iGEM 2021 — Team Edinburgh

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# Team:Edinburgh/Notebook

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[Human Practices](https://2021.igem.org/Team%3AEdinburgh/Human_Practices)
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# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Team:Edinburgh/Engineering

The SuperGrinder

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  + [Stakeholder Engagement](https://2021.igem.org/Team%3AEdinburgh/Human_Practices/Stakeholder)
  + [Entrepreneurship](https://2021.igem.org/Team%3AEdinburgh/Human_Practices/Entrepreneurship)
  + [Sustainable](https://2021.igem.org/Team%3AEdinburgh/Sustainable)
* [VDO](https://video.igem.org/w/fncqg3gMmSeSZpALRQ3zNC)

[Engineering](#backtop)
[Design (1)](#d1)
[Built-Test (1)](#bt1)
[Learn (1)](#l1)
[Design (2)](#d2)
[Built (2)](#b2)
[Test-Learn (2)](#tl2)

# Engineering: Cell free biosynthesis of glutathione with ATP-regenerating system - poultry waste upcycle

## Introduction:

Glutathione (GSH) is a tripeptide consisting of glycine, L-glutamic acid and L- cysteine. The versatility of GSH to the human body contributes to its vast application in the pharmaceutical, food and cosmetics industries, leading to the anticipated market value of US$ 198.2 million by 2027 [1].

In regarding to one of the recalcitrant waste The SuperGrinder is focusing, the poultry waste. The mixture of amino acids from chicken feathers were reported as rich in glycine, glutamic acid and cysteine, which were the precursors of GSH [2]. Therefore the degraded keratin can potentially apply in the glutathione synthesis platform to upcycle this resource.

![](https://static.igem.org/mediawiki/2021/e/e4/T--Edinburgh--eng1.png)

**Figure 1.**Scheme showing glutathione production via (i) GshA and GshB, (ii) GshF.

With the simple composition of GSH, the biosynthesis is simply done by a single bifunctional enzyme, gshF (bifunctional glutathione synthesis enzyme) (Figure 1). However, the bottleneck of the process is the ATP supply which is also the main factor causing the costly production. For this reason, this part of the SuperGrinder looks into the establishment of a glutathione bioproduction platform, furnished with ATP-regeneration in the cell-free system (Figure 2).

![](https://static.igem.org/mediawiki/2021/a/ab/T--Edinburgh--eng2.png)

**Figure 2.**Multiple enzyme ATP regeneration with glutathione production introduced in this study (GCS = g-L-glutamyl-L-cysteine, GSH = glutathione).

1. ## 1. Design:

![](https://static.igem.org/mediawiki/2021/0/01/T--Edinburgh--eng3.png)

**Figure 3.**Graphical overview of computational analysis pipeline of this study. (A) The pathway analysis pipeline of both MDF and ECM analysis. (B) GSH-ATP simulation and optimization pipeline.

The cell-free system was designed as depicted in figure 2. However, before deciding to go forward with these enzymes, the pathway was analysed by Equilibrator [3] to observe the feasibility and potential bottleneck of the whole cascade, along with the free energy analysis under physiological conditions. The result indicates that the overall pathway is feasible but acetate kinase (AckA) is the limiting step (Figure 4).

![](https://static.igem.org/mediawiki/2021/4/40/T--Edinburgh--eng4.png)

**Figure 4.**The pathway analysis results from Equilibrator. R000000 and R000001 are gshF reaction, R000002 is the pyruvate oxidase (pox5) and R000003 is the acetate kinase (ackA)

1. ## 2. Built - Test

The GSH-ATP system is then simulated via the kinetic modelling disregarding the physiological condition and using the parameters found from literature search - in vitro condition (Table 1). The simulation applied the Ordinary Differential Equations (ODEs) to create an exploratory model by speculating the system progression and the experimental result [4] (Figure 5).

**Table 1.**Parameters, mechanism and source of organisms used for GSH-ATP modelling

![](https://static.igem.org/mediawiki/2021/1/1d/T--Edinburgh--eng5.png)

![](https://static.igem.org/mediawiki/2021/8/80/T--Edinburgh--eng5_.png)

**Figure 5.**The GSH-ATP system simulated for 300 minutes, 50 timesteps. (A) The overall plot showing all species involved, including intermediates (glu = L- glutamic acid, cys = L-cysteine, gly = glycine, gcs = g-L-glutamyl-L-cysteine, gsh = reduced glutathione, atp = ATP, adp = ADP, pyr = pyruvate, Ppi = orthophosphate, acp = acetyl phosphate, co2 = CO2, h2o2 = H2O2, ace = acetate). (B-C) are subplots of (A) where species involved in GSH production (B) and species in ATP-regeneration (C) were displayed.

1. ## 3. Learn

The exploratory model allows an investigation of each species corresponding to the initial concentration. Observing the model after simulating several initial values and varying limiting agents, we found that pyruvate supply is the limiting substrate of the overall pathway. Additionally, altering parameters like Vmax of each enzyme is also affecting the system differently. The Vmax of enzymes in the pathway is vastly different in magnitude which implies that some enzymes are more active than others. Therefore, the question of balanced enzyme concentration was brought up. Since enzymes are one of the main costs in cell-free production, finding the minimum enzyme needed for the maximum production will make the pathway substantially efficient.

1. ## 4. Design

The next design iteration is based on the prediction of enzyme ratio needed for the maximum titer of GSH. The fabricated model and SciPy (45) library were used to further predict the character of the system under various enzyme levels. For this experiment, the aim is to do concave maximisation, which is simply to search for the optimum Vmax, that can be assumed relevant to the *in vitro* enzyme level, for the maximum glutathione production. However, using the SciPy library, only the minimise function was available. So, the model was transposed into a quasiconvex function by returning the highest negative glutathione flux and report enzyme level at the point.

![](https://static.igem.org/mediawiki/2021/5/5d/T--Edinburgh--eng6.png)

**Figure 6.**Global optimisation heatmap arranges along the x-axis by the descending summation of Vmax. The ODE was set to compute at time 30 minutes where the system reached equilibrium.

The GshF low-level requirement was recommended from the global optimisation (Figure 6). On average, GSH was produced at 10.993 ± 0.005 mM and the minimum total Vmax discovered were 11.62, 725.69 and 1621.17 mM/min of GshF, Pox5 and AckA, respectively. Various Vmax of AckA and Pox5 indicate a low sensitivity to the pathway to some extent. The ratio between VmaxPox5/ VmaxAckA is 1.43 ± 0.9.

1. ## 5. Built

The GSH-ATP system is then simulated via the kinetic modelling again with the initial condition similar to that being used in practice and applying the amount of enzyme as the Vmax suggested.

![](https://static.igem.org/mediawiki/2021/f/f5/T--Edinburgh--eng7.png)

**Figure 7.**The GSH-ATP system simulated for 60 minutes, 50 timesteps.

1. ## 6. Test-Learn

The predicted condition was demonstrated experimentally using the exact initial concentration of substrate and incubation time as found in the in silico model. However, due to the time constraints of this project, enzymes applied in the experiment are from the clarified cell lysates but not the purified enzyme as predicted.

The conclusion of whether the system regenerates ATP cannot be clear cut because reaction 8, another positive control, is only giving 0.43% yield (calculated from theoretical product titre predicted from the model). Therefore, GSH production in sample 6, which contain lower ATP, might be hindered by the detection limit. Interestingly, sample 7 with higher pyruvate is showing a slightly higher GSH. Even though the GSH amount in reactions 6 and 7 is not significantly different, this unidentical result could possibly indicate ATP regeneration, which is limited by pyruvate. The low level of GSH obtained is possibly caused by the endogenous enzyme. Therefore, the experiment should be perform again with either an excessive amount of pyruvate or a more purified enzyme.

![](https://static.igem.org/mediawiki/2021/1/11/T--Edinburgh--eng8.png)

**Figure 8.**GSH titer from GSH production-coupled ATP regeneration system. Refer to the table for the composition in each setting. Reactions 1, 2, 3, and 5 are Negative control are reactions 1, 2, 3 and 5, while reactions 4 and 8 are positive controls. Reaction 6 demonstrate the system with moderate pyruvate while reaction 7 was supplied with a high concentration of pyruvate [available, available in high level, not available/negative control]. The error bar shows the standard deviation of duplicated GSH detection noted that reactions 1-3 are single replicate.

*References:*

[1] Acumen Research and Consulting. Glutathione Market Value Anticipated To Reach US$ 198.2 Million By 2027: Acumen Research And Consulting. 2021; [online]; IntradoGlobeNewswire. [Updated 2021 Mar 11, Cited 2021 Aug 17]. Available from: https://www.globenewswire.com/news-release/2021/03/11/2191543/0/en/Glutathione-Market-Value-Anticipated-To-Reach-US-198-2-Million-By-2027-Acumen-Research-And-Consulting.html

[2] Taskin M. A new strategy for improved glutathione production from Saccharomyces cerevisiae: use of cysteine- and glycine-rich chicken feather protein hydrolysate as a new cheap substrate. J Sci Food Agric. 2013;93(3):535-41.

[3] Flamholz A, Noor E, Bar-Even A, Milo R. eQuilibrator--the biochemical thermodynamics calculator. Nucleic Acids Res. 2012;40(Database issue):D770-5.

[4] Lewis DD, Villarreal FD, Wu F, Tan C. Synthetic biology outside the cell: linking computational tools to cell-free systems. Front Bioeng Biotechnol. 2014;2:66.

[5] Janowiak BE, Griffith OW. Glutathione synthesis in Streptococcus agalactiae. One protein accounts for gamma-glutamylcysteine synthetase and glutathione synthetase activities. J Biol Chem. 2005;280(12):11829-39.

[6] Pratumsuwan S. In silico design and optimisation of a metabolic pathway for cell-free protein synthesis. University of Edinburgh; 2021 May 20.

[7] Singh R, Wiseman B, Deemagarn T, Jha V, Switala J, Loewen PC. Comparative study of catalase-peroxidases (KatGs). Arch Biochem Biophys. 2008;471(2):207-14.

[8] Cornish-Bowden A. Fundamentals of enzyme kinetics. 4th completely rev. and greatly enl. ed. Weinheim: Wiley-VCH; 2012.

[9] Tittmann K, Wille G, Golbik R, Weidner A, Ghisla S, Hubner G. Radical phosphate transfer mechanism for the thiamin diphosphate- and FAD-dependent pyruvate oxidase from Lactobacillus plantarum. Kinetic coupling of intercofactor electron transfer with phosphate transfer to acetyl-thiamin diphosphate via a transient FAD semiquinone/hydroxyethyl-ThDP radical pair. Biochemistry. 2005;44(40):13291- 303.

[10] Skarstedt MT, Silverstein E. Escherichia coli acetate kinase mechanism studied by net initial rate, equilibrium, and independent isotopic exchange kinetics. J Biol Chem. 1976;251(21):6775-83.

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# Team:Edinburgh/Experiments

The SuperGrinder

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* [VDO](https://video.igem.org/w/fncqg3gMmSeSZpALRQ3zNC)

[Experiments](#backtop)

**PROTOCOLS**

[Cloning and Transformation](#clone)
[Protein expression and Quatification](#prot)
[Enzymatic assays](#enz)

# **Experiments**

## Overall experimental plan:

1. Assembly Design

2. Molecular Cloning (JUMP assembly)

3. Enzymes overexpression

4. Explore enzymes to degrade and upcycle recalcitrant waste

    a. Chicken feather degradation: Keratinase production and immobilisation

    b. Chicken feather upcycle: Glutathione production with ATP-regenerating system

    c. Crustaceans wastes upcycle: Chitin extraction

    d.Biomass degradation: Cellulase directed evolution, Cellulase immobilisation

    e. PET plastic degradation: PETase expression and immobilisation

5. Enzymatic assay

6. Protein immobilisation (sfGFP)

7. Protein stability test after immobilisation

8. Enzyme immobilisation

9. Enzyme stability test and activity assay

## **Protocols**

### **Cloning and Transformation:**

**Chemically competent cells**

1. Inoculate a single colony of appropriate cells into 10ml LB media and culture overnight at 37°C, 200 rpm

2. Inoculate 1% of the overnight culture into a fresh LB media (100 mL)

3. Incubate at 37°C, 200 rpm until OD600 = 0.3-0.6 (approx. 2 hours)

4. Transfer to 2 x 50 mLtubes and leave on ice for 30 minutes

5. Centrifuge at 4000 x g for 5 minutes at 4°C

6. Gently resuspend pellet in 25 mL ice cold 0.1 M MgCl2 and keep on ice for 30 minutes

7. Centrifuge at 4000 xg, 5 min for 5 minutes at 4°C

8. Gently resuspend pellet in 25 mL ice-cold 0.1 M CaCl2 and incubate on ice for 30 minutes

9. Centrifuge at 4000 xg, 5 min for 5 minutes at 4°C

10. Gently resuspend pellet in 1.25 mL ice cold CaCl2 with 15% Glycerol solution

11. Aliquot 100 uL and flash freeze with liquid nitrogen. Store at – 80°C until required

**Colony PCR**

1. Prepare 50 uL of sterile water in a PCR tubes

2. Pick a single colony and resuspend in 50 uL water, then draw 5 uL out into a new tube

3. Freeze 5 uL cell suspension at -20°C for at least 10 minutes

4. Prepare the master mix and add with 5 uL cell suspension (DNA template) and PCR according to the condition in the table below:
following the table below:

![](https://static.igem.org/mediawiki/2021/a/aa/T--Edinburgh--prot1.png)

Run the PCR product on 1% agarose gel with 100 V for 30 minutes and visualise the result under UV exposure.

**Heat shock transformation**

1. Defrost chemically competent cells on ice.

2. Add 2ul of plasmid DNA or 10 uL of assembly reaction to 100 uL competent cells

3. Flick the tube to mix and incubate on ice for 30 minutes

4. Heat shock at 42°C for 30 seconds

5. Incubate on ice for 2 minutes

6. Add 1 mL SOC media and recover cellsat 37°C, 200 rpm for 1 hour

7. Spread 100 uL on an appropriate antibiotic LB/agar plate

8. Spin the rest of the cells at 4000 rpm, remove 900 uL of the media, resusupend and spread onto another LB/agar plate

9. Incubate plates at 37°C overnight

**JUMP assembly: Golden Gate assembly platform**

1. Determine the concentration of the assembly parts with Nanodrop

2. Use DNA calculator (from Promega) to calculate fmol of each part

3. Prepare the assembly parts in the concentration of 20 fmol/uL

4. Set up 20 uL JUMP assembly reaction using 1 uL of all parts (or 20 fmol)

5. Add 2 uL of 10X T4 ligase reaction buffer, 1 uL of either BsmBI (for Level0 assembly) or BsaI-HF (for Level 1 assembly), and 0.25 uL of T4 ligase

6. Add sterile water to adjust the volume up to 20 uL

7. Incubate the reaction following the tables below

![](https://static.igem.org/mediawiki/2021/e/e2/T--Edinburgh--prot2.png)

8. Transform the reaction directly to the competent cells

## **Protein expression and quantification**

**Bradford assay:**

1. Make BSA protein standard dilutions of 2.0 µg/mL, 1.5 µg/mL, 1.0 µg/mL, 0.5 µg/mL, 0.25 µg/mL and 0.0 µg/mL (dH2O).

2. Prepare an eppendorf tube per BSA standard as well as protein sample and add 1 mL 1x Bradford reagent to the tubes.

3. Transfer 5 µL of each BSA standard as well as protein sample to the respective eppendorf tubes and incubate at room temperature for 10 minutes.

4. Calibrate a spectrophotometer with a blank of distilled water

5. Measure A595

6. Make a standard curve using standards and use this to determine protein concentration of samples.

**Enzyme immobilization:**

1. Inoculate 1% of overnight cultures of E. coli BL21(DE3) with level 1 constructs encoding gene with Si-tag and untagged level 1 constructs (as a negative control) in 200ml of fresh LB media containing 50μg/ml of Kanamycin and incubate it at 37°C in a shaking incubator until the OD reaches to 0.5 at 600 nm measured by a spectrophotometer.

2. Then, add IPTG to a final concentration of 1mM and incubate overnight at 18°C.

3. After overnight incubation, centrifuge the cultures in a centrifuge at 4000rpm for 10 min at 4°C, weigh the cell pellets and store them at -20°C.

4. Resuspend the cell pellets to 0.1 g/mL in the lysis buffer (100mM Tris-HCl pH 8.0 or 100 mM Sodium Phosphate buffer pH 6.5, with 0.5 mM EDTA and 100 uM PMSF)

5. Lyse cell by sonication on ice in 5s pulses at 55% amplitude for 10 minutes.

6. Centrifuge the lysates at 4500 rpm for 20 min and at 4°C

7. Prepare 50 mg/mL of the Celite beads in the buffer (100 mM Tris-HCl pH 8.0 or 100 mM Sodium phosphate buffer pH 6.5).

8. Add samples to the beads solution in the amount that dilute sample 10 times

9. Incubate all samples overnight at 4°C on the rotating mixer.

10. After overnight incubation, centrifuge the tubes at 4000 rpm for 5 minutes to settle the beads.

11. Resuspend the beads in 1 mLl of the buffer, spin the tubes at 3500g for 3 minutes and discard the wash buffer.

12. Perform two more washes following the same protocol.

13. After removing the third wash buffer, resuspend the beads with the starting volume of the buffer.

14. Use 100 uL of the bead suspension to measure fluorescence or enzymatic activity.

15. For the stability test, incubate the immobilised and free enzyme at 37°C on the rotating mixer, then draw sample out at various time point to measure the fluorescence or assay activity

**Protein expression and secretion:**

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## **Enzymatic Assay**

**Cex Assay:**

1. Methylumbelliferyl-cellobioside

    - Take 20μl of the 200μl samples from the immobilisation experiment

    - Add methylumbelliferyl-cellobioside (MUC) to a final concentration of 0.2mM into the 20μl samples.

    - Take 1ml of phosphate-buffered saline in a UV cuvette and measure the absorbance by Jenway 7305 spectrophotometer at 348nm and then blank the spectrophotometer with the same buffer.

    - Incubate the samples at 37°C for an hour.

    - Place the samples on ice to stop the reaction and measure the absorbance at 348nm.

    - To determine the enzymatic activity of Cex in the sample, take the difference between the absorbance before and after incubation and relate to a standard curve of 4-methylumbelliferone.

2. O-nitrophenol-xylopyranoside

    - Add 60μl the sample to 300μl of 0.5% w/v O-nitrophenyl-xylopyranoside (ONPX), 150μl sodium acetate (pH 5.0) and 90μl.

    - Add 200μl of this reaction to the 800μl sodium phosphate buffer (pH 12.0) and transfer to a UV cuvette.

    - Measure the absorbance at 405 nm.

    - Place the remaining reaction in a thermomixer set at 300rpm for 2 hours at room temperature.

    - After 2 hours, take 200μl from the reaction and add to a UV cuvette containing 800μl sodium phosphate buffer (pH 12.0) and measure the absorbance at 405 nm.

    - To determine the enzymatic activity of Cex in the sample, relate the difference in absorbance before and after incubation to a standard curve of 4-nitrophenol.

**CenA Assay:**

1. DNS activity assay

    - Evenly mix 400µl crude enzyme lysate of recombinant endoglucanase with 1.6ml CMC-PBS solution (2%CMC with PBS buffer).

    - Prepare the blank control with 400µl crude enzyme and 1.6ml PBS buffer without the substrate.

    - Incubate the samples in a water bath at 37°C for 4h.

    - Add 70µl of the sample to a new 1.5ml microcentrifuge tube containing 70µl of DNS solution.

    - Heat the tube for 12min at 90°C and then put it on ice for 30 min to stop the reaction.

    - Measure the absorbance by spectrophotometer (Genova Nano, JENWAY) at 540 nm, with three parallel samples in each group.

    - Later, plot the glucose standard curve by measuring the absorbance of different glucose concentrations at 540 nm.

    - Firstly, prepare 1mg/ml glucose stock and dilute it to 0, 0.2, 0.4, 0.6, 0.8, 1mg/ml, respectively.

    - Mix all of them with equivalent DNS solution and heat for 12 min and then measure the absorbance at 540 nm.

2.4-MUC activity assay

    - Take 20 µl crude lysate into a 1.5ml microcentrifuge tube.

    - Add 20 µl of 10mM 4-MUC and 960 µl PBS buffer (pH=7.0) and then incubate at 37°C for 1h.

    - Prepare the sample in triplicate.

    - Prepare the blank control the same as each sample except for replacing crude enzyme extract lysate with 20 µl bugbuster master mix solution.

    - Measure the absorbance by spectrophotometer at OD348.

    - Prepare the standard curve of 4-MU with different concentrations.

**Keratinase Assay:**

1. Milk plate screening

    - Autoclave 10% milk solution and LB agar separately, and then cool and mix to a final concentration of 1% milk.

    - Add an appropriate antibiotic before pouring. Plate the transformants onto milk plates and incubate for 48-72 h at 30°C. Halos of clearance around colonies were analysed.

2. Ninhydrin assay

    - Add clarified cell lysate or commercial enzyme solution to the substrate (BSA, or chopped feathers) and incubate (37°C, 100 mM potassium phosphate buffer pH=8, 1000 rpm).

    - The reaction stops at the specified timepoint by addition of equal volume of 10% trichloroacetic acid (TCA).

    - Remove the precipitated protein by centrifugation (10000g, 10 min).

    - Add the supernatant to ninhydrin reagent (Sigma-Aldrich) in a 2:1 ratio and boil for 10 min.

    - Measure the absorbance at 570 nm. Colour generation will be proportional to free amino acids released into the solution.

    - Compare reaction samples to a standard curve prepared with varying concentrations of amino acid (Glycine, L-lysine dihydrochloride, L-cysteine).

3. Keratin azure assay

    - Add 1 mL total volume of clarified cell lysate, supernatant, or commercial enzyme in 50 mM Tris-HCl (pH=8) to 20 mg keratin azure, pre-equilibrated in buffer.

    - Incubate for 60-90 min at 37°C with constant agitation.

    - Centrifuge the samples at 13,500g for 10 min and measure the absorbance of supernatant at 595nm.

**PETase Assay:**

1. para-Nitrophenol butyrate (pNPB)

    - Add 100µg of 2 mM para-Nitrophenol butyrate (pNPB) substrate per mL of the soluble portion of the cell lysates.

    - Add 45 mM Na2HPO4-HCl (pH 7.0), 90 mM NaCl, and 10% (v/v) DMSO and incubate the reaction mixture for 30 minutes at 250 rpm and at 30°C for all PETase-containing constructs and at 72°C for all Leaf-Branch Compost Cutinase-containing constructs

    - Measure the absorbance at 415 nm for the production of para-Nitrophenol (pNP).

2. Absorbance of PET film)

    - Treat the PET film with 70% ethanol and distilled water and air-dried.

    - Incubate 100 µg/mL of the soluble fraction of cell lysates with biaxially 31 orientated two PET films (1 cm × 1 cm) supplied by DuPont Fujifilm Technologies for 3 hours in the buffer containing 50 mM Na2HPO4- HCl (pH 7.0), 50 mM glycine-NaOH (pH 10.0) at 30°C for all PETase-containing enzymes and at 72°C for all LCC-containing enzymes at 200 rpm.

    - Terminate the reaction by heat treatment (85°C for 10 minutes).

    - Collect the supernatant of the reaction mixtures and measure the absorbance at 260 nm for all the degradation products of PET.

    - Due to commercial unavailability of MHET, establish its calibration curve using BHET (Sigma-Aldrich) at varying concentrations (0.01-0.1 mM).

3. Fluorescence of PET film)

    - Adjust the fluorescence filter for excitation at 355 nm and emission at 460 nm by using a microplate reader (GREINER 96 F-BOTTOM).

    - Place TPA (Merck Millipore) solution aliquots (dissolved in 0.1 M NaOH) at the various concentration (0.05-1.00 mM) in a 96-well microplate and add to a final volume of 150 µL with 0.1 M phosphate buffer (pH 8.5).

    - Add 25 µL of 5 mM EDTA (Ethylenediamine tetraacetic acid) from Sigma-Aldrich in each well.

    - Add 25 µL of freshly prepared 10 mM FeSO4 (Sigma-Aldrich) to initiate the hydroxylation of TPA molecule and incubating at room temperature for 20 minutes prior to analysis.

    - To determine the amount of TPA produced by the reaction, add 100 µL of the supernatant from the reaction mixture along with the other chemicals as mentioned (Wei et al., 2012).

**GshF Assay:**

1. GshF can be assayed by the endpoint measurement of the glutathione produced.

2. Take 10 mM MgCl2, 20 mM glycine, 20 mM L-glutamic acid, 20 mM L-cysteine, 10 mM ATP, and 5 mg/mL wet weight cell crude extract in 100 mM Tris-HCl pH 8.0, with a total volume of 1 mL.

3. Take the crude lysate of E. coli Rosetta (DE3) pLysS as the negative control.

4. Add crude extract and incubate at room temperature for 10 minutes to initialise the reaction

5. Draw 25 uL of sample out to determine the GSH.

6. Detect the luminescence signal using Greiner, 96-well, F-bottom microtiter plate with the microplate reader.

7. Determine the amount of GSH following the protocol provided with the detection kit.

**KatG Assay:**

\*KatG was assay based on the peroxidase activity

1. The reaction will take place in two different volumes: 50 uL and 1 mL.

2. Make a 1-mL reaction in UV/VIS cuvette composed of 4 mM 2,2'-Azino-bis (3-Ethylbenzthiazoline-6- Sulfonic Acid) (ABTS), 0.01% w/w H2O2, 0.04 mg/mL BSA, 0.008% v/v TritonX100 and 1 mg/mL wet weight cell crude extract in Sodium acetate buffer pH 5.0.

3. Measure the increment of absorbance at 405 nm with UV/VIS spectrophotometer every 5 or 10 seconds for 4.5 minutes.

4. Set the reaction in a 96-well F-bottom microtiter plate and measure oxidised ABTS produced from an elevated A405 with POLARstar Omega plate reader.

5. Take crude lysate of E. coli BL21 (DE3) as the negative control.

**POX5 Assay:**

1. Perform the reaction in 1 mL and 50 uL; consist of 50 uM sodium pyruvate, 2 mM ABTS, 0.2 mM Thiamine pyrophosphate (TPP), 0.01 mM flavin adenine dinucleotide (FAD), 1 mM EDTA, 10 mM MgCl2, 0.04 mg/mL BSA, 0.008% v/v TritonX-100 and 20 ug/mL of protein in KatG crude extract in 20 mM 24 potassium phosphate buffer pH 5.0.

2. Add 10 uL L. plantarum crude extract for 1 mL-reaction and 10 uL Pox5 from cell-free protein synthesis in 50 uL-reaction.

3. Take crude extract of E. coli BL21 (DE3) and/or as the negative CFPS reaction.

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# Team:Edinburgh/Results

The SuperGrinder

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[Results](#backtop)
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[Other Results](#other)

- Silica-binding tags comparison
- Protein expression and secretion
- Enzyme immobilisation
- Si-tagged enzyme activity
- Structural Modeling

# Results

## What did we aim to do?

We aimed to:

1. Characterise the different silica-binding tags (L2, L2NC, Car9, Sb7)

2. Clone and express enzymes fused with silica-binding tags

3. Successfully immobilise our enzymes to silica surfaces

4. Characterise the effect of tag addition and immobilisation on activity and stability (to conditions/over time)

5. Apply [directed evolution methods](https://2021.igem.org/Team%3AEdinburgh/Directed_Evolution) to increase the activity of our tagged enzymes for application to the SuperGrinder

## What did we find?

We will summarise our findings relating to the immobilisation of the cellulase Cex:

Cex-Car9 showed low solubility at the SDS page assay and reduced activity against the substrate. Indeed, the negative-charges of Car9 seem to hinder the catalytic site (rich in positive charges). On the other hand, L2NC tag seems to increase the solubility of Cex and consequently the activity, even rescuing the non-functional Cex phenotype we came across. L2NC tag very similar results were also observed when fused to LCC and PETase enzymes. This leads to the conclusion that L2NC is the best candidate for the increase, or in any case lack of detrimental effect, of activity for these enzymes.

Although data were insufficient to quantify binding affinities and thus state which tag would bind to the silica beads or glass with the strongest affinity, preliminary qualitative data seem to suggest the L2 tag and the L2NC tag to be the most efficient at binding the acid-scarified glass surface. More studies with a higher number of replicates and normalisation adjustment will be needed in order to define the best candidate. The “proof of concept” page of our wiki shows more detailed data about the immobilisation of the enzymes on glass and silica beads, showcasing promising results.

## Select the section of results you would like to read more about...

**Silica binding tag comparison:**

To first assess the silica-binding tags, we assembled constructs to express GFP under the T7 promoter, with each tag at the C-terminal (except for L2 which was received from Marcos Valenzuela-Ortega as an N-terminal fusion already). We transformed assemblies into E. coli DH5α. As the intended final assembly contained sfGFP, we used a plasmid backbone with a lacZ’ reporter gene (pJUMP29-1A (lacZ’)) to enable **blue-white screening**. After transformation into E. coli DH5α, colonies with an insert replacing the lacZ’ reporter gene were unable to produce functional β-galactosidase to metabolise X-gal to an insoluble blue pigment, thus appeared white (Figure 1). Screening plates had an average ratio of 1:12 blue to white colonies, showing that assembly was successful. Colony PCR performed on selected white colonies and analysed by gel electrophoresis showed a single band of product for each colony matching the expected insert sizes (Figure 2A). Nucleotide sequence was confirmed by Sanger sequencing.

![Blue-white screening](https://static.igem.org/mediawiki/2021/d/d5/T--Edinburgh--Results1.f1.png)

**Figure 1** Example of blue-white screening. 10% cells plated on selective LB agar (kanamycin 50 μg/ml).Left: Uncut backbone transformation (no enzyme control). All colonies blue. Right: selection plate for assembly of sfGFP-linker-L2NC with 19 blue colonies and 239 white.

We used E. coli BL21 (DE3) for expression of constructs, induced with IPTG (Figure 2B). Cells expressing L2-sfGFP (blue line) showed a notably slower growth rate than those expressing the smaller tags, hypothesised to be due to the large size, or some toxic effects of overexpression (Figure 2C). L2-sfGFP cultures also demonstrated lower fluorescence activity. This was supported by observations by Taniguchi et al, 2007 [1] who found that L2 reduced ﬂuorescence activity of GFP by 30% when used as a fusion tag. Insoluble fractions recovered after lysis were also larger (data not shown), suggesting that the L2 tag decreased solubility of the sfGFP, reducing fluorescence activity and perhaps putting higher metabolic burden on cells. This would not be surprising as the L2 tag is larger than sfGFP (273 vs 237 amino acids), so is likely to impact folding. Fluorescence activity and growth seemed to be less impacted by the smaller tags L2NC and Car9.

![Tagging sfGFP](https://static.igem.org/mediawiki/2021/3/34/T--Edinburgh--Results1.f2.png)

**Figure 2** A Colony PCR using PS1/PS2 primers showed expected band sizes for all inserts (3 colonies tested per strain). B. Images post-induction by 1 mM IPTG (top= cell pellet, bottom=cell culture). Cultures with different tags show varying GFP fluorescence levels. C. Growth curves of strains during induction (time of induction marked with blue arrow, OD600=0.5-0.6). Induced cultures are marked by dots/lines, uninduced cultures marked by crosses/dashed lines D. SDS-PAGE analysis of solubility shows tagged GFP present in both soluble and insoluble fractions Expected sizes sfGFP (27.1kDa), L2-sfGFP (57.7kDa), sfGFP-Car9 (28.7kDa), sfGFP-L2NC (41.3kDa). Compared to NEB protein ladder.

Cells were lysed by BugBuster (as a quick-and-easy method), or sonication (allowing us to control the “binding buffer” composition), and lysates incubated with different types of silica beads for different incubation times. Beads were washed using “wash buffer” and then visualised to observe residual GFP fluorescence. We also compared the difference in fluorescence between lysate samples incubated in the presence and absence of silica beads to quantify the amount of GFP removed from solution when beads were present (Table 1).

|  |  |  |
| --- | --- | --- |
|  | **Removal of GFP fluorescence from solution (%)** | |
| **Bead type** | **L2-sfGFP** | **Untagged sfGFP** |
| *500 μm* | 27.0 | 12.0 |
| *Celite 545* | 23.0 | 5.5 |

Proteins successfully immobilised to beads were detected by SDS-PAGE. Beads were boiled in reducing sample buffer to release proteins adsorbed to the surface. Protein bands of the expected sizes were released from bead samples, especially Celite beads, after boiling in reducing sample buffer (Figure 3), although other contaminating bands with lower intensities were also observed in some cases (data not shown). No sfGFP was detectable on beads incubated with untagged samples. This suggests that the reduction in fluorescence observed for untagged sfGFP (5.5-12%) in Table 1 could result from natural reduction in fluorescence activity over time, rather than from immobilisation.

![SDS-PAGE showing immobilised tagged GFP](https://static.igem.org/mediawiki/2021/2/28/T--Edinburgh--Results1.f3.png)

**Figure 3** SDS-PAGE of protein samples released from Celite beads, enabling comparison of silica binding tags. Boxes surround bands of expected sizes.

We also tested removal of fluorescence from solution using different quantities of silica beads, which showed that the fluorescence removal scaled with bead quantity (Figure 4).

![Different bead ratios](https://static.igem.org/mediawiki/2021/1/1c/T--Edinburgh--Results1.f4.png)

**Figure 4** A. GFP removal from solution after incubation of 600 μL clarified cell lysate with three different quantities of beads, overnight at 4°C (low 10 mg, medium 50 mg, high 100 mg). Fluorescence values are normalised to an arbitrary value of 1 in the absence of silica.

Overall our results indicated that the L2NC tag with the addition of the short amino-acid linker was the silica-binding tag with the highest affinity, and the immobilisation worked best with the Celite 545 beads. Further results and comparisons of the tags are demonstrated on the “[Proof of concept](https://2021.igem.org/Team%3AEdinburgh/Proof_Of_Concept)” page.

**References:**

[1] Taniguchi K, Nomura K, Hata Y, Nishimura T, Asami Y, Kuroda A. The Si-tag for immobilizing proteins on a silica surface. Biotechnol Bioeng [Internet]. 2007 Apr 15 [cited 2021 Apr 23];96(6):1023–9. Available from: http://doi.wiley.com/10.1002/bit.21208

**Protein expression and secretion:**

To successfully heterologously express proteases such as KerA, it was necessary to screen different secretion signal peptides. Secretion is important to reduce host toxicity of over-expressed proteases and avoid expression in intracellular inclusion bodies.

### Protein secretion screen

We screened for KerA-secreting clones by comparing zones of clearance on milk agar plates. Whilst hydrolysis of milk does not test specificity toward keratinous substrates, the screen is a quick and easy measure of secreted proteolytic activity. A control plate comparing wild-type strains of *Bacillus subtilis* which had known protease secretion abilities confirmed that the screen worked: zones of clearance were observed around protease secreting strains, but not around those that were deficient in secreted proteases (*B. subtilis* SCK6 and IIG-Bs27) (Figure 1)

![Milk halo protease screen](https://static.igem.org/mediawiki/2021/b/b4/T--Edinburgh--Results2.f1.png)

**Figure 1** Protease secretion from three different strains of Bacillus subtilis, observed by measuring zones of clearance on a milk agar plate (1% skimmed milk powder). Protease secretion was only observed from B. subtilis 168 as expected.

We thus tested the addition of eight different signal peptides to KerA, expressed in *E. coli*. Signal peptides were already assembled in JUMP Level 0 plasmids by Marcos Valenzuela-Ortega (Chris French lab, University of Edinburgh), summarised in Table 1. We also tested secretion of KerA using the native KerA signal prepeptide from *B. licheniformis*. Colony PCR after JUMP assembly and transformation verified that insert sizes of positive transformants were as expected (Figure 2). Nucleotide sequences were confirmed by Sanger sequencing.

![Assemblies of KerA with different SP's: Colony PCR](https://static.igem.org/mediawiki/2021/e/ea/T--Edinburgh--Results2.f2.png)

**Figure 2** Figure 2: Colony PCR from positive transformants using PS1/PS2 primer pair. Gel showed expected insert sizes for KerA assembled with different signal peptides. Signal peptides used are as follows, with expected size in parentheses: **K1**.PelB (1581), **K2**.PelB\_D5 (1599), **K3**.CelCD\_G20 (1587), **K4**.CelCD\_Gdom (2649), **K5**.CelCD\_N29 (1599), **K6**.OsmY (2130), **K7**.YebF (1872), **K8**.Spy (2001), **KK**. KerA native signal peptide (1602), **G**. Empty backbone (pJUMP29-1B) (1161) **-** negative control with no template DNA added (just H2O).

Wild-type (untransformed) *E. coli* BL21 (DE3), and transformants with the empty pJUMP29-1B backbone did not produce a halo, however some assemblies of KerA with signal peptides produced significant zones of clearance on milk plates after incubation (30°C, 48-72 hrs), therefore the screening methodology was considered an appropriate initial selection. Secretion-positive cultures were selected for further assays on proteolytic activity.

The results shown in Figure 3 show the halo screen of both the individually assembled SP-KerA constructs, and a second assembly method in which all 8 signal peptides were added to the same reaction (SP-mix). The assembly product was then directly transformed into *E.coli* BL21 (DE3) to enable expression and screening on the primary transformation plate. The two methods showed slightly different results for unknown reasons. Figure 3E summarises the differences. Overall the secretion signal with most evidence for secretion was Cel-CD\_G20.

![Protease secretion screen results](https://static.igem.org/mediawiki/2021/b/b6/T--Edinburgh--Results2.f3.png)

**Figure 3** **A** Colony PCR using PS1/PS2 primer pair on assemblies of KerA with different signal peptides in *E. coli* DH5a. Expected insert sizes are shown above in bp. Three colonies were selected for each assembly. **B.** Individually assembled KerA with different signal peptides, including green colonies (1G, bottom right) as negative control. Incubated at 30°C, 60 h. **C**. Primary screening plate from mixed signal peptide assembly transformed directly into *E. coli* BL21 (DE3) (1% milk agar plate with kanamycin selection, incubated 28°C, 72 h). **D**. Re-patched colonies selected from primary screening plate, numbered matches labelling on primary screening plate. Three “halo negative” colonies from the primary plate were also selected (1N, 2N, 3N) and a green colony with no insert (1G) as negative controls. Imaged after incubation at 30°C, 72 h. **E.** Table of clones positive for protease secretion on either the individually assembled plate or the mix plate. X signifies that a reproducible halo was observed. (x) signifies that a halo was observed but that it was not reproducible.

### Method discussion:

To screen for secretion from *B. subtilis* we would use *B. subtilis* IIG-Bs27-31 as a host because untransformed cells showed no clearance zone in initial tests, therefore had no background activity. Interestingly, halos were observed after the same period of time regardless of whether IPTG was applied for induction (40 μl to top, 100 mM). This could suggest that the milk in the plate could substitute for IPTG (an analogue of lactose). This finding is potentially useful for lowering production costs of inducible enzymes in industry.

Table 1

|  | Signal Peptide | Sequence | Ref. |
| --- | --- | --- | --- |
| K1 | PelB\_N (*Erwinia carotovora*) | AATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCAGCC | (Wang *et al*, 2016) 22 residues as in pET27b. Added 5’ A |
| K2 | PelB-D5 (*Erwinia carotovora*, modified) | AATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCAGATGATGACGATGATGGAGCC | (Kim *et al*, 2015) 22 residues as in pet27b. Added 5’ DDDDDGA |
| K3 | Cel-CD-Gdom (*Bacillus* sp.) | AATGGAAGGAAACACTCGTGAAGATAATTTTAAACATTTATTAGGTAATGACAATGTTAAACGCCCTTCTGAGGCTGGCGCATTACAATTACAAGAAGTCGATGGACAAATGACATTAGTAGATCAACATGGAGAAAAAATTCAATTACGTGGAATGAGTACACACGGATTACAATGGTTTCCTGAGATCTTGAATGATAACGCATACAAAGCTCTTGCTAACGATTGGGAATCAAATATGATTCGTCTAGCTATGTATGTCGGTGAAAATGGCTATGCTTCAAATCCAGAGCTGATTAAAAGCAGAGTCATTAAAGGAATAGATCTTGCTATTGAAAATGACATGTATGTCATCGTTGATTGGCATGTACATGCACCTGGTGATCCTAGAGATCCCGTTTACGCTGGAGCAGAAGATTTCTTTAGAGATATTGCAGCATTATATCCTAACAATCCACACATTATTTATGAGTTAGCGAATGAGCCAAGTAGTAACAATAATGGTGGAGCTGGGATTCCAAATAATGAAGAAGGTTGGAATGCGGTAAAAGAATACGCTGATCCAATTGTAGAAATGTTACGTGATAGCGGGAACGCAGATGACAATATTATCATTGTGGGTAGTCCAAACTGGAGTCAGCGTCCTGACTTAGCAGCTGATAATCCAATTGATGATCACCATACAATGTATACTGTTCACTTCTACACTGGTTCACATGCTGCTTCAACTGAAAGCTATCCGCCTGAAACTCCTAACTCTGAAAGAGGAAACGTAATGAGTAACACTCGTTATGCGTTAGAAAACGGAGTAGCAGTATTTGCAACAGAGTGGGGAACTAGCCAAGCAAATGGAGATGGTGGTCCTTACTTTGATGAAGCAGATGTATGGATTGAGTTTTTAAATGAAAACAACATTAGCTGGGCTAACTGGTCTTTAACGAATAAAAATGAAGTATCTGGTGCATTTACACCATTCGAGTTAGGTAAGTCTAACGCAACAAGTCTTGACCCAGGGCCAGACCAAGTATGGGTACCAGAAGAGTTAAGTCTTTCTGGAGAATATGTACGTGCTCGTATTAAAGGTGTGAACTATGAGCCAATCGACCGTACAAAATACACGAAAGTACTTGGCGGAGCC | (Gao *et al*, 2016) Domain as used by authors (M + 375 from catalytic domain), +GGA M15743.1 |
| K4 | Cel-CD-G20 (*Bacillus* sp.) | AATGGAAGGAAACACTCGTGAAGATAATTTTAAACATTTATTAGGTAATGACAATGTTAAACGCAAACGCGGAGCC | (Gao *et al*, 2016) Peptide used by authors: M+20 residues from catalytic domain + KRGA GeneBank ID M15743.1 |
| K5 | CelCD-N29 (*Bacillus* sp.) | AATGCTCAGAAAGAAAACAAAGCAGTTGATTTCTTCCATTCTTATTTTAGTTTTACTTCTATCTTTATTTCCGACAGCTCTTGCAGCC | (Gao *et al*, 2016) 29 residue signal peptide of original gene GeneBank ID M15743.1 |
| K6 | OsmY (*E. coli* MG1655) | AATGACTATGACAAGACTGAAGATTTCGAAAACTCTGCTGGCTGTAATGTTGACCTCTGCCGTCGCGACCGGCTCTGCCTACGCGGAAAACAACGCGCAGACTACCAATGAAAGCGCAGGGCAAAAAGTCGATAGCTCTATGAATAAAGTCGGTAATTTCATGGATGACAGCGCCATCACCGCGAAAGTGAAGGCGGCCCTGGTGGATCATGACAACATCAAGAGCACCGATATCTCTGTAAAAACCGATCAAAAAGTCGTGACCCTGAGCGGTTTCGTTGAAAGCCAGGCCCAGGCCGAAGAGGCAGTGAAAGTGGCGAAAGGCGTTGAAGGGGTGACCTCTGTCAGCGACAAACTGCACGTTCGCGACGCTAAAGAAGGCTCGGTGAAGGGCTACGCGGGTGACACCGCCACCACCAGTGAAATCAAAGCCAAACTGCTGGCGGACGATATCGTCCCTTCCCGTCATGTGAAAGTTGAAACCACCGACGGCGTGGTTCAGCTCTCCGGTACCGTCGATTCTCAGGCACAAAGTGACCGTGCTGAAAGTATCGCCAAAGCGGTAGATGGTGTGAAAAGCGTTAAAAATGATCTGAAAACTAAGGGCAGCGGATCAGCC | (Bokinsky *et al*, 2011) Complete gene (201 residues) + GSGSA GeneID: 948895 |
| K7 | YebF (*E. coli* MG1655) | AATGAAAAAAAGAGGGGCGTTTTTAGGGCTGTTGTTGGTTTCTGCCTGCGCATCAGTTTTCGCTGCCAATAATGAAACCAGCAAGTCGGTCACTTTCCCAAAGTGTGAAGATCTGGATGCTGCCGGAATTGCCGCGAGCGTAAAACGTGATTATCAACAAAATCGCGTGGCGCGTTGGGCAGATGATCAAAAAATTGTCGGTCAGGCCGATCCCGTGGCTTGGGTCAGTTTGCAGGACATTCAGGGTAAAGATGATAAATGGTCAGTACCGCTAACCGTGCGTGGTAAAAGTGCCGATATTCATTACCAGGTCAGCGTGGACTGCAAAGCGGGAATGGCGGAATATCAGCGGCGTTCAGCC | (Natarajan *et al*, 2017) Complete gene (118 residues) + SA GeneID: 946363 |
| K8 | Spy (*E. coli* MG1655) | AATGCGTAAATTAACTGCACTGTTTGTTGCCTCTACCCTGGCTCTTGGCGCGGCTAACCTGGCCCATGCCGCAGACACCACTACCGCAGCACCGGCTGACGCGAAGCCGATGATGCACCACAAAGGCAAGTTCGGTCCGCATCAGGACATGATGTTCAAAGACCTGAACCTGACCGACGCGCAGAAACAGCAGATCCGCGAAATCATGAAAGGCCAGCGTGACCAGATGAAACGTCCGCCGCTGGAAGAACGCCGCGCAATGCATGACATCATTGCCAGCGATACCTTCGATAAAGTAAAAGCTGAAGCGCAGATCGCAAAAATGGAAGAACAGCGCAAAGCTAACATGCTGGCGCACATGGAAACCCAGAACAAAATTTACAACATCCTGACGCCGGAACAGAAAAAGCAATTTAATGCTAATTTTGAGAAGCGTCTGACAGAACGTCCAGCGGCAAAAGGTAAAATGCCTGCAACTGCTGAATCAGCC | (Tsirigotaki *et al*, 2017) Complete gene (161 residues) + SA GeneID: 946253 |
| KK | KerA\_SP (Bacillus licheniformis) | ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTCAGCGATTCCGCTTCAGCAGCC | (Lin *et al*, 1995) Annotated prepeptide from native KerA gene |

**Recombinant enzyme immobilisation with silica-binding peptides:**

Immobilisation of tagged enzymes to the beads was mainly detected using activity assays after immobilisation, compared to an untagged version (see next section).

We also began to develop a protocol for high-throughput screening of immobilised enzymes by immobilising tagged enzymes recovered from a directed evolution library to glass slides. This can be done by immersion in lysis solution. The immobilised enzymes on the glass slide could then be used for enzymatic assays in situ, and matched back to the parent colonies from position on the plate. This would be a highly valuable application of these silica-binding tags., enabling screening for hundreds of variants per plate. Moreover, when selecting for enzymes with higher activity, it is beneficial to be able to screen in their immobilised state as this is the condition for their final application.

### Immobilisation to a Glass Surface results

In order to assess whether Si-tagged proteins were immobilising to the glass surface, various Si-tagged sfGFP constructs were tested due to the fluorescent protein’s easy readout in a UV transilluminator. Through iterations, various factors such as pH of the Tris-HCl binding buffer, salt concentration upon binding and the acid-treatment of the glass surface, proved to be important to the effective binding of Si-tagged sfGFP.

![Immobilisation of sfGFP to glass slide](https://static.igem.org/mediawiki/2021/3/33/T--Edinburgh--Results3.glass_immobilisation.png)

**Figure 1 Immobilisation of Si-tagged sfGFP to a Treated Glass Surface.** sfGFP tagged with four different Si-tags (Car9, L2NC-linked, L2NC and L2) as well as a negative control of untagged sfGFP were aliquoted on to a glass slide, washed and a visualised under UV light. After wash acquisition is longer than the others to effectively visualise the fluorescence on the slide.

Figure 1 suggests that there is evidence of the Si-tagged protein binding to the treated glass surface. Different methods were tried, although the final method closely followed Taniguchi *et al*. with some modifications [1]. Infact, increasing the pH of the Tris buffer was believed to be appropriate considering the high prevalence of positively charged amino acid residues in the Si-tags and is more common in the supporting literature [2,3,4]. Additionally, the acid-treatment of the microscope glass slides improved the attachment on the glass slide. Indeed, the exposure to acid removes alkaline salts from the crystalline structure of the glass, being replaced by H+ ions, and increasing silanol groups exposition [5]. A similar experiment was run with the cells lysed by sonication in the 25mM Tris-HCl (pH 8.0) binding buffer without the added salts. None of the samples were able to adhere to the glass. Additionally, an alternative lysing solution, BugBuster, was used instead of B-PER as suggested by Taniguchi and colleagues for cost and availability reasons. No other lysing solutions were tested, and considering BugBuster’s proprietary contents, it may have affected the appropriate binding of the Si-tag to the surface. However, both the lysis method and solutions vary in this experiment and therefore cannot be directly compared, we hypothesized that the added salt increases the specificity of the Si-tag to the silanol group of the glass surface, but more experiments must follow to confirm or deny it.

Eventually, the L2-tagged sfGFP showed the highest fluorescence in comparison to L2NC and Car9 after the wash step, pointing to the conclusion that it most strongly binds to the glass surface. Nonetheless, these results must be considered qualitative as this reflects a degree of fluorescence after a wash step, only. In addition, protein concentrations were not standardised, which may have a large impact on the amount of fluorescence visualised. Instead, the resuspension of the wet cell weight was taken to standardise the samples. Moreover, the samples were only washed in the wash buffer for 15 minutes, whereas Taniguchi and colleagues washed their slide for 24 hours and still visualised their Si-tagged sfGFP constructs [1]. This could be explained by the authors having more sophisticated equipment for their experiment. In addition, longer washings drastically decrease the chance of non-specific binding, allowing for more appropriate conclusions to be drawn.

**Future Perspectives for the High-Throughput Screening of Immobilised Enzymes**

The evidence of immobilisation of Si-tagged proteins on a glass surface is a promising first step towards the high-throughput screening of immobilised enzymes. Future experiments would include binding buffer optimization by varying the concentrations of Tris-HCl (pH 8.0), NaCl and other lysis buffers including the B-PER lysing solution. Indeed, as the lysing solution comprises nearly half of the original mixture, its alteration could have a large impact on the binding capability of the tag.

Additionally, as the treatment of the glass surfaces was suggested to have found a positive impact on protein immobilisation, further treatment techniques are worth pursuing. Infact, increasing the concentration of HCl and exposing the glass slide to the acid for longer amounts of time could further activate the glass slide, thereby increasing the yield of protein immobilisation. Future experiments with this consideration may prove pivotal

**References:**

1. Taniguchi K, Nomura K, Hata Y, Nishimura T, Asami Y, Kuroda A. The Si-tag for immobilizing proteins on a silica surface. Biotechnol Bioeng [Internet]. 2007 Apr 15 [cited 2021 Apr 23];96(6):1023–9. Available from: http://doi.wiley.com/10.1002/bit.21208

2. Kim S, Joo K Il, Jo BH, Cha HJ. Stability-Controllable Self Immobilization of Carbonic Anhydrase Fused with a Silica-Binding Tag onto Diatom Biosilica for Enzymatic CO2Capture and Utilization. ACS Appl Mater Interfaces [Internet]. 2020 Jun 17 [cited 2021 Apr 24];12(24):27055–63. Available from: https://dx.doi.org/10.1021/acsami.0c03804

3. Coyle BL, Baneyx F. A cleavable silica-binding affinity tag for rapid and inexpensive protein purification. Biotechnol Bioeng [Internet]. 2014 Oct 1 [cited 2021 Apr 23];111(10):2019–26.

4. Rauwolf S, Steegmüller T, Schwaminger SP, Berensmeier S. Purification of a peptide tagged protein via an affinity chromatographic process with underivatized silica. Eng Life Sci [Internet]. 2021 [cited 2021 Aug 13]; Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/elsc.202100019 32.

5. Szalóki M, Hegedűs V, Fodor T, Martos R, Radics T, Hegedűs C, et al. Evaluation of the Effect of the Microscopic Glass Surface Protonation on the Hard Tissue Thin Section Preparation. Appl Sci 2020, Vol 10, Page 7742 [Internet]. 2020 Nov 1 [cited 2021 Aug 18];10(21):7742. Available from: https://www.mdpi.com/2076-3417/10/21/7742/htm

**Assessment of enzymatic activity of Si-tagged enzymes:**

Adding fusion tags to an enzyme, and immobilising it to a surface can impact activity. Taniguchi *et al*, 2007 [1] suggested that the largest tag (L2) decreased luciferase activity by 60-70%, suggesting that the large tag may have a greater impact on enzymes which require conformational changes for activity than it had on sfGFP fluorescence activity (demonstrating just 30% reduction). It is important to test the impact on our enzymes to aid our assessment of how feasible the overall SuperGrinder will be based on observed performance. Our preliminary results from activity assays are below, with final results on the [Proof of Concept](https://2021.igem.org/Team%3AEdinburgh/Proof_Of_Concept) page.

### Cex (from *Cellulomonas fimi*) Immobilisation to Silica Beads

The immobilisation protocol was followed with untagged Cex, Cex-Car9 and Cex-L2NC and the enzymatic activity of the supernatant was tested before and after incubation with the beads, the subsequent wash buffers, and the beads themselves. Two assays were performed on the samples to test both the cellulolytic and xylanolytic activity of Cex. In order to assess the activity of the enzyme on the beads, the reaction should ideally be constantly kept in suspension, so as to avoid the sedimentation of the beads to the bottom of the reaction tube. After an incubation period, the reaction should be spun down, and the supernatant taken so as to avoid possible interference of the beads in the spectrophotometer.

**Recombinant Cex Methylumbelliferyl-cellobioside assay**

Methylumbelliferyl-cellobioside (MUC) is a common assay for the activity of exoglucanases. Once digested by the exoglucanase, MUC is converted into cellobiose and 4-methylumbelliferone (4-MU), a fluorogenic compound which has excitation and emission wavelengths at 360/460nm, however the absorbance of which can also be read at 348nm for the monitoring of MU release related to enzyme [2]. The results are displayed in Figure 1. These are interesting preliminary results, but more repeats would allow for more decisive conclusions to be made. These can be viewed on the [Proof of concept](https://2021.igem.org/Team%3AEdinburgh/Proof_Of_Concept) page.

![Cex enzymatic assay](https://static.igem.org/mediawiki/2021/4/4b/T--Edinburgh--Results3.f1.png)

**Figure 1 Enzyme Activity Before and After Immobilisation to MUC.** Enzyme activity was determined by calculating ε of 4-MU = 9954M-1cm-1. 1 U is equal to 1μmol of substrate released per minute. sfGFP-L2NC is a control which can bind Silica beads, but does not impact the screening of the substrate. Cex-L2NC showed increased activity after immobilisation, whereas untagged Cex and Cex-Car9 showed no change. GFP-L2NC results from Wash 3 step cannot be explained.

Nonetheless, while Cex-Car9 appears to be non-functional, as its absorbance is lower than the controls (untagged Cex), the data appear to suggest that the Cex-L2NC can interact with the MUC substrate to produce fluorogenic 4-MU. Curiously, untagged Cex does not appear to have any activity on the substrate. This could be due to protein aggregation after induction with IPTG, thereby nullifying the activity of the enzyme. SDS-PAGE was performed on all samples after 18oC overnight induction, however the results are unclear and inconclusive.We investigated the reasons behind this further using structural modelling.

### PETase and LCC pNPB assays

Para-Nitrophenol butyrate (pNPB) is a model substrate which is hydrolysed by PETase and LCC enzymes into para-Nitrophenol (having a characteristic yellow colour) (Figure 2) with maximum absorbance at 415 nm. This was considered as a preliminary assay to check the presence of active enzymes expressed (3). The calibration curve was then constructed, and the concentration of unknown samples was calculated using the trendline formula.

![PETase assay](https://static.igem.org/mediawiki/2021/c/ca/T--Edinburgh--Results3.f2.png)

**Figure 2 Results of para-Nitrophenol Butyrate assay.** (A) 1-Without level 1 plasmid as
a control, 2- PETase enzyme, 3-PETase tagged with Car9, 4- PETase with L2NC tag,
and 5-PETase with SB7 tag. (B) 1-Tube without any level 1 plasmid as a control, 2- LCC
enzyme, 3-LCC tagged with Car9, 4- LCC with L2NC tag, and 5-LCC with SB7 tag.

![PETase assay graph](https://static.igem.org/mediawiki/2021/7/71/T--Edinburgh--Results3.f3.png)

**Figure 3** para-Nitrophenol (mM) produced by different proteins for the enzymatic
activity analysis.

This experiment demonstrated all the protein constructs to be active. PETase tagged with L2NC was shown to be the most active enzyme, with around four-fold higher enzymatic activity than untagged PETase. Additionally, this test presented a significant increased activity of the PETase enzyme when tags are used. Si-tags might be increasing the enzyme’s affinity for the substrate, resulting in increased activity [4]. This was also true for the LCC enzyme, where all of the tags increased its activity. This can be argued that the tags are contributing to the LCC enzyme’s increased thermal stability (at 72°C), as suggested by [5]. Interestingly, the tube containing cellular proteins without a level 1 plasmid and therefore without cutinases produced a positive pNPB result at 30°C, suggesting the presence of proteins in E. coli BL21 (DE3) cells with cutinase or lipase activity (both of which produce a positive pNPB result).This was significantly enhanced in the presence of a higher temperature (72°C), suggesting that some enzymes are temperature-dependent [6].

The major drawback of this assay is the low structural similarity between the pNPB and PET polymers [3], making it impossible to identify the actual activity on PET, which remains a comparative speculation.

**Assay with PET film**

An absorbance value for all the major degradation products of PET (MHET and TPA), was selected measured with a spectrophotometer at 260 nm absorbance [7]. Unfortunately, since MHET was not commercially available, BHET was used to calculate the calibration curve for its detection,. It is important to note that this method harbours a heavy drawback. Infact, the activity detected might be due to the hydrolysis of molecules on the PET surface rather than degradation of the PET core [3]. For this reason, electron microscopy scanning should be employed in the future for observing the mass loss of the PET film after hydrolysis.

Based on the absorbance profiles of MHET and TPA, they have distinct extinction coefficients at 260 nm of 5500 M−1 cm−1 and 4200 M−1 cm−1, respectively. Concentrations were calculated using the Beer-Lambert Law (Figure 4) [7].

![PETase graph](https://static.igem.org/mediawiki/2021/0/04/T--Edinburgh--Results3.f4.png)

**Figure 4** MHET and TPA (mM) produced by proteins after the degradation of PET film.

According to Figure 4, PETase tagged with L2NC, produced about three times more monomers (MHET and TPA) than PETase alone, indicating that L2NC increased the activity of the PETase enzyme. For the LCC enzyme, all the tags enhanced its activity, although the increase is not statistically significant. These results are debatable since the literature indicates that increasing hydrophobicity of proteins accounts for an increased affinity of enzymes for their substrate [8]. And modelling predicted that, PETase without a tag was the most hydrophobic, while PETase with L2NC was the least one. However, the findings of this assay were the opposite. On the other side, the activity of the LCC enzyme was enhanced in the presence of an SB7 tag that was more hydrophobic than the enzyme alone. However, the findings are not valid for Car9 and L2NC tags. Additionally, as shown by the control reaction (untransformed), there was also an indication of the presence of MHET and TPA produced. Thus, this assay may not be sensitive for detecting these two monomers, as many proteins also absorb at 260 nm and may provide false-positive results after the degradation of PET [7].

**References:**

1. Taniguchi K, Nomura K, Hata Y, Nishimura T, Asami Y & Kuroda A (2007) The Si-tag for immobilizing proteins on a silica surface. Biotechnol. Bioeng. 96: 1023–1029

2. Duedu KO, French CE. Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. 2016 [cited 2021 Aug 13]; Available from: https://doi.org/10.1016/j.enzmictec.2016.08.005

3. Pirillo, V., Pollegioni, L. and Molla, G. (2021) “Analytical methods for the investigation of enzyme-catalyzed degradation of polyethylene terephthalate,” The FEBS Journal. doi: 10.1111/FEBS.15850

4. Costa, S. et al. (2014) “Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system,” Frontiers in Microbiology, 0(FEB), p. 63. doi: 10.3389/FMICB.2014.00063.

5. A, Su et al. (2018) “Immobilized cutinases: Preparation, solvent tolerance and thermal stability,” Enzyme and microbial technology, 116, pp. 33–40. Doi: 10.1016/J.ENZMICTEC.2018.05.010.

6. Peterson, M. E. et al. (2007) “The dependence of enzyme activity on temperature: determination and validation of parameters,” Biochemical Journal, 402(Pt 2), p. 331. doi: 10.1042/BJ20061143

7. Zhong-Johnson, E. Z. L., Voigt, C. A. and Sinskey, A. J. (2021a) “An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films,” Scientific Reports 2021 11:1, 11(1), pp. 1–9. doi: 10.1038/s41598-020-79031-5.

8. Sarkar, A. and Kellogg, G. E. (2010) “Hydrophobicity – Shake Flasks, Protein Folding and Drug Discovery,” Current topics in medicinal chemistry, 10(1), p. 67. Available at: /pmc/articles/PMC2851852/

**Structural modelling:**

### AlphaFold2 prediction of recombinant Cex constructs.

Possible reasons for Cex-Car9’s lack of activity were assessed by using the recent AlphaFold2 package. Images of the models can be viewed in Figure 1.

![Alphafold Cex-Car9 model](https://static.igem.org/mediawiki/2021/9/9c/T--Edinburgh--Results_CexCar9.png)

**Figure 1 3D Structures of Cex-Car9 and Cex-L2NC** Structures generated with the
AlphaFold2 package and visualised on UCSF ChimeraX. A: active site of Cex. B:
Car9 C terminal tag. C: L2NC C-terminal tag.

It is important to note that both tags have a low local distance difference test (lDDT), indicating a low average accuracy of the residue positions. However, if the models in Figure 2 are to be considered, the positively charged Car9 tags are extremely close to the entrance of the negatively charged active site. On the other hand, the much larger L2NC tag coils around the enzyme and away from the active site, possibly explaining its increased activity. The Car9 tag could be impeding the binding of the substrate to the active site, thereby impacting the ability of Cex to catalyse the reaction.

### Further discussion

Another possible explanation for the increased activity of Cex-L2NC is the improved solubility of Cex-L2NC in comparison to Cex-Car9 and untagged Cex. An initial solubility experiment with induction at 37oC was run to assess the solubility of the recombinant protein in E. coli BL21 (DE3). The results of the resulting SDS-PAGE are shown in Figure 2.

![Cex solubility test](https://static.igem.org/mediawiki/2021/e/e5/T--Edinburgh--Results_Cexsolubility.png)

**Figure 2 Solubility test of Cex-Car and Cex-L2NC after incubation overnight at 37oC** Expected weight of Cex-Car9: 49.1 kDa. Expected weight of Cex-L2NC: 61.75 kDa. W: whole cell, S: soluble fraction, I: insoluble fraction.

The acrylamide gel shows that the majority of the recombinant protein is insoluble when induced at 37oC and therefore most likely aggregated and unfunctional. Several repeats of the solubility experiment were attempted with the 18oC however the results were consistently inconclusive. The results of the SDS-PAGE appear to show a greater proportion of the recombinant protein in the soluble fraction for Cex-L2NC than Cex-Car9. It is possible that the highly positively charged residues that comprise the L2NC tag help to act as a solubilising agent, thereby increasing the recombinant enzyme’s activity. Future experiments should continue with L2NC due to its increased solubility, activity and lower dissociation constant [1]

### Hydrophobicity of recombinant PETases and LCCs

For LCC and PETase enzymes, the hydrophobicity of the proteins was calculated with the Kyte and Doolittle scale model which determines it according to the hydropathy index of its amino acids, representing the hydrophobicity of their side chain. According to the graph (Figure 3), L2NC notably increased the solubility of both LCC and PETase, similarly to what previously observed when fused to Cex enzyme.

![PETase hydrophobicity comparison](https://static.igem.org/mediawiki/2021/8/83/T--Edinburgh--Results_PEThydrophobicity.png)

**Figure 3 Hydrophobicity of the proteins as measured by the Kyte and Doolittle scale.**  More negative scores indicate higher hydrophobicity.

**References:**

[1] Taniguchi K, Nomura K, Hata Y, Nishimura T, Asami Y, Kuroda A. The Si-tag for immobilizing proteins on a silica surface. Biotechnol Bioeng [Internet]. 2007 Apr 15 [cited 2021 Apr 23];96(6):1023–9. Available from: http://doi.wiley.com/10.1002/bit.21208