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

(→‎Applications of BBa_K1616005)
(→‎Applications of BBa_K1616005)
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−
 
+
<h3><font style="color:#b22222">Digestion of pDawn and Holin/Endolysin</font></h3>
−
<h3><font style="color:#b22222">Ligation of Gblocks Holin-Endolysin into pDawn </font></h3>
+
  
 
<table id="Mixligation" style="    width: 80%;"><tr>
 
<table id="Mixligation" style="    width: 80%;"><tr>
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</th>
 
</th>
 
<th>  
 
<th>  
−
Water
+
Buffer 2.1
 
</th>
 
</th>
 
<th>  
 
<th>  
−
T4 ligase Buffer
+
DNA
 
</th>
 
</th>
 
<th>  
 
<th>  
−
pDawn
+
EcoRI
 
</th>
 
</th>
 
<th>  
 
<th>  
−
Plasmid
+
Enzyme 2 (0,5 µL)
−
</th><th>
+
−
T4 ligase
+
 
</th>
 
</th>
 
</tr>
 
</tr>
 
 
−
<tr><td><font style="color:#b22222">Holin - Ensolysin </font></td>
+
<tr><td><font style="color:#b22222">pDawn</font></td>
 
<td>  
 
<td>  
−
<center>4,6 µL</center>
+
<center>2 µL</center>
 
</td>
 
</td>
 
 
 
<td>  
 
<td>  
−
<center>1 µL</center>
+
<center>12 µL</center>
 
</td>
 
</td>
 
 
 
 
 
<td>  
 
<td>  
−
<center>1,4 µL</center>
+
<center>0,5 µL</center>
 
</td>
 
</td>
 
 
 
<td>  
 
<td>  
−
<center>2,5 µL</center>
+
<center>NehI</center>
 +
</td>
 +
</tr>
 +
<tr><td><font style="color:#b22222">Holin - Ensolysin </font></td>
 +
<td>
 +
<center>2 µL</center>
 
</td>
 
</td>
 
 
−
+
<td>
 +
<center>12 µL</center>
 +
</td>
 +
 
 
 
<td>  
 
<td>  
 
<center>0,5 µL</center>
 
<center>0,5 µL</center>
 
</td>
 
</td>
−
+
 +
<td>
 +
<center>SpeI</center>
 +
</td>
 
</tr></table>
 
</tr></table>
−
<br>
 
  
−
 
+
<h3><font style="color:#b22222">Ligation of Gblocks Holin-Endolysin into pDawn </font></h3>
−
<h3><font style="color:#b22222">Digestion of pDawn and Holin/Endolysin</font></h3>
+
  
 
<table id="Mixligation" style="    width: 80%;"><tr>
 
<table id="Mixligation" style="    width: 80%;"><tr>
Line 130: Line 136:
 
</th>
 
</th>
 
<th>  
 
<th>  
−
Buffer 2.1
+
Water
 
</th>
 
</th>
 
<th>  
 
<th>  
−
DNA
+
T4 ligase Buffer
 
</th>
 
</th>
 
<th>  
 
<th>  
−
EcoRI
+
pDawn
 
</th>
 
</th>
 
<th>  
 
<th>  
−
Enzyme 2 (0,5 µL)
+
Plasmid
 +
</th><th>
 +
T4 ligase
 
</th>
 
</th>
 
</tr>
 
</tr>
 
 
−
<tr><td><font style="color:#b22222">pDawn</font></td>
+
<tr><td><font style="color:#b22222">Holin - Ensolysin </font></td>
 
<td>  
 
<td>  
−
<center>2 µL</center>
+
<center>4,6 µL</center>
 
</td>
 
</td>
 
 
 
<td>  
 
<td>  
−
<center>12 µL</center>
+
<center>1 µL</center>
 
</td>
 
</td>
 
 
 
 
 
<td>  
 
<td>  
−
<center>0,5 µL</center>
+
<center>1,4 µL</center>
 
</td>
 
</td>
 
 
 
<td>  
 
<td>  
−
<center>NehI</center>
+
<center>2,5 µL</center>
−
</td>
+
−
</tr>
+
−
<tr><td><font style="color:#b22222">Holin - Ensolysin </font></td>
+
−
<td>
+
−
<center>2 µL</center>
+
 
</td>
 
</td>
 
 
−
<td>
+
−
<center>12 µL</center>
+
−
</td>
+
−
+
 
 
 
<td>  
 
<td>  
 
<center>0,5 µL</center>
 
<center>0,5 µL</center>
 
</td>
 
</td>
−
+
−
<td>
+
−
<center>SpeI</center>
+
−
</td>
+
 
</tr></table>
 
</tr></table>
 +
<br>
 +
 +
 
<br><br>
 
<br><br>
 
Room temperature, 30min  heat kill: 80°C, 20min <br>
 
Room temperature, 30min  heat kill: 80°C, 20min <br>

Revision as of 00:33, 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


Aim: Amplification of the part

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


Digestion of pDawn and Holin/Endolysin

Tube

Buffer 2.1

DNA

EcoRI

Enzyme 2 (0,5 µL)

pDawn
2 µL
12 µL
0,5 µL
NehI
Holin - Ensolysin
2 µL
12 µL
0,5 µL
SpeI

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

Culture liquid: pDawn – H/E 20 mL LB + 20 µL Kan, without light;



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

UNIQ947fb0ec75cc82f2-partinfo-00000000-QINU UNIQ947fb0ec75cc82f2-partinfo-00000001-QINU