Team:Goettingen/week18-1

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#1 Selection / Swimming - 18th Week

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V09_1

V09_01_1: Selection of the BL21 tar library, first approach, fourth round

Experiment:
The first approach of the selction of clones that move directly towards our chosen attractants was selected for a fourth time, bacause no chemotaxis could be observed in the third round.
Experimental procedure:
The selection was conducted as described in the methods, but small petridishes (9 cm) were used.
Observation:
On the next day swimming could be observed and the plated were treated as described in the protocol "plating of selected clones"

V09_01_2: Separation assay

Experiment:
One of our goals was to invent es protocol that allows the separation of two different fast strains using the antibiotics marker on an inserted plasmid. The plated prepared at the xx were used to test the separation assay. On these plated cultures of the strain Δtar with J2006 expressing rfp (amp resistance) and the strain Δtar with pSB1C3-tar-QC-18C were mixed 1:1 (according to the OD600) and dropped on 3% tryptone swimming agar plates containing no anibiotic.
Experimental procedure:
- The agar was cut out at three different positions using a to the first mark shortened yellow Eppendorf tip respectively - The first section was cut out at the swimming front (I), and the next two (II, III) in a line behind hte first one - The tips with the agar pieces were inserted into a glas tube filled with 1 ml LB media respectively - The cultures war incubated for 1 h at 37 °C with approx. 180 rpm - a 10^-1 to 10^-4 ditutions series was prepared - 100 µl of the 10^-2 and the 10^-4 dilution was plated on LB agar plates containing either ampicillin or chloramphenicol respecvtively - the plates were incubated in an 33 °C incubator over night
Observation:
The next day the colnies could be counted:
CM containing plates

I: 10^-4: 7 colonies

II: 10^-4: 320 colonies

I: 10^-4: 330 colonies
AMP containing plates

I: 10^-4: 0 colonies

II: 10^-4: 0 colonies

I: 10^-4: 7 colonies
--> the strains could be separated successfully and Δtar with pSB1C3-tar-QC-18C seemed to swimm faster!

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@@ Line 546: / Line 546: @@
 <b>V09_01_2: Separation assay</b><br>
 <ul>
-<li>Experiment: <br>One of our goals was to invent es protocol that allows the separation of two different fast strains using the anitibiotics marker on an inserted plasmid. </li>
+<li>Experiment: <br>One of our goals was to invent es protocol that allows the separation of two different fast strains using the antibiotics marker on an inserted plasmid. The plated prepared at the xx were used to test the separation assay. On these plated cultures of the strain Δ<i>tar</i> with J2006 expressing rfp (amp resistance) and the strain Δ<i>tar</i> with pSB1C3-<i>tar</i>-QC-18C were mixed 1:1 (according to the OD600) and dropped on 3% tryptone swimming agar plates containing no anibiotic.</li>
-<li>Experimental procedure: <br>The selection was conducted as described in the methods, but small petridishes (9 cm) were used.</li>
+<li>Experimental procedure: <br>
-<li>Observation: <br>On the next day swimming could be observed and the plated were treated as described in the protocol "plating of selected clones"</li><br>
+- The agar was cut out at three different positions using a to the first mark shortened yellow Eppendorf tip respectively
+- The first section was cut out at the swimming front (I), and the next two (II, III) in a line behind hte first one
+- The tips with the agar pieces were inserted into a glas tube filled with 1 ml LB media respectively
+- The cultures war incubated for 1 h at 37 °C with approx. 180 rpm
+- a 10^-1 to 10^-4 ditutions series was prepared
+- 100 µl of the 10^-2 and the 10^-4 dilution was plated on LB agar plates containing either ampicillin or chloramphenicol respecvtively
+- the plates were incubated in an 33 °C incubator over night
+</li>
+<li>Observation: <br>The next day the colnies could be counted: <br>
+CM containing plates <br>
+<div style="text-indent:20px;">I: 10^-4: 7 colonies</div>
+<div style="text-indent:20px;">II: 10^-4: 320 colonies</div>
+<div style="text-indent:20px;">I: 10^-4: 330 colonies</div>
+AMP containing plates <br>
+<div style="text-indent:20px;">I: 10^-4: 0 colonies</div>
+<div style="text-indent:20px;">II: 10^-4: 0 colonies</div>
+<div style="text-indent:20px;">I: 10^-4: 7 colonies</div>
+--> the strains could be separated successfully and Δ<i>tar</i> with pSB1C3-<i>tar</i>-QC-18C seemed to swimm faster!
+</li><br>
 </ul>