iGEM 2019 — Team Linkoping_Sweden

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# Team:Linkoping Sweden/Notebook

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* [Home](https://2019.igem.org/Team%3ALinkoping_Sweden)
* Project

  + [Description](https://2019.igem.org/Team%3ALinkoping_Sweden/Description)
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* Lab

  + [Notebook](https://2019.igem.org/Team%3ALinkoping_Sweden/Notebook)
  + [Experiments](https://2019.igem.org/Team%3ALinkoping_Sweden/Experiments)
  + [Results](https://2019.igem.org/Team%3ALinkoping_Sweden/Results)
  + [Characterization](https://2019.igem.org/Team%3ALinkoping_Sweden/Contribution)
  + [Improvement](https://2019.igem.org/Team%3ALinkoping_Sweden/Improve)
  + [Safety](https://2019.igem.org/Team%3ALinkoping_Sweden/Safety)
* People

  + [Team Members](https://2019.igem.org/Team%3ALinkoping_Sweden/Team)
  + [Collaborations](https://2019.igem.org/Team%3ALinkoping_Sweden/Collaborations)
  + [Attributions](https://2019.igem.org/Team%3ALinkoping_Sweden/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3ALinkoping_Sweden/sponsorship)
* Parts

  + [Parts Overview](https://2019.igem.org/Team%3ALinkoping_Sweden/Parts)
  + [Basic Parts](https://2019.igem.org/Team%3ALinkoping_Sweden/Basic_Part)
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  + [Part Collection](https://2019.igem.org/Team%3ALinkoping_Sweden/Part_Collection)+ [Primers](https://2019.igem.org/Team%3ALinkoping_Sweden/Primers)
* Human Practices

  + [Human Practices](https://2019.igem.org/Team%3ALinkoping_Sweden/Human_Practices)
  + [Public Engagement](https://2019.igem.org/Team%3ALinkoping_Sweden/Public_Engagement)

+ [Awards](https://2019.igem.org/Team%3ALinkoping_Sweden/Awards)

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# Notebook

# Notebook

Here you can follow our team's journey from our first meeting to our final week.

## Week 49 2018

We had our first meeting! The team members were introduced to each other and to our PIs. We are all very excited to be apart of LiU iGEM 2019!

## Week 50 2018

We had our second meeting this week. The team was introduced to the different groups they could be apart of. For our team, this includes Laboratory work, Human Practice, Wiki, and Modeling. We also discussed what the next year’s project was going to be.

## Week 51 2018

This week, we have started to look into antimicrobial peptides. Currently, we are planning on having a project that revolves around this. We also talked about how we can integrate *Vibrio natriegens* as a chassis for this year’s project.

## Week 5

We have started to read up on the laboratory methods, and how plasmids are designed.

## Week 6

We have decided to focus our project on antimicrobial peptides.

## Week 7

We have reached out to companies that work with antimicrobial peptides in order to gain feedback on our project. We also had a plasmid design meeting and talked about different parts of plasmids.

## Week 10

We have made our first models for antimicrobial prediction. We have had our first meeting with Dr. Johan Junker from the Center for Disaster Medicine and Traumatology (KMC). We discussed our project and received important feedback.

## Week 11

We have had our first meeting with Mårten Skog, COO from S2Medical, a local company specializing in wound treatment and healing. We have also started to prepare for the Nordic iGEM Conference.

## Week 14

We have talked to first responders (EMTs) in order to learn about burn care. Even though our project focuses more on the healing process it's good to gain an understanding of the entire process of burn care. We have also visited the Burn Care Center (BRIVA) in Linköping and talked to the research experts and staff. They supplied us with a cellulose bandage that we could use in our experiments.

## Week 15

We have decided on which specific peptides we want to analyse. The peptides that were chosen were LL-37, Magainin 2, PlyF307, Pln1 and LysK.

## Week 16

We had our laboratory safety introduction. We have also ordered one of our DNA sequences CBD-asPink.

## Week 21

We attended the Nordic iGEM Conference. Where we met teams from all over the northern part of Europe. During our time there we connected with other teams, exchanged ideas and enjoyed good company. The conference also included an "iGEM-like" competition where the teams presented their project ideas and were judged. Our project managed to win the "Judges' Award" and good times were had. We look forward to meeting the teams again in Boston and seeing their progress.

## Week 23

Summer has begun!! We are thrilled to initiate our lab work!
We started with the cloning process of the CBD-fusion protein and then continued working with the selection plasmid (CBD-pCons AsPink). However, no ligations were successful this week.

## Week 24

We redid all our ligations and tried different restriction enzymes. Still no success. Through Linköping University, some of our team members were involved in leading experiments for young students in the 7th and 8th grades, as part of a Summer Science School Program. The experiment was DNA extraction from bananas and we also talked about synthetic biology and what we aimed to accomplish with our research. During a Skype meeting with [Aalto-Helsinki iGEM Team](https://2019.igem.org/Team%3AAalto-Helsinki) we engaged in some troubleshooting regarding our projects, how to use *V.natriegens*, what antibiotics and backbones to use for *V.natriegens* and much more. We also decided to switch parts for verification.

## Week 25

We have tried new methods this week since we have had problems with the ligation processes. Troubleshooting regarding our failed ligations were conducted. We have also ordered our model peptides from Caslo which they provided us as a sponsorship.

## Week 26

We realized that the overhangs in our inserts were too short and that this created problems for ligation. We therefor ordered new primers with longer overhangs and in the meantime continued with different ligation attempts. We tried "Gibson Assembly" and successfully ligated CBD-AsPink. A large culture was done for protein production. We then ran a cellulose binding assay and thrombin activity assay.

## Week 27

We successfully ligated some of the antimicrobial peptides with the cellulose binding domain (CBD). The first episode of our podcast is out! Take a  [look!](https://play.acast.com/s/attemptingscience/tag%3Asoundcloud%2C2010%3Atracks/648497571)

## Week 28

We have produced the antimicrobial peptides fused with CBD and started to prepare for antimicrobial activity assays. We were contacted by [Bilkent-UNAMBG iGEM Team](https://2019.igem.org/Team%3ABilkent-UNAMBG) regarding a collaboration. They had written a children's book that talked about how synthetic biology can be used to make our lives better. We translated the storybook to Swedish so that the message could reach people and children in Sweden as well. The book has also been translated to other languages and is able to reach people in many different countries.

## Week 29

This week, we have continued experiments with the antimicrobial peptides that had not successfully been ligated. SDS-PAGEs were conducted in order to confirm all the steps in thrombin cleavage assays activity of sfGFP. The protein production of the antimicrobial peptides fused to the cellulose-binding domain was initiated, and the activity of the model peptide Parseq beta was tested in the lab. The second episode of our podcast is out! We also conducted a phone-interview with the former fire fighter Lasse Gustavsson, who survived a major burn trauma. Listening to his stories and experiences was very insightful and humbling. Take a  [look!](https://play.acast.com/s/attemptingscience/tag%3Asoundcloud%2C2010%3Atracks/655189043)

## Week 30

We tested the binding capacity of the cellulose bandage, this was measured with sfGFP as a marker. The antimicrobial activity of PlyF307 was analysed. Our mNG improvement was analysed and compared with the original part. New stocks of the antimicrobial peptides were produced. The activity of the model peptide Parseq alpha was tested in the lab.

## Week 31

This week we tested the antimicrobial activity of Pln1. LL37 and Magainin 2 was purified. Ligation and colony-screening of the model parseq peptides were conducted. Induction series of sfGFP and mNG was conducted. The fluorescence of EforRed was measured. Thrombin cleavage of LL37, Magainin 2, Pln1 and PlyF307 were analysed on an SDS page. LL37, PlyF307, Magainin 2 and Pln1 was cloned into pUC19 and sent to [Aalto-Helsinki iGEM Team](https://2019.igem.org/Team%3AAalto-Helsinki). During this week we also had the wonderful opportunity to visit the Burn Care Center (BRIVA) in Linköping and observe the the care and treatment of burn wounds. During this visit we talked with the staff, observed the changing of wound dressings and watched a transplantation with skin grafts. The third episode of our podcast is out! Take a  [look!](https://play.acast.com/s/attemptingscience/tag%3Asoundcloud%2C2010%3Atracks/662316923)

## Week 32

We have analysed the antimicrobial activity of LL37 and Magainin 2. Induction gradients of sfGFP were conducted. New attempts on LysK cloning was conducted and CHAP was cloned and placed in pSB1C3 and pUC19. The binding of the amorphous cellulose was analysed. The antimicrobial activity of the recombinant parseq peptides was analysed, however these experiments failed.

## Week 33

We investigated the antimicrobial activity LL37, CHAP, Magainin 2, Pln1 and PlyF307 on *B.subtilis* to determine their effect on gram-positive bacteria. A new CBD purification method was investigated. The fourth episode of our podcast is out! Take a  [look!](https://play.acast.com/s/attemptingscience/tag%3Asoundcloud%2C2010%3Atracks/668027834)

## Week 34

We started to analyse the antimicrobial activity of the peptides without the cellulose binding domain attached to the peptide.

## Week 35

We talked about our project and LiU iGEM at the annual welcome fair for new students. During this week some of our team members also headed down to the Linköping City Festival. There we handed out informative pamphlets regarding antibiotic resistance and the threat it poses to medicine and society. It was great to talk to people in the community about this major problem. The fifth episode of our podcast is out! Take a  [look!](https://play.acast.com/s/attemptingscience/tag%3Asoundcloud%2C2010%3Atracks/675533414)

## Week 36

We have started to analyse the activity of the antimicrobial peptides when they are attached to the cellulose bandage. Our assay is meant to mimic the release and activity of our peptides in the wound. Results look dandy!

## Week 37

We have, as all other iGEM teams, handed in our safety from. The sixth and final episode of our podcast is out! Take a  [look!](https://play.acast.com/s/attemptingscience/tag%3Asoundcloud%2C2010%3Atracks/682736748)

## Week 38

We are conducting some of our final experiments and we are working hard on our text for our wiki page.

## Week 41

We have received word from [Aalto-Helsinki iGEM Team](https://2019.igem.org/Team%3AAalto-Helsinki) that they succeeded in verifying our sampels that we sent to them. Unfortunately, their samples did not reach us in time. [Bilkent-UNAMBG iGEM Team](https://2019.igem.org/Team%3ABilkent-UNAMBG) sent us the finished, illustrated storybook and now it can be view on our wiki under collaborations.

## Week 42

We have been working hard on our wiki texts and we have just had a practice presentation of our project at our univeristy. A lot of people showed up and we received great responses and feedback. We had a really good time and now we are very excited for Boston and looking forward to meeting you all!

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# Team:Linkoping Sweden/Experiments

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* Lab

  + [Notebook](https://2019.igem.org/Team%3ALinkoping_Sweden/Notebook)
  + [Experiments](https://2019.igem.org/Team%3ALinkoping_Sweden/Experiments)
  + [Results](https://2019.igem.org/Team%3ALinkoping_Sweden/Results)
  + [Characterization](https://2019.igem.org/Team%3ALinkoping_Sweden/Contribution)
  + [Improvement](https://2019.igem.org/Team%3ALinkoping_Sweden/Improve)
  + [Safety](https://2019.igem.org/Team%3ALinkoping_Sweden/Safety)
* People

  + [Team Members](https://2019.igem.org/Team%3ALinkoping_Sweden/Team)
  + [Collaborations](https://2019.igem.org/Team%3ALinkoping_Sweden/Collaborations)
  + [Attributions](https://2019.igem.org/Team%3ALinkoping_Sweden/Attributions)
  + [Sponsors](https://2019.igem.org/Team%3ALinkoping_Sweden/sponsorship)
* Parts

  + [Parts Overview](https://2019.igem.org/Team%3ALinkoping_Sweden/Parts)
  + [Basic Parts](https://2019.igem.org/Team%3ALinkoping_Sweden/Basic_Part)
  + [Composite Parts](https://2019.igem.org/Team%3ALinkoping_Sweden/Composite_Part)
  + [Part Collection](https://2019.igem.org/Team%3ALinkoping_Sweden/Part_Collection)+ [Primers](https://2019.igem.org/Team%3ALinkoping_Sweden/Primers)
* Human Practices

  + [Human Practices](https://2019.igem.org/Team%3ALinkoping_Sweden/Human_Practices)
  + [Public Engagement](https://2019.igem.org/Team%3ALinkoping_Sweden/Public_Engagement)

+ [Awards](https://2019.igem.org/Team%3ALinkoping_Sweden/Awards)

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# Experiments

# Experiments

## LB-Miller Agar

Agar protocol was extracted from:  [chsProtocols.cshlp.org](http://cshprotocols.cshlp.org/content/2009/3/pdb.rec11683.full)
15 g agar
10 g tryptone
5 g yeast extract
10 g NaCl
Mix with 1 L of water and autoclave at 121 °C

For Low salt agar, add 0.5 g of NaCl instead of 10 g

## LB-Miller

LB-Miller protocol was extracted from: [USBiological](https://www.usbio.net/protocols/lb-broth-miller)
Prepare the following mixture:
10 g tryptone
5 g yeast extract
10 g NaCl
Mix with 1 L of water and autoclave at 121 °C

For Low salt LB-Miller add 0.5 g of NaCl instead of 10 g
This protocol was extracted from: [Sigma-Aldrich](https://www.sigmaaldrich.com/catalog/product/sigma/l3397?lang=en&region=SE)

## PCR

[Q5® Hot Start High-Fidelity](https://international.neb.com/protocols/2012/08/30/pcr-using-q5-hot-start-high-fidelity-dna-polymerase-m0493) from New England Biolabs was used for all PCR reactions.

Prepare the following mixture in a PCR tube:
12.5 µL of Q5 master mix
1.25 µL of forward primer (10 µM)
1.25 µL of reverse primer (10 µM)
1 µL of DNA template
9 µL of water

Place the PCR tube in the thermocycler and run the following cycles:
98°C for 30 sec
98°C for 10 sec
55-72°C for 20 sec
72°C for 30 sec per kb
Repeat from step 2 12-35 times
72°C for 2 minutes

## Agarose gel

Mix 1.3 g of agarose with 100 mL of TAE buffer. Microwave the mixture until boiling then cast the gel in an electrophoresis cast and add a gelcomb for wells.

Sample preparation: mix 8 µl of Loading Dye with 7 µL of DNA sample. Load a DNA ladder as well for reference. Load the gel and run it at 100 V for 60 minutes. Stain with Ethidium bromide for 5 minutes and destain with TAE for 1 minute. Place the gel on a UV table or in a UV camera.

## PCR purification

[Monarch®](https://international.neb.com/products/t1030-monarch-pcr-dna-cleanup-kit-5-ug#Product%20Information) PCR & DNA Cleanup Kit was used for PCR purification.
Dilute the PCR product in DNA Cleanup binding buffer (1:5) ratio. Place the mix in a spin column and centrifuge for 1 minute. Discard flow-through and add 200 µL of DNA wash buffer. Discard the flow-through and add 200 µL DNA wash buffer again. Place the spin column in new Eppendorf tubes and elute with more than 6 µL water. Analyze the samples on a Nanodrop.

## Restriction digest

Restriction enzymes and protocol were provided by [New England Biolabs.](https://international.neb.com/products/restriction-endonucleases)

Prepare the following mixture:
1 µL restriction enzyme
5 µL buffer (either 1.1, 2.1 3.1 or Cutsmart)
1 µg of DNA template
X µL water
Total volume should be set to 50 µL
Place the Eppendorf tubes containing the mixture in a heating block at 37°C for 15-60 minutes. To purify the DNA for later use, utilize a PCR purification kit.

## Ligation

Ligation mixtures were provided by [New England Biolabs.](https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202)

Prepare the following mixture:
2 µL ligation buffer
50 ng vector
37 ng insert (or a molar ratio of 1:7 vector insert )
X µL water
1 µL ligase
Total volume should be set to 20 µL
Incubate the mixture at room temperature for 10 minutes.

## Gibson Assembly

The Gibson assembly kit was provided from [New England Biolabs.](https://international.neb.com/protocols/2012/09/25/gibson-assembly-master-mix-assembly)

Prepare the following mixture:
10 µL Master mix
Add vector and insert in a 1:2 ratio
X µL water
Total volume should be set to 20 µL
Incubate the mixture at 37°C for 15 minutes.

## Chemically competent cell preparation

Culture 25 mL bacteria for 4-6 hours at 37 °C with appropriate antibiotics in a shake incubator. Harvest the cells by centrifuging for 10 minutes at 3000 g. Discard the supernatant and resuspend the pellet in 0.1 M CaCl2. Incubate the cells on ice for 20 minutes then repeat the centrifugation step. Resuspend the cells in 0.1 M CaCl2 with 15 % glycerol. Store the cells in 300 µL fractions in 1.5 mL tubes. Freeze these instantly in liquid nitrogen and store them in -80 °C.

## Heatshock

Add 1-5 µL of plasmid to 50 µL of Heatshock competent cells. Incubate for 30 minutes on ice place the mix on a 42 °C heat block for 45 seconds then incubate the mix on ice for 2 minutes. Add 250 µL LB to the cells and incubate them at 37 °C for 1 hour. Take 100 µL from the mix and plate it on an agar plate with the appropriate antibiotics. Incubate the agar plate at 37 °C for 16 hours.

## Electroporation

Add 1-5 µL plasmids to 50 µL of Electrocompetent cells. Place the mix in a cuvette. Make sure that the mix covers the bottom of the cuvette. Zap the bacteria with approximately 1.8 mV for approximately 5.4 ms. Add 250 µL LB to the mix and place in a new Eppendorf tube. Incubate the tube at 37°C for 1 hour. Take 100 µL from the mix and plate it on an agar plate with the appropriate antibiotics. Incubate the agar plate at 37°C for 16 hours.

## Colony screening

Prepare a PCR reaction without adding the template DNA. Inoculate one colony with a pipet. Dip the inoculated colony in the PCR reaction, then gently place and scrape the tip on a new agar plate with appropriate antibiotics.

## Bacterial culturing

Take 100 µL from a glycerol stock and plate it on an agar plate with the appropriate antibiotics. Incubate the plate overnight.

Add 1 L LB to a culture flask and add the appropriate antibiotics.
Add 20 mL LB to the plate and resuspend the bacteria in it. Add the bacteria mix to the culture flask and incubate it until OD is between 0.4-0.6. Induce the bacteria and place the culture flask in 16°C for 16 hours.

Centrifuge the cells at 3000 g for 30 minutes and extract the supernatant. Resuspend the pellet in 30 mL water and pour the mix in a 50 mL tube. Freeze it in liquid nitrogen and store it at -80°C.

## Plasmid Preparation

[Monarch®](https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010) Plasmid DNA Miniprep Kit Protocol was used for Plasmid Preparation.
Add 5 mL LB-Miller to a 50 mL tube then add the appropriate antibiotics. Inoculate a colony and add it to the mix. Incubate overnight.
Harvest the cell by centrifugation at 3000 g for 5-10 minutes. Extract the supernatant and replace it with 200 µL Plasmid resuspension buffer. Move the mixture to an Eppendorf tube and add 200 µL Plasmid lysis buffer. Mix the sample by inverting it 5-6 times. After 1 minute, add 400 µL Plasmid neutralization buffer then invert the tube 5-6 times and let the samples incubated for 2 minutes at room temperature. Centrifuge the mix for 5 minutes at 16 000 g. Extract the supernatant, place it in a spin column and centrifuge at 16 000 g for 1 minute. Discard the flow through and add 200 µL Plasmid wash buffer 1. Repeat the centrifugation step, then add 400 µL of Plasmid wash buffer 2 and repeat the centrifugation step. Place the spin column in an eppendorf tube and add 30 µL water. Repeat the centrifugation step. Measure the DNA concentration in a Nanodrop and run the sample on a gel electrophoresis to confirm the size.

## Bacterial lysis

There are several different ways to conduct bacterial lysis. We used two types of lysis methods during this project; freeze thaw and sonication. We found varying success, but the most effective for us was sonication.

#### Sonication Take a pellet of harvested cells and resuspend them in the appropriate buffer for your protein. Place the sonication probe in the mix. Run the sonication on 30 % amplitude with 30 seconds on 30 seconds of. Then centrifuge at 12000 g for 20 minutes. Save the supernatant or the insoluble fraction (pellet) if working with insoluble proteins. Freeze thaw Take a pellet of harvested cells and resuspend them in the appropriate buffer for your protein. Place the sample in liquid nitrogen for 1 minute then place the sample on a heat block set to 42 °C for 1 minute. Vortex the sample and repeat all the steps five times.

## CBD binding purification

The CBD cellulose binding purification can be conducted in several ways. Either it can be done in a PD-10 column, in a Falcon tube or on a cellulose bandage.

**-PD10 column purification**
Add 1 mL of protein sample to the column then incubate it on a end-to-end rotator for 1 hour. Wash it with 8 mL of ethanol 1 mL at a time. Elute the sample in 8 mL of water 1 mL at a time. Save all the elution fractions for SDS-PAGE confirmation of purity.

**-50 mL tube purification**
Add cellulose to a protein solution and incubate it on an end-to-end rotator for 1 hour. Add 20 mL of ethanol and incubate it. Then centrifuge the sample att 3000 g for 5 minutes. Extract the ethanol, repeat the centrifugation and extract the rest of the ethanol. Repeat the ethanol wash steps 3 times. Add 5 mL ddH20 and incubate the sample at 37°C for 1 hour. Centrifuge the sample 3000 g for 5 minutes. Extract the supernatant. Run the sample on an SDS-PAGE to confirm the results.

## IMAC Purification

Proteins that are tagged with a His-tag can be purified using IMAC purification.
Add 1 mL nickel agarose to a PD10 column. Add the wash the agarose with 10 mL wash buffer. Add the protein sampel to the column. Wash with 10 mL wash buffer. Add 10 mL elution buffer 1 mL at a time and save all the fractions for gel electrophoresis confirmation.

#### Buffers

Buffer recipe for IMAC Purification was extracted from  [this article](https://experiments.springernature.com/articles/10.1007/978-1-4939-7816-8_10)

**Equilibration: Low Imidazole**
50 mM Tris
300 mM NaCl
500 mM Imidazole
Set the pH to 8

**Elution: High Imidazole**
50 mM Tris
300 mM NaCl
500 mM Imidazole
Set the pH to 7

## Bradford assay

Bradford assay protocol was extracted from: [Bio-Rad](https://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf)
Make a dilution series of BSA.
Take 5 µL sample and add 250 µL Bradford reagents and place the stained sample in a 96 well plate and measure the absorbance at 594 nm. Compare the absorbance to the standard curve and calculate the amount of protein.

## SDS-PAGE

#### Sample preparation and running the gel

Place two gels in the gel electrophoresis tray and add SDS-PAGE running buffer between the gels until it overflows. Then add SDS-PAGErunning buffer in the tray to the appropriate mark.
Mix 10 µL protein sample with 10 µL of 2X sample preparation buffer, add 1 µL 2-mercaptoethanol and mix the sample. Incubate the sample at 95 °C for 5 minutes then load the sample on the SDS-PAGE. Set the voltage to 100 V and run the page until all protein samples have wandered into the gel, the increase the voltage to 200 V. Run the gel until the loading dye has reached the bottom.

#### Staining

Dismantle the SDS-PAGE mold and place the gel in a tray. Rinse the gel with water. Extract all the water and add the staining solution. Incubate the SDS-PAGE with low rotation for 15 minutes. Remove the staining solution, add Destaining solution and incubate the SDS-PAGE with low rotation for 15 minutes. Remove the Destaining solution and add new. Repeat this step until the proteins are seen as visible bands on the gel.

**Polyacrylamide gel recipe**

Prepare the polyacrylamide mold.

For separation gel mix:
2 mL 33 % acrylamide
0.7 mL 1 % Bis-acrylamide
1 mL 2.122 M Tris-HCl buffer pH 9.18
0.7 mL water
0.1 mL 0.22 M ammoniumperoxodisulfate
Let the mixture degas for 5 minutes, then add 11.5 µL TEMED. Quickly cast the gel in the gel mold. Add isopropanol to the gel and let it sit until it solidifies, then extract the isopropanol.

For stacking gel mix:
0.625 mL 33 % acrylamide
1.25 mL 1 % Bis-acrylamide
0.625 mL 0.541 M Tris-HCl buffer pH 6.1
2.4 mL water
0.1 mL 0.22 M ammoniumperoxodisulfate
Let the mixture degas for 5 minutes, then add 10 µL TEMED. Quickly cast the gel in the gel mold. Add the gel combs and let the gel sit until it solidifies.

**Running buffer 10X**

The protocol was extracted from:  [CSHProtocols](http://cshprotocols.cshlp.org/content/2006/1/pdb.rec10475.full?text_only=true)
Prepare the following mix:
30 g Tris
144 g glycine
10 g SDS
1 L ddH20
When using the running buffer, dilute it to 1 X with ddH20

**Laemmli Sample preparation buffer**

The protocol was extracted from:  [USBiological](https://www.usbio.net/protocols/laemmli-sample-buffer-2)
Prepare the following mix:
4 % SDS
20 % glycerol
0,004 % bromphenol blue
0.125 M Tris-Cl, pH 6.8

**Staining solution stock**

Buffer recipe for Staining solution stock was extracted from  [this protocol](https://www.csb.pitt.edu/faculty/clark/files_protocols/coomassie_rapid.pdf)
Mix the following chemicals:
1 g of Coomassie R250
100 mL of Glacial acetic acid
400 mL methanol
500 mL ddH2O

**Destaining solution stock**

Mix the following chemicals:
100 mL of Glacial acetic acid
400 mL methanol
500 mL ddH2O

## CBD binding capability assay

Take 6 mL purified fusion protein containing one carbohydrate binding domain and one part fluorescent protein. The sample need to be in PBS. place a Epiprotect bandage in the solution. Incubate the sample in 37 °C. Take a sample from the supernatant and measure the absorbance at 485 nm. Repeat this every 25 minutes. When no more absorbance is lost from the supernatant the bandage is saturated.

## Thrombin cleavage assay

The protocol was extracted from:  [Novagen](http://wolfson.huji.ac.il/purification/PDF/Protease_fusion_cleavage/NOVAGEN_Thrombin_kit.pdf)
For a total of 50 μL reaction volume mix:
5 μL 10X Thrombin Cleavage Buffer (200 mM Tris-HCl, 1.5 M NaCl, 25 mM CaCl2, pH 8.4)

X μL of target protein

1 μL of 0.5 U thrombin

x μL deionized water

Incubate overnight in either R.T or 37 °C on 200 rpm. (End-to-end rotator works as well).

## Antimicrobial plate reader assay

Dilute bacteria stocks in low salt LB to appropriate concentrations. Add the antimicrobial peptides in respective well. Fill the well to 200 µL with the LB bacteria mix. Place a cover slip over the plate and puncture one hole in the slip for each well. Place the 96 well plate in a plate reader with the incubation temperature 37 °C. Measure OD600 every 15 minutes and shake the plate 1 minute prior to this.

## Bandage activity assay

This assay is carried out to determine the effect of the bandage on bacteria. Prepare the bandage by dipping in cell lysate that contains the CBD fusion protein of your choice. Incubate it for 30 minutes in 4 °C. Remove the lysate and add 70 % ethanol, incubate it for 30 minutes in 4 °C. Repeat this ethanol washing step 3 times. Cut the bandage and place it in a 96 well plate. Add the target bacteria, thrombin cleavage buffer and thrombin. Incubate the plate at 37 °C with shaking in a 96 well plate reader. Measure the absorbance at 600 nm every hour.

## CFU counting

Take 20 µL from the initial stock and dilute it in 180 µL LB. Take 20 µL from the diluted bacteria and add it in 180 µL LB. Repeat this for a series of 10 tubes. Plate 100 µL of bacteria from each dilution on an agar plate with appropriate antibiotics. Incubate the plates at 37 °C for 16 hours. Count the colonies on the first countable plate and the plate after. This will tell you the concentration of the stock.

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# Team:Linkoping Sweden/Results

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Back to the top
 [Cellulose Binding](#Bind)
 [Antimicrobial Effect](#Antimic)
 [Proof of concept](#PoC)

# Results

The results of our project are mainly divided into two sections. These two sections reflect the two main concepts we wanted to try during the project. First, we present the results from our experiments where we attach our carbohydrate binding domain to cellulose as well as other potential wound dressing carbohydrate materials. Secondly, we present the results of how well our antimicrobial agents affect bacterial growth. In this section we also show results of how well the mechanism of action works from start to finish and thereby proving our concept.

# Domain affinity

The binding capacity of the CBD was tested and observed in multiple experiments. In order to be able to follow and observe the binding mechanism of our CBD-construct the green fluorescent protein (sfGFP) was used as a replacement for the antimicrobial agents. Thus, a fusion protein containing sfGFP and CBD was created - mimicking the CBD-peptide/enzyme construct.

The binding strength was confirmed by testing different buffers. The results can be viewed in **Figure 1A**. According to the results, a highly hydrophilic buffer will generate more dissociation between CBD-sfGFP and the cellulose, compared to other less hydrophilic buffers. **Figure 1B** exhibits a large culture of *E. coli* BL21 (DE3) expressing CBD-sfGFP. Lysate containing CBD-sfGFP (via sonication, soluble fraction) from *E. coli* BL21 (DE3) was incubated with Epiprotect for 1 hour and washed thrice with 70 % ethanol. The result can be seen in **Figure 1C**, showing a strong green color for the cellulose which has been incubated with CBD-sfGFP. This indicates that the CBD has a strong affinity for cellulose.

![](https://static.igem.org/mediawiki/2019/3/34/T--Linkoping_Sweden--sfGFPtv%C3%A4tt.png)

![](https://static.igem.org/mediawiki/2019/d/d6/T--Linkoping_Sweden--sfGFPstorodling4.jpeg)

![](https://static.igem.org/mediawiki/2019/4/49/T--Linkoping_Sweden--sfGFPpl%C3%A5ster.jpeg)

**Figure 1.**
**A:** Binding studies of the CBDcipA-sfGFP bound to bacterial cellulose. Washed three times with either 70 % ethanol, PBS or deionized water.
**B:** Induced culture after 16 hours. *E. coli*  BL21 (DE3) cells were grown in the presence of 25 ug/mL chloramphenicol until an OD600 of 0.8 at 37 °C, and later induced with 0.5 mM IPTG. The induced culture was then incubated in 18 °C for 16 hours.
**C:** Left: CBDcipA-sfGFP bound to bacterial cellulose (Epiprotect®2117, S2Medical) in form of a thin film, right: bacterial cellulose (Epiprotect®2117, S2Medical) reference. Binding of CBDcipA-sfGFP was done the same way as the pictures below.

The cellulose bandage (Epiprotect) was incubated with CBD-sfGFP and **Figure 2A** shows the unwashed bandage. **Figure 2B** shows the bandage where the lysate containing CBD-sfGFP has been removed. **Figure 2C** depicts the bandage after one wash with 70 % ethanol for 30 min on an end-to-end rotator. These results show that the CBD has a strong affinity for cellulose.

![](https://static.igem.org/mediawiki/2019/a/a7/T--Linkoping_Sweden--sfGFPr%C3%B6r1.jpeg)

![](https://static.igem.org/mediawiki/2019/2/23/T--Linkoping_Sweden--sfGFPr%C3%B6r2.jpeg)

![](https://static.igem.org/mediawiki/2019/5/5f/T--Linkoping_Sweden--sfGFPr%C3%B6r3.jpeg)

**Figure 2.**
**A:** Lysate containing CBDcipA-sfGFP with bacterial cellulose (Epiprotect®2117, S2Medical) before incubation.
**B:** Lysate (CBDcipA-sfGFP) bound to bacterial cellulose after incubation at room temperature for 30 minutes on an end-to-end rotator.
**C:** Bacterial cellulose after incubation with 70 % ethanol at room temperature for 30 minutes on an end-to-end rotator.
All pictures were taken on a 302 nm UV-table for better visualization of the result.

In **Figure 3**, a cellulose bandage (Epiprotect, S2Medical) was incubated for 1 h at R.T in CBD-sfGFP lysate from BL21 (DE3) and afterwards washed thrice with 1 mL ethanol 70 %. The Negative control (Figure 3D-F) was washed thrice with 70 % ethanol. The bandages were mounted on a microscopy slide and photographed with a epifluorescent microscope. The relative fluorescent units (RFU) were measured in five places with one being a blank (bottom left corner, **Figure 3B** and **E**). The excitation was 485 nm and emission could be plotted in the treatment compared to the negative control (Figure 3C and F). The negative control did not emit due to no CBD-sfGFP being bound, whilst the CBD-sfGFP positive treatment had a clear emission peak matching sfGFP's 510 nm. **Figure 3A** and **D** is broad spectrum photos while **figure 3B** and **E** is only the green spectrum captured.

![Ecolipeptidepenetration](https://static.igem.org/mediawiki/parts/f/f4/T--Linkoping_Sweden--sfgfpmicro.png)

**Figure 3.**
Figure 3A and D are broad spectrum captures of the CBD-sfGFP incubated bandage (A) or the negative control which has not been incubated (D). A capture of only the green spectra at 485 was performed in **Figure 3B** and **E** where five points were measured, including one blank at the corner where the bandage was not present (1-4, Blank). These five points were plotted against wavelength in C and F to measure emission.

In **Figure 4**, the same BL21 CBD-sfGFP lysate was used and the samples were incubated 30 minutes in room temperature. Both the CBD-sfGFP bound agarose and agarose that was not incubated with CBD-sfGFP were washed in 70 % ethanol once. The agarose with bound CBD-sfGFP displayed a yellow tint in comparison to the agarose without bound CBD-sfGFP which was the original transparent color.

![Ecolipeptidepenetration](https://static.igem.org/mediawiki/parts/a/a3/T--Linkoping_Sweden--agarosescbdsfpgf.png)

**Figure 4.**
The negative controls refer to the agarose and agarose powder not being exposed to the CBD-sfGFP containing lysate.
**A & B:** Agarose 2,2%. In **A** the agarose can be observed in normal white light and in **B** the agarose was put on an UV-table (302nm).
**C & D:** Agarose powder was incubated in CBD-sfGFP containing lysate.

![](https://static.igem.org/mediawiki/2019/d/d9/T--Linkoping_Sweden--sfGFPcottonandjelonet.png)

**Figure 5.**
The cotton (**A**) and Jelonet +paraffin and -paraffin (**B**) without sfGFP refer to as negative controls. Both pictures were taken with an UV-table (302nm).
**A:**Cotton was incubated in bacterial lysate containing CBD-sfGFP for 30 minutes and both cotton samples were washed with 70 % ethanol before analyzed on the UV-table.
**B:** The Jelonet bandage was either washed with detergents to remove paraffin (-paraffin) or not (+paraffin). CBD-sfGFP -parafin was incubated for 30 minutes and washed with 70 % ethanol three times.

The samples in **Figure 5** were incubated with the same BL21 (DE3) lysate for 30 minutes and then washed three times with 70 % ethanol.
In **Figure 5A** cotton was incubated with sfGFP whilst in **Figure 5B** a bandage called Jelonet was used. Jelonet contains cotton threads that are covered in paraffin and we discovered that the paraffin inhibited the binding of CBD-sfGFP. Therefore, by using detergents the paraffin could be removed prior to the CBD-sfGFP incubation.

**Figure 1-5** illustrate the modularity of the CBD-construct proving that it not only binds to cellulose, as proved in **Figure 1** and **Figure 2**, but also to other polysaccharide based materials. By binding CBD-sfGFP to Jelonet, **Figure 5B**, we aimed to prove that the construct can be bound to already existing materials in the medical field. Jelonet is commonly used for open wounds such as burn wounds. The bandage both provides some antibacterial barrier as well as preventing the wounds from sticking to gauze used on top of Jelonet.

The bacterial cellulose bandage used in **Figure 1-3** is named Epiprotect®2117 and is produced by S2Medical. Epiprotect®2117 is currently used for wounds such as burn wounds and chronic wounds. These tests were also performed to prove that the CBD-construct can be used in existing medical products. Epiprotect®2117 was also used in later experiments to observe the thrombin cleavage mechanism.

## Purification of CBD-sfGFP

CBD-sfGFP was purified with 2 g cellulose fibers medium length (Whatman, #CF11 medium length cellulose fibers) in a column. In **Figure 6** the fluorescence data of all purification steps are collected. The first striped bar is the flowthrough after adding BL21 (DE3) Gold lysate. This is followed by 8 bars in black representing ethanol washes and thereafter 10 blue bars representing water elution steps are depicted. Lastly, two final steps with ethanol and water are illustrated in red and grey respectively.

The different washes of 70 % ethanol were performed to remove unspecifically bound CBD-sfGFP, leading to only bound CBD-sfGFP remaining on the bandage. This can be seen in the bar graph in **Figure 6** due to the drastic drop of sfGFP elution which means that 70 % ethanol is not useful for CBD-sfGFP elution. The water washes (dH2O, blue bars) indicate that an efficient elution of CBD-sfGFP can be performed within the first 4-6 elutions of dH2O. The last two bars (red - ethanol 70 % and grey - dH2O) indicate the endogenous fluorescent abilities of each solution. It is minimal for ethanol and none for dH2O.

The washes and elutions were later analyzed using SDS-PAGE to verify their pureness. These results can be seen under "SDS-PAGE analysis of cleavage and expression"

![](https://static.igem.org/mediawiki/2019/e/eb/T--Linkoping_Sweden--rening.png)

**Figure 6.**Purification with 2 g cellulose (Whatman, #CF11 medium length cellulose fibres) in a column. The first striped bar is the flowthrough after adding BL21 (DE3) Gold lysate. The black bars are the RFU of the 70 % ethanol washes. The blue bars are the distilled water (dH2O) elutions in which 1 mL dH2O was added until the column stopped dropping. The red and grey ethanol 70 % and dH2O bars illustrate the possible interfering RFU values of each fluid.

## Binding assay of CBD-sfGFP

![](https://static.igem.org/mediawiki/2019/6/6c/T--Linkoping_Sweden--sfGFPbindingassay.jpeg)

**Figure 7.**  This graph depicts the fluorescence of CBD-sfGFP lysate after incubation with cellulose CF11. The volume of bacterial lysate remained constant at 700 ul and the mass of cellulose CF11 was varied between 0-100 mg, which can be seen on the x-axis. The fluorescence was plotted on the y-axis and was measured at emission 510 nm with a gain of 40.

**Figure 7** illustrates that increasing mass of cellulose CF11 fibers leads to elevated of CBD-sfGFP.

To make the binding assay, Eppendorf tubes with varying mass of cellulose (Whatman, #CF11 medium length cellulose fibres) between 0-100 mg was mixed with a constant volume of CBD-sfGFP lysate (700 uL) and vortexed. The tubes were then attached to an end-to-end rotator for 30 minutes. Thereafter, they were placed in a tube holder until the cellulose powder settled in the bottom and the supernatant containing lysate was clear of cellulose. 100 µL from each of the supernatants was then applied on a plate reader measuring the fluorescence of CBD-sfGFP at 485 nm.

## Reporter of successful cleavage and release from the cellulose binding domain

In **Figure 8** the release of sfGFP from our bacterial cellulose bandage (Epiprotect®2117, S2Medical) is visualized over time. The approximate area of the cellulose was 1 cm2. The cellulose-CBD-sfGFP were attached to the sides of a 96-well plate using strings. Allowing the spectrophotometer to measure the supernatant in the center of the well, bottom up (ex. 485 nm, em. 510 nm) for 16 hours. The well further contained 200 uL 1X thrombin cleavage buffer (20 mM Tris-HCl, 150 mM NaCl and 2.5 mM CaCl2). In the positive control experiment, measuring thrombin cleavage (blue), an amount of 0.03 units of human thrombin (Novagen, #69671-3) was added. In the graph (blue), successful release of sfGFP from the CBD can be seen. The maximum amount of fluorescence is reached after about 8 h. In red the negative control experiment can be seen where thrombin buffer was added and the well did not contain any thrombin. No significant amount of fluorescence can be observed. The temperature was set to 37 °C. A visual representation of this can be observed below in **Figure 9**.

![](https://static.igem.org/mediawiki/2019/a/ab/T--Linkoping_Sweden--thrombinkinetic.png)

**Figure 8.** A kinetic experiment of thrombin protease activity. Bacterial cellulose with attached CBDcipA-sfGFP were analyzed spectrophotometrically.

![](https://static.igem.org/mediawiki/2019/c/cb/T--Linkoping_Sweden--Thrombincontrolphoto.jpg)

**Figure 9.** Visual control of human thrombin protease activity. Bacterial cellulose was incubated with CBD-sfGFP for 30 minutes on an end-to-end rotator in room temperature. The samples were incubated with either only thrombin cleavage buffer (negative control) or thrombin and thrombin cleavage buffer (thrombin (+)) overnight on an end-to-end rotator in room temperature.

To the left in **Figure 9** a visual experiment with this part can be seen. After unbound protein had been removed the cellulose was washed three times with 70 % ethanol. To test the activity, 200 uL thrombin cleavage buffer (20 mM Tris-HCl, 150 mM NaCl and 2.5 mM CaCl2) were added along side 0.03 units of human thrombin (Novagen, #69671-3) to the bacterial cellulose (Epiprotect®2117, S2Medical). To the right in **Figure 9**, the successful cleavage of CBD-sfGFP can be seen. The cellulose is to the left of the tube where free (cleaved at the thrombin site) sfGFP can be seen. To the left, the control sample can be seen where no sfGFP can is detected in the supernatant. The picture is taken on a 302 nm UV-table for a better visualization of the results. Both samples were incubated in room temperature over-night on an end-to end rotator.

The sample containing thrombin (thrombin (+)) has a clear cleavage of the linker which can be observed in the tube's supernatant whilst the negative control does not have any sfGFP in its supernatant. Not all sfGFP has been cleaved from the cellulose bandage and this is probably due to the thrombin buffer running out of needed content for the thrombin cleavage.

### SDS-PAGE analysis of cleavage and expression

*E. coli* BL21 (DE3) Gold cells were grown in the presence of 25 ug/mL chloramphenicol until an OD600 of 0.8 at 37 °C was reached, and later induced with 0.5 mM IPTG. The induced culture was then incubated in 18 °C for 16 hours. The bacteria was then lysed with sonication at 30 % for 6 minutes. Most of this part could be found in the soluble fraction. The lysate (1 mL) was then incubated with cellulose (CF11) for 30 minutes in room temperature. Four washes with 70 % ethanol was then conducted, all with a volume of 1 mL. Elution of CBDcipA-sfGFP was done with 1 mL fractions of dH2O. One replicate was cleaved with thrombin instead. Thrombin cleavage buffer (20 mM Tris-HCl, 150 mM NaCl and 2.5 mM CaCl2) was added to the Eppendorf tube with the cellulose at a volume of 500 uL, and 0.03 units was then added to the solution. The cleavage was done in room temperature over 16 hours, with inversion of the tube.

These different solutions were applied to an SDS-PAGE to analyze the effectiveness of the purification as well as of the thrombine cleavage. In **Figure 10** the resulting gel can be observed.

![](https://static.igem.org/mediawiki/2019/1/1d/T--Linkoping_Sweden--sfGFPSDS-PAGE.png)

**Figure 10.** SDS-PAGE analysis of CBD-sfGFP and thrombin cleavage product. The gel to the left is a 4–20% Mini-PROTEAN® TGX™ by BioRad and the ladder is a PageRuler™ Prestained Protein Ladder. The gel was run at 200 V for 30 minutes. Lane 1 contains the ladder, lane 2 bacterial lysate with CBD-sfGFP, lane 3-6 contain four washes with 70 % ethanol, lane 7-10 contain four elution fractions with deionized water. Lane 11 contains sfGFP subsequent to cleavage with thrombin.

Other bacterial proteins are washed away with the ethanol which can be observed by the other fragments observed in lanes 3-6, all of the proteins can also be observed in the bacterial lysate lane 2. In lanes 7-10 water (dH2O) elutions can be observed, they also contain some of the other bacterial proteins though they are very faint and the last lane is almost only CBD-sfGFP. These elutions were the same as those in **Figure 6**. Lane 11 only contains sfGFP which has been cleaved from the CBD-construct, proving a successful cleavage of the linker. The effectiveness of this cleavage is depicted in **Figure 9**.

### Expression in *Vibrio natriegens*

The antibiotic concentrations used were taken from [iGEM18 Marburg](https://2018.igem.org/Team%3AMarburg). For more information of chemical competent cells, transformation and medium please see the original article: [Gibson et al. 2016](https://www.ncbi.nlm.nih.gov/pubmed/27571549).

In order to see if CBD-sfGFP worked in *Vibrio natriegens* using the strain Vmax, pUC19-CBD-sfGFP [BBa\_K3182108](http://parts.igem.org/Part%3ABBa_K3182108) and pUC19-CBD-pCons-AsPink [BBa\_K3182100](http://parts.igem.org/Part%3ABBa_K3182100) was heat shocked into Vmax. Thereafter, the bacteria was spread onto LB-Miller V2 agar plates with 200 µg/ml carbenicillin and incubated in 37 °C for 16 hours. Both plates were illuminated on an UV-table at 302 nm light (**Figure 11 A**). **Figure 11 A** shows CBD-sfGFP in Vmax, emitts a strong green fluorescence, in comparison to the control CBD-pCons-AsPink in Vmax. This indicates that pUC19-CBD-sfGFP was able to replicate and the protein could be expressed.
To measure the protein expression of this part in different bacteria and carbenicillin concentrations. *E. coli* BL21 (DE3) and  *Vibrio natriegens* , using the strain Vmax, was grown in Falcon tubes to 0.5 OD600. Vmax was grown with two different carbenicillin concentrations, 200 and 600 µg/mL, while BL21 (DE3) had 100 µg/mL carbenicillin. The bacteria was induced with 1 mM IPTG and placed in a 96-well plate in 4 replicates with 200 µL per well. A spectrometry experiment was conducted and measured the fluorescence (excitation 470 nm, emission 515 nm) during 28 hours in 37 °C. The results seen in **Figure 11 B** shows that expression in Vmax with 600 µg/mL carbenicillin gave the highest protein yield. Another important factor was the use of an optimal concentration of carbenicillin (600 µg/mL) for Vmax which retained the plasmid more efficiently than Vmax at 200 µg/mL carbenicillin.

![](https://static.igem.org/mediawiki/2019/e/e7/T--Linkoping_Sweden--CombinedVibrio.png)

**Figure 11.** **A** This biobrick expressed in *V.natriegens* Vmax was incubated for 16 h in 37 °C on an LB-Miller V2 Agar plate. The control expressed the biobrick BBa\_K3182100 and express no fluorescent protein. The figure is illuminated on an UV-table in 302 nm light. **B** CBD-sfGFP expression in different chassis. The orange line represent Vmax at 600 µg/mL carbenicillin, blue represents Vmax at 200 µg/mL carbenicillin, and green represents  *E. coli*  BL21 at 100 µg/mL carbenicillin. The y-axis depicts relative fluorescent units and the x-axis represents the time over 28 hours.

# Results of antimicrobial effect

The goal of our project was to functionalize a cellulose wound dressing with antimicrobial agents to help fight infections. We engineered *E. coli* and *V. natriegens* to express antimicrobial peptides and enzymes linked to a carbohydrate binding domain (CBD). These fusion proteins were then purified by attaching them to a polysaccharide based material (e.g. cellulose) which after the purification could act as an antimicrobial bandage. The CBD enabled the attachment of antimicrobial agents to the polysaccharide material while it also inactivated the agents when being expressed. Later, when the bandage came in contact with thrombin the antimicrobial agents would be activated. This is due to thrombin cutting the linker between the antimicrobial agents and CBD which contains a thrombin cleavage site.

### Expression

Here we provide proof of successful expression of agents in *E. coli*. The agents could be found in small amounts in the soluble fraction after sonication. Most of the fusion proteins could be found in the insoluble fraction (bacterial membranes), where we used Triton X-100 1% (non-ionic detergent) to solubilize the pellets and release our agents. Our agents can be seen in the insoluble fraction in **Figure 12C**. **Figure 12** proves that the agents could be expressed (**Figure 12C**), that the agents could be bound to cellulose and cleaved from the cellulose with thrombin, (**Figure 12A** and **Figure 12B**.

In **Figure 12B**  CBD-Pln1 has been purified with cellulose and then eluted with water, proving its binding capacity and its relative purity.

In **Figure 12A**  CBD-Pln1 has been cleaved by thrombin and the cellulose was run in this lane which still contained both uncleaved and cleaved CBD-Pln1. The figure also displays Pln1 not bound to the CBD after thrombin cleavage.

![SDSpage](https://static.igem.org/mediawiki/2019/8/86/T--Linkoping_Sweden--sdspagecomp.png)

**Figure 12:** SDS-PAGE gel showing bacterial expression.
**A:** CBD-Pln1 purified and cleaved with thrombin run on an SDS-PAGE 4-20 % gel. Lane 1 contains CBD-Pln1 previously bound to cellulose and treated with thrombin, which explains the multiple bands. Lane 2 contains the cleaved Pln1 from lane 1, which was found in the supernatant from the cleavage along with thrombin (not visible). Lane 3 contains a protein ladder called LMW by GE Healthcare.
**B:** CBD-Pln1 run on an SDS-PAGE 4-20 % gel after purification. Lane 1 contains a protein ladder called Precision Plus Dual color by BioRad. Lane 2 contains purified CBD-Pln1 in fraction 1 after elution with water. Lane 3 contains purified CBD-Pln1 in fraction 2 after elution with water. Lane 4-5 are empty. Lane 6 contains *E. coli* BL21 (DE3) lysate from induced cells which carrie the plasmid expressing CBD-Pln1.
**C:** Bacterial pellet of BL21 (DE3) expressing PlyF307 and Pln1 were lysated and the insoluble fraction was centrifuged and analyzed in this result. Lane 1 contains a GE healthcare LW protein ladder, lane 2 contains the insoluble fraction of PlyF307 expressing culture and lane 3 contains the insoluble fraction of bacteria expressing Pln1.

## Antimicrobial activity agents in different states

All experiments below used *Escherichia coli* BL21 (DE3) and/or *Bacillus Subtilis* in concentrations of 10 000 CFU/mL to test antimicrobial activity. The usage of the two bacteria verified and explored the agent's activity to both gram negative and positive bacteria, respectively. This dilution was performed in order to get starting cultures of 0 OD600. This method used spectrometry to measure the time until the bacteria started growing, mimicking early stages of a wound, instead of showing the killing capability on high optical density cultures.

### CBD bound agents

An experiment to test the antimicrobial activity of our agents still bound to the CBD was conducted, which can be seen in **Figure 13**. This was done in order to test our hypothesis that the presence of a CBD would yield inactive agents. Six technical replicates of *E. coli* BL21 (DE3)(160 µL in low salt LB-media, 0.4 g/L NaCl) were added to a 96-well (Eppendorf). To this 40 µL water was added to function as a negative control (**Figure 13**) due to the CBD-bound agents being eluted in water. To 6x6 wells a concentration gradient was added in the same way, constantly adding 160 µL (in low salt LB-media) *E. coli* BL21 (DE3) and 40 µL of the unbound agents including additional water for diluted agents in order to achieve 40 µL as a final volume (Figure 1). The experiment was run for 16 hours at 37 °C, and before each measurement a quick shake of 200 rpm, 10 seconds was done. The absorbance at 600 nm was measured, from bottom-up in the center of the well every hour.

![](https://static.igem.org/mediawiki/2019/b/b0/T--Linkoping_Sweden--CBDboundall.png)

## Importance of testing the CBD-bound agents

Our hypothesis, regarding the inactivation of the antimicrobial agent bound to the CBD, was only partially true. The results shown in **Figure 13** showed many interesting findings. Firstly, that the agents still had antimicrobial activity. This result indicated that if the bandage is applied to a patient who is not bleeding or is supplied by extrinsic thrombin, the bandage will still have a partial antimicrobial activity. Another conclusion that can be drawn from this, is that an equilibrium of the growth phase occurs and settles in lower than for the negative control. This means that even though the antimicrobial agents could not inhibit the growth fully, the growth was slowed and the stationary phase was reached earlier (**Figure 13**). The results also explain the difficulty that was experienced during expression of the CBD-agents, the CBD was not effective enough to inactivate the agents completely, leading to a toxic effect on the bacterial chassis. It also explains why they can be found in the insoluble fraction because the agents are still bound to bacterial membranen.

### Unbound agents

To approximate the agents' efficiency after they were released from a bandage (thus not bound to the CBD) we purified the agents. CBD-agents were bound to microcrystalline cellulose and thrombin together with thrombin cleavage buffer was added. *E. coli *and*  B. subtilis* was used as test subjects since they are gram negative and gram positive, respectively.
After over-night incubation in 37 °C at 200 rpm, the microcrystalline cellulose was centrifuged down, creating a supernatant containing thrombin and the unbound agent. This concept and solution is what was tested below in **Figure 14**.

This experiment acts as a positive control before starting our experiments that would show the actual proof of concept with the Novosite CBD-agent-bandage. The experimental setup was the same as the earlier experiment but instead of adding water to the negative control, thrombin (0.5 U, final concentration 0.6 µM) and thrombin cleavage buffer (40 µL, 20 mM Tris-HCl, 150 mM NaCl and 2.5 mM CaCl2) was added. This was because the agent solution contained thrombin and cleavage buffer. The negative control showed to not affect the bacterial growth, but rather increased it due to the added salt in in the cleavage buffer.

The results confirm our theory that the antimicrobial agents will have an increased effect against bacteria after the CBD is cleaved. Pln1, PlyF307 and CHAP had a high activity against *E. coli*. Whilst CHAP had a much higher activity against *B. subtilis*. Pln1 is an antimicrobial peptide (AMP) which has been reported to have effect against both gram positive and gram negative bacteria [1]. According to our results, Pln1 was more effective against the gram negative *E. coli* in comparison to *B. subtilis* which is gram positive. This may be due to the gram negative wall being more easy to penetrate and therefore creating pores, whereas the gram positive wall is thicker and can act as a partial trap for Pln1 [2]. PlyF307 is an antimicrobial enzyme (lysin) derived from an *A. baumannii* bacteriophage [3]. On its C-terminal is an AMP allowing the lysin to easily find its target [3]. PlyF307 is more effective against *A. baumanii* than *E.coli* [3], however, because both bacteria are gram negative a high activity was still retained as seen in **Figure 14C**. CHAP is also a lysin, but it is from an *S. aureus* bacteriophage [4]. *S. aureus* is a gram positive bacteria and due to lysins' generally specific nature, it can be seen in **Figure 14D** and **13E** that CHAP is much stronger against *B. subtilis* **(13E)** in comparison to *E.coli* **(13D)**. **Figure 14E** shows that CHAP completely inhibited *B. subtilis* growth, while still being effective against *E. coli* (likely due to it being able to affect the bacteria during cell division). All of the agents tested in **Figure 14** had a strong activity against at least gram positive or gram negative bacteria. This proved that the agents can be used to kill bacteria. The different activities against gram positive or gram negative bacteria also shows that the Novosite construct can be used modularly to preserve one of these groups if wished. By getting promising results from this "positive control" we could further move on to the proof of concept.

![](https://static.igem.org/mediawiki/2019/2/22/T--Linkoping_Sweden--peptidersnamme.png)

## Parseq peptide activity

The Parseq peptides derived from our developed Parseq model was also tested against the same bacteria as described previously in **Figure 14**. These two Parseq-α and β peptides were chemically synthesised by CASLO through a sponsorship. They were therefore not thrombin cleaved. As a control, the peptide buffer (negative control) Urea was used. the results of these tests can be seen in **Figure 15**. The two peptides performs best against *E.coli* where they both totaly inhibit growth at 30 µg/ml for α and 100 µg/ml for β which is shown in **A** and **B**. In **C** and **D** their effect on *B.subtilis* is shown. Neither peptide were able to totaly inhibit the growth at 300 µg/ml even thou the growth is decreased so the peptides have had a desired effect. In all four graph, especially **A**, **C** and **D**, the concentrations to low to totaly inhibit bacerial growth have delayed the exponential growth phase showing that the peptides still are affecting the bacteria.

![](https://static.igem.org/mediawiki/2019/1/15/T--Linkoping_Sweden--parseqsus.png)

### Immobilization and release on a cellulose bandage *- Proof of concept*

To prove that the concept of iGEM19 Linköping's project worked, the antimicrobial agents were fused to a cellulose bandage (Epiprotect by S2Medical). The Epiprotect bandage, developed by S2Medical, is used clinically for both chronic wounds and burns, further displaying Novosite's potential as a usable product.

The cellulose was first incubated with *E. coli* BL21 (DE3) lysate (20 mL) containing CBD-Pln1 or CBD-PlyF307 for 2 hours in 4 °C. After immobilization of the agent on cellulose, the cellulose was washed three times with carbohydrate binding module buffer (CBM-buffer, 10 mL, Tris–HCl pH 7.0, 20 mM NaCl, 5 mM CaCl2). After washing, small pieces of bandage were cut out to fit into a well in a 96-well plate. Added to these wells was also low salt LB-media (160 µL, 0.4 g/L NaCl) containing *E. coli* BL21 (DE3) at 0 OD600.

Thrombin cleavage buffer (40 µL, 20 mM Tris-HCl, 150 mM NaCl and 2.5 mM CaCl2) was added to all the wells. The negative control, in addition to thrombin cleavage buffer, contained a cellulose bandage without any bound agents. In the Pln1/PlyF307 bandage (triangles, **Figure 15A** and **14B**) the cellulose with bound agents was added. The filled boxes in **Figure 15** below (Pln1/PlyF307 bandage + thrombin) contained the agents bound to cellulose together with thrombin (0.5 U thrombin, final concentration in well 0.6 µM), recreating the mechanism of action.

Noticeably, the thrombin-released agents inhibited *E. coli* growth the most, in comparison to the other combinations tested. In both **Figure 15A** and **14B** the non-released agents bound to cellulose exhibited an antimicrobial activity. This correlates with the results in **Figure 13** which indicated that bound agents exhibited a moderate antimicrobial effect. Pln1 bound and unbound had a similar activity with the unbound being slightly more effective. PlyF307 showed a strong antimicrobial activity in both a bound and unbound state. The unbound/released PlyF307 completely inhibited the growth of *E. coli*. These results proved that the of concept of iGEM19 Linköping's main mechanism worked and that these types of wound dressings have the potential to be used clinically.

![](https://static.igem.org/mediawiki/2019/d/d1/T--Linkoping_Sweden--plasterboth.png)

### References

1. Meng F, Zhao H, Zhang C, Lu F, Bie X, Lu Z. Expression of a novel bacteriocin—the plantaricin Pln1—in Escherichia coli and its functional analysis. Protein Expr Purif. 2016;119:85–93.

2. Jenssen H, Hamill P, Hancock REW. Peptide Antimicrobial Agents. Clin Microbiol Rev. 2006;19(3):491–511.

3. Thandar M, Lood R, Winer BY, Deutsch DR, Euler CW, Fischetti VA. Novel engineered peptides of a phage lysin as effective antimicrobials against multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother. 2016;

4. Horgan M, O’Flynn G, Garry J, Cooney J, Coffey A, Fitzgerald GF, et al. Phage Lysin LysK Can Be Truncated to Its CHAP Domain and Retain Lytic Activity against Live Antibiotic-Resistant Staphylococci. Appl Environ Microbiol. 2009 Feb 1;75(3):872–4.