Protocols, media and devices we used throughout the iGEM project

Assays

TCA vs Acetone Precipitation

This protocol is to test whether Acetone or TCA is a more suitable reagent for precipitating secreted protein.

A preculture of one HA-protein tagged secreting Chlamydomonas culture, a negative control (e.g., UVM4) and a positive control (L5) were prepared in TAP-NH₄ medium. Cultures were incubated under continuous light (70 μmol photons m⁻²s⁻¹) with orbital shaking at 120 rpm for seven days.
Cell density was determined and the HA-protein tagged secreting Chlamydomonas strain was diluted into 2 Erlenmeyer flasks to a density of 2×10⁵ cells/ml. Two negative controls and one positive control were included.
After six days, the cultures were harvested and 5 technical replicates were precipitated, one half with acetone and the other half with TCA.
20 μl of SDS sample from the acetone-precipitated protein and ~4.1 μl from the TCA precipitated protein sample were loaded onto an SDS-PAGE gel to ensure correct ratio of protein concentration.
SDS-PAGE and Western Blotting were performed as described in the standard protocols.

**Figure 1:** SDS-PAGE loading scheme for the TCA vs. Acetone precipitation test. 3.5 μl PageRuler™ Plus Prestained Protein Ladder is used as the marker. For the HA-protein tagged secreting Chlamydomonas strain and negative control (UVM4), 4.1 μl of TCA-precipitated sample and 20 μl of acetone precipitated sample were loaded. PKL5 was included as a positive control. Created with Biorender.

Volume test

This assay is designed for the initial scale-up of protein-secreting strains. It allows an estimation of the culture volume that yields the highest protein amount.

A preculture of one HA-protein tagged secreting Chlamydomonas culture was prepared in TAP-NH₄ medium. Cultures were incubated under continuous light (70 μmol photons m⁻²s⁻¹) with orbital shaking at 120 rpm for seven days.
Cell density was determined, and the test strain was diluted into 1000 mL adjusting to a final density of 2×10⁵. The flasks from 6 mL to 400 mL were filled with this dilution. Dilution was repeated for the 1000 mL flask. Cultures were incubated again for six more days.
After six days, the supernatant was harvested, and proteins were precipitated with ice-cold acetone.
SDS-PAGE and Western blotting were performed as described in the standard protocols.

**Figure 2:** Volume test. **(A)** Erlenmeyer flasks with culture volumes ranging from 6 mL to 100 mL. **(B)** SDS-PAGE loading scheme for the volume test. 3.5 μl PageRuler™ Plus Prestained Protein Ladder is used as the marker. 10 μl of acetone precipitated sample from each culture volume (6-1000 mL) were loaded. Created with Biorender.

ST-Test (secretion-time-Test)

This assay is designed to test the required amount of time for the Chlamydomonas cultures to accumulate the secreted scFv in the media. It allows optimization of the incubation time for subsequent experiments by determining when sufficient protein accumulation has occurred.

Liquid Chlamydomonas cultures of CetFv and HumcetFv and a negative control (e.g. UVM4) were prepared. Cultures were incubated under continuous light (70 μmol photons m⁻²s⁻¹) with orbital shaking at 120 rpm for several days.
Three 25 ml flasks were inoculated with the respective Chlamydomonas cultures, and the final cell density was adjusted to 2 × 10⁵ cells/ml.
Samples of the CetFv and HumcetFv cultures were harvested continuously for six days, starting at day zero after inoculation.
The proteins were precipitated after each harvest using the acetone precipitation method and the acetone-precipitated proteins were frozen at –20°C.
The acetone precipitation protocol was continued and SDS-PAGE followed by Western blotting was performed according to protocols.

**Figure 3:** Schematic overview of the ST-test workflow showing daily sampling from Chlamydomonas cultures over six days, followed by acetone precipitation and protein analysis via SDS-PAGE and Western blotting. Created with Biorender.

Pulldown-Interaction Assay

This interaction assay is designed to determine whether the single-chain fragment can specifically interact with its corresponding receptor epitope (e.g., EGF receptor).

Liquid cultures of CetFv, HumcetFv and a negative control (e.g., PETase) were prepared in TAP-NH₄ medium. Cultures were incubated under continuous light (70 μmol photons m⁻²s⁻¹) with orbital shaking at 120 rpm for several days. note: test proteins must not carry a His-tag.
Cultures were harvested several days after stationary phase in 50 ml Falcon tubes.
Cultures were centrifuged 2x at 4,000g for 4 min to harvest supernatant.
Proteins were concentrated by ammonium sulfate precipitation followed by dialysis in PBS overnight.
66 μl Ni-NTA were equilibrated in PBS in a reaction tube.
20 μl of the EGFR (50 ng/μl) were mixed with 300 μl of ammonium sulfate-concentrated protein and incubated for 30 min at 37°C.
A sample for Western blot analysis was taken, and the volume was adjusted to 1 ml by adding PBS.
Storage buffer was removed from Ni-NTA by plugging a white tip into a blue one. The complete sample was loaded into each Ni-NTA reaction tube and 40 μl of cOmplete™ protease inhibitor were added.
Samples were incubated for 30 min under constant inversion, then placed on ice for 10 min.
Samples were centrifuged at 100 g for 30 sec at 4 °C.
A sample was collected, the supernatant was discarded, and the beads were washed with PBS and 5 mM imidazole.
Samples were centrifuged at 100 g for 30 sec at 4°C.
A sample was taken, the wash buffer was discarded, and 50 μl elution buffer with PBS and 500 mM imidazole were added. Samples were incubated for 2 min.
Samples were centrifuged at 100 g for 30 sec at 4 °C.
The supernatant was collected, and all samples were precipitated with ice-cold acetone.
SDS-PAGE and Western blotting were performed according to standard protocols.

**Figure 4:** Pulldown-Interaction Assay. In the first reaction tube, EGFR and scFv interact with each other. After incubation, the mixture is transferred into a new reaction tube with Ni-NTA beads. The His-tagged EGFR binds to Ni- NTA, keeping the scFv from being washed away. Created with BioRender.

Resazurin Assay

This assay is designed to evaluate the cytotoxic effects of various treatments on cell viability using a resazurin-based metabolic activity indicator.

40,000 cells in triplicates per well in cell culture medium for the 24h incubation period and 10,000 cells in triplicates per well in cell culture medium for the 72h incubation period were plated. Cells were incubated for 24h.
Solutions containing the amount of drug to be tested were prepared. The Cetuximab, scFv, 5-FU and saponin solutions were diluted in a volume of 700 μl for both plates.
Resazurin stock solution was prepared according to the manufacturer's protocol.
After the 24h incubation period, the medium from the wells containing the cells was aspirated and 100 μl of the respective solution were added to the wells in triplicates. Cells were incubated again, and the starting time of the treatment was noted down.
600 μl of the prepared resazurin stock solution were mixed with 5.4 ml of cell culture medium.
The medium from the cells was aspirated and 100 μl of the resazurin mixture were added to the cells. Cells were incubated for exactly 90 minutes.
After the incubation, cells were taken out, and the fluorescence intensity was measured at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.
Steps 5 – 7 were repeated for the 72h incubation plate.

**Figure 5: (A)** 96-well plate after initial treatment with the resazurin mix before the 90-minute incubation period **(B)** 96-well plate expectations after the 90 minutes incubation period, validating the cytotoxic effects of the commercial cetuximab and the scFv **(C)** results after the 90 minutes incubation period, validating the survival of the cetuximab and scFv treated cells.

General Protocols

DNA Protocols

Modular Cloning

Calculate the volume needed for a final concentration of 40 fmol for each part. Knowing the size of the part allows you to calculate the amount of DNA (in ng) equaling 40 fmol. (For this you can use online tools.) Mix the components in a small reaction tube, add the enzymes (BsaI and T4 DNA Ligase) last. Place tubes into a thermocycler.

Component	Amount	Concentration	Protocol
MoClo Parts (n) Part 1 Part 2 Part 3 ... Destiniation vector	1 µl	40 fmol	37°C 2 min 16°C 5 min repeat 45 x 50°C 5 min 80°C 10 min 4°C ∞
T4 DNA Ligase buffer	2 µl	1x
BsaI-HFv2® or BbsI-HF	0.5 µl	10 U
T4 DNA Ligase	0.5 µl	10 U
Milli-Q	To 20 µl	-

Transformation in Escherichia coli

Thaw One Shot® TOP10 (NEB) E. coli cells on ice.
Carefully pipette 10 µl of your MoClo reaction onto the cells, mix, and incubate for 30 min on ice.
→ Preheat the thermomixer to 42°C for the subsequent heat shock.
Heat shock E. coli cells for 50 s at 42°C. Recover for 5 min on ice.
Work sterile: Add 600 µl LB-medium and incubate cells for 45 min at 37°C in the incubator.
Work sterile: Spread out 300 µl onto LB-plates containing spectinomycin (level 0) or kanamycin (level 2). Let plates dry under the clean bench.
Incubate plates overnight at 37°C.

Isolation of Plasmids

Work sterile: Prepare sterile test tubes with 5 ml LB-medium each containing spectinomycin (level 0) or kanamycin (level 2). For each transformation plate, prepare three tubes.
Take out your plates and gently pick a single white colony with a yellow pipette tip, which will then be dropped into the respective test tube. Repeat for two more positive colonies of your transformation plate.
Incubate test tubes overnight at 37°C.
Prepare Eppendorf tubes for harvesting the E. coli cultures and for their respective plasmids, which will be eluted later.
Harvest 2 ml by centrifugation at 4000 rpm for two minutes. Discard supernatant.
Use the plasmid prep kit provided.

Resuspend pellet in 150 µl Buffer A1, vortex.
Add 250 µl Buffer A2 and invert five times. Incubate for 2 min at RT.
Add 350 µl Buffer A3, invert until sample turns colorless.
Centrifuge at full speed (> 12,000 g) for 3 min. Pipette supernatant (750 µl) onto the column provided with the kit.
Centrifuge columns at 2000 g for 30 s. Discard flow through.
Add 450 µl Buffer AQ, centrifuge at full speed (> 12,000 g) for 1 min.
Transfer column to the prepared Eppendorf tube. Add 50 µl Buffer AE onto the column membrane, incubate at least 1 min at RT.
Centrifuge 1 min at full speed to elute plasmids from the column.

Measure DNA concentration at the Nanodrop. Pipette 1 µl of Buffer AE onto the sample spot for blank measurement. Then pipette 1 µl of your plasmid and measure the DNA concentration.

Test Digest

Mix components in Eppendorf tube and incubate at 37°C for 45 min.
Prepare 1% agarose gels with 1x TAE buffer. Dissolve 1 g agarose in 100 ml 1x TAE. Heat solution in microwave and let cool down to 60°C. Then add 5 µl HD Green. Pour agarose gels.
Add 4 µl of 6x loading dye to your test digest samples and load 10 µl onto the agarose gel.
Separate DNA fragments by gel electrophoresis (110 V, 45 – 55 min).
Analyze your gels using the Intas device. Compare your observed fragments with the theoretical in silico test digest.

Nuclear Transformation via Glass Beads

Work sterile! Chlamy clean bench!

For each transformation process:
Linearize 1 µg DNA in a final volume of 10 µl (1 µg DNA, 1 µl CutSmart buffer, 0.5 µl enzyme, to 10 µl with MQ)
→ Use enzyme, which only cuts the backbone of your construct (Check in silico)!
Digest/linearize your construct for 45 min at 37°C.
Measure cell density with the CoulterCounter
For each transformation 5*107 cells are needed, calculate the volume according to the cell density of your culture. Always include a negative control without DNA!
Centrifuge cultures at 4000 rpm for 2 minutes (use sterile 50 ml Falcon tubes).
Discard supernatant and resuspend cells in 330 µl fresh TAP medium.
Add 330 µl of your cell suspension to the sterile reaction tubes filled with glass beads, pipette 10 µl of the linearized DNA to the side of the tube → for the negative control, do not add any DNA.
Take two tubes and vortex for 15 s
Plate out 330 µl onto your plates containing the appropriate antibiotic (avoid glass beads on your plates!)
→ This works best if you pipette at the bottom of the tube and remove the remaining beads on the lid.
Let plates dry under the hood, seal with Parafilm and place into the dark overnight.
Place plates into the light in the next morning.
Wait several days until single colonies appear. Transfer single colonies to fresh agar plates.

Site-Directed Mutagenisis

Pipette the following reagents into PCR tubes:

Reagent	Volume
Q5 Buffer	10 µl
dNTPs	1 µl
Primer forward	2.5 µl
Primer reversed	2.5 µl
Template DNA	10 µl
Q5 Polymerase	0.5 µl
GC Enhancer	10 µl
MQ	13.5 µl

Start the PCR with the following cycles

STEP	TEMP	TIME
Initial Denaturation	98 °C	30 sec
10 Cycles	98 °C 63°-72° gradient 72 °C	07 sec 25 sec 2 min
25 Cycles	98 °C 72 °C 72 °C	07 sec 25 sec 2 min
Final Extension	72 °C	2 min
Hold	4 °C	∞

PCR Clean-Up

NucleoSpin® Gel and PCR Clean-up Kit and protocol by MACHEREY-NAGEL were used

Adjust DNA binding conditions

For very small sample volumes < 30 μL adjust the volume of the reaction mixture to 50–100 μL with water.
Mix 1 volume of sample with 2 volumes of Buffer NTI (e.g., mix 100 μL PCR reaction and 200 μL Buffer NTI).

Bind DNA

Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 mL) and load up to 700 μL sample.
Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube.
Load remaining sample if necessary and repeat the centrifugation step.

Wash silica membrane

Add 700 μL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up Column. Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube.
Recommended: Repeat previous washing step to minimize chaotropic salt carry-over and improve A260/A230 values (see section 2.7 for detailed information).

Dry silica membrane

Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.

Elute DNA

Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 mL microcentrifuge tube (not provided). Add 15–30 μL of hot Buffer NE and incubate at 70 °C for 5 min. Centrifuge for 1 min at 11,000 x g

Protein Analysis & Quantification

TCA Precipitation of Chlamydomonas Supernatant

Harvest 1.9 ml liquid culture of the transformants of interest in a reaction vessel. Culture growth time should be at least 3 days (6 days would be optimal)
Centrifuge at 1,700 g for 4 min, transfer 1.8 ml supernatant into a new reaction vessel
Centrifuge again at 4,000 g for 4 min, transfer 1.7 ml supernatant into a new 2 ml reaction tube
Add 230 µl ice-cold TCA (be careful TCA is caustic!), mix your samples thoroughly and them on ice for 30 minutes.
Cetrifuge all samples at 15,000 g for 15 min at 4 °C
Precipitated protein now remains in the sediment. Carefully remove the supernatant using a white tip plugged into a blue one
Add 100 µl PBS onto the sediment and vortex the sample thoroughly
Add 600 µl ice-cold acetone and vortex samples thoroughly again. Incubate samples overnight at – 20 °C. (Make sure freezer is suitable for storing volatile substances (explosion-proof freezer)!)
Centrifuge samples for 5 min at 15,000 g and 4 °C.
Remove supernatant using a white tip plugged into a blue one.
Dry sediment by incubating the samples with an open lid at 37 °C for about 3 min. Make sure that all acetone is evaporated. Do not overdry the sediment!
Add 20 µl 1x SDS loading buffer (make sure that DTT is added to your SDS loading buffer)
Repeat the following steps multiple times:

Vortex samples thoroughly
Incubate samples at 95 °C for 5 min
Vortex samples thoroughly
Sonicate samples in water bath for 5 min
Inspect if pellet is fully resuspended

Carefully load 10-12 µl onto an SDS gel (high viscosity!) and perform SDS-PAGE usual

Acetone Precipitation of Chlamydomonas Supernatant

Harvest 800 µl liquid culture of the transformants of interest in a reaction vessel. Culture growth time should be at least 3 days (6 days would be optimal).
Centrifuge at 1,700 g for 4 min, transfer 700 µl supernatant into a new reaction vessel.
Centrifuge again at 4.000 g for 4 min, transfer 350 µl supernatant into a new 2 ml reaction tube.
Add 1575 µl ice cold acetone, mix your samples thoroughly and incubate samples overnight at – 20 °C. (Make sure freezer is suitable for storing volatile substances (explosion-proof freezer)!)
Cetrifuge samples for 5 min at 15,000 g and 4 °C.
Remove supernatant using a white tip plugged into a blue one.
Dry sediment by incubating the samples with an open lid at 37 °C for about 3 min. Make sure that all acetone is evaporated. Do not over dry the sediment!
Add 20 µl 1x SDS loading buffer (make sure that DTT is added to your SDS loading buffer)
Repeat the following steps multiple times:

Vortex samples thoroughly
Incubate samples at 95 °C for 5 min
Vortex samples thoroughly
Sonicate samples in water bath for 5 min
Inspect if pellet is fully resuspended

Carefully load 10-12 µl onto a SDS gel (high viscosity!) and perform SDS-PAGE usual

SDS-Gel Electrophoresis

Work with gloves (unpolymerized acrylamide is toxic and keratin gives contaminating bands)!

Wash glass plates thoroughly with sponge and soap, rinse well with VE water and wipe with 70% ethanol. Assemble glass plates into gel pouring device.
Prepare solutions for 4x separation and 4x stacking gels in 50-ml Falcons (don’t add APS and TEMED).
Add APS and TEMED to separating gel solution and mix well.
Pour gel solution between the glass plates in the pouring device and overlay with isopropanol using a Pasteur pipette. Let the gel polymerize for 45 - 60 min.
Decant isopropanol and rinse gels thoroughly with distilled water and remove remaining water on top of the gel with Whatman paper (avoid touching the gel while doing this!).
Add APS and TEMED to the stacking gel solution and mix well.
Pour solution onto the separating gel and slide-in combs; avoid bubbles!
Let the gel polymerize for 30 - 45 min. (Optionally seal the gels in a plastic bag/wrap and store at 4° until use (possible for several days).)
Carefully remove combs and mark the pockets on the outer glass plate.
Assemble glass plates with gels into the running device and place it into the running chamber.
Fill-in electrode buffer into the interior chamber until the buffer level is between outer and inner glass plates; pour remaining buffer into the outer chamber.
Load 10-12 µl of the samples using a 20-µl pipette and load 3 µl of protein marker (M).
Conduct electrophoresis at 150 V for ~60 min; in the meanwhile, prepare Whatman paper and nitrocellulose membranes for blotting.

Western Blotting

Work with gloves!

Cut 10 Whatman papers and one Nitrocellulose membrane to 9 x 6 cm (this stuff is expensive, avoid wasting; never touch the membrane without gloves!); label membrane with a pencil at the upper right corner, this will become the side to which proteins are blotted.
In one corner of a large glass plate, soak 3 Whatman papers with buffer T3 and squeeze out bubbles by rolling over the stack with a test tube; add a little more buffer 3 and place the three filter papers onto the anode of a semi-dry blotting device.
In another corner of the glass plate, soak 2 Whatman papers with buffer T1 and squeeze out bubbles with a fresh test tube; add a little more buffer T1.
Place gel left side right and without bubbles onto the two Whatman papers.
Soak Nitrocellulose membrane in buffer T2 and place it onto the gel such that the pencil mark faces the gel.
Soak 2 more Whatman papers in buffer T2 and place them onto the membrane; squeeze out bubbles with a test tube starting from the middle into all directions (don’t apply too much pressure!).
Take the entire stack, turn it upside-down and place it onto the three Whatman papers soaked with buffer T3 that are already on the anode.
In another corner of the glass plate, soak 3 Whatman papers with buffer T1 and squeeze out bubbles with a fresh test tube; then place the three papers onto the sandwich on the anode.
Close blotting device by placing the cathode on top and conduct transfer for 60 min by applying a current of 0.8 mA/cm2 per gel surface.

Ponceau-Staining

Work with gloves!

Disassemble the sandwich after the transfer and incubate membrane for 1 min in Ponceau–S solution (touch membrane only with forceps! Be careful, it is very fragile!)
Pour the Ponceau solution back into its container (it is re-used several times) and wash membrane with distilled water until bands appear brightly without background.
Take an image of the membrane using the FUSION device.
Allow the membrane to dry on a Whatman paper.

ECL Detection

Blocking: Incubate the membrane in a plastic container with 15 ml PBS-TM for 30 min; shake gently and ensure that the membrane is always covered with solution.
Remove blocking solution.
Primary antibody: Depending on the antibody titer and the abundance of the target protein, dilute primary antibody by 1:1000 to 1:5000 in 15 ml PBS-TM and incubate the membrane with the solution for 1 h with gentle shaking.
Collect the primary antibody solution in a Falcon tube.
Rinse membrane twice with a little PBS-T and wash membrane 3x 5 min with 15 ml PBS-T.
Secondary antibody: Dilute secondary antibody (usually goat anti-rabbit coupled to horseradish peroxidase) at a 1:10000 dilution in 15 ml PBS-TM and incubate the membrane with this solution for 1 h with gentle shaking.
Discard the secondary antibody solution.
Rinse membrane twice with a little PBS-T and wash membrane 3x 5 min with 15 ml PBS-T.
Cut Whatman paper to a size a little larger than the membrane and place it onto the FUSION detection tray.
For each mini-gel blot mix 2 ml of ECL solution I and 2 ml of ECL solution II in a test tube and vortex well (careful: do not cross-contaminate ECL solutions, this will destroy them). Work with gloves.
Distribute the mixed ECL solutions evenly across the Whatman filter paper; squeeze out bubbles with a test tube.
Place membranes with the protein side up onto the Whatman filter paper.
Detect the chemiluminescence signal with the FUSION device.

Coomassie Staining

Take the gel out of the glass plates and place it for 2 min in a clean tray in destaining solution on a shaker.
Remove the destainer, then add the staining solution covering the gel and place it on a shaker for ca. 1 h.
Remove the staining solution and add destainer, shake for 5 min and replace the destainer (you can put some sheets of paper tissue next to the gel into the destainer; this will absorb the Coomassie faster).
Stop when the bands are clearly visible, and the background color is faint (takes 1-4 h).

Protein Precipitation & Purification

Ammonium Sulfate Precipitation for Protein Sample Concentration

Avanti centrifuge

Transfer the supernatant into centrifuge bottles. Use the scale to balance them precisely!
Centrifuge using the Avanti centrifuge at 4,000 × g for 5 minutes (Set the centrifuge to “Yes” and ID: 14. Choose the correct rotor. If necessary, remove any liquid from the rotor.)
Open the centrifuge using the foot pedal. Transfer the supernatant to a new centrifuge bottle and repeat step 2.
4. Transfer the supernatant into a large measuring cylinder.
Add 53.3 g of ammonium sulfate per 100 ml of solution and stir for approx. 30 minutes.
Fill the liquid into centrifuge bottles (balance carefully!) and centrifuge at 12,000 × g for 15 minutes (Grease the rim of the centrifuge lid to ensure a vacuum seal.)
6. After centrifugation, discard the supernatant. Resuspend the pellet in approximately 1% of the original volume using Equilibrator buffer.

Dialysis

Prepare 5 liters of Equilibrator (or PBS) buffer in a large measuring cylinder:

12.11 g Tris (0.02 M)
87.66 g NaCl (0.3 M)
Add 4.5 liters of deionized water, adjust pH to 8 with HCl (32%), then top up to 5 liters
(It is recommended to dilute HCl before use (e.g., 1:2). Caution: corrosive fumes – work under a fume hood! Wear lab coat, gloves, and safety goggles!)

Fill the resuspended protein pellet into a dialysis tubing (don’t forget to clamp it!).
Place 800 ml of Equilibrator buffer into a new beaker. Immerse the sealed dialysis tubing under gentle stirring and incubate for a few hours at 4°C.
Afterwards, transfer the dialysis tubing into the remaining ~4.2 liters of buffer and incubate overnight at 4°C (Very gentle stirring can help.)
The next day, open the dialysis tubing, transfer the contents into Falcon tubes, and begin purification if necessary.

Protein Purification

Add ~1.5 ml - 2 ml of Nickel-NTA beads to the column (ensure the resin is well resuspended before loading)
Prepare the protein sample: either by ammonium sulfate precipitation followed by dialysis, or by harvesting the liquid culture of interest and centrifuging at 4.000 g for 5 min. Transfer the supernatant to a new vessel and repeat this step. Make sure to add NaCl to a final concentration of 300 mM.
Remove most of the storage buffer from the Ni-NTA. Add 10 bed volumes (bv) equilibration buffer and allow the majority of the fluid to drip out of the column.
Add dialyzed or centrifuged supernatant to the column and allow it to flow through by gravity.
Add ~10 bv Wash buffer and allow it to drain by gravity.
Elute the bound protein by adding 2 ml Elution buffer. Incubate the resin with the elution buffer for 1-2 min. collect the complete eluate in two 1.5 ml reaction vessels. Repeat the elution step a second time.

Wash and Regeneration of Nickel-NTA

protocol from cube-biotech

Remove the majority of the fluid in the column containing the Ni-NTA or Ni-IDA matrix. Add 10 bv dd water and allow the majority of the volume to drip out of the column.
Add 10 bv 100 mM EDTA to the column and allow the entire volume to flow through the matrix. Rinse the column again with 10 bv dd water.
Add 10 bv NaOH Wash Buffer to the column and allow the entire volume to flow through the matrix.
Rinse the column with 10 bv dd water.
Add 10 bv 10m M NiSO4 to recharge the matrix. Allow the volume to drip through the column by gravity.
Rinse the column with 5 bv dd water
Add 10 bv of 20% (v/v) ethanol and allow the majority of the volume to drip out of the column. The matrix is now ready to be re-used.

Media

E. coli Media

LB Medium

Ingredient	For 1 Liter
NaCl	10 g
Bacto yeast extract	5 g
Bacto trypton	10 g
ddH₂O	Fill up to 1 L
For Agar plates: add 13 g Agar-Agar (1.3%)

Ingredients of LB for Agar Plates for MoClo Transformation

Level	Ingredient	Stock concentration	Solvent	Concentration in medium
L0	Spectinomycin IPTG X-Gal	100 mg/ml 0.5 M 40 mg/ml	ddH₂O ddH₂O Dimethylformamide	100 µg/ml 0.5 mM 40 µg/ml
L2	Kanamycin	50 mg/ml	ddH₂O	50 µg/ml

Antibiotics for E. coli Cuture Medium

Level	Antibiotic	Solvent	Stock concentration	Concentration in medium
0	Spectinomycin	ddH₂O	100 mg/ml	100 µg/ml
2	Kanamycin	ddH₂O	50 mg/ml	50 µg/ml

Chlamydomonas Medium

TAP-NH₄ Medium

Ingredient	For 1 l	For 5 l
Tris	2.42 g	12.1 g
4xBeijerinck-NH4	25 ml	125 ml
1M posphate-buffer ph7	1 ml	5 ml
„Special K“ Trace elements:
EDTA-Na₂	1 ml	5 ml
(NH₄)₆Mo₇O₂₄	1 ml	5 ml
Na₂SeO₃	1 ml	5 ml
Zn EDTA	1 ml	5 ml
Mn EDTA	1 ml	5 ml
Fe EDTA	1ml	5 ml
Cu EDTA	1 ml	5 ml
Acetic acid	1 ml	5 ml
ddH₂O	Fill up to 1 l	Fill up to 5 l
Adjust with acetic acid to pH 7
For Agar plates: add 13 g Agar-Agar per liter (1,3 %)

HMP-Medium

Ingredient	For 1 l	For 5 l
HEPES	4,77 g	23.38 g
4xBeijerinck-NH4	25 ml	125 ml
1M posphate-buffer ph7	1 ml	5 ml
„Special K“ Trace elements:
EDTA-Na₂	1 ml	5 ml
(NH₄)₆Mo₇O₂₄	1 ml	5 ml
Na₂SeO₃	1 ml	5 ml
Zn EDTA	1 ml	5 ml
Mn EDTA	1 ml	5 ml
Fe EDTA	1ml	5 ml
Cu EDTA	1 ml	5 ml
Acetic acid	1 ml	5 ml
ddH₂O	Fill up to 1 l	Fill up to 5 l
Adjust with KOH to pH 7
For Agar plates: add 13 g Agar-Agar per liter (1,3 %)

Antibiotics for Chlamydomonas Agar Plates

	Antibiotic	Solvent	Stock concentration	Concentration in medium
Spec plates	Spectinomycin	ddH₂O	100 mg/ml	100 µg/ml
Amp plates	Ampicillin	ddH₂O	100 mg/ml	100 µg/ml
Decontamination plates	Ampicillin	ddH₂O	500 mg/ml	500 µg/ml
	Cetotaxime	ddH₂O	100 mg/ml	100 µg/ml
	Carbendazime	50% Ethanol	40 mg/ml	40 µg/ml

General Buffers

Equilibrator

Reagent	Final Concentration	For 500ml
3 M NaCl	0,3 M	50 ml
0,1 M Tris pH 8	0,02 M	100 ml
ddH₂O	/	350 ml

Wash Buffer

Reagent	Final Concentration	For 500 ml
3M NaCl	0.3 M	50 ml
0.1 M Tris pH 8	0.02 M	100 ml
2M Imidazole	0.02 M	5 ml
ddH₂O	/	345 ml

Elution Buffer

Reagent	Final Concentration	For 100 ml
3 M NaCl	300 mM	50 ml
0,1 M Tris pH 8	20 mM	100 ml
2 M Imidazol	500 mM	5 ml
ddH₂O	/	345 ml

EDTA Buffer

Reagent	Final Concentration	For 250 ml
Disodium EDTA dihydrate	100 mM	9.3 g
ddH₂O	/	Fill up to 250 ml

NaOH Wash Buffer

Reagent	Final Concentration	For 250 ml
5 M NaCl	2 M	100 ml
1 M NaOH	500 mM	125 ml
ddH₂O	/	25 ml

NiSO₄-Buffer

Reagent	Final Concentration	For 250 ml
NiSO₄ * 6xddH₂O	10 mM	0.657 g
ddH₂O	/	Fill up to 250 ml

TAE Buffer

50x TAE buffer	1x TAE buffer
242 g Tris 57.1 ml acetic acid 100 ml 0.5 M EDTA pH 8	20 ml 50 x TAE
Fill up to 1 l with ddH₂O

Stacking & Separation Gels

Reagent	Stacking gel	Separation gel
1.5 M Tris-HCL pH 8.8	-	5 ml
0.5 M Tris-HCL pH 6.8	2.5 ml	-
30% acrylamide	1.1 ml	6.8 ml
ddH₂O	6.1 ml	8 ml
20% SDS	50 µl	100 µl
0.5 M EDTA pH 8	50 µl	100 µl
APS (100 mg/ml)	100 µl	200 µl
THEMED	20 µl	12 µl
final	10 ml	20 ml

1x Laemmli Running Buffer

Reagent	Final Concentration	For 2 l
5x SDS-Page Stock	24.8 mM Tris 134.2 mM Glycin	400 ml
20% SDS	0.1 %	10 ml
500 mM EDTA	1 mM	4 ml
ddH₂O	/	Fill up to 2 L

4x Loading buffer

Weigh in:	0.6 g SDS pellets 4 g sucrose
Add: 0.5 M Tris-HCl pH 6.8	5 ml
Fill up to 8 ml with	ddH₂O
Bevor use: add 20% 1M DTT

Destaining Solutions

Reagent	For 1 l
40 % Ethanol	400 ml
7 % Ac-COOH	70 ml
Fill up to 1 l

Staining Solution

Destaining solution + 0.125 % Coomassie brilliant blue R-250

Blotting Solutions

Reagent	Buffer T3	Buffer T2
ddH₂O	500 ml	775 ml
1 M Tris-HCl pH 10.4	300 ml	25 ml
Isopropanol	200 ml	200 ml

Buffer T1
Buffer T2	100 ml
ε-aminocaproic acid	525 mg

Blocking & Washing Buffer

Reagent	10x PBS	PBS-T	PBS-TM
NaCl KCl Na₂HPO₄ KH₂PO₄	80 g 2 g 14.6 g 2 g	100 ml 10x PBS	100 ml PBS-T
20% Tween-20	-	5 ml	-
Milk powder	-	-	3 g
ddH₂O	Fill up to 1 l	895 ml	-

ECL Detection Buffer

Reagent	ECL solution I	ECL solution II
1 M Tris-HCl pH 8.5	5 ml	5 ml
250 mM luminol in DMSO	500 µl	/
90 mM p-coumaric acid in DMSO	220 µl	/
30 % H₂O	/	30.5 µl
Final	50 ml	50 ml

Premixed Buffers, Enzymes, etc.

HDGreen Plus DNA Stain	INTAS science imaging
rCutSmartTM Buffer	New England Biolabs _inc.
Gel Loading Dye Purple (6x)	New England Biolabs _inc.
Quick-Load® Purple 1kb Plus Ladder	New England Biolabs _inc.
EcoRV-HF®	New England Biolabs _inc.
BsaI-HFv2®	New England Biolabs _inc.
Hi-T4TM DNA Ligase	New England Biolabs _inc.
10x Buffer for T4 DNA Ligase with 10 mM ATP	New England Biolabs _inc.
BamHI-HF ®	New England Biolabs _inc.
BbsI-HF	New England Biolabs _inc.
SapI	New England Biolabs _inc.
Q5 High GC Enhancer	New England Biolabs _inc.
Q5 DNA Polymerase	New England Biolabs _inc.
Q5 Reaction Buffer	New England Biolabs _inc.
PageRuler™ Plus Prestained Protein Ladder	Thermo Scientific
10 mM dNTP Mix	Thermo Scientific
KpnI	Fermentas
His Epitope Tag Mouse mAB igG1	Dianova

Devices

Primus 96 Plus 96-Well PCR Thermocycler	MWG Biotech AG
FastGene Ultra Cycler Gradient (96-well)	NIPPON Genetics
BX53 microscope	Olympus
U-HSCBM	Olympus
X-Cite 120PC Q	Lumen Dynamics
Ultrospec 2100 pro	Amersham Biosciences
PJ3000 scale	Mettler
Toledo FE20 pH meter	Mettler
AE 160 precision scale	Mettler
NanoDrop 2000 Spectrophotometer	Thermo scientific
Gelimager Chemostar ECL	Intas
Pioneer PX323/E precision scale	Ohaus
Transferpette S	Brand
Unimax 1010 shaker	heidolph
Vortexer VX100	Labnet
Small shaker VM3	CAT
Centrifuge 5810 R	Eppendorf
Centrifuge 5910 R	Eppendorf
Dri-Block® FDB03DD	Techne
Fusion-FX7 Imaging system	Vilber
Electrophoresis Power Supply – EPS 301	GE Healthcare
RCT magnetic stirrer	IKA
HERAsafe® KSP18	Kendro
Sorvall Lynx 6000 Centrifuge	Thermo scientific
Avanti J-26S XP Centrifuge	Beckman Coulter
Solaris Shaker	Thermo scientific
SONOREX Super RK 514 BH	Bandelin
CERTOMAT® RM	Sartorius stedim biotech
SCANLAF MARS	LaboGene
Celldrop FL Fluorescence Cell Counter	DeNovix®
Micro centrifuge	Carl Roth
Drying oven	memmert