Team:NTNU-Trondheim/Notebook

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    <title>Trondheim iGEM 2013 </title>
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{{#calendar: year=2013 | title=Team:NTNU-Trondheim }}
 
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<div id="top-section3"><center><img src="https://static.igem.org/mediawiki/2013/2/21/Logo2_NTNU.png" alt="header" border="0"  usemap="#igemmap" width="1000" height="400"></center></div>
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<div id='cssmenu'>
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<ul>
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  <li class='active'><a href='https://2013.igem.org/Team:NTNU-Trondheim'><span>Home</span></a></li>
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  <li class='has-sub'><a href='#'><span>Project</span></a>
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      <ul>
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        <li><a href='https://2013.igem.org/Team:NTNU-Trondheim/Project'><span>Project description</span></a></li>
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        <li><a href='https://2013.igem.org/Team:NTNU-Trondheim/novelapproach'><span>A novel approach</span></a></li>
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        <li><a href='https://2013.igem.org/Team:NTNU-Trondheim/Model'><span>Modelling</span></a></li>
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        <li><a href='https://2013.igem.org/Team:NTNU-Trondheim/Experiments_and_Results'><span>Experiments and Results</span></a></li>
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        <li><a href='https://2013.igem.org/Team:NTNU-Trondheim/Parts'><span>BioBrick Parts</span></a></li>
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        <li class='last'><a href='https://2013.igem.org/Team:NTNU-Trondheim/Acknowledgements'><span>Acknowledgements</span></a></li>
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      </ul>
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  </li>
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  <li class='has-sub'><a href='#'><span>Technical Stuff</span></a>
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      <ul>
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        <li><a href='https://2013.igem.org/Team:NTNU-Trondheim/Notebook'><span>Notebooks</span></a></li>
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        <li><a href='https://2013.igem.org/Team:NTNU-Trondheim/Protocols'><span>Protocols</span></a></li>
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        <li><a href='https://2013.igem.org/Team:NTNU-Trondheim/Primers'><span>Primers</span></a></li>
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        <li class='last'><a href='https://2013.igem.org/Team:NTNU-Trondheim/Safety'><span>Safety</span></a></li>
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      </ul>
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  </li>
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  <li class='has-sub'><a href='#'><span>Team</span></a>
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      <ul>
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        <li><a href='https://2013.igem.org/Team:NTNU-Trondheim/Team'><span>Meet the team</span></a></li>
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        <li class='last'><a href='https://igem.org/Team.cgi?id=1082'><span>Official Team Profile</span></a></li>
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      </ul>
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  </li>
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  <li class='has-sub'><a href='https://2013.igem.org/Team:NTNU-Trondheim/Outreach'><span>Outreach</span></a>
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  </li>
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    <li class='has-sub'><a href='#'><span>Judging</span></a>
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    <ul>
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    <li><a href='https://2013.igem.org/Team:NTNU-Trondheim/Achievements'><span>Achievements</span></a></li>
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    <li class='last'><a href='https://2013.igem.org/Team:NTNU-Trondheim/Medalcriteria'><span>Medal criteria</span></a></li>
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    </ul>
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    </li>
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  <li class='last'><a href='https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker'><span>Matchmaker</span></a></li>
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</ul>
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</div>
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<div> <div class ="row-end"> </div>
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</div>
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</div>
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<div id="Default">
 
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== Notebook ==
 
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<div class="row">
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<div class="col12" id="spacer3"></div>
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<div class="row-end"> </div>
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[https://2013.igem.org/Team:NTNU-Trondheim/TransformedBricks Transformed BioBricks]
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<div class="col8" align = "center" style="background-color:#C3A7F3;>
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<p style="text-align:center; color:black; "> Notebook</p> </div>
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<div class ="row-end"> </div>
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</div>
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====Tuesday 18.06.13====
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<a href="https://2013.igem.org/Team:NTNU-Trondheim/Notebook/June">June</a><br>
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<a href="https://2013.igem.org/Team:NTNU-Trondheim/Notebook/July">July</a><br>
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<a href="https://2013.igem.org/Team:NTNU-Trondheim/Notebook/August">August</a><br>
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<a href="https://2013.igem.org/Team:NTNU-Trondheim/Notebook/September">September</a><br>
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<a href="https://2013.igem.org/Team:NTNU-Trondheim/Notebook/October">October</a><br>
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<br></br>
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<a href="https://2013.igem.org/Team:NTNU-Trondheim/TransformedBricks">Transformed Biobrick</a><br>
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Started transforming BioBricks. Transformed some composite BioBricks consisting of constitutive promotor, RBS, a fluorescent protein and a terminator. Transformed the parts Promoter+RBS+RFP+term (<partinfo>BBa_I13521</partinfo>), Promoter+RBS+GFP+term (<partinfo>BBa_I13522</partinfo>), Promoter+RBS+CFP+term (<partinfo>BBa_I13600</partinfo>) and P<sub>m</sub>+RBS+mCherry+term to competent ''E.coli'' DH5&alpha; cells, to have some colorful cells to show the camera crew from NRK (Norwegian broadcasting), who will visit our lab tomorrow :-)
 
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For our transformation protocol, see the [https://2013.igem.org/Team:NTNU-Trondheim/Protocols protocol page].
 
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----
 
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====Wednesday 19.06.13====
 
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We transformed BioBricks consisting of fluorescent proteins. Transformed the parts YFP (<partinfo>BBa_E0030</partinfo>), CFP (<partinfo>BBa_E0020</partinfo>), RFP (<partinfo>BBa_E1010</partinfo>), GFP (<partinfo>BBa_E0040</partinfo>), BFP (<partinfo>BBa_K592100</partinfo>) and SYFP (<partinfo>BBa_K864100</partinfo>).
 
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We transferred mCherry from agar plate to liquid medium and incubated at 37&deg; C.
 
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----
 
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====Thursday 20.06.13====
 
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We transferred the transformed cells from yesterday to liquid medium and incubated at 37&deg;C [https://2013.igem.org/Team:NTNU-Trondheim/Protocols protocol].
 
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We isolated the DNA from the transformed mCherry cells by using the [https://2013.igem.org/Team:NTNU-Trondheim/Protocols miniprep protocol] and measured the concentration by [http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf NanoDrop ND-1000 Spectrophotometer].
 
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{|border="1"
 
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|Sample
 
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|Concentration (ng/&micro;l)
 
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|-
 
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|mCherry1
 
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|5,6
 
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|-
 
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|mCherry2
 
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|7,3
 
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|}
 
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----
 
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====Friday 21.06.13====
 
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We isolated the DNA from the transformed cells by using the [https://2013.igem.org/Team:NTNU-Trondheim/Protocols miniprep protocol] and measured the concentration by [http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf NanoDrop ND-1000 Spectrophotometer].
 
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{|border="1"
 
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|Sample
 
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|Concentration (ng/&micro;l)
 
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|-
 
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|CFP1
 
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|8,59
 
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|-
 
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|CFP2
 
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|7,04
 
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|-
 
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|BFP
 
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|7,06
 
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|-
 
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|SYFP
 
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|5,42
 
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|-
 
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|RFP1
 
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|5,04
 
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|-
 
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|RFP2
 
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|7,52
 
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|-
 
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|YFP
 
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|6,79
 
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|-
 
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|GFP
 
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|5,27
 
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|}
 
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====Monday 24.06.13 - Friday 28.06.13====
 
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This week we have established protocols for further work and designed primers for PCR.
 
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====Monday 01.07.13====
 
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We transformed some BioBricks: pBAD promoter (<partinfo>BBa_K206000</partinfo>), RBS (<partinfo>BBa_J61101</partinfo>) and a plasmid backbone  (<partinfo>BBa_J01101</partinfo>).
 
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====Tuesday 02.07.2013====
 
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This day we started making the small scale vesicle preparation according to our vesicle isolation protocol.
 
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Overnight cell cultures of ''E.coli'' ER2566 and ''E.coli'' BW27784 transformed with the pUM9 plasmid were preprared. The pUM9 plasmid has amp^R induces a stress respons in ''E.coli'' when arabinose is present. According to the litterature (link?) stress in ''E.coli'' increases vesicle production.
 
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The ER2566 cell were cultured in plain LB, while the BW27784 cells were cultured in LB with ampenicillin (100 &micro;g/mL).
 
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We transferred the samples of RBS, pBAD and the plasmid backbone (pTet) from agar plate to liquid medium and incubated at 37&deg; C.
 
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----
 
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====Wednesday 03.07.2013====
 
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The small-scale vesicle preparation continued with completion of step 2-7 in the vesicle isolation protocol. There where made three samples in step 2; ''E.coli'' ER2566 in LB, ''E.coli'' BW27784 in LB with ampenicillin (100 &micro;g/mL) (ara-) and ''E.coli'' BW27784 in LB with ampenicillin (100 &micro;g/mL) with the addition of arabinose (0.5 %) after 1 hour of incubtion (ara+).
 
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Optical denisty (OD) at 600 nm was mesured as indicated in the table below.
 
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{|border="l"
 
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|Sample
 
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|OD<sub>600</sub>
 
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|-
 
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|ER2566
 
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|0.9805
 
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|-
 
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|ara-
 
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|0.1263
 
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|-
 
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|ara+
 
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|0.0471
 
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|}
 
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We isolated the DNA from the transformed cells (with RBS, pBAD and pTet) by using the [https://2013.igem.org/Team:NTNU-Trondheim/Protocols miniprep protocol] and measured the concentration by [http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf NanoDrop ND-1000 Spectrophotometer].
 
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{|border="1"
 
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|Sample
 
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|Concentration (ng/&micro;l)
 
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|-
 
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|RBS
 
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|4,91
 
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|-
 
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|pTet1
 
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|8,99
 
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|-
 
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|pTet2
 
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|4,39
 
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|-
 
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|pBAD1
 
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|3,54
 
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|-
 
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|pBAD2
 
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|9,24
 
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|}
 
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====Thursday 04.07.2013====
 
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Continuation of the small-scale vesicle preparation according to the vesicle isolation protocol. Step 8-12 was completed with the exception that the pellet was resuspended in 0.5 mL DPBSS insted of 100 &micro;L in step 11 and that the check for sterility in step 12 was not performed. The SDS-PAGE showed no signs of vesicles.
 
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----
 
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====Monday 08.07.10====
 
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An overnight culture was started for vesicle isolation as desribed for tuesday 02.07.10.
 
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====Tuesday 09.07.2013====
 
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Today we ran PCR on 4 FP's <partinfo>Bba_K864100</partinfo>, <partinfo>Bba_K592100</partinfo>, <partinfo>Bba_E0040</partinfo>, <partinfo>Bba_E1010</partinfo> and 1 backbone <partinfo>Bba_J01101</partinfo>. We used the Phusion DNA Polymerase protocol from New England Biolabs [https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530] for 50 ul sample(without DMSO). All amplicons were run on a GelGreen 0.8% agarose gel. We only got a band on our RFP <partinfo>Bba_E1010</partinfo>, and it was not the expected size. We will adjust the PCR parameters and run them again tomorrow.
 
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----
 
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====Wednesday 10.07.2013====
 
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We ran the PCR again with different parameters. Increased annealing- and elongationtime and a separate program for the backbone due to its larger size. We still only got a band for RFP <partinfo>Bba_E1010</partinfo>. In the prosess of rechecking the primers it was discovered that the primers for RFP are incorrect which explains the discrepancy in productsize. The other primers should be working so tomorrow we will redo the dilutions of the primers, increase the elongation time and lower the annealing temperature a little, add DMSO, add a bit larger templatesample and use 1-step PCR.
 
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====Thursday 11.07.2013====
 
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Today we ran PCR again. This time we omitted the RFP, since we discovered that one of the primers was incorrect. We diluted the primers one more time and changed the parameters for the PCR. This time we only did one step, with 30 cycles. We also decreased the annealing temperature and increased the elongationtime. The results were more uplifting this time around after running gel electrophoreses (picture coming soon). We got a clear band on the backbone and faint bands on the GFP and SYFP. Since there was a faint band on the backbone of a larger size than the one we wanted, we added 1 &micro;l of Dpn1 to the amplicon and put it in a 37 &deg;C waterbath for an hour. We used the QIAGEN PCR purification kit to isolate the DNA and measured the cioncentration by  [http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf NanoDrop ND-1000 Spectrophotometer].
 
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{|border="1"
 
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|Sample
 
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|Concentration (ng/&micro;l)
 
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|-
 
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|SYFP
 
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|13,16
 
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|-
 
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|GFP
 
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|55,62
 
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|-
 
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|BB
 
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|55,50
 
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|}
 
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Latest revision as of 22:45, 4 October 2013

Trondheim iGEM 2013

header
Mercury
Notebook

June
July
August
September
October


Transformed Biobrick


Retrieved from "http://2013.igem.org/Team:NTNU-Trondheim/Notebook"