Team:FAU Erlangen/Notebook

iGEM Erlangen


Biology

June




06-28-2016

Generated competent cells from W3110ΔCsgA (gift from team Marburg).
Transformed the cells with pPickUp, pPickUp2, pGlow.
Directly inoculated overnight cultures



06-29-2016

Stored cultures @ 4°C

July




07-21-2016

Set up experiment for biofilm growth on rotary shaker.
Microscope glass slide in petri dish covered with LB+Kan, inoculated with W3110ΔCsgA/pPickUp2,
incubated overnight



07-22-2016

Attempted crystal violet staining of biofilm. No difference to negative control.


07-28-2016

Set up biofilm growth on rotary shaker with W3110ΔCsgA/pPickUp2.


07-31-2016

Attempted crystal violet staining of biofilm. Negative result.

August




08-01-2016

Set up biofilm growth experiment on rocking shaker.


08-02-2016

Attempted CV staining. Negative result. However, little growth was observed in culture. More time necessary?


08-05-2016

Set up biofilm growth experiment with W3110ΔCsgA/pPickUp2 over the weekend on rocking shaker.


08-09-2016

CV staining biofilm. Positive result. Rocking shaker works.


08-12-2016

Set up biofilm growth over weekend on rocking shaker with square petri dish and 6 FTO glass slides.


08-15-2016

Moved biofilms to 4°C


08-24-2016

Attempted to make solar cells from biofilms with TiO2. No voltage current measurable.


08-26-2016

Set up biofilms for growth over weekend in square petri dish on rocking shaker.


08-31-2016

Built solar cells, but no current/voltage was measurable.


09-01-2016

Solar cell test: with/without biofilm. Very low current/voltage, but with biofilm seems to be slightly better.

September

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



09-21-2016

Incubated colonies from plates for miniprep in 2 mL LB+Amp each.
Inoculated square petri dish with FTO glass plates and LB+Kan with W3110dCsgA/ pPickUp2 E.coli for biofilm growth.
→ Incubation on rocking shaker at room temperature


09-22-2016

Miniprep of the overnight cultures
Test digest with EcoRI/PstI
Fragments inspected via agarose gel → poorly conclusive gel, but fragments nowhere near the expected sizes
New digest of pQE-9, CsgA-ZnS, CsgA-ZnO
Purification via gel extraction
Wrong sizes of bands
→ Ordered new sequences from IDT
Retransformation of NEB-5-alpha with pQE-9 miniprep, no plating, directly inoculated in 2x approx. 3 mL of LB+Amp



09-23-2016

Minipreped pQE-9 from overnight cultures
Test digest with EcoRI/PstI
Test gel → wrong bands



09-24-2016

Digested pQE-9 Minipreps with EcoRI/PstI for gel extraction
Agarose gel electrophoresis → apparently wrong bands again
Bands were excised, stored at -20 °C
Started mineralization experiment with Biofilms and 80, 40, 20, 10, 5 mM Zn(OH)2 in petri dish on rotary shaker at room temperature


09-26-2016

Gel test: linearized pQE-9 and linearized parts registry backbones
→ ALL bands wrong sizes
→ DNA ladder with wrong band sizes?
Inoculated new cultures (pPickUp2) in petri dishes with FTO glass slides for biofilm growth



09-27-2016

New digest of minipreps from 09-16-2016
Gel: Tried another tube with NEB 2-log DNA ladder (left side of gel, right side: same one as before)
Correct Band sizes still CsgA-ZnO constructs may be positive.
Mineralization experiment failed; No adhesion of ZnO-biofilm to glass slide



09-28-2016

Gel test, linearized pQE-9, pSB1C3, gBlock CsgA-ZnO, gblock CsgA-ZnS
Bands have wrong sizes again. However, there is a second, fainter ladder visible, which appears to indicate the correct sizes.
Something is wrong with the ladder in our setup. Maybe due to adding gel stain prior to casting the gel?



09-29-2016

New test: ran two gels, one stained precast, one unstained
Stained: wrong sizes, unstained, correct sizes → earlier fragments were likely correct
Ligation: CsgA-Zno and CsgA-ZnS with pQE-9 and pSB1C3 overnight at 4 °C
Made solar cells from biofilms with TiO2 nanoparticles, sintered 4 minutes with Bunsen burner,
measured with sunlight and UV transilluminator → detectable current/voltage in both cases



09-30-2016

Measured solar cells again – higher values than the day before
Transformed NEB-5-alpha cells with the Ligations
Cells were plated on LB Agar with the appropriate antibiotic for selection


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