iGEM 2016 — Team FAU_Erlangen

[![](https://static.igem.org/mediawiki/2016/8/8b/HQ_page_logo.jpg)](https://2016.igem.org)

# Team:FAU Erlangen/Notebook

iGEM Erlangen

# Notebook

"Either write something worth reading or do something worth writing."
 *Benjamin Franklin*

[![](https://static.igem.org/mediawiki/2016/4/48/Team_Erlangen_FBLogo.png)](https://www.facebook.com/IGEM-FAU-Erlangen-522447454463293/)
[![](https://static.igem.org/mediawiki/2016/b/b0/Team_Erlangen_YTLogo.png)](https://www.youtube.com/channel/UC4lwWonhs3Dug5bASoDXf5w)
[![](https://static.igem.org/mediawiki/2016/5/5f/Team_Erlangen_IGLogo.png)](https://2016.igem.org/Main_Page)
[![](https://static.igem.org/mediawiki/2016/f/fd/Team_Erlangen_FAULogo.png)](https://2016.igem.org/Team%3AFAU_Erlangen)

* ☰Menu
* [Project](https://2016.igem.org/Team%3AFAU_Erlangen/Description)
  [+ Inspiration](https://2016.igem.org/Team%3AFAU_Erlangen/Description#Inspiration)
  [+ Biofilm](https://2016.igem.org/Team%3AFAU_Erlangen/Description#Biofilm)
  [+ Grätzel Cell](https://2016.igem.org/Team%3AFAU_Erlangen/Description#BioSolar)
  [+ Parts](https://2016.igem.org/Team%3AFAU_Erlangen/Description#GCell)
  [+ References](https://2016.igem.org/Team%3AFAU_Erlangen/Description#Parts)
* [Results](https://2016.igem.org/Team%3AFAU_Erlangen/Results)
  [+ Growing Biofilms](https://2016.igem.org/Team%3AFAU_Erlangen/Results#Inspiration)
  [+ Binding of Heavy Metals](https://2016.igem.org/Team%3AFAU_Erlangen/Results#Biofilm)
  [+ ZnO Mineralization](https://2016.igem.org/Team%3AFAU_Erlangen/Results#BioSolar)
  [+ Solar cell results](https://2016.igem.org/Team%3AFAU_Erlangen/Results#GCell)
  [+ References](https://2016.igem.org/Team%3AFAU_Erlangen/Results#Parts)
* [Notebook](https://2016.igem.org/Team%3AFAU_Erlangen/Notebook)
  [+ Biology](https://2016.igem.org/Team%3AFAU_Erlangen/Notebook#Inspiration)
  [+ Chemistry/Physics](https://2016.igem.org/Team%3AFAU_Erlangen/Notebook#Biofilm)
  [+ References](https://2016.igem.org/Team%3AFAU_Erlangen/Notebook#BioSolar)
* [Human Practices](https://2016.igem.org/Team%3AFAU_Erlangen/Human_Practices)
  [+ School laboratory](https://2016.igem.org/Team%3AFAU_Erlangen/Human_Practices#Inspiration)
  [+ Science Day](https://2016.igem.org/Team%3AFAU_Erlangen/Human_Practices#Biofilm)
* [Collaborations](https://2016.igem.org/Team%3AFAU_Erlangen/Collaborations)
  [+ iGEM Team Aachen](https://2016.igem.org/Team%3AFAU_Erlangen/Collaborations#Inspiration)
  [+ iGEM Team Munich](https://2016.igem.org/Team%3AFAU_Erlangen/Collaborations#Biofilm)
  [+ iGEM Team Marburg](https://2016.igem.org/Team%3AFAU_Erlangen/Collaborations#BioSolar)
* [Safety](https://2016.igem.org/Team%3AFAU_Erlangen/Safety)
  [+ Killswitch](https://2016.igem.org/Team%3AFAU_Erlangen/Safety#Inspiration)
  [+ Binding of Heavy Metals](https://2016.igem.org/Team%3AFAU_Erlangen/Safety#Biofilm)
  [+ References](https://2016.igem.org/Team%3AFAU_Erlangen/Safety#BioSolar)
* [Achievements](https://2016.igem.org/Team%3AFAU_Erlangen/Achievements)
* [Team](https://2016.igem.org/Team%3AFAU_Erlangen/Team)

[* Biology](#Inspiration)
[* Chemistry](#Biofilm)
[* References](#BioSolar)

# 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)

![](https://static.igem.org/mediawiki/2016/4/4a/2016--Team_Erlangen--notebook-image1.png)

![](https://static.igem.org/mediawiki/2016/8/88/2016--Team_Erlangen--notebook-table1.png)

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

![](https://static.igem.org/mediawiki/2016/0/03/2016--Team_Erlangen--notebook-image2.png)

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.

![](https://static.igem.org/mediawiki/2016/d/d1/2016--Team_Erlangen--notebook-image3.png)

 **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](https://www.pdf-archive.com/2016/10/20/protocols-pdf/)

# 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

▲

[![](https://static.igem.org/mediawiki/2016/f/f8/2016--Team_Erlangen--sponsors-biolabs.png)](http://www.neb-online.de/)
[![](https://static.igem.org/mediawiki/2016/1/1e/2016--Team_Erlangen--sponsors-efi.png)](https://www.fau.de/forschung/forschungseinrichtungen/emerging-fields-initiative/)
[![](https://static.igem.org/mediawiki/2016/e/e2/2016--Team_Erlangen--sponsors-fau.png)](https://www.fau.de/)
[![](https://static.igem.org/mediawiki/2016/d/de/2016--Team_Erlangen--sponsors-gatc.png)](https://www.gatc-biotech.com/de/index.html)
[![](https://static.igem.org/mediawiki/2016/0/07/2016--Team_Erlangen--sponsors-gdch.png)](https://www.gdch.de/)

[![](https://static.igem.org/mediawiki/2016/a/a7/2016--Team_Erlangen--sponsors-lauf.png)](http://www.stwl.lauf.de/)
[![](https://static.igem.org/mediawiki/2016/4/43/2016--Team_Erlangen--sponsors-omnilab.png)](https://www.omnilab.de/)
[![](https://static.igem.org/mediawiki/2016/0/01/2016--Team_Erlangen--sponsors-roth.png)](https://www.carlroth.com/de/de)
[![](https://static.igem.org/mediawiki/2016/6/6f/2016--Team_Erlangen--sponsors-promega.png)](https://www.promega.de/)

#

**Contact:** Department Biology Friedrich-Alexander-University Erlangen-Nuremberg Staudtstr. 5 91058 Erlangen igem2016fau@gmail.com

[![](https://static.igem.org/mediawiki/2016/b/bf/HQ_menu_logo.jpg)](https://2016.igem.org)