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V08_06
V08_06_1 Motility assay with MG1655 and RP437
- Experiment:
Overnight cultures of MG1655 and RP437 were adjusted to an OD600 of 0,1. A dilution series up to 10^-4 was prepared. 200 µl of each dilution was plated out on tryptone swimming agar. Plates were incubated over night at 30°C.
- Observations & Results:
Plating out on semisolid tryptone agar appears to be difficult. Glass pearls sink in tryptone swimming agar.
V08_06_2 Overnight cultures
- Experiment:
5ml LB-medium containing amp was inoculated with group 2 constructs FliC (DH10B and Salmonella), MotA, MotB, yhjH and the control puc18 in BL21 and DH10B. To test over night growth in other media, also M9-minimal media and 1% tryptone media were inoculated with the same strains. 5 ml LB-medium was inoculated with MG1566 and RP437 were in
- Observations & Results:
DH5a and XL1 blue was not used anymore to test the constructs, because these E.coli stems show very low motility.
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V08_07
V08_07_1 Motility assay with MG1655 and RP437 of V08_03_2
- Experiment:
MG1655 and RP437 were picked of plates from V08_03_2 and solved in LB-agar. 1 µl of the strains were dropped in a grit with 1 cm distance to each next drop on fresh 0,3% LB-swimming plates. The experiment was conducted in two replicates.
- Observations & Results:
Almost all drops grew well. Few MG1655 drops showed swimming.
V08_07_2 Motility assay with MG1655 and RP437 from over night cultures
- Experiment:
Over night cultures from V08_06_2 were used to prepare a dilution series with LB-medium up to 10^-3. The dilution series were dropped in a grit with 1 cm distance to each next drop on fresh 0,3% LB-swimming plates. The strains BL21_rfp and BL21_FlhDC were also dropped out to have a motility reference of putative slower E.coli strains.
- Observations & Results:
Almost all drops grew well. Few MG1655 drops showed swimming.
V08_07_3 Motility assay with BL21 and DH10B strains from over night cultures V08_06_2
- Experiment:
The OD600 of the different over night cultures was measured. Fresh LB- and M9-Medium was inoculated in order to reach a OD600 of 0,1. All strains were grown for 6 hours at 37°C. Only the strains incubated in LB-medium were used in the following steps. The cultures were spun down for 5 mins at 5k X g. The supernatant was discarded. Afterwards, replicates were treated in different ways: One charge was washed with CT-buffer and centrifuged again for 5 min at 5k X g, the other one was dropped immeadiatly. 5 µl of the strains were dropped on fresh M9-minimal plates with 100 µl in Whatman Paper in the center of the plate. Plates were incubated over night at 30°C.
- Observations & Results:
Almost now growth after wash treatment with CT-buffer. The washing process has a very negative influence. Also, the density of the cultures might not have been big enough for growth. Some of the non-washed BL21 cultures displayed growth and minor swimming after 2 days. M9-medium is not suitable for over night cultures!
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V08_08
V08_08_1 Chemical transformation of tar-18C in E .coli DH10B Δtar
- Experiment:
The transformation was performed together with group 2 and is described in their notebook.
- Observations & Results:
J62001 was used as a plasmid backbone because it offers a amp resistance. Also both constructs, for the tar-rescue construct behind the constitutive promoter and der rfp reporter gene behind the same promoter are on the same plasmid. Therefore, these constructs can be used in a separation assay.
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V08_09
V_08_09_1 Separation assay
- Experiment:
Newly transformed strains Δtar_18C_tar and Δtar_18C_rfp were used in the morning to inoculate 5 ml LB-medium with ampicillin and shaken at 37°C and 180 rpm. 0,3% M9-minimal plates including methionine and amp were poured. 1.5 ml of the cultures were centrifuged for 5 mins at 5 X g after 7 hours of growth. The cell pellet was resuspended in the rest media after discarding the supernatant and 5 µl of the strains were dropped on the M9 plates with all three attractants: aspartate, tryptone and AS-mix. The experiment was conducted in two replicates.
- Observations & Results:
Growth visible after 72h. Chemotaxis visible for the attractants tryptone and AS-mix, but not for aspartate. Maybe we should increase the concentration of asparate in solution. Note: Over night cultures lead to a better bacterial density. Seven hours of growth are probably not enough for a sufficient bacterial density.
V08_09_2 Overnight cultures
- Experiment:
5ml LB-medium with ampicillin was inoculated with Δtar_18C_tar and Δtar_18C_rfp.
XL1 blue was not used anymore to test the constructs, because these E.coli stems show very low motility.
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V08_10
V_08_10_1 Separation assay
- Experiment:
Repetition of V_08_09_1. Fresh plates were dried under the clean bench with open lid for 5 min.
- Observations & Results:
Plates showed much less condensation water. But no swimming on the plates visible.
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