Team:UGent/Labjournal
From 2013.igem.org
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<h1> Journal</h1> | <h1> Journal</h1> | ||
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<h2> July </h2> | <h2> July </h2> | ||
</html>{{:Team:UGent/Templates/ToggleBoxStart}}Week 4: july 22 - 28 {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}<html> | </html>{{:Team:UGent/Templates/ToggleBoxStart}}Week 4: july 22 - 28 {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}<html> | ||
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</ul> | </ul> | ||
</html>{{:Team:UGent/Templates/ToggleBoxEnd}}<html> | </html>{{:Team:UGent/Templates/ToggleBoxEnd}}<html> | ||
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<h2> August </h2> | <h2> August </h2> | ||
</html>{{:Team:UGent/Templates/ToggleBoxStart}}Week 1: july 29 - august 4 {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}{{:Team:UGent/Templates/ToggleBoxEnd}}{{:Team:UGent/Templates/ToggleBoxStart}}Week 2: august 5 - 11 {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}<html> | </html>{{:Team:UGent/Templates/ToggleBoxStart}}Week 1: july 29 - august 4 {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}{{:Team:UGent/Templates/ToggleBoxEnd}}{{:Team:UGent/Templates/ToggleBoxStart}}Week 2: august 5 - 11 {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}<html> | ||
<ul> | <ul> | ||
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<li> Experiment 1 </li> | <li> Experiment 1 </li> | ||
<ul class="a"> | <ul class="a"> | ||
- | <li> <b>Inoculate</b> <i> E. coli </i>DH5a + pTGD-ccdA-Pmb1GFP-CmFRT </li> | + | <li> <b>Inoculate</b> <i> E. coli </i>DH5a + pTGD-<i>ccdA</i>-Pmb1GFP-CmFRT </li> |
- | <li> <b>Purify plasmid pTGD-ccdA-Pmb1GFP-CmFRT</b> using Qiagen spin mini kit: Nanodrop -- <b>362.4 ng/µL</b> </li> | + | <li> <b>Purify plasmid pTGD-<i>ccdA</i>-Pmb1GFP-CmFRT</b> using Qiagen spin mini kit: Nanodrop -- <b>362.4 ng/µL</b> </li> |
<li> <b>Preparative Restriction Digest (RD)</b> of purified plasmid: BspHI & BstAPI: expected fragments of 5541bp and 903 bp </li> | <li> <b>Preparative Restriction Digest (RD)</b> of purified plasmid: BspHI & BstAPI: expected fragments of 5541bp and 903 bp </li> | ||
<li> <b>Gel purify RD-fragment of 5541 bp</b> using Qiagen Qiaquick gel extraction kit: Nanodrop -- <b>70.4 ng/µL</b></li> | <li> <b>Gel purify RD-fragment of 5541 bp</b> using Qiagen Qiaquick gel extraction kit: Nanodrop -- <b>70.4 ng/µL</b></li> | ||
- | <li> <b>PCR on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT with MDM588 & MDM589</b> | + | <li> <b>PCR on RD (HR-<i>ccdA</i>-Pmb1GFP-HR)and on plasmid pTGD-<i>ccdA</i>-PMbAFGP-CmFRT with MDM588 & MDM589</b> |
<BR> ->HiFi PCR using Primestar polymerase. Analytical gel: <b>negative </b> | <BR> ->HiFi PCR using Primestar polymerase. Analytical gel: <b>negative </b> | ||
<BR> ->PCR using Q5 polymerase. Analytical gel: <b> negative </b> | <BR> ->PCR using Q5 polymerase. Analytical gel: <b> negative </b> | ||
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<BR> ->Touchdown PCR using Q5 polymerase. Analytical gel: <b> negative </b> </li> | <BR> ->Touchdown PCR using Q5 polymerase. Analytical gel: <b> negative </b> </li> | ||
- | <li> <b>Control plasmid pTGD-ccdA-PMbAFGP-CmFRT: RD with AclI.</b> Expected fragments: 758 bp, 1730 bp and 3956 bp. Analytical gel: <b> negative </b> --> <u> Problem with plasmid pTGD-ccdA-PMbAFGP-CmFRT </u></li> | + | <li> <b>Control plasmid pTGD-<i>ccdA</i>-PMbAFGP-CmFRT: RD with AclI.</b> Expected fragments: 758 bp, 1730 bp and 3956 bp. Analytical gel: <b> negative </b> --> <u> Problem with plasmid pTGD-<i>ccdA</i>-PMbAFGP-CmFRT </u></li> |
- | <li> <b> Transformation</b>: Knock in with <u> | + | <li> <b> Transformation</b>: Knock in with <u>linear back-up</u> DNA by electroporation. Incubation of the transformed cells and transfer the culture on a Cm plate. </li> |
<li> Colony PCR on 48 colonies using crimson taq polymerase with two primer pairs: MDM0141/MDM0010 and MDM0046/MDM123. Expected fragments: 550bp & 2450 bp. Analytic gel: <b> 4 positives </b> | <li> Colony PCR on 48 colonies using crimson taq polymerase with two primer pairs: MDM0141/MDM0010 and MDM0046/MDM123. Expected fragments: 550bp & 2450 bp. Analytic gel: <b> 4 positives </b> | ||
<img src="https://static.igem.org/mediawiki/2013/0/02/UGent_2013_CPCR_KI.jpg" width="300"/></a></li> | <img src="https://static.igem.org/mediawiki/2013/0/02/UGent_2013_CPCR_KI.jpg" width="300"/></a></li> | ||
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<li> Experiment 2 </li> | <li> Experiment 2 </li> | ||
<ul class="a"> | <ul class="a"> | ||
- | <li> Inoculate <i>E. coli</i> DH5a + p5SpFRT-T7<i>ccdB</i> <BR> Inoculate <i>E. coli</i> DH5a + p10SpFRT-T7<i>ccdB</i> <BR> Inoculate <i>E. coli</i> DH5a + p20SpFRT-T7<i>ccdB</i> </li> | + | <li> Inoculate <i>E. coli</i> DH5a; + p5SpFRT-T7<i>ccdB</i> <BR> Inoculate <i>E. coli</i> DH5a; + p10SpFRT-T7<i>ccdB</i> <BR> Inoculate <i>E. coli</i> DH5a; + p20SpFRT-T7<i>ccdB</i> </li> |
<li> Purify plasmids using Qiagen spin mini kit: nanodrop <BR> p5SpFRT-T7<i>ccdB</i>: 119.6 ng/µl <BR> p10SpFRT-T7<i>ccdB</i>: 156.3 ng/µl <BR> p20SpFRT-T7<i>ccdB</i>: 392.9 ng/µl </li> | <li> Purify plasmids using Qiagen spin mini kit: nanodrop <BR> p5SpFRT-T7<i>ccdB</i>: 119.6 ng/µl <BR> p10SpFRT-T7<i>ccdB</i>: 156.3 ng/µl <BR> p20SpFRT-T7<i>ccdB</i>: 392.9 ng/µl </li> | ||
<li> <b>CcdB operon</b>: | <li> <b>CcdB operon</b>: | ||
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<BR> -> Plate pSB4A5 and pSB6A1 on ampicillin plate and pSB3T5 on tetracyclin | <BR> -> Plate pSB4A5 and pSB6A1 on ampicillin plate and pSB3T5 on tetracyclin | ||
and grow overnight at 37°C | and grow overnight at 37°C | ||
- | <BR> -> Plates with transformants: pSB6A1 lots of colonies, pSB3T5 sufficient colonies and pSB4A5 no colonies </li> | + | <BR> -> Plates with transformants: pSB6A1 lots of colonies, pSB3T5 sufficient colonies and pSB4A5 no colonies |
+ | </li> | ||
<li> <B>Vectors pSB4A5 </B>(plate 5, well 5I): | <li> <B>Vectors pSB4A5 </B>(plate 5, well 5I): | ||
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</li> | </li> | ||
<li> Inoculate pSB3T5 and pSB6A1 again and in warm chamber (37°C). </li> | <li> Inoculate pSB3T5 and pSB6A1 again and in warm chamber (37°C). </li> | ||
- | <li> Inoculation colonies of pSB4A5 (plate 2, well 2J) and pSB6A1 (plate 2, well 2L) (2 times: one for further work and one for cryovials) | + | <li> Inoculation colonies of pSB4A5 (plate 2, well 2J) and pSB6A1 (plate 2, well 2L) (2 times: one for further work and one for cryovials) |
<li> Purification of plasmids pSB3T5, pSB6A1 and pSB4A5 </li> | <li> Purification of plasmids pSB3T5, pSB6A1 and pSB4A5 </li> | ||
<li> Generation of <b>restriction</b> digest fragments of T7-<i>ccdB</i> insert (from p5SpFRT-T7<i>ccdB</i> and p20SpFRT-T7<i>ccdB</i>) and vector (pSB3T5 (2x), pSB4A5 (from plate 2, well 2J) and pSB6A1 (1x from plate 5, well 1K and 1x from plate 2, well 2L) with XbaI and PstI-HF (gel purify RD-fragments using Qiagen Qiaquick gel extraction kit (expected fragment of 2157 bp for T7-<i>ccdB</i>, 3229 bp for pSB3T5, 3372 bp for pSB4A5 and 3999 bp for pSB6A1): Nanodrop | <li> Generation of <b>restriction</b> digest fragments of T7-<i>ccdB</i> insert (from p5SpFRT-T7<i>ccdB</i> and p20SpFRT-T7<i>ccdB</i>) and vector (pSB3T5 (2x), pSB4A5 (from plate 2, well 2J) and pSB6A1 (1x from plate 5, well 1K and 1x from plate 2, well 2L) with XbaI and PstI-HF (gel purify RD-fragments using Qiagen Qiaquick gel extraction kit (expected fragment of 2157 bp for T7-<i>ccdB</i>, 3229 bp for pSB3T5, 3372 bp for pSB4A5 and 3999 bp for pSB6A1): Nanodrop | ||
<br>p5: 43.7 ng/µl | <br>p5: 43.7 ng/µl | ||
<br>p20: 24.2 ng/µl | <br>p20: 24.2 ng/µl | ||
- | <br> | + | <br>pSB3T5 (inoculation 1): 23.5 ng/µl |
- | <br> | + | <br>pSB3T5 (inoculation 2): 23 ng/µl |
- | <br> | + | <br>pSB4A5: 40.5 ng/µl |
- | <br> | + | <br>pSB6A1 (plate 2, 2L): 40.1 ng/µl |
- | <br> | + | <br>pSB6A1 (plate 5, 1K): 30.4 ng/µl </li> |
- | <li> <b>Ligation</b> of T7<i>ccdB</i> from p5SpFRT-T7<i>ccdB</i> and pSB6A1 (1x from plate 5, well 1K and 1x from plate 2, well 2L), p20SpFRT-T7<i>ccdB</i> and pSB3T5, p20SpFRT-T7<i>ccdB</i> and pSB4A5 (from plate 2, well 2J) (3(insert)/1(plasmid) ratio) </li> | + | <li> <b>Ligation</b> of T7-<i>ccdB</i> from p5SpFRT-T7<i>ccdB</i> and pSB6A1 (1x from plate 5, well 1K and 1x from plate 2, well 2L), p20SpFRT-T7<i>ccdB</i> and pSB3T5, p20SpFRT-T7<i>ccdB</i> and pSB4A5 (from plate 2, well 2J) (3(insert)/1(plasmid) ratio) </li> |
- | <li> <b>Transformation</b> in <i>E.coli</i> Top10 (heat shock)</li> | + | <li> <b>Transformation</b> in <i>E. coli</i> Top10 (heat shock)</li> |
<li> Plate transformation on ampicillin (pSB4A5 and pSB6A1) and tetracycline plate (pSB3T5) and grow overnight at 37°C (2 plates: 10<sup>-2</sup>, 10<sup>-1</sup>) | <li> Plate transformation on ampicillin (pSB4A5 and pSB6A1) and tetracycline plate (pSB3T5) and grow overnight at 37°C (2 plates: 10<sup>-2</sup>, 10<sup>-1</sup>) | ||
<br> -> Plates with transformants: no colonies</li> | <br> -> Plates with transformants: no colonies</li> | ||
- | <li> New <b>transformation</b> in <i>E.coli</i> Top10 from pSB4A5 and pSB6A1 using heat shock | + | <li> New <b>transformation</b> in <i>E. coli</i> Top10 from pSB4A5 and pSB6A1 using heat shock |
<br> -> Plates with transformants: pSB6A1 2 colonies, pSB4A5 few colonies </li> | <br> -> Plates with transformants: pSB6A1 2 colonies, pSB4A5 few colonies </li> | ||
<li> New <b>ligation</b> with RD-fragments created before (vectors pSB3T5, pSB4A5, pSB6A1 (plate 5, well 1K) and insert T7-<i>ccdB</i> created in experiment 6), at 22.5°C o/n) </li> | <li> New <b>ligation</b> with RD-fragments created before (vectors pSB3T5, pSB4A5, pSB6A1 (plate 5, well 1K) and insert T7-<i>ccdB</i> created in experiment 6), at 22.5°C o/n) </li> | ||
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<br> -> Purify plasmids using Qiagen Qiaprep Spin minikit: nanodrop: 200,2 ng/µl</li> | <br> -> Purify plasmids using Qiagen Qiaprep Spin minikit: nanodrop: 200,2 ng/µl</li> | ||
<li> <b>Restriction</b> of CcdB operon and pSB1C3 with XbaI and PstI | <li> <b>Restriction</b> of CcdB operon and pSB1C3 with XbaI and PstI | ||
+ | <br> Preparative gel: <img src="https://static.igem.org/mediawiki/2013/9/90/Preparatieve_gel_week2_aug.jpg" width="100"/></a> | ||
+ | <br> restriction digest of T7-<i>ccdB</i> (expected to be 2044bp) and restriction digest of backbone of pSB1C3 (expected length is 2232 bp) | ||
<br> -> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | <br> -> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | ||
<br> Restriction digest of T7-<i>ccdB</i>: 16.9 ng/µl | <br> Restriction digest of T7-<i>ccdB</i>: 16.9 ng/µl | ||
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</ul> | </ul> | ||
</ul> | </ul> | ||
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</html>{{:Team:UGent/Templates/ToggleBoxEnd}}{{:Team:UGent/Templates/ToggleBoxStart}}Week 3: august 12 - 18 {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}<html> | </html>{{:Team:UGent/Templates/ToggleBoxEnd}}{{:Team:UGent/Templates/ToggleBoxStart}}Week 3: august 12 - 18 {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}<html> | ||
<ul> | <ul> | ||
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<br>p20: 21.1 ng/µl</li> | <br>p20: 21.1 ng/µl</li> | ||
<li><b>cPCR</b> with Taq polymerase of colonies pSB6A1 (primers: MDM0096 and CLG0019) and pSB4A5 (primers: MDM0095 and CLG0019): <b>negative</b> | <li><b>cPCR</b> with Taq polymerase of colonies pSB6A1 (primers: MDM0096 and CLG0019) and pSB4A5 (primers: MDM0095 and CLG0019): <b>negative</b> | ||
- | <li> New <b>transformation</b> with pSB3T5, pSB4A5 and pSB6A1 using heat shock in <i>E.coli</i> <b>DH5a</b> | + | <li> New <b>transformation</b> with pSB3T5, pSB4A5 and pSB6A1 using heat shock in <i>E. coli</i> <b>DH5a</b> |
<br> -> Plate on normal plates and glucose plates (100 ml 2% glucose, to avoid leaky expressing of <i>ccdB</i>) | <br> -> Plate on normal plates and glucose plates (100 ml 2% glucose, to avoid leaky expressing of <i>ccdB</i>) | ||
<br> -> Plates with transformants: pSB6A1 some colonies, pSB4A5 some colonies and pSB3T5 no colonies </li> | <br> -> Plates with transformants: pSB6A1 some colonies, pSB4A5 some colonies and pSB3T5 no colonies </li> | ||
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<li>New <b>ligation</b> with rest of RD-fragments of plasmids pSB3T5 (2 times) and pSB4A5 and CcdB from experiment 6 and <b>transformation</b> in DH5a using heat shock</li> | <li>New <b>ligation</b> with rest of RD-fragments of plasmids pSB3T5 (2 times) and pSB4A5 and CcdB from experiment 6 and <b>transformation</b> in DH5a using heat shock</li> | ||
<li>Due to lack of success: <b>start from the beginning</b>.</li> | <li>Due to lack of success: <b>start from the beginning</b>.</li> | ||
- | <li><b>Inoculation</b> of pSB3T5, pSB4A5, p5SpFRT-T7<i>ccdB</i>, p10SpFRT-T7<i>ccdB</i> and p10SpFRT-T7<i>ccdB</i> from cryovials. </li> <li>Also resuspend pSB3T5 (plate 5, well 7C) from iGEM kit, <b>transform</b> in <i>E. coli</i> DH5a using heat shock, plate on tetracycline plates and incubate overnight at 37°C | + | <li><b>Inoculation</b> of pSB3T5, pSB4A5, p5SpFRT-T7<i>ccdB</i>, p10SpFRT-T7<i>ccdB</i> and p10SpFRT-T7<i>ccdB</i> from cryovials. |
+ | <br> <img src="https://static.igem.org/mediawiki/2013/e/ee/UGent_2013_plasmids_for_purification.png" width="200"/></a></li> | ||
+ | <li>Also resuspend pSB3T5 (plate 5, well 7C) from iGEM kit, <b>transform</b> in <i>E. coli</i> DH5a using heat shock, plate on tetracycline plates and incubate overnight at 37°C | ||
<br> -> no colonies | <br> -> no colonies | ||
<br> -> transform again in DH5a using heat shock | <br> -> transform again in DH5a using heat shock | ||
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<br>pSB4A5 (1): +/- 400 ng/µl | <br>pSB4A5 (1): +/- 400 ng/µl | ||
<br>pSB4A5 (2): +/- 350 ng/µl </li> | <br>pSB4A5 (2): +/- 350 ng/µl </li> | ||
- | <li>PCR on <b>CcdB operon</b> using Q5 polymerase (primers: MDM0586_Fw-Trc-<i>ccdB</i>-G00000 and MDM0587_Rv-Trc-ccdB-G00001) </li> | + | <li>PCR on <b>CcdB operon</b> using Q5 polymerase (primers: MDM0586_Fw-Trc-<i>ccdB</i>-G00000 and MDM0587_Rv-Trc-<i>ccdB</i>-G00001) </li> |
<li><b>cPCR</b> on pSB3T5 colonies from old plates using Taq polymerase (primers: MDM0602 and CLG0019, expected fragment of 503 bp): <b>negative</b></li> | <li><b>cPCR</b> on pSB3T5 colonies from old plates using Taq polymerase (primers: MDM0602 and CLG0019, expected fragment of 503 bp): <b>negative</b></li> | ||
<li><b>Restriction</b> of ccdB operon, pSB3T5 and pSB4A5 using PstI-HF and XbaI (gel purify RD-fragments using Qiagen Qiaquick gel extraction kit (expected fragment of 2157 bp for T7-<i>ccdB</i>, 3229 bp for pSB3T5 and 3372 bp for pSB4A5): concentrations deduced from gel</li> | <li><b>Restriction</b> of ccdB operon, pSB3T5 and pSB4A5 using PstI-HF and XbaI (gel purify RD-fragments using Qiagen Qiaquick gel extraction kit (expected fragment of 2157 bp for T7-<i>ccdB</i>, 3229 bp for pSB3T5 and 3372 bp for pSB4A5): concentrations deduced from gel</li> | ||
- | <li><b>Ligation</b> of pSB3T5 and T7<i>ccdB</i> (from p5SpFRT-T7<i>ccdB</i>), pSB4A5 and T7<i>ccdB</i> (from p5SpFRT-T7<i>ccdB</i>) and pSB4A5 and T7<i>ccdB</i> (from p20SpFRT-T7<i>ccdB</i>) with T4 DNA ligase at 16°C overnight</li> | + | <li><b>Ligation</b> of pSB3T5 and T7-<i>ccdB</i> (from p5SpFRT-T7<i>ccdB</i>), pSB4A5 and T7-<i>ccdB</i> (from p5SpFRT-T7<i>ccdB</i>) and pSB4A5 and T7-<i>ccdB</i> (from p20SpFRT-T7<i>ccdB</i>) with T4 DNA ligase at 16°C overnight</li> |
<li><b>Transformation</b> of ligation mixtures in DH5a using heat shock | <li><b>Transformation</b> of ligation mixtures in DH5a using heat shock | ||
<br>-> no colonies</li> | <br>-> no colonies</li> | ||
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<ul class="a"> | <ul class="a"> | ||
<li> Inoculation KI-strain 5 & 8 from experiment 1 </li> | <li> Inoculation KI-strain 5 & 8 from experiment 1 </li> | ||
- | <li> <b> Transformation of pSB6A1 - | + | <li> <b> Transformation of pSB6A1 - T7-<i>ccdB</i> </b> in KI-strains 5 and 8 </li> |
<li> <b> Colony PCR </b>on kolonies from transformed strains using emerald polymerase with out primers to check KI: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: <b> positive </b> </li> | <li> <b> Colony PCR </b>on kolonies from transformed strains using emerald polymerase with out primers to check KI: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: <b> positive </b> </li> | ||
<li> <b> Colony PCR </b>on kolonies from transformed strains using emerald polymerase with MDM0096 & CLG0019 to check if plasmid is present. Expected fragment: 437 bp. Analytic gel: <b> positive </b><br> | <li> <b> Colony PCR </b>on kolonies from transformed strains using emerald polymerase with MDM0096 & CLG0019 to check if plasmid is present. Expected fragment: 437 bp. Analytic gel: <b> positive </b><br> | ||
<img src="https://static.igem.org/mediawiki/2013/b/b7/UGent_2013_CPCR_KI%2Bplasmid.png" width="150"></li> | <img src="https://static.igem.org/mediawiki/2013/b/b7/UGent_2013_CPCR_KI%2Bplasmid.png" width="150"></li> | ||
</ul> | </ul> | ||
- | + | <br> | |
<li> Experiment 6 </li> | <li> Experiment 6 </li> | ||
<ul class="a"> | <ul class="a"> | ||
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<br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>negative</b> | <br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>negative</b> | ||
<li> <b>CcdB operon</b>: HiFi PCR of plasmids p20SpFRT-T7<i>ccdB</i> with MDM0586/MDM0587 to amplify CcdB operon</li> | <li> <b>CcdB operon</b>: HiFi PCR of plasmids p20SpFRT-T7<i>ccdB</i> with MDM0586/MDM0587 to amplify CcdB operon</li> | ||
- | <li> New <b>restriction</b> of CcdB operon and pSB1C3 with | + | <li> New <b>restriction</b> of CcdB operon and pSB1C3 with Xba |
+ | I and PstI | ||
<br> -> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | <br> -> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | ||
<br> Restriction Digest of T7-<i>ccdB</i>: 16.6 ng/µl | <br> Restriction Digest of T7-<i>ccdB</i>: 16.6 ng/µl | ||
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</html>{{:Team:UGent/Templates/ToggleBoxEnd}}{{:Team:UGent/Templates/ToggleBoxStart}}Week 4: august 19 - 25 {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}<html> | </html>{{:Team:UGent/Templates/ToggleBoxEnd}}{{:Team:UGent/Templates/ToggleBoxStart}}Week 4: august 19 - 25 {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}<html> | ||
<ul> | <ul> | ||
- | |||
<li> Experiment 2 </li> | <li> Experiment 2 </li> | ||
<ul class="a"> | <ul class="a"> | ||
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<li> <b> Colony PCR with higher annealing temperature to minimize false priming</b> on kolonies from transformed strains using emerald polymerase with out primers to check KI: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: <b> positive </b> </li> | <li> <b> Colony PCR with higher annealing temperature to minimize false priming</b> on kolonies from transformed strains using emerald polymerase with out primers to check KI: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: <b> positive </b> </li> | ||
<li> <b> HiFi PCR using Q5 polymerase with two primer pairs generating overlapping fragments of the ccdA.gfp.cat construct </b>: MDM0046 (out) & MDM0123: 2130 bp and MEMO1237 & MDM0010 (out): 3400 bp. Analytic gel: <b>negative</b> </li> | <li> <b> HiFi PCR using Q5 polymerase with two primer pairs generating overlapping fragments of the ccdA.gfp.cat construct </b>: MDM0046 (out) & MDM0123: 2130 bp and MEMO1237 & MDM0010 (out): 3400 bp. Analytic gel: <b>negative</b> </li> | ||
- | |||
</ul> | </ul> | ||
<br> | <br> | ||
Line 245: | Line 251: | ||
-> Add phage lysate (10,20,40 & 80 µL)<br> | -> Add phage lysate (10,20,40 & 80 µL)<br> | ||
-> Wait untill lysis visible </li> | -> Wait untill lysis visible </li> | ||
- | <li> <b> Perform CIChE with 8 | + | <li> <b> Perform CIChE with 8 + pSB6A1 & 5 + pSB6A1 </b> (we will do 2 strains parallel): 0,0mM - 0,05mM IPTG </li> |
<li> <b> Transduction</b> of CIChe strains: no positive colonies after UV-test </li> | <li> <b> Transduction</b> of CIChe strains: no positive colonies after UV-test </li> | ||
</ul> | </ul> | ||
Line 261: | Line 267: | ||
<br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>negative</b> </li> | <br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>negative</b> </li> | ||
<li> <b>CcdB operon</b>: HiFi PCR of plasmids p20SpFRT-T7<i>ccdB</i> with MDM0586/MDM0587 to amplify CcdB operon | <li> <b>CcdB operon</b>: HiFi PCR of plasmids p20SpFRT-T7<i>ccdB</i> with MDM0586/MDM0587 to amplify CcdB operon | ||
- | <br> because we have no more T7<i>ccdB</i> fragments</li> | + | <br> because we have no more T7-<i>ccdB</i> fragments</li> |
<li> New <b>restriction</b> of CcdB operon and pSB1C3 with XbaI and PstI | <li> New <b>restriction</b> of CcdB operon and pSB1C3 with XbaI and PstI | ||
<br> -> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | <br> -> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | ||
Line 271: | Line 277: | ||
<li> <b>Colony PCR</b> (Taq): <b>negative</b> | <li> <b>Colony PCR</b> (Taq): <b>negative</b> | ||
<br> It seems that restriction-ligation does not work, thus we will use another technique, called Gibson Assembly </li> | <br> It seems that restriction-ligation does not work, thus we will use another technique, called Gibson Assembly </li> | ||
- | <li> Designing primers for Gibson Assembly for the insert T7<i>ccdB</i> and the backbone pSB1C3 </li> | + | <li> Designing primers for Gibson Assembly for the insert T7-<i>ccdB</i> and the backbone pSB1C3 </li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
Line 284: | Line 290: | ||
<br>p10pFRT-T7<i>ccdB</i>: 62.8 ng/µl | <br>p10pFRT-T7<i>ccdB</i>: 62.8 ng/µl | ||
<br>p20pFRT-T7<i>ccdB</i>: 286.1 ng/µl</li> | <br>p20pFRT-T7<i>ccdB</i>: 286.1 ng/µl</li> | ||
- | < | + | <li><b>PCR on CcdB operon</b> using Q5 polymerase (primers: MDM0586_Fw-Trc-<i>ccdB</i>-G00000 and MDM0587_Rv-Trc-<i>ccdB</i>-G00001): nanodrop |
<br>p10: 29.7 ng/µl | <br>p10: 29.7 ng/µl | ||
<br>p20: 31.4 ng/µl</li> | <br>p20: 31.4 ng/µl</li> | ||
Line 311: | Line 317: | ||
<li> Experiment 3 </li> | <li> Experiment 3 </li> | ||
<ul class="a"> | <ul class="a"> | ||
- | <li> <b> | + | <li> <b> Transformation </b> of plasmids:p5SpFRT-T7<i>ccdB</i>, p10SpFRT-T7<i>ccdB</i>, p20SpFRT-T7<i>ccdB</i> in KI-strain (8) and back-up strain 5 </u> </li> |
- | < | + | <li><b>Colony PCR</b> on 5 + pSB6A1/p5/p10/p20 using emerald polymerase with primerpairs MDM0046/MDM010 to check KI. Expected fragment: 5500bp. Analytic gel: <b> positive </b> <br><img src="https://static.igem.org/mediawiki/2013/8/88/UGent_2013_CPCR_MDMcm.jpg" width="150"></li> |
- | <li><b>Colony PCR</b> on | + | <li><b>Colony PCR</b> on 5 + pSB6A1/p5/p10/p20 using emerald polymerase with primerpairs MDM0096/CGL0019 for pSB6A1 and MDM0039/MDM0060 for p5/p10/p20. Expected fragment: 437bp & 910 bp. Analytic gel: <b> positive for p5/p10/p20</b> </li> |
- | <li><b>Colony PCR</b> on | + | <li><b> New transformation </b> 5 +pSB6A1 and check KI + plasmid as described above. Analytic gel: <b> positive </b>.</li> |
- | <li><b> New transformation </b> | + | |
</ul> | </ul> | ||
<br> | <br> | ||
Line 321: | Line 326: | ||
<li> Experiment 4 </li> | <li> Experiment 4 </li> | ||
<ul class="a"> | <ul class="a"> | ||
- | <li> <b> Abort CIChe with 8 | + | <li> <b> Abort CIChe with 8+pSB6A1 & 5+pSB6A1 </b> because of mutation in pSB6A1 </li> |
<li> Lysate preparation </li> | <li> Lysate preparation </li> | ||
- | + | <li> <b> Transduction: </b> on CIChE strains described above </li> | |
- | <li> <b> Transduction: </b> on CIChE strains described above | + | <li> Test lysate by performing transduction on strain 5 with different concentrations of lysate: <b>negative</b> </li> |
- | + | ||
- | <li> Test lysate by performing transduction on | + | |
- | + | ||
</ul> | </ul> | ||
<br> | <br> | ||
Line 334: | Line 336: | ||
<li> Experiment 6 </li> | <li> Experiment 6 </li> | ||
<ul class="a"> | <ul class="a"> | ||
- | <b | + | <li><b>While waiting for the Gibson Assembly primers </b></li> |
+ | <ul class="a"> | ||
<li> Again <b>restriction</b> of CcdB operon and pSB1C3 with XbaI and PstI | <li> Again <b>restriction</b> of CcdB operon and pSB1C3 with XbaI and PstI | ||
<br> -> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | <br> -> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | ||
Line 343: | Line 346: | ||
<br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>positive</b> </li> | <br> -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>positive</b> </li> | ||
<li> <b>Colony PCR</b> (Taq) of these colonies: <b>negative</b> | <li> <b>Colony PCR</b> (Taq) of these colonies: <b>negative</b> | ||
- | < | + | </ul> |
- | < | + | <li> <b>After receiving the primers for Gibson Assembly</b> |
- | <b | + | <ul class="a"> |
<li> <b>Q5 PCR</b> of CcdB operon and pSB1C3, using the designed Gibson primers | <li> <b>Q5 PCR</b> of CcdB operon and pSB1C3, using the designed Gibson primers | ||
<br> -> Purification of PCR-product, after DpnI treatment, by using minElute reaction cleanup kit | <br> -> Purification of PCR-product, after DpnI treatment, by using minElute reaction cleanup kit | ||
Line 360: | Line 363: | ||
<br> Strange phenomena: colonies are red, let’s control it </li> | <br> Strange phenomena: colonies are red, let’s control it </li> | ||
<li> <b>Colony PCR</b> (Taq) of colonies derived from Gibson Assembly: <b>negative</b> | <li> <b>Colony PCR</b> (Taq) of colonies derived from Gibson Assembly: <b>negative</b> | ||
- | <br> We think that some original plasmid has to be present | + | <br> We think that some of the original plasmid has to be present |
- | <br> and the bacteria love that plasmid more than the plasmid with <i>ccdB</i></li> | + | <br> and the bacteria love that plasmid more than the plasmid with T7-<i>ccdB</i></li> |
- | + | </ul> | |
Line 375: | Line 378: | ||
<li> <b>Restriction</b> of CcdB operon and pSB3T5 with EcoRI and SpeI | <li> <b>Restriction</b> of CcdB operon and pSB3T5 with EcoRI and SpeI | ||
<br>-> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | <br>-> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop: | ||
- | <br>Restriction Digest of T7-ccdB: 14.4 ng/µl | + | <br>Restriction Digest of T7-<i>ccdB</i>: 14.4 ng/µl |
<br>Restriction Digest of pSB3T5 : 7.2 ng/µl | <br>Restriction Digest of pSB3T5 : 7.2 ng/µl | ||
<li> <b>Restriction</b> of CcdB operon and pSB4A5 with EcoRI and SpeI | <li> <b>Restriction</b> of CcdB operon and pSB4A5 with EcoRI and SpeI | ||
Line 391: | Line 394: | ||
<li> <b>Transformation</b> in <i>E. coli</i> Top10 subcloning cells, using electroporation (Time Constant: 4.4) | <li> <b>Transformation</b> in <i>E. coli</i> Top10 subcloning cells, using electroporation (Time Constant: 4.4) | ||
<br>-> Plate on ampicillin plate and grow overnight at 37°C:<b>negative</b> </li> | <br>-> Plate on ampicillin plate and grow overnight at 37°C:<b>negative</b> </li> | ||
- | |||
- | |||
</ul> | </ul> | ||
<br> | <br> | ||
+ | |||
<li> Experiment 4 </li> | <li> Experiment 4 </li> | ||
<ul class="a"> | <ul class="a"> | ||
Line 409: | Line 411: | ||
<br> -> Wild type | <br> -> Wild type | ||
<br> -> DH5a + pTGD-ccd-Pmb1GFP-CmFRT | <br> -> DH5a + pTGD-ccd-Pmb1GFP-CmFRT | ||
- | <br> -> Strains with GFP: 8/ | + | <br> -> Strains with GFP: 8 / 5 + pSB6A1/p5/p10/p20 -- 0mM & 0.01 mM IPTG </li> |
<li> Test evaluation GFP with EZrich-medium: | <li> Test evaluation GFP with EZrich-medium: | ||
<br> -> Wild type | <br> -> Wild type | ||
<br> -> DH5a + pTGD-ccd-Pmb1GFP-CmFRT | <br> -> DH5a + pTGD-ccd-Pmb1GFP-CmFRT | ||
- | <br> -> Strains with GFP: 8/ | + | <br> -> Strains with GFP: 8 / 5 + pSB6A1/p5/p10/p20 -- 0mM/0.01mM/0.05mM & 0.2mMM IPTG </li> |
</ul> | </ul> | ||
<br> | <br> | ||
Line 451: | Line 453: | ||
-> Plate on chloramphenicol plate and grow overnight at 37°C: <b>positive</b> => many colonies</li> | -> Plate on chloramphenicol plate and grow overnight at 37°C: <b>positive</b> => many colonies</li> | ||
<li> <b>Colony PCR</b> (Crimson): | <li> <b>Colony PCR</b> (Crimson): | ||
- | + | <br> We tested 48 colonies (using primers MDM0606 and MDM0607) and 4 colonies show the right fragment (2520 bp) | |
+ | <br> The first 24 colonies <img src=" https://static.igem.org/mediawiki/2013/1/13/UGent_2013_pSB1C3_cPCR_w1_septI.png | ||
+ | " width="200"/></a>, and the second 24 colonies <img src=" https://static.igem.org/mediawiki/2013/0/0d/UGent_2013_pSB1C3_cPCR_w1_septII.png | ||
+ | " width="200"/></a> | ||
<br>-> Inoculate these 4 good colonies</li> | <br>-> Inoculate these 4 good colonies</li> | ||
<li> Plasmid purification of the 4 colonies, using Qiagen Qiaprep Spin minikit: nanodrop: | <li> Plasmid purification of the 4 colonies, using Qiagen Qiaprep Spin minikit: nanodrop: | ||
Line 463: | Line 468: | ||
<li> pSB1C3-T7<i>ccdB</i> of these 3 colonies sent for <b>sequencing</b> with primers MDM0606, MDM0607, MDM0060 and MDM0039 | <li> pSB1C3-T7<i>ccdB</i> of these 3 colonies sent for <b>sequencing</b> with primers MDM0606, MDM0607, MDM0060 and MDM0039 | ||
<br>-> sequence shows a mutation in the stop-codon, so we have to start over again</li> | <br>-> sequence shows a mutation in the stop-codon, so we have to start over again</li> | ||
+ | |||
+ | |||
+ | |||
</ul> | </ul> | ||
Line 471: | Line 479: | ||
<li> Experiment 2 </li> | <li> Experiment 2 </li> | ||
<ul class="a"> | <ul class="a"> | ||
- | <li> <b>Q5 PCR</b> for amplification of T7<i>ccdB</i>-fragment, using the designed Gibson primers, on p5SpFRT-T7<i>ccdB</i>, p10SpFRT-T7<i>ccdB</i> and p20SpFRT-T7<i>ccdB</i> | + | <li> <b>Q5 PCR</b> for amplification of T7-<i>ccdB</i>-fragment, using the designed Gibson primers, on p5SpFRT-T7<i>ccdB</i>, p10SpFRT-T7<i>ccdB</i> and p20SpFRT-T7<i>ccdB</i> |
<br> -> Using DpnI at 37°C for 1 hour | <br> -> Using DpnI at 37°C for 1 hour | ||
<br> -> Purification of PCR-product by using minElute reaction cleanup kit | <br> -> Purification of PCR-product by using minElute reaction cleanup kit | ||
<br> -> Control on gel: <b>negative</b> | <br> -> Control on gel: <b>negative</b> | ||
+ | |||
+ | <li> Plasmid pSB3T5</li> | ||
+ | <ul class="a"> | ||
<li> <b>Gibson Assembly</b> (1h at 50°C) of pSB3T5 and CcdB operon that we already amplified before</li> | <li> <b>Gibson Assembly</b> (1h at 50°C) of pSB3T5 and CcdB operon that we already amplified before</li> | ||
<li> <b>Transformation</b> in <i>E. coli</i> Top10 subcloning cells, using electroporation (Time Constant: 3.0) | <li> <b>Transformation</b> in <i>E. coli</i> Top10 subcloning cells, using electroporation (Time Constant: 3.0) | ||
<br>-> Plate on tetracyclin plate and grow overnight at 37°C: <b> negative</b>, there are oly red colonies</li> | <br>-> Plate on tetracyclin plate and grow overnight at 37°C: <b> negative</b>, there are oly red colonies</li> | ||
+ | </ul> | ||
+ | <li> Plasmid pSB4A5</li> | ||
+ | <ul class="a"> | ||
<li> <b>Gibson Assembly</b> (1h at 50°C) of pSB4A5 and CcdB operon that we already amplified before</li> | <li> <b>Gibson Assembly</b> (1h at 50°C) of pSB4A5 and CcdB operon that we already amplified before</li> | ||
<li> <b>Transformation</b> in <i>E. coli</i> Top10 subcloning cells, using electroporation (Time Constant: 4.0) | <li> <b>Transformation</b> in <i>E. coli</i> Top10 subcloning cells, using electroporation (Time Constant: 4.0) | ||
<br>-> Plate on ampicillin plate and grow overnight at 37°C: <b>positive</b> </li> | <br>-> Plate on ampicillin plate and grow overnight at 37°C: <b>positive</b> </li> | ||
- | <li> <b>Colony PCR</b> (Crimson): there is only 1 colony. | + | <li> <b>Colony PCR</b> (Crimson): there is only 1 colony. We tested this colony (using primers MDM0606 and MDM0607) and it show the right fragment (2486 bp) on gel |
- | + | <br><img src="https://static.igem.org/mediawiki/2013/3/31/UGent_2013_Ene_colonie_4A5.jpg" width="50"/> | |
- | <br>-> Inoculate these | + | <br>-> Inoculate these colonies</li> |
- | <li> Plasmid purification of the | + | <li> Plasmid purification of the colonies, using Qiagen Qiaprep Spin minikit</li> |
+ | <li> Seqenate this plasmid: the result is a totally different sequence than expected </li> | ||
+ | </ul> | ||
+ | <li> Plasmid pSB6A1</li> | ||
+ | <ul class="a"> | ||
+ | <li> We controled the pSB6A1 on mutations (by sequencing) and we found also a mutation on in the <i>ccdB</i> gene</li> | ||
+ | <li> Try to design Gibson primers to undo the mutations | ||
+ | <br>(We will use primers to amplify the biggest part of the plasmid (PCR) and afterwards using Gibson Assembly with oligos, which undo the founded mutation) </li> | ||
+ | <li> <b>Q5 PCR</b> for amplification of biggest part of the plasmid pSB6A1 (using the designed Gibson primers). | ||
+ | <br> -> Using DpnI at 37°C for 1 hour | ||
+ | <br> -> Purification of PCR-product by using minElute reaction cleanup kit | ||
+ | <br> -> Control on gel: <b>positive</b> (fragment length is 6192bp)</li> | ||
+ | <li> <b>Gibson Assembly</b> | ||
+ | <br> -> melt oligos first: 5 minutes at 95°C | ||
+ | <br> -> 6192bp-fragment and oligos at 50°C (1 h)</li> | ||
+ | <li> <b>Transformation</b> in <i>E. coli</i> Top10 subcloning cells, using electroporation (Time Constant: 2.4) | ||
+ | <br>-> Plate on ampicillin plate and grow overnight at 37°C: <b> positive</b> </li> | ||
+ | <li> We made our own competent cells which have the <i>ccdA</i> gene in the genome, so they will be more tolerant to CcdB. We hope on no mutations this time. | ||
+ | <br>-> <b>Q5 PCR</b>: Control of Knock-In | ||
+ | <br> -> Control on gel: <b>positive</b> | ||
+ | <br> <img src="https://static.igem.org/mediawiki/2013/0/08/UGent_2013_Control_ccda_competente_cellen.jpg" width="50"/> | ||
+ | <br> -> Further on we will call these cells competent strain 8 | ||
+ | </li> | ||
+ | <li> Second <b>transformation</b> in competent strain 8, using electroporation (Time Constant: 1.6) | ||
+ | <br>-> Plate on ampicillin plate and grow overnight at 37°C: <b> negative</b> </li> | ||
+ | <li> <b>Gibson Assembly</b> | ||
+ | <br> -> melting oligos first: 5 minutes at 95°C + infinite at 50°C | ||
+ | <br> -> 6192bp-fragment and oligos at 50°C (1 h)</li> | ||
+ | <li> <b>Transformation</b> in competent strain 8, using electroporation (Time Constant: 1.7) | ||
+ | <br>-> Plate on ampicillin plate and grow overnight at 37°C: <b> negative</b> </li> | ||
+ | </ul> | ||
+ | |||
Line 491: | Line 536: | ||
<li> Experiment 4 </li> | <li> Experiment 4 </li> | ||
<ul class="a"> | <ul class="a"> | ||
+ | <li> CIChE with strain 8 + p5/p10/p20: | ||
+ | <br> -> STEP: 0mM - 0.1mM IPTG | ||
+ | <br> -> JUMP: 0mM - 0.5mM IPTG </li> | ||
+ | <li> CIChE with strains 5 + p5/p10/p20: | ||
+ | <br> -> STEP + JUMP: 0 - 0.01mM </li> | ||
</ul> | </ul> | ||
<br> | <br> | ||
- | |||
<li> Experiment 5 </li> | <li> Experiment 5 </li> | ||
<ul class="a"> | <ul class="a"> | ||
- | <li> Evaluation GFP 4 colonies/strain - 3 repeats | + | <li> <b> MTP experiment: version 1 (using precultures) </b> |
+ | <br> (Evaluation GFP 4 colonies/strain - 3 repeats) | ||
<br> -> 8+pSB6A1 - sequential: 0mM - 0.5mM IPTG | <br> -> 8+pSB6A1 - sequential: 0mM - 0.5mM IPTG | ||
<br> -> 8+pSB6A1 - strains grown without antibiotics: 0mM - 0.5mM IPTG | <br> -> 8+pSB6A1 - strains grown without antibiotics: 0mM - 0.5mM IPTG | ||
<br> -> 8+p10 - sequential: 0mM - 0.05mM IPTG </li> | <br> -> 8+p10 - sequential: 0mM - 0.05mM IPTG </li> | ||
+ | <li> Measure the OD and the GFP (using FLUOstar)</li> | ||
</ul> | </ul> | ||
Line 507: | Line 558: | ||
<ul class="a"> | <ul class="a"> | ||
- | <li> <b>Q5 PCR</b> for amplification of T7<i>ccdB</i>-fragment, using the designed Gibson primers, on p5SpFRT-T7<i>ccdB</i>, p10SpFRT-T7<i>ccdB</i> and p20SpFRT-T7<i>ccdB</i> | + | <li> <b>Q5 PCR</b> for amplification of T7-<i>ccdB</i>-fragment, using the designed Gibson primers, on p5SpFRT-T7<i>ccdB</i>, p10SpFRT-T7<i>ccdB</i> and p20SpFRT-T7<i>ccdB</i> |
<br> -> Using DpnI at 37°C for 1 hour | <br> -> Using DpnI at 37°C for 1 hour | ||
<br> -> Purification of PCR-product by using minElute reaction cleanup kit | <br> -> Purification of PCR-product by using minElute reaction cleanup kit | ||
<br> -> Control on gel: <b>negative</b> | <br> -> Control on gel: <b>negative</b> | ||
- | <li> <b>Gibson Assembly</b> (1h at 50°C), using a T7<i>ccdB</i> we already amplified before</li> | + | <li> <b>Gibson Assembly</b> (1h at 50°C), using a T7-<i>ccdB</i> we already amplified before</li> |
<li> <b>Transformation</b> in <i>E. coli</i> Top10 subcloning cells, using electroporation (Time Constant: 4.0) | <li> <b>Transformation</b> in <i>E. coli</i> Top10 subcloning cells, using electroporation (Time Constant: 4.0) | ||
<br>-> Plate on chloramphenicol plate and grow overnight at 37°C: <b> negative</b> </li> | <br>-> Plate on chloramphenicol plate and grow overnight at 37°C: <b> negative</b> </li> | ||
<li> Try to design Gibson primers to undo the mutations, found in the <i>ccdB</i> gene | <li> Try to design Gibson primers to undo the mutations, found in the <i>ccdB</i> gene | ||
<br>(We will use primers to amplify the biggest part of the plasmid (PCR) and afterwards using Gibson Assembly with oligos, which undo the founded mutation) </li> | <br>(We will use primers to amplify the biggest part of the plasmid (PCR) and afterwards using Gibson Assembly with oligos, which undo the founded mutation) </li> | ||
- | <li> <b>Q5 PCR</b> for amplification of biggest part of the plasmid (using the designed Gibson primers). We will do this on the 2 best colonies of last time. | + | <li> <b>Q5 PCR</b> for amplification of biggest part of the plasmid (using the designed Gibson primers). |
+ | <br> We will do this on the 2 best colonies of last time. | ||
<br> -> Using DpnI at 37°C for 1 hour | <br> -> Using DpnI at 37°C for 1 hour | ||
<br> -> Purification of PCR-product by using minElute reaction cleanup kit | <br> -> Purification of PCR-product by using minElute reaction cleanup kit | ||
Line 526: | Line 578: | ||
<br>(Time Constant for colony 1: 4.0, for colony 2: 4.1) | <br>(Time Constant for colony 1: 4.0, for colony 2: 4.1) | ||
<br>-> Plate on chloramphenicol plate and grow overnight at 37°C: <b> positive</b> </li> | <br>-> Plate on chloramphenicol plate and grow overnight at 37°C: <b> positive</b> </li> | ||
- | <li> We made our own competent cells which have the <i>ccdA</i> gene in the genome, so they will be more tolerant to CcdB. | + | <li> We made our own competent cells which have the <i>ccdA</i> gene in the genome = strain 8, so they will be more tolerant to CcdB. |
<br> We hope on no mutations this time | <br> We hope on no mutations this time | ||
<br>-> <b>Q5 PCR</b>: Control of Knock-In | <br>-> <b>Q5 PCR</b>: Control of Knock-In | ||
- | <br> -> Control on gel: <b>positive</b></li> | + | <br> -> Control on gel: <b>positive</b> |
- | <li> Second <b>transformation</b> in | + | <br> <img src="https://static.igem.org/mediawiki/2013/0/08/UGent_2013_Control_ccda_competente_cellen.jpg" width="50"/> |
+ | </li> | ||
+ | <li> Second <b>transformation</b> in competent strain 8, using electroporation | ||
<br>(Time Constant for colony 1: 1.5, for colony 2: 1.4) | <br>(Time Constant for colony 1: 1.5, for colony 2: 1.4) | ||
<br>-> Plate on chloramphenicol plate and grow overnight at 37°C: <b> positive</b> </li> | <br>-> Plate on chloramphenicol plate and grow overnight at 37°C: <b> positive</b> </li> | ||
Line 536: | Line 590: | ||
<br> -> melting oligos first: 5 minutes at 95°C + infinite at 50°C | <br> -> melting oligos first: 5 minutes at 95°C + infinite at 50°C | ||
<br> -> 4233bp-fragment and oligos at 50°C (1 h)</li> | <br> -> 4233bp-fragment and oligos at 50°C (1 h)</li> | ||
- | <li> <b>Transformation</b> in | + | <li> <b>Transformation</b> in competent strain 8, using electroporation (Time Constant for colony 1: 1.5, for colony 2: 1.4) |
<br>-> Plate on chloramphenicol plate and grow overnight at 37°C: <b> positive</b> </li> | <br>-> Plate on chloramphenicol plate and grow overnight at 37°C: <b> positive</b> </li> | ||
Line 542: | Line 596: | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
- | </html>{{:Team:UGent/Templates/ToggleBoxEnd}}{{:Team:UGent/Templates/ToggleBoxStart}}Week 3: september 16 - | + | </html>{{:Team:UGent/Templates/ToggleBoxEnd}}{{:Team:UGent/Templates/ToggleBoxStart}}Week 3: september 16 - 22 {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}<html> |
<ul> | <ul> | ||
- | <li> Experiment | + | <li> Experiment 2 </li> |
<ul class="a"> | <ul class="a"> | ||
- | + | <li> Plasmid pSB3T5</li> | |
- | + | <ul class="a"> | |
+ | <li> <b>Transformation</b> in competent strain 8, using electroporation (Time Constant: 1.4) | ||
+ | <br>-> Plate on tetracyclin plate and grow overnight at 37°C: <b> negative</b></li> | ||
+ | <li> <b>Transformation</b> in competent strain 8, using electroporation | ||
+ | <br> (Time Constant: pSB3T5+T7-<i>ccdB</i> of p10: 4.1, pSB3T5+T7-<i>ccdB</i> of p20: 2.2) | ||
+ | <br>-> Plate on tetracyclin plate and grow overnight at 37°C: <b> positive</b>, but also red colonies </li> | ||
<li> <b>Colony PCR</b> (Crimson): | <li> <b>Colony PCR</b> (Crimson): | ||
- | <br> We tested 24 colonies (using primers MDM0606 and MDM0607) and we found many good colonies | + | <br> We tested 15 colonies (using primers MDM0606 and MDM0607) : <b> negative</b> (expected fragment: 2486 bp)</li> |
- | + | </ul> | |
+ | <li> Plasmid pSB4A5</li> | ||
+ | <ul class="a"> | ||
+ | <li> <b>Transformation</b> in competent strain 8, using electroporation (Time Constant: 1.5) | ||
+ | <br>-> Plate on ampicillin plate and grow overnight at 37°C: <b> negative</b></li> | ||
+ | <li> <b>Transformation</b> in competent strain 8, using electroporation | ||
+ | <br> (Time Constant: pSB3T5+T7-<i>ccdB</i> of p10: 2.7, pSB3T5+T7-<i>ccdB</i> of p20: 2.6) | ||
+ | <br>-> Plate on ampicillin plate and grow overnight at 37°C: <b> positive</b>, but also red colonies </li> | ||
+ | <li> <b>Colony PCR</b> (Crimson): | ||
+ | <br> We tested the few colonies, we had (using primers MDM0606 and MDM0607) : <b> negative</b> (expected fragment: 2486 bp)</li> | ||
+ | </ul> | ||
+ | <li> Plasmid pSB6A1</li> | ||
+ | <ul class="a"> | ||
+ | <li> <b>Colony PCR</b> (Crimson): | ||
+ | <br> We tested 24 colonies of pSB6A1(using primers MDM0606 and MDM0607) and we found many good colonies | ||
+ | <br> <img src=" https://static.igem.org/mediawiki/2013/9/9d/UGent_2013_pSB6A1_cPCR_w3_sept.png | ||
+ | " width="200"/></a> (expected length is 4387 bp) | ||
<br>-> We chose 4 colonies to sequenate | <br>-> We chose 4 colonies to sequenate | ||
<br>-> Inoculate these 4 good colonies</li> | <br>-> Inoculate these 4 good colonies</li> | ||
<li> Plasmid purification of the 4 colonies, using Qiagen Qiaprep Spin minikit: nanodrop: | <li> Plasmid purification of the 4 colonies, using Qiagen Qiaprep Spin minikit: nanodrop: | ||
- | <br>-> | + | <br>-> pSB6A1 strain 4 : 399.4 ng/µl |
- | <br>-> | + | <br>-> pSB6A1 strain 13: 412.0 ng/µl |
- | <br>-> | + | <br>-> pSB6A1 strain 16: 365.5 ng/µl |
- | <br>-> | + | <br>-> pSB6A1 strain 19: 420.3 ng/µl</li> |
- | <li> <b>Sequencing</b> shows the | + | <li> <b>Sequencing</b> shows in 2 colonies other mutations |
+ | <br>and the other 2 colonies shows the right sequence | ||
+ | <br>-> send this 2 good strains to iGEM (<b>BioBrick</b>)</li> | ||
</ul> | </ul> | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | <li> Experiment 3 </li> | ||
+ | <ul class="a"> | ||
+ | <li>Transformation | ||
+ | </ul> | ||
+ | <br> | ||
+ | <li> Experiment 4 </li> | ||
+ | <ul class="a"> | ||
+ | <li> CIChE with strains 8 + p5/p10/p20: | ||
+ | <br> -> STEP: 0.1mM - 1.0mM IPTG | ||
+ | <br> -> JUMP: 0.5mM - 3mM IPTG </li> | ||
+ | <li> CIChE with strains 5 + p5/p10/p20: | ||
+ | <br> -> STEP: 0.01mM - 0.5mM IPTG | ||
+ | <br> -> JUMP: 0.5mM - 1.0mM IPTG </li> | ||
+ | <li> CIChE with strains 8 + pSB6A1 (New plasmids from colony 4 & 16 (exp 2)): | ||
+ | <br> -> STEP: 0mM - 0.2mM IPTG | ||
+ | <br> -> JUMP: 0.5mM - 5.0mM IPTG </li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <li> Experiment 6 </li> | ||
+ | <ul class="a"> | ||
+ | <li> <b>Colony PCR</b> (Crimson): We tested 24 colonies (using primers MDM0606 and MDM0607) and we found many good colonies | ||
+ | <br> <img src=" https://static.igem.org/mediawiki/2013/2/25/UGent_2013_pSB1C3_cPCR_w3_sept.png | ||
+ | " width="200"/></a> (expected length is 2520 bp) | ||
+ | <br>-> We chose 4 colonies to sequenate | ||
+ | <br>-> Inoculate these 4 good colonies</li> | ||
+ | <li> Plasmid purification of the 4 colonies, using Qiagen Qiaprep Spin minikit: nanodrop: | ||
+ | <br>-> pSB1C3 strain 10: 181.1 ng/µl | ||
+ | <br>-> pSB1C3 strain 19: 169.1 ng/µl | ||
+ | <br>-> pSB1C3 strain 20: 184.8 ng/µl | ||
+ | <br>-> pSB1C3 strain 24: 184.5 ng/µl</li> | ||
+ | <li> <b>Sequencing</b> shows the same mutation at the end codon again</li> | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
- | </html> | + | </html>{{:Team:UGent/Templates/ToggleBoxEnd}}{{:Team:UGent/Templates/ToggleBoxStart}}Week 4: september 23 - 29 {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}<html> |
+ | <ul> | ||
+ | <li> Experiment 4 </li> | ||
+ | <ul class="a"> | ||
+ | <li> CIChE with strains 5 + p5/p10/P20: | ||
+ | <br> -> STEP: 1.0mM - 4.0mM IPTG</li> | ||
+ | <li> CIChE with strain 8 + pSB6A1: | ||
+ | <br> -> STEP: 0.2mM - 2.0mM IPTG | ||
+ | <br> -> JUMP: 5mM - 120mM IPTG</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <li> Experiment 5 </li> | ||
+ | <ul class="a"> | ||
+ | <li> <b> MTP experiment: version 1 (using precultures) </b> | ||
+ | <br> -> Plate1: 8 + p5;STEP; 0mM Day1 - 0.5mM Day2 | ||
+ | <br> -> Plate2: 8 + p10;STEP; 0mM Day1 - 0.5mM Day2 | ||
+ | <br> -> Plate3: 8 + p20;STEP; 0mM Day1 - 0.5mM Day2 | ||
+ | <br> -> Plate4: 8 + pSB6A1-16;STEP; 0mM Day1 - 0.05mM Day2 | ||
+ | <br> -> Plate5: 8 + p5;JUMP; 0mM Day1 - 2mM Day2 | ||
+ | <br> -> Plate6: 8 + p10;JUMP; 0mM Day1 - 2mM Day2 | ||
+ | <br> -> Plate7: 8 + p20;JUMP; 0mM Day1 - 2mM Day2 | ||
+ | </li> | ||
+ | <li> Measure the OD and the GFP of all previous plates (using FLUOstar)</li> | ||
+ | <li> <b> MTP experiment: Test version 2 </b> | ||
+ | <br> (to know how long they have to grow until they reach a sufficient OD: 4 - 5 hours) | ||
+ | </li> | ||
+ | <li> <b> MTP experiment: version 2 </b> | ||
+ | <br> -> Plate1: 8 + p5;STEP; 0mM Day1 - 0.5mM Day2 | ||
+ | <br> -> Plate2: 8 + p10;STEP; 0mM Day1 - 0.5mM Day2 | ||
+ | <br> -> Plate3: 8 + p20;STEP; 0mM Day1 - 0.5mM Day2 | ||
+ | <br> -> Plate4: 8 + pSB6A1-16;STEP; 0mM Day1 - 0.05mM Day2 | ||
+ | <br> -> Plate5: 8 + p5;JUMP; 0mM Day1 - 2mM Day2 | ||
+ | <br> -> Plate6: 8 + p10;JUMP; 0mM Day1 - 2mM Day2 | ||
+ | <br> -> Plate7: 8 + p20;JUMP; 0mM Day1 - 2mM Day2 | ||
+ | <br> -> Plate8: 8 + pSB6A1-16;STEP; 0.1mM Day1 - 0.2mM Day1 & JUMP;0mM Day1 - 5mM Day1 | ||
+ | <br> -> Plate9: 8 + pSB6A1-4;STEP; 0mM Day1 - 0.5mM Day2 | ||
+ | <br> -> Plate10: 8 + pSB6A1-4;STEP; 1.0mM Day1 - 2.0mM Day1 & JUMP; 0mM Day1 - 1.5mM Day1 | ||
+ | <br> -> Plate11: 8 + pSB6A1-4;JUMP; 1.5mM Day2 - 30.0mM Day2 | ||
+ | <br> -> Plate12: 8 + pSB6A1-4;JUMP; 60.0mM Day2 - 120.0mM Day1 <b>&</b> 5 + p5;STEP;0mM Day1 - 0.2mM Day1 | ||
+ | <br> -> Plate13: 5 + p5;STEP; 0.2mM Day2 - 3.0mM Day2 | ||
+ | <br> -> Plate14: 5 + p5;STEP; 3.5mM Day1 & JUMP;0mM Day1- 1mM Day1 | ||
+ | <br> <b> & </b> 5 + p10;STEP;0mM Day1 - 0,01mM Day1 | ||
+ | <br> -> Plate15: 5 + p10;STEP; 0.01mM Day2 - 1.0mM Day2 | ||
+ | <br> -> Plate16: 5 + p10;STEP; 2mM Day1 - 3.5mM Day1 & JUMP;0mM Day1 - 0.1mM Day1 | ||
+ | <br> -> Plate17: 5 + p10;JUMP; 0.1mM Day2 - 1.0mM Day1 <b> & </b> 5 + p20;STEP; 0mM Day1 - 0.1mM Day2 | ||
+ | <br> -> Plate18: 5 + p20;STEP; 0.2mM Day1 - 3.0mM Day1 | ||
+ | <br> -> Plate19: 5 + p20;STEP; 3.0mM Day1 - 3.5mM Day1 & JUMP;0mM Day1 - 1mM day1 | ||
+ | </li> | ||
+ | <li> Measure the OD and the GFP of all plates (using FLUOstar)</li> | ||
+ | </ul | ||
+ | </ul> | ||
+ | </html>{{:Team:UGent/Templates/ToggleBoxEnd}}<html> | ||
+ | |||
+ | |||
+ | <h2> October </h2> | ||
+ | </html>{{:Team:UGent/Templates/ToggleBoxStart}}Week 1: september 30 - october 4 {{:Team:UGent/Templates/ToggleBoxStart1}}{{:Team:UGent/Templates/ToggleBoxStart2}}<html> | ||
+ | <ul> | ||
+ | <li> Experiment 4 </li> | ||
+ | <ul class="a"> | ||
+ | <li> CIChE with strains 5 + p5/p10/P20: | ||
+ | <br> -> STEP: 4.0mM - 5.0mM IPTG </li> | ||
+ | <li> CIChE with strain 8 + pSB6A1: | ||
+ | <br> -> STEP: 2.0mM - 3.0mM IPTG | ||
+ | <br> -> JUMP: 120.0mM - 240mM IPTG</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <li> Experiment 5 </li> | ||
+ | <ul class="a"> | ||
+ | <li> <b> MTP experiment: version 2 </b> | ||
+ | <br> -> Plate20: 8 + pSB6A1-4;STEP; 2.5mM Day2 - 3.0mM Day2 & JUMP; 120mM Day2 - 240mM Day2 | ||
+ | <br> -> Plate21: 5 + p5;STEP; 3.5mM Day2 - 5mM Day2 <b> & </b> 5 + p10;STEP; 3.5mM Day2 - 5mM Day2 | ||
+ | <br> -> Plate22: 5 + p20;STEP; 3.5mM Day2 - 5mM Day2 <b> & </b> 5 + p5;STEP; 1mM Day1 and 3mM Day1 | ||
+ | <br><b> & </b> 5 + p10;STEP; 1mM Day1 and 3mM Day1<b> & </b> 5 + p20;STEP; 1mM Day1 and 3mM Day1 | ||
+ | </li> | ||
+ | <li> Measure the OD and the GFP of the 3 plates (using FLUOstar)</li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | </html>{{:Team:UGent/Templates/ToggleBoxEnd}} | ||
+ | |||
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Latest revision as of 01:35, 5 October 2013
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