Team:Goettingen/week20-1

From 2012.igem.org

(Difference between revisions)
Line 30: Line 30:
<b>V09_14_1: BL21 <i>tar</i>-Library selection: fourth approach, first step: application of the library</b><br>
<b>V09_14_1: BL21 <i>tar</i>-Library selection: fourth approach, first step: application of the library</b><br>
<ul>
<ul>
-
<li>Experiment: <br>The library was applied to the 0.3% tryptone swimming agar for selection</li>
+
<li>Experiment: <br>The library was applied to the 0.3% tryptone swimming agar for selection</br>
-
<li>Experimental procedure: <br>View <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.</li>
+
View <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.</li>
<li>Observations: <br>
<li>Observations: <br>
09_16: Because of group internal misscommunication the library was not further selected but applied to LB-plates containing chloramphenicol. Continuation: V09_16_1.</li>
09_16: Because of group internal misscommunication the library was not further selected but applied to LB-plates containing chloramphenicol. Continuation: V09_16_1.</li>
Line 39: Line 39:
<b>V09_14_2: Separation assay</b><br>
<b>V09_14_2: Separation assay</b><br>
<ul>
<ul>
-
<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 plates prepared at the V09_14_2 were used to test the separation assay. On these plates 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 0.3% tryptone swimming agar plates containing no anibiotic.</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 plates prepared at the V09_14_2 were used to test the separation assay. On these plates 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 0.3% tryptone swimming agar plates containing no anibiotic.</br>
-
<li>Experimental procedure: The cultures were mixed and applied to 0.3% tryptone swimming agar containing different amounts of aspartate respectively (2 x 10 µl, 2 x 25 µl, 2 x 100 µl). The mix was dropped on the plates two times and  Δtar with pSB1C3-tar-QC-18 and Δtar with J2006 expressing rfp once each.</li>
+
The cultures were mixed and applied to 0.3% tryptone swimming agar containing different amounts of aspartate respectively (2 x 10 µl, 2 x 25 µl, 2 x 100 µl). The mix was dropped on the plates two times and  Δtar with pSB1C3-tar-QC-18 and Δtar with J2006 expressing rfp once each.</li>
<li>Observations: <br>
<li>Observations: <br>
Continuation: V09_16_1</li>
Continuation: V09_16_1</li>
Line 48: Line 48:
<b>V09_14_3: Does the rfp expression influence the cell density measurement?</b><br>
<b>V09_14_3: Does the rfp expression influence the cell density measurement?</b><br>
<ul>
<ul>
-
<li>Experiment: <br>We suspected that the rfp expression might influence the cell density measurement and to invest this the following experiment was conducted.</li>
+
<li>Experiment: <br>We suspected that the rfp expression might influence the cell density measurement and to invest this the following experiment was conducted.</br>
-
<li>Experimental procedure: The strains Δtar with J2006 expressing rfp and Δtar with pSB1C3-tar-QC-18C were inoculated in the morning and diluted (10<sup>-1</sup>, 10<sup>-2</sup>, 10<sup>-3</sup>, 10<sup>-4</sup>, 10<sup>-5</sup>) in the afternoon. The OD600 was measured and the cultures subsequently plated on LB agar plates.</li>
+
The strains Δtar with J2006 expressing rfp and Δtar with pSB1C3-tar-QC-18C were inoculated in the morning and diluted (10<sup>-1</sup>, 10<sup>-2</sup>, 10<sup>-3</sup>, 10<sup>-4</sup>, 10<sup>-5</sup>) in the afternoon. The OD600 was measured and the cultures subsequently plated on LB agar plates.</li>
<li>Observations: <br>
<li>Observations: <br>
In general the number of colonies of the rfp expressing strain is always much lower than in the other strain even though the measured OD600 is much higher! The rfp expression influences the OD600 measurement!!!</li>
In general the number of colonies of the rfp expressing strain is always much lower than in the other strain even though the measured OD600 is much higher! The rfp expression influences the OD600 measurement!!!</li>
 +
</ul>
 +
<br>
 +
 +
<b>V09_14_3: Does the amount of aspartate influence the swimming behaviour of pSB1C3-tar-QC-18C?</b><br>
 +
<ul>
 +
<li>Experiment: <br>It wa ssuspected, that the amount of aspartate influences the swimming behaviour of the <i>tar</i> rescue strain<br>
 +
The strains Δtar with J2006 expressing rfp and Δtar with pSB1C3-tar-QC-18C were dropped on plates containing 0.3 % tryptone swimming agar and whatmanpaper soaked with different volumes of the aspartate solution (10µl, 25 µl, 100µl)<br>
 +
View <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.</li>
 +
<li>Observations: <br>
 +
09_15: no different tendencies could be observed.</li>
</ul>
</ul>
<br>
<br>
Line 66: Line 76:
<b>V09_16_1: BL21 <i>tar</i>-Library selection: fourth approach, fourth step: plating of the selected clones,continuation of V09_14_1</b><br>
<b>V09_16_1: BL21 <i>tar</i>-Library selection: fourth approach, fourth step: plating of the selected clones,continuation of V09_14_1</b><br>
<ul>
<ul>
-
<li>Experiment: <br>Because of group internal misscommunication the library was not further selected but applied to LB-plates containing chloramphenicol.r</li>
+
<li>Experiment: <br>Because of group internal misscommunication the library was not further selected but applied to LB-plates containing chloramphenicol</br>
-
<li>Experimental procedure: <br>View <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.</li>
+
<br>View <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.</li>
<li>Observations: <br>
<li>Observations: <br>
09_17: Growth was observed on every plate. They were placed in the fridge. No clones were picked for sequencing.</li>
09_17: Growth was observed on every plate. They were placed in the fridge. No clones were picked for sequencing.</li>
Line 76: Line 86:
<b>V09_16_2: Testing of the effect of the promotor strength on the swimming behaviour of the strains Δtar and BL21 with pSB1C3_tar_QC with different promotors</b><br>
<b>V09_16_2: Testing of the effect of the promotor strength on the swimming behaviour of the strains Δtar and BL21 with pSB1C3_tar_QC with different promotors</b><br>
<ul>
<ul>
-
<li>Experiment: <br>In order to qualtify the swimming behaviour the strains Δtar and BL21 with pSB1C3_tar_QC with different promotors are used for a standard swimming/cemotaxis assay</li>
+
<li>Experiment: <br>In order to qualtify the swimming behaviour the strains Δtar and BL21 with pSB1C3_tar_QC with different promotors are used for a standard swimming/cemotaxis assay</br>
-
<li>Experimental procedure: 100 µl Aspartate solution on the whatmanpaper was used as an attractant and 0.3% tryptone swimming agar was used. <br>View <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.</li>
+
100 µl Aspartate solution on the whatmanpaper was used as an attractant and 0.3% tryptone swimming agar was used. <br>View <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.</li>
<li>Observations: <br>
<li>Observations: <br>
09_17: No swimming could be observed.</li>
09_17: No swimming could be observed.</li>
Line 85: Line 95:
<b>V09_16_3: Swimming behaviour of the unmodified strains MG, BL21; XL blue and DH10B on 0.3% tryptone swimming agar </b><br>
<b>V09_16_3: Swimming behaviour of the unmodified strains MG, BL21; XL blue and DH10B on 0.3% tryptone swimming agar </b><br>
<ul>
<ul>
-
<li>Experiment: <br>In order to qualtify the swimming behaviour of the unmodified strains they are placed on swimming agar</li>
+
<li>Experiment: <br>In order to qualtify the swimming behaviour of the unmodified strains they are placed on swimming agar</br>
-
<li>Experimental procedure: The strains were placed on 0.3% tryptone swimming agar and M9 agar containing either aspartate, the aminoacid mix or the tryptone solution in the whatmanpaper (concentrations: view <a href="https://2012.igem.org/Team:Goettingen/Project/Materials">materials</a>. The standard swimming/chemotaxis assay was applied, view <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.<br>
+
The strains were placed on 0.3% tryptone swimming agar and M9 agar containing either aspartate, the aminoacid mix or the tryptone solution in the whatmanpaper (concentrations: view <a href="https://2012.igem.org/Team:Goettingen/Project/Materials">materials</a>. The standard swimming/chemotaxis assay was applied, view <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.<br>
<li>Observations: <br>
<li>Observations: <br>
09_17: No swimming could be observed.</li>
09_17: No swimming could be observed.</li>
Line 94: Line 104:
<b>09_16_4: Separation assay, continuation of V09_14_2</b><br>
<b>09_16_4: Separation assay, continuation of V09_14_2</b><br>
<ul>
<ul>
-
<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 plates prepared at the V09_14_2 were used to test the separation assay. On these plates 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 0.3% tryptone swimming agar plates containing no anibiotic.</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 plates prepared at the V09_14_2 were used to test the separation assay. On these plates 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 0.3% tryptone swimming agar plates containing no anibiotic.</br>
-
<li>Experimental procedure:<br>
+
- The agar was cut out at the swimming front using a to the first mark shortened yellow Eppendorf tip respectively. Two different mixed cultures and one containing pSB1C3-tar-QC-18C or J2006 expressing rfp only.<br>
- The agar was cut out at the swimming front using a to the first mark shortened yellow Eppendorf tip respectively. Two different mixed cultures and one containing pSB1C3-tar-QC-18C or J2006 expressing rfp only.<br>
- The tips with the agar pieces were inserted into an e-cup filled with 1 ml LB media respectively<br>
- The tips with the agar pieces were inserted into an e-cup filled with 1 ml LB media respectively<br>

Revision as of 00:36, 26 September 2012

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#1 Selection / Swimming - 20th Week

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09_14


V09_14_1: BL21 tar-Library selection: fourth approach, first step: application of the library
  • Experiment:
    The library was applied to the 0.3% tryptone swimming agar for selection
    View methods.
  • Observations:
    09_16: Because of group internal misscommunication the library was not further selected but applied to LB-plates containing chloramphenicol. Continuation: V09_16_1.

V09_14_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 plates prepared at the V09_14_2 were used to test the separation assay. On these plates 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 0.3% tryptone swimming agar plates containing no anibiotic.
    The cultures were mixed and applied to 0.3% tryptone swimming agar containing different amounts of aspartate respectively (2 x 10 µl, 2 x 25 µl, 2 x 100 µl). The mix was dropped on the plates two times and Δtar with pSB1C3-tar-QC-18 and Δtar with J2006 expressing rfp once each.
  • Observations:
    Continuation: V09_16_1

V09_14_3: Does the rfp expression influence the cell density measurement?
  • Experiment:
    We suspected that the rfp expression might influence the cell density measurement and to invest this the following experiment was conducted.
    The strains Δtar with J2006 expressing rfp and Δtar with pSB1C3-tar-QC-18C were inoculated in the morning and diluted (10-1, 10-2, 10-3, 10-4, 10-5) in the afternoon. The OD600 was measured and the cultures subsequently plated on LB agar plates.
  • Observations:
    In general the number of colonies of the rfp expressing strain is always much lower than in the other strain even though the measured OD600 is much higher! The rfp expression influences the OD600 measurement!!!

V09_14_3: Does the amount of aspartate influence the swimming behaviour of pSB1C3-tar-QC-18C?
  • Experiment:
    It wa ssuspected, that the amount of aspartate influences the swimming behaviour of the tar rescue strain
    The strains Δtar with J2006 expressing rfp and Δtar with pSB1C3-tar-QC-18C were dropped on plates containing 0.3 % tryptone swimming agar and whatmanpaper soaked with different volumes of the aspartate solution (10µl, 25 µl, 100µl)
    View methods.
  • Observations:
    09_15: no different tendencies could be observed.



09_16


V09_16_1: BL21 tar-Library selection: fourth approach, fourth step: plating of the selected clones,continuation of V09_14_1
  • Experiment:
    Because of group internal misscommunication the library was not further selected but applied to LB-plates containing chloramphenicol

    View methods.
  • Observations:
    09_17: Growth was observed on every plate. They were placed in the fridge. No clones were picked for sequencing.

V09_16_2: Testing of the effect of the promotor strength on the swimming behaviour of the strains Δtar and BL21 with pSB1C3_tar_QC with different promotors
  • Experiment:
    In order to qualtify the swimming behaviour the strains Δtar and BL21 with pSB1C3_tar_QC with different promotors are used for a standard swimming/cemotaxis assay
    100 µl Aspartate solution on the whatmanpaper was used as an attractant and 0.3% tryptone swimming agar was used.
    View methods.
  • Observations:
    09_17: No swimming could be observed.

V09_16_3: Swimming behaviour of the unmodified strains MG, BL21; XL blue and DH10B on 0.3% tryptone swimming agar
  • Experiment:
    In order to qualtify the swimming behaviour of the unmodified strains they are placed on swimming agar
    The strains were placed on 0.3% tryptone swimming agar and M9 agar containing either aspartate, the aminoacid mix or the tryptone solution in the whatmanpaper (concentrations: view materials. The standard swimming/chemotaxis assay was applied, view methods.
  • Observations:
    09_17: No swimming could be observed.

09_16_4: Separation assay, continuation of V09_14_2
  • 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 plates prepared at the V09_14_2 were used to test the separation assay. On these plates 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 0.3% tryptone swimming agar plates containing no anibiotic.
    - The agar was cut out at the swimming front using a to the first mark shortened yellow Eppendorf tip respectively. Two different mixed cultures and one containing pSB1C3-tar-QC-18C or J2006 expressing rfp only.
    - The tips with the agar pieces were inserted into an e-cup filled with 1 ml LB media respectively
    - The cultures were 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 cultures containing pSB1C3-tar-QC-18C or J2006 expressing rfp only were only plated on cm or amp plates.
    - the plates were incubated in an 33 °C incubator over night.
  • Observations:
    - Mix #1:
    Cm: 10-2: 1284
    Cm: 10-4: -
    Amp: 10-2: 1460
    Amp: 10-4: -
    - Mix 2#:
    Cm: 10-2: 14
    Cm: 10-4: 1
    Amp: 10-2: -
    Amp: 10-4: -
    - pSB1C3-tar-QC-18C only
    Cm: 10-4: 76
    - J2006 expressing rfp only
    Cm: 10-4: 112



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