Difference between revisions of "Part:BBa K1616005:Experience"
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===Applications of BBa_K1616005===
 
===Applications of BBa_K1616005===
 +
<h2> Notebook </h2>
 +
Gblocks: ordered of Holin/ Endolysin fragments from IDT <br>
 +
BBa_K1616005 has been characterized into pDawn plasmid, however, the submitted part was into <b>pSB1C3</b>
 +
<br><br>
 +
<h3><font style="color:#b22222">PCR of Holin/Endolysin parts</font></h3> <br>
 +
<i>Aim: Amplification of the part</i>
 +
<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>
  
−
<h2><font style="color:#b22222"> Notebook </font></h2>
 
  
 +
<h3><font style="color:#b22222">Digestion of pDawn and Holin/Endolysin</font></h3>
  
 +
<table id="Mixligation" style="    width: 80%;"><tr>
 +
<th>
 +
Tube
 +
</th>
 +
<th>
 +
Buffer 2.1
 +
</th>
 +
<th>
 +
DNA
 +
</th>
 +
<th>
 +
EcoRI
 +
</th>
 +
<th>
 +
Enzyme 2 (0,5 µL)
 +
</th>
 +
</tr>
 +
 +
<tr><td><font style="color:#b22222">pDawn</font></td>
 +
<td>
 +
<center>2 µL</center>
 +
</td>
 +
 +
<td>
 +
<center>12 µL</center>
 +
</td>
 +
 +
 +
<td>
 +
<center>0,5 µL</center>
 +
</td>
 +
 +
<td>
 +
<center>NehI</center>
 +
</td>
 +
</tr>
 +
<tr><td><font style="color:#b22222">Holin - Ensolysin </font></td>
 +
<td>
 +
<center>2 µL</center>
 +
</td>
 +
 +
<td>
 +
<center>12 µL</center>
 +
</td>
 +
 +
 +
<td>
 +
<center>0,5 µL</center>
 +
</td>
 +
 +
<td>
 +
<center>SpeI</center>
 +
</td>
 +
</tr></table>
  
−
<h2><font style="color:#b22222"> Results </font></h2>
+
<h3><font style="color:#b22222">Ligation of Gblocks Holin-Endolysin into pDawn </font></h3>
  
−
<center>[[Image:Graph-HE.png|center|600px|]] </center>
+
<table id="Mixligation" style="    width: 80%;"><tr>
 +
<th>
 +
Tube
 +
</th>
 +
<th>
 +
Water
 +
</th>
 +
<th>
 +
T4 ligase Buffer
 +
</th>
 +
<th>
 +
pDawn
 +
</th>
 +
<th>
 +
Plasmid
 +
</th><th>
 +
T4 ligase
 +
</th>
 +
</tr>
 +
 +
<tr><td><font style="color:#b22222">Holin - Ensolysin </font></td>
 +
<td>
 +
<center>4,6 µL</center>
 +
</td>
 +
 +
<td>
 +
<center>1 µL</center>
 +
</td>
 +
 +
 +
<td>
 +
<center>1,4 µL</center>
 +
</td>
 +
 +
<td>
 +
<center>2,5 µL</center>
 +
</td>
 +
 +
 +
 +
<td>
 +
<center>0,5 µL</center>
 +
</td>
 +
 +
</tr></table>
 +
<br>
 +
 
 +
 
 +
<br><br>
 +
Room temperature, 30min  heat kill: 80°C, 20min <br>
 +
 
 +
Transformation of E.coli DH5 alpha competent cells <br>
 +
 
 +
Culture liquid: pDawn – H/E  20 mL LB + 20 µL Kan, without light;
 +
 
 +
<h3><font style="color:#b22222">Measurement of the OD600 of liquid culture</font></h3>
 +
When the OD600 has reached 0.6 or more, the volume of culture has been divided into 3 erlenmeyers:
 +
<ul>
 +
<li>1 erlenmeyer without light exposition, 37°C </li>
 +
<li>1 erlenmeyer with light exposition, 37°C </li>
 +
<li>1 erlenmeyer with light exposition, room temperature </li>
 +
</ul>
 +
Measurement of the OD<sub>600</sub> every hour <br>
 +
<i>3 culture on 5have shown a bacterial development</i>
 +
<center>[[Image:Graph-HE.png|500px|]]  [[Image:Graph-150915 2.png|500px|]]  [[Image:Graph-150915 3.png|500px|]] </center>
 +
<br>
 +
 
 +
 +
<br><br>
 +
<h2>Results </h2>
 +
 
 +
<center>[[Image:Graph-HE.png|center|600px|]]</center>
  
  

Latest revision as of 01:36, 23 September 2015

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Applications of BBa_K1616005

Notebook

Gblocks: ordered of Holin/ Endolysin fragments from IDT
BBa_K1616005 has been characterized into pDawn plasmid, however, the submitted part was into pSB1C3

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;

Measurement of the OD600 of liquid culture

When the OD600 has reached 0.6 or more, the volume of culture has been divided into 3 erlenmeyers:

  • 1 erlenmeyer without light exposition, 37°C
  • 1 erlenmeyer with light exposition, 37°C
  • 1 erlenmeyer with light exposition, room temperature

Measurement of the OD600 every hour
3 culture on 5have shown a bacterial development

Graph-HE.png Graph-150915 2.png Graph-150915 3.png





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

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