Team:Goettingen/week18-2

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<ul>
<ul>
<li>Experiment:  <br>
<li>Experiment:  <br>
-
The retransformation was performed as described in the standard protocol.<br>
+
The retransformation was performed as described in the standard <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>.<br>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
The retransformation was successful since numerous colonies could be obtained except for the empty negative control. </li>
The retransformation was successful since numerous colonies could be obtained except for the empty negative control. </li>
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<ul>
<ul>
<li>Experiment:  <br>
<li>Experiment:  <br>
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For we aimed to investigate the effect of our constructs on cells that are used to swim competent MG1655 cells were transformed with the following constructs: <br>
+
We aimed to investigate the effect of our constructs on cells that are used to swim. Therefore competent MG1655 cells were transformed with the following constructs: <br>
 +
<br>
<i>fliC</i> (DH10B) <br>
<i>fliC</i> (DH10B) <br>
<i>fliC</i> (<i>Salmonella</i>) <br>  
<i>fliC</i> (<i>Salmonella</i>) <br>  
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18O-<i>flhDC</i> <br>
18O-<i>flhDC</i> <br>
18C-<i>flhDC</i></li>
18C-<i>flhDC</i></li>
 +
<br>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
The transformation was not successful. The next morning only one plate (the positive control) featured any visible colonies. However, after several additional hours of incubation also some very small colonies appeared at some other plates. Nevertheless, the efficiency was very low. We suggest that this is normal since MG1655 is no cloning strain and might have thus a much lower transformation efficiency. This idea was supported by our inquiries. </li>
The transformation was not successful. The next morning only one plate (the positive control) featured any visible colonies. However, after several additional hours of incubation also some very small colonies appeared at some other plates. Nevertheless, the efficiency was very low. We suggest that this is normal since MG1655 is no cloning strain and might have thus a much lower transformation efficiency. This idea was supported by our inquiries. </li>
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<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
In oder to perform a Real-Time Quantitative Reverse Transcription PCR over night cultures of the <i>tar</i>-promoter constructs in BL21 were prepared.<br></li>
+
In oder to perform a Real-Time Quantitative Reverse Transcription PCR, over night cultures of the <i>tar</i>-promoter constructs in BL21 were prepared.<br></li>
</ul><br>  
</ul><br>  
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<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
The flhDC-promoter constructs as well as the vector pSB1C3 still including <i>tar</i> were digested with <i>EcoRI</i> and <i>PstI</i> as described in the protocol. Afterwards, the fragments of the desired size were cut out and frozen in order to extract the DNA from the gel slices the following day. </li>
+
The flhDC-promoter constructs as well as the vector pSB1C3 still including <i>tar</i> were digested with <i>EcoRI</i> and <i>PstI</i> as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. Afterwards, the fragments of the desired size were cut out and frozen in order to extract the DNA from the gel slices the following day. </li>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
The gel featured clearly visible bands of the expected length and thus the digestion was successful. </li>
The gel featured clearly visible bands of the expected length and thus the digestion was successful. </li>
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<ul>
<ul>
<li>Experiment:  <br>
<li>Experiment:  <br>
-
The ligation of the digested <i>flhDC</i>-promoter constructs with pSB1C3 was conducted as described in the protocol. <br>
+
The ligation of the digested <i>flhDC</i>-promoter constructs with pSB1C3 was conducted as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. <br>
</ul><br>
</ul><br>

Latest revision as of 17:18, 22 September 2012

Deutsch  / English 

#2 Speed Improvement - 18th week

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V08_27


V08_27_1 Miniprep of the tar-promoter constructs
  • Experiment:
    The Minipreps were conducted using the PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.

V08_27_2 Chemical retransformation of the tar-promoter constructs into E. coli BL21
  • Experiment:
    The retransformation was performed as described in the standard protocol.
  • Observations & Results:
    The retransformation was successful since numerous colonies could be obtained except for the empty negative control.

V08_27_3 Chemical transformation of different motility increasing constructs into E. coli MG1655
  • Experiment:
    We aimed to investigate the effect of our constructs on cells that are used to swim. Therefore competent MG1655 cells were transformed with the following constructs:

    fliC (DH10B)
    fliC (Salmonella)
    motA
    motB
    yhjH

    18M-flhDC
    18O-flhDC
    18C-flhDC

  • Observations & Results:
    The transformation was not successful. The next morning only one plate (the positive control) featured any visible colonies. However, after several additional hours of incubation also some very small colonies appeared at some other plates. Nevertheless, the efficiency was very low. We suggest that this is normal since MG1655 is no cloning strain and might have thus a much lower transformation efficiency. This idea was supported by our inquiries.

V08_27_4 Preparation of over night cultures
  • Experiment:
    In oder to perform a Real-Time Quantitative Reverse Transcription PCR, over night cultures of the tar-promoter constructs in BL21 were prepared.



V08_28


Preparative double digestion of the flhDC-promoter constructs and pSB1C3
  • Experiment:
    The flhDC-promoter constructs as well as the vector pSB1C3 still including tar were digested with EcoRI and PstI as described in the protocol. Afterwards, the fragments of the desired size were cut out and frozen in order to extract the DNA from the gel slices the following day.
  • Observations & Results:
    The gel featured clearly visible bands of the expected length and thus the digestion was successful.


V08_29


V08_19_1 Purification of the the flhDC-promoter constructs and pSB1C3
  • Experiment:
    The restriction product was purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.

V08_29_2 Insertion of the flhDC-promoter constructs into pSB1C3
  • Experiment:
    The ligation of the digested flhDC-promoter constructs with pSB1C3 was conducted as described in the protocol.


V08_30


Miniprep of the flhDC-promoter constructs in pUC18
  • Experiment:
    In order to obtain further plasmid material Minipreps were conducted using the PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.


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