Difference between revisions of "Team:Bielefeld-CeBiTec/Notebook/Mutation"

Line 46: Line 46:
 
<div id="6" class="collapse">  
 
<div id="6" class="collapse">  
 
<br>
 
<br>
−
Transformation in DH5&alpha;:
+
<ul>
 +
<li>Transformation in DH5&alpha;:</li>
 
<ul>
 
<ul>
 
<li>pHSG-WTPolI</li>
 
<li>pHSG-WTPolI</li>
Line 54: Line 55:
 
<li>psBIA3:E0044</li>
 
<li>psBIA3:E0044</li>
 
</ul>
 
</ul>
−
Everything besides pSBIA3:E0044 grew. Taking overnight cultures at 37&deg;C.
+
</ul>
−
Plating provided JS200 strain out.
+
<li>Everything besides pSBIA3:E0044 grew. Taking overnight cultures at 37&deg;C.</li>
 +
<li>Plating provided JS200 strain out.</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
Line 90: Line 92:
 
<li>Transformation in DH5&alpha;:</li>
 
<li>Transformation in DH5&alpha;:</li>
 
<ul>
 
<ul>
−
<li>psBIC3:E1010</li>
+
<li>pSBIC3:E1010</li>
−
<li>psBIC3:K731722</li>
+
<li>pSBIC3:K731722</li>
 
</ul>
 
</ul>
 
<li>Transformation in JS200;:</li>
 
<li>Transformation in JS200;:</li>
Line 109: Line 111:
 
<ul>
 
<ul>
 
<li> Overnight cultures of transformation from week 9.</li>
 
<li> Overnight cultures of transformation from week 9.</li>
−
<li>Plasmid isolation & restriction with SpeI. </li>
+
<li>Plasmid isolation & restriction with Spe&thinsp;I. </li>
 
<li>Gel</li>
 
<li>Gel</li>
−
<li>Idea: BioBrick site directli behind pMBi ori of psBIK3</li>
+
<li>Idea: BioBrick site directly behind pMBi ori of psBIK3</li>
 
<ul>
 
<ul>
 
<li>1. Deletion of BioBrick site</li>
 
<li>1. Deletion of BioBrick site</li>
Line 118: Line 120:
 
<ul>
 
<ul>
 
<li>Template: pSBIK3:J04450</li>
 
<li>Template: pSBIK3:J04450</li>
−
<li>part 1 with primer CH07/CH21, tm=56&deg;C</li>
+
<li>Part 1 with primer CH07/CH21, temp=56&deg;C</li>
−
<li>part 2 with primer CH08/CH20, tm=57&deg;C</li>
+
<li>Part 2 with primer CH08/CH20, temp=57&deg;C</li>
−
<li>-> part 1 worked; part 2 not</li>
+
<li>-> Part 1 worked; part 2 not</li>
 
</ul>
 
</ul>
 
<li>Transformation in DH5&alpha; of pSBIC3:K592100</li>
 
<li>Transformation in DH5&alpha; of pSBIC3:K592100</li>
Line 135: Line 137:
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li>Restriction of pSBIK3:J04450 with SPEI and Xba</li>
+
<li>Restriction of pSBIK3:J04450 with Spe&thinsp;I and Xba&thinsp;I</li>
−
<li>colony PCR of K592100</li>
+
<li>Colony PCR of K592100</li>
−
<li>gradient PCR of part 1 & part 2</li>
+
<li>Gradient PCR of part 1 & part 2</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
Line 146: Line 148:
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li>Q5 PCR at 67&deg;C:</li>
+
<li>Q5 PCR at 67&thinsp;&deg;C:</li>
 
<ul>
 
<ul>
 
<li>template: PSBIK3:J04450; primer PS02/PS03</li>
 
<li>template: PSBIK3:J04450; primer PS02/PS03</li>
Line 152: Line 154:
 
<li>template: PSBIC3:K592100; primer PS00/PS01</li>
 
<li>template: PSBIC3:K592100; primer PS00/PS01</li>
 
</ul>
 
</ul>
−
<li>Gibson-Assembly: 15 &mu; Gibson Mastermix + 3.5 &mu; pSBIK3 Backbone + 1.5 &mu; BFP-Insert</li>
+
<li>Gibson-Assembly: 15 &mu;l Gibson Mastermix + 3.5 &mu;l pSBIK3 Backbone + 1.5 &mu;l BFP-Insert</li>
 
<li>Transformation in DH5&alpha;</li>
 
<li>Transformation in DH5&alpha;</li>
 
<li>12 clones on LB-Kan plate that glow blue!</li>
 
<li>12 clones on LB-Kan plate that glow blue!</li>
Line 177: Line 179:
 
<ul>
 
<ul>
 
<li>Plasmid isolation of the overnight cultures</li>
 
<li>Plasmid isolation of the overnight cultures</li>
−
<li>Restriction with EcoRI/ PstI</li>
+
<li>Restriction with EcoR&thinsp;I/ Pst&thinsp;I</li>
−
<li>Sequencing of AraProm, dam-SeqA, emrR</li>
+
<li>Sequencing of AraProm, dam-seqA, emrR</li>
 
<li>Transformation in DH5&alpha; of pSBIC3:K516132</li>
 
<li>Transformation in DH5&alpha; of pSBIC3:K516132</li>
 
<li>Repeated colony PCR of dnaQ, ugi-cda1 -> only empty plasmids -> new primer</li>
 
<li>Repeated colony PCR of dnaQ, ugi-cda1 -> only empty plasmids -> new primer</li>
 
<li>overnight culture of pSBIC3:K516132</li>
 
<li>overnight culture of pSBIC3:K516132</li>
 
<li>Plasmid isolation of pSBIC3:K516132</li>
 
<li>Plasmid isolation of pSBIC3:K516132</li>
−
<li>Q5 PCR with template pSBIC3:K516132, primer CH30/CH31, tm = 67 &deg;C</li>
+
<li>Q5 PCR with template pSBIC3:K516132, primer CH30/CH31, temp = 67 &deg;C</li>
−
<li>DpnI digestion of pcr product pSBIC3:K516132, 1h 37&deg;C</li>
+
<li>DpnI digestion of pcr product pSBIC3:K516132, 1h 37&thinsp;&deg;C</li>
−
<li>1% Agarosegel & purification of the 2kb band -> new backbone</li>
+
<li>1% Agarosegel & purification of the 2&thinsp;kb band -> new backbone</li>
 
<li>repeated Gibson assembly with the new backbone and ugi-cda1/ dnaQ</li>
 
<li>repeated Gibson assembly with the new backbone and ugi-cda1/ dnaQ</li>
 
<li>Transformation in DH5&alpha;</li>
 
<li>Transformation in DH5&alpha;</li>
Line 218: Line 220:
 
<li>Q5 PCR</li>
 
<li>Q5 PCR</li>
 
<ul>
 
<ul>
−
<li>template pSBIC3:dnaQ, primer CH35/CH36, tmp = 59.9&deg;</li>
+
<li>template pSBIC3:dnaQ, primer CH35/CH36, temp = 59.9&thinsp;&deg;C</li>
−
<li> template pSBIC3:dam-seqA, primer CH37/CH38, tmp = 58.3&deg;C</li>
+
<li>template pSBIC3:dam-seqA, primer CH37/CH38, temp = 58.3&thinsp;&deg;C</li>
−
<li> template pSBIC3:dam-seqA, primer CH39/CH40, tmp = 57&deg;</li>
+
<li>template pSBIC3:dam-seqA, primer CH39/CH40, temp = 57&thinsp;&deg;</li>
−
<li>template pHSG-WTPolI, primer CH42/CH43, tmp = 58.4&deg;C</li>
+
<li>template pHSG-WTPolI, primer CH42/CH43, temp = 58.4&thinsp;&deg;C</li>
−
<li>template pHSG-EPPolI, primer CH42/CH43, tmp = 58.4&deg;C </li>
+
<li>template pHSG-EPPolI, primer CH42/CH43, temp = 58.4&thinsp;&deg;C</li>
 
</ul>
 
</ul>
 
<li>DpnI digestion, gel extraction</li>
 
<li>DpnI digestion, gel extraction</li>
−
<li>Restriction of pSBIC3:RFPcd with Xba/SpeI, gel extraction of 2 kb band</li>
+
<li>Restriction of pSBIC3:RFPcd with Xba&thinsp;I/Spe&thinsp;I, gel extraction of 2&thinsp;kb band</li>
 
</ul>
 
</ul>
 
</ul>
 
</ul>
Line 235: Line 237:
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li>Gibson of dnaQ, dam, seqA, C3-BB</li>
+
<li>Gibson of dnaQ, dam, seqA, C3 backbone</li>
 
<li>Transformation in KRX compis</li>
 
<li>Transformation in KRX compis</li>
 
<li>Colony-PCR with the Gibson clones -> no bands</li>
 
<li>Colony-PCR with the Gibson clones -> no bands</li>
 
<li>Q5 PCR</li>
 
<li>Q5 PCR</li>
 
<ul>
 
<ul>
−
<li>template pSBIC3:dnaQ, primer CH35/36, tmp = 59.9&deg;C</li>
+
<li>template pSBIC3:dnaQ, primer CH35/36, temp = 59.9&thinsp;&deg;C</li>
−
<li> template pSBIC3:dam-seqA, primer CH37/38, tmp = 58.3&deg;C </li>
+
<li> template pSBIC3:dam-seqA, primer CH37/38, temp = 58.3&thinsp;&deg;C </li>
−
<li> template pSBIC3:dam-seqA, primer CH39/40, tmp = 57&deg;C </li>
+
<li> template pSBIC3:dam-seqA, primer CH39/40, temp = 57&thinsp;&deg;C </li>
−
<li> template pSBIK3:J04450, primer CH30/31, tmp = 67&deg;C </li>
+
<li> template pSBIK3:J04450, primer CH30/31, temp = 67&thinsp;&deg;C </li>
 
</ul>
 
</ul>
 
<li>DpnI distriction and gel extraction of pSBIK3 backbone</li>
 
<li>DpnI distriction and gel extraction of pSBIK3 backbone</li>
Line 256: Line 258:
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li>colony-PCR of mutagenisis assembly I with VR/VF -> plating positive clones out</li>
+
<li>colony-PCR of ASI with VR/VF -> plating positive clones out</li>
 
<li>Sequencing of positive clones</li>
 
<li>Sequencing of positive clones</li>
 
<li>Q5 PCR</li>
 
<li>Q5 PCR</li>
Line 267: Line 269:
 
</ul>
 
</ul>
 
<li>PCR clean up of the Q5 PCR</li>
 
<li>PCR clean up of the Q5 PCR</li>
−
<li>Gibson assembly of emrR, ugi, cda1 in pSBIK3-BB (ASII)</li>
+
<li>Gibson assembly of emrR, ugi, cda1 in pSBIK3 backbone (ASII)</li>
−
<li>Gibson assembly of EPPolI/ WTPolI in pSBIK3-BB</li>
+
<li>Gibson assembly of EPPolI/ WTPolI in pSBIK3 backbone</li>
 
<li>Transformation in DH5&alpha;</li>
 
<li>Transformation in DH5&alpha;</li>
 
</ul>
 
</ul>
Line 278: Line 280:
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li>Colony PCR -> mutagen assembly II did not work</li>
+
<li>Colony PCR -> ASII did not work</li>
 
<li>Repeated colony PCR -> sequencing of positive clones</li>
 
<li>Repeated colony PCR -> sequencing of positive clones</li>
 
</ul>
 
</ul>
Line 298: Line 300:
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li>Sequencing results: mutagenesis AS I has mutations, AS II did not work</li>
+
<li>Sequencing results: mutagenesis ASI has mutations, ASII did not work</li>
 
<li>Transformation of ASII Gibson</li>
 
<li>Transformation of ASII Gibson</li>
 
<li>Plasmid isolation of ASI/ ASII clones</li>
 
<li>Plasmid isolation of ASI/ ASII clones</li>
Line 304: Line 306:
 
<li>Restriction of positive clones of ASII</li>
 
<li>Restriction of positive clones of ASII</li>
 
<li>Repeated colony PCR of ASI with cPCR_dnaQ_fw/VR -> plating out right clones of pSBIC3:dnaQ, pSBIC3:emrR</li>
 
<li>Repeated colony PCR of ASI with cPCR_dnaQ_fw/VR -> plating out right clones of pSBIC3:dnaQ, pSBIC3:emrR</li>
−
<li>Restriction of right ASII clones with Eco/PstI</li>
+
<li>Restriction of right ASII clones with EcoR&thinsp;I/Pst&thinsp;I</li>
−
<li>Restriction of right ASI clone with Eco/PstI</li>
+
<li>Restriction of right ASI clone with EcoR&thinsp;I/Pst&thinsp;I</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
Line 317: Line 319:
 
<li>PCR for coloning ASI into pSBIC3</li>
 
<li>PCR for coloning ASI into pSBIC3</li>
 
<ul>
 
<ul>
−
<li>template pSBIK3:dnaq/dam/seqA, primer CH35/40, tmp = 58.6&deg;C</li>
+
<li>template pSBIK3:dnaq/dam/seqA, primer CH35/40, temp = 58.6&thinsp;&deg;C</li>
−
<li>template pSBIC3:RFPcd, primer CH30/31, tmp = 67&deg;C</li>
+
<li>template pSBIC3:RFPcd, primer CH30/31, temp = 67&thinsp;&deg;C</li>
−
<li>template pSBIC3:AraC-Pbad, primer CH54/52, tmp = 58.6&deg;C</li>
+
<li>template pSBIC3:AraC-Pbad, primer CH54/52, temp = 58.6&thinsp;&deg;C</li>
 
</ul>
 
</ul>
 
<li>DpnI digestion</li>
 
<li>DpnI digestion</li>
−
<li>Restriction with Eco/Pst</li>
+
<li>Restriction with EcoR&thinsp;I/Pst&thinsp;I</li>
 
<ul>
 
<ul>
 
<li>pSBIK3:J04450</li>
 
<li>pSBIK3:J04450</li>
Line 329: Line 331:
 
</ul>
 
</ul>
 
<li>Plasmid isolation of positive emrR clones and positive ASII clone, strong RFP</li>
 
<li>Plasmid isolation of positive emrR clones and positive ASII clone, strong RFP</li>
−
<li>Restriction with EcoRI/PstI of  ASII and pSBIC3:Strong RFP</li>
+
<li>Restriction with EcoR&thinsp;I/Pst&thinsp;I of  ASII and pSBIC3:Strong RFP</li>
 
<li>Ligation of ASII and strong RFP</li>
 
<li>Ligation of ASII and strong RFP</li>
 
<li>Transformation of the ligation into DH5&alpha;</li>
 
<li>Transformation of the ligation into DH5&alpha;</li>
Line 345: Line 347:
 
<li>Colony PCR</li>
 
<li>Colony PCR</li>
 
<ul>
 
<ul>
−
<li>template pSBIC3:AraCPbad-nonleaky, primer VF/CH53, tmp = 58&deg;C</li>
+
<li>template pSBIC3:AraCPbad-nonleaky, primer VF/CH53, temp = 58&thinsp;&deg;C</li>
−
<li> template pSBIC3:ASI, primer VR/VF_rev, tmp = 58&deg;C </li>
+
<li> template pSBIC3:ASI, primer VR/VF_rev, temp = 58&thinsp;&deg;C </li>
  
 
</ul>
 
</ul>
 
<li>Q5 PCR</li>
 
<li>Q5 PCR</li>
 
<ul>
 
<ul>
−
<li> template A68T_A, primer CH20/A68T_rev, tmp = 58.6&deg;C (AA)</li>
+
<li> template A68T_A, primer CH20/A68T_rev, temp = 58.6&thinsp;&deg;C (AA)</li>
−
<li> template A68T_B, primer A68T_B/CH21, tmp = 58.5&deg;C (AB)</li>
+
<li> template A68T_B, primer A68T_B/CH21, temp = 58.5&thinsp;&deg;C (AB)</li>
−
<li> template C379A_A, primer CH20/C379A_rev, tmp = 57.7&deg;C (BA)</li>
+
<li> template C379A_A, primer CH20/C379A_rev, temp = 57.7&thinsp;&deg;C (BA)</li>
−
<li> template C379A_B, primer C379A_fw/CH21, tmp = 58&deg;C (BB)</li>
+
<li> template C379A_B, primer C379A_fw/CH21, temp = 58&thinsp;&deg;C (BB)</li>
−
<li> template A380T_A, primer CH20/ A380T_rev, tmp = 58.1&deg;C (CA)</li>
+
<li> template A380T_A, primer CH20/ A380T_rev, temp = 58.1&thinsp;&deg;C (CA)</li>
−
<li> template A380T_B, primer A380T_fw/CH21, tmp = 58.1&deg;C (CB)</li>
+
<li> template A380T_B, primer A380T_fw/CH21, temp = 58.1&thinsp;&deg;C (CB)</li>
 
</ul>
 
</ul>
 
<li>Gel extraction</li>
 
<li>Gel extraction</li>
Line 363: Line 365:
 
<li>Sequencing of pSBIK3:EPPolI, pSBIK3:WTPolI with CH61,63,64</li>
 
<li>Sequencing of pSBIK3:EPPolI, pSBIK3:WTPolI with CH61,63,64</li>
 
<li>Colony PCR of AraC-Pbad (non leaky), ASI</li>
 
<li>Colony PCR of AraC-Pbad (non leaky), ASI</li>
−
<li>Q5 PCR template ASI, primer dnaQ_Gib_fw/ seqA_Gib_rev, tmp = 57.5&deg;C</li>
+
<li>Q5 PCR template ASI, primer dnaQ_Gib_fw/ seqA_Gib_rev, temp = 57.5&thinsp;&deg;C</li>
 
<li>Sequencing of pSBIC3:emrR-1/-2 with VR/VF</li>
 
<li>Sequencing of pSBIC3:emrR-1/-2 with VR/VF</li>
−
<li>Colony PCR of pSBIK3:K608010_A/B/C with CH50/VR, tmp = 50&deg;C</li>
+
<li>Colony PCR of pSBIK3:K608010_A/B/C with CH50/VR, temp = 50&thinsp;&deg;C</li>
 
<li>Plasmid isolation of pSBIC3:AraC-Pbad (non-leaky), pSBIK3:608010</li>
 
<li>Plasmid isolation of pSBIC3:AraC-Pbad (non-leaky), pSBIK3:608010</li>
−
<li>Restriction of C3 backbone with Eco/Pst</li>
+
<li>Restriction of C3 backbone with EcoR&thinsp;I/Pst&thinsp;I</li>
 
<li>Transformation of pSBIC3:K516031 in KRX</li>
 
<li>Transformation of pSBIC3:K516031 in KRX</li>
 
<li>cPCR of pSBIC3:K608010_StopA/B/C with VR/VF</li>
 
<li>cPCR of pSBIC3:K608010_StopA/B/C with VR/VF</li>
Line 382: Line 384:
 
<li>Restriction</li>
 
<li>Restriction</li>
 
<ul>
 
<ul>
−
<li>pSBIC3:AraCPbad with SpeI, PstI</li>
+
<li>pSBIC3:AraCPbad with Spe&thinsp;I, Pst&thinsp;I</li>
−
<li> pSBIC3:AraCPbad(non-leaky) with SpeI, PstI </li>
+
<li> pSBIC3:AraCPbad(non-leaky) with Spe&thinsp;I, Pst&thinsp;I </li>
−
<li> pSBIC3:RFP Generator with Xba, PstI </li>
+
<li> pSBIC3:RFP Generator with Xba&thinsp;I, Pst&thinsp;I </li>
−
<li> pSBIK3:MP6-I-Klon 4 with SpeI, PstI </li>
+
<li> pSBIK3:MP6-I-Klon 4 with Spe&thinsp;I, Pst&thinsp;I </li>
−
<li> pSBIK3:MP6-II-Klon with Xba, PstI </li>
+
<li> pSBIK3:MP6-II-Klon with Xba&thinsp;I, Pst&thinsp;I </li>
 
</ul>
 
</ul>
 
<li>Designing of new primer</li>
 
<li>Designing of new primer</li>
Line 397: Line 399:
 
<li>C3 + MutII</li>
 
<li>C3 + MutII</li>
 
</ul>
 
</ul>
−
<li>Repeat of Restriction of ASII: template K3:ASII, EcoRI-Hf/PstI</li>
+
<li>Repeat of Restriction of ASII: template K3:ASII, EcoR&thinsp;I-Hf/Pst&thinsp;I</li>
 
<li>Gel extraction of the restriction</li>
 
<li>Gel extraction of the restriction</li>
 
<li>Ligation</li>
 
<li>Ligation</li>
Line 403: Line 405:
 
<li>cPCR with VR/VR of C3:ASII, C3:AraC-Pbad-RBS-RFP, C3:AraC-Pbad(non-leaky)-RBS-RFP, K3:ASI+ASII</li>
 
<li>cPCR with VR/VR of C3:ASII, C3:AraC-Pbad-RBS-RFP, C3:AraC-Pbad(non-leaky)-RBS-RFP, K3:ASI+ASII</li>
 
<li>Plating out C3:AraC-Pbad-RBS-RFP with arabinose or glucose</li>
 
<li>Plating out C3:AraC-Pbad-RBS-RFP with arabinose or glucose</li>
−
<li>Q5 PCR, tmp = 57&deg;C</li>
+
<li>Q5 PCR, temp = 57&thinsp;&deg;C</li>
 
<ul>
 
<ul>
 
<li>template C3:AraC-Pbad(non)-RBS-RFP, primer CH60/CH61= </li>
 
<li>template C3:AraC-Pbad(non)-RBS-RFP, primer CH60/CH61= </li>
Line 417: Line 419:
 
<li>Gibson assembly</li>
 
<li>Gibson assembly</li>
 
<ul>
 
<ul>
−
<li>damSeqA + C3</li>
+
<li>damseqA + C3</li>
 
<li>ugi-cda1 + C3</li>
 
<li>ugi-cda1 + C3</li>
 
<li>WTPolI-Part + C3</li>
 
<li>WTPolI-Part + C3</li>
Line 426: Line 428:
 
</ul>
 
</ul>
 
<li>Transformation</li>
 
<li>Transformation</li>
−
<li>cPCR with VR/VF, tmp = 57&deg;C</li>
+
<li>cPCR with VR/VF, temp = 57&thinsp;&deg;C</li>
 
<ul>
 
<ul>
 
<li>C3:AraC-Pbad(tight)-B0031-WTPolI</li>
 
<li>C3:AraC-Pbad(tight)-B0031-WTPolI</li>
Line 448: Line 450:
 
<ul>
 
<ul>
 
<li>cPCR of ugi-cda1 -> Sequencing of right clones</li>
 
<li>cPCR of ugi-cda1 -> Sequencing of right clones</li>
−
<li>Q5 PCR, tmp = 58&deg;C</li>
+
<li>Q5 PCR, temp = 58&thinsp;&deg;C</li>
 
<ul>
 
<ul>
−
<li>template C3:damSeqA, primer CH71/72</li>
+
<li>template C3:damseqA, primer CH71/72</li>
 
<li>template C3:StoppGFP Stop A Stop B, primer CH20/66</li>
 
<li>template C3:StoppGFP Stop A Stop B, primer CH20/66</li>
−
<li>template C3:damSeqA, primer CH73/74</li>
+
<li>template C3:damseqA, primer CH73/74</li>
 
<li>template K3:Stopp GFP A+B, primer CH64/21</li>
 
<li>template K3:Stopp GFP A+B, primer CH64/21</li>
 
</ul>
 
</ul>
Line 458: Line 460:
 
<li>Gel extraction of Stop A, Stop B</li>
 
<li>Gel extraction of Stop A, Stop B</li>
 
<li>Gibson assembly stop A + stop B</li>
 
<li>Gibson assembly stop A + stop B</li>
−
<li>Restriction with Eco/Pst ASI+ASII, GFP_Astop_2, GFP_Astop_2</li>
+
<li>Restriction with EcoR&thinsp;I/Pst&thinsp;I ASI+ASII, GFP_Astop_2, GFP_Astop_2</li>
 
<li>Transformation of C3:PRha, C3:PRha-GFPGen in KRX</li>
 
<li>Transformation of C3:PRha, C3:PRha-GFPGen in KRX</li>
 
<li>Transformation of PRha-GFPGen in top10</li>
 
<li>Transformation of PRha-GFPGen in top10</li>
Line 470: Line 472:
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li>Q5 PCR, tmp = 58&deg;C</li>
+
<li>Q5 PCR, temp = 58&deg;C</li>
 
<ul>
 
<ul>
 
<li>template C3:dnaQ, primer 54/55</li>
 
<li>template C3:dnaQ, primer 54/55</li>
Line 484: Line 486:
 
<li>Gel extraction</li>
 
<li>Gel extraction</li>
 
<li>Gibson assembly of seqA</li>
 
<li>Gibson assembly of seqA</li>
−
<li>Transformation in DH5&alpha; of C3:SeqA, K3:StopA+B</li>
+
<li>Transformation in DH5&alpha; of C3:seqA, K3:StopA+B</li>
−
<li>Q5 PCR of C3:damSeqA with CH71/72, tmp = 58&deg;C</li>
+
<li>Q5 PCR of C3:damseqA with CH71/72, temp = 58&thinsp;&deg;C</li>
 
<li>DpnI digestion</li>
 
<li>DpnI digestion</li>
−
<li>Gibson assembly< of dnaQ_Gen + BB, WTPolI_Gen + BB, EPPol + BB, Mp6_Gen + BB</li>
+
<li>Gibson assembly of dnaQ_Gen + BB, WTPolI_Gen + BB, EPPol + BB, Mp6_Gen + BB</li>
 
<li>Transformation of the Gibson assembly in DH5&alpha;</li>
 
<li>Transformation of the Gibson assembly in DH5&alpha;</li>
 
<li>Overnight cultures of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP </li>
 
<li>Overnight cultures of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP </li>
−
<li> Cultures (+ 50mM Arabinose) of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP </li>
+
<li>Cultures (+ 50mM Arabinose) of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP </li>
 
<li>cPCR of dnaQ-Gen, MP6-Gen with VR/VF and CH77/VR</li>
 
<li>cPCR of dnaQ-Gen, MP6-Gen with VR/VF and CH77/VR</li>
 
<li>Overnight cultures of B0031-RFP-Ter, K808000, B0032-RFP-Ter</li>
 
<li>Overnight cultures of B0031-RFP-Ter, K808000, B0032-RFP-Ter</li>
−
<li><Q5 PCR of C3:damSeqA, primer 71/72, gradient 55-59&deg;C/li>
+
<li>Q5 PCR of C3:damseqA, primer 71/72, gradient 55-59&thinsp;&deg;C/li>
 
<li>Sequencing of stop A+B</li>
 
<li>Sequencing of stop A+B</li>
−
<li>Restriction of K3:ASII, C3:Terminator with EcoRI, PstI</li>
+
<li>Restriction of K3:ASII, C3:Terminator with EcoR&thinsp;I, Pst&thinsp;I</li>
 
<li>Gibson assembly of M6 in RFPGen -> Transformation</li>
 
<li>Gibson assembly of M6 in RFPGen -> Transformation</li>
 
<li>Test cultivation of a 96-well plate</li>
 
<li>Test cultivation of a 96-well plate</li>
 
<li>cPCR of M6-Gen -> Plasmid isolation</li>
 
<li>cPCR of M6-Gen -> Plasmid isolation</li>
−
<li>Restriction of dnaQ-Gen with Xba/Pst</li>
+
<li>Restriction of dnaQ-Gen with Xba&thinsp;I/Pst&thinsp;I</li>
−
<li>araE-PCR with tmp 58&deg;C</li>
+
<li>araE-PCR with temp 58&thinsp;&deg;C</li>
 
<li>Gibson assembly of araE</li>
 
<li>Gibson assembly of araE</li>
 
<li>PCR of DNA, primer 81/82 and C3:med RFP 83/84</li>
 
<li>PCR of DNA, primer 81/82 and C3:med RFP 83/84</li>
Line 507: Line 509:
 
<li>DpnI digestion</li>
 
<li>DpnI digestion</li>
 
<li>PCR clean up</li>
 
<li>PCR clean up</li>
−
<li>Gibson of araE-Insert + araE-BB</li>
+
<li>Gibson of araE-Insert + araE-backbone</li>
 
<li>Transformation of the ligation in KRX</li>
 
<li>Transformation of the ligation in KRX</li>
−
<li>Restriction of C3:M6-Gen with Eco/Xba, weak/RFP Gen with Xba/Pst</li>
+
<li>Restriction of C3:M6-Gen with EcoR&thinsp;I/Xba&thinsp;I, weak/RFP Gen with Xba&thinsp;I/Pst&thinsp;I</li>
 
<li>cPCR of C3:araE-Device</li>
 
<li>cPCR of C3:araE-Device</li>
−
<li>Gibson C3 BB + ugi_cda1, C3 BB + ASII</li>
+
<li>Gibson C3 backbone + ugi_cda1, C3 backbone + ASII</li>
 
<li>Transformation of the Gibson assembly in DH5&alpha;</li>
 
<li>Transformation of the Gibson assembly in DH5&alpha;</li>
 
<li>cPCR the transformation, Prha-B0031-dnaQ-Ter with VR/VF</li>
 
<li>cPCR the transformation, Prha-B0031-dnaQ-Ter with VR/VF</li>
Line 522: Line 524:
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li>Restriction of M6-Generator with Xba/Pst, C3:K808000, C3:Prha with Spe/Pst</li>
+
<li>Restriction of M6-Generator with Xba&thinsp;I/Pst&thinsp;I, C3:K808000, C3:Prha with Spe&thinsp;I/Pst&thinsp;I</li>
 
<li>Clean up</li>
 
<li>Clean up</li>
−
<li>Ligation with BB</li>
+
<li>Ligation with backbone</li>
−
<li>Transformation of Ligation, C3:AraCPbad(tight)-dnaQ in Top10, plA230 in JS200 with EPPolI/WTPolI</li>
+
<li>Transformation of ligation, C3:AraCPbad(tight)-dnaQ in Top10, plA230 in JS200 with EPPolI/WTPolI</li>
−
<li> Transformation of plA230 in JS200 with EPPolI/WTPolI; overnight culture with 10 ml Cm, Kan, 37&deg;C</li>
+
<li> Transformation of plA230 in JS200 with EPPolI/WTPolI; overnight culture with 10 ml Cm, Kan, 37&thinsp;&deg;C</li>
 
<li>Transformation of C3:AraC(tight)dnaQ + plA230 in Top10</li>
 
<li>Transformation of C3:AraC(tight)dnaQ + plA230 in Top10</li>
 
<li>Sequencing of C3:AraCPbad-dnaQ, C3:PRha-dnaQ</li>
 
<li>Sequencing of C3:AraCPbad-dnaQ, C3:PRha-dnaQ</li>
 
<li>Overnight culture of JS200 + pHSG-Pol + pLA230; Top 10 C3:AraCPbad-dnaQ</li>
 
<li>Overnight culture of JS200 + pHSG-Pol + pLA230; Top 10 C3:AraCPbad-dnaQ</li>
−
<li>inoculation of Top 10 + C3:AraCPbad-dnaQ to OD 0.1; 1-2h growth, +25mM arabinose/25mM glycose</li>
+
<li>Inoculation of Top 10 + C3:AraCPbad-dnaQ to OD 0.1; 1-2&thinsp;h growth, +25mM arabinose/25mM glycose</li>
 
<li>Transformation of JS200 EP/WT with plA230</li>
 
<li>Transformation of JS200 EP/WT with plA230</li>
 
<li>cPCR of AraC-Pbad-M6; Prha-M6</li>
 
<li>cPCR of AraC-Pbad-M6; Prha-M6</li>
Line 547: Line 549:
 
<br>
 
<br>
 
<ul>
 
<ul>
−
<li>Transformation of Pbad(med)dnaQ + plA230; Pbad-M6 + plA230 in Top10</li>
+
<li>Transformation of Pbad(med)dnaQ + plA230; Pbad-M6 + pLA230 in Top10</li>
−
<li>Transformation of JS200 + EP+ plA230; JS200 + WT+plA230 -> liquid culture</li>
+
<li>Transformation of JS200 + EP + plA230; JS200 + WT + pLA230 -> liquid culture</li>
 
<li>Isolation of JS200 + PolI+plA230</li>
 
<li>Isolation of JS200 + PolI+plA230</li>
−
<li>Inoculation of Top10 + plA230 + Pbad(med)-dnaQ; Top10 + plA230 + Pbad-M6 -> growth till OD 0.7336 (M6)/ 0.568 (dnaQ)</li>
+
<li>Inoculation of Top10 + pLA230 + Pbad(med)-dnaQ; Top10 + plA230 + Pbad-M6 -> growth till OD 0.7336 (M6)/ 0.568 (dnaQ)</li>
 
<li>Inoculation of with 1) 25mM arabinose; 2) + 25mM glucose 3) isolation</li>
 
<li>Inoculation of with 1) 25mM arabinose; 2) + 25mM glucose 3) isolation</li>
−
<li>Restriction of the MiSeq probes: WT, EP, plA230, plA230+dnaQ; plA230+M6</li>
+
<li>Restriction of the MiSeq probes: WT, EP, plA230, pLA230+dnaQ; pLA230+M6</li>
 
<li>Cloning of Pbad(med)WT/EP in pSB4C5; araE-dev + Pbad-GFP; B0032-RFP + Pbad; M6 + Pbad(med)</li>
 
<li>Cloning of Pbad(med)WT/EP in pSB4C5; araE-dev + Pbad-GFP; B0032-RFP + Pbad; M6 + Pbad(med)</li>
 
<li>Transformation of K3:K608010 –WT; - StopA; -StopB; - StopC in Top10</li>
 
<li>Transformation of K3:K608010 –WT; - StopA; -StopB; - StopC in Top10</li>
Line 560: Line 562:
 
<li>Q5 PCR</li>
 
<li>Q5 PCR</li>
 
<ul>
 
<ul>
−
<li>template C3:ugi-cda, primer 67/68, tmp = 58&deg;C</li>
+
<li>template C3:ugi-cda, primer 67/68, temp = 58&thinsp;&deg;C</li>
−
<li>template C3:cda, primer 69/70, tmp = 60&deg;C</li>
+
<li>template C3:cda, primer 69/70, temp = 60&thinsp;&deg;C</li>
−
<li>template A3, primer CK06/05, tmp = 59.9&deg;C</li>
+
<li>template A3, primer CK06/05, temp = 59.9&thinsp;&deg;C</li>
−
<li>template reporter, primer CK07/08, tmp = 59&deg;C</li>
+
<li>template reporter, primer CK07/08, temp = 59&thinsp;&deg;C</li>
  
 
</ul>
 
</ul>
Line 575: Line 577:
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li>Transformation of C3:dnaQ Dev + plA230, C3:M6 Dev + plA230 in Top10</li>
+
<li>Transformation of C3:dnaQ Dev + pLA230, C3:M6 Dev + pLA230 in Top10</li>
 
<li>Reversions assay</li>
 
<li>Reversions assay</li>
 
<li></li>
 
<li></li>
Line 590: Line 592:
 
<br>  
 
<br>  
 
<ul>
 
<ul>
−
<li>Analysis reversion assay</a>
+
<li>Analysis reversion assays</li>
 +
<li>Analysis of MiSeq data</li>
 
</ul>
 
</ul>
 
</div>
 
</div>

Revision as of 11:21, 19 October 2016



lab notebook.

Mutation


Firstly, we researched for literature on issues involving in vivo mutagenesis and error prone polymerase. In this phase we read the papers from Prof. Dr. Manel Camps.


Discussing the results of the literature research and creating a first draft of what must be done in the laboratory. Further literature research.


Looking for suitable parts that can be used for our mutagenesis system. Creating plasmid circuits in geneious. Extension of the first draft.


Designing of first primers and getting to know the laboratory. Also literature research.


Contacting the leading expert for the error prone polymerase via e-mail. Dividing the laboratory tasks and creating milestones.


  • Transformation in DH5α:
    • pHSG-WTPolI
    • pHSG-EPPolI
    • pLA230
    • psBIK3:J04450
    • psBIA3:E0044
  • Everything besides pSBIA3:E0044 grew. Taking overnight cultures at 37°C.
  • Plating provided JS200 strain out.


    • Plasmid isolation of:
      • pHSG-WTPolI
      • pHSG-EPPolI
      • pLA230
      • psBIK3:J04450
    • Overnight cultures from JS200.
    • JS200 glycerin stocks (500 μ culture + 200 μ 86% glycerin)


    Rethinking of our plan.


    • Transformation in DH5α:
      • pSBIC3:E1010
      • pSBIC3:K731722
    • Transformation in JS200;:
      • pHSG-EPPolI & plA230
      • pHSG-WTPolI & plA230
    • Platting out pSBIC3:Isopropanolpathway from iGEM Bielefeld 2014


    • Overnight cultures of transformation from week 9.
    • Plasmid isolation & restriction with Spe I.
    • Gel
    • Idea: BioBrick site directly behind pMBi ori of psBIK3
      • 1. Deletion of BioBrick site
        • Q5 PCR
          • Template: pSBIK3:J04450
          • Part 1 with primer CH07/CH21, temp=56°C
          • Part 2 with primer CH08/CH20, temp=57°C
          • -> Part 1 worked; part 2 not
        • Transformation in DH5α of pSBIC3:K592100
      • 2.: New BioBrick site behind ori


    • Restriction of pSBIK3:J04450 with Spe I and Xba I
    • Colony PCR of K592100
    • Gradient PCR of part 1 & part 2


    • Q5 PCR at 67 °C:
      • template: PSBIK3:J04450; primer PS02/PS03
      • template: PSBIC3:E1010; primer CH30/CH31
      • template: PSBIC3:K592100; primer PS00/PS01
    • Gibson-Assembly: 15 μl Gibson Mastermix + 3.5 μl pSBIK3 Backbone + 1.5 μl BFP-Insert
    • Transformation in DH5α
    • 12 clones on LB-Kan plate that glow blue!


    • Gene synthesis is there (AraProm, dnaQ, dam-seqA, emrR, ugi-cda1)! Resolve in TE-buffer
    • Gibson assembly of the gene synthesis in pSBIC3 backbone
    • Transformation in NEB electro competent cells
    • Colony PCR with VR/VF
    • Overnight culture from AraProm, dam-seqA, emrR


    • Plasmid isolation of the overnight cultures
    • Restriction with EcoR I/ Pst I
    • Sequencing of AraProm, dam-seqA, emrR
    • Transformation in DH5α of pSBIC3:K516132
    • Repeated colony PCR of dnaQ, ugi-cda1 -> only empty plasmids -> new primer
    • overnight culture of pSBIC3:K516132
    • Plasmid isolation of pSBIC3:K516132
    • Q5 PCR with template pSBIC3:K516132, primer CH30/CH31, temp = 67 °C
    • DpnI digestion of pcr product pSBIC3:K516132, 1h 37 °C
    • 1% Agarosegel & purification of the 2 kb band -> new backbone
    • repeated Gibson assembly with the new backbone and ugi-cda1/ dnaQ
    • Transformation in DH5α
      • pSBIA3:J04450
      • psBIC3:J06702
      • GFP cd
      • pSBIC:E2050
      • dnaQ Gibson
      • ugi-cda1 Gibson
    • Overnight culture of DH5α with pSBIK3:RFPcd/ pSBIK3:BFPcd
    • Testing of the fluorescent proteins RFRP and BFP with the Teacan
    • Colony PCR of pSBIC3:dnaQ with VR/VF
    • Overnight culture of good clones, pGFPuv, pSBIA3:J04450, pSBIC3:J06702


    • Primer design for genesynthesis
    • Plasmid isolation of the overnight cultures
    • Restriction of pSBIC3:dnaQ, mCherry, E2050-cds
    • Sequencing of dnaQ clones with VR/VF
    • Colony-PCR with CH34/VR on ugi-cda1
    • Sequencing of ugi-cda1
    • Assembly of dnaQ, dam, seqA
      • Q5 PCR
        • template pSBIC3:dnaQ, primer CH35/CH36, temp = 59.9 °C
        • template pSBIC3:dam-seqA, primer CH37/CH38, temp = 58.3 °C
        • template pSBIC3:dam-seqA, primer CH39/CH40, temp = 57 °
        • template pHSG-WTPolI, primer CH42/CH43, temp = 58.4 °C
        • template pHSG-EPPolI, primer CH42/CH43, temp = 58.4 °C
      • DpnI digestion, gel extraction
      • Restriction of pSBIC3:RFPcd with Xba I/Spe I, gel extraction of 2 kb band


    • Gibson of dnaQ, dam, seqA, C3 backbone
    • Transformation in KRX compis
    • Colony-PCR with the Gibson clones -> no bands
    • Q5 PCR
      • template pSBIC3:dnaQ, primer CH35/36, temp = 59.9 °C
      • template pSBIC3:dam-seqA, primer CH37/38, temp = 58.3 °C
      • template pSBIC3:dam-seqA, primer CH39/40, temp = 57 °C
      • template pSBIK3:J04450, primer CH30/31, temp = 67 °C
    • DpnI distriction and gel extraction of pSBIK3 backbone
    • Gibson assembly of dnaQ, dam, seqA in K3 backbone (ASI)
    • Transformation in DH5α


    • colony-PCR of ASI with VR/VF -> plating positive clones out
    • Sequencing of positive clones
    • Q5 PCR
      • template pSBIC3:emrR, primer BF1/2
      • template pSBIC3:ugi-cda1, primer BF4/5
      • template pSBIC3:emrR, primer BF6/7
      • template pHSG:EPPolI, primer CH42/43
      • template pHSG:WTPolI, primer CH42/43
    • PCR clean up of the Q5 PCR
    • Gibson assembly of emrR, ugi, cda1 in pSBIK3 backbone (ASII)
    • Gibson assembly of EPPolI/ WTPolI in pSBIK3 backbone
    • Transformation in DH5α


    • Colony PCR -> ASII did not work
    • Repeated colony PCR -> sequencing of positive clones


    • Transformation of pSBIC3:K584001 & K608010
    • Planning of reversion experiments


    • Sequencing results: mutagenesis ASI has mutations, ASII did not work
    • Transformation of ASII Gibson
    • Plasmid isolation of ASI/ ASII clones
    • Colony PCR of ASII
    • Restriction of positive clones of ASII
    • Repeated colony PCR of ASI with cPCR_dnaQ_fw/VR -> plating out right clones of pSBIC3:dnaQ, pSBIC3:emrR
    • Restriction of right ASII clones with EcoR I/Pst I
    • Restriction of right ASI clone with EcoR I/Pst I


    • Sequencing of dnaQ-dam-seqA (ASI), emrR_ugi_cda1 (ASII)
    • PCR for coloning ASI into pSBIC3
      • template pSBIK3:dnaq/dam/seqA, primer CH35/40, temp = 58.6 °C
      • template pSBIC3:RFPcd, primer CH30/31, temp = 67 °C
      • template pSBIC3:AraC-Pbad, primer CH54/52, temp = 58.6 °C
    • DpnI digestion
    • Restriction with EcoR I/Pst I
      • pSBIK3:J04450
      • pSBIC3:K608010
      • pSBIC3:K584001
    • Plasmid isolation of positive emrR clones and positive ASII clone, strong RFP
    • Restriction with EcoR I/Pst I of ASII and pSBIC3:Strong RFP
    • Ligation of ASII and strong RFP
    • Transformation of the ligation into DH5α
    • Gel extraction of pSBIK3, K608010, K584001
    • Ligation of pSBIK3 + K608010
    • Overnight culture of the ligation
    • Gel extraction of PCR products & PCR clean up
    • Gibson assembly
      • pSBIC3:AraC-Pbad (non-leaky)
      • C3 backbone + ASI clone 4
      • C3 backbone + ASI clone 15
    • Ligation & Transformation in DH5α
    • Colony PCR
      • template pSBIC3:AraCPbad-nonleaky, primer VF/CH53, temp = 58 °C
      • template pSBIC3:ASI, primer VR/VF_rev, temp = 58 °C
    • Q5 PCR
      • template A68T_A, primer CH20/A68T_rev, temp = 58.6 °C (AA)
      • template A68T_B, primer A68T_B/CH21, temp = 58.5 °C (AB)
      • template C379A_A, primer CH20/C379A_rev, temp = 57.7 °C (BA)
      • template C379A_B, primer C379A_fw/CH21, temp = 58 °C (BB)
      • template A380T_A, primer CH20/ A380T_rev, temp = 58.1 °C (CA)
      • template A380T_B, primer A380T_fw/CH21, temp = 58.1 °C (CB)
    • Gel extraction
    • Gibson assembly
    • Transformation in KRX
    • Sequencing of pSBIK3:EPPolI, pSBIK3:WTPolI with CH61,63,64
    • Colony PCR of AraC-Pbad (non leaky), ASI
    • Q5 PCR template ASI, primer dnaQ_Gib_fw/ seqA_Gib_rev, temp = 57.5 °C
    • Sequencing of pSBIC3:emrR-1/-2 with VR/VF
    • Colony PCR of pSBIK3:K608010_A/B/C with CH50/VR, temp = 50 °C
    • Plasmid isolation of pSBIC3:AraC-Pbad (non-leaky), pSBIK3:608010
    • Restriction of C3 backbone with EcoR I/Pst I
    • Transformation of pSBIC3:K516031 in KRX
    • cPCR of pSBIC3:K608010_StopA/B/C with VR/VF
    • Sequencing A with VF, B with VR and C with VR


    • Plasmid isolation of Stop-GFPs -> Sequencing
    • Restriction
      • pSBIC3:AraCPbad with Spe I, Pst I
      • pSBIC3:AraCPbad(non-leaky) with Spe I, Pst I
      • pSBIC3:RFP Generator with Xba I, Pst I
      • pSBIK3:MP6-I-Klon 4 with Spe I, Pst I
      • pSBIK3:MP6-II-Klon with Xba I, Pst I
    • Designing of new primer
    • Gel extraction of the Restriction
    • Ligation
      • C3:AraC-Pbad + RFP Gen
      • C3:AraC-Pbad(non-leaky) + RFP Gen
      • K3:Mut-I + Mut II
      • C3 + MutII
    • Repeat of Restriction of ASII: template K3:ASII, EcoR I-Hf/Pst I
    • Gel extraction of the restriction
    • Ligation
    • Transformation of the Ligation, pHSG-EPPolI, pHSG-WTPolI in KRX
    • cPCR with VR/VR of C3:ASII, C3:AraC-Pbad-RBS-RFP, C3:AraC-Pbad(non-leaky)-RBS-RFP, K3:ASI+ASII
    • Plating out C3:AraC-Pbad-RBS-RFP with arabinose or glucose
    • Q5 PCR, temp = 57 °C
      • template C3:AraC-Pbad(non)-RBS-RFP, primer CH60/CH61=
      • template C3:AraC-Pbad(non)-RBS-RFP, primer CH56/CH57
      • template C3:dnaQ, primer CH54/CH55
      • template pHSG-WTPolI, primer CH58/C59
      • template pHSG-WTPolI, primer CH42/C43
      • template pHSG-EPPolI, primer CH58/C59
      • template pHSG-EPPolI, primer CH42/C43
    • DpnI digestion
    • Gel extraction
    • Gibson assembly
      • damseqA + C3
      • ugi-cda1 + C3
      • WTPolI-Part + C3
      • EPPolI-Part + C3
      • dnaQ-Insert + dnaQ-BB
      • WTPolI-Expression + PolI-BB
      • EPPolI-Expression + PolI-BB
    • Transformation
    • cPCR with VR/VF, temp = 57 °C
      • C3:AraC-Pbad(tight)-B0031-WTPolI
      • C3:ugi-cda1
      • C3:AraC-Pbad(tight)-B0031-dnaQ
      • C3:EPPolI
      • C3:AraC-Pbad(tight)-B0031-EPPolI
      • C3:seqA
      • C3:WTPolI
    • Sequencing
    • Cultur of KRX with C3:AraC-Pbad(tight)-B0031-E1010 with L-Arabinose -> repeat with Top10
    • Transformation of C3:AraC-Pbad(tight)-B0031-E1010 in Top10


    • cPCR of ugi-cda1 -> Sequencing of right clones
    • Q5 PCR, temp = 58 °C
      • template C3:damseqA, primer CH71/72
      • template C3:StoppGFP Stop A Stop B, primer CH20/66
      • template C3:damseqA, primer CH73/74
      • template K3:Stopp GFP A+B, primer CH64/21
    • DpnI digestion of the Q5 PCR
    • Gel extraction of Stop A, Stop B
    • Gibson assembly stop A + stop B
    • Restriction with EcoR I/Pst I ASI+ASII, GFP_Astop_2, GFP_Astop_2
    • Transformation of C3:PRha, C3:PRha-GFPGen in KRX
    • Transformation of PRha-GFPGen in top10
    • Transformation of StoppA+B in DH5α


    • Q5 PCR, temp = 58°C
      • template C3:dnaQ, primer 54/55
      • template C3:WTPolI (CWP1), primer 58/59
      • template C3:EPPolI (CEP1), primer 58/59
      • template C3:ASI+ASII, primer 54/76
      • template C3:K516031, primer 56/57
      • template C3: K516031, primer 60/61
      • template C3: K516031, primer 60/61
      • template C3: K516031, primer 75/57
    • DpnI digestion
    • Gel extraction
    • Gibson assembly of seqA
    • Transformation in DH5α of C3:seqA, K3:StopA+B
    • Q5 PCR of C3:damseqA with CH71/72, temp = 58 °C
    • DpnI digestion
    • Gibson assembly of dnaQ_Gen + BB, WTPolI_Gen + BB, EPPol + BB, Mp6_Gen + BB
    • Transformation of the Gibson assembly in DH5α
    • Overnight cultures of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP
    • Cultures (+ 50mM Arabinose) of KRX Pbad-GFP, KRX PRha-GFP, Top10 Pbad-GFP, Top10 PRha-GFP
    • cPCR of dnaQ-Gen, MP6-Gen with VR/VF and CH77/VR
    • Overnight cultures of B0031-RFP-Ter, K808000, B0032-RFP-Ter
    • Q5 PCR of C3:damseqA, primer 71/72, gradient 55-59 °C/li>
    • Sequencing of stop A+B
    • Restriction of K3:ASII, C3:Terminator with EcoR I, Pst I
    • Gibson assembly of M6 in RFPGen -> Transformation
    • Test cultivation of a 96-well plate
    • cPCR of M6-Gen -> Plasmid isolation
    • Restriction of dnaQ-Gen with Xba I/Pst I
    • araE-PCR with temp 58 °C
    • Gibson assembly of araE
    • PCR of DNA, primer 81/82 and C3:med RFP 83/84
    • Gel extraction of dnaQ-Gen
    • Ligation with C3:AraC-Pbad/C3:PRha
    • DpnI digestion
    • PCR clean up
    • Gibson of araE-Insert + araE-backbone
    • Transformation of the ligation in KRX
    • Restriction of C3:M6-Gen with EcoR I/Xba I, weak/RFP Gen with Xba I/Pst I
    • cPCR of C3:araE-Device
    • Gibson C3 backbone + ugi_cda1, C3 backbone + ASII
    • Transformation of the Gibson assembly in DH5α
    • cPCR the transformation, Prha-B0031-dnaQ-Ter with VR/VF


    • Restriction of M6-Generator with Xba I/Pst I, C3:K808000, C3:Prha with Spe I/Pst I
    • Clean up
    • Ligation with backbone
    • Transformation of ligation, C3:AraCPbad(tight)-dnaQ in Top10, plA230 in JS200 with EPPolI/WTPolI
    • Transformation of plA230 in JS200 with EPPolI/WTPolI; overnight culture with 10 ml Cm, Kan, 37 °C
    • Transformation of C3:AraC(tight)dnaQ + plA230 in Top10
    • Sequencing of C3:AraCPbad-dnaQ, C3:PRha-dnaQ
    • Overnight culture of JS200 + pHSG-Pol + pLA230; Top 10 C3:AraCPbad-dnaQ
    • Inoculation of Top 10 + C3:AraCPbad-dnaQ to OD 0.1; 1-2 h growth, +25mM arabinose/25mM glycose
    • Transformation of JS200 EP/WT with plA230
    • cPCR of AraC-Pbad-M6; Prha-M6
    • Transformation of plA230 in JS00 EP/WT
    • Rifampicillin assay
    • beta-lactamase Reversions assay
    • Transformation of plA230 + C3:Pbad(med)-dnaQ in Top10
    • Overnight cultures



    • Transformation of Pbad(med)dnaQ + plA230; Pbad-M6 + pLA230 in Top10
    • Transformation of JS200 + EP + plA230; JS200 + WT + pLA230 -> liquid culture
    • Isolation of JS200 + PolI+plA230
    • Inoculation of Top10 + pLA230 + Pbad(med)-dnaQ; Top10 + plA230 + Pbad-M6 -> growth till OD 0.7336 (M6)/ 0.568 (dnaQ)
    • Inoculation of with 1) 25mM arabinose; 2) + 25mM glucose 3) isolation
    • Restriction of the MiSeq probes: WT, EP, plA230, pLA230+dnaQ; pLA230+M6
    • Cloning of Pbad(med)WT/EP in pSB4C5; araE-dev + Pbad-GFP; B0032-RFP + Pbad; M6 + Pbad(med)
    • Transformation of K3:K608010 –WT; - StopA; -StopB; - StopC in Top10
    • Transformation of C3:Pbad(med)dnaQ & K3:K608010 –WT; - StopA; -StopB; - StopC in Top10
    • Reversions assay
    • Restriction of E/P of pSB4C5(E/P)
    • Q5 PCR
      • template C3:ugi-cda, primer 67/68, temp = 58 °C
      • template C3:cda, primer 69/70, temp = 60 °C
      • template A3, primer CK06/05, temp = 59.9 °C
      • template reporter, primer CK07/08, temp = 59 °C



    • Transformation of C3:dnaQ Dev + pLA230, C3:M6 Dev + pLA230 in Top10
    • Reversions assay


    • Analysis reversion assays
    • Analysis of MiSeq data