Team:DTU Denmark/notebookredoxilator

From 2009.igem.org

(Difference between revisions)

Latest revision as of 02:31, 22 October 2009

Welcome to the DTU iGEM wiki!

Notebook

Activities relating to our two sub-projects:

- The Redoxilator
- The USER^TM assembly standard
- Biobricks
- Protocols

Day-to-day activities

Activities relating to the redoxilator
For more detailed information see results.

Thursday 15/10/2009
BioLector experiment initiated! Lets get some results!

Wednesday 14/10/2009
To have inoculum for the BioLector experiment, colonies were picked from our plates and grown till tomorrow.

Monday 5/10/2009
The yeasts were streak diluted onto a new plate to be sure that we have a single transformant.

Thursday 1/10/2009
Our redoxilator construct and leaky construct were ready for yeast transformation into CEN PK 113 5D (Ura-) using the URA marker present these plasmids.

Wednesday 30/9/2009
The resulting vectors were purification. The presence of our leaky redoxilator construct was verified by digestion with XmaI.

Tuesday 29/9/2009
Liquid over night colonies were prepared. Cultures were randomly picked using a sterile pipette tip from the transformation plate and incubated into liquid media containing kanamycin.

Monday 28/9/2009
To test how leaky our redoxilator construct was, the redoxilator part was removed by NotI digested followed by gel-purification. The backbone missing the redox sensor/activator was ligated and transformed into E. coli.

Friday 25/9/2009
Colonies seen!

Thursday 24/9/2009
Our dear redoxilator construct arrieved from California (DNA 2.0) The DNA was extracted from filter paper using MilliQ water and transformed into E. Coli.

June 2009
The genetic design is now ready, and it should work according to the newly finished model.

Since January 2009...
Figuring out the project idea took a while and meetings among the team members started early this year.

Work process

Our team works parallel in smaller sub-teams. Some of us work hard in the lab, while others are in the process of developing software and the in silico model of the Redoxilator system. However, we constantly keep each other updated, and meet often to exchange ideas and take turns at the different tasks, thus exhausting all of our combined knowledge in every aspect of this project.

Comments or questions to the team? Please Email us -- Comments of questions to webmaster? Please Email us

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      <a href="https://2009.igem.org/Team:DTU_Denmark/notebookredoxilator" CLASS=leftbar>- The Redoxilator</a><br>
      <a href="https://2009.igem.org/Team:DTU_Denmark/notebookuserfusion" CLASS=leftbar>- The USER<sup>TM</sup> assembly standard</a><br>
+    <a href="https://2009.igem.org/Team:DTU_Denmark/notebookbiobrick" CLASS=leftbar>- Biobricks</a><br>
+    <a href="https://2009.igem.org/Team:DTU_Denmark/protocols" CLASS=leftbar>- Protocols</a><br>
     </font>
    </td>
@@ Line 180: / Line 183: @@
    </font>
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 <!-- INSERT MAIN TEXT HERE! (formatting: <b>bold</> <i>italic> <h4>header</h4>) -->
-<font size="4"><b>Activities relating to the redoxilator</b></font><br><br>
+<font size="4"><b>Activities relating to the redoxilator</b></font><br>
+For more detailed information see <a href="https://2009.igem.org/Team:DTU_Denmark/results"> results</a>. <br><br>
-<font size="3"><b>July 20th</b></font><br>
+<font size="3"><b> Thursday 15/10/2009 </b></font><br>
-<p align="justify">Construction of biobricks: The restriction products were evaluated on a 1% agarose gel (see figure 2).</p><br>
+BioLector experiment initiated! Lets get some results!
+<br><br>
-</html>
+<font size="3"><b> Wednesday 14/10/2009 </b></font><br>
-[[image:DTUgel1.jpg|300px|center|]]
+To have inoculum for the BioLector experiment, colonies were picked from our plates and grown till tomorrow.
-<html>
+<br><br>
-<p align="center"><i>Figure 2: Restriction analysis of the three biobricks.</i><br><br></p>
-<font size="3"><b>July 19th</b></font><br>
+<font size="3"><b> Monday 5/10/2009 </b></font><br>
-<p align="justify">Construction of biobricks: The three plasmids were purified according to the protocol (link to protocol). Following the purification of the plasmids, a restriction analysis was performed using SpeI enzyme (10U) and incubated at 37ºC overnight.</p><br>
+The yeasts were streak diluted onto a new plate to be sure that we have a single transformant.
+<br><br>
-<font size="3"><b>July 18th</b></font><br>
+<font size="3"><b> Thursday 1/10/2009 </b></font><br>
-<p align="justify">Construction of biobricks: Some colonies were picked from the transformation plates and made overnight cultures in liquid media.</p><br>
+Our redoxilator construct and <i>leaky</i> construct were ready for yeast transformation into CEN PK 113 5D (Ura-) using the URA marker present these plasmids.<br><br>
-<font size="3"><b>July 17th</b></font><br>
+<font size="3"><b> Wednesday 30/9/2009 </b></font><br>
-<p align="justify">Construction of biobricks: Following purification, all of the PCR product was digested with <i>Xba</i>I (60U) and <i>Spe</i>I (30U). The mixtures were incubated at 37ºC. After 8 hours of incubation, more enzyme was added (<i>Xba</i>I (20 U) and <i>Spe</i>I (10U)) to ensure maximum digestion. Following enzymatic purification a gel was run to estimate the concentration of the vector and the PCR product (see figure 1).</p><br>
+The resulting vectors were purification. The presence of our leaky redoxilator construct was verified by digestion with XmaI.<br><br>
-</html>
+<font size="3"><b> Tuesday 29/9/2009 </b></font><br>
-[[image:DTUgel2.jpg|300px|center|]]
+Liquid over night colonies were prepared. Cultures were randomly picked using a sterile pipette tip from the transformation plate and incubated into liquid media containing kanamycin. <br><br>
-<html>
-<p align="center"><i>Figure 1: Electrophoresis gel picture of the PCR products of the three biobricks and the vector. The band 11P at 1500 bp corresponds to the vector; the three other bands at 537 bp, 207 bp and 125 bp correspond to the fragments Cln, GFP and Cln_GFP respectively.</i><br><br>
-<p align="justify">To meet the 3:1 ratio of fragment to vector 12μl fragment was mixed with 2μl vector, 2μl ligase buffer (10x), 1μl ligase, 3 μl Mq H20. The ligation of the fragment and the vector was carried out on PCR machine using a 3:1 ratio for 1.5 hours using the idea by Lund, A. H., M. Duch, and F. S. Pedersen. 1996. Increased cloning efficiency by temperature-cycle ligation. Nucleic Acids Res. 24:800-801. doi:l50400 [pii].
-The final product was finally transformed into competent E coli cells.</p><br>
-<font size="3"><b>July 16th</b></font><br>
+<font size="3"><b> Monday 28/9/2009 </b></font><br>
-<p align="justify">Construction of biobricks: The 357bp Cln2 fragment was amplified by PCR using the primers BGHA351/BGHA362 and Phusion polymerase. Primers BGHA368/369 were used to amplify the GFP fragment and finally, primers BGHA368/353 were used to amplify the fragment containing the GFP fused with the Cln. Following amplification the PCR products were run on a 1% agarose gel and purified  The primers added tails to the PCR product containing an <i>Xba</i>I site in the forward primer and a <i>Spe</i>I in the reverse primer to meet the “Silver fusion” standard BBF RFC 23.<br>
+To test how leaky our redoxilator construct was, the redoxilator part was removed by NotI digested followed by gel-purification. The backbone missing the redox sensor/activator was ligated and transformed into <i>E. coli</i>.<br><br>
-<a href="http://dspace.mit.edu/bitstream/handle/1721.1/32535/PhillipsSilverFusion.pdf?sequence=1">Article: "A New Biobrick Assembly Strategy Designed for Facile Protein Engineering"</a><br>
-</p><br>
-<font size="3"><b>July 15th</b></font><br>
+<font size="3"><b> Friday 25/9/2009 </b></font><br>
-<p align="justify">Construction of biobricks: The plasmid was purified according to the protocol (link to protocol).
+Colonies seen!<br><br>
-Following purification, the plasmid was digested using two enzymes, XbaI (20U) and SpeI (10U). The mixture was incubated at 37ºC. 8 hours after incubation, more enzyme was added to ensure maximum digestion as well as Antartic phosphatase (25U). After one hour the mixture was run on a 1% agarose gel and the 2kb backbone was purified (link to protocol).<br><br>
-Once we had the backbone where all three biobricks were going to be cloned, there was nothing left missing to start working on the biobricks.
-<br><br>
-All three biobricks were constructed by PCR from the pSVA13, a plasmid which was kindly obtained from Dr. Simon V. Avery at the University of Nottingham.
-</p><br>
-<font size="3"><b>July 14th</b></font><br>
+<font size="3"><b> Thursday 24/9/2009 </b></font><br>
-<p align="justify">Construction of biobricks: Some colonies were picked from the transformation plate and made overnight cultures in liquid media.</p><br>
+Our dear redoxilator construct arrieved from California (DNA 2.0)
+The DNA was extracted from filter paper using MilliQ water and transformed into <i>E. Coli</i>.<br><br>
-<font size="3"><b>July 13th</b></font><br>
+<font size="3"><b> June 2009 </b></font><br>
-<p align="justify">Construction of biobricks: We chose <a href="http://partsregistry.org/Part:pSB1A2">pSB1A2 </a>  from iGEM spring 2009 DNA collection Kit Plate (well 11P). The crystallized DNA was ressuspended and transformed into competent E. coli cells (link to protocol)
+The genetic design is now ready, and it should work according to the newly finished model. <br><br>
-</p><br>
+<font size="3"><b> Since January 2009... </b></font><br>
+Figuring out the project idea took a while and meetings among the team members started early this year.