iGEM 2022 — Team nthu-taiwan


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Engineering

Avenue ahead to our dream.

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* [Outline](#heading1)
* [Experiment](#heading2)
* [Model](#heading3)
* [Reference](#heading4)

# Outline

We designed a system combined antimicrobial peptides and microneedles, hoping to cure skin diseases at an early stage. After cycles of brainstorming and modifying, we figure out the best way to reach our expectations. In fact, the engineering cycle helps us a lot to put our ideas into practice and the cycle can be found in every part of our project.

Fig.1 shows that our experiment went through five stages and the Model has four stages in the project. In the end, we combined them to make our project much better.

![](https://static.igem.wiki/teams/4336/wiki/wiki/engineering/eng1.png)

Fig.1 From the left, experiment , model engineering cycle

# Experiment

Our project has two parts: AMPC and MN. This part mainly describes the evolution of AMPC, and Fig.2 is the evolution diagram of the microneedle. For the detailed evolution process, please visit [Hardware](https://2022.igem.wiki/nthu-taiwan/hardware).

![](https://static.igem.wiki/teams/4336/wiki/wiki/engineering/eng2.png)

Fig.2 The evolution of microneedles

### Design

Aiming at specific detection and sterilization of *S. aureus*, we designed an antimicrobial peptide complex using the V8 protease cleavage site as a linker between our antimicrobial peptides. V8 protease is an extracellular protease of *S. aureus* that cleaves peptide bonds exclusively on the aspartate (D) and glutamic acid (E) amino residues. In our antimicrobial peptide complex, we combine one kind of antimicrobial peptide. The main antimicrobial peptide we planned to use the human AMPs, such as [hBD-3, HNP-1, or LL-37](https://2022.igem.wiki/nthu-taiwan/experiments#heading2).

### Redesign

However, IDT company can’t produce repetitive sequences. Interestingly, we found that two different AMPs have the synergistic effect for better sterilization in the literature, so we redesign that with two different AMPs. Besides, we selected an AMP called lysostaphin to be the main AMP component, which can hydrolyze the cell wall of *S. aureus*. We also combined it with other AMP, at this time, not only [hBD-3, HNP-1, and LL-37, but Histatin 5 and Ranalexin](https://2022.igem.wiki/nthu-taiwan/experiments#heading2) became the AMP component. Different from the first design, we tried to use different AMP and nucleic acid to increase the randomness, making our sequences work.

![](https://static.igem.wiki/teams/4336/wiki/wiki/engineering/eng3.png)

Fig.3 The evolution of AMPC design (a) first designed AMPC (b) redesigned AMPC

### Build & Test

We put the bacterium into 37°C incubator. However, we found that the protein expression was not obvious, so the sample was changed to be incubated at 16°C to facilitate stable expression of the protein. According to the SDS-PAGE analysis results, we divided protein expression into three categories, soluble , insoluble and not induced. Therefore, we try to purify the protein by the method of solubilization of inclusion body to break this barrier, so that it could reach the bacteria and function.

### Learn

At the purification part, we tried hard to get the proteins and do the disc diffusion assays. We have tried eight proteins and found that protein No.3 and No.7 being the most effective. Next, we focus on protein No.3 and No.7, cultivating and purifying them repeatedly. After that, we adjusted their concentration and redesigned the experiment in order to enhance the antimicrobial effect.

See [Result - Microneedle page](https://2022.igem.wiki/nthu-taiwan/results_mns) and [Result - AMPC page](https://2022.igem.wiki/nthu-taiwan/results_amp) for detail information.

# Model

### Design

Our model purpose was to **predict the effective time** of our system. That is, we’re trying to see how long the AMP will be effective after the AMPC release from the microneedle and be cleaved by V8 protease. We’ve tried to get the reaction rate constant of the reaction of that AMPC to become single AMPs, the minimum inhibitory concentration of different single AMP, and assume a value to the degradation rate of a single AMP according to experiments. Last, we transfer the chemical reaction to a differentiated equation and use python code to draw the drug **concentration-time curve**.

![](https://static.igem.wiki/teams/4336/wiki/wiki/model/cleavage-site-2.png)

We designed the formula and assumed two different reaction rate constants for the different site. we assumed k\_1 is the first V8 protease cleavage site, and k\_2 is the second one.

![](https://static.igem.wiki/teams/4336/wiki/wiki/engineering/engin-math1.png)

Preference sites are represented EL, ED, EF, and other sites are EG, EA, and EK.
(’’EL’’ means E and L are P1 and P1’ sites respectively.)
Next, we would need two more variables for our model. One is the MIC values of each AMP, the other one is the degradation rate of a single AMP.

### Bulid

##### MIC Values

We found the minimum inhibitory concentration of each AMP, we check whether the MIC is reached to judge whether our drug is efficient.

| AMP | Molecular Weight (Da) | Minimum Inhibitory Concentraion (MIC) |
| --- | --- | --- |
| IcCCL28-25 | 8500 | 6.25-50 μg/mL(0.735-5.88μM) |
| HNP1 | 3442 | 2.2 - 7.9 μg/mL(0.639-2.29μM) |
| Ranalexin | 2618 | 4 - 16 mg/L(1.9-7.6μM) |
| LL37 | 4493 | 2.9 - 3.6 μg/mL(0.645-0.801μM) |
| hBD3 | 5157 | 100 μg/mL(19.39μM) |
| Lysostaphin | 27000 | 0.003 - 2 μg/mL(0.0001-0.074μM) |
| Histatin5 | 3037 | >400 μg/mL(>131.7μM) |

Table.1 AMPs’ molecular weight and minimum inhibitory concentration

##### Degradation Rate

Because we couldn’t find more related research about degradation rate, we refer to the research paper that LL37’s half-life time is 1 hour in cell as the AMPs half-life time. Then, set the parameter in the differential equation to draw the **concentration-time curve**.

##### Python Code

In fact, we’ve completed the code and tried to set the default value randomly based on the 2021 CCU\_Taiwan team’s result and some papers before we get the real data of each reaction rate from the experiment to see if the result is reasonable or not. However, we think there’s another way to simulate realistic circumstances. We then talked with seniors and redesigned our model. Full source code can be found in our GitLab and more design details can be seen on the Model page.

### Learn

Che-Kang Chang who is NTHU 2016 member advised us to apply the enzyme kinetics in our model, making our model more persuasive. We looked into some tutorials and examples to start redesigning our work.

### Redesign

We modified our model design and applied the Enzyme Kinetics formula.

![](https://static.igem.wiki/teams/4336/wiki/wiki/engineering/engin-math2.png)

Enzyme Kinetics has four hypotheses:

1. [ES] is constant. So k\_1[E][S] = k\_2[ES] + k\_3[ES] always holds.
2. All enzyme concentration [E\_t] = exist alone [E\_f] + [ES]
3. The reaction rate is decided by k\_3[ES].
4. The maximum reaction rate V\_max is the situation in which all the enzymes are turned into [ES]. V\_max = k\_3[E\_t]

Keeping the MIC values and the assumption of degradation rate in the original version, we modified more in the reaction rate constant part among the three main variables in our model.

### Bulid

We referred to the paper about V8 protease to set the k\_3. Because [S] is much larger than [E], we consider that k\_3 is much larger than k\_2 and assume the k\_2 value is 0. Therefore, we can get the k\_1 value by definition K\_max = (k2+k3)/k1.

| Reaction rate const | Value(hr^-1) |
| --- | --- |
| k\_1 | 0.037 |
| k\_2 | 0 |
| k\_3 | 1044 |

Table.2 Reaction rate constant we assumed.

In the new version of python code, the skeleton was basically the same but was changed the equations and had three reaction rate constants this time. Please check out the full source code in our GitLab.

### Conclusion

According to the above, we knew the reaction rate is controlled by k\_3[ES], and we got the k\_3 value from the research paper. In order to reach our purpose, we hoped our system could release the drug slowly and stably. Therefore, we expect our system can reach the MIC of AMPs keeping for a few hours. By assuming the enzyme concentration, we estimate how much initial concentration of the drug we need to achieve the minimum inhibitory concentration, and estimate how much concentration of the drug we need.

See [Model page](https://2022.igem.wiki/nthu-taiwan/model) for detail information.

# Reference

1. Domhan C, Uhl P, Kleist C, Zimmermann S, Umstätter F, Leotta K, Mier W, Wink M. Replacement of l-Amino Acids by d-Amino Acids in the Antimicrobial Peptide Ranalexin and Its Consequences for Antimicrobial Activity and Biodistribution. Molecules. 2019 Aug 17;24(16):2987. doi: 10.3390/molecules24162987. PMID: 31426494; PMCID: PMC6720431.
2. Yang XY, Li CR, Lou RH, Wang YM, Zhang WX, Chen HZ, Huang QS, Han YX, Jiang JD, You XF. In vitro activity of recombinant lysostaphin against *Staphylococcus aureus* isolates from hospitals in Beijing, China. J Med Microbiol. 2007 Jan;56(Pt 1):71-76. doi: 10.1099/jmm.0.46788-0. PMID: 17172520.
3. Singh D, Vaughan R, Kao CC. LL-37 peptide enhancement of signal transduction by Toll-like receptor 3 is regulated by pH: identification of a peptide antagonist of LL-37. J Biol Chem. 2014 Oct 3;289(40):27614-24. doi: 10.1074/jbc.M114.582973. Epub 2014 Aug 4. PMID: 25092290; PMCID: PMC4183800.
4. Su J, Li H, Hu J, Wang D, Zhang F, Fu Z, Han F. LcCCL28-25, Derived from Piscine Chemokine, Exhibits Antimicrobial Activity against Gram-Negative and Gram-Positive Bacteria In Vitro and In Vivo. Microbiol Spectr. 2022 Jun 29;10(3):e0251521. doi: 10.1128/spectrum.02515-21. Epub 2022 May 26. PMID: 35616397; PMCID: PMC9241943.
5. Wanmakok M, Orrapin S, Intorasoot A, Intorasoot S. Expression in Escherichia coli of novel recombinant hybrid antimicrobial peptide AL32-P113 with enhanced antimicrobial activity in vitro. Gene. 2018 Sep 10;671:1-9. doi: 10.1016/j.gene.2018.05.106. Epub 2018 May 30. PMID: 29859288.
6. Komatsu T, Salih E, Helmerhorst EJ, Offner GD, Oppenheim FG. Influence of histatin 5 on Candida albicans mitochondrial protein expression assessed by quantitative mass spectrometry. J Proteome Res. 2011 Feb 4;10(2):646-55. doi: 10.1021/pr100861k. Epub 2010 Dec 6. PMID: 21080726; PMCID: PMC3033980.
7. Midorikawa K, Ouhara K, Komatsuzawa H, Kawai T, Yamada S, Fujiwara T, Yamazaki K, Sayama K, Taubman MA, Kurihara H, Hashimoto K, Sugai M. *Staphylococcus aureus* susceptibility to innate antimicrobial peptides, beta-defensins and CAP18, expressed by human keratinocytes. Infect Immun. 2003 Jul;71(7):3730-9. doi: 10.1128/IAI.71.7.3730-3739.2003. PMID: 12819054; PMCID: PMC162002.

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Experiments

Experience is the best teacher.

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* [Overview](#heading1)
* [Antimicrobial Peptides Selection](#heading2)
* [Construction of AMPC](#heading3)
* [Major Experiments & Results](#heading4)
* [Reference](#heading5)

# Overview

In our mission to address *S. aureus* skin infection, we decided to provide a new treatment for the disease. Inspired by existing treatments using the antimicrobial peptides (AMPs) to target the *S. aureus*. Moreover, we came up with **the antimicrobial peptides complex**, abbreviated to **AMPC**, and designed the specific cleavage sites to change inactive and active states for the AMP.

![](https://static.igem.wiki/teams/4336/wiki/wiki/exp/exp1.jpg)

Fig.1 The AMPC is inactive, but can be activated upon cleavaged by V8 protease.

# Antimicrobial Peptides Selection

When choosing AMP, we give priority to two major directions. First, we look for the original AMP of human beings. Next, we considered AMP that is not possessed by humans, but has a good antimicrobial effect on *S. aureus*. Furthermore, has a synergistic effect to increase the antimicrobial effect.

### Information of Antimicrobial Peptides (AMPs)

1. Cathelicidin LL-37
   AMP of human origin can reduce undesired immune response, and have good activity against *S. aureus* biofilm.
2. Human Beta Defensin-3 (hBD3)
   AMP of human origin can reduce undesired immune response, and reasonable effective, well characterized, two Glu residues can reduce the chance of non-specific digestion by V8 protease.
3. Human neutrophil peptide 1 (HNP1)
   AMP of human origin can reduce undesired immune response, and reasonable effective, well characterized, one Glu residues can reduce the chance of non-specific digestion by V8 protease.
4. Histatin 5 (Hst 5)
   AMP of human origin can reduce undesired immune response, and show a high bactericidal effect on MRSA .
5. Lysostaphin
   AMP of non-human origin can be produced by *S. aureus* simulans. Lysostaphin is a single polypeptide chain (approximately 27 kDa, aa 248-452) , and it shows synergistic effect after combined with some AMP.
6. Ranalexin
   AMP from the skin of the bullfrog, which shows a high antimicrobial effect on MRSA. Also it shows synergistic anti-staphylococcal activity after combined with Lysostaphin .
7. LcCCL28
   AMP of non-human origin is antimicrobial peptide from shrimp Litopenaeus vannamei , it can be showed good antimicrobial activity.

# Construction of AMPC

To attain the goal of specific detection and sterilization of *S. aureus*, first, we designed the HAMA coating on microneedles to release the AMP while encountering *S. aureus*. Consistent with the specificity design of the microneedle, the professor suggested us to change the construction of AMP inside to let it also have specific anti-*S. aureus* efficacy. Here is where the concept of AMPC comes from. We decided to combine AMP with the linker, which is the cleavage site of a protease secreted by *S. aureus*. Hence, we expect AMPC achieves the following purpose:

1. It can be **produced by E. coli without causing any detrimental effects**. It might be difficult for E. coli to produce simple AMP effectively while AMP might kill E. coli or be eaten as a nutrient..
2. **AMPC will remain inactive if the linker exists**. The antimicrobial effect appears only when encountering *S. aureus*.
3. The specificity of AMP release can **reduce multi-drug resistance and normal microflora disruption**.
4. AMPC has the property of **slow drug release**, it may prolong the half-life of AMP and improve the convenience for patient usage so that patients don’t need to change their medicament often. Additionally, at the point of safety, AMPC can avoid large amounts of harmful antimicrobial peptide released at the same time if AMP has negative effect on the patient.
5. The high positive charges of AMPC allow the absorption of toxic microbial components such as lipoteichoic acid to reduce inflammation of the wounds.

We investigated the protease which only secreted by S. aureus, for example, Staphopain A, V8 protease, SplA, SplB, SplD, and aureolysin. Considering the working environment and the protease is common, we finally chose the V8 protease cleavage site as linker of AMPC. V8 protease specifically cleaves peptides at glutamate residues in ammonium bicarbonate buffer, pH 7.8. So we use ammonium bicarbonate buffer to dialysis the purified protein, hoping to increase the success probability of our experiment.

### AMPC Gene Construction

In order to express protein better, we chose pET28a as a plasmid vector, which carries T7 promoter, lac operator, and poly-histidine purification tag coding sequence. Separating the design into a few parts, the first is the linker sequence design. As just mentioned, we chose glutamate residues, which is the V8 protease cleavage site, as the linker. Also, we checked that there aren’t any EN, EE, and EP sequence at the cleavage site, which dislikes by V8 protease.

We ordered the sequence from IDT company as the pattern of linker [AMP sequence] linker [AMP sequence] linker [AMP sequence]. However, the redundancy sequence can’t be produced by IDT company. In addition, we found that some AMPs such as LL37 and Ranalexin have synergistic anti-staphylococcal activity. Therefore, we designed AMPC by combining two different AMPs together. Detail sequence is mentioned on the [Parts](https://2022.igem.wiki/nthu-taiwan/parts) page.

See [Parts page](https://2022.igem.wiki/nthu-taiwan/parts) for detail information.

The main AMP in AMPC is lysostaphin, and by linking it with other AMP as mentioned previously such as LL37, we made eight combinations of AMPC. Also, we used the same AMP but synonymous codons to avoid repetitive sequences and NdeI and XhoI restriction sites for digestion. Using NdeI and XhoI restriction enzyme, we digested our target gene from the pUCIDT (Amp) Golden Gate vector provided by IDT company. Afterward, we inserted it into the pET28a vector and transformed the plasmid into E. coli TOP10 for storage.

![](https://static.igem.wiki/teams/4336/wiki/wiki/exp/exp2.jpg)

Fig.2 The flowchart of gene cloning.

### Protein Production, Purification and Antimicrobial Activity Test

After cloning, we inserted target gene into pET28a vector and transformed the plasmid into E. coli TOP10 for storage. Then we extracted the plasmid and transformed it into pET28a. Induce the bacteria by IPTG overnight for protein expression, we purified the protein by nickel column. Lastly, we test the antimicrobial activity by disc diffusion assay.

![](https://static.igem.wiki/teams/4336/wiki/wiki/exp/exp3.jpg)

Fig.2 The flowchart of gene cloning.

# Major Experiments & Results

See [Result - AMPC page](https://2022.igem.wiki/nthu-taiwan/results_amp) for detail information.

* Cloning
* Induction test for Protein Expression
* Protein purification
* Disc Diffusion assay

# Reference

1. Carmona C, Gray GL. Nucleotide sequence of the serine protease gene of *Staphylococcus aureus*, strain V8. Nucleic Acids Res. 1987 Aug 25;15(16):6757. doi: 10.1093/nar/15.16.6757. PMID: 3306605; PMCID: PMC306157.
2. Houmard J, Drapeau GR. Staphylococcal protease: a proteolytic enzyme specific for glutamoyl bonds. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3506-9. doi: 10.1073/pnas.69.12.3506. PMID: 4509307; PMCID: PMC389807.
3. Sharma R, Sharma PR, Choudhary ML, Pande A, Khatri GS. Cytoplasmic expression of mature glycylglycine endopeptidase lysostaphin with an amino terminal hexa-histidine in a soluble and catalytically active form in Escherichia coli. Protein Expr Purif. 2006 Jan;45(1):206-15. doi: 10.1016/j.pep.2005.07.025. Epub 2005 Aug 19. PMID: 16181789.
4. Sadeghi S, Bakhshandeh H, Ahangari Cohan R, Peirovi A, Ehsani P, Norouzian D. Synergistic Anti-Staphylococcal Activity Of Niosomal Recombinant Lysostaphin-LL-37. Int J Nanomedicine. 2019 Dec 10;14:9777-9792. doi: 10.2147/IJN.S230269. PMID: 31849468; PMCID: PMC6911324.
5. Chen S, He FT, Dong YL, Li RF, Gao HG, Chen M, Peng JH. [The cloning, high level expression in Escherichia coli of human beta-defensin 3 and its antimicrobial activity analysis]. Sheng Wu Gong Cheng Xue Bao. 2004 Jul;20(4):490-5. Chinese. PMID: 15968976.

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