Team:TU Munich/lab/notebook/part2

Light sensor systems and AND-Gate cloning Part II Alex/Susan

09-08-2011

daily work (Alex H. and Susan)

Sequencing of following parts was done by Kathi: T7promotor-lacZ (I712074-I732017), Terminator-blue light sensor (B0015-K238013), T7 Pol-RBS (K228000-J44001) --> Results: not clear! smaller parts seem to be missing in the sequence (but: these parts were already on the plasmid which were used for ligation)

THEREFORE: try again!!! and repeat ligations from 21/7 and 12/7

Restriction digests: part 8a (RBS-T7Pol) (X,P), part 6c (Terminator-BlueLight) (S,P), SupDtRNA (X,P), B0015 (E,S), K238013 (E,X), J44001 (S,P), K228000 (X,P) SupDtRNA (E,X) parts 8a and 6c might be switched, we will confirm it on the gel tomorrow), SupDtRNA was restricted twice in order to try out different ligation strategies

Due to power cut tomorrow no overnight cultures can be done

10-08-2011

Daily work (Alex H. and Susan)

Gel electrophoresis: An Agarose gel electrophoresis (1%, 1x TAE buffer) of the eight digested samples from yesterday was performed (running time approximately 1.5 h).

Transformation: In order to yield further DNA of part 2b (lacZ-T7 promotor), 1 ul of part 2b was mixed with 40 ul electrocompetent BL21 bacteria. Transformation was done according to the protocol. After incubation for 2 h in SOC-medium, bacteria were plated onto Amp-plates and incubated at 37 °C over night.

Test of electrocompetent cells: DH5a cells were inoculated on LB plates (A, K or Cm) and incubated over night at 37 °C.

Results

Gel electrophoresis: We expect the following bands to appear on the gel: part 8a (X,P): ca. 2.7 kbp (insert: T7 pol-rbs) and ca. 2 kbp (backbone) part 6c (S,P): ca. 2.2 kbp (backbone including blue light promotor-terminator) SupDtRNA (X,P): ca. 0.15 kbp (insert: SupDtRNA) and ca. 2 kbp (backbone) B0015 (E,S): ca. 0.15 kbp (insert: terminator) and ca. 3.1 kbp (backbone) K238013 (E,X): ca. 2.1 kbp (backbone including K238013) J44001 (S,P): ca. 2.1 kbp (backbone including J4401) K228000 (X,P): ca. 2.7 kbp (K228000) and ca. 2 kbp (backbone) SupDtRNA (E,X): ca. 2.1 kbp (backbone including SupDtRNA)

If 6c and 8a were exchanged by mistake (see above), the following bands should appear: part 8a (S,P): ca. 4.7 kbp (backbone including T7 pol-rbs) part 6c (X,P): ca. 0.2 kbp (insert: blue light promotor-terminator) and ca. 2 kbp (backbone)

The picture of the gel was named 100811_GelVerdau0809_SusanAlex. Digestion was successful for SupDtRNA (X,P), SupDtRNA (E,X), J4401 (S,P) and K228000 (X,P). Digestion of K238013 (E,X) yielded a band of about 2.7 kbp, which contradicts our expectations. part 8a and part 6c were exchanged by mistake, as they show the bp calculated for the exchanged parts. DNA was extracted using freeze and squeeze.

11-08-2011

Daily work (Susan and Alex)

Gene synthesis: We approached Invitrogen and ShineGene for possible gene synthesis of our plasmids, in case we do not make it in time to synthesize them.

New electrocompetent cells: We inoculated new DH5a cells in 20 ml LURIA medium, as our own ones seem to be contaminated.

Test of electrocompetent cells: Miniprep with Metabion Kit of DH5a cells in LB (overnight culture from 10-8-2011)

Test of lacZ expression in plasmid 2 with T7 promotor (miniprep 2b from 14-7-2011): colony picking and growth in 5 ml LB-medium with 5ul Amp (Start 9:45h) OD measurement: at 16:00h finally 0.775 IPTG induction: 40ul IPTG (0.1M) in 2ml cells (since cells didn't grow as fast as expected experiment is repeated tomorrow) --> 4 new cultures are raised overnight in 5ml LB with Amp (two from 100ul Transformation culture, 2 from 100ul concentrated culture)

Before induction, cells were spread on two amp-LB plates: negative control with 16ul (should be 20mg/ml) SGal (50mg/ml), and test for lacZ expression with 16ul SGal and 40ul (IPTG (0.1M in H2O)

Ligation and transformation: Ligation of the digested parts from yesterday: J44001 (rbs) with K228000 (T7 Pol) and K322127 (red light sensor) with K228001 (tRNA). After ligation, the plasmids were transformed by electroporation into competent DH5alpha E. coli (by Andrea, not our own stock).

Digestion: Repetition of the digestions from yesterday for B0015 (E,S; double terminator; was conducted with two different lots) and K238013 (E,X; blue light promotor).

Results

Test of electrocompetent cells: Colonies have appeared on ampicillin, kanamycin and chloramphenicol plates. Thus, our DH5a stock is most likely contaminated...