Moritz P95 (Talk | contribs) |
Moritz P95 (Talk | contribs) |
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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.}} | 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.}} | ||
− | {{Heidelberg/templateus/Tablebox| | + | {{Heidelberg/templateus/Tablebox||{{#tag:html|<table class="table table-bordered mdl-shadow--4dp" XSSCleaned="overflow-x: scroll !important"> |
− | + | <thead style="background-color: #005493 !important;"> <tr> | |
− | + | <th>TIme,Date,Operator,Sample,Action,DONE ToDo:,OD Turbidostat 2,Stock ID,TO DOs:</th> </tr> | |
− | + | </thead><tbody> <tr> | |
− | <thead> | + | <td>12:00,24/10/2017,MP,-,Started filling the turbidostat (T2) with medium ,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <tr> | + | <tr> |
− | <th> | + | <td>13:30,24/10/2017,MP,-,Inoculation of turbidostat 2 with 50 ml Stock culture ,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | < | + | <tr> |
− | < | + | <td>14:00,24/10/2017,MP,-,Measured OD,,"0,18",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | < | + | <tr> |
− | < | + | <td>15:00,24/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | < | + | <tr> |
− | < | + | <td>16:00,24/10/2017,MP,-,Measured OD,,"0,511",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | </tr> | + | <tr> |
− | </ | + | <td>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),</td> </tr> |
− | < | + | <tr> |
− | <tr> | + | <td>18:00,24/10/2017,MP,1,Inoculation with 1 ml phage supernatant,,"0,514",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td | + | <td>19:00,24/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td | + | <tr> |
− | <td> | + | <td>20:00,24/10/2017,MS,1,"Measured OD, Took samples L1, T1 - 1",,"0,637",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td> | + | <td>21:00,24/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | </tr> | + | <td>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),</td> </tr> |
− | <tr> | + | <tr> |
− | <td> | + | <td>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),</td> </tr> |
− | <td | + | <tr> |
− | <td | + | <td>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</td> </tr> |
− | <td> | + | <tr> |
− | <td> | + | <td>01:00,25/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td> | + | <td>02:00,25/10/2017,MS,,Measured OD,,"0,789",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | </tr> | + | <tr> |
− | <tr> | + | <td>03:00,25/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td | + | <td>04:00,25/10/2017,MS,3,"Measured OD, Took samples L1, T1 - 3",,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td | + | <tr> |
− | <td> | + | <td>05:00,25/10/2017,,,,,"0,792",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td> | + | <td>06:00,25/10/2017,MS,,Measured OD,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | </tr> | + | <td>07:00,25/10/2017,,,,,"0,817",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <tr> | + | <tr> |
− | <td> | + | <td>08:00,25/10/2017,TB,4,"Measured OD, Took samples L1, T1 - 4",Took samples,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td | + | <tr> |
− | <td | + | <td>09:00,25/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td> | + | <td>10:00,25/10/2017,TB,,Measured OD,,"0,816",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td> | + | <td>11:00,25/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | </tr> | + | <tr> |
− | <tr> | + | <td>12:00,25/10/2017,TB,,Measured OD,Done PCR,"0,81",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),TO DO: Phage-PCR</td> </tr> |
− | <td> | + | <tr> |
− | <td | + | <td>13:00,25/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td | + | <tr> |
− | <td> | + | <td>14:00,25/10/2017,PP,,Measured OD,,"0,843",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td> | + | <td>15:00,25/10/2017,,,,inoculated growth for plaque assay,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | </tr> | + | <td>16:00,25/10/2017,PP,5,"Measured OD, Took samples L1, T1 - 5",,"0,833",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <tr> | + | <tr> |
− | <td> | + | <td>17:00,25/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td | + | <tr> |
− | <td | + | <td>18:00,25/10/2017,PP,,Measured OD,,"0,836",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td> | + | <td>19:00,25/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td> | + | <td>20:00,25/10/2017,MP,,Measured OD,,"0,862",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),TO DO: Plaque Assay</td> </tr> |
− | </tr> | + | <tr> |
− | <tr> | + | <td>21:00,25/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td | + | <td>22:00,25/10/2017,MP,,Measured OD,,"0,81",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td | + | <tr> |
− | <td> | + | <td>23:00,25/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td> | + | <td>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</td> </tr> |
− | <td> | + | <tr> |
− | </tr> | + | <td>01:00,26/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <tr> | + | <tr> |
− | <td> | + | <td>02:00,26/10/2017,CG,,Measured OD,,"0,845",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td | + | <tr> |
− | <td | + | <td>03:00,26/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td> | + | <td>04:00,26/10/2017,CG,,Measured OD,,"0,864",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | <td> | + | <tr> |
− | <td> | + | <td>05:00,26/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | </tr> | + | <tr> |
− | </tbody> | + | <td>06:00,26/10/2017,CG,,Measured OD,,"0,836",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> |
− | </table> | + | <tr> |
− | + | <td>07:00,26/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | |
− | + | <tr> | |
− | + | <td>08:00,26/10/2017,TB,7,"Measured OD, Took samples L1, T1 - 7",,"0,831",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | |
− | + | <tr> | |
− | + | <td>09:00,26/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | |
− | + | <tr> | |
− | + | <td>10:00,26/10/2017,TB,,Measured OD, ,"0,827",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | |
− | + | <tr> | |
+ | <td>11:00,26/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>12:00,26/10/2017,TB,,Measured OD,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),TO DO: Phage-PCR</td> </tr> | ||
+ | <tr> | ||
+ | <td>13:00,26/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>14:00,26/10/2017,TB,,Measured OD,,"0,738",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>15:00,26/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>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),</td> </tr> | ||
+ | <tr> | ||
+ | <td>17:00,26/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>18:00,26/10/2017,,,Measured OD,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>19:00,26/10/2017,MS,,,,"0,582",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>20:00,26/10/2017,,,Measured OD,Plaque assay was performed successfully ,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),TO DO: Plaque Assay</td> </tr> | ||
+ | <tr> | ||
+ | <td>21:00,26/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>22:00,26/10/2017,MS,,Measured OD,,"0,633",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>23:00,26/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>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</td> </tr> | ||
+ | <tr> | ||
+ | <td>01:00,27/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>02:00,27/10/2017,MS,,Measured OD,,"0,692",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>03:00,27/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>04:00,27/10/2017,MS,,Measured OD,,"0,937",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>05:00,27/10/2017,,,,,"0,945",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>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),</td> </tr> | ||
+ | <tr> | ||
+ | <td>07:00,27/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>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),</td> </tr> | ||
+ | <tr> | ||
+ | <td>09:00,27/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>10:00,27/10/2017,PP,,Measured OD,,"0,903",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>11:00,27/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>12:00,27/10/2017,LP,,Measured OD,see above,"0,924",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),TO DO: Phage-PCR</td> </tr> | ||
+ | <tr> | ||
+ | <td>13:00,27/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>14:00,27/10/2017,MP,,Measured OD,,"0,942",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>15:00,27/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>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),</td> </tr> | ||
+ | <tr> | ||
+ | <td>17:00,27/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>18:00,27/10/2017,MP,,Measured OD,,"0,822",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>19:00,27/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>20:00,27/10/2017,MK,,Measured OD,,"0,59",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),TO DO: Plaque Assay</td> </tr> | ||
+ | <tr> | ||
+ | <td>21:00,27/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>22:00,27/10/2017,CG,,Measured OD,,"0,61",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>23:00,27/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>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</td> </tr> | ||
+ | <tr> | ||
+ | <td>01:00,28/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>02:00,28/10/2017,CG,,Measured OD,,"0,809",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>03:00,28/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>04:00,28/10/2017,MP,,Measured OD,PCR: negative - phage washout,"0,820",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>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),</td> </tr> | ||
+ | <tr> | ||
+ | <td>06:00,28/10/2017,MP,,Measured OD,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>07:00,28/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>08:00,28/10/2017,MP,13,"Measured OD, Took samples L1, T1 - 13",,"0,667",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>09:00,28/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>10:00,28/10/2017,PP,,Measured OD,,"0,62",199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>11:00,28/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>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</td> </tr> | ||
+ | <tr> | ||
+ | <td>13:00,28/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>14:00,28/10/2017,PP,,Measured OD,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>15:00,28/10/2017,,,,,,199 (pMax69 aka AP-SplitT7 + GenIII+YFP + MP4),</td> </tr> | ||
+ | <tr> | ||
+ | <td>16:00,28/10/2017,PP,14,"Measured OD, Took samples L1, T1 - 14",FINAL PROBES,,,</td> </tr> | ||
+ | </tbody></table> | ||
+ | }}|}} | ||
}} | }} | ||
}} | }} |
Revision as of 15:13, 1 November 2017
PACE
Phage-assisted continous evolution
![](
https://static.igem.org/mediawiki/2017/3/32/T--Heidelberg--Team_Heidelberg_2017_MP_PACEprinicipalfigure.png)
Introduction
Ave Caesar![](
https://static.igem.org/mediawiki/2017/8/82/T--Heidelberg--Team_Heidelberg_2017_MP_pictureTurbidostat.png)
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.
![](
https://static.igem.org/mediawiki/2017/9/97/T--Heidelberg--Team_Heidelberg_2017_MP_pictureTurbidostat2.png)
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)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![](
https://static.igem.org/mediawiki/2017/6/6b/T--Heidelberg--Team_Heidelberg_2017_graphical_abstract_JMu.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.
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,,, |