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

October

10-01-2016

no colonies on the plates, except pUC-19 control (1 colony)
plated rest of the transformations
Picked colonies from old plates with CsgA-ZnS and CsgA-ZnO constructs
prepared square petri dishes with 6 FTO glass slides each and approx. 15-20ml LB/Kan.
Inoculated with W3110ΔCsgA/pPickUp2. Incubated overnight at 37°C on shaker.

10-02-2016

Miniprep of Overnight cultures of pQE-9 with CsgA-ZnS (3) and CsgA-ZnO (4) constructs and pQE-9 (2)
Test digest with EcoRI/PstI
Bad contrast for lower part of dna ladder
One clone from CsgA-ZnO (4) may actually be positive. Further investigation needed.
additionally, pQE-9 minis
EcoRI and PstI single digests
Just in case, inoculated petri dish with FTO glass slides for biofilm growth with positive(?) clone
----------------------------------------------------------
Inoculated culture for competent cells. Followed protocol from
https://www.embl.de/pepcore/pepcore_services/cloning/cloning_methods/chemically_compet ent/.

10-03-2016

New test digest of overnight cultures with EcoRI/PstI. Gel looks like the last one, but better contrast.
Band sizes look to be in the correct range.
Made solar cells with and without biofilm for comparison. Measured values have high variance, inconclusive. Inoculated new biofilms overnight @ 37°C

10-04-2016

Test digest of positive CsgA-ZnO with XbaI/SpeI to confirm that it is a BioBrick. Also digested two empty pQE-9 preps for comparison and loaded EcoRI/PstI digested pQE-9 for reference.
Bands have the correct size; clone is very likely positive.
Biofilms did grow, however, they are not ideal.
Made new solar cells with ZnO and TiO2. Once again, measurable current and voltage, high standard deviation.

10-05-2016

New ligation of ZnS-CsgA with pQE-9 and pSB1C3.
Incubated 1h @ room temperature.
Transformed NEB-5-alpha with 5µl ligation each, plated on LB+Amp / LB+Cam agar.
Incubation overnight @ 37°C
Rest of ligation stored at 4°C

10-06-2016

Few colonies grown on plates (CsgA-ZnS in pSB1C3 only).
Picked 8 clones.
Incubation overnight @ 37°C

10-07-2016

4 cultures grew.
Miniprepped overnight cultures.
Test digest with EcoRI/PstI
Loaded all samples on agarose gel with linearized pQE-9 for reference.
Correct insert sizes.
Preparative digest with EcoRI/PstI of ZnoTag, mApple, mApple expression construct, CsgA-Zno, CsgAZnS.
Samples were loaded on gel. Bands were excised and frozen @ -20°C

10-08-2016

Started mineralization experiment with 100, 50, 25 mM ZnOH and ZnO binding biofilm.
Preparative digest of pQE-9, pSB1C3, new CsgA-ZnO, ZnO SpyC, new CsgA-ZnS.
Due to inattentiveness, the gel was shaken before loading and vector samples spilled over to to other wells. Due to this, the vectors were not excised.
The other three bands were excised and purified together with the five samples from the day before.
The purified fragments were than ligated with pQE-9 or pSB1C3 backbones.
NEB-5-alpha cells were transformed with the ligations, plated and incubated overnight @ 37°C.

10-09-2016

Colonies on most plates except mApple.
Transformed NEB-5-alpha with HaloTag E. coli vector. Plated part of the transformation, inoculated larger part in 3ml LB+Amp.
Incubated overnight @ 37°C

10-10-2016

HaloTag plasmid was miniprepped.
Plasmid and a SpyCatcher constructed for ligation into the HaloTag vector (synthesized by IDT) were digested with XbaI/PstI and purified by gel extraction.
Vector and insert were ligated overnight @ 4°C.
Picked colonies from the plates from the day before. Inoculated in 2ml LB + antibiotic, incubated @ 37°C overnight

10-11-2016

Made competent cells E. coli BL21(DE3) for expression of HaloTag-SpyCatcher construct.
Transformation of competent BL21(DE3) cells with HaloTag ligation.
Plated, incubated overnight @ 37°C
Miniprepped the overnight cultures, test digest with EcoRI/PstI. Gel was allowed to run a bit too far,
so some of the constructs are not visible.
Found colony on mApple plate. Inoculated overnight culture.

10-12-2016

No colonies on plate.
Mineralization: discarded supernatant, rinsed FTOs: White layer sticks to FTO surface with biofilm, easy to wipe off everything else.
Made solar cells from mineralized biofilms. Compared sintered vs not sintered. Sintering still improves efficiency. Sintered 100 mM mineralization yields by far the highest current and voltage yet.
New test digest of minipreps from 10-11. Most clones are positive.
New ligation of HaloTag vector with SpyCatcher insert at 4°C overnight.
Prepped mApple culture. Test digest with EcoRI/PstI.

10-13-2016

Made new competent BL21(DE3).
Transformed with ligation. This time, also with positive control pUC19

10-14-2016

No colonies on the plate
Set up new biofilm growth, this time with CsgA-ZnO curlis and also with CsgA-ZnS curlis, for heavy metal binding.

10-16-2016

Set up Biofilm mineralization experiment. With 3x 100 mM, 3x 50 mM ZnOH

10-18-2016

stopped mineralization by discarding supernatant. Sintered slides. protocol

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

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Team:FAU Erlangen/Notebook

Notebook