Biology

09-05-2016

Diluted gBlocks from IDT to 10 ng/µL
→ RBS_mApple_His, CsgA_ZnOTag, CsgA_ZnSPeptide, CC_SpyC, ZnoTag_SpyC, Dissolved in Water

Also digested linearized (BamHI/SalI) pQE-9

→15min at 37°C
→20min at 80°C

Ligation:
Each gBlock with pQE-9, one hour at room temperature →Frozen at -20°C


09-08-2016

Heat shock transformation of NEB-5-alpha cells with the ligations from Monday 09-05-2016. 35 µL cells + 2 µL ligation reaction.
Additionally: heat shock transformation of W3110dCsgA with ligations of CsgA-ZnO tag and CsgA-ZnS tag

100 µL cells + 5 µL ligation


09-09-2016

Colonies found on NEB-5 alpha plates, no colonies on W3110dCsgA plates → Moved plates to 4 °C.


09-15-2016

Picked three colonies from each pQE-9 plate
Incubated overnight at 37 °C


09-16-2016

Miniprep: Overnight cultures using standard protocol from biochemistry division
Restriction digest: EcoRI/PstI
Agarose gel to check minis
Gel was stained with Ethidium Bromide (EtBr)

Expected bands: pQE-9: 3.4 kb, Inserts range from 300-700 bp
Most bands do not appear to even remotely correspond to expected sizes


09-19-2016

New test digest of 09-16 minis.
This time: one hour digest time

Result: Inserts are not correct. Vectors are not correct. Back to the drawing board


09-20-2016

New start:
Second time digested
CsgA-ZnO tag, CsgA-ZnS tag, 10 ng/µL each
Vector pQE-9 (linearized) as 1:2 dilution

20 µL digest volume
0.5µL EcoRI-HF
0.5µL PstI
2 µL Buffer 2.1
7 µL water
10 µL DNA

One hour at 37 °C
20 min at 80 °C
Stored at -20 °C

Chemistry/Physics

Zn(NO₃)₂ x 4 H₂O, Zn(Ac)₂, Cetyltrimethylammonium bromide (C₁₆TAB), NaOH and KOH were purchased from Sigma-Aldrich. All experiments were carried out in ultrapure water. The synthesis of ZnO microflowers is similar to a synthesis performed by Zhang et al. (2004). 0.5 g of Zn(Ac)₂ were added to 110 mL of a saturated C₁₆TAB aqueous solution under stirring. After 10 min of stirring, 10 mL of a 2 M NaOH aqueous solution were added under stirring, resulting in a white aqueous solution maintained at 110 °C for 1 h under reflux. The white solid was centrifuged, washed with distilled water and ethanol until no foam was present after shaking. Finally, the white sediment was dried at 60 °C under vacuum.

The mineralization process was related to a process developed by Umetsu et al. (2005) and used by Tomizaki et al. (2012) to produce ZnO nanofibers. A 0.1 M zinc nitrate solution was mixed with the same volume of 0.2 M KOH. The produced Zn(OH)₂ was sonicated and centrifuged. The supernatant was discarded and the sediment was resuspended with distilled water of the same volume. Using the 0.1 M Zn(OH)₂ stock solution, solutions with a concentration of 0.02 M and 0.05 M were prepared. Solutions of 0.1 M, 0.05 M and 0.02 M were added to the freshly prepared biofilms (for preparation description, see the biological part). After three days, the biofilms were separated from the solution, washed with distilled water and dried at room temperature.

References

• Tomizaki, K. Y., Kubo, S., Ahn, S. A., Satake, M., & Imai, T. (2012). Biomimetic alignment of zinc oxide nanoparticles along a peptide nanofiber. Langmuir, 28(37), 13459-13466. doi: 10.1021/la301745x



• Umetsu, M., Mizuta, M., Tsumoto, K., Ohara, S., Takami, S., Watanabe, H., ... & Adschiri, T. (2005). Bioassisted Room‐Temperature Immobilization and Mineralization of Zinc Oxide—The Structural Ordering of ZnO Nanoparticles into a Flower‐Type Morphology. Advanced Materials, 17(21), 2571-2575. doi: 10.1002/adma.200500863



• Zhang, H., Yang, D., Ji, Y., Ma, X., Xu, J., & Que, D. (2004). Low temperature synthesis of flowerlike ZnO nanostructures by cetyltrimethylammonium bromide-assisted hydrothermal process. The Journal of Physical Chemistry B, 108(13), 3955-3958. doi: 10.1021/jp036826f