iGEM 2023 — Team virginia

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**Prior to lab**
**Week of May 29**
**Week of June 5**
**Week of June 12**
**Week of June 19**
**Week of June 26**
**Week of July 3**
**Week of July 10**

**Week of July 17**
**Week of July 24**
**Week of July 31**
**Week of August 7**
**Week of August 14**
**Week of August 21**
**Week of August 28**
**Week of September 4**
**Week of September 11**
**Week of September 18**
**Week of September 25**
**Week of October 2**
**Week of October 9**

**Click on one of the buttons at the top to diplay a notebook page.**

**Any lab pictures associated with the entry will appear here.**

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* [Initial Design](#topic1)
* [Protein Expression](#topic2)

+ [Build](#topic3)
+ [Test](#topic4)
+ [Re-Design](#topic5)
+ [Re-Test](#topic6)

* [Protein Purification](#topic7)

+ [Design](#topic8)
+ [Build](#topic9)
+ [Test](#topic10)
+ [Re-Design](#topic11)
+ [Re-Test](#topic12)
+ [Re-Re-design](#topic13)

# Initial Design

To produce active nisin, the *nisA* gene is not the only one that needs to be expressed. NisA has to undergo several post-translational modifications to reach its fully active form. Previous iGEM teams have attempted to produce nisin; however, no team has been able to successfully do so. In 2014, the Groningen team aimed to produce active nisin. In their constructs, they incorporated the nisin immunity genes, *nisR* and *nisK*, and two genes required for post-translational modifications of nisin, *nisB* and *nisC*. They used fluorescent genes to indicate successful transformation of *E. coli*. In their results, they were able to transform cells with all gene transformants except for *nisB*, *nisR* and *nisK*. However, the team was unable to successfully express any proteins related to nisin biosynthesis. A few years later, the iGEM 2022 Calgary team attempted to produce another nisin homolog: nisin Q. However, *nisQ* was the only gene they considered, not accounting for the necessary post-translational modifications required to produce active nisin.

To become fully active nisin, NisA must be post-translationally modified in three ways: dehydration, cyclization, and cleavage. NisB is a dehydratase responsible for the first modification. NisB dehydrates eight serines and threonines to dehydroalanine and dehydrobutyrine, respectively. NisC is a cyclase that is responsible for the second modification. NisC forms five thioether bonds between dehydroamino acids and cysteines, giving nisin its distinct structure. NisP is an extracellular protease responsible for the third modification. NisP cleaves the leader peptide (23 of the 57 amino acids making up NisA), leaving fully modified and active nisin. In the naturally occurring producer, *Lactococcus lactis*, there are other proteins that play a role in the immunity of the host to nisin and the extracellular transport of NisA, but these proteins do not influence the actual production of nisin. We concluded that NisB and NisC are required to produce nisin. For our project, we wanted to be able to activate fully modified nisin at the site of infection, so we used the leader cleavage site to our advantage. By altering the cleavage site, NisP would not be required. Instead, we utilized the V8 protease, an extracellular protease exclusively produced by *Staphylococcus aureus*, one of the main pathogens contributing to cellulitis infections. Using the V8 cleavage site, NiSkin will remain in an inactive form until it reaches the site of infection.

To help NiSkin reach the site of infection, we employed the use of the skin-penetrating peptide, TD-1. From literature and consultations with experts, we knew that TD-1 would work best when fused to the protein of interest. However, TD-1 has never been fused to NisA, so the most effective way of linking the two proteins is unknown. Therefore, we designed three experimental fusion proteins. The first construct consists of a truncated leader, with TD-1 and a V8 cleavage site between the leader and the nisin precursor peptide of NisA. The second construct consists of TD-1 fused to the full NisA primary structure with a V8 site between the leader peptide and the nisin precursor. The third construct consists of TD-1 and NisA, with a glycine-serine linker in between the two and a V8 cut site between the leader peptide and the nisin precursor. Along with our fusion protein constructs, we wanted to confirm that we had the correct machinery to produce fully modified nisin, so we made two constructs without TD-1: the first is NisA with a V8 protease site separating the leader peptide and the nisin precursor, and the second is NisA with its original NisP cleavage site. An outline of all of our constructs can be found in Figure 2 on the Experiments Page of our wiki.

# Protein Expression

## Build

We decided to use *E. coli* BL21 (DE3) as our chassis, with a two-plasmid system. Our plan was to insert *nisA* and *nisB* into the open reading frame of the high-copy vector pRSFDuet-1 and insert *nisC* into the low-copy vector pACYCDuet-1. Once we received the control constructs, we amplified the sequences using PCR. We transformed our plasmids into *E. coli* DH5α and amplified our plasmids. We confirmed insertion with restriction digests/gel electrophoresis and Sanger sequencing. Then, we transformed our plasmids into *E. coli* BL21 (DE3) cells to begin protein expression. In total, we created 14 DH5α strains and 17 BL21 (DE3) strains with our genes, which can be seen in Table 1 of the Results page on our wiki.

## Test

Once the transformation of pRSFDuet-1-*nisA*-*nisB* and pACYCDuet-1-*nisC* into BL21 (DE3) cells was confirmed, the next step was to express our proteins. Since our vectors use a T7 promoter, we used IPTG induction to over-express our proteins of interest. Our initial protein sample protocol involved incubating a culture with 0.4 mM IPTG for 2 hours, centrifuging 0.5 mL of culture and resuspending in Tris-Cl, adding sample buffer, and boiling the sample for 3 minutes. For our SDS-PAGE gels, we used 13% Tris-glycine gels to test our protein samples. We calculated the predicted molecular weights of our proteins using the amino acid sequence. Our first step was to confirm the expression of NisA, NisB, and NisC on their own. Therefore, using the protocol listed above, we made protein samples for BL21 (DE3) cells containing pRSFDuet-1-*nisA*, pRSFDuet-1-*V8*-*nisA*, pRSFDuet-1-*nisB*, and pACYCDuet-1-*nisC*. The first protein that we confirmed expression of was NisC, as we saw a band around the predicted molecular weight of 48 kDa. We were able to confirm expression of V8-NisA, NisA, and NisB, by using an 8-18% Tris-glycine SDS-PAGE, as it allowed us to better visualize bands at the top and bottom of the gels. We saw NisB at its predicted molecular weight of 118 kDa. For V8-NisA and NisA, we did not see bands at the predicted molecular weights of 5.81 kDa, 5.84 kDa, respectively, but we saw clear indication of a protein being induced by IPTG at 11-12 kDa, and we later confirmed through Western Blot. After confirming that the proteins were being expressed on their own, the next step was to express the three other NisA constructs and to co-express all three of our proteins (NisA, NisB, and NisC) in the same chassis. We were able to confirm the expression of our other three constructs: TL-TD1-V8-NisA, TD1-V8-NisA, and TD1-linker-V8-NisA. However, we were having difficulties in co-expressing all three proteins. The results we were getting did not have a clear indication of the presence of bands at the predicted molecular weights, with the induced sample and non-induced examples mostly looking identical.

## Re-Design

After remaking protein samples from the whole cell lysate of BL21 (DE3) cells containing the *nisA*, *nisB*, and *nisC* genes multiple times and still getting no results on our gel, we decided to modify our protein sample protocol to get better expression of NisB and NisC. After consulting multiple advisors well-versed in protein expression, we suspected that the short incubation period, the concentration of IPTG being used to induce the samples, the amount of culture we use, and the temperature at which we are inducing the cells could be factors that determine how well NisB and NisC are expressed and how they interact with NisA. The original incubation time we used was 2 hours. We were advised to have a longer incubation time, anywhere from 4 to 6 hours. The concentration we were originally using was 0.4 mM IPTG. Using a lower amount of IPTG could help express NisB and NisC, as it has been shown to improve folding and solubility of proteins. Temperature also has a similar effect; incubating at a lower temperature for a longer duration could improve the folding of NisB and NisC and allow for interactions between the three proteins.

## Re-Test

To test which factor(s) affected the expression of our three proteins, we made modifications to our protein sample protocol using V8-NisA expression, changing one variable at a time. First, we tested different incubation times, inducing the cultures with 0.4 mM IPTG and letting samples incubate for 2 hours, 4 hours, 6 hours, and 8 hours before centrifuging. We found that 4 hours was the most effective incubation time. Secondly, we varied the amount of IPTG used to induce, and we found that 0.4 mM had the same effect as 0.1 mM IPTG, so we continued using the same amount as before. Third, we increased the amount of culture we centrifuged from 0.5 mL to 1.5 mL, which gave us larger bands at the predicted molecular weight of our protein. Fourth, we decreased incubation temperature from 37°C to 18°C and extended incubation time to 24 hours. We used BL21 (DE3) cells containing both pRSFDuet-1-*V8*-*nisA*-*nisB* and pACYCDuet-1-*nisC*. This method was the most effective at co-expressing the three proteins: we saw both distinct bands at the predicted molecular weights of NisB and NisC and an increase in molecular weight of NisA. Such an increase might be due to successful post-translational modifications which would most likely impede movement through the gel.

# Protein Purification

## Design

After designing the initial NisA and TD-1 fusion proteins, we realized that there needed to be some mechanism for isolating the product from the device. To this end, we installed a His6 tag at the product’s N-terminus and conjugated the tag to our fusion protein with a WELQ sequence. This proteolytic cleavage site is cut at the carboxyl side of the glutamine residue. Thus, we planned to purify NisA protein variants via the His6 tag and then remove the amino acids needed for purification, leaving behind pure product.

## Build

The pRSFDuet-1 plasmids, which served as vectors for our constructs, contain a translational start codon upstream of our first restriction site. Between the start codon and the restriction site where *nisA* is inserted is sequence coding for a His6 tag. Thus, we biobricked a sequence coding for the WELQ cut site onto the 5’ end of our *nisA* constructs so that we are able to cleave the His6 tag after it is no longer needed. Assembly of this His6-WELQ-NisA system required only the two restriction enzymes needed to insert *nisA*. We verified correct insertion of *nisA* with restriction site mapping and Sanger sequencing outsourced to Azenta. We verified correct expression of His6-tagged NisA with a Western blot using anti-His6 antibodies.

## Test

His6-tagged NisA was purified with nickel-nitrilotriacetic acid beads on a nickel column. Results were verified with an SDS-PAGE gel of the elution fractions. However, the 20 μg/mL yield following purification was too low for downstream applications, considering that yield would further decrease after cleavage of the His6 tag.

## Re-design

Our initial design had successfully purified nisin, but in too small an amount to be useful. Thus, we brainstormed ways to increase yield with our given design. We could increase the volume of cell culture expressing nisin, because more protein would bind the column and more would be eluted; we could increase the bed volume of the column to decrease the loss of nisin in the flow through; we could increase the concentration of imidazole used to elute nisin off the column; finally, we could decrease the elution volume to concentrate the protein that leaves the column.

## Re-test

We repeated purifications of His6-tagged NisA and varied protocol elements. When the cell culture volume was increased 25-fold, we found that no nisin was eluted. The SDS-PAGE gel of the fractionation steps showed that nisin was not present in the high-speed supernatant applied to the column, with the predicted band disappearing after sonication. Fractionation was most likely unsuccessful because the sonicator settings were not altered to account for a denser sample being sonicated. Next, increasing the bed volume during purification actually decreased nisin yield. In the same elution volume as before, less nisin would elute because of a higher binding affinity to the resin. When the imidazole concentration was increased, no change in nisin yield was observed. No nisin had been detected in the final elution with the highest imidazole concentration, so increasing the imidazole concentration of the middle elution at which nisin elutes was unlikely to have an effect. Lastly, we have yet to test whether decreasing the elution volume increases nisin yield.

## Re-re-design

After optimizing our method to purify His6-tagged nisin, we realized that we would have no way of purifying nisin from the reaction mixture following cleavage of the His6 tag with WELQut protease. Thus, we next plan to order an immobilized version of WELQut protease which can bind the column and cleave nisin on the column such that pure nisin elutes. We could further increase the concentration of our nisin yield with a low molecular weight cut off centrifugal filter that allows solvent to collect at the bottom of the tube while keeping nisin at the top.

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* [Designing NiSkin](#topic1)
* [Building Our Device](#topic2)
* [Protein Expression and Confirmation](#topic3)
* [Tests of Our Engineered Proteins](#topic4)
* [Protocols](#topic5)

+ [Preparation of Competent *E. coli*](#itemA)
+ [Polymerase Chain Reaction (PCR)](#itemB)
+ [Restriction Digest](#itemC)
+ [DNA Gel Electrophoresis](#itemD)
+ [Ligation](#itemE)
+ [Transformation of Chemically Competent Cells](#itemF)
+ [Preparing Glycerol Stocks of Strains](#itemG)
+ [Protein Expression](#itemH)
+ [Cell Fractionation](#itemI)
+ [SDS-PAGE](#itemJ)
+ [Western Blot](#itemK)
+ [Affinity Purification](#itemL)
+ [Disk Diffusion Assay](#itemM)
+ [Franz Diffusion Assay](#itemN)
+ [Buffers, Reagents, and Other Materials](#itemO)

* [References](#topic6)

# Designing NiSkin

Although nisin is an antimicrobial peptide, *nisA* does not encode fully active nisin. The primary structure of NisA must be post-translationally modified in three critical ways before it shows antimicrobial activity.

![niaABC catalytic activity](https://static.igem.wiki/teams/4853/wiki/pages-design/experiments/experiments-fig1.png)

**Figure 1**. Diagrams showing enzymatic activity of the three post-translational modifications required to produce active nisin. Made with BioRender. **A** NisB is depicted dehydrating serine into dehydroalanine. **B** NisC is depicted forming a thioether bond between dehydroalanine and cysteine. **C** NisP cleaves the leader peptide between R23 and I24. **D** Structure of active nisin with three letter abbreviations for amino acids. ABU, DHB, and DHA are aminobutyric acid, dehydrobutyrine, and dehydroalanine, respectively.

First, the dehydratase NisB dehydrates certain residues on NisA (Fig. 1A). Second, the cyclase NisC forms five thioether bonds, giving nisin its distinctive structure with five rings (Fig. 1B). Both NisB and NisC specifically recognize the leader peptide sequence (23 of 57 total amino acids in NisA, on the N-terminus side). Third, the extracellular peptidase NisP cleaves the leader (Fig. 1C), leaving fully modified and active nisin (Fig. 1D) [1]. While NisB and NisC are required to produce active nisin and therefore must be co-expressed in our chassis, we altered the leader cleavage site to our advantage. The V8 protease is an extracellular protease produced by *Staphylococcus aureus*, one of the main pathogens in cellulitis infections. By changing the NisP cleavage site to a V8 cleavage site, we can make the pathogen work for us and against itself – during the production of NiSkin, we do not have to worry about the cleavage step. Instead, NiSkin will remain in an inactive form until it reaches the site of infection, where the V8 protease being released by *S. aureus* will cleave the leader peptide, leaving nisin with antibacterial activity.

![construct diagram](https://static.igem.wiki/teams/4853/wiki/pages-design/experiments/figure-2.jpeg)

**Figure 2**. Gene diagrams. **A** *V8*-*nisA*, *nisB* operon. **B** *nisA*, *nisB* operon. **C** *TL*-*TD1*-*V8*-*nisA*, *nisB* operon. TL = truncated leader. **D** *TD1*-*V8*-*nisA*, *nisB* operon. **E** *TD1*-*linker*-*V8*-*nisA*, *nisB* operon. **F** *nisC* operon.

From the literature, we know that TD-1 works best when fused to the N-terminus of the protein of interest [2]. However, TD-1 has never been tested with nisin, so the most effective way of linking the two proteins is unknown. To find the best method, we designed five different *nisA* genes. The first two are controls: one using the original primary structure found in *Lactococcus lactis* but with a V8 cleavage site and one with the original NisA primary structure (Figure 2A, 1B). The other three are variants of TD-1 fused with NisA: the first uses a truncated leader with TD-1 embedded between a truncated leader and the rest of NisA (Figure 2C); the second has TD-1 conjugated to the full leader (Figure 2D); the third has TD-1 conjugated to a glycine-serine linker conjugated to the full leader sequence (Figure 2E). All three TD-1-NisA fusion proteins use the V8 cleavage site for the leader. Additionally, all five proteins have an N-terminal His6 tag for purification purposes. With the sequences coding for NisB (Figure 2A-E) and NisC (Figure 2F), we have a total of seven different gene sequences. Because NisB and NisC do not serve any additional functions after modifying NisA and because epitope tags might interfere with enzymatic activity, NisB and NisC are untagged. All sequences were codon optimized for *E. coli* BL21(DE3).

# Building Our Device

All of our sequences were synthesized then amplified using PCR. *nisA* and *nisB* were inserted into the same open reading frame (ORF) on the pRSFDuet-1 vector. *nisC* was inserted into the pACYCDuet-1 vector. pRSFDuet-1 contains an RSF1030 replicon and pACYCDuet-1 contains a P15A replicon, making them compatible for transformation into the same chassis [3]. Plasmids were first transformed into *E. coli* DH5α cells for plasmid amplification, which was verified with restriction digests/gel electrophoresis and Sanger sequencing of the plasmids. Both plasmids were then transformed into *E. coli* BL21(DE3) cells, so we were ready to co-express the NisA variants alongside NisB and NisC.

# Protein Expression and Confirmation

After confirming proper plasmid insertion of pRSFDuet-1-*nisA*-*nisB* (and the other four variants) and pACYCDuet-1-*nisC* with a restriction digest and Sanger sequencing, the next step was to express our engineered NisA, NisB, and NisC. Both vectors use a T7 promoter system, allowing us to chemically induce transcription with IPTG and regulate the expression of our proteins. Protein samples were analyzed with SDS-PAGE, which confirmed that we are expressing proteins when *E. coli* BL21(DE3) cultures are induced with IPTG.

Analyzing our proteins by Western blot was not simple; there are no available antibodies against NisA, NisB, or NisC. However, for purification purposes, all of our NisA proteins have an N-terminal His6 tag. Thus, we then analyzed our NisA constructs on a Western blot using anti-His6 antibodies (Invitrogen, MA1-4806) and confirmed that we are producing NisA constructs that have the His6 tag.

# Tests of Our Engineered Proteins

We defined two criteria by which we wanted to quantitatively test our engineered nisin: antibacterial activity and skin permeability. An effective topically applied drug must both have strong antibacterial activity and be able to reach the site of infection at the correct dosage. After purifying our engineered nisin via affinity purification with the His6 tag, we could begin testing.

The disk diffusion assay is a well-proven method for studying antibacterial activity of different antibiotics. An agar plate is grown with an even lawn of bacteria and disks soaked with specific concentrations of antibiotic are placed on the plate. A ring of inhibition (no growth) will form around the disk; the larger the ring, the more effective the antibiotic. Nisin has already demonstrated strong activity against *Staphylococcus aureus*, one of the main pathogens in cellulitis infections [4,5]. A successful test will demonstrate that our engineered nisin is able to achieve a comparable level of antibacterial activity.

To test skin permeability, we are using a Franz diffusion assay. Using porcine skin to model human skin, we can measure how much of our engineered nisin is able to diffuse across the skin and to the theoretical site of infection. By comparing the permeation of nisin against our engineered nisin with TD-1, we can quantify how much TD-1 aids permeation. Additionally, this assay will tell us what concentration of NiSkin is necessary in a skin cream to deliver the correct dosage of nisin into the dermis.

# Protocols

## Preparation of Competent *E. coli*

1. Inoculate 2 mL of LB with a single *E. coli* colony. Incubate the preculture overnight at 37°C and 250 RPM.
2. In the morning, inoculate 500 mL of LB with 1 mL of saturated preculture. Incubate at 37°C and 250 RPM until OD600 = 0.5.
3. Every step from now on should be completed on ice in a 4°C cold room. Transfer the culture to two prechilled sterile 250 mL centrifuge tubes.
4. Centrifuge at 2700*g* and 4°C for 10 minutes. Discard the supernatant and place the pellets on ice.
5. Resuspend each pellet in 10 mL cold 0.1 M CaCl2. Pool into one prechilled sterile 50 mL centrifuge tube.
6. Centrifuge the cells at 2000*g* and 4°C for 5 minutes. Discard the supernatant and resuspend in 10 mL CaCl2. Set on ice for 30 minutes.
7. Centrifuge the cells at 2000*g* and 4°C for 5 minutes. Discard the supernatant and resuspend in 2 mL CaCl2. Leave at 4°C overnight.
8. The following day, add 70 µL of dimethyl sulfoxide (DMSO) and gently mix. Using liquid nitrogen, snap-freeze 50 µL aliquots of competent cells. Store at -80°C.

## Polymerase Chain Reaction (PCR)

1. Combine 300 ng DNA, 25 µL 2x PCR Master Mix, 1 µL of 10 µM forward primer, 1 µL of 10 µM reverse primer and PCR-grade water up to 50 µL in thermal cycler snap tubes (ThermoFisher, AB2005).
   1. Add water first, then master mix, then primers, and lastly DNA.
   2. Make 100 µM stocks of primers in 10 mM Tris-Cl (pH 8) when they arrive, and then dilute tenfold with water for 10 µM working stocks.
   3. We used Quick-Load Taq 2x Master Mix, which contains Taq DNA Polymerase, its buffer, and dNTPs.
2. Place reaction mixture in thermal cycler under the following conditions:
   1. Initial Denaturation: 95°C for 30 seconds.
   2. 35 cycles of
      1. Denaturation: 95°C for 30 seconds.
      2. Annealing: 62°C for 60 seconds (varies with primers).
      3. Extension: 68°C for 120 seconds.
   3. Final extension: 68°C for 5 minutes.
   4. Hold: 4°C indefinitely.
3. All thermal cycler conditions change with the DNA polymerase, primers, and template. After testing our primers with a low-fidelity enzyme master mix, we used a high-fidelity enzyme master mix to amplify our sequences. The high-fidelity enzyme had higher accuracy but lower processivity, so it needed longer extension times and higher temperatures.
   1. Use this site to calculate optimal annealing temperatures (<https://tmcalculator.neb.com/#!/main>).
4. PCR products were purified from reaction mixtures with PCR Cleanup Kit (Qiagen, 28104).

## Restriction Digeset

1. Add 300 ng DNA, 3 µL 10x Enzyme Buffer, 1 µL of each restriction enzyme (20,000 U/mL), and nuclease free water up to 30 µL total reaction volume.
   1. Use the NEBuffer Activity/Performance Chart with Restriction Enzymes to find the optimal buffer for the enzyme(s) being used, i.e. r2.1 for NdeI.
   2. Calculate volumes to add beforehand; add water first, then buffer, DNA, and lastly enzymes.
2. Let the reaction mixture incubate at 37°C for one hour, then store at -20°C until use.
   1. Ideally, digestion products should be used immediately to avoid star activity.

## DNA Gel Electrophoresis

1. Prepare gel tray by adding end gates via lab tape, using your nails to create a firm seal.
2. Create a 1% w/v agarose gel solution by adding 0.5 g agarose to 50 mL 1x TAE solution. Place in a lab microwave until the solution begins to boil (about 50 seconds). When the solution has cooled to room temperature, add 5 µL 10,000x GelRed (Biotium, 41003) and swirl to mix.
   1. For smaller DNA fragments (less than 1 kb), we used a 1.3% agarose gel for clearer band separation.
3. Pour agarose gel solution into gel tray. Add comb to create wells. Let cool until solidified (about 30 minutes).
   1. Check after 10 minutes that end gate tape seal is holding.
4. Remove tape seal from gel tray and place gel tray in the buffer chamber. Pour into buffer chamber enough running buffer solution (TAE) until top of gel is covered.
5. Prepare samples to load by spotting 1 µL 6x Purple Gel Loading Dye (New England Biolabs, B7024S) onto parafilm, pipetting 12 µL sample onto the spot, mixing, and pipetting 12 µL sample into a well.
   1. We initially followed manufacturer recommendation of 1 µL dye per 5 µL sample but found that our sample bands were still clear with less dye!
   2. We used 1Kb Plus DNA Ladder (Invitrogen, 10787018) diluted 1:4 with water as a DNA ladder in all our gels.
6. Run the gel by covering buffer chamber with lid such that the anode (red) (positive charge) is oriented at the bottom of the gel with the wells at the top. Turn on the power supply and set to constant voltage at 60 mV.
   1. A good indication that voltage is applied to the gel is a stream of bubbles appearing in the running buffer solution at the cathode side (black) (negative charge).
   2. Check back after 5 minutes to ensure that dye bands have moved from wells towards bottom of gel.
7. When the dye bands reach ¾ of the way towards the bottom, turn off power supply and remove gel from buffer chamber. Visualize DNA bands on a UV table with low intensity (high UV provides a clearer image but induces mutations faster), or image DNA bands on a gel imager, such as a Gel Doc EZ Imager (Bio-Rad).

## Ligation

1. Perform restriction digest on insert and vector as described above.
2. Use a DNA Cleanup Kit (Monarch, T1030S) to purify DNA from reaction.
3. Characterize concentration of digested fragments with NanoDrop (Thermo Scientific, 13-400-519).
4. Combine digested insert, digested vector, 2 µL 2x ligation buffer (New England Biolabs, B0202S), 1 µL DNA ligase (400,000 U/mL) (New England Biolabs, M0202S), and water up to 20 µL final volume in a 50 µL Eppendorf tube.

* Add pre-calculated volume of water first, then buffer, then DNA, then enzyme.
* Insert and vector should have been digested with the same pair of enzymes.
* Insert should be added in a 3:1 molar ratio to vector.

5. For example, if there is a 10 ng/µL digested insert solution and 30 ng/µL digested vector solution, and the insert and vector are 1 kb and 3 kb respectively, then 3 µL insert for every 1 µL vector.
6. Let reaction mixture incubate for 3 hours at 16°C in thermal cycler set to continuous temperature.
7. Directly transform competent cells with 5 ng DNA from ligation mixture.

* Competent cells with lower transformation efficiency will require more DNA to observe transformant colonies.

## Transformation of Chemically Competent Cells

1. Thaw competent cells on ice. When barely thawed, split cells into 25-50 µL volumes in 1.5 mL sterile tubes. Add 1 pg to 100 ng (< 5 µL) of plasmid DNA to the cells and mix by gently flicking 4-5 times.
2. Place the cells on ice for 30 minutes. Do not mix.
3. Heat shock the cells at 42°C for 45 seconds. Immediately place on ice for 2 minutes.
4. Add room-temperature SOC outgrowth medium (New England Biolabs, B9020S) to 1 mL total volume per tube. Incubate at 37°C and 250 RPM for 1 hour.
5. While incubating, warm antibiotic selection plates to 37°C.
6. Depending on the transformation efficiency of the cells, it may be necessary to dilute the cells in SOC. Using sterile glass beads, spread 50-200 µL of cells onto the warmed plates.
7. Incubate at 37°C for 16-20 hours.
8. Transformation efficiency is calculated as the number of distinct colonies plated per µg of DNA.

## Preparing Glycerol Stocks of Strains

1. Select colony from plate with pre-sterilized inoculator loop or flame-sterilized wooden dowel, and dissolve in 2 mL Luria-Bertani Medium (LB) to create an overnight culture.
   1. Add antibiotic if cell line contains a plasmid to prevent plasmid loss.
2. Let inoculated culture incubate in 37°C incubator shaking at 250 RPM for 12 hours or overnight.
3. Into a cryotube, pipette 1 mL overnight culture and 1 mL 50% glycerol. Label cryotube and place in –80°C freezer for storage.
   1. Final glycerol concentration can range from 15-50%. 25% is ideal for scraping for future use.

## Protein Expression

1. Start overnight culture of strain containing plasmid to be expressed in LB medium at 37°C.
2. In the morning (after ~12 hours), dilute overnight culture 1:10 by adding 400 µL overnight culture to 4 mL LB broth, as well as 1x final concentration antibiotic to which plasmid contains resistance.
3. Periodically check OD600 using spectrophotometer blanked with LB medium from the same bottle used to prepare the culture. When OD600 = 0.6, divide culture in half into separate culture tubes (2 mL each). Induce protein expression in one tube by adding 1 µL 0.8 M isopropyl thiogalactopyranoside (IPTG) for final concentration 0.4 mM IPTG.
   1. Our two plasmids use a T7-inducible promoter system, so we induce with IPTG. Different inducible promoter systems may use different molecules.
   2. The non-induced tube serves as a negative control.
4. After four hours, pellet cells by centrifuging at 13,000*g* for 1 minute and remove supernatant. Use in downstream applications.
   1. An inducer concentration, temperature, and induction period scan can be used to determine optimal protein expression. In separate experiments, vary the amount of inducer used, the temperature at which protein is expressed, and the amount of time for which protein is expressed. For example, we adapted our protocol from a different expression system and found that we could produce more protein by inducing with less IPTG at a lower temperature for longer periods of time. You can also try varying the OD600 at which you induce expression.

## Cell Fractionation

1. Dilute overnight culture 1:10 into 14 mL culture. When OD600 = 0.6, divide culture into 2 mL (negative control) and 12 mL portions. Induce protein expression at OD600 = 0.6.
2. Take 1 mL of cells from induced and non-induced cultures. Pellet each separately at 13,000*g* for 1 minute at room temperature. Remove supernatant and resuspend pellets in 30 µL 1% w/v SDS and 102 µL 2x Sample Buffer. Heat for 3 minutes in boiling water. Label tubes WC-I and WC+I, respectively, and store at –20°C.
   1. Use a vacuum or micropipette to aspirate off all supernatant.
3. Pellet 9 mL induced culture, remove supernatant, and resuspend in 1 mL Sonication Buffer.
4. Sonicate 3 x 15 seconds on ice with 20 second pause between sonication cycles. Power at 24 W. Microprobe used.
   1. Save 5 µL pre-sonicate to compare against sonicate with phase-contrast microscopy. Use this technique to guarantee cell lysis before continuing.
   2. Inclusion bodies will appear as clear circles that refract light. If your protein is present in inclusion bodies, you will need to modify the purification protocol from what is given here.
5. Create protein sample from sonicate by adding 75 µL sonicate to 25 µL 1% w/v SDS and 250 µL 2x Sample Buffer. Heat for 3 minutes in boiling water and label tube Son. When loading onto SDS-PAGE gel, use 2.38x the volume of this sample relative to volumes of samples prepared in other steps. Store at –20°C.
6. Centrifuge remaining sonicate at 4°C for 10 minutes at 13,000*g*. Remove 875 µL supernatant and keep on wet ice. Aspirate or pipet away remaining supernatant.
7. Create protein sample of supernatant by adding 25 µL supernatant to 25 µL 2x sample buffer and heating for 3 minutes in boiling water.
8. Create protein sample of pellet by resuspending it in 875 µL 0.3% w/v SDS, adding 875 µL 2x sample buffer, and boiling in water for 3 minutes.
9. Spin down the 875 µL saved supernatant in ultracentrifuge at 100,000*g* at 4°C for 15 minutes.
   1. Pre-chill the rotor. Use this site (<https://www.sciencegateway.org/tools/rotor.htm>) to calculate the correct RPM for your chosen rotor. For example, 100,000*g* in a TLA-120.1 Beckman rotor corresponds to 53,000 RPM.
   2. Don’t forget to use ultracentrifuge tubes that won’t shatter at high g-force.
10. Collect high speed supernatant and save for downstream application.
11. Create protein sample of high-speed supernatant by adding 25 µL high speed supernatant to 25 µL 2x sample buffer and boiling in water for 3 minutes.
12. Create protein sample of high-speed pellet by resuspending in 850 µL 2x sample buffer and adding 425 µL 0.3% w/v SDS. Boil in water for 3 minutes.
13. Run an SDS-PAGE gel of each protein sample made in this protocol. Monitor the presence of your expressed protein through each step and verify it is the portion that you use in downstream applications.

## SDS-PAGE

**Electrophoresis:**

1. Electrode buffer should completely cover the openings at the top and the bottom of the plates, in order to properly allow current to flow through the gel.
2. Load up to 20 µL of protein sample per well. For the colorimetric ladder, load less than 5 µL.
   1. For the ladder, we used Precision Plus Protein Dual Color Standards (Bio-Rad, 161-0374).
3. Start electrophoresis at 5 mA per gel.
4. When the protein has passed through the stacking gel, turn the current up to 15-18 mA per gel.
5. Wait until the dye front has passed all the way through the gel to allow for maximum separation of protein.

**Staining (skip if using gel for a Western blot):**

1. After opening the plates, carefully cut off the stacking gel with a razor and dispose of it. Use round-tipped tweezers to gently place the running gel in a small box or tray. To save staining and destaining solution, the bottom of the box should not be much larger than the gel.
2. Add enough Fairbanks I to cover the gel. Place on rocker overnight.
3. In the morning, replace Fairbanks I with Fairbanks II. Place on rocker for 6 hours.
4. Replace Fairbanks II with the destaining solution. Place on rocker for an hour.
5. Replace destaining solution with fresh destaining solution. Place on rocker for another hour.
6. Image.

**Note:** Acrylamide is a potent neurotoxin and potential carcinogen. Observe proper handling and disposal practices.

## Western Blot

**After SDS-PAGE:**

1. Assemble the cassette in the following order after soaking each piece in transfer buffer: sponge, filter paper x2, nitrocellulose membrane, SDS-PAGE gel, filter paper x2, sponge.
   1. The membrane should only be touched with tweezers to prevent unintentional transfer of protein from external sources.
2. Fill the electrophoresis box with transfer buffer. When placing the cassette in the box, the nitrocellulose should be on the cathode side (the protein is negatively charged).
3. In a cold room (4°C) with a stir bar in the electrophoresis box, run at 60 V for 4 hours.
4. Carefully remove the nitrocellulose membrane and place into a plastic box/tray. Add Ponceau S stain and place on rocker for 5 minutes. This will quickly show any protein on the membrane.
5. Remove the Ponceau S and use a squirt bottle with purified water to get off any excess stain.
6. Using a soft pencil, mark ladder and lanes. Cut away unnecessary membrane with a razor blade. Place the membrane into the smallest container it will fit into, to conserve reagents.

**For all wash steps: use enough solution to cover the membrane and place on rocker.**

7. Wash for 5 minutes with Tris buffered saline (TBS). Wash with 5% w/v instant milk (Carnation brand) for 30 minutes to block the membrane. Wash twice with Tris buffered saline with Tween 20 (TBST) for 5 minutes each.
8. Add primary antibody to 5 mL of TBST. Wash for 2 to 3 hours minimum (overnight is fine).
   1. Use manufacturer recommendations for antibody dilution.
9. Wash with TBS, then TBST twice, then TBS for 5 minutes each.
10. For every wash step from now on, cover in tinfoil to prevent photobleaching of the fluorophore conjugated to the secondary antibody.
11. Add secondary antibody to 5 mL of TBST. Wash for 30 minutes – more than 30 minutes risks having too bright a background.
    1. Use manufacturer recommendations for antibody dilution.
12. Wash with TBS, then TBST twice, then TBS for 5 minutes each.
13. Wash with TBS twice for 10 minutes each.
14. Image on Gel Doc EZ Imager with protein gel and correct stain selected.

## Affinity Purification

*(Adapted from Roche Ni NTA protocol [https://www.studocu.com/row/document/](https://www.studocu.com/row/document/universite-badji-mokhtar-de-annaba/biologie-moleculaire/roche-ni-nta-protocol-affinity-tag/39669072))*

1. In 4°C cold room, add 1 mL Ni-NTA resin slurry to the column. Equilibrate the column by adding 10 mL equilibration buffer and pipetting to mix with the beads. Wait 15 minutes for beads to settle back down to the bottom of the column.
2. Under gravity flow, apply sample to the column when equilibration buffer nearly reaches the resin bed. Make sure that the resin bed never runs dry.

1. Save a small portion of the sample as a control.
2. If sample has not been ultracentrifuged, it might clog the column.

3. After the sample has entered the resin, add 10 mL wash buffer. Begin collecting 2 mL fractions of effluent, starting with the flow through (obtained after adding sample to column but before adding wash buffer).
4. Elute nonspecifically bound proteins with 10 mL elution buffer 1.
5. Elute specifically bound protein with 10 mL elution buffer 2.
6. Elute all bound proteins with 10 mL elution buffer 3.

1. The elution buffer series contains an increasing concentration of imidazole, a molecule which replaces the His6 tag that binds the Ni2+ ions.

7. Run a SDS-PAGE gel of all fractions to identify fractions containing pure protein of interest.

1. You can optimize elution conditions for your specific protein by changing the concentration of imidazole in each elution buffer. For example, increase the imidazole concentration in elution buffer 2 if you notice there is protein of interest in elution buffer 3, or decrease the imidazole concentration in elution buffer 1 if you notice protein of interest is eluting too early in elution buffer 1.

8. Once protein of interest is confirmed in elution fraction, remove imidazole by dialysis.

1. Re-hydrate MWCO 1000 dialysis tubing (Spectrum Laboratories, Inc., 086802C) in dH2O for 15 minutes.
2. Add fraction containing protein of interest to tubing and remove air bubbles before clamping off tubing.
3. Place in 2 L of 5 mM Sodium Phosphate Buffer (pH 7), made by diluting 33.3 mL 0.6 M Sodium Phosphate Buffer (pH 7) in up to 2 L of dH2>O.
4. After 4 hours, replace solution with another 2 L 5 mM Sodium Phosphate Buffer pH 7.
5. After another 4 hours or overnight, remove sample from solution.

9. Snap freeze by adding 5% glycerol v/v and placing in liquid nitrogen. Store in –80°C.

## Disk Diffusion Assay

1. Prepare M16 + 5% glucose (pH 6) plates.
   * Include recipe for these plates.
   * Store plates at 4°C.
2. Prepare antibiotic disks by adding 10 µL solution to sterile disks (Becton, Dickinson and Co., 231039) and letting the disks dry in a 37°C incubator for 4 hours.
   * Use solvent as negative control.
   * Use 5 mg/mL kanamycin as positive control.
   * 5 mg nisin (Sigma Aldrich, N5704) was dissolved in 1 mL 50 mM Sodium Phosphate Buffer to prepare 5 mg/mL stocks.
3. Let plates dry for one hour in a 37°C incubator by placing them upside down with the agar plate itself placed at an angle in the dish, such that the lid is cracked open.
4. Prepare 1 mL of cell culture by dissolving scraped *M. luteus* colonies from a LB plate in 0.9% NaCl solution.
   * Measure the absorbance with a spectrophotometer and dilute the sample until OD600 = 0.2.
5. Pipette 800 µL of cell culture onto dried plates, use a sterilized spreader to distribute the liquid equally across the plate, and let the plate incubate right side up in a 37°C incubator for 2 hours or until the liquid has been absorbed.
6. Sterilize metal forceps with ethanol and flame before picking up antibiotic disks and placing them onto the plates. Let the plates incubate in a 37°C incubator for 24-48 hours, or until zones of inhibition can be measured.
7. Measure zones of inhibition with a ruler by taking three diameter measurements for each zone and averaging them.
   * If no zone of inhibition forms, record value as 0 cm and not the diameter of the disk itself.

## Franz Diffusion Assay

1. Allow porcine skin (Stellen Medical L LLC, Fisher Scientific, NC1275387) to thaw at room temperature for 30 minutes.
2. Cut a 2.5 x 2.5 cm2 skin sample using a sterile scalpel.
3. Measure skin resistivity using a multimeter

1. Place one electrical probe on the stratum corneum side of the skin.
2. Place the other electrical probe on the dermal side of the skin.
3. Resistivity values less than 187.5 kΩ (30 kΩ/cm2) were considered unfit for further experimentation.

4. Place a stir bar in the receptor compartment of the Franz Diffusion cell (Figure 2).
5. Stretch the skin over top of the receptor compartment ensuring there is little to no folding on the surface of the skin. Then, place the donor compartment over the skin and clamp together.
6. Load 9 mL of Phosphate-Buffered Saline (PBS) (pH 7.4) ensuring that no air bubbles form underneath the surface of the skin.

1. Tip: Tilt the clamped Franz Diffusion cell such that the sampling port is pointing upwards.

7. Place a dish with stir bar and water on a stir plate in an incubator.
8. Turn on stir plate and allow temperature of the water to increase to 37°C.
9. Place the Franz Diffusion cell, fitted with porcine skin and PBS, in the dish in the incubator.
10. Wait 30 minutes to allow temperatures to increase to 37°C.
11. Apply 1 mL of donor solution to the donor compartment.
12. Take 20 µL samples from sampling port at regular intervals for 24 hours.
13. Measure the absorbance of the sample at 274 nm.
14. Determine concentration of receptor compartment using known absorbance values.
15. Replace the extracted volume with PBS, once again ensuring that no air bubbles form underneath the surface of the skin.

![Franz diffusion cell in water bath](https://static.igem.wiki/teams/4853/wiki/figure-3.png)

**Figure 3**. Franz diffusion cell components. (1) Sampling port, where samples are extracted from the receptor compartment. (2) Porcine skin, serves as the membrane through which permeable substances move between the donor and receptor compartments. (3) Donor compartment, where the donor solution is applied. (4) Clamp, used to hold the skin in place between donor and receptor compartments. (5) Receptor compartment, where substances that permeate the membrane are collected. (6) Stir bar, used to ensure that the receptor compartment concentration is uniform.

![Franz diffusion cell in water bath](https://static.igem.wiki/teams/4853/wiki/pages-design/experiments/figure-4.png)

**Figure 4**. Experimental setup of the Franz diffusion cell. Placed in a water bath on a stir plate inside an incubator. Cork used to keep the cell upright during the experiment.

## Buffers, Reagents, and Other Materials

**50x Running Buffer (50x TAE)**

In a beaker dissolve 242 g Tris base in 500 mL milliQ water, add 57.1 mL glacial acetic acid and 100 mL 0.5 M EDTA pH 8, transfer to graduated cylinder and add milliQ water until final volume is 1 L. Dilute in milliQ water for 1x working solution. The 1x working solution is 40 mM Tris/acetate and 1 mM EDTA.
 *Adapted from Cold Spring Harbor (<http://m.cshprotocols.cshlp.org/content/2006/1/pdb.rec8644.full>).*

**Luria-Bertani Medium**

To 950 mL of dH2O, add: 10 g tryptone, 5 g yeast extract, 10 g NaCl. Shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (~0.2 mL). Adjust the volume of the solution to 1 L with d H2O. Sterilize by autoclaving for 20 min at 15 psi on liquid cycl (From Sambrook and Russel. *Molecular Cloning: A Laboratory Manual*, 3rd e.d., Volume 3).

**Fairbanks I**

* 10% acetic acid
* 25% isopropanol
* 0.05% w/v Coomassie Blue R (0.5g/L)
Stir for 24 hours before use.
**Fairbanks II**

* 10% acetic acid
* 25% isopropanol
* 0.005% w/v Coomassie Blue R (0.05g/L)
Stir for 24 hours before use.
**Destaining solution**

* 10% v/v glacial acetic acid
* 20% v/v methanol

**2x Sample Buffer**

* 2.5 mL 0.5 M Tris (pH 6.8)
* 2.0 mL 10% w/v sodium dodecyl sulfate
* 2.0 mL glycerol
* 1.0 mL water
* 1.0 mL β-mercaptoethanol
* 1.5 mL 0.1% bromophenol blue

**Electrode Buffer**

In 900 mL dH2O, dissolve 144 g glycine, 30 g Tris base, and 5 g SDS. Add dH2O up to 1 L.

**Transfer Buffer**

In 700 mL dH2O, dissolve 3.03 g Tris base and 14.4 g glycine. Add dH2O up to 800 mL. Add 200 mL methanol for final concentration 20% v/v and final volume 1 L.

**Sonication Buffer**

* 10 mM Sodium Phosphate Buffer pH 7
* 1 mM phenylmethylsulphonyl fluoride (PMSF)

Make a 100 mM stock of PMSF in ethanol. Store at 4°C.
Make Sonication Buffer fresh for each fractionation. To make 5 mL Sonication Buffer, add 83.3 µL 0.6 M sodium phosphate buffer pH 7 and 50 µL 100 mM PMSF to 4.867 mL milliQ water.

**Equilibration Buffer**

* 20 mM Tris
* 200 mM NaCl
* pH 7.5

**Wash Buffer**

* 20 mM Tris
* 200 mM NaCl
* 5 mM imidazole
* pH 7.5

**Elution Buffer 1**

* 20 mM Tris
* 200 mM NaCl
* 20 mM imidazole
* pH 7.5

**Elution Buffer 2**

* 20 mM Tris
* 200 mM NaCl
* 200 mM imidazole
* pH 7.5

**Elution Buffer 3**

* 20 mM Tris
* 200 mM NaCl
* 500 mM imidazole
* pH 7.5

**Tris-Buffered saline**

Dissolve 8 g of NaCl, 0.2 g KCl, and 3 g of Tris base in 800 mL of dH2O. Add 0.015 g of phenol red and adjust the pH to 7.4 with HCl. Add dH2O to 1 L. Sterilize by autoclaving for 20 minutes at 15 psi on liquid cycle. Store buffer at room temperature. (From Sambrook & Russell. *Molecular Cloning: A Laboratory Manual*, 3rd e.d., Volume 3). To create Tris-Buffered Saline with Tween 20 (TBST), add 0.01% v/v Tween 20 to TBS.

**Ponceau S Stain**

* 0.2% w/v Ponceau S
* 3% v/v Trichloroacetic acid
* 3% w/v sulfosalicylic acid

**0.6 M Sodium Phosphate Buffer (pH 7)**

Prepare 1 M Dibasic Sodium Phosphate and 1 M Monobasic Sodium Phosphate solutions. To create Buffer of pH 7, add 57.7% Dibasic Sodium Phosphate v/v and 42.3% v/v Monobasic Sodium Phosphate. Dilute with dH2O to create lower concentration buffers.

**M16 + 5% glucose Agar Plates (pH 6)**

In a 2 L Erlenmeyer flask, dissolve 5 g Pancreatic Digest of Casein, 5 g Soy Peptone, 5 g Beef Extract, 2.5 g Yeast Extract, 0.5 g Ascorbic Acid, 0.25 g MgSO4, 0.25 g Disodium-B-glycerophosphate, and 11 g Agar in dH2O up to final volume 861 mL. Adjust pH to 6 with HCl. Heat with stirring and boil for 1 minute to completely dissolve reagents. Autoclave the solution for 20 minutes at 15 psi on liquid cycle. When solution has cooled to 50°C, add 50 mL sterile 10% lactose solution and 89 mL sterile 25% w/v glucose. Pour solution into plates and let solidify overnight. Store upside down at 4°C. (Adapted from *Difco and BBL Manual of Microbiological Culture Media*, 2nd e.d.).

**Phosphate-Buffered Saline (PBS)**

Dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2PO4, and 0.24 g of KH2PO4 in 800 mL of dH2O. Adjust the pH to 7.4 with HCl. Add dH2O to 1 L. Sterilize by autoclaving for 20 minutes at 15 psi on liquid cycle or by filter sterilization. Store the buffer at room temperature. (From Sambrook & Russell. *Molecular Cloning: A Laboratory Manual*, 3rd e.d., Volume 3).

# Reference

1. Lubelski, J., Rink, R., Khusainov, R., Moll, G. N., & Kuipers, O. P. (2008). Biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin. Cellular and Molecular Life Sciences, 65(3), 455–476. https://doi.org/10.1007/s00018-007-7171-2
2. Kumar, S., Zakrewsky, M., Chen, M., Menegatti, S., Muraski, J., Mitragotri, S. Peptides as skin penetration enhancers. *Journal of Controlled Release* 2015, 199, 168-178. <https://doi.org/10.1016/j.jconrel.2014.12.006>
3. Novagen User Protocol TB340 Rev. F 0211JN. 2011.
4. Okuda, K., Zendo, T., Sugimoto, S., Iwase, T., Tajima, A., Yamada, S., Sonomoto, K., & Mizunoe, Y. (2013). Effects of bacteriocins on methicillin-resistant *Staphylococcus aureus* biofilm. Antimicrobial agents and chemotherapy, 57(11), 5572–5579. <https://doi.org/10.1128/AAC.00888-13>
5. Yehia, H. M., Alkhuriji, A. F., Savvaidis, I., & Al-MASOUD, A. H. (2022). Bactericidal effect of nisin and reuterin on methicillin-resistant *Staphylococcus aureus* (MRSA) and *S. aureus* ATCC 25937. Food Science and Technology, 42, e105321. <https://doi.org/10.1590/fst.105321>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

* [Cloning](#topic1)
* [Protein Expression](#topic2)
* [Purification](#topic3)
* [Activity and Permeation](#topic4)
* [References](#topic5)

# Cloning

We first cloned *nisB* into the pRSFDuet-1 vector using standard cloning techniques, digested with the restriction enzymes NdeI and XhoI. Gel electrophoresis, using highly purified low melting temperature agarose (BioWhittaker Molecular Applications, 50070), was used to isolate the desired fragments. An *in agaro* ligation generated a new plasmid. The ligation reaction mixture was directly transformed into *E. coli* DH5α cells for amplification. Plasmid was recovered by miniprep (Qiagen, 27106). Successful insertion was verified with restriction site mapping. This pRSFDuet-1-*nisB* plasmid became the foundation for five more plasmids, each containing a variation of *nisA*.

Alleles of *nisA* were inserted into pRSFDuet-1-*nisB* as described above, except restriction enzymes BamHI and HindIII were used for digestion. All five *nisA* alleles are approximately the same size; length ranges from 200 to 250 base pairs. Furthermore, they were all inserted into the same site on pRSFDuet-1-*nisB*. *nisC* was inserted into pACYCDuet-1 with restriction enzymes NdeI and XhoI. Figures 1 and 2 show the restriction site mapping used to verify insertion for the six plasmids necessary to our project: the five pRSFDuet1-*nisA*-*nisB* constructs and pACYCDuet-1-*nisC*. The backbone vectors pRSFDuet-1 and pACYCDuet-1 were used as negative controls.

![pACYCDuet-1-<i>nisC</i>](https://static.igem.wiki/teams/4853/wiki/results/figure-1-1.png)

**Figure 1**. Restriction digest verification of pACYCDuet-1-*nisC*. pACYCDuet-1 and pACYCDuet-1-*nisC* were digested with SspI. **A** 1% agarose gel with digested plasmids. **B** Plasmid maps with SspI cut sites are shown.

![pRSFDuet-1](https://static.igem.wiki/teams/4853/wiki/results/figure-2-2.png)

**Figure 2**. Restriction digest verification of five plasmids containing *nisB* and a *nisA* allele. **A** 1.3% agarose gel in which pRSFDuet-1 and each *nisA* plasmid were digested with BamHI and HindIII. Gamma = 0.4. Excised *nisA* bands are indicated with a red arrow. TL = truncated leader. **B** Plasmid maps with BamHI and HindIII cut sites are shown.

The experimental data matched our predictions for fragment size, suggesting successful plasmid creation. SspI cuts twice in pACYCDuet-1, generating fragments of 1727 and 2281 bp; SspI cuts three times pACYCDuet-1-*nisC*, generating fragments of 1405, 1727, and 2079 bp. Digesting with BamHI and HindIII generates fragments of ~200 and 6700 bp in pRSFDuet-1-*nisA*-*nisB* constructs and of 37 and 3792 bp in pRSFDuet-1. We further verified plasmid creation by outsourcing sequencing. We additionally made five more plasmids in DH5α strains by inserting the five *nisA* variants into pRSFDuet-1 directly, without *nisB*. We anticipated that removing *nisB* as another variable in protein expression might aid future troubleshooting.

Altogether, we made 31 different strains: 14 DH5α and 17 BL21(DE3), five of which were double transformants. Table 1 lists them by strain number and genotype. Transformation was verified in every strain by restriction site mapping at a minimum.

### Table 1. List of Strains

![table of strains](https://static.igem.wiki/teams/4853/wiki/results/results-table1-1.png)

# Protein Expression

![protein expression](https://static.igem.wiki/teams/4853/wiki/results/figure-3-1.png)

**Figure 3**. NisA protein samples (induced with 0.4 mM IPTG at 37°C) compared to NisA, NisB, and NisC protein samples (induced with 0.4 mM IPTG at 18°C). All samples were expressed in *E. coli* BL21(DE3). **A** Coomassie-Blue stained 8-18% Tris-glycine SDS-PAGE. Bands of interest are marked with red asterisks. **B** Western blot on a nitrocellulose membrane with anti-His6 primary antibodies.

With our devices made, we next verified that the two-plasmid system was able to express NisA. Figure 3A shows an SDS-PAGE gel of induced cultures of cells with one of the *nisA* alleles and the *nisB* and *nisC* genes, alongside induced cultures of cells containing only the *nisA* alleles. The T7 promoter shows leaky expression when non-induced, so non-transformed *E. coli* BL21(DE3) cells serve as a negative control instead of non-induced cells with the genes. The predicted molecular weights of the five NisA variants are shown in Table 2. These proteins migrate anomalously slowly through an SDS-PAGE gel, appearing at a higher molecular weight (11-12 kDa) than their predicted molecular weights. These bands are visible on the corresponding Western blot (Figure 3). This anomalous migration of NisA is consistent with previous data from Chen and Kuipers (2021) [1].

### Table 2. Predicted Molecular Weights of NisA Variants

![table of weights](https://static.igem.wiki/teams/4853/wiki/table2.png)

The predicted molecular weight of NisB is 118 kDa, while the predicted molecular weight of NisC is 48 kDa. Bands at this weight can be seen on an SDS-PAGE of protein samples of induced cultures of BL21(DE3) cells containing the *nisA*, *nisB*, and *nisC* genes (Figure 3A). Corresponding bands can be seen on a Western blot using anti-His6 antibodies for the same samples, along with a distinct band at 75 kDa and several low-intensity bands in between those three bands (Figure 3B). However, only the NisA variants have a His6 tag, suggesting some alteration to the migration of each of the NisA proteins through an SDS-PAGE gel is due to interactions between NisA and the post-translational modifiers NisB and NisC.

One possible explanation of the reduced mobility through an SDS-PAGE gel is the post-translational modifications themselves: NisC creates rings in the NisA primary structure through the five thioether bonds, creating a condensed secondary structure in NisA (see Experiments Figure 1D for active nisin structure). Another possible explanation is incomplete post-translational modification: during the enzymatic reactions catalyzed by NisB and NisC, transient covalent bonds are formed between NisA and the active site of each enzyme. Incomplete reactions could result in denaturation-stable NisA-NisB and NisA-NisC complexes that appear near the predicted molecular weights of NisB and NisC (Figure 3B). It is also possible that some NisA dissociates from NisB or NisC before the enzymatic reactions are complete. Eight serines and threonines are dehydrated by NisB and five thioether bonds are created by NisC; if less than eight of the residues are dehydrated or partially dehydrated, or if fewer than five thioether bonds are created, it would create variability in the migration of modified NisA through an SDS-PAGE gel. The distinct band that appears at 75 kDa may be NisA that has been fully modified by NisB and NisC, while the several bands of low intensity between the 48 kDa and 118 kDa bands may be partially modified NisA.

While the exact cause of the retarded migration of NisA through an SDS-PAGE gel in the presence of NisB and NisC is unknown, all five variants of NisA are affected similarly. NisB and NisC recognize the leader peptide of NisA, but it appears that the addition of TD-1, both on the N-terminus of the leader and between the leader and the nisin precursor, does not affect the specificity of NisB or NisC for NisA.

# Purification

We first determined if NisA is a soluble or insoluble protein by fractionating IPTG-induced culture of BL21(DE3) cells containing a pRSFDuet-1-*nisA* plasmid. Figure 4 shows an SDS-PAGE gel with protein samples from each step of fractionation. We concluded that both soluble and insoluble forms of NisA exist from its presence in the pellet and supernatant. NisA being partially insoluble was further supported by the observation of inclusion bodies in the sonicate, under phase-contrast microscopy.

![purification1](https://static.igem.wiki/teams/4853/wiki/results/results-fig4.png)

**Figure 4**. Monitoring effectiveness of cell fractionate with Coomassie Blue-stained 8-18% Tris-glycine SDS-PAGE gel. With BL21(DE3) containing pRSFDuet-1-*nisA* culture, expression was induced with 0.4 mM IPTG for 4 hours at 37°C (Lane 1). Half the culture was not induced (Lane 2). Cells harvested from induced culture were sonicated (Lane 3) and then centrifuged at 13,000*g*. Protein samples were made of medium-speed supernatant (Lane 4) and medium-speed pellet (Lane 5). NisA bands are labeled with a red arrow.

Because NisA is partially soluble, we moved forward with immobilized metal affinity chromatography to purify unmodified soluble NisA. To prevent column clogging, the medium speed supernatant was centrifuged at 100,000*g* and the high-speed supernatant collected. High-speed supernatant was applied to nickel-nitrilotriacetic acid beads and fractions were collected from the flow through, wash, and three elution fractions.

![purification2](https://static.igem.wiki/teams/4853/wiki/results/results-fig5.png)

**Figure 5**. Coomassie Blue-stained 8-18% Tris-glycine SDS-PAGE gel of elution fractions after purifying unmodified NisA from high-speed supernatant of fractionation on Nickel-NTA column. Bands of interest are marked with a red asterisk. MW of ladder bands have been labeled in kD.

Figure 5 shows an SDS-PAGE gel of protein samples made from each fraction. The solitary bands in the Elution Buffer 2 fractions are of the predicted molecular weight of unmodified NisA. We pooled the eluate fractions containing pure unmodified NisA and characterized them with a BCA Pierce Kit at 20 µg/mL.

We have not yet purified the modified NisA proteins from a BL21(DE3) strain containing the *nisA*, *nisB*, and *nisC* genes. Early attempts showed that the protocols for induction and fractionation needed to be modified to accommodate the metabolic burden of NisB and NisC being produced alongside NisA.

# Activity and Permeation

Once modified NisA is produced, we need a method to verify its antimicrobial properties. A disk diffusion assay was adapted for this need, but first this assay had to be optimized for nisin. We found that *E. coli* was an inadequate positive control strain for nisin because the antibiotic is only active against Gram-positive bacteria. Thus, we changed to *M. luteus* for plating. *M. luteus* does not grow optimally on LB agar, so we replaced the LB agar plates with M16 + 5% glucose plates. We found that nisin poorly diffuses through agar; zones of inhibition were significantly smaller for nisin than kanamycin at equivalent concentrations (Figure 6). To increase the limit of detection of active nisin for our assay, we supplemented our nisin stock with 0.01% Tween 20. We posited that the non-ionic detergent would better solubilize the amphipathic nisin in the agar. Figure 6 shows the culmination of these efforts. Adding Tween 20 greatly increases nisin’s zone of inhibition without demonstrating antimicrobial activity itself.

![disk diffusion](https://static.igem.wiki/teams/4853/wiki/results/results-fig6-2.png)

**Figure 6**. Disk diffusion asay of nisin on M16 + 5% glucose agar plate covered in *M. luteus*. Disks were loaded with 10 µL solution. Kanamycin at 5 mg/mL was used as a positive control. Nisin was dissolved at 5 mg/mL in a Sodium Phosphate Buffer pH 6.0, used as a negative control (Buffer). Tween 20 was added to labeled samples to final concentration 0.01% v/v. Zones of inhibition are labeled in cm.

When we purify modified NisA variants, we will add Tween 20 to raise the detection threshold for antimicrobial activity. We also plan to change the strain used in plating from *M. luteus* to S. aureus to better model our device’s efficacy.

To test permeation, we utilized the Franz diffusion assay. Before using the assay with nisin, its reliability must be tested with a positive control to establish a functional assay for the experimental variants. Caffeine was chosen as a positive control based on literature [2]. Caffeine absorbs light with a peak at 274 nm, so we first plotted the absorbance of caffeine at 274 nm against its concentration to calculate an extinction coefficient. Figure 7 shows that our line of best fit for this data demonstrates strong linearity.

![caffeine absorbance](https://static.igem.wiki/teams/4853/wiki/caff.png)

**Figure 7**. Calculating the extinction coefficient of caffeine. Concentrations were prepared in triplicate. The error bars shown represent one standard deviation from the mean. Absorbances were measured with a NanoDrop Spectrophotometer.

Next, the permeation of caffeine across skin was measured. Figure 8 shows that caffeine did accumulate over time in the receptor compartment, suggesting permeation through the skin. After 6 hours, 74.4% of the caffeine had diffused through the skin. We applied a linear fit to the curve according to diffusion equations generated with modeling; the data showed a strong fit. Our next step will be to repeat this experiment to validate the results.

![caffine over time](https://static.igem.wiki/teams/4853/wiki/perm.png)

**Figure 8**. Cumulative mass of permeated caffeine over time. 1 mL of caffeine at 250 µg/mL in PBS was loaded into the donor compartment of the Franz diffusion cell. 20 µL samples of the receptor compartment were taken at 1.5, 2, 4, and 6 hours. The absorbances of the samples were measured and the linear model was used to calculate the caffeine concentration of the samples. To calculate total permeated caffeine, the concentration was multiplied by the total volume of the receptor compartment, 9 mL.

Once we have purified modified NisA from our device, we will use this functional assay to measure permeation. To measure the concentration of NisA in the receptor compartment, we will switch from a spectrophotometer to a BCA Pierce Protein Concentration Kit. The four relevant experimentals are V8-NisA, TL-TD1-V8-NisA, TD1-V8-NisA, and TD1-linker-V8-NisA. We predict that the addition of TD-1 will increase permeation through the skin.

# References

1. Chen, J., & Kuipers, O. P. (2021). Isolation and Analysis of the Nisin Biosynthesis Complex NisBTC: Further Insights into Their Cooperative Action. mBio, 12(5), e02585-21. https://doi.org/10.1128/mBio.02585-21
2. van de Sandt, J. J., van Burgsteden, J. A., Cage, S., Carmichael, P. L., Dick, I., Kenyon, S., Korinth, G., Larese, F., Limasset, J. C., Maas, W. J., Montomoli, L., Nielsen, J. B., Payan, J. P., Robinson, E., Sartorelli, P., Schaller, K. H., Wilkinson, S. C., & Williams, F. M. (2004). In vitro predictions of skin absorption of caffeine, testosterone, and benzoic acid: a multi-centre comparison study. Regulatory toxicology and pharmacology : RTP, 39(3), 271–281. https://doi.org/10.1016/j.yrtph.2004.02.004

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