Team:Goettingen/week4-2

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#2 Speed Improvement - 4th week

V05_23_1 Preparing BioBrick plasmids with constitutive promoters

Experiment:
J23100 – 18C – x2547
J23114 – 20 I – x256
J23112 – 20 E – x1
resuspended in 10 µl water and stored at -20°C

V05_23_2 Chemical transformation of P1 18C and P1 20I in E. coli (DH10B)

Experiment:
For the chemical transformations the standard protocol was followed.
Observations & Results:
On all plates bacterial growth could be observed, even on the negative control plate. We suggest that the plates are all right, but the competent cells are not. The over night culture seems to have been prepared with cells already containing puc18 and thus an ampicillin resistance. The experiment has to be repeated with new competent cells.

Preparation of over night cultures

Experiment:
Over night cultures for the preparation of new competent cells were prepared using E. coli (DH10B) cells without any resistance.

V05_25_1 Preparation of chemically competent cells of E. coli (DH10B)

Experiment:
The preparation of the competent cells was performed according to the following protocol. This time cells without any resistance were utilized for inoculation.

V05_25_2 Chemical transformation of competent E. coli (DH10B)

Experiment:
For the chemical transformation the standard protocol was followed. The following constructs were transformed into
E. coli
J23112 - 20E - x1
J23113 - 20G - x21
J23109 - 2G - x106
J23114 - 20I - x256
J23105 - 18M - x623
J23106 - 18O - x1185
J23104 - 18K - x 1831
J23100 - 18 C - x 2547
I0500 - 14N - pBAD/araC promoter
Observations & Results:
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 prepared, were 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.
The strain (DH10B) grows very fast, thus try not to incubate too long to prevent formation of satellite colonies.
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.

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.