Difference between revisions of "Team:Bielefeld-CeBiTec/Notebook/Mutation"
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<li>template: pSBIC3:K592100; primer PS00/PS01</li> | <li>template: pSBIC3:K592100; primer PS00/PS01</li> | ||
</ul> | </ul> | ||
| − | <li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a>: 15 μl Gibson Mastermix + 3.5 μl pSBIK3 Backbone + 1.5 μl BFP-Insert</li> |
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5α</li> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5α</li> | ||
<li>12 clones on LB-Kan plate that glow blue!</li> | <li>12 clones on LB-Kan plate that glow blue!</li> | ||
| Line 174: | Line 174: | ||
<ul> | <ul> | ||
<li>Gene synthesis is there (AraProm, dnaQ, dam-seqA, emrR, ugi-cda1)! Resolve in TE-buffer</li> | <li>Gene synthesis is there (AraProm, dnaQ, dam-seqA, emrR, ugi-cda1)! Resolve in TE-buffer</li> | ||
| − | <li>Gibson assembly of the gene synthesis in pSBIC3 backbone</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of the gene synthesis in pSBIC3 backbone</li> |
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in NEB electro competent cells</li> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in NEB electro competent cells</li> | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | ||
| − | with VR/VF</li> | + | with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li> |
<li>Overnight culture from AraProm, dam-seqA, emrR</li> | <li>Overnight culture from AraProm, dam-seqA, emrR</li> | ||
</ul> | </ul> | ||
| Line 203: | Line 203: | ||
of pcr product pSBIC3:K516132, 1h 37 °C</li> | of pcr product pSBIC3:K516132, 1h 37 °C</li> | ||
<li>1% Agarosegel & purification of the 2 kb band -> new backbone</li> | <li>1% Agarosegel & purification of the 2 kb band -> new backbone</li> | ||
| − | <li>repeated Gibson assembly with the new backbone and ugi-cda1/ dnaQ</li> | + | <li>repeated <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> with the new backbone and ugi-cda1/ dnaQ</li> |
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5α</li> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5α</li> | ||
<ul> | <ul> | ||
| Line 216: | Line 216: | ||
<li>Testing of the fluorescent proteins RFP and BFP with the Teacan</li> | <li>Testing of the fluorescent proteins RFP and BFP with the Teacan</li> | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | ||
| − | of pSBIC3:dnaQ with VR/VF</li> | + | of pSBIC3:dnaQ with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VR</a>/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li> |
<li>Overnight culture of good clones, pGFPuv, pSBIA3:J04450, pSBIC3:J06702</li> | <li>Overnight culture of good clones, pGFPuv, pSBIA3:J04450, pSBIC3:J06702</li> | ||
</ul> | </ul> | ||
| Line 231: | Line 231: | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a> | ||
of pSBIC3:dnaQ, mCherry, E2050-cds</li> | of pSBIC3:dnaQ, mCherry, E2050-cds</li> | ||
| − | <li>Sequencing of dnaQ clones with VR/VF</li> | + | <li>Sequencing of dnaQ clones with VR/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li> |
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | ||
with CH34/VR on ugi-cda1</li> | with CH34/VR on ugi-cda1</li> | ||
| Line 260: | Line 260: | ||
<br> | <br> | ||
<ul> | <ul> | ||
| − | <li>Gibson of dnaQ, dam, seqA, C3 backbone</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of dnaQ, dam, seqA, C3 backbone</li> |
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in KRX compis</li> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in KRX compis</li> | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | ||
| Line 275: | Line 275: | ||
and <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">gel extraction</a> | and <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">gel extraction</a> | ||
of pSBIK3 backbone</li> | of pSBIK3 backbone</li> | ||
| − | <li>Gibson assembly of dnaQ, dam, seqA in K3 backbone (ASI)</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of dnaQ, dam, seqA in K3 backbone (ASI)</li> |
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5α</li> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5α</li> | ||
</ul> | </ul> | ||
| Line 286: | Line 286: | ||
<ul> | <ul> | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | ||
| − | of ASI with VR/VF -> plating positive clones out</li> | + | of ASI with VR/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a> -> plating positive clones out</li> |
<li>Sequencing of positive clones</li> | <li>Sequencing of positive clones</li> | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a> | ||
| Line 299: | Line 299: | ||
<li>PCR clean up of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a> | <li>PCR clean up of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a> | ||
</li> | </li> | ||
| − | <li>Gibson assembly of emrR, ugi, cda1 in pSBIK3 backbone (ASII)</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of emrR, ugi, cda1 in pSBIK3 backbone (ASII)</li> |
| − | <li>Gibson assembly of EPPolI/ WTPolI in pSBIK3 backbone</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of EPPolI/ WTPolI in pSBIK3 backbone</li> |
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5α</li> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5α</li> | ||
</ul> | </ul> | ||
| Line 382: | Line 382: | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a> | ||
of PCR products & PCR clean up</li> | of PCR products & PCR clean up</li> | ||
| − | <li>Gibson assembly</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a></li> |
<ul> | <ul> | ||
<li>pSBIC3:AraC-Pbad (non-leaky)</li> | <li>pSBIC3:AraC-Pbad (non-leaky)</li> | ||
| Line 392: | Line 392: | ||
</li> | </li> | ||
<ul> | <ul> | ||
| − | <li>template pSBIC3:AraCPbad-nonleaky, primer VF/CH53, temp = 58 °C</li> | + | <li>template pSBIC3:AraCPbad-nonleaky, primer <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>/CH53, temp = 58 °C</li> |
<li>template pSBIC3:ASI, primer VR/VF_rev, temp = 58 °C </li> | <li>template pSBIC3:ASI, primer VR/VF_rev, temp = 58 °C </li> | ||
</ul> | </ul> | ||
| Line 407: | Line 407: | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a> | ||
</li> | </li> | ||
| − | <li>Gibson assembly</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a></li> |
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in KRX</li> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in KRX</li> | ||
<li>Sequencing of pSBIK3:EPPolI, pSBIK3:WTPolI with CH61,63,64</li> | <li>Sequencing of pSBIK3:EPPolI, pSBIK3:WTPolI with CH61,63,64</li> | ||
| Line 414: | Line 414: | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a> | ||
template ASI, primer dnaQ_Gib_fw/ seqA_Gib_rev, temp = 57.5 °C</li> | template ASI, primer dnaQ_Gib_fw/ seqA_Gib_rev, temp = 57.5 °C</li> | ||
| − | <li>Sequencing of pSBIC3:emrR-1/-2 with VR/VF</li> | + | <li>Sequencing of pSBIC3:emrR-1/-2 with VR/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li> |
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | ||
of pSBIK3:K608010_A/B/C with CH50/VR, temp = 50 °C</li> | of pSBIK3:K608010_A/B/C with CH50/VR, temp = 50 °C</li> | ||
| Line 423: | Line 423: | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of pSBIC3:K516031 in KRX</li> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of pSBIC3:K516031 in KRX</li> | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | ||
| − | of pSBIC3:K608010_StopA/B/C with VR/VF</li> | + | of pSBIC3:K608010_StopA/B/C with VR/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li> |
| − | <li>Sequencing A with VF, B with VR and C with VR</li> | + | <li>Sequencing A with <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>, B with VR and C with VR</li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 480: | Line 480: | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a> | ||
</li> | </li> | ||
| − | <li>Gibson assembly</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a></li> |
<ul> | <ul> | ||
<li>damseqA + C3</li> | <li>damseqA + C3</li> | ||
| Line 492: | Line 492: | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a></li> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a></li> | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | ||
| − | with VR/VF, temp = 57 °C</li> | + | with VR/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>, temp = 57 °C</li> |
<ul> | <ul> | ||
<li>C3:AraC-Pbad(tight)-B0031-WTPolI</li> | <li>C3:AraC-Pbad(tight)-B0031-WTPolI</li> | ||
| Line 528: | Line 528: | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a> | ||
of Stop A, Stop B</li> | of Stop A, Stop B</li> | ||
| − | <li>Gibson assembly stop A + stop B</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> stop A + stop B</li> |
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a> | ||
with EcoR I/Pst I ASI+ASII, GFP_Astop_2, GFP_Astop_2</li> | with EcoR I/Pst I ASI+ASII, GFP_Astop_2, GFP_Astop_2</li> | ||
| Line 558: | Line 558: | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a> | ||
</li> | </li> | ||
| − | <li>Gibson assembly of seqA</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of seqA</li> |
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5α of C3:seqA, K3:StopA+B</li> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> in DH5α of C3:seqA, K3:StopA+B</li> | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a> | ||
| Line 564: | Line 564: | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">DPN1 digestion</a> | ||
</li> | </li> | ||
| − | <li>Gibson assembly of dnaQ_Gen + BB, WTPolI_Gen + BB, EPPol + BB, Mp6_Gen + BB</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of dnaQ_Gen + BB, WTPolI_Gen + BB, EPPol + BB, Mp6_Gen + BB</li> |
| − | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the Gibson assembly in DH5α</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> in DH5α</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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | ||
| − | of dnaQ-Gen, MP6-Gen with VR/VF and CH77/VR</li> | + | of dnaQ-Gen, MP6-Gen with VR/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a> 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><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Q5 PCR</a> | ||
| Line 576: | Line 576: | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a> | ||
of K3:ASII, C3:Terminator with EcoR I, Pst I</li> | of K3:ASII, C3:Terminator with EcoR I, Pst I</li> | ||
| − | <li>Gibson assembly of M6 in RFPGen -> <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a></li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of M6 in RFPGen -> <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a></li> |
<li>Test cultivation of a 96-well plate</li> | <li>Test cultivation of a 96-well plate</li> | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | ||
| Line 584: | Line 584: | ||
of dnaQ-Gen with Xba I/Pst I</li> | of dnaQ-Gen with Xba I/Pst I</li> | ||
<li>araE-PCR with temp 58 °C</li> | <li>araE-PCR with temp 58 °C</li> | ||
| − | <li>Gibson assembly of araE</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> 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> | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gel extraction</a> | ||
| Line 592: | Line 592: | ||
</li> | </li> | ||
<li>PCR clean up</li> | <li>PCR clean up</li> | ||
| − | <li>Gibson of araE-Insert + araE-backbone</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of araE-Insert + araE-backbone</li> |
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">ligation</a> in KRX</li> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">ligation</a> in KRX</li> | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Restriction digestion</a> | ||
| Line 598: | Line 598: | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | ||
of C3:araE-Device</li> | of C3:araE-Device</li> | ||
| − | <li>Gibson C3 backbone + ugi_cda1, C3 backbone + ASII</li> | + | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Gibson assembly</a> of C3 backbone + ugi_cda1, C3 backbone + ASII</li> |
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the Gibson assembly in DH5α</li> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Transformation</a> of the Gibson assembly in DH5α</li> | ||
<li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | <li><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">Colony PCR</a> | ||
| − | the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">transformation</a>, Prha-B0031-dnaQ-Ter with VR/VF</li> | + | the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Protocols">transformation</a>, Prha-B0031-dnaQ-Ter with VR/<a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 653: | Line 653: | ||
</li> | </li> | ||
<ul> | <ul> | ||
| − | <li>template C3:ugi-cda, primer | + | <li>template C3:ugi-cda, primer <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Experiments/Primers">VF</a>/68, temp = 58 °C</li> |
<li>template C3:cda, primer 69/70, temp = 60 °C</li> | <li>template C3:cda, primer 69/70, temp = 60 °C</li> | ||
<li>template A3, primer CK06/05, temp = 59.9 °C</li> | <li>template A3, primer CK06/05, temp = 59.9 °C</li> | ||
Revision as of 18:07, 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 μl culture + 200 μl 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
- Plasmid isolation & restriction digestion 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 digestion 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 digestion 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
- DPN1 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:RFP-cd/ pSBIK3:BFP-cd
- Testing of the fluorescent proteins RFP 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 digestion 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
- DPN1 digestion , Gel extraction
- Restriction digestion of pSBIC3:RFP-cd with Xba I/Spe I, gel extraction of 2 kb band
- Gibson assembly 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
- DPN1 digestion 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
- 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 digestion of positive clones of ASII
- Repeated colony PCR of ASI with primer cPCR_dnaQ_fw/VR -> plating out right clones of pSBIC3:dnaQ, pSBIC3:emrR
- Restriction digestion of right ASII clones with EcoR I/Pst I
- Restriction digestion 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:RFP-cd, primer CH30/31, temp = 67 °C
- template pSBIC3:AraC-Pbad, primer CH54/52, temp = 58.6 °C
- DPN1 digestion
- Restriction digestion with EcoR I/Pst I
- pSBIK3:J04450
- pSBIC3:K608010
- pSBIC3:K584001
- Plasmid isolation of positive emrR clones and positive ASII clone, strong RFP
- Restriction digestion 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 digestion of C3 backbone with EcoR I/Pst I
- Transformation of pSBIC3:K516031 in KRX
- Colony PCR 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 digestion
- 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 digestion
- Ligation
- C3:AraC-Pbad + RFP-Generator
- C3:AraC-Pbad(non-leaky) + RFP-Generator
- K3:Mut-I + Mut II
- C3 + MutII
- Repeat of restriction digestion of ASII: template K3:ASII, EcoR I-Hf/Pst I
- Gel extraction of the restriction digestion
- Ligation
- Transformation of the ligation, pHSG-EPPolI, pHSG-WTPolI in KRX
- Colony PCR 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
- DPN1 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
- Colony PCR 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
- Colony PCR 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
- DPN1 digestion of the Q5 PCR
- Gel extraction of Stop A, Stop B
- Gibson assembly stop A + stop B
- Restriction digestion with EcoR I/Pst I ASI+ASII, GFP_Astop_2, GFP_Astop_2
- Transformation of C3:PRha, C3:PRha-GFP-Generator in KRX
- Transformation of PRha-GFP-Generator 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
- DPN1 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
- DPN1 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
- Colony PCR 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 digestion of K3:ASII, C3:Terminator with EcoR I, Pst I
- Gibson assembly of M6 in RFPGen -> Transformation
- Test cultivation of a 96-well plate
- Colony PCR of M6-Gen -> Plasmid isolation
- Restriction digestion 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
- DPN1 digestion
- PCR clean up
- Gibson assembly of araE-Insert + araE-backbone
- Transformation of the ligation in KRX
- Restriction digestion of C3:M6-Gen with EcoR I/Xba I, weak/RFP Gen with Xba I/Pst I
- Colony PCR of C3:araE-Device
- Gibson assembly of C3 backbone + ugi_cda1, C3 backbone + ASII
- Transformation of the Gibson assembly in DH5α
- Colony PCR the transformation, Prha-B0031-dnaQ-Ter with VR/VF
- Restriction digestion 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
- Colony PCR 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 digestion 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 digestion of E/P of pSB4C5(E/P)
- Q5 PCR
- template C3:ugi-cda, primer VF/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
