<b>V06_05_2 Chemical transformation of competent <i>E. coli</i> (DH10B) </b><br>
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<b>V06_05_2 Insertion of <i>flhDC</i> into the BioBrick plamids</b><br>
<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
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For the chemical transformation the standard protocol was followed. The following constructs were transformed into <br>
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The ligation of the digested flhDC with the BioBrick plasmids was conducted as described in the protocol. <br> </li>
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<i>E. coli:</i> <br>
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J23112 - 20E - x1 <br>
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J23113 - 20G - x21 <br>
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J23109 - 2G - x106 <br>
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J23114 - 20I - x256 <br>
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J23105 - 18M - x623 <br>
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J23106 - 18O - x1185 <br>
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J23104 - 18K - x 1831 <br>
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J23100 - 18 C - x 2547 <br>
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I0500 - 14N - pBAD/araC promoter </li>
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<li>Observations & Results: <br>
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The transformation was successful. Numerous colonies on all plates as well as on the positive control. Now growth could be observed on the negative control plate, so the competent cells are preparation of the competent cells was correct this time. Additionally, no growth could be observed at the 14N plate. This is the only vector containing a kanamycin resistance instead of ampicillin. Eventually, the kanamycin concentration was too strong and inhibited growth.<br>
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The strain (DH10B) grows very fast, thus try not to incubate too long to prevent formation of satellite colonies. <br>
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The cells hosting plasmids with strong promoters are colored red due to the rfp gene fused to the promoter. This phenomenon can be used to verify correct transformation.<br> </li>
Chemical retransformartion of the flhDC-promoter constructs into E. coli (DH10B)
Experiment:
For the chemical retransformation the standard protocol for transformation was followed. The following constructs were transformed into
E. coli:
20E - #2
20E - #3
18M - #2
18M - #3
18C - #1
18C - #2
Observations & Results:
The retransformation was successful since all plates showed numeours colonoes except the negative control.
V06_05
V06_05_1 Preparative double digestion of flhDC and plasmids containing further promoter
Experiment:
In order to clone flhDC behind further promoters, the purified PCR product was digested with XbaI and PstI, whereas the BioBrick plasmids were cut with SpeI and PstI. The digestion was performed as described in the protocol. This time the follwing promoters were used:
J23113 - 20G - x21
J23109 - 2G - x106
J23114 - 20I - x256
J23106 - 18O - x1185
J23104 - 18K - x 1831
V06_05_2 Insertion of flhDC into the BioBrick plamids
Experiment:
The ligation of the digested flhDC with the BioBrick plasmids was conducted as described in the protocol.
V05_27
Preparation of over night cultures
Experiment:
In order to isolate the plasmids, over night cultures of the E. coli (DH10B) cells transformed with the different constitutive promoters were prepared.
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