Team:Heidelberg/Pace

iGEM TEAM HEIDELBERG 2017

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PACE

Phage-assisted continous evolution

Lorem ipsum ...

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.

The Idea

To prove our hypothesis, we planned a circuit for the directed evolution of PAM specificity of Cas9 (Fig. 1). The main challenge was to link transcription activation to the binding of Cas9. We chose a system, which contains a dCas9 fused to a RNA polymerase Ω subunit (rpoZ) BIKARDETAL..2013. The rpoZ is able to recruit the transcription machinery to a poromoter and therefore activate gene expression. In our scenario, the nuclease targets a region upstream of a minimal promoter. In case the dCas9 is able to bind the DNA, the fused rpoZ activates gene expressio expreession. The J23117 promoter from the registry was chosen for activation BIKARDETAL..2013. It stands our through its low background activity and can be activated by the factor of 23 when rpoZ binds. Upstream of the promoter sequence, a spacer with a wildtype NGG PAM, that was previously used to target RFP was placed QIETAL..2013. If the Cas9 is able to bind to the spacer, transcription is activated. By changing the PAM sequence or generating PAM libraries, it is possible to induce a selection pressure on the randomly mutating protein. As a result, proteins with a weaker PAM specificity evolve, which can be used, no matter if the NGG motif is present exactly at the desired position. Of course, this circuit was designed according to our cloning standard by Gibson assembly.

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.

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,,,

References