Team:Hong Kong HKUST/wetlab/notebook

From 2014.igem.org

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<p>The wet lab started at the beginning of summer and lasted for three months. Our wet lab work is divided into four parts, namely:  
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(1) (Detecting Module) ComD & ComE, (2) (Detecting Module) P<sub><i>comCDE</i></sub>, (3) (Regulation Module) ComX & ComW, and  
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(4) (Regulation Module) combox. During the summer, lab work such as primer design, digestion, and ligation was carried out extensively
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and useful data were collected. These can be retrieved from Notebook and Protocols pages for details of experimental flows.
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The BioBrick, researcher, environmental and university safety issues can be found in Safety page. In terms of project process,
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some experiments could not be completed due to time constrains and they are outlined in Future Work page.  
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<div id="flight">
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<div id="satu" align="center"> <h1>June 2013</h1>
 +
 
 +
      <div>
 +
      <a href="#" class="head">Week 4</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>June 24</b><br>
 +
Wet lab <br>
 +
·      Inoculation of pBlueScript KS(+) for training <br>
 +
·      Autoclave basic materials <br>
 +
·      Preparing LB <br>
 +
·      LB-Ampicillin plates poured
 +
Dry lab <br>
 +
Protocols review <br>
 +
 
 +
<br><b>
 +
June 25 </b><br>
 +
Wet lab <br>
 +
·  Plasmid extraction of pBlueScript KS(+)  <br>
 +
·      LB-Chloramphenicol plates poured <br>
 +
Dry lab <br>
 +
·      Primers design for FABP1 promoter, PPAR-alpha promoter, GRP78 promoter <br>
 +
·      Informal meeting <br>
 +
 
 +
<br><b>
 +
June 26 </b><br>
 +
Wet lab <br>
 +
·      Extraction of gDNA from HepG2 Cells for FABP1 promoter and PPAR-alpha promoter <br>
 +
·      Transformation of pEGFP-N1 and <a href="http://parts.igem.org/Part:BBa_K817002">BBa_K817002</a> (P<i>fadBA</i>) <br>
 +
 
 +
<br><b>
 +
June 27 </b><br>
 +
Wet lab <br>
 +
·      Inoculation of pEGFP-N1 and BBa_K817002 (P<i>fadBA</i>) for plasmid extraction <br>
 +
 
 +
<br><b>
 +
June 28</b> <br>
 +
Wet lab <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 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>
 +
·      Ran 0.8% gel for pEGFP-N1 and 2% gel for BBa_K817002 (P<i>fadBA</i>) <br>
 +
 
 +
<br><b>
 +
June 30 </b><br>
 +
Dry lab <br>
 +
Experiment planning and protocols revision for next week work <br><br>
 +
 
 +
      </div>
 +
      </div>
 +
 
 +
</div>
 +
</div>
 +
<div id="satu" align="center"> <h1><center>July 2013</center></h1>
 +
 
 +
  <div>
 +
      <a href="#" class="head">Week 1</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br> <b> July 2 </b> <br>
 +
Wet lab <br>
 +
·      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>
 +
·      Inoculation of pEGFP-N1 for plasmid extraction <br>
 +
·      Transformation of P<i>fadBA</i> due chloramphenicol plates contamination <br>
 +
 
 +
<br> <b> July 3 </b> <br>
 +
·      Extraction of pEGFP-N1 plasmids by miniprep <br>
 +
·      PCR for PPAR-alpha promoter amplification <br>
 +
·      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>
 +
Wet lab<br>
 +
·      Inoculation of BBa_K817002  P<i>fadBA</i><br>
 +
·      PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br>
 +
·      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>
 +
Wet lab<br>
 +
·      Extraction of BBa_K817002 (P<i>fadBA</i>) <br>
 +
·      Inoculation of pEGFP-N1<br>
 +
·      PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br>
 +
·      Gel check, 0.8% gel for previously gDNA extraction<br>
 +
·      Ran gel for PCR check of PPAR-alpha promoter and FABP1 promoter<br>
 +
·      Poured new LB-Kanamycin plates<br>
 +
·      Transformation of <a href="http://parts.igem.org/Part:BBa_J176171">BBa_J176171</a><br>
 +
Dry lab<br>
 +
·      Discussion about re-design constructions, possible change for BBa_J176171 as mammalian expression vector<br>
 +
·      Primer redesign for PPAR-alpha promoter and FABP1 promoter<br><br>
 +
 
 +
 
 +
      </div>
 +
  </div>
 +
 
 +
  <div>
 +
      <a href="#" class="head">Week 2</a>
 +
      <div class="content" align="left">
 +
<br> <b> July 8</b> <br>
 +
Wet lab<br>
 +
·      gDNA extraction from HepG2 cells<br>
 +
·      Restriction of BBa_K817002 P<i>fadBA</i><br>
 +
·      Gel check for gDNA extraction<br>
 +
·      Inoculation of BBa_J176171, BBa_K817002 (P<i>fadBA</i>), pEGFP-N1 for plasmid extraction<br>
 +
·      New primers for PPAR-alpha promoter and FABP1 promoter arrival<br>
 +
·      Ran gel for BBa_K817002 P<i>fadBA</i> restriction check, gel extraction and purification<br>
 +
 
 +
<br> <b> July 9</b> <br>
 +
Wet lab<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 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>
 +
·      Ran gel for pEGFP-N1 and BBa_J176171 restriction products<br>
 +
·      PCR PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br>
 +
·      <a href="http://parts.igem.org/Part:BBa_J52034">BBa_J52034</a> (<i>fad</i>R) transformation<br>
 +
 
 +
<br> <b> July 10</b> <br>
 +
Wet lab<br>
 +
·      Ran gel for PCR check of PPAR-alpha promoter and FAPB1 promoter<br>
 +
·      PCR for FABP1 promoter, PCR replication<br>
 +
·      BBa_J176171 vector dephosphorylation  by antartic phosphatase<br>
 +
·      Ligation of BBa_K817002 and P<i>fadBA</i> and BBa_J176171 <br>
 +
·      BBa_J52034 (<i>fad</i>R) inoculation<br>
 +
·      Digestion for BBa_K817002 P<i>fadBA</i> extraction<br>
 +
Gel check for BBa_K817002 P<i>fadBA</i> extraction<br>
 +
 
 +
<br> <b> July 11</b> <br>
 +
Wet lab<br>
 +
·      Gel purification for BBa_K817002 P<i>fadBA</i> extraction<br>
 +
·      Gel check for FABP1 promoter PCR product<br>
 +
·      PCR clean-up<br>
 +
·      BBa_J52034 (<i>fad</i>R) miniprep<br>
 +
·      BBa_J52034 restriction by Pst1 HF and Not1 HF<br>
 +
·      PCR  for PPAR-alpha promoter<br>
 +
Dry lab<br>
 +
Re-design constructions, now using BBa_J176171 as mammalian expression vector for all related constructions<br>
 +
 
 +
<br> <b> July 12</b> <br>
 +
Wet lab<br>
 +
·  Digestion of FABP1 promoter PCR product<br>
 +
·      Ran gel for PCR check of PPAR-alpha promoter<br>
 +
·      Digest pEGFP-n1 for BBa_J52034 <i>fad</i>R<br>
 +
·      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 P<i>fadBA</i><br>
 +
Dry Lab<br>
 +
Primers design for pCMV cloning for <i>fad</i>R expression<br><br>
 +
      </div>
 +
  </div>
 +
 
 +
  <div>
 +
      <a href="#" class="head">Week 3</a>
 +
      <div class="content" align="left">
 +
<br><b>July 15</b> <br>
 +
Wet lab<br>
 +
·      Ligation of FABP1 promoter, EGFP, and BBa_J176171, using 3 pieces ligation<br>
 +
·      Transformation of  FABP1 promoter, EGFP, and BBa_J176171 ligation<br>
 +
·      FABP1 promoter+EGFP +BBa_J176171 ligation restriction check<br>
 +
 
 +
<br><b>July 16</b> <br>
 +
Wet Lab<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 <i>fad</i>R<br>
 +
·      BBa_K817002 P<i>fadBA</i> promoter extraction by EcoR1 and Pst1 HF<br>
 +
·      BBa_J52034 restriction by EcoR1 and Pst1 HF<br>
 +
·      Inoculations for full construct of FABP1 promoter and pEGFP-N1<br>
 +
·      Streak colonies containing the right construct for FABP1 promoter<br>
 +
 
 +
<br><b>July 17</b> <br>
 +
Wet lab<br>
 +
·      Plasmid extraction for FABP1 promoter+EGFP+BBa_J176171 by miniprep<br>
 +
·      Repeat <i>fad</i>R and P<i>fadBA</i> constructs<br>
 +
·      Digest pEGFP-n1 and BBa-J176171 for <i>fad</i>R<br>
 +
·      Digestion for BBa_K817002 (P<i>fadBA</i>) extraction<br>
 +
·      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>
 +
Wet lab<br>
 +
·      Gel purification for all digested products<br>
 +
·      FABP1 promoter digestion check for whole construct prior transfection<br>
 +
·      P<i>fadBA</i> vector de phosphorylation and ligation with EGFP and BBa_J176171<br>
 +
·      Transformation of P<i>fadBA</i>+EGFP+BBa_J176171<br>
 +
 
 +
<br><b>July 19</b> <br>
 +
Wet lab<br>
 +
·      FABP1 promoter preparation for transfection<br>
 +
·      Ligation of P<i>fadBA</i> and BBa_J176171 followed by transformation<br>
 +
·      PCR for PPAR-alpha promoter cloning<br>
 +
Dry lab<br>
 +
·      Design for characterization of the transfected cells with FABP1 construct<br>
 +
·      Review for Multiple Sites Mutagenesis<br>
 +
·      Ordered primers for pCMV cloning from pEGFP-N1<br><br>
 +
 
 +
      </div>
 +
  </div>
 +
 
 +
  <div>
 +
      <a href="#" class="head">Week 4</a>
 +
      <div class="content" align="left">
 +
<br><b>July 22</b> <br>
 +
Wet lab<br>
 +
·  FABP1 construct given for characterization and transfection<br>
 +
·  Further colony screening for FABP1 construct, inoculations<br>
 +
·  Inoculation of P<i>fadBA</i> and BBa_J176171<br>
 +
·  Ran gel for PPAR-alpha promoter PCR products<br>
 +
 
 +
<br><b>July 23</b> <br>
 +
Wet lab<br>
 +
·  Primers arrival for pCMV cloning<br>
 +
·  PCR for pCMV cloning<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>
 +
·  Digestion of pEGFP-N1 for <i>fad</i>R, followed by gel check and extraction<br>
 +
 
 +
<br><b>July 24</b> <br>
 +
Wet lab<br>
 +
·      DNA purification from gel extraction for digested pEGFP-N1<br>
 +
·      Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter<br>
 +
·      Ran gel for pCMV cloning from pEGFP-N1<br>
 +
·      PCR clean up for pCMV cloning from pEGFP-N1<br>
 +
·      PCR for PPAR-alpha promoter with new primers using Taq polymerase<br>
 +
·      Transformation of mutagenesis products<br>
 +
·      Inoculation of P<i>fadBA</i> ligation colonies<br>
 +
 
 +
<br><b>July 25</b> <br>
 +
Wet lab<br>
 +
·      Ran gel check PPAR-alpha promoter PCR cloning with new primers using Taq polymerase<br>
 +
·      Minprep of P<i>fadBA</i>+EGFP+BBa_J176171 ligation colonies, followed by digestion check <br>
 +
 
 +
<br><b>July 26</b> <br>
 +
Wet lab<br>
 +
·      Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter<br>
 +
·      Ran gel before parental string digestion of mutagenesis products<br>
 +
·      Digestion of pEGFP-N1 by Apa1 and Xba1 followed by gel check<br>
 +
·      Plasmids preparation according to transfection requirements, construct and controls<br><br>
 +
 
 +
      </div>
 +
  </div>
 +
 
 +
  <div>
 +
      <a href="#" class="head">Week 5</a>
 +
      <div class="content" align="left">
 +
<br><b>July 30</b> <br>
 +
Wet lab<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>
 +
·      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>
 +
Dry lab<br>
 +
·      Discussion and re assessment of constructs related to pEGFP-N1<br>
 +
 
 +
 
 +
<br><b>July 31</b> <br>
 +
·      Ran gel check for PPAR-alpha promoter PCR cloning with new primers using Vent polymerase<br>
 +
·      PCR for pCMV cloning from pEGFP-N1, followed by gel check and PCR clean up<br>
 +
·      PCR for PPAR-alpha promoter with reference primers using Vent polymerase<br>
 +
·      Ran gel check for PPAR-alpha promoter PCR cloning with reference primers using Vent polymerase<br><br>
 +
 
 +
      </div>
 +
  </div>
 +
</div>
 +
 
 +
<div id="satu" align="center"> <h1>August 2013</h1>
 +
 
 +
<div>
 +
      <a href="#" class="head">Week 1</a>
 +
      <div class="content" align="left">
 +
<br><b>August 2</b> <br>
 +
Wet lab<br>
 +
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
 +
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
 +
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
 +
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
 +
 
 +
      </div>
 +
    </div>
<table class= "site_map_table" align= "center">
<table class= "site_map_table" align= "center">
<tr>
<tr>

Revision as of 03:43, 1 October 2014

Notebook

June 2013

Week 4

July 2013

Week 1
Week 2

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

Week 3

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

Week 4

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

Week 5

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

Week 1

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

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