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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>
 +
 +
 +
<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>
 +
 +
<h3><font style="color:#b22222">Ligation of Gblocks Holin-Endolysin into pDawn </font></h3>
 +
 +
<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>
 +
 +
 +
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===
 
===User Reviews===

Latest revision as of 01:36, 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
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

UNIQ7c96cdec8e527dd6-partinfo-00000000-QINU UNIQ7c96cdec8e527dd6-partinfo-00000001-QINU