iGEM 2020 — Team Lund

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Lund/Notebook

iGEM Lund 2019

* [Home](/Team%3ALund)
* Project

  + [Description](/Team%3ALund/Description)
  + [Design](/Team%3ALund/Design)
  + [Experiments](/Team%3ALund/Experiments)
  + [Notebook](/Team%3ALund/Notebook)
  + [Contribution](/Team%3ALund/Contribution)
  + [Results](/Team%3ALund/Results)
  + [Proof of Concept](/Team%3ALund/Proof_Of_Concept)
  + [Model](/Team%3ALund/Model)
  + [Engineering](/Team%3ALund/Engineering)
  + [Attributions](/Team%3ALund/Attributions)
  + [Safety](/Team%3ALund/Safety)
* People

  + [Team](/Team%3ALund/Team)
  + [Collaborations](/Team%3ALund/Collaborations)
  + [Attributions](/Team%3ALund/Attributions)
* Parts

  + [Overview](/Team%3ALund/Parts)
  + [Basic Parts](/Team%3ALund/Basic_Part)
  + [Composite Parts](/Team%3ALund/Composite_Part)
* Practices

  + [Human Practices](/Team%3ALund/Human_Practices)

[Proposed Implementation](/Team%3ALund/Implementation)

![](https://static.igem.org/mediawiki/2020/4/4b/T--Lund--LogoPNG1.png)

![](https://static.igem.org/mediawiki/2020/4/4b/T--Lund--LogoPNG1.png)

# Lab Notebook

Here follows a weekly summary of our notebook, briefly describing the work we did and what we accomplished each week. For more information about the protocols we followed and the experiments performed, see [Experiments](/Team%3ALund/Experiments).

### Week 1 (June 15-19)

LB medium, an ampicillin stock and petri dishes with solid LB medium were prepared, with and without ampicillin. LB medium, pipette tips, eppendorf tubes, glycerol, CaCl2-solution and RNAse free water were autoclaved. E. coli TG1, TG1 (pET-11a), and BL21 (DE3) were plated, incubated and stored in a cold room.

### Week 2 (June 22-26)

Our supervisor introduced us to the department's centrifuges, spectrophotometers and plasmid purification kit. Competent TG1 cells were prepared and stored at -80°C. TG1 (pET-11a) cells were incubated overnight, the plasmids were purified and DNA concentration was measured using a spectrophotometer. A test digestion with restriction enzymes NdeI and BamHI was performed to test appropriate reaction times for each enzyme. Digested plasmids were dephosphorylated with FastAP and loading dye was then added.

### Week 3 (June 29- July 3)

A gel electrophoresis was performed with the plasmid samples from the previous week. NdeI seemed to not have digested the plasmid fully, while BamHI had started to digest the plasmid unspecifically, due to too long reaction time. New test digestions were performed with longer reaction times for NdeI (samples taken at 60-130 min) and shorter for BamHI (samples taken at 6-15 min). After examining the gel of another test it was decided to run the reactions for 60 min with NdeI and 5-10 min with BamHI.

### Week 4 (July 6-10)

Double-digestion and dephosphorylation of the purified pET-11a was done, then a gel electrophoresis was performed with the samples, showing that the digestion was complete. A gel extraction was performed and the digested plasmid was eluted in an elution buffer, then stored at -20°C. E. coli BL21(DE3) was incubated overnight and prepared to make competent cells, then stored at -80°C.

### Week 5 (July 13-17)

We had finished all the preparations that we could do and waited for IDT’s free 20k bp, which were delayed. We then decided we could not wait for the free bps and ordered part of our gBlocks ourselves. We ordered all AMPs with Trx as a fusion partner (Trx-gblocks). GST-Scarabaecin was accidentally ordered instead of Trx-Scarabaecin. Previously ordered primers arrived from IDT.

### Week 6 (July 20-24)

A new plasmid purification was performed after an overnight incubation of TG1 (pET-11a) and DNA concentration was measured.

A test amplification of gBlocks was performed using the primers on one of the inserts. The PCR machine’s temperature gradient was used to find the best annealing temperature of the primers. After examining the agarose gel of the PCR product we decided to use an annealing temperature of 66°C. The Trx-gblocks (and GST-Scarabaecin) were amplified using that annealing temperature.

### Week 7 (July 27-31)

Amplified Trx-gblocks were double digested with NdeI and BamHI, they were run on a gel and purified, in the final step eluting them in nuclease free water. Concentrations of all the samples were measured. Ligation of pET-11a and Trx-gblocks was performed and the product was transformed into E. coli TG1. Cells were spread on plates with LB medium containing ampicillin. A positive control with TG1 transformed with purified undigested pET-11a and a negative control using TG1 transformed with digested pET-11a without any insert was also made.

After incubation some colonies were present for MsrA2, EAFP1, Ec-AMP-D1 and Temporin A. Colony PCR was performed and run on gel, results were not convincing. Two more ligations and transformations were performed as above, however no colonies were present except for on the positive control. Digestion with NdeI and BamHI was tested again to ensure this was not an error and it did not seem to be a problem.

### Week 8 (August 3-7)

A fourth ligation and transformation was performed, this time using more ligase in the ligation mixture and prolonging the ligation time from 30min to >120min. Cells were also incubated with the ligated plasmids for a longer time. Still no colonies were found after incubation on plates.

A new double digestion with NdeI and BamHI was performed for the amplified Trx-gblocks. They were purified from a gel and DNA concentrations were measured. TG1 (pET-11a) was incubated overnight and plasmids were again purified and digested by NdeI and BamHI, DNA concentration was measured.

A fifth ligation and transformation was performed, using the new materials and prolonged ligation and incubation times. This time colonies were present for all different g-blocks, there was no growth on the negative control and plenty of growth on the positive control.

A new set of gBlocks arrived from IDT (Delayed free kbp), which contained all AMPs fused to GST as well as Temporin A and Scarabaecin fused to Trx and also our Enterokinase composite part.

### Week 9 (August 10-14)

After a first failed colony PCR, another colony PCR was performed picking five colonies per Trx-insert. All inserts had at least 3 colonies that appeared to have the correctly ligated plasmid. Before the PCR, the colonies were also plated on a masterplate and incubated. From the masterplate, clones of each insert were incubated and their plasmids were purified. Plasmids were sent to Eurofin for sequencing and the received data was analysed, showing that almost all gblocks had been inserted correctly without mutations occurring.

The new set of gblocks (GST-inserts) was amplified and purified.

### Week 10 (August 17-21)

The recombinant plasmids were transformed into BL21, TB-medium and IPTG-stock were prepared. Inoculations from master plates were incubated overnight (one per Trx-insert) and protein expression cultivations were started the day after for the recombinants. Cultivations were induced using IPTG and samples were taken during a period of 24h. A test SDS-page was performed to learn the technique.

The GST-inserts were double digested with NdeI and BamHI. The enterokinase part was amplified and digested. The digestion reactions were run on a gel and extracted, eluted in water and DNA concentrations were measured. Transformation was made into TG1 cells and colonies were formed for all inserts except GST-Rs-AFP1. A colony PCR and masterplate was performed, however something went wrong with the reaction.

### Week 11 (August 24-28)

Samples from cultivation had been centrifuged and were resuspended to obtain the same OD. SDS-page was performed for the samples but did not give any clear results, it did however seem like the proteins had not been expressed for all samples except one. In a GST-scarabaecin sample there was a thick band not present in controls or other samples.

A second protein expression cultivation was performed according to the same protocol as the previous one. Samples were taken and OD was measured during the 24h period, and at the end of expression all cells were harvested by centrifugation, storing the cell pellets on ice. New SDS-pages were made for samples taken during the expression, but something went wrong with the staining. The harvested cell pellets were resuspended and sonicated, pellets and supernatants were stored separately.

### Week 12 (September 1-5)

SDS-page was carried out for the pellets and supernatants of the harvested, sonicated cells. After examining these gels and the gels from the previous week, we had a strong belief that GST-Scarabaecin had been expressed. This was the only insert with GST instead of Trx and we started having suspicions that GST might yield higher expression than Trx.

### Week 13 (September 8-13)

Pre-inoculation was prepared for a third protein expression cultivation.

### Week 14 (September 14-20)

The third cultivation was started and was performed the same way as the previous tries. The harvested cells were treated in three different ways to try and solubilize potential proteins. The treatments were resuspension in SDS loading dye (as before), resuspension in urea and then SDS loading dye, and lastly resuspension in guanidine hydrochloride and then SDS loading dye. SDS-pages of the supernatant (after sonication) and the three treatments were carried out, however the target proteins did not appear present.

### Week 15 (October 5-10)

For the second set of parts, a new colony PCR was performed for the clones of the previously made masterplate. However no convincing results of successful transformation were obtained, and it was decided that we no longer had time to continue the work on these parts.

For the first set, a new protein cultivation was started to try and repeat the expression of GST-Scarabaecin (and possibly others). After making SDS-pages of samples taken at 6h and 8h after IPTG induction results showed that there had been no significant protein expression. We decided that we would have to call an end to the lab work, and try to settle with the results that we had gotten.

### Week 16 (October 13-17)

The last SDS-pages were for the protein cultivation done the week before. The SDS-pages finally showed what appeared to be protein expression! Things were cleaned up in the lab.

![](https://static.igem.org/mediawiki/2019/4/40/T--Lund--lund.png)
![](https://static.igem.org/mediawiki/2020/3/38/T--Lund--slu.JPG)
![](https://static.igem.org/mediawiki/2020/7/7d/T--Lund--lth.JPG)
![](https://static.igem.org/mediawiki/2020/1/11/T--Lund--protecto.JPG)
![](https://static.igem.org/mediawiki/2020/2/2b/T--Lund--GransboLogo.png)

[* ![](https://static.igem.org/mediawiki/2019/b/be/T--Lund--instagram.png)@igem\_lund](https://www.instagram.com/igem_lund/)
[* ![](https://static.igem.org/mediawiki/2019/5/5b/T--Lund--facebook.png)fb.com/igemlund](https://www.facebook.com/igemlund/)
[* ![](https://static.igem.org/mediawiki/2019/8/83/T--Lund--github.png)igemlund](https://github.com/igemlund)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Lund/Engineering

iGEM Lund 2019

* [Home](/Team%3ALund)
* Project

  + [Description](/Team%3ALund/Description)
  + [Design](/Team%3ALund/Design)
  + [Experiments](/Team%3ALund/Experiments)
  + [Notebook](/Team%3ALund/Notebook)
  + [Contribution](/Team%3ALund/Contribution)
  + [Results](/Team%3ALund/Results)
  + [Proof of Concept](/Team%3ALund/Proof_Of_Concept)
  + [Model](/Team%3ALund/Model)
  + [Engineering](/Team%3ALund/Engineering)
  + [Attributions](/Team%3ALund/Attributions)
  + [Safety](/Team%3ALund/Safety)
* People

  + [Team](/Team%3ALund/Team)
  + [Collaborations](/Team%3ALund/Collaborations)
  + [Attributions](/Team%3ALund/Attributions)
* Parts

  + [Overview](/Team%3ALund/Parts)
  + [Basic Parts](/Team%3ALund/Basic_Part)
  + [Composite Parts](/Team%3ALund/Composite_Part)
* Practices

  + [Human Practices](/Team%3ALund/Human_Practices)

[Proposed Implementation](/Team%3ALund/Implementation)

![](https://static.igem.org/mediawiki/2020/4/4b/T--Lund--LogoPNG1.png)

![](https://static.igem.org/mediawiki/2020/4/4b/T--Lund--LogoPNG1.png)

# Engineering

On this page, we will showcase the engineering success behind our project

![](https://static.igem.org/mediawiki/2020/8/8d/T--Lund--engineering.png)

Figure 1. A flowchart showing the engineering cycle, with comments regarding how each step affected our project.

We were only available to achieve protein expression, but the following paragraphs.
One of the reasons, apart from wanting to test combinations, for why we express multiple AMPs is because we expect to gain new knowledge along with the experiments. Many of them have never been previously expressed in bacteria, much less in E. coli. We need a system able to produce a gene product according to at least two quality parameters: a significant yield of soluble fusion protein and a usable yield of protease digested AMPs.

For producing the soluble protein, we try multiple fusion partners, Thioredoxin A (Trx A) and Glutathione S-Transferase (GST). Since many of the peptides are cysteine rich, we expect the disulphide bridge formation catalyzing Trx A to have the greatest solubility increase, but this hypothesis requires experimental validation. Given that we got protein expression with both of the fusion partners, we will have to test the solubility of both fusion partners before deciding on the most suitable one. We plan on doing this by sonicating the cells and then run SDS-PAGEs to see whether the protein is in the soluble or insoluble phase. There may also be other reasons for why a particular antimicrobial peptide is not expressed properly, and using a wider selection hedges us against unexpected issues.

When it comes to the AMP yield, meaning the yield from fusion partner digestion, we require experimental validation. The protease, enterokinase (EK), is a naturally occuring protease in mammals (humans, bovine). The region it recognizes and cleaves is hydrophilic, so it should be accessible for EK. However, the substrate data from Brenda indicates that while enterokinase is selective, it’s specificity varies with the chains, such that experimental validation is the most feasible approach within the scope of the project.

When we have produced cell extract containing AMPs, we will screen for which ones produce the strongest antimicrobial effect. Based on this, we will conduct a factorial experiment trying to determine if we can get a significant synergistic effect of combination therapy. We believe that with these objectives along with the proper research, design, building and testing, we will follow the engineering cycle in a way that will benefit the project.

![](https://static.igem.org/mediawiki/2019/4/40/T--Lund--lund.png)
![](https://static.igem.org/mediawiki/2020/3/38/T--Lund--slu.JPG)
![](https://static.igem.org/mediawiki/2020/7/7d/T--Lund--lth.JPG)
![](https://static.igem.org/mediawiki/2020/1/11/T--Lund--protecto.JPG)
![](https://static.igem.org/mediawiki/2020/2/2b/T--Lund--GransboLogo.png)

[* ![](https://static.igem.org/mediawiki/2019/b/be/T--Lund--instagram.png)@igem\_lund](https://www.instagram.com/igem_lund/)
[* ![](https://static.igem.org/mediawiki/2019/5/5b/T--Lund--facebook.png)fb.com/igemlund](https://www.facebook.com/igemlund/)
[* ![](https://static.igem.org/mediawiki/2019/8/83/T--Lund--github.png)igemlund](https://github.com/igemlund)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Lund/Experiments

iGEM Lund 2019

* [Home](/Team%3ALund)
* Project

  + [Description](/Team%3ALund/Description)
  + [Design](/Team%3ALund/Design)
  + [Experiments](/Team%3ALund/Experiments)
  + [Notebook](/Team%3ALund/Notebook)
  + [Contribution](/Team%3ALund/Contribution)
  + [Results](/Team%3ALund/Results)
  + [Proof of Concept](/Team%3ALund/Proof_Of_Concept)
  + [Model](/Team%3ALund/Model)
  + [Engineering](/Team%3ALund/Engineering)
  + [Attributions](/Team%3ALund/Attributions)
  + [Safety](/Team%3ALund/Safety)
* People

  + [Team](/Team%3ALund/Team)
  + [Collaborations](/Team%3ALund/Collaborations)
  + [Attributions](/Team%3ALund/Attributions)
* Parts

  + [Overview](/Team%3ALund/Parts)
  + [Basic Parts](/Team%3ALund/Basic_Part)
  + [Composite Parts](/Team%3ALund/Composite_Part)
* Practices

  + [Human Practices](/Team%3ALund/Human_Practices)

[Proposed Implementation](/Team%3ALund/Implementation)

![](https://static.igem.org/mediawiki/2020/4/4b/T--Lund--LogoPNG1.png)

![](https://static.igem.org/mediawiki/2020/4/4b/T--Lund--LogoPNG1.png)

# Protocols and Experiments

## Protocols

### Medium Preparation:

#### LB Media

 For each 1 L reaction mixture, mix the following:

* 5 g Yeast Extract
* 10 g Tryptone
* 10 g NaCl

#### Preparation:

1. Weigh ingredients individually and put them in a schott bottle with a magnetic stirrer
2. Fill with 1 L of distilled water
3. Stir until all powder is dissolved

#### TB Media

 For each 1 L reaction mixture, mix the following:

* 12 g Peptone
* 24 g Yeast Extract
* 4 mL Glycerol
* 2.3 g KH2PO4
* 12.5 g K2HPO4

#### Preparation:

1. Weigh and later autoclave Potassium salts, glycerol separately and peptone along with yeast extract to avoid precipitation
2. Dissolve all the components in a sufficient amount of distilled water but not exceeding the total 1 L volume

#### LB Agar Media

 For each 1 L reaction mixture, mix the following:

* 5 g Yeast Extract
* 10 g Tryptone
* 10 g NaCl
* 150 g agarose

#### Preparation:

1. Weigh ingredients individually and put them in a schott bottle with a magnetic stirrer
2. Fill with 1 L of distilled water
3. After autoclaving, cool the media and add 100 μg/ml of antibiotic of choice (ampicillin) depending on the application
4. Pour into sterile Petri dishes

### PCR - Amplification of gBlocksMasterMix  For each 50 µL reaction mixture, mix the following in this order: * 38.25 µL MilliQ water * 10 µL HF buffer * 0.25 µL of each dNTP * 0.25 µL Forward Primer * 0.25 µL Reverse Primer * 0.5 µL Phusion High Fidelity DNA Polymerase Divide the MasterMix into 10 PCR tubes and add 0.5 µL Template DNA (total volume of 20 µL in each tube) and arrange them in the thermocycler. Thermal Cycler Settings: 1. Initial denaturation: 98 ℃ for 30 seconds 2. Denaturation: 98 ℃ for 10 seconds 3. Annealing: 67.4 ℃ (for Trx 61.1 ℃) for 20 seconds (15 cycles) 4. Extension: 72 ℃ for 20 seconds 5. Final extension: 72 ℃ for 7minutes 6. Hold: 4 ℃ until loading Gel ElectrophoresisGel preparation: 1. Prepare 2 % agarose gel for gel electrophoresis (0.8 grams agarose powder in 40 mL TAE buffer) 2. Heat the mixture in the microwave until the solution is clear. 3. Add 2 µL GelRed after the solution has cooled down. 4. Fix comb and add the solution into the casket for the gel to solidify. Ladder & samples: 1. Add 2 µL of 100 bp ladder (GeneRuler). 2. Mix 10 µL of PCR samples with 2 µL loading dye. Electrophoresis conditions: 1. Remove the comb and move the solidified gel to the electrophoresis set up. 2. Fill the chamber with TAE buffer until all the wells are covered completely. 3. Load the gel and run it for 90 minutes at 100 V (NOTE: start at 80 V until the samples in the wells move into the gel and then increase to 100 V). Colony PCRTemplate preparation and Master Plate 1. Divide an Agar-plate into four parts using a marker pen. 2. Inoculate one colony from the transformed TG1 onto one part of the agar plate. Make sure to spread the bacteria within the marked area, and watch out for droplets of water which may spread the bacteria around the plate. 3. Directly after spreading the bacteria on the plate, dip the inoculation loop in a prepared PCR tube to inoculate this as well. 4. Incubate plates at 37 ℃ for a few hours or until a suitable amount of growth is seen. 5. While waiting, continue with Colony PCR. 6. Inoculate positive transformants from the masterplate in 10ml tubes and incubate at 37 ℃ over night. This is for Sanger Sequencing. PCR reaction mixture For each 20 µL reaction mixture, mix the following: * 2 µL dNTP mix * 2 µL Forward primer * 2 µL Reverse primer * 0.1 µL Taq DNA Polymerase * 2 µL Taq buffer * 3 µL MgCl2 * 12 µL MilliQ water Thermal Cycler Settings: 1. Initial denaturation: 95 ℃ for 180 seconds 2. Denaturation: 95 ℃ for 30 seconds 3. Annealing: 60 ℃ for 30 seconds (35 cycles) 4. Extension: 72 ℃ for 20 seconds 5. Final extension: 72 ℃ for 10 minutes 6. Hold: 4 ℃ until loading Transformations  For TG1 and BL21-DE3 cells (Heat Shock): 1. Thaw the competent cells on ice. 2. Add 10 μL of ligation mixture to 200 μL of cells on ice (maximum concentration of plasmid allowed is 100 ng/mL). 3. Swirl gently to mix the contents. 4. Incubate on ice for 1 hour. 5. Transfer tubes to a heat block with a temperature of 42 ℃ for 45 seconds. 6. Place samples back on ice and let them cool for 5 minutes. 7. Add 400 μL of LB media without antibiotics. 8. Incubate for 45 min in the shaking incubator at 37°C, for the cells to revive. 9. Centrifuge the tubes in a tabletop centrifuge for 1 minute and remove medium so that only 50 μL is left. Resuspend the cells in the 50 μL that are left. 10. Plate 50 μL of the culture on LB agar with ampicillin antibiotic and incubate overnight at 37 °C. SDS-PAGEReagents: 1. Make 50 mM sodium acetate buffer at pH 5.0 2. Pre-casted gel (Bio-Rad) 3. Loading buffer (sample buffer): 1. 3.3 ml of TRIS 0.5 M pH 6.8 2. 8 ml of SDS 10% w/v 25 3. 4 ml of glycerol 4. 1 ml of bromophenol blue 0.2% w/v 5. 2.62 g of DTT (dithiotreithol) 6. Adjust volume to 20 ml using MilliQ-water- Electrophoresis (Running) buffer: 1. 7.5 g of Tris 2. 36 g of glycine 3. 2.5 g of SDS 4. Adjust pH to 8.3 using HCl and make the final volume up to 500 ml using MilliQ-water. Dilute the buffer 5 times - Molecular weight marker- Staining solution: 1. 1 g of Coomassie blue 0.2% 2. 200 ml of MeOH 40% 3. 50 ml of HAc 10% 4. 260 ml of MilliQ-water - De-staining solution: 1. 40% methanol 2. 10% acetic acid 3. 50% MilliQ-water

#### Sample Preparation:

1. Check OD and normalize all the samples to OD 2.
2. Resuspend cell pellet in 20 µl of 2x SDS loading dye.
3. Boil samples at 100°C for 10 min.
4. Load the prepared samples into the gel (Bio Rad plates, 15 well of 15μL, 4 - 20% gradient gel).
5. After loading, fill the apparatus with the buffer solution between the glass plate until it reaches the maximum volume.

#### Running the Gel:

1. Run the gel at 100 V until the dye front migrates into the running gel (~10 min).
2. Increase to 150 V until the dye front reaches the bottom of the gel (~30 min).

#### Staining & Destaining the Gel:

1. Remove the gel from the apparatus.
2. Remove the spacers and glass plates, by using the metal spatula.
3. Place the gel in a plastic container.
4. Add ~20 ml staining solution (50% methanol, 10% HAc, 0.1% Coomassie Brilliant Blue) and stain for 1 hour on a shaking plate.
5. Pour off the staining solution back to the Schott bottle.
6. Add ~5 mL destaining solution (50% Methanol, 10% HAc) and destain briefly  for ~1 min.
7. Pour off  the destaining solution in the active carbon containing Schott bottle.
8. Add ~ 10 mL of destaining solution.
9. Destain in a shaking plate until the gel is visibly destained (overnight).
10. Pour off  the destain solution in the active carbon containing Schott bottle.
11. Rinse with ddH2O.

### Plamid Isolation

 The protocol according to [Nucleospin plasmid kit](https://static.igem.org/mediawiki/2019/9/9d/T--Lund--Plasmid_Isolation_Kit.pdf) was followed for Isolation of plasmid with low copy number.

### DNA Purification The protocol according to [Nucleospin Gel and PCR clean-up kit](https://static.igem.org/mediawiki/2019/3/37/T--Lund--PCR_%26_Gel_clean-up.pdf) was followed for DNA purification. Restriction Enzyme Cloning 1. Premix the following in an eppendorf tube: * 11 µL MilliQ water * 2 µL FD buffer * 5 µL Plasmid DNA 2. Add 1 µL Nde I, mix gently and spin down. 3. Incubate for 60 min at 37 ℃. 4. Add 1 µL BamH I, mix gently and spin down. 5. Incubate for 7 min at 37 ℃. 6. Incubate for 5 min at 80 ℃ to inactivate enzymes. 7. 7Add 1 µL of FastAP and incubate for 10 min at 37 ℃. 8. Incubate for 5 min at 75 ℃ to inactivate enzyme. 9. Store on ice or at -20 ℃. PCR product digestion 1. Combine the reagents in the following order at room temperature * 16 μL MilliQ water * 2 μL FastDigest buffer * 10 μL DNA (~0,2 μg) 2. Mix gently and spin down 3. Add 1 μL Nde1 and incubate at 37°C for 2 hours. 4. 4Add 1 μL BamH1 and incubate for 10 minutes Ligation 1. Prepare the reaction mixture: * 1 : 4 molar ratio (plasmid : gblock) * 2 µL Ligase Buffer * 1 µL T4 DNA ligase * MilliQ water to 20 µL 2. Incubate for at least 2 hours at room temperature. 3. Use 10 uL of ligation mixture in the transformation of 200 uL bacteria. Cultivation- Incolulate transformed BL21 in 5ml LB media with antibiotic and incubate at 37 °C overnight. - Put the samples on ice and measure optical density of the incubated BL21 - Pipette 0.5ml of culture with OD = 3 at 600nm into the flask containing 100 mL TB media with antibiotic - Incubate at 37 °C for 3-4 hours at 160rpm (Check the OD at fixed intervals) - Take control samples for SDS-page - When OD at 600nm ≈ 1, add 1mM IPTG - Grow for 24h and take samples for SDS-page after 2, 4, 8, 16 and 24 hours - Pour culture into tubes, centrifuge and save cell pellets

![](https://static.igem.org/mediawiki/2019/4/40/T--Lund--lund.png)
![](https://static.igem.org/mediawiki/2020/3/38/T--Lund--slu.JPG)
![](https://static.igem.org/mediawiki/2020/7/7d/T--Lund--lth.JPG)
![](https://static.igem.org/mediawiki/2020/1/11/T--Lund--protecto.JPG)
![](https://static.igem.org/mediawiki/2020/2/2b/T--Lund--GransboLogo.png)

[* ![](https://static.igem.org/mediawiki/2019/b/be/T--Lund--instagram.png)@igem\_lund](https://www.instagram.com/igem_lund/)
[* ![](https://static.igem.org/mediawiki/2019/5/5b/T--Lund--facebook.png)fb.com/igemlund](https://www.facebook.com/igemlund/)
[* ![](https://static.igem.org/mediawiki/2019/8/83/T--Lund--github.png)igemlund](https://github.com/igemlund)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Lund/Results

iGEM Lund 2019

* [Home](/Team%3ALund)
* Project

  + [Description](/Team%3ALund/Description)
  + [Design](/Team%3ALund/Design)
  + [Experiments](/Team%3ALund/Experiments)
  + [Notebook](/Team%3ALund/Notebook)
  + [Contribution](/Team%3ALund/Contribution)
  + [Results](/Team%3ALund/Results)
  + [Proof of Concept](/Team%3ALund/Proof_Of_Concept)
  + [Model](/Team%3ALund/Model)
  + [Engineering](/Team%3ALund/Engineering)
  + [Attributions](/Team%3ALund/Attributions)
  + [Safety](/Team%3ALund/Safety)
* People

  + [Team](/Team%3ALund/Team)
  + [Collaborations](/Team%3ALund/Collaborations)
  + [Attributions](/Team%3ALund/Attributions)
* Parts

  + [Overview](/Team%3ALund/Parts)
  + [Basic Parts](/Team%3ALund/Basic_Part)
  + [Composite Parts](/Team%3ALund/Composite_Part)
* Practices

  + [Human Practices](/Team%3ALund/Human_Practices)

[Proposed Implementation](/Team%3ALund/Implementation)

![](https://static.igem.org/mediawiki/2020/4/4b/T--Lund--LogoPNG1.png)

![](https://static.igem.org/mediawiki/2020/4/4b/T--Lund--LogoPNG1.png)

# Results and Discussion

This is the page were we present our results.

## Overview

The laboratory experiments were started with the restriction cloning of the parts with the pET11a vector and followed with heat shock transformation in TG1 cells. The successful cloning was confirmed primarily using colony PCR and later by Sanger sequencing. The plasmids were transformed to BL21 DE3 and were used in protein expression. The expressed proteins were identified using SDS-PAGE.
The protocols followed in each experiment are detailed in the Protocol and Experiments

The following eight parts were successfully transformed into E. coli BL21 (DE3):

* **Trx-EAFP1: [BBa\_K3619014](http://parts.igem.org/Part%3ABBa_K3619014)**
* **Trx-Ec-AMP-D1: [BBa\_K3619015](http://parts.igem.org/Part%3ABBa_K3619015)**
* **Trx-MsrA2: [BBa\_K3619016](http://parts.igem.org/Part%3ABBa_K3619016)**
* **Trx-Rs-AFP1: [BBa\_K3619017](http://parts.igem.org/Part%3ABBa_K3619017)**
* **Trx-nmDEF02: [BBa\_K3619018](http://parts.igem.org/Part%3ABBa_K3619018)**
* **Trx-Temporin A: [BBa\_K3619020](http://parts.igem.org/Part%3ABBa_K3619020)**
* **GST-Scarabaecin: [BBa\_K3619028](http://parts.igem.org/Part%3ABBa_K3619028)**
* **Thioredoxin: [BBa\_K3619031](http://parts.igem.org/Part%3ABBa_K3619031)**

Protein expression was successfully performed for all these parts except Trx-Ec-AMP-D1.

## Parts

Several basic parts were added to the registry and 17 composite parts were designed to express AMPs and the protease enterokinase as fusion proteins. For more information about the design of the parts see Project Design, and for a list of the basic and composite parts used see the Parts page.

## Transformation and Isolation of BL21 (DE3):

The cloned pET11a plasmids containing the antimicrobial peptide sequences were transformed into TG1 competent cells by heat shock transformation method. Five single colonies were picked up from each of the agar plates having the transformants. These colonies were used for preparing colony PCR as well as master plate. The gel image of the sample obtained from the PCR colony is given in Figure 1.

![](https://static.igem.org/mediawiki/2020/3/3c/T--Lund--cpcr.png)

**Figure 1:** Gel showing the results of colony PCR with the 100 bp DNA ladder and Thioredoxin (Trx). The band marked with red arrow are the samples which showed 100% sequence identity in sanger sequencing and were further transformed into BL21 (DE3).

## Sequencing of Composite parts:

Plasmid purification was made for the transformants and three samples of each of the eight inserts were sent to Eurofins Genomics for sanger sequencing. Four samples of each insert for the first batch of composite parts were sequenced and all had at least one sample of 100% sequence identity with the constructed parts, see analysed data here and raw data here. This proves that the correct sequences have been transformed for composite parts presented in table 1.

↓ Table 1: amount of transformants with 100% identity

| **Insert** | **Parts** | **Number of transformants with 100% identity** |
| --- | --- | --- |
| ***Trx-EAFP1*** | [BBa\_K3619014](http://parts.igem.org/Part%3ABBa_K3619014) | 1 |
| ***Trx-Ec-AMP-D1*** | [BBa\_K3619015](http://parts.igem.org/Part%3ABBa_K3619015) | 2 |
| ***Trx-MsrA2*** | [BBa\_K3619016](http://parts.igem.org/Part%3ABBa_K3619016) | 3 |
| ***Trx-nmDEF02*** | [BBa\_K3619018](http://parts.igem.org/Part%3ABBa_K3619018) | 3 |
| ***Trx-Rs-AFP1*** | [BBa\_K3619017](http://parts.igem.org/Part%3ABBa_K3619017) | 2 |
| ***Trx-Temporin A*** | [BBa\_K3619020](http://parts.igem.org/Part%3ABBa_K3619020) | 3 |
| ***GST-Scarabaecin*** | [BBa\_K3619028](http://parts.igem.org/Part%3ABBa_K3619028) | 3 |
| ***Thioredoxin*** | [BBa\_K3619031](http://parts.igem.org/Part%3ABBa_K3619031) | 3 |

## Cultivation and Protein expression:

The 8 constructs which were successfully transformed into the BL21 (DE3) cells were cultivated in TB media and the proteins were expressed with the induction of 1mM IPTG.

The growth curve (figure 2) depicts that all the transformed cells except for Trx- EAFP 1, are in their log phase until roughly 6 hours after induction and then enter the stationary phase. Whereas Trx- EAFP 1 continues to follow the log phase. So the E. coli cells accepted our AMP gene and were able to reproduce successfully without our AMPs inhibiting its metabolic processes.

![](https://static.igem.org/mediawiki/2020/9/97/T--Lund--growth.png)

**Figure 2:** the growth curve of the BL21 (DE3) cells after the induction of IPTG.

SDS-page samples of the cultivation were taken every 2 hours after IPTG induction and protein expressions were seen in all hour samples but the 8 hours samples (late log phase) showed a higher amount of protein comparatively when visualized through SDS-page. From the total of 8 constructs we successfully transformed, one was Thioredoxin and the other seven were our AMPs with Thioredoxin as fusion partner. Six of our seven AMPs showed very good desired protein expression, see figure 3. This means that E. coli was a very good expression system and also our gene design for the different AMPs worked well and can be repeated for future works.

↓ Table 2: Size of the associated gene product of the insert

| **Insert** | Trx-Temporin A | Trx-Msr A2 | Trx-NmDEF02 | Gst-Scarabaecin | Trx-Ec-AMP-01 | Trx-EAFP1 | Trx-Rs-AFP1 | Thioredoxin |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Size (kDa)** | 14.73 | 16.63 | 18.47 | 28.47 | 18.21 | 17.56 | 19.03 | 13.35 |

![](https://static.igem.org/mediawiki/2020/2/24/T--Lund--ProofofConcpet.png)

**Figure 3.** SDS PAGE gel showing the proteins obtained from the 8 constructs after 8 hours after IPTG induction and the control sample with its respective ladder size.

## Conclusion

We successfully designed and expressed almost all of the proteins with Thioredoxin as a fusion partner as well as one construct with GST as a fusion partner in E. coli BL21 and the results are promising. We also optimised the expression time and determined it to be 8 hours after induction of IPTG.

## Future plans

In the future, more testing needs to be conducted in order to show the effectiveness of the different peptides, and to be able to decide on the most potent combination of peptides towards treating late blight. We plan on first testing the cell lysate on isolated P. infestans, and after evaluating the effectiveness of the peptides, we would continue with the most promising ones and test them on potato leaves, to better simulate reality. We would also test the peptides on whole potato plants to simulate as natural an environment as possible. This would enable us to conduct tests during a longer period of time and see if there is any harm to the plant from the lysate. We would also be able to test how often the cell lysate has to be reapplied and how environmental conditions such as rain affects the application frequency.

As of now, we transformed but neither sequenced nor expressed the constructs with GST as a fusion partner, but this is something we would like to do. Scarabaecin fused to GST showed very good expression, so more experiments with GST should be done before finally deciding on which fusion partner is to be used.

When having decided on the most potent mixture of peptides and the most suitable fusion partner, we plan on trying the whole two plasmid-system with Enterokinase in one plasmid and the peptides in another plasmid. Making this work, with Enterokinase cleaving away the fusion partner and separating the different peptides, would be critical to obtain an economically viable product. The kill switch would also have to be tested in our system, to ensure that the product is completely safe to the environment and that no living GMMs are released.

We did not have time to try the peptides WHIPR and MAFP-1 that the model created, but gaining some experimental data could greatly improve and validate the model. By having a model that can continuously come up with effective antifungal peptides, the problem of P. infestans becoming resistant to the peptide mixture would be avoided since one or more of the peptides could be switched to another one suggested by the model.

In conclusion, we have gained positive results so far and would be able to continue developing the project.

![](https://static.igem.org/mediawiki/2019/4/40/T--Lund--lund.png)
![](https://static.igem.org/mediawiki/2020/3/38/T--Lund--slu.JPG)
![](https://static.igem.org/mediawiki/2020/7/7d/T--Lund--lth.JPG)
![](https://static.igem.org/mediawiki/2020/1/11/T--Lund--protecto.JPG)
![](https://static.igem.org/mediawiki/2020/2/2b/T--Lund--GransboLogo.png)

[* ![](https://static.igem.org/mediawiki/2019/b/be/T--Lund--instagram.png)@igem\_lund](https://www.instagram.com/igem_lund/)
[* ![](https://static.igem.org/mediawiki/2019/5/5b/T--Lund--facebook.png)fb.com/igemlund](https://www.facebook.com/igemlund/)
[* ![](https://static.igem.org/mediawiki/2019/8/83/T--Lund--github.png)igemlund](https://github.com/igemlund)