iGEM 2018 — Team Stuttgart

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# Team:Stuttgart/Notebook

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[HOME](https://2018.igem.org/Team%3AStuttgart)

TEAM

[Team Members](https://2018.igem.org/Team%3AStuttgart/Team)
[Collaborations](https://2018.igem.org/Team%3AStuttgart/Collaborations)
[Sponsoring](https://2018.igem.org/Team%3AStuttgart/Sponsoring)

PROJECT

[Description](https://2018.igem.org/Team%3AStuttgart/Description)
[Design](https://2018.igem.org/Team%3AStuttgart/Design)
[Experiments](https://2018.igem.org/Team%3AStuttgart/Experiments)
[Notebook](https://2018.igem.org/Team%3AStuttgart/Notebook)
[InterLab](https://2018.igem.org/Team%3AStuttgart/InterLab)
[Model](https://2018.igem.org/Team%3AStuttgart/Model)
[Results](https://2018.igem.org/Team%3AStuttgart/Results)
[Demonstrate](https://2018.igem.org/Team%3AStuttgart/Demonstrate)
[Improve](https://2018.igem.org/Team%3AStuttgart/Improve)
[Attributions](https://2018.igem.org/Team%3AStuttgart/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AStuttgart/Parts)
[Basic Parts](https://2018.igem.org/Team%3AStuttgart/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AStuttgart/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AStuttgart/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AStuttgart/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AStuttgart/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AStuttgart/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AStuttgart/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AStuttgart/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AStuttgart/Hardware)
[Measurement](https://2018.igem.org/Team%3AStuttgart/Measurement)
[Model](https://2018.igem.org/Team%3AStuttgart/Model)
[Plant](https://2018.igem.org/Team%3AStuttgart/Plant)
[Software](https://2018.igem.org/Team%3AStuttgart/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=Stuttgart)

# Notebook

## Abbreviations

Team Rhamnolipid: Rhl **-** Team Nisin: Nis **-** Team Chitosan: Chi **-** Team Linkage: Link **-** Team Modeling: Mod

## Timeline

### 15.10. - 19.10.2018

Preparation for Giant Jamboree

**Nis:** Repeated SDS-PAGE of nisin and staining with silver nitrate, to obtain a more clearly visible result.

### 08.10. - 12.10.2018

Last week in the lab / Wiki time

**Rhl:** Wiki was completed.

**Nis:** Additionally, a SDS-PAGE of the purified nisin was performed and the gel was stained with silver nitrate.

**Chi/Nis:** Disruption of cells from last weeks expression was performed by high pressure homogenization (EmulsiFlex C5). Protein purification was done by HPLC using a His-Trap column (GE-Healthcare). Proteins (COD, NotC, NisA) were analyzed by SDS-PAGE and Coomassie staining.

**Mod:**The text for the wiki was written and last analyzes were performed.

### 01.10. - 05.10.2018

Last few days of our project

**Rhl:** Temperature gradient PCR of *rhlC* with GC-Enhancer due to the fact that the gene contains high GC content. After this, the project was dropped because time was running out.

**Nis:** Restriction digestion of the Nisin-Composite part with XbaI and BbsI. Subsequent gel-extraction, religation and transformation into DH5 alpha *E. coli*. was performed to obtain a proper Basic Biobrick. DNA samples were isolated via Mini-Prep-Kit and were sent to sequencing.

**Chi:** Alternative strategy for construction of BBa\_K2380002 was tested by digesting BBa\_K28480001 with BbsI and XbaI, gelextraction of the 3.4 kb fragment and religation.

Expression of BBa\_K2380043 and BBa\_K28480001 at 30 °C in 250 mL. Cells were harvested by centrifugation, resuspended in phosphate buffer and stored at -20 °C.

**Link:** In this week we coated a door handle with our polymer and started writing the wiki.

**Mod:**All MD simulation was started again under npt condition to achieve a simulation time of 200 ns.

### 24.09. - 28.09.2018

Last Steps

**Rhl:** Transformation of *E. coli* DH5α with BB\_B0034 (ribosome binding site), BBa\_K1331006 (*rhlAB*) and a vector carrying a kanamycin resistance. Overnight cultivation thereof, Miniprep and digestion with EcoRI, SpeI or XbaI, respectively. This plan was dropped since the yield was not satisfying.

**Nis:** Sequencing confirmed the Composite Biobrick for Nisin, containing our modified Nisin with different tags, a RBS and an arabinose promotor. In order to include the whole processing cluster of Nisin Gibson Assembly was performed with NisA\_LY, NisBTCIP and pSB1C3.

**Chi:** Construction of BBa\_K28480001 (NodC+COD-HisTag) from BBa\_K2380002 (NodC, SpeI) and the PCR product from BBa\_K2380043 (XbaI/SpeI). Multiple colonies were picked and the orientation of the COD fragment in the construct was tested by colonyRCA and restriction digestion using MscI.

Activity assay was performed for the chitin synthase NodC using the UDP Glo Kit (Promega) and UDP-GlcNAC as substrate. Activity was compared for NodC from BBa\_K2380002 and BBa\_K28480000.

**Link:**The company dispendix provided us their iDot to print bacteria suspension on agar plate. With this we conducted experiments for antimicrobial.

**Mod:**The first equilibration run successfully but for the production run with npt condition the equilibration time was to short, so the step size for equilibration was extended activity.

**German Red Cross:** Two team members interviewed Michael Teutsch about the requirements of an antimicrobial surface in practice.

### 17.09. - 21.09.2018

Work in the lab

**Rhl:** Order of new optimized primers. Temperature gradient PCR with different template concentrations.

**Nis:**The vector pSB1C3 with the araBAD promotor was amplified using new PCR components. Finally it worked. Gibson Assembly was performed for both our NisA inserts and the products were transformed into  *E. coli* .

**Chi:**Adding a His6-Tag to COD by PCR using BBa\_K2380043, and -044 respectively, as templates. PCR was checked by agarose gel electrophoresis.

Testing of a chitosan assay in the presence of proteins. Standard calibration curves for chitosan and BSA in the range of 20-100 µg mL-1 were recorded.

### 10.09. - 14.09.2018

Work in the lab

**Rhl:** Temperature gradient PCR of all templates with different concentrations.

**Nis:** We decided for a new approach: cloning our NisA-inserts into pSB1C3 that already contains an arabinose promoter and a RBS (araBAD promotor). Design of new primers for our construct. PCRs were performed for our inserts and the vector + araBAD promotor and checked on an agarose gel. Again only the amplification of the NisA inserts worked.

**Chi:**Expression of BBa\_K2380002/-042 and the combined construct BBa\_K2848000 was repeated using a higher culture volume (100 mL). Cells were disrupted in phosphate buffer by high pressure homogenization using an EmulsiFlex C5 (Avestin, ATA Scientific). Proteins were purified by their His-Tag using HPLC (HisTrap column, ÄKTA, GE Healthcare).

**Link:**IR analysis of samples from chemical linkage.

### 03.09. - 07.09.2018

Work in the lab

**Rhl:** Overnight cultivation of transformed *E. coli* DH5α and Miniprep for higher DNA concentrations. Control of templates by restriction digest of biobricks with EcoRI and SpeI. *RhlC* was loaded without digestion since it does not have the same restriction sites. Sizes of fragments accorded with theoretical values leading to the conclusion of having the correct templates. To verify these results, all biobricks were sequenced with positive results. Sequencing of *rhlC*.

**Nis:** We changed every parameter possible in our PCR to amplify pSB1C3 for Gibson cloning but nothing worked.

**Chi:**Expression of BBa\_K2380002/-042 and the combined construct BBa\_K2848000. Wt DH5α expression was performed in parallel. Cells were disrupted by ultrasound and purified using a Ni-NTA agarose column. Purified fractions were analyzed by SDS-PAGE and Coomassie staining.

All contructs from Darmstadt 2017 were tested by XbaI+PstI double digestion and agarose gel electrophoresis.

**Link:**Analyisis of enzymatic linkage and chemical approaches for coupling.

**Mod:**A lot of problem solving was performed in the simulation team to get a running md simulation.

### 27.08. - 31.08.2018

Work in the lab

**Rhl:** Temperature gradient PCR with *araC*, *rhlA*, *rhlB* and *rhlC*.

**Nis:** Since assembly by restriction cloning didn’t work, we decided for a new cloning strategy and switched to GibsonAssembly. Design of new primers. PCRs were performed on our NisA inserts and pSB1C3 and checked on an agarose gel.

**Chi:**Contruction of BBa\_K2848000 from BBa\_K2380002 (NodC, SpeI + PstI) and BBa\_K2380042 (NodB, NheI + PstI). BBa\_K2848000 contains NodC and NodB under the control of a pBAD arabinose promotor (BBa\_K808000). Each ORF is flanked by its own RBS (BBa\_K2380024) and terminator (BBa\_B0015).
An alternative clone was also constructed using BBa\_K2380041, resulting in a plasmid without a second RBS for the NodB ORF.

Expression of BBa\_K2380002/-042 and -043 was performed again and the proteins analyzed by SDS-PAGE and Coomassie staining.

**Link:**In this week we performed further different chemical and enzymatic approaches for coupling chitosan.

**Mod:**A code was adapted from the group of Pleiss to perfume the equilibration and production simulation on multi cluster.

### 20.08. - 24.08.2018

Work in the lab

**Rhl:** PCR with plasmids containing *araC*, *rhlA*, *rhlB* and *rhlC*.

**Nis:** To obtain pSB1C3 for cloning we transformed two biobricks ([BBa\_K398326](http://parts.igem.org/wiki/index.php?title=Part:BBa_K398326) and [BBa\_K398331](http://parts.igem.org/wiki/index.php?title=Part:BBa_K398331)) from the distribution Kit 2018 into *E. coli*. We isolated both plasmids and digested them with SpeI and XbaI before ligating them to both our NisA gBlocks. Screening was done with colony PCR but no positive clones were detected.

**Chi:**Expression of NodC (BBa\_K2380001) and COD (BBa\_K2380043) at 30 °C and room temperature in triplicates each. Proteins were analyzed by SDS-PAGE and Coomassie staining.

**Link:**Further animicrobial assays to check for the antimicrobial potential of TAGC. We also tested different ratios of reactants for chemical linkage of rhamnolipids with chitosan. Differnt methods of precipitation of the product were tested. The different batches of enzymatic coupling were analyzed.

### 13.08. - 17.08.2018

Work in the lab

**Rhl:** Overnight cultivation of transformed *E. coli* DH5α and Miniprep to amplify the plasmids. Control of concentration with Nanodrop.

**Nis:**We finally started working in the lab. To clone our ordered NisA (NisA\_LY and NisA\_YL) into the iGEM backbone vector pSB1C3, we performed several PCRs on the inserts and pSB1C3 and checked them on agarose gels. We managed to amplify both our inserts. However, though we changed various parameters of our PCR protocol like annealing temperature, number of cycles or elongation time we didn’t manage to amplify pSB1C3. Digest of both NisA inserts with SpeI and XbaI.

**Link:**First antimicrobial assays to prove the efficacy of our chemical produced surface. We tried different approaches of enzymatic coupling of GFP with chitosan by tyrosinase. Those approaches were analysed by PAGE.

### 06.08. - 10.08.2018

Work in the lab

**Rhl:** Transformation of *E. coli* DH5α with existing biobricks BBa\_K1331001 (*rhlA)*, BBa\_K1331002 (*rhlB*), BBa\_K808000 (araC) and BBa\_J04450 (mRFP) as well as pVLT33 containing *rhlC*.

**Link:**Further tyrosinase based experiments for linking GFP and chitosan. We met with experts of the institute of plastics engineering.

**Mod:**A few tutorials were read and the setup for the simulation of md model were performed .

### 30.07. - 03.08.2018

Work in the lab

**Rhl:** Transformation of *E. coli* DH5α with existing biobricks BBa\_K1331001 (*rhlA*), BBa\_K1331002 (*rhlB*) and BBa\_K808000 (*araC*).

**Link:**Chemical reactions to link rhamnolipids to chitosan. First enzymatic approach to couple GFP with chitosan, by tyrosinase reaction.

### 23.07. - 27.07.2018

Work in the lab

**Rhl:**Overnight cultivation of transformed *E. coli* DH5α and Miniprep to amplify the plasmids. A stock in 30 % glycerol was generated, too.

**Chi:**Expression of NodC (BBa\_K2380001), NodB (BBa\_K2380042) and Wild Type *E. coli* DH5α at 30 °C and room temperature in triplicates each. Proteins were analyzed by SDS-PAGE and Western Blot using an anti-His antibody coupled to horseradish peroxidase. Detection was performed with luminol.

**Link:**Chemical reactions to couple rhamnolipids with chitosan.

### 16.07 - 20.07.2018

Work in the lab

**Rhl:** Transformation of *E. coli* DH5α with pVLT33 containing *rhlC*.

**Link:**Production of GFP: We purified GFP from a GFP expressing *E. coli*culture by high-pressure homogenisation and affinity chromatography. We planned to couple GFP to chitosan by enzymatic (tyrosinase) reaction. This sould work as a proof of concept for coupling nisin to chitosan. Improvement of the chemical reaction to link rhamnolipids to chitosan.

**European iGEM Meetup** Four team members attended the European iGEM Meetup in Munich, Germany. There, we presented our poster and visited some interesting presentations and workshops.

### 09.07. - 13.07.2018

Work in the lab

**Link:**Further experiments to couple rhamnolipids with chitosan.

### 02.07. - 06.07.2018

Work in the lab

**Rhl:** Contact to Prof. Dr. Blank and Dr. Germer from RWTH Aachen for scientific support and request for a plasmid containing rhamnosyltransferase II.

**Link:**First chemical reaction for linking rhamnolipid to chitosan was conducted.

### 25.06. - 29.06.2018

Work in the lab

**Rhl:** Pre-culture of *Pseudomonas putida* KT2440 and storage in 50 % glycerol stocks.

**Chi:**Expression of NodC (BBa\_K2380001) and NodB (BBa\_K2380042) at 30 °C and room temperature in triplicates each. Proteins were analyzed by SDS-PAGE and Coomassie staining.

**InterLab:** We started the iGEM InterLab experiments.

**Science day:**

A few team members presented our project to the people.

### 18.06. - 22.06.2018

Work in the lab

**Rhl:** Request of composite part BBa\_K1331006 from the regsitry and we contacted Prof. Dr. Syldatk from KIT for scientific support.

**Link:**The ordered tyrosinase was tested for activity by enzyme activity assay.

**Mod:**Two members of the team attended a three week practical course provided by Prof. Dr. Pleiss about MD simulations.

**German iGEM Meetup:** Our Team visited the German iGEM Meetup at Marburg, Germany. Here we presented our poster and got an impress of the work of the other German iGEM Teams.

### 11.06 - 15.06.2018

Work in the lab

**Chi:**Transformation of Darmstadt BioBricks in *E. coli* DH5α. Test transformation of BBa\_J04450 in pSB1C3 for the calculation of transformation efficiency. Transformation efficiency was calculated to about 107 cfu µg-1.

**Mod:**Two members of the team attended a three week practical course provided by Prof. Dr. Pleiss about MD simulations.

### 04.06. - 08.06.2018

Work in the lab

**Rhl:** Order of missing chemicals and TLC-layers.

**Chi:**Preparation of chemical ultra-competent *E. coli* DH5α. Protocol is based on Inoue et al (1990), Gene, 96:23-28, with modifications ([check here](http://staceylab.missouri.edu/preparation-of-ultra-competent-e-coli-cells-for-transformation/ "protocol ultra-competent <i>E. coli</i>")).

**Link:**We ordered chemicals and talked with Dr. Claasen about IR analysis of our chemical reaction products. The amount of chemicals and substrate for the reactions was calculated.

**Mod:**Two members of the team attended a three week practical course provided by Prof. Dr. Pleiss about MD simulations.

### 28.05. - 01.06.2018

First steps in the lab

**Rhl:** Preparation of media and agar plates.

### 21.05. - 25.05.2018

Some lab experience

**Rhl:** Primer Design and order, planning of cloning and gene assembly.

**Chi:**BioBricks from Darmstadt 2017 were requested: BBa\_K238000/-01/-02/-41/-42/-43/-44.

**Link:** Preparation of buffers, media and enzyme stock solutions.

**Mod:**organising the task and to performe a literature search.

### January - May 2018

Beginning of the iGEM Stuttgart 2018 team

The old team of 2017 recruited students for the 2018 iGEM Stuttgart team. We started brain storming for new possible projects. Different subteams were created for the different task (e.g. Financing/Sponsoring, Lab-management, Homepage). In parallel we did research for our project (literature and experts), developed experiments and planned our lab work. We met weekly to report our progress and discuss with our Advisors and PIs.


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Stuttgart/Experiments

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[![](https://static.igem.org/mediawiki/2018/b/bb/T--Stuttgart--Logo.jpg)](https://2018.igem.org/Team%3AStuttgart)
[HOME](https://2018.igem.org/Team%3AStuttgart)

TEAM

[Team Members](https://2018.igem.org/Team%3AStuttgart/Team)
[Collaborations](https://2018.igem.org/Team%3AStuttgart/Collaborations)
[Sponsoring](https://2018.igem.org/Team%3AStuttgart/Sponsoring)

PROJECT

[Description](https://2018.igem.org/Team%3AStuttgart/Description)
[Design](https://2018.igem.org/Team%3AStuttgart/Design)
[Experiments](https://2018.igem.org/Team%3AStuttgart/Experiments)
[Notebook](https://2018.igem.org/Team%3AStuttgart/Notebook)
[InterLab](https://2018.igem.org/Team%3AStuttgart/InterLab)
[Model](https://2018.igem.org/Team%3AStuttgart/Model)
[Results](https://2018.igem.org/Team%3AStuttgart/Results)
[Demonstrate](https://2018.igem.org/Team%3AStuttgart/Demonstrate)
[Improve](https://2018.igem.org/Team%3AStuttgart/Improve)
[Attributions](https://2018.igem.org/Team%3AStuttgart/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AStuttgart/Parts)
[Basic Parts](https://2018.igem.org/Team%3AStuttgart/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AStuttgart/Composite_Part)

[SAFETY](https://2018.igem.org/Team%3AStuttgart/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AStuttgart/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AStuttgart/Public_Engagement)

AWARDS

[Entrepreneurship](https://2018.igem.org/Team%3AStuttgart/Entrepreneurship)
[Model](https://2018.igem.org/Team%3AStuttgart/Model)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=Stuttgart)

# Experiments

### Media

LB-Medium

5 g/L yeast extract, 10 g/L tryptone, 0.5 g/L NaCl, 15 g/L Agar (for solid media), in ddH2O

Yeast medium

10 g/L yeast extract, 20 g/L tryptone, 20 g/L glucose (autoclave separately), 0.5 g/L NaCl, in ddH2O

SOB

5 g/L yeast extract, 20 g/L tryptone, 0.6 g/L NaCl, 0.2 g/L KCl, after autoclavation 10 mM MgCl2 and 10 mM MgSO4 were aseptically added, in ddH2O

SOC

5 g/L yeast extract, 20 g/L tryptone, 0.6 g/L NaCl, 0.2 g/L KCl, after autoclavation 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose were aseptically added, in ddH2O

2xTY

10 g/L yeast extract, 16 g/L tryptone, 5 g/L NaCl, dissolved in ddH2O

### Buffers and solutions

50 mM phosphate buffer PB pH 6.0

6.8 mM Na2HPO4, 43.2 mM NaH2PO4, in ddH2O

50 mM phosphate buffer PB pH 6.5

17.9 mM Na2HPO4, 32.1 mM NaH2PO4, in ddH2O

Phosphate buffered saline PBS

137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, in ddH2O

50 mM TRIS buffer

38.4 mM TRIS-HCl, 11.6 mM TRIS base, adjust pH to 7.6, add 500 mM imidazol for elution buffer

TRIS buffer for Polyacrylamide gel

1.5 M TRIS-HCl, add NaOH to pH 8.8 in ddH2O

0.5 M TRIS-HCl, add NaOH to pH 6.6 in ddH2O

Sodium dodecyl sulfate

10 % SDS in ddH2O

Ammoniumperoxodisulfate

10 % APS in ddH2O

12.5 % Polyacrylamide gel

separating gel: 2.5 mL 30 % acryl amide, 1.5 mL 4x TG buffer, 1.94 mL H2O, 60 µL 10 % APS, 6 µL TEMED

staking gel (4 %): 260 µL 30 % acryl amide, 500 µL 4x TG buffer, 1.22 mL H2O, 40 µL 10 % APS, 4 µL TEMED

For higher separating gel percentage, increase the amount of acryl amide.

4x Tris-Glycine buffer

12 g Tris base, 57.6 g glycine, ad 1000 mL H2O

2x Laemmli loading buffer

62.5 mM Tris/HCl pH 6.8, 3 % (w/v) SDS, 5 % (w/v) β-Mercaptoethanol, 10 % (v/v) glycerol, 0.025 mg/mL bromphenol blue

Coomassie staining solution

0.5 % (w/v) Coomassie Brilliant Blue R250, 50 % (v/v) ethanol, 7 % (v/v) acetic acid, 42.5 % (v/v) H2O

Coomassie destaining solution

40 % (v/v) ethanol, 10 % (v/v) acetid acid, 50 % (v/v) H2O

Chloramphenicol

Stock solution: 25 mg/mL Chloramphenicol in EtOH
Dilution of 1:1000 in media

Kanamycine

Stock solution: 50 mg/mL kanamycine in ddH2O
Dilution of 1:1000 in media

Ampicillin

Stock solution: 100 mg/mL ampicillin in ddH2O
Dilution of 1:1000 in media

Reagent solution for chitosan assay

14 mL 0.2 M borate buffer, pH 8.9
+ 500 µL 0.11 M *o*‑phthalaldehyde (in ethanol)
+ 500 µL 0.071 M N‑acetyl cysteine (in ethanol)

Tyrosinase

25 kU lyophylized tyrosinase dissolved in 10 mL 50 mM PB pH 6

Tyrosine

2 mM L-Tyrosin in 100 mL ddH2O

Chitosan

1.6 % (w/v) chitosan (MW: 100.000-200.000 g/mol) in 50 mM PB pH 6, add 2 M HCl to pH 2

Silver staining

* Fixative:

+ 20 mL methanol, 18.2 mL formaldehyde-solution (37% v/v), 11.8 mL ddH2O

* Sodium thiosulfate solution 0.02 % (w/v):

+ 0.05 g Na2S2O3, ad 250 mL ddH2O

* Silver nitrate solution 0.1% (w/v):

+ 0.25 g AgNO3, ad 250 mL ddH2O

* Developer solution:

+ 7.5 g Na2CO3, 125 µL formaldehyde-solution (37% v/v), 200 µL Sodium thiosulfate solution (0.02 % w/v), ad 250 mL ddH2O

* Stop solution:

+ 18.6 g EDTA, ad 1 L ddH2O

Marker for SDS-PAGE

Roti®-Mark TRICOLOR 10-245 kDa (Carl Roth)

Marker for Agarosegel

1 kb Plus DNA Ladder (NEB)

### Enzymes

Enzymes provided by New England Biolabs® Inc.:

* PstI
* PstI-HF
* XbaI
* SpeI
* MscI
* NheI-HF

Enzymes provided by Thermo Fisher Scientific:

* BpiI

Enzymes provided by Sigma-Aldrich®:

* tyrosinase

### Protocols Chitosan

Pre-culture

* 3 mL SOB media + antibiotics inoculate with one colony from agar plate

* incubate at 37 °C, 180 rpm, over night

Culturing

* Inoculate 1:200 with pre-culture, incubate at 30 °C or room temperature, 180 rpm, over night

* Harvest cells by full speed centrifugation at 4 °C

* resuspend the pellet in phosphate buffer

* store at -20 °C until cell disruption or proceed directly

Induction

* Grow of culture to OD600 = 0.6

* add 1 mM IPTG or 10 mM arabinose to induce production of COD or NodC/NodB

Digestion

|  |  |
| --- | --- |
| 1 µg | DNA |
| 5 µL | 10x buffer |
| 1 U | enzyme 1 |
| 1 U | enzyme 2 |
| ad 50 µL | ddH2O |
|

* Double-digest the DNA at 37 °C for 1 h
* Load sample to an 1 % agarose gel containing ethidium bromide
* Run electrophoresis at 90 V, 1 h
* For gel purification cut out respective bands
* Purification of DNA with ZymocleanTM Gel DNA Recovery Kit (Zymo Research)

Ligation 3:1

|  |  |
| --- | --- |
| 50 ng | vector |
| 25 ng | insert |
| 1 µL | 10x ligation buffer |
| 1 µL | T4 ligase |
| ad 10 µL | ddH2O |
|

* Ligate the vector and insert at 4 °C over night

Transformation

Organisms used: *E. coli* DH5α or BL21 (DE3)

* Add 10 µL ligation approach or 100 ng purified DNA to 100 µL competent cells
* Incubate on ice for 15 min
* Heatshock (30 s, 42 °C)
* 2 min on ice
* Add 900 µL SOB medium, incubate at 37 °C, 180 rpm for 1 h
* Plate 100 µL to a LB-plate containing antibiotics, incubate at 37 °C over night
* Check for colonies the next day

Miniprep

* Spin down 1.5 mL of pre-culture, full speed, 2 min
* Purify plasmids with Monarch® Plasmid Miniprep Kit from New England Biolabs® Inc. (NEB)

His-tag PCR

To improve BBa\_K2380043 and BBa\_K2380044 (COD, Darmstadt 2017) PCR using Q5 High-Fidelity DNA Polymerase (NEB) was performed to add a C-terminal His-tag.

Primers

* VF2: 5'‑tgccacctgacgtctaagaa‑3'
* COD\_rev+His6+SpeI: 5'‑GCCGCTACTAGTATTAGTGGTGATGGTGATGATGcagagcagtaaacagcgtattctggg‑3'

| 5x Reaction Master Mix | |
| --- | --- |
| dd H2O | 78.75 µL |
| Q5 Reaction Buffer (5x) | 25 µL |
| Primers (10 µM) | 6.25 µL each |
| dNTPs (10 mM each) | 2.5 µL |
| Q5 DNA Polymerase (2 U/µL) | 1.25 µL |

Add 24 µL Master Mix to a fresh PCR tube and add 1 µL template DNA (1‑2 ng/µL).

Run PCR using the following profile:

| Temperature | Duration | |
| --- | --- | --- |
| 98 °C | 2 min | |
| 98 °C | 10 s | 25 cycles |
| 67 °C | 30 s |
| 72 °C | 1 min |
| 72 °C | 3 min | |

Check PCR products by agarose gel electrophoresis, expected fragment size = 1522 bp.

colonyRCA

For quick analysis of the orientation of the insert COD + His-tag in the vector BBa\_K2380002 (NodC), a colonyRCA was conducted.

* Reaction was prepared as described in the TempliPhiTM Kit from GE Health care
* Digest the sample with MscI
* Load the digested sample to a 1 % agarose gel with ethidium bromide
* Run electrophoresis at 100 V, 1 h
* Check the bands to identify the right colonies

Cell disruption

Cell disruption was conducted with the EmulsiFlex C5 (Avestin) for purification with HisTrap column (GE Healthcare). Cells were resuspended
in 50 mL phosphate buffer containing 2 mM EDTA. Lysis was processed in 3 cycles at 1000 bar.

Purification of His-tag proteins by affinity chromatography (ÄKTA)

* Load cell lysate to a 5 mL His-trap column (GE Healthcare)
* Elution with 50 mM TRIS-HCl, 250 mM imidazol
* Collect protein fraction and check by SDS-PAGE

SDS-PAGE

* Load 20 µL of samples (diluted with 2x loading buffer)
* Run 12.5 % polyacrylamide-gel at 100 V, 90 min
* Stain the gel with Coomassie staining solution for at least 1 h
* Destain the gel until you can see protein bands (eventually over night)

UDP-Glo assay

Verification of NodC activity was done with the UDP‑Glo assay. Solutions were used from the UDP‑Glo kit from Promega. UDP‑GlcNAC was used as a substrate for NodC. Luminescense was measured with TECAN Microplate reader, integration time was 1 s.

Chitosan assay

To detect produced chitosan in cell extracts an assay by Larionova *et al.* [[C.1]](#reflC.1) was tested.

* 1 mL sample in 0.25 M NaCl
* add 1 mL reagent solution (freshly prepared)
* incubate 60 min at room temperature
* measure absorbance at 340 nm

Standard curves for chitosan and BSA were recorded using the following stock solutions:

* chitosan: 1 mg/mL in 0.01 M HCl
* BSA: 1 mg/mL in 0.2 M borate buffer, pH 8.9

| Reaction Scheme | | | | | |
| --- | --- | --- | --- | --- | --- |
| c [mg/mL] | 0.9 | 0.8 | 0.6 | 0.4 | 0.2 |
| stock [µL] | 900 | 800 | 600 | 400 | 200 |
| buffer [µL] | 0 | 100 | 300 | 500 | 700 |
| 2.5 M NaCl [µL] | 100 | 100 | 100 | 100 | 100 |

### Protocols Nisin

PCR

Amplification of the different constructs was performed using Q5 polymerase (Table 2) or ready‑to‑use Q5 High-Fidelity 2X Master Mix (Table 3). The standard program in Table 1 was used.

**Table 1:** Standard PCR program.

![](https://static.igem.org/mediawiki/2018/9/9b/T--Stuttgart--PCRProgramm.jpeg)

**Table 2:** Reaction components for PCR amplicifation using Q5 polymerase.

![](https://static.igem.org/mediawiki/2018/9/99/T--Stuttgart--PCR-Q5.jpeg)

**Table 3:** Reaction components for PCR amplicifation using Q5 High-Fidelity 2X Master Mix.

![](https://static.igem.org/mediawiki/2018/8/8b/T--Stuttgart--PCR-MM.jpeg)

The conditions and used primers for successful amplification of our constructs is shown in table 4.

**Table 4:** PCR conditions for successful amplification.

![](https://static.igem.org/mediawiki/2018/e/e8/T--Stuttgart--PCR-successful.jpeg)

Primers (5'-3') used for successful amplification:

* Vector\_gib\_ara\_f: TACTAGTAGCGGCCGCTG
* Vector\_gib\_ara\_r: CTAGTAGTCTTCTTTCTCCTCTTTGCTAGTAG
* araBAD\_NisA\_YL\_f: GCAAAGAGGAGAAAGAAGACTACTAGATGTATGCGGCGTATCATC
* araBAD\_NisA\_r: TGCAGCGGCCGCTACTAGTATTATTTGCTTACGTGAATACTACAATG
* araBAD\_NisA\_LY\_f: GCAAAGAGGAGAAAGAAGACTACTAGATGAGTACAAAAGATTTTAACTTGGATTTG
* araBAD\_NisA\_r: TGCAGCGGCCGCTACTAGTATTATTTGCTTACGTGAATACTACAATG
* araBAD\_NisA\_LY\_f: GCAAAGAGGAGAAAGAAGACTACTAGATGAGTACAAAAGATTTTAACTTGGATTTG
* araBAD\_NisA\_B\_overhang\_r: CTAACAAAATACTATCCTTTGATTTGGTTATTTGCTTACGTGAATACTACAATG
* araBAD\_NisBTCIP\_f: CCAAATCAAAGGATAGTATTTTGTTAG
* araBAD\_NisBTCIP\_r: TGCAGCGGCCGCTACTAGTACATTACTTCATTATACCATTCATTCTG

Templates we used in the PCRs listed above to get our constructs:

Vector-ara-pBAD-RBS: [BBa\_K2848000](http://parts.igem.org/Part%3ABBa_K2848000)

Nis-YL: gblock\_Promoterlong\_\_YAAY\_leader\_his6\_NisA

GATTCTAGAGAGACATACTTGAATGACCTAGTCTTATAACTATACTGACAATAGAAACATTAACAAATCTAAAACAGTCT
TAATTCTATCTTGAGAAAGTATTGGTAATAATATTATTGTCGATAACGCGAGCATAATAAACGGCTCTGATTAAATTCTG
AAGTTTGTTAGATACAATGATTTCGTTCGAAGGAACTACAAAATAAATTATAAGGAGGCACTCAAAATGTATGCGGCGTA
TCATCATCATCATCATCATAGTACAAAAGATTTTAACTTGGATTTGGTATCTGTTTCGAAGAAAGATTCAGGTGCATCAC
CACGCATTACAAGTATTTCGCTATGTACACCCGGTTGTAAAACAGGAGCTCTGATGGGTTGTAACATGAAAACAGCAACT
TGTCATTGTAGTATTCACGTAAGCAAATAaACTAGTTAG

NisA-LY and NisA-LY-NisB-overhang: gblock\_Promoter-long\_leader\_YAAY\_his6\_NisA

GATTCTAGAGAGACATACTTGAATGACCTAGTCTTATAACTATACTGACAATAGAAACATTAACAAATCT
AAAACAGTCTTAATTCTATCTTGAGAAAGTATTGGTAATAATATTATTGTCGATAACGCGAGCATAATAAACGGCTCTGA
TTAAATTCTGAAGTTTGTTAGATACAATGATTTCGTTCGAAGGAACTACAAAATAAATTATAAGGAGGCACTCAAAATGA
GTACAAAAGATTTTAACTTGGATTTGGTATCTGTTTCGAAGAAAGATTCAGGTGCATCACCACGCATCTATGCGGCGTAT
CATCATCATCATCATCATATTACAAGTATTTCGCTATGTACACCCGGTTGTAAAACAGGAGCTCTGATGGGTTGTAACAT
GAAAACAGCAACTTGTCATTGTAGTATTCACGTAAGCAAATAaACTAGTTAG

NisBTCIP: *Lactococcus lactis* NZ9700 gDNA

Agarose gel electrophoresis

For testing which amplification was successful, agarose gel electrophoresis in horizontal chambers was used. The electrophoresis was performed using 1% TAE agarose (Agarose LE, Genaxxon) gels with ethidium bromide in 1x TAE buffer. The gel was loaded with 4 µL of the amplification product and 1x Gel Loading Dye Purple (NEB). As marker served the Quick-Load Purple 1 kb Plus DNA Ladder (NEB). The running conditions were between 4-5 V/cm. The results were documented with the DIANAII raytest DARKROOM system.

Gibson assembly

The final constructs were built using Gibson assembly. For that purpose, the amplicons of vector and inserts with their overhangs respectively were purified using Monarch PCR & DNA Cleanup Kit (NEB) following manufacturer's protocol. For Gibson assembly 10 µL Gibson Assembly Master Mix 2x (NEB) with 100 ng vector and insert(s) in 3x molar excess to vector was used. The reaction was done in a total volume of 20 µL. The mix was then incubated at 50 °C for 1 h.

Transformation

For heat shock transformation chemically competent *E. coli* DH5α or BL21 (DE3) were used.
5 µL of the Gibson reaction were added to 50 µL of competent cells. The mix was then incubated on ice for 30 min. Subsequently the cells were heat shocked for exactly 30 s at 42 °C and placed on ice for 5 min. The transformation mix was then resuspended in SOB medium incubated at 37 °C, 180 rpm for 1 h. The whole mix was then plated on LBCm-plates and incubated at 37 °C for 24 h.

Cultivation

For pre-culture 2 mL LBCm medium was inoculated with single colonies and incubated at 37 °C for 20-24 h.

Mini prep

Pre-culture was used for mini prep with Monarch Plasmid Miniprep Kit (NEB) following manufacturer's protocol and concentrations were measured using NanoDrop.

Sequencing

To check successful cloning of the final constructs, 5 µL of each purified plasmid DNA with concentrations ranging from 70-100 ng/µL were sequenced with 5 µL of sequencing primers (5 µM) in a total volume of 15 µL using LightRun (GATC, Eurofins).

Primers used for sequencing:

* VF2: 5'‑TGCCACCTGACGTCTAAGAA‑3'
* VR: 5'‑ATTACCGCCTTTGAGTGAGC‑3'

Generation of basic BioBrick

For generation of the basic BioBrick our composite BioBrick [BBa\_K2848004](http://parts.igem.org/Part%3ABBa_K2848004) was source. 1 µg [BBa\_K2848004](http://parts.igem.org/Part%3ABBa_K2848004) was digested with 1 µL XbaI (NEB) and BbsI respectively in 1x Buffer G (both from Thermo Fisher) at 37 °C for 1 h. The digest was checked with agarose gel electrophoresis (as described above). The upper band was cut out and cleaned up with Zymoclean Gel DNA Recovery Kit (Zymogen). Afterwards 2 µL of the cleaned up fragment was ligated with 2 µL T4 DNA Ligase in 1x T4 Buffer (both NEB) in a total volume of 20 µL. The mix was incubated for 45 min at room temperature and 5 µL were transformed, plasmid-DNA purified and sequenced as described above, resulting in our basic BioBrick [BBa\_K2848003](http://parts.igem.org/Part%3ABBa_K2848003).

Nisin expression

[BBa\_K2848004](http://parts.igem.org/Part%3ABBa_K2848004) was transformed into chemically competent *E. coli* DH5α. A single colony was inoculated in 2 mL LBCm and did grow for 1 h. This was used as inoculum for 200 mL SOBCm and 200 arabinose (1 g/L) was added to induce Nisin expression. This was cultivated at 37 °C and 180 rpm over night.

SDS-PAGE

* Load 20 µL of samples (diluted with 5x loading buffer)
* Run 8 % polyacrylamide-gel at 100 V, 120 min
* Stain the gel with Coomassie staining solution for at least 1 h
* Destain the gel until you can see protein bands (eventually over night)

Western-Blot

* Clean polyacrylamide gel with ddH2O
* Blotting:
  + Add 3 Whatman-paper soaked in blot-buffer to the blotting machine
  + Soak nitrocellulose membrane in blot-buffer and add on top of the Whatman-Paper
  + Add polyacrylamide gel
  + Add 3 Whatman-paper soaked in blot-buffer
  + Remove airbubbles with roller
* Run blotting machine (110 mA per gel, 10-12 V)
* Block the nitrocellulose membrane in Roche-Block Solution for 30 min
* Incubate membranes with antibody in Roche-Block Solution for 1 hour
* Wash in wash-buffer 3 x 3 min

Silver-staining

* 15 min incubation of the gel in fixative
* Wash two times with ddH2O for 5 min
* Incubate in sodium thiosulfate solution for 1 min
* Wash three times with ddH2O for 20 s
* Incubation in silver nitrate solution for 10 min
* Wash three times with ddH2O for 20 s
* After the washing steps incubate in developer solution until protein bands are visible
* Stop the reaction immediately with stop solution

### Protocols Rhamnolipid

Pre-culture: of *Pseudomonas putida* K2440 and  *E. coli* storage in 50 % glycerol stocks

Transformation: of Biobricks *rhlA* (BBa\_K1331001, Plate 5 Well 9L), *rhlB* (BBa\_K1331002, Plate 5 Well 9N), *araC* (BBa\_K808000, Plate 2 Well 7E), *mRFP* (BBa\_J04450) as well as *rhlC* (pVLT33) in  *E. coli* DH5α as described in the iGEM Protocol:

* Resuspend the DNA and add to the competent cells
* Incubate on ice for 30 min, heat shock at 42 °C for 45 s and again incubate on ice for 5 min
* Add YT media and incubate at 37 °C for 1 h
* Pipette 100 µL onto petri plates
* Spin down the cells for 3 min, discard the supernatant and resuspend the cells in remaining 1
* Again pipette onto petri plates and incubate everything overnight (14-18 h) at 37 °C

Cultivation: of each transformation in LB media by picking a single colony and cultivate overnight at 37°C

Miniprep: for plasmid cleanup (Jena Bioscience)

* Centrifuge overnight culture and resuspend
* Add Lysis Buffer, invert and incubate for 1-2 min
* Add neutralization buffer, invert and centrifuge for 5 min
* Activate the Binding column with activation buffer
* Transfer supernatant on the activated column, shortly centrifuge, add wash buffer (with Ethanol) and centrifuge again
* Wash again, centrifuge and put column in a clean 1.5 mL microtube
* Add Elution Buffer or dd-H2O, incubate, centrifuge for 1 min and store at -80 °C

PCR: using a Q5 polymerase

* Primer concentration of 10 µM, amount of DNA between 1 pg\*µL-1 and 3 ng\*µL-1

Program as described below:

* 98 °C for 1 min 30 s
* cycle: 35 times in a row
* 98 °C for 10 s
* Temperature gradient starting 5 degree below Tm for 45 s
* 72 °C for 60 s
* 72 °C for 2 min

### Protocols Linkage

#### Chemical linkage

The experiments for chemical linking of rhamnolipids to chitosan were conducted following protocols established by Hinner *et al.* 2016 [[L.1]](#reflL.1) and personal communication with the first author.

![](https://static.igem.org/mediawiki/2018/3/39/T--Stuttgart--Chemical_linkage-reac.jpeg)

All reactions are carried out at a 80 °C in ionic liquid 1-ethyl-3-methyl-imidazolium acetate ([EMIM]OAc) under continuous stirring.

General procedure:

* Dissolve 1 % (w/v) Chitosan in [EMIM]OAc at 80 °C for 30 min
* Add rhamnolipid to the chitosan and dissolve for 30 min
* To start the reaction, add the divinyladipate to the mixture
* Stop the reaction by adding H2O after 2 h
* The product precipitated overnight at 4 °C* Afterwards, dialyze the polymer overnight against 1 L of PBS
  * Antimicrobial effects can be examined by antimicrobial assay

Antimicrobial assay:

* Inoculate overnight cultures of *E. coli*, *C. glutamicum*, *L. lactis*, *B. subtilis* and *S. cerevisiae* in 5 mL appropriate medium (LB or Yeast medium).
* Plate 100 µL of overnight culture on LB agar plates
* Divided the plate into different areas (e.g. control, TAGC, foil, ...) and place the substances in the plate
* After incubation, evaluate the growth of the microorganisms
* Remove the tested surfaces and incubate the plates again
* Afterwards, check the plate for growth of new colonies on previous covered spots

It was first planned to analyze the reaction product by IR spectometry. During the experiments, the IR spectometry had not proved as an appropriate method for analyzing. We were not able to identify the successful ester formation in the spectograph. Therefore, different reaction controls were investigated.

The reaction was optimized to yield the best polymer properties and antimicrobial effect. Therefore, the molecular ratios between AGU (anhydro glucose unit), divinyladipate and rhamnolipids were varied (1:1:1, 1:2:1, 1:2:2).

#### Enzymatic linkage

There exist diverse publications (e.g. Freddi *et al.* (2006) [[L.2]](#reflL.2), Chen *et al.* 2003 [[L.3]](#reflL.3)), describing the linkage of proteins to chitosan by tyrosinase. Hereby, the tyrosinase oxidizes tyrosine residues from the proteins. The oxidized tyrosine residues can react with the amino group of the chitosan by formation of a covalent bond.

![](https://static.igem.org/mediawiki/2018/a/a0/T--Stuttgart--Enzymatic_linkage-reac.jpg)

Tyrosinase activity assay:

The tyrosinase was ordered [CAS: 9002-10-2](https://www.sigmaaldrich.com/catalog/product/sigma/t3824?lang=en&region=US) and tested for activity by enzyme assay.

* Dilute tyrosine solution in a 96-well plate to different final concentrations (2, 1, 0.5, 0.25, 0.125 mM) with 50 mM phosphate buffer pH 6.0.
* Start the reaction by adding 100 µL of tyrosinase (1 U) to the 100 µL tyrosine in the well
* Measure the absorbance at 280 nm with a plate reader every minute for 30 min in total
* Plot the data and calculate the reaction parameters

Linkage of GFP to chitosan:

To establish the linkage of proteins to chitosan by tyrosinase in our lab, the experiments were conducted with GFP as protein. Our antimicrobial peptide posesses no tyrosine residue in it's native form. Two different protocols were tested for linkage, according to the publications above.

Protocol 1:

* Dilute chitosan stock solution 1:2 in phosphate buffer pH 6.5 and adjust pH to 6.5 with 0.5 M NaOH
* Add 0.5 mL GFP (14 mg/mL) and 100 µL tyrosinase to 4 mL chitosan solution
* Incubate the mixture at room temperature under continuous shaking for 3 days (open vessel for oxygen!)
* Take samples at different time points (start, 24 h, 48 h, end)
* Centrifuge samples and analyze supernatant by PAGE

Protocol 2:

* Dilute chitosan stock solution 1:2 in phosphate buffer pH 6.0 and adjust pH to 5.8 with 0.5 M NaOH
* Add 50 µL tyrosinase to 500 µL GFP and incubate for 3 days at room temperature, shaking (open vessel for oxygen!)
* Afterwards, add 2 mL of chitosan solution (mentioned above) to the batch. Incubate the mixture subsequently for 3 more days
* Take samples at different time points
* Centrifuge samples and analyze supernatant by PAGE

GFP purification:

* Defrost GFP containing *E. coli* filtrated cell suspension and resuspend in PBS
* Disrupt cells by high-pressure homogenizer (3 cycles)
* Centrifuge to separate protein containing supernatant from debris
* Purificate GFP by his-tag chromatography(ÄKTA). Use TRIS buffer (50 mM, pH 7.6) was for equilibration, loading and washing steps. Bound protein can be eluted by 50 mM TRIS-buffer pH 7.6 containing 500 mM imidazole
* Subsequently, dialyze the GFP containing fraction against 3 L phosphate buffer (50 mM, pH 6.5) overnight
* Determine protein concentration by nano drop

### References

[[C.1]](#refC.1) Colorimetric assay of chitosan in presence of proteins and polyelectrolytes by using o-phthalaldehyde. N.I. Larionova, D.K. Zubaerova, D.T. Guranda, M.A. Pechyonkin, N.G. Balabushevich. *Carbohydrate Polymers*. 2009. **75**, Issue 4, pp 724-727, doi.org/10.1016/j.carbpol.2008.10.009.

[[L.1]](#refL.1) Homogeneous vinyl ester-based synthesis of different cellulose derivatives in 1-ethyl-3-methyl-imidazolium acetate. L. P. Hinner , J. L. Wissner , A. Beurer , B. A. Nebel and B. Hauer. *Green Chemistry*. 2016. Volume 18, pp 6099-6107.

[[L.2]](#refL.2) Tyrosinase-catalyzed modification of *Bombyx mori* silk fibroin: grafting of chitosan under heterogeneous reaction conditions. G. Freddi, A. Anghileri, S. Sampaio, J. Buchert, P. Monti, P. Taddei. *Journal of Biotechnology*. 2006. Volume 125, Issue 2, pp 281-294.

[[L.3]](#refL.3) Enzyme-catalyzed gel formation of gelatin and chitosan: potential for in situ applications. T. Chen, H.D. Embree, E.M. Brown, M.M. Taylor, G.F. Payne. *Biomaterials*. 2013. Volume 24, Issue 17, pp 2831-2841.

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# Team:Stuttgart/Results

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# Results

## Chitosan

Aim of the chitosan subproject was the production of chitosan in an expression system. To achieve this, the chitin synthase NodC and one of the chitin deacetylases NodB or COD had to be encoded on a plasmid.

Already existing BioBricks from the iGEM Team Darmstadt 2017 ([ChiTUcare](https://2017.igem.org/Team%3ATU_Darmstadt)) were used for the cloning of the enzymes in a plasmid. BioBrick BBa\_K2380002 encoding NodC combined with an arabinose inducible promoter was used as vector.

After having recieved plasmid DNA from iGEM Team Darmstadt transformation was successfully conducted and plasmid DNA isolated using Monarch Plasmid Miniprep Kit (NEB). In parallel, 10 ng of pUC19 plasmid DNA were transformed to evaluate the transformation efficiency of the prepared competent *E. coli* DH5α cells. Transformation efficiency proofed to be about 107 cfu/µg.

Plasmids were checked by restriction digestion using XbaI and PstI to excise the inserts from the pSB1C3 backbone. The respective agarose gel is shown in figure C.1.

![](https://static.igem.org/mediawiki/2018/3/31/T--Stuttgart--Chi-XbaI-PstI_BBs_180904_v2.png
)

*Figure C.1:* 1 % agarose gel of plasmid DNA after restriction digestion with XbaI and PstI.

Except for BBa\_K2380044 (COD basic part) all fragment sizes matched the expections.

Construction of combined BioBrick containing *nodC* and *nodB*

BBa\_K2380002 was opened at the BioBrick suffix using SpeI and PstI. The COD ORF including the preceeding RBS was excised from BBa\_K2380042 by double digestion using NheI and PstI. After agarose gel electrophoresis of the COD sample the respective band at about 1400 bp was cut out and eluted in 10 µL ddH2O using ZymocleanTM Gel DNA Recovery Kit (Zymo Research). The NodC restriction reaction was purified using Monarch PCR & DNA Cleanup Kit (NEB). After ligation and transformation in *E. coli* DH5α colonies were picked for overnight culture and plasmid DNA checked by double digestion with XbaI and PstI and agarose gel electrophoresis (Figure C.1, rightmost lane). DNA sequence and plasmid map can be found in the registry ([BBa\_K2848000](http://parts.igem.org/Part%3ABBa_K2848000)).

Expression of BBa\_K2380002/-042 and our combined construct BBa\_K2848000 was performed using a culture volume of 100 mL. Cells were disrupted in phosphate buffer by high pressure homogenization using an EmulsiFlex C5 (Avestin, ATA Scientific). Proteins were purified using immobilized metal affinity chromatography (HisTrap column, ÄKTA, GE Healthcare) and analyzed by SDS-PAGE and Coomassie staining (Figure C.2).

![](https://static.igem.org/mediawiki/2018/c/cb/T--Stuttgart--SDS-PAGE_Chi_180913_Aekta3.png)

*Figure C.2:* SDS-PAGE of purified protein fractions after expression using BBa\_K2380002 (NodC), BBa\_K2380042 (NodB) and BBa\_K2848000 (NodC+NodB). Red arrow: NodC.

As successful expression of NodC was noticed, enzyme activity was tested by UDP Glo assay. Results of the corresponding luminescence measurements are displayed in figure C.3.

![](https://static.igem.org/mediawiki/2018/6/6c/T--Stuttgart--NodC_Activity_Assay_new.jpeg)

*Figure C.3:* Activity assay of purified NodC after expression using BBa\_K2380002 (NodC) and BBa\_K2848000 (NodC + NodB). Assay was performed using the UDP-Glo Kit (Promega) and UDP-GlcNAC as substrate. Activity was measured by detecting luminescence with a TECAN microplate reader, integration time = 1 s.

In figure C.3 the activity of NodC expressed from our combined BioBrick has a much higher activity than NodC expressed from BBa\_K2380002. As there was no change in the sequence of the NodC expression cassette the reason for this higher activity is not clear. Further experiments have to be conducted to validate the activity of NodC.

With respect to the complicated purification of NodB from inclusion bodies built during expression which team Darmstadt 2017 has already described. We exchanged the chitin deacetylase from NodB to COD. Prior to cloning by restriction and ligation, we added a His-tag to COD ([BBa\_K2380043](http://parts.igem.org/Part%3ABBa_K2380043)) by PCR. In addition to the C-terminal His-tag a BioBrick suffix compatible SpeI restriction site was added.

After successful PCR (Figure C.4), product was digested using XbaI and SpeI. BBa\_K2380002 was again used as vector. After opening the vector by SpeI restriction digestion and dephosphorylation by using alkaline phosphatase, ligation of COD + His-tag into the vector was performed overnight. The COD fragment could be inserted in either orientation. Therefore, 16 colonies were picked and colonyRCA was performed for amplifying circular (plasmid) DNA. The correct orientation was approved by restriction digestion using MscI and agarose gel electrophoresis (Figure C.5)

![](https://static.igem.org/mediawiki/2018/0/0c/T--Stuttgart--Chi-COD-His-tag-PCR_180920.png)

*Figure C.4:* 1% agarose gel of His-tag PCR using BBa\_K2380043 as template. Expected fragment size is 1522 bp as indicated by red arrow.

![](https://static.igem.org/mediawiki/2018/1/18/T--Stuttgart--Chi-colonyRCA-MscI_180927_v2.png
)

*Figure C.5:* 1% agarose gel of RCA reaction after restriction digestion with MscI to check correct orientation of the COD ORF. Expected fragments are 2590 bp, 2355 bp and 318 bp.

Expression of BBa\_K2380043 and BBa\_K2848001 was performed using a culture volume of 250 mL. Cells were disrupted in phosphate buffer by high pressure homogenization using an EmulsiFlex C5 (Avestin, ATA Scientific). Proteins were purified using immobilized metal affinity chromatography (HisTrap column, ÄKTA, GE Healthcare) and analyzed by SDS-PAGE and Coomassie staining (Figure C.6).

![](https://static.igem.org/mediawiki/2018/5/53/T--Stuttgart--SDS-PAGE_NodC%2BCOD_%C3%84kta.jpeg)

*Figure C.6:* 12.5 % SDS-PAGE after His-tag purification of cell lysates and elutions from BBa\_K2848001 and BBa\_K2380043. For both constructs two approaches were analyzed.

NodC (approximately 21 kDa) and COD (approximately 45.5 kDa) could not be detected by SDS-PAGE and Coomassie staining. Further experiments are necessary to evaluate the expression of NodC and COD in the plasmid BBa\_K2848001. Culturing and purification of BBa\_K2380043 acted as a negative control because the COD on this plasmid has no His-tag for purification.

### Outlook

Further experiments for the subproject chitosan are the validation of the new generated BioBricks BBa\_K2848000 and BBa\_K2848001. By varying the amount of IPTG and arabinose induction of expression can be optimized. Purification of NodC and COD from cell culture with a His-Trap column should be analyzable by SDS-PAGE. Eventually the staining method has to be more sensitive (e.g. silver staining, western blot). After proof of function chitosan could be produced by *E. coli* in one culturing step by adding UDP-N-acetylglucosamine (UDP-GlcNAc) to the medium.

The final purification of chitosan from the cell culture has to be investigated. The fact that chitosan is possibly toxic to *E. coli* must be considered. Eventually, induction of chitin synthase and chitin deacetylase has to be separated to protect *E. coli* from early cell death [(see Design)](https://2018.igem.org/Team%3AStuttgart/Design). Otherwise, intracellular chitosan has perhaps no toxic effect, because it interacts with peptidoglycan [[C.1]](#reflC.1).

## Nisin

Team Nisin generated BioBricks that enable the production of a modified nisin in *E. coli*. The template for the construct of nisin (containing two tags: one for purification: His6-tag, one for covalent linkage using tyrosinase: YAAY-tag) was synthesized by IDT. This template then was used for successful amplifications of the NisA inserts (Figure N.1).

![](https://static.igem.org/mediawiki/2018/0/07/T--Stuttgart--NisACompositeGel.jpeg)

***Figure N.1*: Agarose gel of the PCR products of the araBAD\_NisA insert.** As marker we used the 1 kb Plus DNA ladder from NEB.
YL: NisA with YAAY and His6-tag at the N-Terminus.
LY: NisA with YAAY and His6-tag after the leader peptide.
OH: amplicons with overhangs for Gibson with vector pSB1C3 and the Genecluster NisBTCIP

The inserts were cloned into the BioBrick [BBa\_K2380002](http://parts.igem.org/Part%3ABBa_K2380002) from Darmstadt using Gibson assembly to replace the chitin synthase *nodC* and preserve the araBAD promoter. This resulted in the composite BioBrick [BBa\_K2848004](http://parts.igem.org/Part%3ABBa_K2848004) encoding the modified nisin with the inducible araBAD promoter and a strong RBS confirmed by sanger sequencing (Figure N.2).

![](https://static.igem.org/mediawiki/2018/3/3e/T--Stuttgart--araBAD-NisA-Sequencing.jpeg)

***Figure N.2*:** BioBrick [BBa\_K2848004](http://parts.igem.org/Part%3ABBa_K2848004) confirmed by sanger sequencing.

To obtain the basic BioBrick encoding only *nisA*, [BBa\_K2848004](http://parts.igem.org/Part%3ABBa_K2848004) was digested with BbsI and XbaI and ligated after removal of the promoter and RBS fragment (Figure N.3). This resulted in the basic BioBrick [BBa\_K2848003](http://parts.igem.org/Part%3ABBa_K2848003) confirmed by sanger sequencing (Figure N.4).

![](https://static.igem.org/mediawiki/2018/8/8b/T--Stuttgart--NisABasicGel.jpeg)

***Figure N.3*: Agarose gel of the pSB1C3\_araBAD\_NisA\_LY construct digested
with XbaI and BbsI.** As marker we used the 1 kb Plus DNA ladder from NEB.
The product containing the araBAD promoter is 1242 bp long. The product
holding pSB1C3 and the protein coding region for NisA is 2275 bp long.

![](https://static.igem.org/mediawiki/2018/6/63/T--Stuttgart--BasicBioBrick-NisA-new.jpeg)

***Figure N.4*:** BioBrick [BBa\_K2848003](http://parts.igem.org/Part%3ABBa_K2848003) confirmed by sanger sequencing

To confirm the production of pre-nisin in *E. coli* we used western blot (Figure N.5) for visualization (pre-nisin having higher molecular weight then natural nisin).

![](https://static.igem.org/mediawiki/2018/f/f9/T--Stuttgart--NisWesternBlot.png)

***Figure N.5*: Western Blot of pre-nisin.** As marker we used Roti-Mark Standard Protein Marker. Pre-nisin has a molecular weight of 7.37 kDa and was detected using an alphaHis antibody. The red arrow indicates NisA.

Since we aimed for a visually more pleasing confirmation, we used SDS-PAGE of pre-nisin with silver staining in figure N.6.

![](https://static.igem.org/mediawiki/2018/9/9b/T--Stuttgart--Silbernitrat2.png)

***Figure N.6*: SDS-PAGE of pre-nisin with silver staining.** As marker Roti-Mark Standard Protein Marker was used. The black arrow indicates NisA with a molecular weight of 7.37 kDa.

### Outlook

We are currently able to produce pre-nisin with the required tags for purification and linkage but for further application nisin has to be fully processed in order to enable its complete antimicrobial effects. Therefore, we cloned the processing enzyme cluster *nisB*, *nisT*, *nisC*, *nisI* and *nisP* (required for post-translational modification, resistance and export) in our BioBrick [BBa\_K2848004](http://parts.igem.org/Part%3ABBa_K2848004) downstream of *nisA*. This theoretically enables us to produce fully processed nisin containing the YAAY- and His6-tag C-terminal in a resistant *E. coli* which also exports nisin to the surrounding medium. Unfortunately, the construct still contains two forbidden SpeI restriction sites so it cannot be registered as a BioBrick. Those restriction sites will have to be removed using *site directed mutagenesis*.
Due to secretion of nisin and its inducible expression, production of large amounts of nisin is possible in the future using for example a bioreactor in a perfusion process.

## Rhamnolipid

The transformation of *E. coli* DH5α with existing BioBricks BBa\_K1331001 (*rhlA*), BBa\_K1331002 (*rhlB*), BBa\_K808000 (*araC*) and BBa\_J04450 (mRFP) as well as pVLT33 containing *rhlC* was successfully carried out. Unfortunately not all required PCRs worked and it was not possible to amplify *rhlC*. However, the restriction digest provided the right sizes of fragments accorded with theoretical values leading to the conclusion of having the correct templates (Figure R.1). Since most of the PCRs were not successful (with temperature gradient, GC Enhancer and different concentrations) the project was dropped because time was running out. A possible reason could be the high complexity of the gene and especially the high GC-content. This could lead to an inhibition of the proofreading DNA polymerase [[R.1]](#reflR.1). The content of *rhlC* is indicated by about 82 % in a window of 100 bases starting at base 843 (IDT).

![](https://static.igem.org/mediawiki/2018/6/62/T--Stuttgart--Verdau_RL.png)

*Figure R.1*: Results of agarose gel electrophoresis: AraC = PCR of araC at given temperature, *Rhl A* = PCR of *rhlA* at given temperature, *Rhl B* = PCR of *rhlB* at given temperature, *Rhl C* = PCR of *rhlC* at given temperature, *RhlC* BB = PCR of *rhlC* at given temperature with second primer pair for direct creation of a basic part BioBrick, *Rhl* A digest: digestion of pSB1C3 containing *rhlA* with EcoRI and SpeI, *Rhl* B digest: digestion of pSB1C3 containing *rhlB* with EcoRI and SpeI, AraC digest: digestion of pSB1C3 containing araC with EcoRI and SpeI, *Rhl C*: pVLT33 containing *rhlC* without digestion, mRFP digest: digestion of pSB1C3 containing mRFP with EcoRI and SpeI

### Outlook

Although production of rhamnolipids in *P. putida* could not be carried out successfully here, it has been demonstrated that establishing thereof is feasible. For example, the iGEM teams from Uppsala 2015 produced rhamnolipids in *E. coli* and University of Columbia 2016 in *P. putida*. Wittgens *et al.* also reported the construction of new production strains for rhamnolipid synthesis in *P. putida* KT2440 [[R.2]](#reflR.2).

Heterologous production of di-rhamnolipids is feasible as previous mentioned examples show, since the molecular biological techniques are all standard methods nowadays. As *rhlC* could not be added to the registry as sought, this task is still open for following teams.

## Linkage

The objective of our experiments was the chemical and enzymatic linkage of the produced compounds. Through linkage of the antimicrobial agents to the chitosan scaffold we generated our anti germ coating.

### Chemical linkage

The aim of the experiment was the linking of the antimicrobial agent rhamnolipid to the chitosan scaffold by divinyladipate [[L.1]](#reflL.1). Thereby, divinyladipate acts as a linker. By transesterfication an ester bond is formed between the components and they become connected.

First, we tried to verify the successful linkage by IR-spectroscopy. No specific peak was found to identify the formed ester bond. There was an ester peak in each spectrograph of the product. This peak likely originates from the ester bond in the rhamnolipid. Therefore, IR-spectroscopy was no appropriate method to verify the reaction and we tried an other approach to prove the success of the reaction.

We analyzed different mixtures of the educts as controls for the reaction (Figure L.1).

![](https://static.igem.org/mediawiki/2018/4/4a/T--Stuttgart--Chemical_linkage-controls.jpeg)

*Figure L.1:* Control reactions for chemical linkage. All reactions were executed according to the same protocol. Top left to right: chitosan-chitosan in ionic liquid, chitosan+rhamnolipid+divinyladipate-all educts were dissolved in ionic liquid, rhamnolipid-rhamnolipid in ionic liquid. Bottom left to right: chitosan+divinyladipate-chitosan and divinyladipate were dissolved, chitosan+rhamnolipid-chitosan and rhamnolipid in ionic liquid, rhamnolipid+divinyladipate-rhamnolipid and divinyladipate were dissolved in ionic liquid.

Figure L.1 indicates that a solid compound only precipitated if all educts were added to the reaction. In the control approaches no solid precipitated. Following, the reaction was successful when all educts where mixed.

In order to find the ideal molecular ratios between the educts we tried different amounts of the substances. After diverse approaches we concluded that a ratio of one part AGU (anhydro glucose unit) chitosan per two molecules of rhamnolipid and divinyladipate (1:2:2) resulted in an optimal surface. This ratio is equivalent to 1 % (w/v) chitosan and 0.93 mM of rhamnolipid and divinyladipate. The resulting surface showed the best properties regarding precipitation and antimicrobial effect. This surface was used for [antimicrobial tests and "Demonstrate"](https://2018.igem.org/Team%3AStuttgart/Demonstrate).

### Enzymatic linkage

For enzymatic linkage of nisin to chitosan we used a tyrosinase. This enzyme is described in diverse publications to link proteins to chitosan ([[L.2]](#reflL.2), [[L.3]](#reflL.3)).

Prior to further experiments the activity of the tyrosinase was tested. Figure L.2 shows the activity of the substrate turnover of different tyrosine concentrations by tyrosinase.

![](https://static.igem.org/mediawiki/2018/2/22/T--Stuttgart--Enzymatic_linkage-enzymeactivity.jpeg)

*Figure L.2:* Enzyme activity of tyrosinase EC(1.14.18.1). The absorbance 280 nm was plotted over time in minutes. Different tyrosine concentrations were diluted in phosphate buffer. The reaction was started by adding 1 Unit tyrosinase.

In Figure L.2 you can see the increase of absorbance at 280 nm in time indicating the oxidization of tyrosine. By oxidization of tyrosine the electron system of the aromatic ring is changed leading to a shift in the absorbance. The graph shows a sigmoidal curve which is typical for an enzyme reaction. The higher the tyrosine concentration the higher the final absorbance. Following the tyrosinase works as expected.

Proof of concept:

For linkage of nisin to chitosan a tyrosine residue has to be introduced first. While the Nisin-team was working on the modification of nisin we linked the tyrosine containing green fluourescence protein (GFP) to chitosan as a proof of concept. Therefore we tried two different [protocols](https://2018.igem.org/Team%3AStuttgart/Experiments) for the reaction and analyzed the reaction product by SDS-PAGE subsequently. Figure L.3 depicts the SDS-PAGE results for protocol 1 and 2. There were loaded samples from supernatants of different time points on the gel. For protocol 1 chitosan was mixed with GFP and tyrosinase and incubated for 3 days. In protocol 2 GFP and tyrosinase were incubated for 3 days before adding the chitosan solution.

![](https://static.igem.org/mediawiki/2018/6/62/T--Stuttgart--Enzymatic_linkage-GFP.jpeg)

*Figure L.3:* SDS-PAGE of tyrosinase based linkage of GFP to chitosan. Sample supernatants of different time points were loaded. A: Sample supernatants of time points (0, 24, 48, 72 h) of control and sample treated acording to protocol 1. B: Sample supernatants of time points (0, 72(1), 72(2), 144 h) of control and sample treated acording to protocol 2.

The supernatant was loaded after short centrifugation of the samples. After centrifugation the chitosan was found in the pellet. Therefore bound GFP was also in the pellet. A decrease of protein in the supernatant indicates a linkage of GFP with chitosan. Figure L.3.A shows that the GFP band (~25 kDa) of samples of time points 24, 48 and 72 h is smaller and less intense in contrast to the control. This indicates a decrease of GFP in the supernatant and therefore a successful linkage of GFP to chitosan. In addition you can see a new band (~63 kDa) for the samples at 24 and 48 h. This band likely originates from cross linked GFP. The oxydized tyrosine can form a covalent bond with the amine group of the chitosan but also with the amine group of the amino acids of the GFP. This side reaction was stronger for protocol 2 (Figure L.3.B). In this case the tyrosine of the GFP was first oxidized before adding the chitosan. Whilst oxidation the oxidized tyrosine residues reacted with amine groups of GFP because no chitosan was present. Nevertheless the connection of GFP to chitosan was successful, too. The GFP band (~25 kDa) of sample after 144 h was smaller and less intense in comparison to the control. However, the protocol 1 leaded to better results and less side products. Thus we suggest protocol 1 for linking proteins to chitosan.

### Outlook

We showed successfully the production of a version of TAGC by chemical linkage. Tyrosine-containing proteins could be linked enzymatically to chitosan.

The procedure of chemical linkage can be improved by varying the ratios of the educts. The following precipitation and wash steps can be optimized as well. To remove remaining solute (ionic liquid) and ease the processing of the polymer a liquid-liquid extraction might be helpful. This step enables also the recycling of the ionic liquid. The ionic liquid is quite expensive. Therefore you can save lot of money and resources by recycling of the reaction solute. Moreover the liquid-liquid extraction provides further possibilities to analyze the polymer (e.g. NMR-spectroscopy). To improve linkage and the features of TAGC other linkers than divinyladipate might be utilized in the reaction.

Basically linkage of proteins to chitosan is possible. The next objective is the linkage of nisin to chitosan. Hence varying ratios of nisin, chitosan and tyrosinase need to be examined for finding best degree of substitution (DS). Besides analyzing the reaction by PAGE, MALDI-TOF is a suitable high-end analyzing method. Precipitation of the generated polymer is an important step for processing. Further research and development are necessary to apply the product as coating.

The major goal for future development of TAGC is the final linkage of all components (rhamnolipid and nisin) to chitosan. Due to the synergistic effect of rhamnolipid and nisin we expect stronger antimicrobial effects.

## References

[[C.1]](#ref1) Evaluation of the antimicrobial activity of chitosan and its quaternized derivative on *E. coli* and *S. aureus* growth. Rejane C. Goy, Sinara T.B. Morais Odilio B.G. Assis. *Brazilian Journal of Pharmacognosy*. 2016. Volume 26. Page 122-127.

[[R.1]](#reflR.1) Guanine-rich sequences inhibit proofreading DNA polymerases, Zhu et al. 2016. *SCIENTIFIC REPORTS*. DOI: 10.1038/srep28769.

[[R.2]](#reflR.2) Growth independent rhamnolipid production from glucose using the non-pathogenic *Pseudomonas putida* KT2440. A. Wittgens, T. Tiso, T.T. Arndt, P. Wenk, J. Hemmerich, C. Müller, R. Wichmann, B. Küpper, M. Zwick, S. Wilhelm, R. Hausmann, C. Syldatk, F. Rosenau, L.M. Blank. 2011. *Microb Cell Fact*, 10:80.

[[L.1]](#reflL.1) Homogeneous vinyl ester-based synthesis of different cellulose derivatives in 1-ethyl-3-methyl-imidazolium acetate, Hinner et al., 2016, Green Chemistry, DOI: 10.1039/C6GC02005D

[[L.2]](#reflL.2) Tyrosinase-catalyzed modification of Bombyx mori silk fibroin: grafting of chitosan under heterogeneous reaction conditions, Freddi et al., 2006, Journal of Biotechnology, DOI: 10.1016/j.jbiotec.2006.03.003

[[L.3]](#reflL.3) Enzyme-catalyzed gel formation of gelatin and chitosan: potential for in situ applications. Chen et al. 2003. *Biomaterials*. DOI: 10.1016/S0142-9612(03)00096-6.