes gene III, the aforementioned essential M13 gene required for phage replication. Importantly, gene III expression from the AP is made dependent on the function of the phage-encoded POI, e.g. via a synthetic circuit. In other words: the AP induces a selection pressure towards optimizing POI for the function required to activate gene III expression. In the simple case of evolving a transcription factor as depicted in Figure 1, gene III would simply be expressed from the promoter (“pTarget”) the transcription factor should be optimized for.

PACE employs a custom bioreactor setup and complex flow controls to achieve continuous in vivo directed evolution of the phage gene pool (Figure 2). The PACE setup comprises four major compartments: medium tank, turbidostat, lagoon and waste tank. The turbidostat thereby which contain “fresh” E. coli host cells (transformed with AP and MP) cultivated at constant cell density and in the absence of arabinose, so that the toxic MP-encoded genes are not expressed. The “lagoon” is the compartment in which the actual evolution takes place, i.e. where the phages infect the host cells and propagate, provided the encoded POI activates gene III on the AP. In addition, the medium in the lagoon is supplemented by arabinose via a corresponding inducer supply line. The lagoon is thereby kept under constant flux (typically ~1 lagoon volume per hour), i.e. fresh host cells are introduced from the turbidostat the identical volume is simultaneously going to the waste line. Thereby, phages which are inefficiently propagating (i.e. due to low fitness of their encoded POI) are washed out, while highly propagating (i.e. fit) variants take over the gene pool and further evolve.

Figure 2: Simplified schematics of in vivo evolution with PACE.
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PACE Methods

Figure 3 shows a detailed, interactive PACE scheme. You can klick on the different components to receive information about the individual components of our PACE setup. Figure 4 and 5 show the different components of our custom-made PACE setup as it is standing in our lab right now. The accompanying video introduced you to our PACE apparatus in further detail.
Figure 3: Interactive schematics of the PACE setup.
Video
Figure 4: PACE apparatus - Part 1.
This image shows our PACE device, with two turbidostats and two lagoons. All tubings are color coded and equivalent to the tubings shown in the scheme in Fig. 3.
Figure 5: PACE apparatus - Part 2
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.

Our PACE protocol

Before starting a PACE run, several prerequisites have to be fulfilled. 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.
  1. 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).
  2. 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.
  3. 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.
  4. 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.
  5. 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 sterile filtrated.
  6. 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.
  7. After connecting the media line of the turbidostat to the medium container, the turbidostat should be filled with medium until a volume of 1.5 l is reached, by starting the media pump.
  8. 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.
  9. 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.
  10. 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
  11. 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 1010 PFU/ml to the lagoon and start the existing PACE run
  12. 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. Also, 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.

Results – evolving split T7 polymerase toward improved auto-reassembly

We set out to employ PACE for evolving a split T7 polymerase towards increased auto-reassembly. Details on the how we obtained the particular split variant used in this PACE experiment can be found on our Protein Interaction subpage. In brief, based on the paper by Tiun Han et al. (ACS Synthetic Biology, 2017Han.2017), we tested M13 phages encoding 4 different split T7 variants for their ability to propagate on an E. coli selection strain transformed with a pT7-geneIII accessory plasmid. The variant encoding the split site at T7 residues 564/565 showed highest propagation and was therefore chosen for further optimization via PACE. We generated a PACE strain, by co-transforming our T7-gene III AP and MP4 (Badran et al, Nature Communications, 2015RN159). Then, by using our custom-build setup, we performed 30 hours of in vivo directed evolution via PACE (Figure 6).
Figure 6: Concept for evolution of split T7 towards improved auto-reassembly by PACE
Phages are continuously propagated on a mutagenic E. coli selection strain, carrying an AP comprising of a T7 promoter driving gene III. Thereby, variants should evolve, which show improved split T7 function.
During the PACE run continuously monitored the phage titer as well as optical density of our E. coli hosts (Figure 7). The used flow rate was approximately 1 lagoon volume per hour and adapted according to the optical density measurements.
Figure 7: Monitoring of Phage Titer and Optical Density during the Split T7 PACE run.
(A) Phage titer was estimated by manual sampling every 5 hours followed by plague essay. (B) Samples were taken every two hours and OD600 was determined using a nanophotometer. During the whole PACE run, the optical density is located in the range of 0.5 to 1, which is equivalent to the reaction conditions reported in the literature (Esvelt et al, Nature, 2011RN44').
After having finished our PACE run, we performed plague assays and PCR amplified the Split T7 genes from 5 different plagues followed by sanger-sequenced. As hoped, we observed a recurrent, coding mutation (T877P) present in three out of the five sequenced split T7 variants (Figure 8).
Figure 8: Mutational pattern of the evolved split T7 variant.
Following three days of in vivo evolution with PACE, a plague assay was performed and the split T7 insert of five individual phage clones was analyzed by sanger-sequencing. We observed a recurrent mutation (T877P) in three out of the five clones, suggesting an evolutionary advantage (i.e. increased fitness) of the corresponding split T7 mutant as compared its non-mutated counterpart.
Interestingly this residue is located very close to the interaction surface of the two split T7 domains, suggesting a possible role in T7 auto-reassembly (Figure 9).

References