Difference between revisions of "Team:Goettingen/Notebook/reporterSystems September"

 
(One intermediate revision by the same user not shown)
Line 4: Line 4:
 
         <p class="notebook-head_date">14.09.18</p>
 
         <p class="notebook-head_date">14.09.18</p>
 
     </div>
 
     </div>
−
<div class="notebook-content">
+
    <div class="notebook-content">
−
<p>A PCR was performed to fuse the promotors P<sub>yciAB</sub> and P<sub>yciC</sub> to <em>xylR</em>.</p>
+
        <p>A PCR was performed to fuse the promotors P<sub>yciAB</sub> and P<sub>yciC</sub> to <em>xylR</em>.</p>
 
         <div class="notebook-table article_table">
 
         <div class="notebook-table article_table">
 
             <table>
 
             <table>
Line 46: Line 46:
 
             </table>
 
             </table>
 
         </div>
 
         </div>
−
<div class="notebook-table article_table">
+
        <div class="notebook-table article_table">
 
             <table>
 
             <table>
 
                 <tr>
 
                 <tr>
Line 60: Line 60:
 
                     <td class="article-table_right"><em>P<sub>yciC</sub></em></td>
 
                     <td class="article-table_right"><em>P<sub>yciC</sub></em></td>
 
                 </tr>
 
                 </tr>
−
    </table>
+
            </table>
−
</div>
+
        </div>
−
<p>The annealing temperature was set for 54°C and lasted for 1 minute. The elongatin settings were 2 min and 72°C.</p>
+
        <p>The annealing temperature was set for 54°C and lasted for 1 minute. The elongatin settings were 2 min and 72°C.</p>
−
<div class="article_picture">
+
        <div class="article_picture">
−
    <img src="https://static.igem.org/mediawiki/2018/6/6d/T--Goettingen--PCR_170918.png">
+
            <img src="https://static.igem.org/mediawiki/2018/6/6d/T--Goettingen--PCR_170918.png">
−
    <p>Lane 1 and 3:<em>P<sub>yciC</sub>-xylR</em>, lane 2 <em>P<sub>yciAB</sub>-xylR</em></p>
+
            <p>Lane 1 and 3:<em>P<sub>yciC</sub>-xylR</em>, lane 2 <em>P<sub>yciAB</sub>-xylR</em></p>
−
</div>
+
        </div>
 +
    </div>
 
</div>
 
</div>
 
<div class="notebook-item">
 
<div class="notebook-item">
Line 73: Line 74:
 
         <p class="notebook-head_date">19.09.18</p>
 
         <p class="notebook-head_date">19.09.18</p>
 
     </div>
 
     </div>
−
<div class="notebook-content">
+
    <div class="notebook-content">
−
<div class="notebook-table article_table">
+
        <div class="notebook-table article_table">
 
             <table>
 
             <table>
 
                 <tr>
 
                 <tr>
Line 107: Line 108:
 
             </table>
 
             </table>
 
         </div>
 
         </div>
−
<p>The sample was purified with the DNA purification kit and eluted in 32&nbsp;&mu;L. Also, the DNA concentration was measured with the Nanodrop device.</p>
+
        <p>The sample was purified with the DNA purification kit and eluted in 32&nbsp;&mu;L. Also, the DNA concentration was measured with the Nanodrop device.</p>
−
<div class="notebook-table article_table">
+
        <div class="notebook-table article_table">
 
             <table>
 
             <table>
 
                 <tr>
 
                 <tr>
Line 136: Line 137:
 
             </table>
 
             </table>
 
         </div>
 
         </div>
−
<p>1.5&nbsp;&mu;L of the digested plasmid were put on an agarose gel next to a not digested plasmid sample.</p>
+
        <p>1.5&nbsp;&mu;L of the digested plasmid were put on an agarose gel next to a not digested plasmid sample.</p>
−
<div class="article_picture">
+
        <div class="article_picture">
−
    <img src="https://static.igem.org/mediawiki/2018/6/61/T--Goettingen--180918_restriktionsverdau.png">
+
            <img src="https://static.igem.org/mediawiki/2018/6/61/T--Goettingen--180918_restriktionsverdau.png">
−
    <p>Lane 1: undigested pAC7, lane 2: digested pAC7</p>
+
            <p>Lane 1: undigested pAC7, lane 2: digested pAC7</p>
−
</div>
+
        </div>
−
<div class="notebook-table article_table">
+
        <div class="notebook-table article_table">
 
             <table>
 
             <table>
 
                 <tr>
 
                 <tr>
Line 169: Line 170:
 
             </table>
 
             </table>
 
         </div>
 
         </div>
−
<p>The reaction was inactivated for 5 min at 80°C. Afterwards 1&nbsp;&mu;L fast AP was added and the reaction was then incubated for 10 min at 37°C. The sample was purified with the DNA purification kit and eluted in 32&nbsp;&mu;L water. The framgemts were ligated with pAC7 and a religation was performed along side the ligation reaction.</p>
+
        <p>The reaction was inactivated for 5 min at 80°C. Afterwards 1&nbsp;&mu;L fast AP was added and the reaction was then incubated for 10 min at 37°C. The sample was purified with the DNA purification kit and eluted in 32&nbsp;&mu;L water. The framgemts were ligated with pAC7 and a religation was performed along side the ligation reaction.</p>
−
<div class="notebook-table article_table">
+
        <div class="notebook-table article_table">
 
             <table>
 
             <table>
 
                 <tr>
 
                 <tr>
Line 202: Line 203:
 
             </table>
 
             </table>
 
         </div>
 
         </div>
−
<div class="notebook-table article_table">
+
        <div class="notebook-table article_table">
 
             <table>
 
             <table>
 
                 <tr>
 
                 <tr>
Line 230: Line 231:
 
             </table>
 
             </table>
 
         </div>
 
         </div>
−
<p>Competent DH5&alpha; cells were transformed with the ligation samples and the cells were plated on LB Amp plates containing x-gal.</p>
+
        <p>Competent DH5&alpha; cells were transformed with the ligation samples and the cells were plated on LB Amp plates containing x-gal.</p>
 +
    </div>
 
</div>
 
</div>
 
<div class="notebook-item">
 
<div class="notebook-item">
Line 237: Line 239:
 
         <p class="notebook-head_date">20.09.18</p>
 
         <p class="notebook-head_date">20.09.18</p>
 
     </div>
 
     </div>
−
<div class="notebook-content">
+
    <div class="notebook-content">
−
<p>Inoculation of 15 ml LB over night cultures of three colonies each that showed a blue coulor. </p>
+
        <p>Inoculation of 15 ml LB over night cultures of three colonies each that showed a blue coulor. </p>
 +
    </div>
 
</div>
 
</div>
 
<div class="notebook-item">
 
<div class="notebook-item">
Line 245: Line 248:
 
         <p class="notebook-head_date">21.09.18</p>
 
         <p class="notebook-head_date">21.09.18</p>
 
     </div>
 
     </div>
−
<div class="notebook-content">
+
    <div class="notebook-content">
−
    <p>A plasmid prep was performed and the DNA concentration was measured using the nanodrop.</p>
+
        <p>A plasmid prep was performed and the DNA concentration was measured using the nanodrop.</p>
−
    <div class="notebook-table article_table">
+
        <div class="notebook-table article_table">
 
             <table>
 
             <table>
 
                 <tr>
 
                 <tr>
Line 279: Line 282:
 
             </table>
 
             </table>
 
         </div>
 
         </div>
−
    <p>A test digest was performed.</p>
+
        <p>A test digest was performed.</p>
−
    <div class="notebook-table article_table">
+
        <div class="notebook-table article_table">
 
             <table>
 
             <table>
 
                 <tr>
 
                 <tr>
Line 312: Line 315:
 
             </table>
 
             </table>
 
         </div>
 
         </div>
−
<p>The whole digestion sample was transferred to an agarosegel.</p>
+
        <p>The whole digestion sample was transferred to an agarosegel.</p>
−
    <div class="article_picture">
+
        <div class="article_picture">
−
    <img src="https://static.igem.org/mediawiki/2018/4/40/T--Goettingen--200918.png">
+
            <img src="https://static.igem.org/mediawiki/2018/4/40/T--Goettingen--200918.png">
−
    <p>Lane 1 to 3: <em>P<sub>yciAB</sub>-xylR</em> clone 1 to 3, lane 4 to 6 <em>P<sub>yciC</sub>-xylR</em> clone 1 to 3.</p>
+
            <p>Lane 1 to 3: <em>P<sub>yciAB</sub>-xylR</em> clone 1 to 3, lane 4 to 6 <em>P<sub>yciC</sub>-xylR</em> clone 1 to 3.</p>
−
</div>
+
        </div>
−
    <p>The colones 1 and 3 of <em>P<sub>yciAB</sub>-xylR</em> and clone 3 of :<em>P<sub>yciC</sub>-xylR</em> were send away for sequnecing. Clone 3 of :<em>P<sub>yciAB</sub>-xylR</em> came back positiv.</p>
+
        <p>The colones 1 and 3 of <em>P<sub>yciAB</sub>-xylR</em> and clone 3 of :<em>P<sub>yciC</sub>-xylR</em> were send away for sequnecing. Clone 3 of :<em>P<sub>yciAB</sub>-xylR</em> came back positiv.</p>
 +
    </div>
 
</div>
 
</div>
 
<div class="notebook-item">
 
<div class="notebook-item">
Line 324: Line 328:
 
         <p class="notebook-head_date">24.09.18</p>
 
         <p class="notebook-head_date">24.09.18</p>
 
     </div>
 
     </div>
−
<div class="notebook-content">
+
    <div class="notebook-content">
−
    <p>The strain BP270 was made competent. Next, the competent cells were transformed with pAC7 containing <em>P<sub>yciAB</sub>-xylR</em> isolated from clone 3. In a second reaction BP270 cells were transformed using only the empty pAC7 vector</p>
+
        <p>The strain BP270 was made competent. Next, the competent cells were transformed with pAC7 containing <em>P<sub>yciAB</sub>-xylR</em> isolated from clone 3. In a second reaction BP270 cells were transformed using only the empty pAC7 vector</p>
 +
    </div>
 
</div>
 
</div>
 
<div class="notebook-item">
 
<div class="notebook-item">
Line 332: Line 337:
 
         <p class="notebook-head_date">24.09.18</p>
 
         <p class="notebook-head_date">24.09.18</p>
 
     </div>
 
     </div>
−
<div class="notebook-content">
+
    <div class="notebook-content">
−
    <p>6 colonies of each transformation were streaked out on agar plates to gain single colonies. </p>
+
        <p>6 colonies of each transformation were streaked out on agar plates to gain single colonies. </p>
 +
    </div>
 
</div>
 
</div>
 
<div class="notebook-item">
 
<div class="notebook-item">
−
    <div class="notebook-head">
+
<div class="notebook-head">
−
        <h3 class="notebook-head_title"></h3>
+
    <h3 class="notebook-head_title"></h3>
−
        <p class="notebook-head_date">25.09.18</p>
+
    <p class="notebook-head_date">25.09.18</p>
−
    </div>
+
</div>
−
<div class="notebook-content">
+
<div class="notebook-content">
−
<p>To test whether the correct integartion of the construct was successfull, the &alpha; amylase-activity was determined using starch agar plates. Here we determined that all picked clones of the transformation using only the empty plasmid were correct while only one of the 6 picked clones was correct for the transformation with the whole construct. The correct clone was used for further experiments. </p>
+
    <p>To test whether the correct integartion of the construct was successfull, the &alpha; amylase-activity was determined using starch agar plates. Here we determined that all picked clones of the transformation using only the empty plasmid were correct while only one of the 6 picked clones was correct for the transformation with the whole construct. The correct clone was used for further experiments. </p>
 +
</div>
 
</div>
 
</div>

Latest revision as of 11:44, 15 October 2018

14.09.18

A PCR was performed to fuse the promotors PyciAB and PyciC to xylR.

Component Volume in [µL]
5x HF buffer 10
dNTPs (12.5 mM) 2
fwd Primer: iGEM2018_83 or iGEM2018_85 2
rev Primer: iGEM2018_90 2
B. subtilis SP1 chromosomal DNA 1
Phusion DNA polymerase 0.25
sterile H2O 32.75
Total 50
Primers Product
iGEM2018_83 + iGEM2018_90 PyciAB
iGEM2018_85 + iGEM2018_90 PyciC

The annealing temperature was set for 54°C and lasted for 1 minute. The elongatin settings were 2 min and 72°C.

           <img src="T--Goettingen--PCR_170918.png">

Lane 1 and 3:PyciC-xylR, lane 2 PyciAB-xylR

19.09.18

Compound Volume in [μL]
Fragment 20
BamHI 2
EcoRI 2
FD buffer 3
H2O 3
30 min at 37°C

The sample was purified with the DNA purification kit and eluted in 32 μL. Also, the DNA concentration was measured with the Nanodrop device.

Compound Volume in [μL]
pAC7 20
BamHI 3
FD buffer 3
H2O 4
45 min at 37°C

1.5 μL of the digested plasmid were put on an agarose gel next to a not digested plasmid sample.

           <img src="T--Goettingen--180918_restriktionsverdau.png">

Lane 1: undigested pAC7, lane 2: digested pAC7

Compound Volume in [μL]
pAC7 20
EcoRI 3
FD buffer 3
H2O 4
30 min at 37°C

The reaction was inactivated for 5 min at 80°C. Afterwards 1 μL fast AP was added and the reaction was then incubated for 10 min at 37°C. The sample was purified with the DNA purification kit and eluted in 32 μL water. The framgemts were ligated with pAC7 and a religation was performed along side the ligation reaction.

Compound Volume in [μL]
Vector (pAC7) 1
Insert (PyciAB/C-xylR) 2 (AB) 1 (C)
T4 buffer 2
T4 ligase 2
H2O 13 (AB) 14 (C)
at least 2 h at RT
Compound Volume in [μL]
Vector (pAC7) 1
T4 buffer 2
T4 ligase 2
H2O 15
at least 2 h at RT

Competent DH5α cells were transformed with the ligation samples and the cells were plated on LB Amp plates containing x-gal.

20.09.18

Inoculation of 15 ml LB over night cultures of three colonies each that showed a blue coulor.

21.09.18

A plasmid prep was performed and the DNA concentration was measured using the nanodrop.

Plasmid DNA concentration in [ng/µL]
PyciAB-xylR sample 1 250.1
PyciAB-xylR sample 2 317.0
PyciAB-xylR sample 3 269.4
PyciC-xylR sample 1 250.4
PyciC-xylR sample 2 245.2
PyciC-xylR sample 3 242.7

A test digest was performed.

Compound Volume in [µL]
BamHI 1
EcoRI 1
FD buffer 1
Plasmid 2
H2O 5
Incubate for 15 min at 37°C

The whole digestion sample was transferred to an agarosegel.

           <img src="T--Goettingen--200918.png">

Lane 1 to 3: PyciAB-xylR clone 1 to 3, lane 4 to 6 PyciC-xylR clone 1 to 3.

The colones 1 and 3 of PyciAB-xylR and clone 3 of :PyciC-xylR were send away for sequnecing. Clone 3 of :PyciAB-xylR came back positiv.

24.09.18

The strain BP270 was made competent. Next, the competent cells were transformed with pAC7 containing PyciAB-xylR isolated from clone 3. In a second reaction BP270 cells were transformed using only the empty pAC7 vector

24.09.18

6 colonies of each transformation were streaked out on agar plates to gain single colonies.

25.09.18

To test whether the correct integartion of the construct was successfull, the α amylase-activity was determined using starch agar plates. Here we determined that all picked clones of the transformation using only the empty plasmid were correct while only one of the 6 picked clones was correct for the transformation with the whole construct. The correct clone was used for further experiments.