Difference between revisions of "Part:BBa K1616005:Experience"

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===Applications of BBa_K1616005===
 
===Applications of BBa_K1616005===
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<h2> Notebook </h2>
 
<h2> Notebook </h2>
 +
Gblocks: ordered of Holin/ Endolysin fragments from IDT <br>
 +
<br><br>
 +
<h3><font style="color:#b22222">PCR of Holin/Endolysin parts</font></h3> <br>
 +
<table id="Mixligation" style="    width: 80%;"><tr>
 +
<th>
 +
 +
</th>
 +
<th>
 +
MQ Water
 +
</th>
 +
<th>
 +
RB Buffer
 +
</th>
 +
<th>
 +
Mg<sup>2+</sup>
 +
</th>
 +
<th>
 +
dNTP 10 µM
 +
</th>
 +
<th>
 +
Primer Fwd 25 µM
 +
</th>
 +
<th>
 +
Primer Rev 25 µ
 +
</th>
 +
<th>
 +
DNA
 +
</th><th>
 +
Taq Pol enzyme
 +
</th>
 +
</tr>
 +
 +
<tr><td><font style="color:#b22222">Holin - Ensolysin </font></td>
 +
<td>
 +
<center>39 µL</center>
 +
</td>
 +
 +
<td>
 +
<center>5 µL</center>
 +
</td>
 +
 +
 +
<td>
 +
<center>1,5 µL</center>
 +
</td>
 +
 +
<td>
 +
<center>1 µL</center>
 +
</td>
 +
 +
<td>
 +
<center>1µL pSB1C3 Fwd</center>
 +
</td>
 +
<td>
 +
<center>1µL pSB1C3 Rev</center>
 +
</td>
 +
<td>
 +
<center>1 µL</center>
 +
</td>
 +
<td>
 +
<center>0,5 µL</center>
 +
</td>
 +
 +
</tr></table>
 +
 +
 +
  
 +
<br><br>
 
<h3><font style="color:#b22222">Ligation of Gblocks Holin-Endolysin into pDawn </font></h3>
 
<h3><font style="color:#b22222">Ligation of Gblocks Holin-Endolysin into pDawn </font></h3>
  

Revision as of 00:13, 23 September 2015

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1616005

Notebook

Gblocks: ordered of Holin/ Endolysin fragments from IDT


PCR of Holin/Endolysin parts


MQ Water

RB Buffer

Mg2+

dNTP 10 µM

Primer Fwd 25 µM

Primer Rev 25 µ

DNA

Taq Pol enzyme

Holin - Ensolysin
39 µL
5 µL
1,5 µL
1 µL
1µL pSB1C3 Fwd
1µL pSB1C3 Rev
1 µL
0,5 µL





Ligation of Gblocks Holin-Endolysin into pDawn

Tube

Water

T4 ligase Buffer

pDawn

Plasmid

T4 ligase

Holin - Ensolysin
4,6 µL
1 µL
1,4 µL
2,5 µL
0,5 µL



Room temperature, 30min heat kill: 80°C, 20min

Transformation of E.coli DH5 alpha competent cells



Results

Graph-HE.png


Bacteria expressing pDawn-HE were prepared in a pre-culture until DO600 reached a value between 0,6 and 0,8. Then, one condition was illuminated with white-light and incubated at 37°C, another one was kept in the dark and incubated at 37°C and the last condition was illuminated with white-light and incubated at room temperature. The value of DO was taken regularly to observe the kinetic of growth under each condition.

In absence of light, and at 37°C, bacteria growth was normal but light illumination was enough to slow-down the growth of bacteria. Incubation at room temperature with white-light illumination allowed better folding of HE and had a stronger effect on the growth of bacteria.

User Reviews

UNIQ283f2de985baa8b9-partinfo-00000000-QINU UNIQ283f2de985baa8b9-partinfo-00000001-QINU