Team:Goettingen/week16-2


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#2 Speed Improvement - 16th week

Preparation of over night cultures

Experiment:
In order to perform another test we used methionine containing agar and determined the effect of new over night cultures of tar-18C and RFP-18C in Δtar as well as MG1655 and RP437 without any construct.

V08_13_1 Performance of motility assay

Experiment:
This time methionine was added to the prepared M9 agar plates and the Whatman Filter Paper were imbued with an amino acid mixture solution as attractant. Here again the over night cultures were dropped onto the plates directly.
Observations & Results:
The following day (14th August) already swimming could be observed on all plates. In general MG1655 featured the strongest motility, followed by tar-18C. This pattern intensified over the following days, but at all no chemotactic behavior could clearly be identified.

V08_13_2 Preparation of over night cultures

Experiment:
For testing fliC (DH10B), fliC (Salmonella), motA, motB and yhjH again, over night cultures of the constructs in E. coli DH10B, BL21, DH5α and XL1 Blue were prepared. Furthermore, cultures of pUC18 in the different strains as a reference were made.

Performance of motility assay

Experiment:
Here again methionine was added to the prepared M9 agar plates and the Whatman Filter Paper were imbued with an amino acid mixture solution as an attractant. The over night cultures were dropped onto the plates directly.
Observations & Results:
Two days after motility assay preparation (16th of August) first stirrings of swimming were visible. In general BL21 shows the strongest motility followed by DH10B. XL1 Blue and DH5α are rather poor. The strongest effect can be attributed to fliC (especially the gene originating from DH10B) and motB. However, the comparison to the reference pUC18 is for all strains rather disillusioning. Whereas pUC18 lead to rather small halos in comparison to fliC and motB at some plates, on other plates pUC18 caused the biggest ones. Thus, the results cannot unambiguously be evaluated.

Preparation of over night cultures

Experiment:
In order to reproduce the motility assay from the day before new over night cultures of fliC (DH10B), fliC (Salmonella), motA, motB, yhjH and pUC18 in E. coli DH10B, BL21, DH5α and XL1 Blue were prepared.
Observations & Results:
The growing of the culture again posed a problem to us since some cultures grew quite nice, whereas others multiplied only poorly to not at all. Therefore, we were not able to conduct the assay.

Preparative double digestion of 18M-flhDC, 18O-flhDC and 18C-flhDC

Experiment:
The flhDC-promoter constructs were digested with XbaI 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 sizes when irradiated with UV rays.

Purification of the flhDC-promoter constructs

Experiment:
The restriction products were purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.