Team:Hong Kong HKUST/notebook/mod2
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<ol class="pos_fixed"> | <ol class="pos_fixed"> | ||
+ | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li> | ||
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod1">Module 1</a></li> | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod1">Module 1</a></li> | ||
+ | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod2">Module 2</a></li> | ||
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod3">Module 3</a></li> | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod3">Module 3</a></li> | ||
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod4">Module 4</a></il> | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod4">Module 4</a></il> | ||
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a> | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a> | ||
<ul> | <ul> | ||
- | |||
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li> | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li> | ||
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">Protocols</a></li> | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">Protocols</a></li> | ||
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</div> | </div> | ||
<br><br><br><br><br><br><br><br> | <br><br><br><br><br><br><br><br> | ||
- | <div id="title"><h3>FA Sensing Mechanism Module's Notebook</h3></div> | + | <div id="title"><center><h3>FA Sensing Mechanism Module's Notebook</h3></center></div> |
<div id="flight"> | <div id="flight"> | ||
<div id="satu" align="center"> <h1>June 2013</h1> | <div id="satu" align="center"> <h1>June 2013</h1> | ||
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Wet lab <br> | Wet lab <br> | ||
· Extraction of pEGFP-N1 and BBa_K817002 (P<i>fadBA</i>) plasmids by miniprep <br> | · Extraction of pEGFP-N1 and BBa_K817002 (P<i>fadBA</i>) plasmids by miniprep <br> | ||
- | · Restriction of pEGFP-N1 Restriction digestion by Ase1 and | + | · Restriction of pEGFP-N1 Restriction digestion by Ase1 and BamH1 for PPAR-alpha promoter; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1 promoter, Pst1 and Not1; followed by gel check, extraction and purification <br> |
· Restriction of c(P<i>fadBA</i>) by EcoR1 and Pst1 <br> | · Restriction of c(P<i>fadBA</i>) by EcoR1 and Pst1 <br> | ||
· Ran 0.8% gel for pEGFP-N1 and 2% gel for BBa_K817002 (P<i>fadBA</i>) <br> | · Ran 0.8% gel for pEGFP-N1 and 2% gel for BBa_K817002 (P<i>fadBA</i>) <br> | ||
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<br> <b> July 2 </b> <br> | <br> <b> July 2 </b> <br> | ||
Wet lab <br> | Wet lab <br> | ||
- | · Digestion of pEGFP-N1 by Ase1 and | + | · Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter <br> |
· LB-Chloramphenicol plates (the previous ones were found contaminated) <br> | · LB-Chloramphenicol plates (the previous ones were found contaminated) <br> | ||
· Inoculation of pEGFP-N1 for plasmid extraction <br> | · Inoculation of pEGFP-N1 for plasmid extraction <br> | ||
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· Extraction of pEGFP-N1 plasmids by miniprep <br> | · Extraction of pEGFP-N1 plasmids by miniprep <br> | ||
· PCR for PPAR-alpha promoter amplification <br> | · PCR for PPAR-alpha promoter amplification <br> | ||
- | · Digestion of pEGFP-N1 by Ase1 and | + | · Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter <br> |
<br> <b> July 4</b> <br> | <br> <b> July 4</b> <br> | ||
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· Inoculation of BBa_K817002 P<i>fadBA</i><br> | · Inoculation of BBa_K817002 P<i>fadBA</i><br> | ||
· PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br> | · PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br> | ||
- | · Ran gel for digestion of pEGFP-N1 by Ase1 and | + | · Ran gel for digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; Xba1 and BamH1 for FABP1 promoter; Ase1 and Xhol1 for GRP78<br> |
<br> <b> July 5</b> <br> | <br> <b> July 5</b> <br> | ||
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Wet lab<br> | Wet lab<br> | ||
· Extraction of BBa_J176171, BBa_K817002 P<i>fadBA</i> and pEGFP-N1 by miniprep<br> | · Extraction of BBa_J176171, BBa_K817002 P<i>fadBA</i> and pEGFP-N1 by miniprep<br> | ||
- | · Restriction of pEGFP-N1 Restriction digestion by Ase1 and | + | · Restriction of pEGFP-N1 Restriction digestion by Ase1 and BamH1 for PPAR-alpha promoter; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1 promoter, Pst1 and Not1; followed by gel check, extraction and purification<br> |
· Restriction of BBa_J176171 for P<i>fadBA</i> Ase1 and BamH1; FABP1 promoter by Xba1 and Not1<br> | · Restriction of BBa_J176171 for P<i>fadBA</i> Ase1 and BamH1; FABP1 promoter by Xba1 and Not1<br> | ||
· Ran gel for pEGFP-N1 and BBa_J176171 restriction products<br> | · Ran gel for pEGFP-N1 and BBa_J176171 restriction products<br> | ||
· PCR PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br> | · PCR PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br> | ||
- | · <a href="http://parts.igem.org/Part:BBa_J52034">BBa_J52034</a> ( | + | · <a href="http://parts.igem.org/Part:BBa_J52034">BBa_J52034</a> (<i>fad</i>R) transformation<br> |
<br> <b> July 10</b> <br> | <br> <b> July 10</b> <br> | ||
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· BBa_J176171 vector dephosphorylation by antartic phosphatase<br> | · BBa_J176171 vector dephosphorylation by antartic phosphatase<br> | ||
· Ligation of BBa_K817002 and P<i>fadBA</i> and BBa_J176171 <br> | · Ligation of BBa_K817002 and P<i>fadBA</i> and BBa_J176171 <br> | ||
- | · BBa_J52034 ( | + | · BBa_J52034 (<i>fad</i>R) inoculation<br> |
· Digestion for BBa_K817002 P<i>fadBA</i> extraction<br> | · Digestion for BBa_K817002 P<i>fadBA</i> extraction<br> | ||
Gel check for BBa_K817002 P<i>fadBA</i> extraction<br> | Gel check for BBa_K817002 P<i>fadBA</i> extraction<br> | ||
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· Gel check for FABP1 promoter PCR product<br> | · Gel check for FABP1 promoter PCR product<br> | ||
· PCR clean-up<br> | · PCR clean-up<br> | ||
- | · BBa_J52034 ( | + | · BBa_J52034 (<i>fad</i>R) miniprep<br> |
· BBa_J52034 restriction by Pst1 HF and Not1 HF<br> | · BBa_J52034 restriction by Pst1 HF and Not1 HF<br> | ||
· PCR for PPAR-alpha promoter<br> | · PCR for PPAR-alpha promoter<br> | ||
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· Digestion of FABP1 promoter PCR product<br> | · Digestion of FABP1 promoter PCR product<br> | ||
· Ran gel for PCR check of PPAR-alpha promoter<br> | · Ran gel for PCR check of PPAR-alpha promoter<br> | ||
- | · Digest pEGFP-n1 for BBa_J52034 | + | · Digest pEGFP-n1 for BBa_J52034 <i>fad</i>R<br> |
- | · Ran gel for pEGFP-n1 for BBa_J52034 | + | · Ran gel for pEGFP-n1 for BBa_J52034 <i>fad</i>R followed by gel purification<br> |
- | · BBa_J176171 vector dephosphorylation by Antarctic phosphatase for FABP1 promoter and | + | · BBa_J176171 vector dephosphorylation by Antarctic phosphatase for FABP1 promoter and P<i>fadBA</i><br> |
Dry Lab<br> | Dry Lab<br> | ||
- | Primers design for pCMV cloning for | + | Primers design for pCMV cloning for <i>fad</i>R expression<br><br> |
</div> | </div> | ||
</div> | </div> | ||
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<br><b>July 15</b> <br> | <br><b>July 15</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
- | · Ligation of FABP1 promoter, | + | · Ligation of FABP1 promoter, EGFP, and BBa_J176171, using 3 pieces ligation<br> |
- | · Transformation of FABP1 promoter, | + | · Transformation of FABP1 promoter, EGFP, and BBa_J176171 ligation<br> |
- | · FABP1 promoter+ | + | · FABP1 promoter+EGFP +BBa_J176171 ligation restriction check<br> |
<br><b>July 16</b> <br> | <br><b>July 16</b> <br> | ||
Wet Lab<br> | Wet Lab<br> | ||
· Miniprep for full construct of FABP1 promoter and pEGFP-N1<br> | · Miniprep for full construct of FABP1 promoter and pEGFP-N1<br> | ||
- | · | + | · <i>fad</i>R and pEGFP-N1 Backbone parts ligation <br> |
- | · Plasmids extraction for pCMV cloning for | + | · Plasmids extraction for pCMV cloning for <i>fad</i>R<br> |
· BBa_K817002 P<i>fadBA</i> promoter extraction by EcoR1 and Pst1 HF<br> | · BBa_K817002 P<i>fadBA</i> promoter extraction by EcoR1 and Pst1 HF<br> | ||
· BBa_J52034 restriction by EcoR1 and Pst1 HF<br> | · BBa_J52034 restriction by EcoR1 and Pst1 HF<br> | ||
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<br><b>July 17</b> <br> | <br><b>July 17</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
- | · Plasmid extraction for FABP1 promoter+ | + | · Plasmid extraction for FABP1 promoter+EGFP+BBa_J176171 by miniprep<br> |
- | · Repeat | + | · Repeat <i>fad</i>R and P<i>fadBA</i> constructs<br> |
- | · Digest pEGFP-n1 and BBa-J176171 for | + | · Digest pEGFP-n1 and BBa-J176171 for <i>fad</i>R<br> |
· Digestion for BBa_K817002 (P<i>fadBA</i>) extraction<br> | · Digestion for BBa_K817002 (P<i>fadBA</i>) extraction<br> | ||
- | · Gel check and gel extraction for BBa_J176171 for | + | · Gel check and gel extraction for BBa_J176171 for <i>fad</i>R and BBa_K817002 (P<i>fadBA</i>) <br> |
<br><b>July 18</b> <br> | <br><b>July 18</b> <br> | ||
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· Gel purification for all digested products<br> | · Gel purification for all digested products<br> | ||
· FABP1 promoter digestion check for whole construct prior transfection<br> | · FABP1 promoter digestion check for whole construct prior transfection<br> | ||
- | · P<i>fadBA</i> vector de phosphorylation and ligation with | + | · P<i>fadBA</i> vector de phosphorylation and ligation with EGFP and BBa_J176171<br> |
- | · Transformation of P<i>fadBA</i>+ | + | · Transformation of P<i>fadBA</i>+EGFP+BBa_J176171<br> |
<br><b>July 19</b> <br> | <br><b>July 19</b> <br> | ||
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· Digestion and gel for construction check for FABP1 promoter<br> | · Digestion and gel for construction check for FABP1 promoter<br> | ||
· P<i>fadBA</i> gel extraction and purification, followed by vector dephosphorylation BBa_J176171 and ligation, then transformation<br> | · P<i>fadBA</i> gel extraction and purification, followed by vector dephosphorylation BBa_J176171 and ligation, then transformation<br> | ||
- | · Digestion of pEGFP-N1 for | + | · Digestion of pEGFP-N1 for <i>fad</i>R, followed by gel check and extraction<br> |
<br><b>July 24</b> <br> | <br><b>July 24</b> <br> | ||
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Wet lab<br> | Wet lab<br> | ||
· Ran gel check PPAR-alpha promoter PCR cloning with new primers using Taq polymerase<br> | · Ran gel check PPAR-alpha promoter PCR cloning with new primers using Taq polymerase<br> | ||
- | · Minprep of P<i>fadBA</i>+ | + | · Minprep of P<i>fadBA</i>+EGFP+BBa_J176171 ligation colonies, followed by digestion check <br> |
<br><b>July 26</b> <br> | <br><b>July 26</b> <br> | ||
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· PPAR-alpha promoter PCR cloning with new primers using Vent polymerase<br> | · PPAR-alpha promoter PCR cloning with new primers using Vent polymerase<br> | ||
· Restriction check for pEGFP-N1, using EcoR1, Xba1, Spe1 and Pst1<br> | · Restriction check for pEGFP-N1, using EcoR1, Xba1, Spe1 and Pst1<br> | ||
- | · BBa_J176171 vector de phosphorylation, followed by ligation with | + | · BBa_J176171 vector de phosphorylation, followed by ligation with EGFP using T4 Ligase, then transformation into E. Coli strain DH10b<br> |
· PCR for PPAR-alpha promoter with new primers using Vent polymerase<br> | · PCR for PPAR-alpha promoter with new primers using Vent polymerase<br> | ||
Dry lab<br> | Dry lab<br> | ||
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Wet lab<br> | Wet lab<br> | ||
· Gel Check for PPAR-alpha promoter PCR promoter cloning, using temperature gradient and every available set of primers designed. <br> | · Gel Check for PPAR-alpha promoter PCR promoter cloning, using temperature gradient and every available set of primers designed. <br> | ||
- | · Digestion of BBa_J176171 using single restriction site, Xba1, vector dephosphorylation and ligation with | + | · Digestion of BBa_J176171 using single restriction site, Xba1, vector dephosphorylation and ligation with <i>fad</i>R followed by transformation<br> |
- | · Ligation check of P<i>fadBA</i>+BBa_J176171+ | + | · Ligation check of P<i>fadBA</i>+BBa_J176171+EGFP using Age1<br> |
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Taq polymerase<br> | · PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Taq polymerase<br> | ||
· gDNA Gel check<br> | · gDNA Gel check<br> | ||
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Wet lab<br> | Wet lab<br> | ||
· Ran gel for PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Taq polymerase<br> | · Ran gel for PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Taq polymerase<br> | ||
- | · Inoculation of | + | · Inoculation of <i>fad</i>R+BBa_J176171<br> |
<br><b>August 7</b> <br> | <br><b>August 7</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
- | · Digestion check of BBa_J176171+ | + | · Digestion check of BBa_J176171+EGFP by Not1 and Pst1<br> |
- | · Digestion check of BBa_J176171+ | + | · Digestion check of BBa_J176171+<i>fad</i>R by Xba1 and HindIII<br> |
· Ran gel for digestion check<br> | · Ran gel for digestion check<br> | ||
- | · Inoculation of | + | · Inoculation of <i>fad</i>R+BBa_J176171, colony screening<br> |
<br><b>August 8</b> <br> | <br><b>August 8</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
- | · Miniprep for | + | · Miniprep for <i>fad</i>R+BBa_J176171, colony screening<br> |
- | · Digestion check of BBa_J176171+ | + | · Digestion check of BBa_J176171+<i>fad</i>R by Xba1 and HindIII<br> |
· Ran gel for digestion check<br> | · Ran gel for digestion check<br> | ||
- | · Inoculation of | + | · Inoculation of <i>fad</i>R+BBa_J176171, colony screening<br> |
- | · pCMV from cloned from pEGFP-N1adding restriction sites for ligation with | + | · pCMV from cloned from pEGFP-N1adding restriction sites for ligation with EGFP and BBa_J176171, followed by transformation<br> |
<br><b>August 9</b> <br> | <br><b>August 9</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
· Primers arrival for BBa_J04450-PSB1C3 for site direct mutagenesis<br> | · Primers arrival for BBa_J04450-PSB1C3 for site direct mutagenesis<br> | ||
- | · Digestion check of BBa_J176171+ | + | · Digestion check of BBa_J176171+<i>fad</i>R Xba1 and HindIII, Xba1 and Spe1<br> |
· Ran gel for digestion check<br> | · Ran gel for digestion check<br> | ||
· Site direct Mutagenesis for BBa_J04450-PSB1C3 removing Xba1 restriction site, followed by gel check before parental string digestion and transformation<br> | · Site direct Mutagenesis for BBa_J04450-PSB1C3 removing Xba1 restriction site, followed by gel check before parental string digestion and transformation<br> | ||
- | · Inoculation of pCMV from cloned from pEGFP-N1adding restriction sites for ligation with | + | · Inoculation of pCMV from cloned from pEGFP-N1adding restriction sites for ligation with EGFP and BBa_J176171<br><br> |
</div> | </div> | ||
</div> | </div> | ||
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<p><strong>August 12 </strong><br /> | <p><strong>August 12 </strong><br /> | ||
Wet lab <br /> | Wet lab <br /> | ||
- | · Miniprep of pCMV+ | + | · Miniprep of pCMV+EGFP+ BBa_J176171, followed by digestion check and gel <br /> |
· PCR for pCMV cloning from pEGFP-N1 using Vent polymerase, followed by gel check and PCR clean up <br /> | · PCR for pCMV cloning from pEGFP-N1 using Vent polymerase, followed by gel check and PCR clean up <br /> | ||
- | · Digestion check of BBa_J176171+ | + | · Digestion check of BBa_J176171+<i>fad</i>R by Xba1, HindIII and Spe1 <br /> |
· Ran gel for digestion check <br /> | · Ran gel for digestion check <br /> | ||
· pEGFP-N1 digestion for GRP78, extraction pCMV using Ase1 and Xho1, followed by gel check and DNA purification <br /> | · pEGFP-N1 digestion for GRP78, extraction pCMV using Ase1 and Xho1, followed by gel check and DNA purification <br /> | ||
- | · Digestion of BBa_J176171 using single restriction site, Xba1, vector dephosphorylation and ligation with | + | · Digestion of BBa_J176171 using single restriction site, Xba1, vector dephosphorylation and ligation with <i>fad</i>R followed by transformation <br /> |
· Site direct mutagenesis for BBa_J04450-PSB1C3, followed by gel check before parental digestion with Dpn1 and transformation</p> | · Site direct mutagenesis for BBa_J04450-PSB1C3, followed by gel check before parental digestion with Dpn1 and transformation</p> | ||
<p> </p> | <p> </p> | ||
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Wet lab <br /> | Wet lab <br /> | ||
· P<i>fadBA</i>+pEGFP-N1 ligation, first vector dephosphorylation using Antarctic phosphatase followed by ligation by T4Ligase and transformation into <em>E. Coli</em> SURE strain. <br /> | · P<i>fadBA</i>+pEGFP-N1 ligation, first vector dephosphorylation using Antarctic phosphatase followed by ligation by T4Ligase and transformation into <em>E. Coli</em> SURE strain. <br /> | ||
- | · pCMV+ | + | · pCMV+EGFP+BBa_J176171 digestion check, followed by gel check <br /> |
· Repeat GRP78+pEGFP-N1 ligation, followed by transformation into <em>E. Coli</em> DH10b strain. <br /> | · Repeat GRP78+pEGFP-N1 ligation, followed by transformation into <em>E. Coli</em> DH10b strain. <br /> | ||
- | · Digestion check for | + | · Digestion check for <i>fad</i>R+BBa_J176171 usign Xba1 and HindIII <br /> |
- | · Inoculations of | + | · Inoculations of <i>fad</i>R+ BBa_J176171, pCMV+EGFP+ BBa_J176171, P<i>fadBA</i>+pEGFP-N1, P<i>fadBA</i>+EGFP+ BBa_J176171 followed by gel check</p> |
<p> </p> | <p> </p> | ||
<p><strong>August 16 </strong><br /> | <p><strong>August 16 </strong><br /> | ||
Wet lab <br /> | Wet lab <br /> | ||
- | · Miniprep of | + | · Miniprep of <i>fad</i>R+ BBa_J176171, pCMV+EGFP+ BBa_J176171, P<i>fadBA</i>+pEGFP-N1, P<i>fadBA</i>+EGFP+ BBa_J176171 <br /> |
- | · Restriction check for | + | · Restriction check for <i>fad</i>R+ BBa_J176171 and P<i>fadBA</i>+pEGFP-N1 followed by gel check <br /> |
- | · Inoculations of | + | · Inoculations of <i>fad</i>R+ BBa_J176171, P<i>fadBA</i>+pEGFP-N1, P<i>fadBA</i>+EGFP+ BBa_J176171 <br /> |
· DH105alpha <em>E. Coli</em> competent cells preparation</p> | · DH105alpha <em>E. Coli</em> competent cells preparation</p> | ||
<p> </p> | <p> </p> | ||
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Wet lab <br /> | Wet lab <br /> | ||
· Repeat GRP78+pEGFP-N1 ligation, followed by transformation into <em>E. Coli</em> DH10b strain. <br /> | · Repeat GRP78+pEGFP-N1 ligation, followed by transformation into <em>E. Coli</em> DH10b strain. <br /> | ||
- | · Miniprep of | + | · Miniprep of <i>fad</i>R+ BBa_J176171, P<i>fadBA</i>+pEGFP-N1, P<i>fadBA</i>+EGFP+ BBa_J176171 <br /> |
· Restriction check for P<i>fadBA</i>+pEGFP-N1 followed by gel check <br /> | · Restriction check for P<i>fadBA</i>+pEGFP-N1 followed by gel check <br /> | ||
- | · Restriction check for | + | · Restriction check for <i>fad</i>R+ BBa_J176171 and P<i>fadBA</i>+pEGFP-N1 followed by gel check <br /> |
- | · Streak colony with the right | + | · Streak colony with the right <i>fad</i>R+BBa_J176171 construct <br /> |
· DH5alpha <em>E. Coli </em>competent cells preparation <br /> | · DH5alpha <em>E. Coli </em>competent cells preparation <br /> | ||
· <br /> | · <br /> | ||
Line 722: | Line 744: | ||
· Digestion check P<i>fadBA</i>+pEGFP-N1 with Pst1, Xba1. Followed by gel check <br /> | · Digestion check P<i>fadBA</i>+pEGFP-N1 with Pst1, Xba1. Followed by gel check <br /> | ||
· Digestion check for damaged plates of GRP78+pEGFP-N1 using Xba1 <br /> | · Digestion check for damaged plates of GRP78+pEGFP-N1 using Xba1 <br /> | ||
- | · Digestion check of P<i>fadBA</i>+ | + | · Digestion check of P<i>fadBA</i>+EGFP+ BBa_J176171 using Age1HF, HindIII and Xba1 <br /> |
· Site Direct Mutagenesis for illegal restriction sites for FABP1 promoter<br /> | · Site Direct Mutagenesis for illegal restriction sites for FABP1 promoter<br /> | ||
- | · Inoculate GRP78+pEGFP-N1, pCMV+ | + | · Inoculate GRP78+pEGFP-N1, pCMV+EGFP+BBa_J176171 <br /> |
· DH10b <em>E. Coli</em> competent cells preparation</p> | · DH10b <em>E. Coli</em> competent cells preparation</p> | ||
<p> </p> | <p> </p> | ||
Line 730: | Line 752: | ||
Wet lab <br /> | Wet lab <br /> | ||
· Miniprep GRP78+pEGFP-N1, followed by digestion check and gel check using Spe1 <br /> | · Miniprep GRP78+pEGFP-N1, followed by digestion check and gel check using Spe1 <br /> | ||
- | · Miniprep pCMV+ | + | · Miniprep pCMV+EGFP+BBa_J176171 followed by digestion check using Nde1 and Age1HF <br /> |
- | · Digestion check of P<i>fadBA</i>+ | + | · Digestion check of P<i>fadBA</i>+EGFP+BBa_J176171 using Nde1 , <i>fad</i>R+ BBa_J176171 using Age1 <br /> |
- | · Site direct Mutagenesis for FABP1 promoter+ | + | · Site direct Mutagenesis for FABP1 promoter+EGFP+BBa_J176171 <br /> |
· PCR for GRP78 promoter cloning from pDRIVE_hGRP78, followed by gel check PCR clean up, digestion using Ase1 and Xho1 and DNA purification</p> | · PCR for GRP78 promoter cloning from pDRIVE_hGRP78, followed by gel check PCR clean up, digestion using Ase1 and Xho1 and DNA purification</p> | ||
<p> </p> | <p> </p> | ||
Line 738: | Line 760: | ||
Wet lab <br /> | Wet lab <br /> | ||
· DH10b <em>E. Coli</em> competent cells preparation <br /> | · DH10b <em>E. Coli</em> competent cells preparation <br /> | ||
- | · Site direct Mutagenesis for FABP1 promoter+ | + | · Site direct Mutagenesis for FABP1 promoter+EGFP+BBa_J176171 with phosphorylated primers, followed by gel check before Dpn1 parental string digestion <br /> |
· Ligation of GRP78 and pEGFP-N1, first vector dephophorylation and ligation with T4Ligase. Transformed into <em>E. Coli</em> DH10b strain <br /> | · Ligation of GRP78 and pEGFP-N1, first vector dephophorylation and ligation with T4Ligase. Transformed into <em>E. Coli</em> DH10b strain <br /> | ||
· Transformation of pBlueScript KS (+) in to SURE <em>E. Coli</em> <br /> | · Transformation of pBlueScript KS (+) in to SURE <em>E. Coli</em> <br /> | ||
Line 756: | Line 778: | ||
Wet lab <br /> | Wet lab <br /> | ||
· FABP1 promoter assessment under PCR conditions <br /> | · FABP1 promoter assessment under PCR conditions <br /> | ||
- | · BBa_J176171+ | + | · BBa_J176171+EGFP assessment under PCR conditions <br /> |
- | · | + | · BBa_J176171 assessment under PCR conditions <br /> |
- | · Temperature gradient for FABP1 promoter+BBa_J176171+ | + | · Temperature gradient for FABP1 promoter+BBa_J176171+EGFP construct <br /> |
· Inoculate pBlueScript KS (+)</p> | · Inoculate pBlueScript KS (+)</p> | ||
<p> </p> | <p> </p> | ||
Line 767: | Line 789: | ||
<p><strong>August 28 </strong><br /> | <p><strong>August 28 </strong><br /> | ||
Wet lab <br /> | Wet lab <br /> | ||
- | · | + | · BBa_J176171 assessment under PCR conditions <br /> |
- | · Temperature gradient for FABP1 promoter+BBa_J176171+ | + | · Temperature gradient for FABP1 promoter+BBa_J176171+EGFP construct <br /> |
· PCR for FABP1 promoter cloning from gDNA <br /> | · PCR for FABP1 promoter cloning from gDNA <br /> | ||
· GRP78 ligation to dephosphorylated pEGFP-N1, ligation done using T4 Ligase, transformed into <em>E.Coli</em> DH10b <br /> | · GRP78 ligation to dephosphorylated pEGFP-N1, ligation done using T4 Ligase, transformed into <em>E.Coli</em> DH10b <br /> |
Latest revision as of 23:14, 27 September 2013
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FA Sensing Mechanism Module's Notebook
June 2013
June 24
Wet lab
· Inoculation of pBlueScript KS(+) for training
· Autoclave basic materials
· Preparing LB
· LB-Ampicillin plates poured Dry lab
Protocols review
June 25
Wet lab
· Plasmid extraction of pBlueScript KS(+)
· LB-Chloramphenicol plates poured
Dry lab
· Primers design for FABP1 promoter, PPAR-alpha promoter, GRP78 promoter
· Informal meeting
June 26
Wet lab
· Extraction of gDNA from HepG2 Cells for FABP1 promoter and PPAR-alpha promoter
· Transformation of pEGFP-N1 and BBa_K817002 (PfadBA)
June 27
Wet lab
· Inoculation of pEGFP-N1 and BBa_K817002 (PfadBA) for plasmid extraction
June 28
Wet lab
· Extraction of pEGFP-N1 and BBa_K817002 (PfadBA) plasmids by miniprep
· Restriction of pEGFP-N1 Restriction digestion by Ase1 and BamH1 for PPAR-alpha promoter; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1 promoter, Pst1 and Not1; followed by gel check, extraction and purification
· Restriction of c(PfadBA) by EcoR1 and Pst1
· Ran 0.8% gel for pEGFP-N1 and 2% gel for BBa_K817002 (PfadBA)
June 30
Dry lab
Experiment planning and protocols revision for next week work
July 2013
July 2
Wet lab
· Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter
· LB-Chloramphenicol plates (the previous ones were found contaminated)
· Inoculation of pEGFP-N1 for plasmid extraction
· Transformation of PfadBA due chloramphenicol plates contamination
July 3
· Extraction of pEGFP-N1 plasmids by miniprep
· PCR for PPAR-alpha promoter amplification
· Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter
July 4
Wet lab
· Inoculation of BBa_K817002 PfadBA
· PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· Ran gel for digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; Xba1 and BamH1 for FABP1 promoter; Ase1 and Xhol1 for GRP78
July 5
Wet lab
· Extraction of BBa_K817002 (PfadBA)
· Inoculation of pEGFP-N1
· PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· Gel check, 0.8% gel for previously gDNA extraction
· Ran gel for PCR check of PPAR-alpha promoter and FABP1 promoter
· Poured new LB-Kanamycin plates
· Transformation of BBa_J176171
Dry lab
· Discussion about re-design constructions, possible change for BBa_J176171 as mammalian expression vector
· Primer redesign for PPAR-alpha promoter and FABP1 promoter
July 8
Wet lab
· gDNA extraction from HepG2 cells
· Restriction of BBa_K817002 PfadBA
· Gel check for gDNA extraction
· Inoculation of BBa_J176171, BBa_K817002 (PfadBA), pEGFP-N1 for plasmid extraction
· New primers for PPAR-alpha promoter and FABP1 promoter arrival
· Ran gel for BBa_K817002 PfadBA restriction check, gel extraction and purification
July 9
Wet lab
· Extraction of BBa_J176171, BBa_K817002 PfadBA and pEGFP-N1 by miniprep
· Restriction of pEGFP-N1 Restriction digestion by Ase1 and BamH1 for PPAR-alpha promoter; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1 promoter, Pst1 and Not1; followed by gel check, extraction and purification
· Restriction of BBa_J176171 for PfadBA Ase1 and BamH1; FABP1 promoter by Xba1 and Not1
· Ran gel for pEGFP-N1 and BBa_J176171 restriction products
· PCR PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· BBa_J52034 (fadR) transformation
July 10
Wet lab
· Ran gel for PCR check of PPAR-alpha promoter and FAPB1 promoter
· PCR for FABP1 promoter, PCR replication
· BBa_J176171 vector dephosphorylation by antartic phosphatase
· Ligation of BBa_K817002 and PfadBA and BBa_J176171
· BBa_J52034 (fadR) inoculation
· Digestion for BBa_K817002 PfadBA extraction
Gel check for BBa_K817002 PfadBA extraction
July 11
Wet lab
· Gel purification for BBa_K817002 PfadBA extraction
· Gel check for FABP1 promoter PCR product
· PCR clean-up
· BBa_J52034 (fadR) miniprep
· BBa_J52034 restriction by Pst1 HF and Not1 HF
· PCR for PPAR-alpha promoter
Dry lab
Re-design constructions, now using BBa_J176171 as mammalian expression vector for all related constructions
July 12
Wet lab
· Digestion of FABP1 promoter PCR product
· Ran gel for PCR check of PPAR-alpha promoter
· Digest pEGFP-n1 for BBa_J52034 fadR
· Ran gel for pEGFP-n1 for BBa_J52034 fadR followed by gel purification
· BBa_J176171 vector dephosphorylation by Antarctic phosphatase for FABP1 promoter and PfadBA
Dry Lab
Primers design for pCMV cloning for fadR expression
July 15
Wet lab
· Ligation of FABP1 promoter, EGFP, and BBa_J176171, using 3 pieces ligation
· Transformation of FABP1 promoter, EGFP, and BBa_J176171 ligation
· FABP1 promoter+EGFP +BBa_J176171 ligation restriction check
July 16
Wet Lab
· Miniprep for full construct of FABP1 promoter and pEGFP-N1
· fadR and pEGFP-N1 Backbone parts ligation
· Plasmids extraction for pCMV cloning for fadR
· BBa_K817002 PfadBA promoter extraction by EcoR1 and Pst1 HF
· BBa_J52034 restriction by EcoR1 and Pst1 HF
· Inoculations for full construct of FABP1 promoter and pEGFP-N1
· Streak colonies containing the right construct for FABP1 promoter
July 17
Wet lab
· Plasmid extraction for FABP1 promoter+EGFP+BBa_J176171 by miniprep
· Repeat fadR and PfadBA constructs
· Digest pEGFP-n1 and BBa-J176171 for fadR
· Digestion for BBa_K817002 (PfadBA) extraction
· Gel check and gel extraction for BBa_J176171 for fadR and BBa_K817002 (PfadBA)
July 18
Wet lab
· Gel purification for all digested products
· FABP1 promoter digestion check for whole construct prior transfection
· PfadBA vector de phosphorylation and ligation with EGFP and BBa_J176171
· Transformation of PfadBA+EGFP+BBa_J176171
July 19
Wet lab
· FABP1 promoter preparation for transfection
· Ligation of PfadBA and BBa_J176171 followed by transformation
· PCR for PPAR-alpha promoter cloning
Dry lab
· Design for characterization of the transfected cells with FABP1 construct
· Review for Multiple Sites Mutagenesis
· Ordered primers for pCMV cloning from pEGFP-N1
July 22
Wet lab
· FABP1 construct given for characterization and transfection
· Further colony screening for FABP1 construct, inoculations
· Inoculation of PfadBA and BBa_J176171
· Ran gel for PPAR-alpha promoter PCR products
July 23
Wet lab
· Primers arrival for pCMV cloning
· PCR for pCMV cloning
· Digestion and gel for construction check for FABP1 promoter
· PfadBA gel extraction and purification, followed by vector dephosphorylation BBa_J176171 and ligation, then transformation
· Digestion of pEGFP-N1 for fadR, followed by gel check and extraction
July 24
Wet lab
· DNA purification from gel extraction for digested pEGFP-N1
· Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter
· Ran gel for pCMV cloning from pEGFP-N1
· PCR clean up for pCMV cloning from pEGFP-N1
· PCR for PPAR-alpha promoter with new primers using Taq polymerase
· Transformation of mutagenesis products
· Inoculation of PfadBA ligation colonies
July 25
Wet lab
· Ran gel check PPAR-alpha promoter PCR cloning with new primers using Taq polymerase
· Minprep of PfadBA+EGFP+BBa_J176171 ligation colonies, followed by digestion check
July 26
Wet lab
· Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter
· Ran gel before parental string digestion of mutagenesis products
· Digestion of pEGFP-N1 by Apa1 and Xba1 followed by gel check
· Plasmids preparation according to transfection requirements, construct and controls
July 30
Wet lab
· PPAR-alpha promoter PCR cloning with new primers using Vent polymerase
· Restriction check for pEGFP-N1, using EcoR1, Xba1, Spe1 and Pst1
· BBa_J176171 vector de phosphorylation, followed by ligation with EGFP using T4 Ligase, then transformation into E. Coli strain DH10b
· PCR for PPAR-alpha promoter with new primers using Vent polymerase
Dry lab
· Discussion and re assessment of constructs related to pEGFP-N1
July 31
· Ran gel check for PPAR-alpha promoter PCR cloning with new primers using Vent polymerase
· PCR for pCMV cloning from pEGFP-N1, followed by gel check and PCR clean up
· PCR for PPAR-alpha promoter with reference primers using Vent polymerase
· Ran gel check for PPAR-alpha promoter PCR cloning with reference primers using Vent polymerase
August 2013
August 2
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol
August 5
Wet lab
· Gel Check for PPAR-alpha promoter PCR promoter cloning, using temperature gradient and every available set of primers designed.
· Digestion of BBa_J176171 using single restriction site, Xba1, vector dephosphorylation and ligation with fadR followed by transformation
· Ligation check of PfadBA+BBa_J176171+EGFP using Age1
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Taq polymerase
· gDNA Gel check
Dry lab
· Primers design for BBa_J04450-PSB1C3 Xba1 restriction site removal by site direct mutagenesis
August 6
Wet lab
· Ran gel for PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Taq polymerase
· Inoculation of fadR+BBa_J176171
August 7
Wet lab
· Digestion check of BBa_J176171+EGFP by Not1 and Pst1
· Digestion check of BBa_J176171+fadR by Xba1 and HindIII
· Ran gel for digestion check
· Inoculation of fadR+BBa_J176171, colony screening
August 8
Wet lab
· Miniprep for fadR+BBa_J176171, colony screening
· Digestion check of BBa_J176171+fadR by Xba1 and HindIII
· Ran gel for digestion check
· Inoculation of fadR+BBa_J176171, colony screening
· pCMV from cloned from pEGFP-N1adding restriction sites for ligation with EGFP and BBa_J176171, followed by transformation
August 9
Wet lab
· Primers arrival for BBa_J04450-PSB1C3 for site direct mutagenesis
· Digestion check of BBa_J176171+fadR Xba1 and HindIII, Xba1 and Spe1
· Ran gel for digestion check
· Site direct Mutagenesis for BBa_J04450-PSB1C3 removing Xba1 restriction site, followed by gel check before parental string digestion and transformation
· Inoculation of pCMV from cloned from pEGFP-N1adding restriction sites for ligation with EGFP and BBa_J176171
August 12
Wet lab
· Miniprep of pCMV+EGFP+ BBa_J176171, followed by digestion check and gel
· PCR for pCMV cloning from pEGFP-N1 using Vent polymerase, followed by gel check and PCR clean up
· Digestion check of BBa_J176171+fadR by Xba1, HindIII and Spe1
· Ran gel for digestion check
· pEGFP-N1 digestion for GRP78, extraction pCMV using Ase1 and Xho1, followed by gel check and DNA purification
· Digestion of BBa_J176171 using single restriction site, Xba1, vector dephosphorylation and ligation with fadR followed by transformation
· Site direct mutagenesis for BBa_J04450-PSB1C3, followed by gel check before parental digestion with Dpn1 and transformation
August 13
Wet lab
· Primers phosphorylation for FABP1 promoter Multi-site Mutagenesis using T4 PNK
· Primers phosphorylation for Site Direct Mutagenesis for BBa_J04450-PSB1C3 using T4 PNK
· Site Direct Mutagenesis for BBa_J04450-PSB1C3 using phosphorylated primers and non phosphorylated primers
· Overnight culture for Competent cells
· pDRIVE_hGRP78 arrival, transformation into E. Coli strain DH10b
August 14
Wet lab
· Nothing done due the typhoon
August 15
Wet lab
· PfadBA+pEGFP-N1 ligation, first vector dephosphorylation using Antarctic phosphatase followed by ligation by T4Ligase and transformation into E. Coli SURE strain.
· pCMV+EGFP+BBa_J176171 digestion check, followed by gel check
· Repeat GRP78+pEGFP-N1 ligation, followed by transformation into E. Coli DH10b strain.
· Digestion check for fadR+BBa_J176171 usign Xba1 and HindIII
· Inoculations of fadR+ BBa_J176171, pCMV+EGFP+ BBa_J176171, PfadBA+pEGFP-N1, PfadBA+EGFP+ BBa_J176171 followed by gel check
August 16
Wet lab
· Miniprep of fadR+ BBa_J176171, pCMV+EGFP+ BBa_J176171, PfadBA+pEGFP-N1, PfadBA+EGFP+ BBa_J176171
· Restriction check for fadR+ BBa_J176171 and PfadBA+pEGFP-N1 followed by gel check
· Inoculations of fadR+ BBa_J176171, PfadBA+pEGFP-N1, PfadBA+EGFP+ BBa_J176171
· DH105alpha E. Coli competent cells preparation
August 19
Wet lab
· Repeat GRP78+pEGFP-N1 ligation, followed by transformation into E. Coli DH10b strain.
· Miniprep of fadR+ BBa_J176171, PfadBA+pEGFP-N1, PfadBA+EGFP+ BBa_J176171
· Restriction check for PfadBA+pEGFP-N1 followed by gel check
· Restriction check for fadR+ BBa_J176171 and PfadBA+pEGFP-N1 followed by gel check
· Streak colony with the right fadR+BBa_J176171 construct
· DH5alpha E. Coli competent cells preparation
·
August 20
Wet lab
· Digestion check PfadBA+pEGFP-N1 with Pst1, Xba1. Followed by gel check
· Digestion check for damaged plates of GRP78+pEGFP-N1 using Xba1
· Digestion check of PfadBA+EGFP+ BBa_J176171 using Age1HF, HindIII and Xba1
· Site Direct Mutagenesis for illegal restriction sites for FABP1 promoter
· Inoculate GRP78+pEGFP-N1, pCMV+EGFP+BBa_J176171
· DH10b E. Coli competent cells preparation
August 21
Wet lab
· Miniprep GRP78+pEGFP-N1, followed by digestion check and gel check using Spe1
· Miniprep pCMV+EGFP+BBa_J176171 followed by digestion check using Nde1 and Age1HF
· Digestion check of PfadBA+EGFP+BBa_J176171 using Nde1 , fadR+ BBa_J176171 using Age1
· Site direct Mutagenesis for FABP1 promoter+EGFP+BBa_J176171
· PCR for GRP78 promoter cloning from pDRIVE_hGRP78, followed by gel check PCR clean up, digestion using Ase1 and Xho1 and DNA purification
August 22
Wet lab
· DH10b E. Coli competent cells preparation
· Site direct Mutagenesis for FABP1 promoter+EGFP+BBa_J176171 with phosphorylated primers, followed by gel check before Dpn1 parental string digestion
· Ligation of GRP78 and pEGFP-N1, first vector dephophorylation and ligation with T4Ligase. Transformed into E. Coli DH10b strain
· Transformation of pBlueScript KS (+) in to SURE E. Coli
Dry lab
· Discuss about FABP1 construct vanishing under PCR conditions
August 26
Wet lab
· FABP1 promoter assessment under PCR conditions
· BBa_J176171+EGFP assessment under PCR conditions
· BBa_J176171 assessment under PCR conditions
· Temperature gradient for FABP1 promoter+BBa_J176171+EGFP construct
· Inoculate pBlueScript KS (+)
August 27
Wet lab
· DH10b E. Coli competent cells preparation
August 28
Wet lab
· BBa_J176171 assessment under PCR conditions
· Temperature gradient for FABP1 promoter+BBa_J176171+EGFP construct
· PCR for FABP1 promoter cloning from gDNA
· GRP78 ligation to dephosphorylated pEGFP-N1, ligation done using T4 Ligase, transformed into E.Coli DH10b
· Miniprep pBlueScript KS (+)
· Digest pBlueScript KS (+) with Xba1 and BamH1HF
· Restreak BBa_J176171 and inoculate it
August 29
Wet lab
· Miniprep estreaked BBa_J176171
· GRP78 ligation to dephosphorylated pEGFP-N1, ligation done using T4 Ligase, transformed into E.Coli DH10b
· Ligation of FABP1 promoter in to pBlueScript KS (+) using T4 Ligase followed by transformation into DH10b E. Coli
· Transformation of BBa_J176171 into SURE E. Coli
August 30
Wet lab
· Inoculate GRP78+pEGFP-N1
· Inoculate FABP1 promoter+pBlueScript KS (+)
· Inoculate BBa_J176171
· PCR conditions check for BBa_J176171 looking for heat degradation
August 31
Wet lab
· Miniprep of FABP1 promoter+pBlueScript KS (+), BBa_J176171
· PCR conditions check for BBa_J176171 looking for heat degradation
September 2013
September 1
Wet lab
· Digestion check for FABP1 promoter+pBlueScript KS (+) using EcoR1. Followed by gel check.
September 2
Wet lab
· Digestion check for FABP1 promoter+pBlueScript KS (+) using EcoR1. Followed by gel check.
September 3
Wet lab
· Restriction check from the damaged plates GRP78+pEGFP-N1 using Xba1
· Repeat FABP1 promoter and pBlueScript KS (+) ligation, vector Dephosphorylation by Antartic Phosphatase and ligation using T4 Ligase, followed by transformation into DH10b E. Coli
September 4
Wet lab
· GRP78 promoter PCR Cloning from pDRIVE_hGFRP78, followed by PCR cleanup using kit. Then restriction check using Xba1
· pEGFP-N1 restriction for GRP78 assembly, digested by Ase1 and BamH1, then Gel check, Gel extraction and purification, vector dephosphorilation and ligation by T4 ligase. Transformed into DH10b E. Coli
· Inoculation of FABP1 promoter+ pBlueScript KS (+)
September 5
Wet lab
· Miniprep of FABP1 promoter+pBlueScript KS (+) then restriction check using Ecor1, followed by gel check.
· Site Direct Mutagenesis for FABP1 promoter+pBlueScript KS (+), Gel Check
September 6
Wet lab
· Restriction of GRP78 PCR product by Ase1 and Xho1
· PCR for PPAR-alpha promoter cloning
Dry lab
· Review on previous Mutagenesis attempts and troubleshooting
September 9
Wet lab
· Ran gel for PPAR-alpha promoter cloning
· pEGFP-N1 digestion for PPAR-alpha promoter and vector dephosphorylation
September 10
Wet lab
· Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check
· Site Direct Mutagenesis FABP1 promoter+pBlueScript KS(+) then Gel Check
September 11
Wet lab
· Site Direct Mutagenesis FABP1 promoter+pBlueScript KS(+) then Gel Check
· Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check
· Digest mutant FABP1 promoter+ pBlueScript KS(+) with Dpn1 then transformed into SURE E. Coli
September 12
Wet lab
· GRP78 PCR product digested by Ase1 and Xho1, ligated with dephosphorylated pEGFP-N1 vector and ligated by T4 ligase, transformed into DH10b E. Coli
· Inoculations of mutant FABP1 promoter+pBlueScript KS(+)
September 13
Wet lab
· Miniprep of Mutant FABP1 promoter+ pBlueScript KS(+), then restriction check using Ecor1, ran gel
· Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check
September 14
Wet lab
· Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check