iGEM Leiden | 2019

S.P.L.A.S.H.

Suckerin Polymer Layer to Achieve Sustainable Health

Experiments

During the development of our project, experimentation has been a key part. For the production of a suckerin-based hydrogel, we set up numerous necessary experiments, with the use of various established protocols. Along the way, experimental settings had to be adjusted to optimize the results. All these processes, are described in the subdivisions of the page, namely experiments, documentation, and protocols. For easy navigation, you can use the side menu on the right to jump to the desired section.

Experiments

To conduct this project successfully several experiments were performed. All experiments described below are the initially planned experiments. Hyperlinks will be present to send you to the corresponding protocol, part(s) or results. If kits were in use the full kit name will be displayed together with the supplier. The experiments are ordered per organism and will follow a chronological order.

Of all known suckerins, suckerin-19 (also known as suckerin-39) has been most extensively studied, since this protein is most abundant in nature and has a classical representation of the suckerin protein family [1]. Besides suckerin-19, we focus on the production of suckerin-12, as it has previously been studied in relation to hydrogel production [2], as well as suckerin-8 and -9, which have a higher abundance in module 1 or 2, respectively, thereby representing different flexibility and rigidity [1,3].

Suckerin production in E. coli

Suckerin-8, -9, and -12

In order to establish stable suckerin production in E. coli multiple experiments were performed. First, genes encoding for suckerin-8 (BBa_K3041000), suckerin-9 (BBa_K3041001) and suckerin-12 were (BBa_K3041002) codon optimized for E. coli according to the IDT codon optimization tool, and subsequently the prefix and suffix were added to construct them into the biobrick format. These sequences were then synthesized by IDT and after arrival cloned into PBS1A3 using digestion with EcoRI and PstI, gel purification and ligation. The ligation mixture was transformed into the E. coli strain DH5α to obtain high plasmid concentrations. Cells were inoculated on LB agar supplemented with the appropriate selection antibiotic and successful transformants were then inoculated in liquid LB. After incubation, the plasmid was purified from the culture using either the Promega plasmid purification kit or the New England Biolabs Monarch kit. The plasmids were verified by digestion and thereafter the P_Lac expression cassette was added in front of the suckerins for inducable production (BBa_K3041015, BBa_K3041016 and BBa_K3041017). When successful, these plasmids were transformed into the production host Rosetta. Rosetta is an E. coli strain optimized for production of eukaryotic proteins. For production, the transformants were grown in liquid LB to an OD₆₀₀ between 0.6 and 0.8. Next, protein production was induced using 1 mM IPTG followed by additional growth for approximately 7 hours at both 18°C and 37°C. The cultures were spun down and used for protein purification.

Suckerin-19

To produce suckerin-19 we appealed to the expertise of the Miserez group from the Nanyang Technical University in Singapore. They kindly provided us with advice and their pQE80-L-STR19 plasmid containing the suckerin-19 gene. This plasmid was transformed into Rosetta to produce suckerin-19. After the protein was verified using a SDS-gel, primers were designed to create a biobrick of suckerin-19. This biobrick was placed in the PSB1A3 plasmid under control of the P_Lac expression cassette. In this way, production can be induced as described above.

Suckerin production in S. cerevisiae

For production in S. cerevisiae suckerin-6, -8, -9, and -12 were synthesized in biobrick format by IDT. These suckerins were cloned into pYES2 and pMU-His (His-tag) by digestion with EcoRI and SpeI, whilst the plasmids were digested with EcoRI and XbaI. This resulted in inducible plasmids for production in S. cerevisiae . After ligation, the mixture was first transformed in E. coli DH5α, the plasmid was purified with the Promega plasmid purification kit or the New England Biolabs Monarch kit. After verification, the plasmids were transformed into S. cerevisiae and protein production was induced using 1 mM Galactose.

Inducible system

To make the hydrogel adjustable to everyone’s needs, an inducible system was developed. All suckerin proteins are composed of a certain ratio of module 1 (M1) and 2 (M2). M1 is rich in alanine, valine, histidine, serine and threonine resulting in rigidity and strength (BBa_K3041007). Module 2, on the other hand, mainly consists of glycine and provides the protein with a flexible phenotype (BBa_K3041008). This system provides the possibility to synthesize a suckerin protein which consists of the perfect combination of M1 and M2, which are under the regulation of the inducible promoters lactose and arabinose, respectively. This allows for a customizable system in which characteristics can be adjusted as desired, thereby creating an on-demand artificial suckerin synthesis system.

Protein purification

Two approaches of protein purification were examined. First, His-tag purification using the His-tag purification system from Promega and secondly, the salting-out protocol provided by the Miserez group. The purified proteins were loaded on SDS-PAGE to validate their size. In the case that corresponding proteins sizes were observed, production was scaled-up using bioreactors.

Hydrogel formation

Protein purification yielding a high quantity, enables the formation of a hydrogel. According to literature the hydrogel can be formed by pouring the purified suckerin in a cast together with 0.04% hydrogen peroxide (H₂O₂) and hydrogen peroxidase. This enables protein cross-linking and thereby the formation of a hydrogel.

Linker system

As part of the original project the linkers were going to be implemented as a cleavable link between the hydrogel and antimicrobial peptides, numbing agents or wound healing agents to create a versatile system. This offers the potential for controlled delivery and thereby prevents excessive release. In our case we focussed on linkers that can be cleaved by possible pathogenic activity. To achieve this the V8-linker and SplB-linker were proposed. These linkers can be cleaved by the corresponding V8 protease or SplB protease produced by S. aureus . During a pathogenic infection caused by S. aureus, the activity of the V8 and SpIB proteases will result in cleavage of the linkers, thereby releasing the antimicrobial peptides. In our system antimicrobial peptides such as human-derived antimicrobial peptide CAP18 can be released upon infection, thereby inhibiting the development of resistance and unnecessary exposure to antimicrobial peptides.

Documentation

Here we would like to describe the hurdles we faced and how we overcame them. Further, we describe how we kept a mutual notebook.

In an iGEM project, a lot of people need to work together. Therefore the Leiden iGEM team decided to start every morning with coffee, cookies and a discussion on what needed to be done and what difficulties we faced the day before. After this, all tasks were divided among the people present and our lab day would start. This approach gave us a clear insight into the adjustments that needed to be made and the phase we were in. During the project, we suffered a lot of cloning issues. To overcome them we discussed this with our PI’s and experts. To make sure the experiments did not fail due to mutations of mismatches in the plasmids, some were sent for sequencing. Eventually, we managed to construct the needed plasmids allowing us to produce the required protein.

As we induced the production according to the protocol provided by the Miserez group. Most efficient purification conditions were investigated by adjusting IPTG concentrations, incubation time and incubation temperature. From these results, we concluded that a growth period of 8-16 h is needed to achieve the optimal OD for induction (OD₆₀₀ of 0.6-1.0). After induction, the cultures were grown for an additional 7 h. To test if our method would yield protein, the first tests were performed with a suckerin-19 producing Rossetta strain. In this case, the protein was purified by His-link purification.

After production, the protein needed to be purified. Several methods were tested including His-tag purification with added His-tag, His-tag purification based on the histidine residues in the protein, Salting-out and dialysis. Salting-out and dialysis resulted in the highest yield of protein.

These initial production experiments were performed in small volume Erlenmeyer flasks. Therefore, the next step was to scale up production using bioreactors. With the help of Mandy Hulst, we have been able to achieve the successful production of suckerin-12 and suckerin-19. These proteins could now be used to create a suckerin-based hydrogel.

Furthermore, we worked on the linker system. This linker system can add antimicrobial features to the hydrogel antimicrobial, meaning that in times of an infection antimicrobial peptides will be released. A lot of work was conducted, however we have not been able to finish the system due to time constraints. This emphasizes the importance to start long cloning chains earlier in the process.

To make sure all progress was documented we started a SciNote journal where everyone could fill in their own experiments. This allowed us to work separately and together at the same time.

Another measure we took, to make our work as consistent as possible, was the labelling poster. This poster described the labelling rules, which had to be followed by every team member. This made cross experiments easier and the lab work more efficient.

Protocols

Oligo annealing

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

Add:

1 µL of oligo I (100 µM)
1 µL of oligo II (100 µM)
1 µL 10X T4 ligase buffer
1 µL T4 PNK (NEB)
6 µL MilliQ

Anneal oligo's
37 °C for 30 min
95 °C for 5 min. then ramp down to 25 °C at 5°C/min.

Hold at 4 °C until ready to proceed

Gel electrophoresis

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

Note: This protocol uses Ethidium Bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

Heat a 1% agarose TAE buffer solution to dissolve the agarose
Add EtBr to a final concentration of 0.5 μL/mL and transfer the mixture to a gel electrophoresis mold
Allow the gel to solidify
Place the gel into an electrophoresis device filled with TAE buffer and load 4 µL of 1kb gene ruler
Run the gel at 100 V for 20 to 60 minutes (depending on the size of the fragment you want to observe)
Transfer the gel to a Geldock and visualize the DNA bands

Digestion

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

For digestion of a plasmid or fragment consult the corresponding Snapgene file to determine which restriction enzymes to use. For the restriction buffer consult with the supplier which buffer is needed for duo restriction.

DNA	800 ng
Restriction enzyme 1	0.5 µL
Restriction enzyme 1	0.5 µL
10x restriction buffer	1.5 µL

Add H₂O to a final volume of 15 µL
Digest for 2 hours at 37 °C

Gel electrophoresis

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

Heat a 1% agarose TAE buffer solution to dissolve the agarose
Add EtBr to a final concentration of 0.5 μL/mL and transfer the mixture to a gel electrophoresis mold
Allow the gel to solidify
Place the gel into an electrophoresis device filled with TAE buffer and load 4 µL of 1kb gene ruler
Run the gel at 100 V for 20 to 60 minutes (depending on the size of the fragment you want to observe)
Transfer the gel to a Geldock and visualize the DNA bands

DNA fragment isolation from agarose gels

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

Always use a freshly prepared agarose/TAE solution (old agarose reduces the recovery of the DNA significantly)
Cut out fragment (using a long UV wavelength, 365 nm). Collect a maximum of 2 lanes of a mini gel per Eppendorf tube
Add 200 μL water and crush agarose slice as good as possible with a long small piece of plastic
Add 500 μL phenol (saturated with 10 mM Tris pH 7-8) and mix very well
Incubate the sample at -80 °C for at least 10 min.
Centrifuge the sample in an Eppendorf centrifuge for 10 min. at RT and max speed
Take water layer (upper layer) and extract once with chloroform
Take water layer (upper layer), add 40 μL 3 M potassium acetate (KAc) pH 7.0 (~1/10 volume) and 1 mL of 96% EtOH
Incubate overnight at -20 °C
Centrifuge for 10 min. at max rpm and wash pellet with 200 µl of 70% EtOH
Dry pellet at RT (leave open Eppendorf tube on the bench)
Resuspend pellet in 15 μL sterile water

Ligation

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

After gel purification, add 2 µL of ligation buffer, 1 µL of ligase and 2 µL of ATP solution (optional)
Ligate at RT for a minimum of 2 hours

Polymerase chain reaction (PCR)

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

Prepare the PCR reaction as indicated below:

Component	Volume (final concentration)
Taq polymerase buffer (10x)	5 μL
Taq polymerase	1 μL
dNTPs	4 μL (200 μM total)
Forward primer	0.5 μL (0.5 μL)
Reverse primer	0.5 μL (0.5 μL)
MilliQ	39 µL
Total	50 µL

Add 1 μL of genomic DNA (approximately 200 ng) to the reaction mix

Put the sample(s) in a PCR machine and run the following protocol:

Step 1 (1x)	2 min. @ 94 °C
Step 2 (30x)	30 sec. @ 94 °C
	30 sec. @ *°C
	** sec. @ 72 °C
Step 3 (1x)	5 min. @ 72 °C
	hold @ 12 °C

*note: this temperature depends on your primer pair

**note: depends on the length of the amplified piece (around 1 min./kb)

Check the PCR products by performing gel electrophoresis (see protocol gel electrophoresis)

Preparing cool competent cells

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

Inoculate 10-12 medium sized (2-3 mm) colonies in 25 mL of SOB- medium in a 100 mL flask. Incubate overnight at RT with shaking at 150 rpm until an OD₆₀₀ between 0.2-0.6 is reached
Transfer cells to a 50 mL tube and keep on ice for 10 min.
Centrifuge the cells for 10 min. at 2000xg and 4 °C
Resuspend the pellet in 10 mL of ice-cold TB and subsequently put on ice for 10 min.
Centrifuge the cells as mentioned before. Afterwards, resuspend the pellet in 4 mL of TB. Add DMSO with gentle swirling to a final concentration of 7%. Next, put the cells on ice for 10 min.
Pipette aliquots of 100 µL into sterile 1.5 mL Eppendorf tubes and freeze immediately in liquid nitrogen
Tubes should be stored at -80 °C

Media and solutions

TB per liter:

10mM Pipes	3.02 g
15 mM CaCl₂	2.2 g
250 mM KCl	18.64 g

Mix all components except for MnCl2 and adjust pH to 6.7 with KOH
Dissolve MnCl₂
Sterilize the solution by filtration through a 0.2 µm filter
Store at 4 °C

SOB per liter:

2% Bacto-Trypton	20 g
0.5% Bacto-Yeast Extract	5 g
10 mM NaCl	0.5 g
2.5 mM KCl	10 mL of 250 mM KCl

Adjust pH to 7 with NaOH and sterilize by autoclaving.

Before use add:

10 mM MgCl₂	5 mL of 2 M MgCl₂ (sterile)
10 mM MgSO₄	10 mL of 1 M SO₄ (sterile)

Preparing LB agar plates

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

Add LB agar to dH₂O in the prescribed concentration (Sigma LB broth with agar Lennox 35 g/L)
Sterilize by autoclaving
Let the solution cool down to 60 °C and add antibiotics (see concentrations below) when necessary
Pour ~25 mL LB-agar in empty petri dishes
Leave the petri dishes open until the agar is completely solidified (approximately 10 min.)

Final concentration of antibiotics in the agar:

Ampicillin: 100 μg/mL
Chloramphenicol: 25 μg/mL
Kanamycin: 50 μg/mL
Tetracyclin: 10 μg/mL

E. coli transformation

Thaw competent E. coli DH5α cells on ice
Add 10 µL of ligated construct solution or 1 µL of prepped plasmid to 100 µL cells and mix carefully by tapping the tube
Incubate the sample on ice for 30 min.
Incubate the sample for 60 sec. at 42 °C (heat shock) before returning it to ice
Add 1 mL LB medium to the sample and incubate at 37 °C for an hour with constant shaking at 120 rpm
Inoculate 1 plate with 100 µL and 1 plate with the rest ~900 µL of the obtained solution or centrifuge culture for 2 min at 13.4 rpm, then discard 800-900 µL LB and resuspend the pellet in the leftover medium and plate on LB agar plates with appropriate antibiotic(s) and incubate overnight at 37 °C

Preparing Streptomyces protoplasts + transformation

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

Protoplasts:

Inoculate 40 µL of a fresh spore stock in 20 mL YEME with 5 mM MgCl₂ (stock 1 M) and 0.5 % Glycine (stock 20%) and incubate overnight at 30 °C
Transfer the culture to a 50 mL tube and centrifuge for 10 min. at 5000 rpm and 4 °C
Wash the pellet once with 10 mL 10.3% sucrose
Resuspend the pellet in 3 mL P-buffer and add 4 mg/mL lysozyme (filter sterilized) in 1 mL P-buffer
Incubate the cells for 60 min. at 30 °C
Pipette up and down three times with a 5 mL glass/plastic-pipette and incubate for another 15 min. at 30 °C
Add 5 mL P-buffer, pipette up and down three times and put on ice
Filter suspension using a 20 mL syringe containing a piece of cotton wool
Centrifuge the tube for 10 min. at 3000 rpm and 4 °C, then gently resuspend the pellet by pipetting up and down with 200-1000 μL P-buffer (depending on the size of the pellet)
Pipette aliquots of 50 µL into sterile 1.5 mL Eppendorf tubes
Protoplasts can be frozen at -80 °C

Transformation:

Thaw protoplasts on ice (50 μL = ~10¹⁰ protoplasts)
Add up to 5 μL DNA in water and mix gently by tapping
Immediately add 200 μL 25% PEG in P-buffer and mix gently by pipetting up and down three times or by tapping
Inoculate the cells on R5 plates
After incubation of 14-20 hours, overlay the plates with 1 mL water containing the appropriate antibiotic

Final concentration antibiotics in the agar plates:

Thiostrepton 20 μg/mL
Apramycin 50 μg/mL
Hygromycin 50 µg/mL for S. coelicolor or 200 μg/ml for S. lividans

Yeast transformation

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

Inoculate 50 mL MY (Maltose yeast medium) + Uracil (final concentration of 76 mg/L) with a yeast colony overnight at 30 °C.
Measure OD at 620nm and dilute cells in 50 mL YDP to OD₆₂₀ between 0.1 and 0.2
Centrifuge in sterile 50 mL tube and resuspend cell pellet in 25 mL MilliQ
Centrifuge for 3 min. and resuspend pellet in 1.0 mL 100 mM Lithium acetate
Transfer to 1.5 mL Eppendorf tube
Centrifuge 15 sec. and remove supernatant
Resuspend cells in 1 mL of 100 mM Lithium acetate
Add:
- 240 µL PEG 3350 (50% w/v)
- 36 µL 1 M LiAc
- 25 µL single-stranded salmon semen DNA (2.0 mg/mL denaturated by incubation at 100 °C for 10 min.)
- 48 µL H₂O + 2 µL plasmid DNA
Vortex and incubate 30 min. at 30 °C
Heat shock 20 min. at 42 °C
Centrifuge at 6-8000 rpm for 15 sec. and remove supernatant
Add 1 mL MilliQ and resuspend cells by carefully pipetting up and down
Spread samples of 250 µL on selective MY agar plates and incubate 3 days at 30 °C

Protein purification

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

By kit: Protocol of the promega His-tag purification system

By Singapore protcol:

Grow E. coli strain Rosetta pQE80-L-SRT19 in Luria Bertani miller broth medium (LB) to an OD₆₀₀ 0.6-0.8
Induce the target proteins with 1 mM IPTG
Incubate for 7 hours at both RT and 37 °C
Harvest cells by centrifugation at 4000 rpm for 30 min.
Store at -80 °C until further use
Resuspend cell pellet in 40 mL of lysis buffer (50 mM Tris at pH 7.4, 20 mM NaCl, 5 mM DDT and 1 mM PMSF (PMSF is used to prevent the proteins from being degraded by enzymes)
Sonicate cells (20 min., followed by pulse on for 2 min. and off for 5 min.)
Centrifuge cell-disrupted samples at 19000 rpm at 4 °C for 50 min.
Discard the supernatant and wash the cell pellet four times with 50 mL of wash buffer I (2% Triton X-100, 100 mM Tris at pH 7.4, 5 mM EDTA, 2 mM urea and 5 mM DDT)
After each wash step, centrifuge the solution at 6450 rpm for 15 min. and discard the supernatant
Wash cell pellet three times with 50 mL of wash buffer II (100 mM Tris pH 7.4, 5 mM EDTA and 5 mM DDT)
After each wash step, centrifuge the solution at 6450 rpm for 10 min. and discard the supernatant
Re-solubilize the cell pellet in 40 mL of 5% acetic acid and centrifuge the samples at 19000 rpm at 4 °C for 50 min.
Collect the supernatant and discard the pellet
Place the supernatant on SDS-PAGE

Protein Inclusion Body Purification

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

For the purification of suckerin-12 and -19 after large-scale production, we referred to protocols written by Buck et al. and Ding et al., respectively [3, 4].

Suckerin-12 purification

Materials:

Lysis buffer	50 mM Tris pH 8.0
	100 mM NaCl
	100 µg/mL lysozyme
Triton X-100 wash	1% (v/v) Triton X-100
	50 mM Tris pH 8.0
	100 mM NaCl
Wash buffer	50 mM Tris pH 8.0
	100 mM NaCl

Method:

- Cell pellet stored at -80°C

- Resuspend cell pellet in lysis buffer

- Disrupt cells by sonication (20 min, followed by pulse on for 2 mins and off for 5 min.)
- Collect cell-disrupted samples by centrifuging at 1900 rpm for 50 min. and 4°C

- Wash 2x with Triton X-100 wash I
- Wash 1x with wash II
- Resuspend in water
- Adjust pH to 3 with 1 M HCl
- Collect supernatant with 8000 rpm 10 min. and discard pellet
- Adjust pH to 8 with 1 M Tris HCl pH 9, 100 mM NaCl
- Centrifuge
- Wash precipitate 2x with 80% ethanol.
- Dry precipitate
- Resuspend in water at 6% (w/v)
- Adjust pH to 3 with Glacial Acetic acid
- Dialyse against Acetic Acid
- Lyophilize
- Dried protein stored at -80 °C

For direct use:
- Resuspended in 6% (w/v) water
- Adjust pH to 5 with Glacial Acetic Acid.

Suckerin-19 purification

Materials:

Lysis buffer	50 mM Tris pH 7.4
	20 mM NaCl
	5 mM DDT
	1 mM PMSF
Wash buffer I	2% (v/v) Triton X-100
	100 mM Tris pH 7.4
	2 mM urea
	5 mM DDT
Wash buffer II	100 mM Tris pH 7.4
	5 mM EDTA
	5 mM DDT

Method:

- Cell pellet stored at -80°C

- Resuspend cell pellet in lysis buffer

- Disrupt cells by sonication (20 min, followed by pulse on for 2 mins and off for 5 min)
- Collect cell-disrupted samples by centrifuging at 1900 rpm for 50 min and 4°C
- Wash 4x insoluble inclusion bodies with 50 mL Wash buffer I. After each wash step, centrifuge at 6450 rpm for 15 min.

- Wash 3x with 50 mL Wash buffer II. After each wash step, centrifuge at 6450 rpm for 10 min.
- Resolubilize in 40 mL 5% Acetic acid
- Centrifuge at 1900 rpm for 50 min.
- Collect supernatant and discard pellet.

- Dialyze against 5% Acetic acid (2 L) for 48 hours.
- Freeze o/n at -80°C
- Lyophilize

SDS-PAGE

Note: This protocol uses ethidium bromide (EtBr), which is a carcinogenic compound. Materials that come in contact with EtBr need to be handled with a glove.

Preparing the separation gel:

Set the casting frames (clamp two glass plates in the casting frames) on the casting stands
Prepare the gel solution in a separate small beaker Note: The APS and TEMED must be added right before each use (and in the fume hood), since they initiate the gelification
Swirl the solution gently but thoroughly
Pipet appropriate amount of separation gel solution into the gap between the glass plates
To make the top of the separating gel horizontal, add isopropanol to cover the top of the gel
Wait 40 min. to let it gelify

Make the stacking gel:

Discard the isopropanol
Pipet stacking gel until overflow
Insert the well-forming comb without trapping air under the wells. Wait for 40 min. to let it gelify

Approximate volumes of 700 mL SDS-running buffer for 1-2 gel or 1 L for 3-4 gel is needed.

Run at about 40 V, until proteins enter separation gel (~30 min.)
Increase the voltage to 120 V and run for ~40 min.
After electrophoresis (or after fixation), put the gel in a box with hot dd-water (microwave for ~15 sec.)
Shake for 3-5 min., repeat twice, make sure the SDS is completely removed
Add enough staining solution to cover the gel, heat in the microwave for 10 sec. (avoid boiling), then put on a shaker for 30 min.
Pour staining solution off and add dd-water to de-stain to a proper background level
Visualize the protein bands using a Geldock

Gels and buffers

Seperation gel:

H₂O	3123 µL
1.5 M Tris-HCl, pH 8.8	2500 µL
30% A./Bis-acrylamide	4167 µL
10% (w/v) SDS	100 µL
10% (w/v) APS	100 µL
TEMED	5 µL

Stacking gel:

H₂O	2250 µL
0.5 M Tris-HCl, pH 6.8	1000 µL
10% (w/v) SDS	40 µL
30% Acrylamide/Bis-acrylamide (29:1)	666 µL
10% (w/v) APS	40 µL
TEMED	4 µL

5x loading buffer:

10% (w/v) SDS	1 g/10 mL
40% (v/v) Glycerol	4 mL 100% Glycerol/10 mL
0.2 M Tris-HCl, pH 6.8	4 mL 0.5 M Tris-HCl/10 mL
0.05% Bromophenolblue	5 mg/10 mL
H₂O	1 mL
1% Dithiothreitol/β-mercapto-ethanol	10 µL/mL add just before use

SDS-running buffer:

250 mM Tris-HCl	30 g/L
2 M Glycine	144 g/L
1% (w/v) SDS	10 g/L

Staining solution:

Coomassie brilliant blue G-250	30-40 mg
dd-water	500 mL
Stir for 2-4 hours
37% HCl	1.5 mL

References

Guerette P., Hoon S., Ding D., Amini S., Masic A., Ravi V., . . . Miserez A. (2014). Nanoconfined beta-Sheets Mechanically Reinforce the Supra-Biomolecular Network of Robust Squid Sucker Ring Teeth. Acs Nano, 8(7), 7170-7179.
Hiew S., & Miserez A. (2017). Squid Sucker Ring Teeth: Multiscale Structure-Property Relationships, Sequencing, and Protein Engineering of a Thermoplastic Biopolymer. Acs Biomaterials Science & Engineering, 3(5), 680-693.
Buck C., Dennis P., Gupta M., Grant M., Crosby M., Slocik J., . . . Naik R. (2019). Anion‐Mediated Effects on the Size and Mechanical Properties of Enzymatically Crosslinked Suckerin Hydrogels. Macromolecular Bioscience, 19(3), N/a.
Ding D., Guerette P., Hoon S., Kong K., Cornvik T., Nilsson M., . . . Miserez A. (2014). Biomimetic production of silk-like recombinant squid sucker ring teeth proteins. Biomacromolecules, 15(9), 3278-3289.

Team:Leiden/Experiments

S.P.L.A.S.H.

Suckerin Polymer Layer to Achieve Sustainable Health

Experiments

Experiments

Suckerin production in E. coli

Suckerin production in S. cerevisiae

Inducible system

Protein purification

Hydrogel formation

Linker system

Documentation

Protocols

Oligo annealing

Gel electrophoresis

Digestion

Gel electrophoresis

DNA fragment isolation from agarose gels

Ligation

Polymerase chain reaction (PCR)

Preparing cool competent cells

Preparing LB agar plates

E. coli transformation

Preparing Streptomyces protoplasts + transformation

Yeast transformation

Protein purification

Protein Inclusion Body Purification

SDS-PAGE

References