Team:Heidelberg/Pace


PACE
Phage-assisted continous evolution
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Introduction

Ave Caesar
Figue 1: PACE apparatus - Part 1
This image shows the construction of our PACE device, with two turbidostats and two lagoons. All tubings are color coded and equivalent to the tubings shown in the scheme of Fig. xy.
Figue 2: 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.
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.

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.
  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 steril 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 1.5l 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 10 10>sup> 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. 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

      Phage Based in vivo Eolution with GeneVI

      One of the major challenges in the context of transcription activation with help of the rpoZ is leaky expression. This is a setious problem for PACE, because if geneIII is expressed prior to phage infection leads to infection the bactirial cell turns resistant RN44. Fortunately, it was shown that geneVI can be used for phage propagation in the context of directed evolution as well. In contrast to the commonly used geneIII, it has the advantage that leaky geneVI expression has no effect on the infectivity of E. coli by phages BRODELETAL..2016. As a consequence, we decided to adapt this approach and towork with geneVI instead of geneIII. 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).
      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.
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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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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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(RN158). We choose this example of PACE as our role model for testing of this highly complex method, needing the set-up of a continous culture, as well as a flow setup, demanding costly and highly sensitive hardware for its application. In the paper, Pu et al. grasp at evolving a small N-terminal domain of the T7RNAP for efficiently binding its N-terminal domain by help of two leucine zipper, strongly binding each other. Depending on their proximity, the two domains of the RNAP interact and can activate geneIII transcription.
    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 (RN44´). 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:




  

    
    

      
                  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

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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References