Difference between revisions of "Part:BBa K1616005:Experience"
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<h2> Notebook </h2> | <h2> Notebook </h2> | ||
Gblocks: ordered of Holin/ Endolysin fragments from IDT <br> | 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> | <br><br> | ||
<h3><font style="color:#b22222">PCR of Holin/Endolysin parts</font></h3> <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> | <table id="Mixligation" style=" width: 80%;"><tr> | ||
<th> | <th> | ||
Line 71: | Line 73: | ||
+ | <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> | <h3><font style="color:#b22222">Ligation of Gblocks Holin-Endolysin into pDawn </font></h3> | ||
Line 121: | Line 177: | ||
</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> | ||
Line 128: | Line 187: | ||
Culture liquid: pDawn – H/E 20 mL LB + 20 µL Kan, without light; | 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> | <br><br> | ||
<h2>Results </h2> | <h2>Results </h2> | ||
− | <center>[[Image:Graph-HE.png|center|600px|]] </center> | + | <center>[[Image:Graph-HE.png|center|600px|]]</center> |
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 |
|
|
|
|
|
|
|
|
Digestion of pDawn and Holin/Endolysin
Tube |
Buffer 2.1 |
DNA |
EcoRI |
Enzyme 2 (0,5 µL) |
---|---|---|---|---|
pDawn |
|
|
|
|
Holin - Ensolysin |
|
|
|
|
Ligation of Gblocks Holin-Endolysin into pDawn
Tube |
Water |
T4 ligase Buffer |
pDawn |
Plasmid |
T4 ligase |
---|---|---|---|---|---|
Holin - Ensolysin |
|
|
|
|
|
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
Results
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
UNIQe089b03eaec273a6-partinfo-00000000-QINU UNIQe089b03eaec273a6-partinfo-00000001-QINU