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Lab Results

Biochemistry

In this project we were able to produce several parts, but unfortunately we only had enough time to charcterize some of them. To these belong promotor + tired + terminator and promtor + sfp, which were charcterized via kinetic measurements. The measured OD values before and after induction with 400 mM IPTG (+ 200 µg cystein) are shown in tables 1 and 2.

Table 1: OD values before induction
BL21 tired sfp
t = 0 0.1250 0.0364 0.0260
t = 70 0.3473 0.0644 0.1154
t = 120 1.0422 0.3031 0.4058
t = 180 1.5018 0.4356 0.9930

Table 2: OD values after induction
BL21 Tired Control Tired iptg induced Tired iptg + Cysteine sfp Control sfp iptg induced
t = 0.5 0.2428 0.0574 0.555 0.6535 0.1235 0.1386
t = 1 0.2601 0.0748 0.0812 0.0523 0.1847 0.1845
t = 1.5 0.2807 0.1048 0.1046 0.0637 0.2267 0.2300
t = 2 0.3416 0.1423 0.1470 0.0931 0.3073 0.3004
t = 2.5 0.3582 0.1690 0.1701 0.0935 0.3398 0.3318
t = 3 0.4007 0.2066 0.2055 0.1146 0.3715 0.3649

As one can see, all OD values are increasing with time but the values of tired and sfp were always lower than the ones of BL21. It's also noticable, that the ODs of sfp increase faster than the tired's.

An other part we characterized was PchR-GFP, which we used for toxicity tests. ... ...

Furthermore, we produced promotor + dhbE and HMWP2, but because of time issues, we weren't able to characterize them yet.

Chemistry

The products pyochelin, NH-nor-pyochelin and 4,5-Dihydro-2-(2,4-dihydroxyphenyl)thiazole-4(R)-carboxylic acid were synthetized.

The presence and purity of the products NH-nor-pyochelin and 4,5-Dihydro-2-(2,4-dihydroxyphenyl)thiazole-4(R)-carboxylic acid was confirmed with 1H-, 13C- and 2D NMR spectroscopy while the presence of pyochelin was confirmed using only 1H-NMR, since 2D spectra were not available at that point. The pyochelin was contaimnated to a certain degree, but it was nevertheless used for subsequent testings. The amount of pyochelin was not sufficient for purification.

UV-Vis spectra of Pyochelin, NH-nor-pyochelin and 4,5-Dihydro-2-(2,4-dihydroxyphenyl)thiazole-4(R)-carboxylic acid were then recorded. To investigate complex formation with iron(III) and gallium (III), solutions of salts including both metals (gallium(III) nitrate and iron(III) chloride) were mixed with the siderophore solutions. UV-Vis spectra were recorded to investigate the kinetics of the complex formations (see here). Finally, siderophore-gallium complexes were created and used for toxicity tests.

Nanoscience

Sadly our first charge of microfluidic devices broke down when we tried to coat them with an ECM-solution to grow our cells on. And we were not able to produce a second charge before wiki freeze.


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