iGEM 2019 — Team Humboldt_Berlin

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# Team:Humboldt Berlin/Experiments

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# Experiments

## Protocols

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### Chlamydomonas Protocol

### Cultivation

### Media

To be able to work with *C. reinhardtii*, you will need medium. This medium can be in either liquid or solid form and is essential for any experiment regarding C. reinhardtii. The media we used for
*C. reinhardtii* is TAP medium.

| Media | 0,5 L | 1,8L |
| --- | --- | --- |
| **40 x TAP/TA/TAPi** | 12,5 ml | 20 ml |
| **40 x Beijerink Salts** | 12,5 ml | 20 ml |
| **Trace Elements (revised)** | 3,5 ml | 5,6 ml |
| **H2O** | 471,5 ml | 754,4 ml |
| **For Plates: + Agar (1,8 %)** | 7,2 g | 14,4 g |

Tap Medium should be adjusted to 7,0 - 7,2 pH and autoclaved.

### Stocks

| 40 x TAP 1L | autoclave, store at 4°C |
| --- | --- |
| Tris (121,14 g/mol) | 96,8 g (0,8 M) |
| 1 M (K) PO4 | 40 ml (40 mM) |

adjust pH 7.0-7.2 with conc. AcOH (~44ml)

| 40 x TA 1L | autoclave, store at 4°C |
| --- | --- |
| Tris (121,14 g/mol) | 96,8 g (0,8 M) |

adjust pH 7.0-7.2 with conc. AcOH (~44ml)

| 40 x TAPi 1L | autoclave, store at 4°C |
| --- | --- |
| Tris (121.14 g/mol) | 96,8 g (0,8 M) |
| 1 M K2HPO3 120.08 g/mol | 40ml (40mM) |

adjust pH 7.0-7.2 with conc. AcOH (~44ml)

| 1 M (K)PO4 | MW(g/mol) | conc. | 420 ml |
| --- | --- | --- | --- |
| K2HPO4 ∙3 H2O | 228,22 | 1 M; 228 g/l | ≈0,6 M; 136 g/l |
| K2HPO4 | 136,09 | 1 M; 136 g/l | ≈ 0,4 M; 55,08 g/l |

250 ml 1 M K2HPO4 + ca. 170 ml KH2PO4 , adjust to pH 7, autoclave, store at 4°C

| 1 M (K)PO3 | MW(g/mol) | conc. | 1 l |
| --- | --- | --- | --- |
| K2HPO3 | 120.086 | 1 M; 120.086 g/l | ≈1 M; 120.086 g/l |

K2HPO3 was obtained by Haihang co. ltd. (13977-65-6) Autoclave, store at 4°C

| 40 x Beijerinck salts 1 L | autoclave, store at 4°C |
| --- | --- |
| NH 4Cl | 16 g |
| CaCl2 ∙2H 2O | 2 g |
| MgSO 4 ∙7H 2O | 4 g |

**Trace Elements (revised)**

To make the trace elements following publication was used: Kropat, J., Hong-Hermesdorf, A., Casero, D., Ent, P., Castruita, M., Pellegrini, M., …
Malasarn, D. (2011). A revised mineral nutrient supplement increases biomass and growth rate in *Chlamydomonas reinhardtii*.
The Plant Journal, 66(5), 770–780. <http://doi.org/10.1111/j.1365-313X.2011.04537.x>

### Additives

| Additive (optional) | Working Concentration |
| --- | --- |
| Arginin | 100 µg/ml |
| Paromomycin | 10-12 µg/ml |
| Hygromycin | 10 µg/ml |
| Ampicillin | 100-500 µg/ml |

### Cultures

Generally, all *C. reinhardtii* strains we used are stored as stocks in agar-tubes at room temperature and a light intensity of 150µE . To start a liquid culture, these were picked and resuspended in TAP medium, placed in a shaking incubator at 110 rpm and 150 µE. Cultures were subsequently split to remain at a cell density of 107 . Successfully transformed clones were plated on TAP-agar plates (with 10 µg/ml antibiotics) and incubated in a light incubator under continuous light conditions at room temperature.

### Multi Cultivator MC1000

Cultivation experiments conducted with the Multicultivator MC1000 from PSI were set up as followed; All cultivation tubes, hoses, glassware and media were autoclaved at 121°C for 40 min at 4 bar. OD measurements were made at 680 nm and 720 nm. To blank, the media was filled into the cultivation tubes under a biosafty cabinet and then measured. Cultures were measured in a Photometer and normalized to OD 1 [R.U.] and were then given to the blanked media to dilute them to OD ~0,1 [R.U.]. After filling in the cultures, all hoses wer conected as discribed in the MC 1000 manual and the experiments were started.

### Transformation (Electroporation)

For the nuclear transformation of our *C. reinhardtii* strains, we chose the electroporation method. Transforming of L1-constructs was performed as a co-transformation with a plasmid conferring antibiotic resistance to select for cells that were transformed successfully.

1. Grow a *C. reinhardtii* culture in TAP medium, shaking at 110 rpm under alternating temperature and light cycles (14 h 40-60 µmol photons m-2 s-1 at 25 °C and 10 h darkness at 18 °C). Grow cells for 10 days, diluting the culture every 48-72 h to keep them in the logarithmic growth phase. The cell density for transformation should be around 1-2 x 10^6 cells/ml
2. Harvest cells and start transformation 1-2 h before switching from light to dark conditions. Centrifuge cells (2500 g for 10 min at RT) and resuspend pellet in ME-Suc buffer to a cell density of 1 x 10^8 cells/ml. Aliquot 500 µL of the cell suspension in 2 ml Eppendorf tubes.
3. Heat shock cells, then incubate for 30 min at 40ºC in a thermomixer, while gently mixing at 350 rpm. Let cells recover for 30 min at RT.
4. Prepare a 24-well plate with 600 µL TAP medium in each well.

**Electroporation conditions.** List of optimal electroporation parameters for different C. reinhardtii strains for the use with the NEPA21 electroporator.

| Strain | Cell wall | Voltage | pulse length | interval | No. of pulses | decay rate | polarity |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Strain |  |  |  |  |  |  |  |
| CC-3403 | no | 200 V | 8 ms | 50 ms | 2 | 40% | + |
| UVM-4 | no | 200 V | 8 ms | 50 ms | 2 | 40% | + |
| SAG32-11b | yes | 300 V | 12 ms | 50 ms | 1 | 40% | + |
| 2. Transfer pulse (same for all strains) |  |  |  |  |  |  |  |
| *all strains* |  | 20 V | 50 ms | 50 ms | 5 | 40% | +/- |

5. Transformtation

1. Choose electroporation conditions for the desired strain according to Table 1
2. One electroporation requires: 38 µL of heat-shocked cells, 0.5 g of antibiotic-resistance marker plasmid and 0.5 µg of the plasmid to be transformed. Combine cells with plasmids for 5 electroporations (see Table 2 below).

**Electroporation mix.**

|  |  |  |
| --- | --- | --- |
| resistance marker plasmid (1 µg/µl) | 5x 0.5 µg | 2.5 µ |
| L1-construct plasmid (1 µg/µl) | 5x 0.5 µg | 2.5 µ |
| cells (1 x 10 <8 cells/ml) | 5x 38 µl | 190 µl |

3. Fill a 2 mm electroporation cuvette with 40 µL of cell-mixture. Carefully monitor the resistance of the solution and keep it around 400-500 Ω.
4. To collect the transformed cells after electroporation, transfer 600 µL fresh TAP medium from the recovery plate into the cuvette, then place mix back in the well. Repeat this process with the remaining electroporation mix. Use the same cuvette for all 5 electroporations of the mixture.
5. Seal recovery plate with parafilm and incubate overnight with gentle shaking (110 rpm) under continuous light (50 µmol m-2 s-1).

6. The following day, place cells from one well (600 µL) gently onto one TAP-Agar plate with 10 µg/ml antibiotics. Incubate the sealed plates at room temperature under continuous light for 7-14 days until single colonies are visible to the naked eye.

### Colony Screening PCR

To check if a construct was successfully transformed into *C. reinhardtii*, a colony PCR can be used as a screening method. The goal of the colony PCR is to amplify the inserted DNA fragment from the transformed C. reinhardtii genome through a PCR. This allows us to assess if the transformation was successful. If a DNA band is visible with the expected length on the electrophoresis gel, the transformation was probably successful. Sequencing of the amplified DNA fragment is necessary to assure the success of the transformation.

**Preparation of DNA:**

1. For each Plate with Chlamy that you are going to screen, take one V-Bottom 96-well-plate
2. Put 40 µl of resuspended cell solution in each well of the V-Bottom plate
3. Centrifuge at 2500 g for 10 min and discard supernatant immediately
4. Add 20 µl dilution buffer to each well and resuspend the pellet. Incubate 5 min at room temperature.
5. Centrifuge at 4000 g for 10 min

**Preparation of DNA:**

It is important to calculate the mastermix total volume and the volume of the components according to the amount of wells with DNA you want to screen. A volume 18 µl mastermix and 2 µl DNA per well is recommended.

| Total volume | 100 µl |
| --- | --- |
| Phire Plant MM (2x) | 50 µl |
| screen FW primer (100 µM) | 0.5 µl |
| screen RV primer (100 µM) | 0.5 µl |
| Betaine (5M) | 20 µl |
| H2O | 30 µl |

Take a PCR-96-well plate and add 18 µl mastermix to each well. Then add 2 µl DNA to each well.

**PCR:**

| PCR steps |  |  |  |
| --- | --- | --- | --- |
| initial denaturation | 98 °C | 5 min |  |
| [denaturation | 98 °C | 10 s< |  |
| annealing | 65 °C | 10 s |  |
| elongation | 72 °C | 100 s] | x40 |
| final extension | 72 °C | 2 min |  |
| storage | 8 °C | ∞ |  |

The PCR cycles and conditions can be adapted according to the fragment length.
Run an electrophoresis gel following the gel electrophoresis protocol with the PCR probes. If bands are visible at the expected length, sequence the probes.

### Fluorescence Screening

After having obtained successful results of the integration of YFP into a *C. reinhardtii* clone from the colony PCR, it can be screened for fluorescence. If the YFP protein was expressed correctly, the clone should display a signal.

1. For each clone to be screened, place 160 µl of resuspended cell culture in a 96-well plate
2. Measure using a fluorescence plate reader. We used the TECAN Plate Reader Infinite 200 Pro, a monochromator
3. For YFP, measure using an excitation wavelength of 514 nm and read the emission at 527 nm

### *E. coli* Protocols

### Cultivation

### Media & Plates

To be able to work with *E. coli.*, you will need medium. This medium is essential for any experiment regarding *E coli.* The medium we used for *E. coli* is LB medium.

**LB Medium**

For 1 L LB medium: 20 g LB Powder in 1 L dest. water, autoclave

**LB Plates**

For 1 L LB medium with Agar: 25 g LB Powder with 13 g agar-agar in 1 L dest. water, autoclave.

| Antibiotics | Working Concentration |
| --- | --- |
| Spectinomycin | 50 µg/ml |
| Ampicillin | 100 µg/ml |
| Carbenicillin | 100 µg/ml |

### Overnight Cultures

Cultivation of single colonies is done overnight for 8-16 hours, depending on desired culture density. Use a snap-cap culture tube or 25 ml-flasks with cotton tops.

1. Pipet 5 ml of LB-Medium in the culture tube. If required, add the working concentration of antibiotics.
2. Working under sterile conditions, pick single colonies from a growth LB-plate with a sterile toothpick or pipet tip. Drop the toothpick or tip into the culture tube.
3. Place the tube in an incubator at 37°C while shaking at 220 rpm.

### Transformation

1. Thaw 50 µl competent *E. coli* cells on ice and add 1-5 µl plasmid. Add the same amount of water to ONE tube, as a control with no expected growth
2. Mix carefully, do not vortex/resuspend
3. Incubate on ice (20-30 min)
4. Heat shock: 30-60 sec, at 42 °C
5. Incubate on ice (2 min)
6. Suspend the cells in 1 ml LB Medium and incubate for 45 min at 37 °C
7. Centrifuge cells at 100 g for 4 min at 4 °C
8. Dispose most of the supernatant
9. Put 50-100 µl of cell suspension onto a petri dish with LB medium with antibiotics
10. Grow overnight at 37 °C

### DNA-Isolation

### Mini-Prep

Extraction of plasmid DNA from *E. coli* overnight cultures can be done using commercial kits. This step is required for most downstream applications of the plasmid, such as sequencing and transformation. We used Macherey Nagel NucleoSpin ® Plasmid for a rapid plasmid isolation following the provided protocol

1. Use 1–5 mL of a saturated *E. coli* LB culture, pellet cells in a standard benchtop microcentrifuge for 30 sat 11,000 x g. Discard the supernatant and remove as much of the liquid as possible.
2. Resuspend the cell pellet with 250 µl of Resuspension Buffer (A1). Make sure no cell clumps remain before adding lysis Buffer (A2)
   Add 250 μL lysis Buffer (A2). Mix gently by inverting the tube 6–8 times. Do not vortex to avoid shearing of genomic DNA. Incubate at room temperature for up to 5 min or until lysate appears clear.
   Add 300 μL of neutralization Buffer A3. Mix thoroughly by inverting the tube 6–8 times until blue samples turn colorless completely! Do not vortex to avoid shearing of genomic DNA.
3. Centrifuge for 5 min at 11,000 x g at room temperature. Repeat this step in case the supernatant is not clear!
4. Place a NucleoSpin® Plasmid / Plasmid (NoLid) Column in a Collection Tube (2 mL) and decant the supernatant from step 3 or pipette a maximum of 700 μL of the supernatant onto the column. Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the NucleoSpin® Plasmid / Plasmid (NoLid) Column back into the collection tube.
5. Wash silica membrane using 500 µl of preheated (at 50°C) washing buffer (A4) and centrifuge for 1 min at 11.000 x g.
   Add 600 µl of concentrated washing buffer (A4), centrifuge for 1 min at 11.000 x g and discard the flow-through.
6. Dry silica membrane by centrifuging for 2 min at 11,000 x g and discard the collection tube afterwards.
7. Place the NucleoSpin® Plasmid / Plasmid (NoLid) Column in a 1.5 mL microcentrifuge tube and add 50 μL Elution Buffer (AE) preheated to 70 °C. Incubate for 2 min at 70 °C. Centrifuge for 1 min at 11,000 x g

### Pauls MiniMiraPrep

Super High Yield protocol for non-*E.coli*-codon optimzed DNA: Up to 600 ng of plasmid-DNA from 5ml cultures.

1. Cultivate in 5-8ml ON Culture Tubes and harvest bacterial cells 12,000 x g, 30 s
2. Cell lysis

   |  |  |
   | --- | --- |
   |O 150 μL | Buffer A1 |
   | 250 μL | Buffer A2 RT, up to 2 min |
   | 350 μL | Buffer A3 |
3. Clarification of the lysate > 4000 x g, 10 min
4. Collect supernatant in a 2ml eppi. Add 1x volume of 96% ethanol (700µl).
5. Bind DNA:
   Load supernatant on a spin Collum
   1,000–2,000 x g, 30 s
6. Wash and dry silica membrane:
   450 μL Buffer AQ > 12,000 x g, 1 min
   repeat 2 times
7. Elute DNA:
   50 μL Buffer AE preheated to 70°C RT, 1 min
   > 12,000 x g, 1 min
   repeat 2 times

### Colony PCR

To assess if the transformation of a construct into E. coli was successful, a colony PCR can be made. In the colony PCR, cells are put into a PCR cycler with primers of the transformed construct. Thus, if the E. coli contain this DNA fragment, it will be amplified during the PCR and become visible once a gel electrophoresis is performed.

1. Pick isolated colonies with a toothpick and put each colony into 20 µl water.
2. Use 1 µl of the colony-containing water as template for the PCR. Include a negative control just with water as a template, optimally, a positive control containing a template of known fragment length.

   **PCR Mix**

   |  | End Concentration | Total Volume: 20 µl |
   | --- | --- | --- |
   | H2O |  | Fill up to 20 µl |
   | GC Green Buffer (5x) | 1x | 4 µl |
   | dNTP's (10mM) (5x) | 200µM | 0,4 µl |
   | Forward Primer (10 µM) | 0.5 µM | 1 µl |
   | Reverse Primer (10 µM) | 0.5 µM | 1 µl |
   | DMSO | 3% | 0,6 µl |
   | Lab-Phusion Polymerase |  | 0,2 µl |
   | Template DNA (Colony) | <250 ng | 1 µl |

   **PCR Cycles**

   | PCR steps |  |  |  |
   | --- | --- | --- | --- |
   | initial denaturation | 98°C | 3 min |  |
   | denaturation | 98°C | 10s |  |
   | annealing | 67 °C | 20 s |  |
   | elongation | 72 °C | 20 s | ] x30 |
   | final extension | 72 °C | 10 min |  |
   | storage | 8 °C | ∞ |  |
3. Load a 1% agarose gel with 5 µl PCR sample of each reaction and run at 80-100 V for 20-35 min.

### Glycerol Stocks

After successfully growing a transformed E. coli clone, it is important to conserve it for future use and backup. To conserve *E. coli*, glycerol stocks can be made.

1. Make 50% glycerol by adding 100% glycerol and water in equal parts
2. Add 500 µl *E. coli* solution to 500 µl 50% glycerol in a 1.5-2 ml screw top tube
3. Freeze at -80 °C

### Lysis

1. Pellet *E. coli* overnight culture by centrifuging 10 min at 6000 g. Remove supernatant.
2. Resuspend cell pellet in lysis buffer. Use 300 µl buffer per 2 ml overnight cell culture.
3. Add freshly prepared lysozyme. Use 25 µl lysozyme stock solution per 2 ml overnight cell culture.
   (Lysozyme stock solution: 10 mg/ml solved in 10 mM Tris-HCL, pH 8.0)
4. Mix by vortexing a few seconds.
5. Incubate sample at 37 °C for 30 min. After that maintain samples on ice for the remaining protocol.
6. Sonication: 3 times, 30 sec, with 1 pulse per sec. (We used Bandelin Sonopuls GM70.) Allow samples to rest on ice between each sonication.
7. Centrifuge samples at maximum speed for 3 min. to pellet cell debris.
8. Carefully pipet cell lysate (supernatant) into new tube. (If relevant examine cell debris pellet regarding lysis effectiveness.)

**Lysis buffer components:**

| component | conc. |
| --- | --- |
| Tris-HCL (pH 8.0) | 10 mM |
| EDTA | 1 mM |
| NACl | 100 mM |
| Triton™ X-100 | 0.5 % (v/v) |

 Protocol adapted from Liljeruhm, 2014

### Synthesis Protocols

### Sequence & Primer Design

Gene parts that should be a level 0 part must be flanked by a specific syntax. This syntax was added to a gene part by primer within a PCR reaction or when parts were completely synthesized they already contained the syntax. As we are using the Golden Gate cloning strategy we are using the restriction sites for the TypeIIS restriction enzymes *Bpi*I and *Bsa*I.
Primers were designed to attach up to 18 bp to the gene of interest followed by the syntax which is illustrated in the figure.
The *Bpi*I recognition sites are positioned on the outsides of the construct surrounded by two random bases which are represented as “n”. The *Bpi*I cutting site is located two bases downstream. The 3’ end of the gene part are complementary sequences to the 5’ BpiI recognition and cutting
site. The L1 fusion site is responsible for the position of the gene part in a functional transcriptional unit within a level 1 backbone (see our description about GoldenGate cloning). The numbers 1-8 stand for the specific 4 bases guiding the placement of the part according to the MoClo fusion sites defined by Patron in 2015 (Patron et al., 2015). The *Bpi*I cutting site (blue) is similar to the *Bpi*I cutting site on the level 0 backbone enabling the fusion of the parts into the backbone.
Primers were also used to mutate sequences when the DNA parts included a forbidden site. Synthesized parts were codon optimized for transcription in *Chlamydomonas reinhardtii.*

![Gene Arrangement](https://static.igem.org/mediawiki/2019/8/8c/T--Humboldt_Berlin--part_design_experiments.png)

### PCR

**PCR Mix**

The reactions were performed following the recipe below in the table. The cycler program used is depicted in Tab. 8. The annealing temperature depends on the TM of the utilized primers and are calculated by Thermo Fisher Scientific Tm calculator.

|  | End Concentration | Total Volume: [µl] |
| --- | --- | --- |
| H2O |  | Fill up to 20 µl |
| 5x GC Green Buffer (5x) | 1x | 4 µl |
| Trimethylglycine 5M | 1x | 4 µl |
| dNTP's (10mM) (5x) | 200µM | 0,4 µl |
| Forward Primer (10 µM) | 0.5 µM | 1 µl |
| Reverse Primer (10 µM) | 0.5 µM | 1 µl |
| DMSO | 3% | 0,6 µl |
| Lab-Phusion Polymerase | 0.02 U/µl | 0,2 µl |
| Template DNA | <1-2 ng | 2 µl |

**PCR Cycles**

| PCR steps |  |  |  |
| --- | --- | --- | --- |
| initial denaturation | 98°C | 30 s |  |
| [denaturation | 98°C | 10 s |  |
| annealing | varies | 20 s |  |
| elongation | 72 °C | 1 min/kb | ] x30 |
| final extension | 72 °C | 10 min |  |
| storage | 8 °C | ∞ |  |

### Oligo annealing

For short gene parts, complementary, single stranded oligos were ordered and linked to a double stranded fragment by using a thermocycler. Heating, followed by slow cooling, facilitates hybridization. The T4 Polynucleotide Kinase (PNK) phosphorylases the DNA strand. The oligos contain the sequence for the MoClo overhangs which are not combinable and thereby leaving the required single stranded overhangs. Thus, the resulting fragment does not need to be digested by enzymes and can be ligated directly.
The reaction listed in the table must be incubated for 30 minutes at 37 °C, 5 minutes at 95°C and is then slowly cooled down to 25 °C by decreasing the temperature by 5 degrees per minute inside a PCR-cycler (Ran et al., 2013).

| Component | Amount [µl] |
| --- | --- |
| Oligo 1 (100 µl) | 1 |
| Oligo 2 (100 µl) | 1 |
| T4 ligation buffer, 10 x | 1 |
| T4 PNK | 1 |
| ddH2O | 6 |

### Digestion

**Backbone**

1. Plasmid-prep (or overnight culture midi-prep) from backbone (e.g. L1c-RFP)
2. Use at least 2,5 µg backbone
3. You have to use 1µl enzyme per 1 µg Backbone DNA
4. The volume of (all) enzymes can constitute max. 10% of the approach, because glycerol disturbs the reaction
5. Incubate for 30-60 min at 37°C. The minimal incubation time is 15 min, maximum 2 hours

| Component | Amount [µl] | Amount [µl] |
| --- | --- | --- |
| 10x FD *(Fast Digest)* | 6 µl | 2 µl |
| Backbone | 2,5 µg = x µl | 1 µg = x µl |
| *BamH*I | 2,5 µl | 1 µl |
| *Xho*I | 2,5 µl | 1 µl |
| FastAP *[alcaline phosphatase]* (for backbone digest only) | 1 µl | 1 µl |
| H2O |  |  |
| Volume | ∑ 60 µl | ∑ 20 µl |

**Following:**

1. Clean-Up:

1. Gel clean-up: apply everything on gel and cut it out of the gel (best option, but you probably lose some DNA parts)
2. PCR clean up: apply 5µl on gel for control, purify the rest (here the yield is higher, but there may be some uncutted plasmid left in the reaction, which leads to unspecific colonies)

2. (optional: to inactivate enzyme incubate 5-20min at 65-80°C . Depends on enzyme. Not all enzymes can be inactivated by heat. look up needed information!)
3. Measure concentration

**Insert**

1. PCR product (for example selection cassette)

1. PCR with Phusion, 50-100 µl
2. Apply 5 µl on gel as control
3. Digestion with *BamH*I & *Xho*I without purification, directly with PCR product

   | Component | Amount [µl] |
   | --- | --- |
   | 10x FD *(Fast Digest)* | 10 µl |
   | PCR Produkt (Insert) | 50 µl |
   | *BamH*I | 5 µl |
   | *Xho*I | 5 µl |
   | H<2O | 30 µl |
   | Volume | ∑ 100 µl |

   Incubation 30-60 min @37°C

2. Apply everything on gel, purify with kit (gel clean-up)
3. Measure concentration

### Ligation

### General ligation

1. Set up the following reaction in a microcentrifuge tube on ice.
   (T4 DNA Ligase should be added last). Use NEBioCalculator to calculate molar ratios.
   70 ng of Vector DNA is commonly used for our ligations. In order to achieve the 3:1 molar ratio one needs to calculate the volume of the insert DNA solution in relation to the vector DNA.
   Volume Vector-DNA:
   X ng\*µl Plasmid / 70 ng = xx µl
   Volume insert DNA
   70 ng \* x bp insert DNA / x bp vector DNA = X ng of insert DNA
   x ng of insert DNA / x ng/µ insert conc. = xx µl volume of insert DNA
The calculated volumina can then be used in the following reaction

| Component | 20 µl reaction |
| --- | --- |
| T4 DNA Ligase Buffer (10X) | 2 μl |
| Vector DNA | 50-200 ng |
| Insert DNA (3:1 molar ratio) | ~ |
| Nuclease-free water | to 20 μl |
| T4 DNA Ligase | 1 μl |

\* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature
2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
4. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
5. Heat inactivate at 65°C for 10 minutes
6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

### MoClo Ligation

**L0 Cloning**

To assemble L0 Parts prepare a 20µl PCR tube containing:

| Create new L0 part | concentration | total | volume |
| --- | --- | --- | --- |
| L0-Backbone | x ng/µl | x ng | x µl |
| PCR-Insert (molar ratio 3:1) | x ng/µl | x ng | x µl |
| *Bpi*I |  |  | 0.5 µł |
| T4 DNA Ligase buffer (10x) | 10x |  | 2 µl |
| (optional: Bovine Serum Albumin 1 mg/ml) |  |  | (1µl) |
| H2O |  |  | adjust to 20 µl |
| Volume |  |  | ∑ 20µl |

To reach a molar ratio of 3:1 PCR Insert to L0-Backbone the volumes have to be calculated for each PCR-Insert before beginning the protocol. We used an Excel-sheet for those calculations. Creating an automated method for those repetitive calculations is advised. Once finished preparing the tube, transfer tube to a PCR-Cycler and start cycling program below:

| Step |  |  |  |
| --- | --- | --- | --- |
| Step 1 | 37 °C | 3 min |  |
| Step 2 | 16 °C | 4 min | go back to step 1 x15-120 |
| Step 3 | 65 °C | 10 min |  |
| *hold* | 8 °C |  |  |

**L1 Cloning:**

1. Calculate needed volumes to reach a molar ratio (insert to backbone) of 2 to 1 for each L0-Part as well as for the L1-Backbone
2. Set up the L1 Ligation in a 20 µl PCR tube
3. Add ingredients below (with calculated volumes from step 1)

   | Make L1 construct | conc | total | volume |
   | --- | --- | --- | --- |
   | pL1-RFP (2.8 kb) | x ng/µl | y ng | z µl |
   | pL0 parts in 2 (pL0) : 1 (pL1) molar ratios |  |  |  |
   | pL0-part1 Promoter | x ng/µl | y ng | z µl |
   | pL0-part2 Linker | x ng/µl | y ng | z µl |
   | pL0-part3 CDS1 | x ng/µl | y ng | z µl |
   | pL0-part4 CDS2 | x ng/µl | y ng | z µl |
   | pL0-part5 Terminator | x ng/µl | y ng | z µl |
   | Thermo FastDigest Eco31I |  |  | 0.5 µl |
   | Thermo T4 DNA Ligase buffer (10x) | 10x |  | 2 µl |
   | Thermo T4 DNA Ligase (5 U/µL) |  |  | 1 µl |
   | (optional: Bovine Serum Albumin 1 mg/ml) |  |  | (1 µl) |
   | H2O |  |  |  |
   | Volume |  |  | ∑ 20µl |
4. Mix the reaction by gently pipetting up and down
5. Transfer tube to PCR-Cycler and set up cycling program as follows

   | Step |  |  |  |
   | --- | --- | --- | --- |
   | Step 1 | 37 °C | 3 min |  |
   | Step 2 | 16 °C | 4 min | go back to step 1 x26 |
   | Step 3 | 37 °C | 3 min |  |
   | Step 4 | 65 °C | 10 min |  |
   | *hold* | 8 °C |  |  |
6. Transfer 1-5µl of the reaction into 50µl of competente cells

### Sequencing

Our Sequencing was done by LGC Genomics. Their online ordering criteria can be found on their website.
We’ve sent them 20 µl of our purified PCR products at a minimal concentration of 100 ng/µl in standard 1,5 ml Eppendorf tubes.

### General

### Gel Electrophoresis

1. Make a TBE or TAE gel by adding agarose to the buffer. The agarose concentration in the gel varies depending on the DNA fragment length. For example 1% agarose gel for fragments between 500 bp and 2000 bp.
2. Add fluorescent dye to the liquid gel, for example ‘Midori Green’ (5 µl Midori/100 ml gel)
3. Pour the liquid gel into the gel chamber and wait until it becomes solid (20 min)
4. Put the solid gel into the gel sled and load 5 µl probe into each chamber and a gene ruler for reference
5. Add voltage to the sled (10 V/ 1 cm) and let the gel run
6. Take an image of the gel

### Workflow

### Plasmid assembly

### L0-Vector construction

Our L0 destination vector was created starting from the universal L0-acceptor plasmid pAGM9121 containing the recognition sites for the restriction enzymes *Bsa*I and *Bpi*I, spectinomycin-resistance-cassette (*SmR*), the origin of replication (*Ori*) and a LacZ-operon (*lacZ*-alpha). To integrate RFP with its MoClo L0-overhangs, perform classical cloning, in order to not lose the restriction site for *Bpi*I.

**Procedure:**

1. Digest the destination vector and PCR-product of RFP separately. Use the pipetting scheme below.

   |  | RFP | vector |
   | --- | --- | --- |
   | 10x Fast Digest buffer | 2 µl | 2 µl |
   | DNA | 10 µl | 15 µl |
   | *Bpi*I | 2 µl | 2 µl |
   | FastAP, alkaline phosphatase | -- | 1 µl |
   | Distilled Water | 17 µl | -- |
   | Final Volume | 30 | 20 |
2. Cycle at 37°C for 15-60 min in a thermocycler.
3. Heat-inactivate enzymes for 20 min at 65°C in the thermocycler.
4. Purify product using a commercial PCR cleanup kit.
5. Ligate RFP and the destination vector by incubating the following reaction system for 15-30 minutes at room temperature:

   | Component | volume |
   | --- | --- |
   | Plasmid 200 ng | -- |
   | Insert (molar ratio 3:1) | -- |
   | T4 ligase | 1 µl |
   | T4 Buffer 10x | 1 µl |
   | Distilled Water | fill up to 10 µl |
   | Final Volume | 10 µl |

### L0-Part construction

Using the MoClo toolkit for *C. reinhardtii* (as described in the ["Design”](/Team%3AHumboldt_Berlin/Design) section), cloning of the gene parts and modules is done by firstly, identifying the BpiI restriction site needed in this and further cloning steps. Additionally, the specific fusion sites needed for each part to be assembled in their right position in the transcriptional unit have to be determined. Then, the genetic part is provided with overhangs containing these sequence details via polymerase chain reaction (PCR).

**Procedure**

1. Design primers containing overhangs and enzyme recognition sites to be 15-30 bp long and having annealing temperatures between 50°C and 72°C.
2. Pipet reaction mixture into PCR tubes using the below scheme. The reaction volume should be between 10 and 50 µl.

   |  | Reaction concentration |
   | --- | --- |
   | 5x GC Green buffer | 1x |
   | 2 mM dNTP | 200 µM |
   | Primer 1 (100 µM) | 0,5 µM |
   | Primer 2 (100 µM) | 0,5 µM |
   | Template DNA | 1 pg–10 ng per 50 µl or "dipping" pipet tip into colony |
   | Phusion polymerase | 0,02 U/µl |
   | Betaine 5M | 1 M |
   | DMSO | 3% |
   | distilled water | fill up to final reaction volum |
3. Perform the cycling in a thermal cycler using the following parameters:

   | Step | Temp. | Time | Repeats |
   | --- | --- | --- | --- |
   | 1 | 98 °C | 30 s |  |
   | 2 | 98 °C | 5-10 s | 30 s |
   | 3 | Annealing ∓65 °C | 10 - 30 s |  |
   | 4 | 72 °C | 15-30 s/kb product length | go to step 2, repeat: x 25-35 |
   | 5 | 72 °C | 5-10 min |  |
4. Optional: Perform PCR product cleaning using a commercial kit for this purpose

### L0-Ligation

The advantage of the MoClo design is that digest and ligation can occur in a single step, as opposed to classical cloning. For a level 0 ligation, BpiI is used as the restriction enzyme for both components, the vector and the insert. In this protocol, the ATP needed for the restriction enzyme to act is provided by the T4 ligase buffer.

**Procedure**

1. Pipet reaction components into a PCR reaction tube.

   |  | conc. | total | volume |
   | --- | --- | --- | --- |
   | pL0-RFP (3.1 kb) | 70 ng/µl | 50 ng | 0.7 µl |
   | PCR-Insert (3:1) molar ratio | ~15 ng/µl | 42 ng | 3 µl |
   | Thermo FastDigest *Bpi*I |  |  | 0.5 µl |
   | Thermo T4 DNA Ligase buffer (10x) | 10x |  | 2 µl |
   | Thermo T4 DNA Ligase (5 U/µL) |  |  | 1 µl |
   | (optional: Bovine Serum Albumin 1 mg/ml) |  |  | 1 µl |
   | H*2*O |  |  | fill up |
   | Volume |  |  | ∑ 20 µl |
2. Perform the cycling in a thermal cycler using the following parameters:

   | Temp. | Time | Function |
   | --- | --- | --- |
   | [37 °C | 5 min |  |
   | 16 °C | 5 min | Activation of enzyme and ligase x15-120] |
   | 55 °C | 15 min | Enzyme inactivation |
   | 85 °C | 20 min | ligase inactivation |
   | 4 °C | hold |  |
3. Transform 2-3 µl into 50 µl of competent *E. coli* cells (use Spectinomycin LB-plates) or store at -20 °C.

### L0-Plasmid selection

1. Pick white colonies from the plate, 13-15 hours after transformation.
2. Pick 8 colonies and perform [Colony PCR](#EColonyPCR).
3. Analyse colony PCR, take a picture of the gel and place well-looking clones in an overnight culture.
4. *Next day*: store back-up plate at 4°C.
5. Make [glycerol stocks](#GlycerolStock) of overnight cultures, labeled with clone number and store at -80°C.
6. [Plasmid prep](#PlasmidPrep) of positive clones, and send in for sequencing.

### L1-Vector construction

According to iGEM and Golden Gate standards, we need a backbone containing RFP, an ampicillin resistance and an origin of replication (Ori) for plasmid replication in E. coli. RFP and Amp/Ori are amplified from their respective plasmids and ligated using a Golden Gate cloning protocol. The Golden Gate enzymes *Bsa*I and *Bpi*I flanking RFP are attached in opposite order compared to a L0 plasmid. This is important to maintain the alternating usage of these enzymes.

1. Amplify RFP from the L0-RFP using primers that attach restriction sites for *Bsa*I, *Bpi*I, *BamH*I and *Spe*I and the L1 fusion sites. Use the below scheme.

   |  | Reaction concentration |
   | --- | --- |
   | 5x GC Green buffer | 1x |
   | 2 mM dNTP | 200 µM |
   | Primer 1 (100 µM) | 0,5 µM |
   | Primer 2 (100 µM) | 0,5 µM |
   | Template DNA | 1 pg–10 ng per 50 µl |
   | Phusion polymerase | 0,02 U/µl |
   | 5x GC Green buffer | 1x |
   | Trimethylglycine | 1M |
   | DMSO | 3% |
   | distilled water | fill up to final reaction volume |
2. Amplify the ampicillin resistance and Ori from the pICH47732 plasmid via PCR, creating a single Amp/Ori PCR product. Add *BamH*I, *Xho*I and *Xba*I recognition sites during the PCR-reaction.
3. Perform a MoClo Digest-Ligation.

### MoClo Digest-Ligation: Golden Gate

1. Pipet reaction components into an annotated PCR reaction tube.

   |  | conc. | total | volume |
   | --- | --- | --- | --- |
   | Vector | 70 ng/µl | 50 ng | 0.7 µl |
   | Insert (1:1) molar ratio | ~15 ng/µl | 42 ng | 3 µl |
   | Thermo FastDigest *Bpi*I |  |  | 0.5 µl |
   | Thermo FastDigest Buffer (10x) *Bpi*I |  |  | 2 µl |
   | Thermo T4 DNA Ligase buffer (10x) | 10x |  | 2 µl |
   | Thermo T4 DNA Ligase (5 U/µL) |  |  | 1 µl |
   | H*2*O |  |  | fill up |
   | Volume |  |  | ∑20 µl |
2. Perform the cycling in a thermal cycler using the following parameters:

   | Temp. | Time | Function |
   | --- | --- | --- |
   | [37 °C | 5 min |  |
   | 16 °C | 5 min | Activation of enzyme and ligase x15-120] |
   | 55 °C | 15 min | Enzyme inactivation |
   | 85 °C | 20 min | ligase inactivation |
   | 4 °C | hold |  |
3. Transform 2-3 µl into 50 µl of competent E. coli cells (use Ampicillin LB-plates) or store at -20 °C.

### Documentation

[This](https://static.igem.org/mediawiki/2019/6/65/T--Humboldt_Berlin--AirtableWorkflow.pdf) is our structured overview of importent experiments and their order. It's sorted by workflow topic (Synthesis, *E. coli*, Chlamydomonas and general)

### Learn more...

[![results preview](https://static.igem.org/mediawiki/parts/9/91/Humboldt_Berlin_Contr_chromo-lysate-plate.jpg)](/Team%3AHumboldt_Berlin/Contribution)
[![experiments preview](https://static.igem.org/mediawiki/2019/3/3c/T--Humboldt_Berlin--Demonstrate_header.jpg)

#### Demonstrate

Following the iGEM guidelines we demostrated that our engineered system works.](/Team%3AHumboldt_Berlin/Demonstrate)

![](https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg) Go to top

Liljeruhm, J., Gullberg, E., Forster, A.C., (2014) “Synthetic Biology: A Lab Manual”
DOI: 10.1142/9061, page 118 - 119.

Patron, N. J., Orzaez, D. , Marillonnet, S. , et al. (2015), Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol, 208: 13-19. doi:10.1111/nph.13532

Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature protocols, 8(11), 2281–2308. doi:10.1038/nprot.2013.143

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# Team:Humboldt Berlin/Results

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# Results

[![Establish Chlamydomonas](https://static.igem.org/mediawiki/2019/f/fe/T--Humboldt_Berlin--chlamy_to_igem_menu.png)](#Establish)

[![PETDegradation](https://static.igem.org/mediawiki/2019/d/dd/T--Humboldt_Berlin--chlamy_pet_degrade_menu.png)](#PET)

[![](https://static.igem.org/mediawiki/2019/6/67/T--Humboldt_Berlin--chlamy_industry_menu.png)](#Industry)

## 1. Establishing Chlamy in the iGEM competition

### Synthesis of L0 and L1 Constructs

We created a basic set of genetic parts for the use in *C. reinhardtii*. We chose to use the [MoClo assembly standard](https://2019.igem.org/Team%3AHumboldt_Berlin/Design#GoldenGate) for two reasons: First, *C. reinhardtii* is a eukaryotic organism with full transcription units that comprise more elements than those of bacterial systems (promoter, 5'UTR, signal peptides, CDS, 3'UTR, terminator). To assemble multiple units through the BioBrick RFC[10] assembly is time consuming, while other assembly standards like the Golden Gate based MoClo syntax allow the cloning of multiple parts in a one-step reaction. Second, *C. reinhardtii* has only been [rarely used as chassis in iGEM](http://parts.igem.org/Collections/Plants#Chlamydomonas_reinhardtii) so far, so there is a need to add more parts to the iGEM Registry. For us this was also the chance to start over with a more advanced cloning standard like **MoClo**.

,

We created a set of parts, ([our Chlamy-HUB Collection](https://2019.igem.org/Team%3AHumboldt_Berlin/Part_Collection)), including basic regulatory sequences, reporter genes and epitope-tags. Most importantly, we created new MoClo backbones to comply with the iGEM standard (by exchanging the lacZ cloning reporter by an RFP insert). Furthermore, we designed L1 and L2 backbones that include a *C. reinhardtii* selection cassette. This means L1-constructs can directly be used for transformations of *C. reinhardtii*.

All parts were synthesized or amplified by PCR. Successful amplifications were verified by an agarose gel showing a band at the expected size and were ligated into the backbones using Golden Gate cloning. Vectors were then transformed into *E. coli*. Those containing the L0 part formed white colonies. Transformants containing empty backbones grew red since the backbones contain RFP as a selection marker. Correct transformants were confirmed by DNA sequencing. L1 plasmids that were successfully transformed into  *C. reinhardtiii*  were monitored by Colony PCR. Successful amplification-, transformation- and sequencing procedures are marked by a tick.

#### Examples for successful Results of L0 parts

| Successful Amplification | Successful Transformation into *E. coli* | Successful Sequencing |
| --- | --- | --- |
| ![](https://static.igem.org/mediawiki/2019/c/ce/T--Humboldt_Berlin--results_L0_1_1.png) | ![](https://static.igem.org/mediawiki/2019/8/8a/T--Humboldt_Berlin--results_L0_2.png) | ![](https://static.igem.org/mediawiki/2019/9/93/T--Humboldt_Berlin--results_L0_3.png) |

#### Cloning Results of L0 parts

| Part Name | Registry Name | Description | Fusion Sites | Successful Amplification | Successful Transformation into *E. coli* | Successful Sequencing |
| --- | --- | --- | --- | --- | --- | --- |
| L0 - Backbone | [BBa\_K2984010](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984010) | L0 backbone (plasmid with RFP insert) | - | ✓ | ✓ | ✓ |
| L0-AR | [BBa\_K2984009](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984009) | L0 backbone with AR promoter | A1-A3 | ✓ | ✓ | ✓ |
| L0-AR | [BBa\_K2984025](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984025) | L0 backbone with AR promoter | A1-B1 | ✓ | ✓ | ✓ |
| L0-PsaD | [BBa\_K2984022](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984022) | L0 backbone with PsaD promoter | A1-A3 | ✓ | ✓ | ✓ |
| L0-PsaD | [BBa\_K2984008](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984008) | L0 backbone with PsaD promoter | A1-B1 | ✓ | ✓ | ✓ |
| L0-bleR+scp | [BBa\_K2984045](http://parts.igem.org/Part%3ABBa_K2984045) | Bleomycin resistance fused to self cleaving peptide | B1-B1 | ✓ | ✓ | ✓ |
| L0-ARS1 | [BBa\_K2984000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984000) | L0 backbone with 3'UTR Arylsulfatase1 secretion signal | B2-B2 | ✓ | ✓ | ✓ |
| L0-GLE | [BBa\_K2984001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984001) | L0 backbone with 3'UTR gametolysin secretion signal | B2-B2 | ✓ | ✓ | ✓ |
| L0-PETase | [BBa\_K2984011](http://parts.igem.org/wiki/index.php?title=Part:BBa_K29840011) | L0 backbone with PETase CDS | B3-B4 | ✓ | ✓ | ✓ |
| L0-Hyg | [BBa\_K2984012](http://parts.igem.org/wiki/index.php?title=Part:BBa_K29840012) | L0 backbone with Hygromycin B resistance CDS | B3-B4 | ✓ | ✓ | ✓ |
| L0-Paro | [BBa\_K2984006](http://parts.igem.org/wiki/index.php?title=Part:BBa_K29840006) | L0 backbone with Paromomycin resistance CDS | B3-B4 | ✓ | ✓ | ✓ |
| L0-ptxD | [BBa\_K2984026](http://parts.igem.org/wiki/index.php?title=Part:BBa_K29840026) | L0 backbone with phosphite oxidoreductase CDS | B3-B3 | ✓ | ✓ | ✓ |
| L0-ptxD | [BBa\_K2984013](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984013) | L0 backbone with phosphite oxidoreductase CDS | B3-B4 | ✓ | ✓ | ✓ |
| L0-YFP | [BBa\_K2984017](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984017) | L0 backbone with yellow fluorescent protein CDS | B3-B4 | ✓ | ✓ | ✓ |
| L0-YFP | [BBa\_K2984024](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984024) | L0 backbone with yellow fluorescent protein CDS | B4-B4 | ✓ | ✓ | ✓ |
| L0-YFP | [BBa\_K2984020](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984020) | L0 backbone with yellow fluorescent protein CDS | B5-B5 | ✓ | ✓ | ✓ |
| L0-6xHis tag | [BBa\_K2984014](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984014) | L0 backbone with 6x histidine tag | B5-B5 | ✓ | ✓ | ✓ |
| L0-3xHis tag | [BBa\_K2984015](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984015) | L0 backbone with 3x histidine tag | B5-B5 | ✓ | ✓ | ✓ |
| L0-SP20 | [BBa\_K2984016](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984016) | L0 backbone with glycomodule for secretion enhancement | B5-B5 | ✓ | ✓ | ✓ |
| L0-RbcS2 | [BBa\_K2984018](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984018) | L0 backbone with RuBisCo terminator | B5-C1 | ✓ | ✓ | ✓ |
| L0-RbcS2 | [BBa\_K2984021](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984021) | L0 backbone with RuBisCo terminator | B6-C1 | ✓ | ✓ | ✓ |
| L0-Linker | [BBa\_K2984034](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984034) | L0 backbone with Linker sequence | B2-B2 | ✓ | ✓ | ✓ |
| L0-Ble | [BBa\_K2984040](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984040) | L0 backbone with Bleomycin resistance | B1-B1 | ✓ | ✓ | ✓ |
| L0-scp | [BBa\_K2984044](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984044) | L0 backbone with Bleomycin resistance | B1-B1 | ✓ | ✓ | ✓ |
| L0-PETase | [BBa\_K2984049](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984049) | L0 backbone with PETase | B3-B3 | ✓ | ✓ | ✓ |
| L0-PsaDIntron | [BBa\_K2984046](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984046) | L0 backbone with PsaDIntron | A1-A3 | - | - | - |
| L0-RFP | [BBa\_K2984004](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984004) | L0 backbone with RFP | - | ✓ | ✓ | ✓ |
| L0-amp | [BBa\_K2984007](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984007) | L0 backbone with ampilicin resistance | - | ✓ | ✓ | ✓ |
| L0-amp | [BBa\_K29858](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984058) | Linker L1c -> L0 | - | ✓ | ✓ | ✓ |
| L0 Linker | [BBa\_K29860](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984060) | Linker L0 -> L1a | - | ✓ | ✓ | ✓ |
| L0 Linker | [BBa\_K29861](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984061) | Linker L0 -> L1b | - | ✓ | ✓ | ✓ |
| L2 Linker | [BBa\_K29862](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984062) | Linker L2 -> L1b | - | ✓ | ✓ | ✓ |
| Linker sequence | [BBa\_K29834](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984034) | Linker sequence | B2-B2 | ✓ | ✓ | ✓ |

#### Examples for Successful Results of L1 and L2 Backbones

| Successful Amplification | Successful Transformation into *E. coli* | Successful Sequencing |
| --- | --- | --- |
| ![](https://static.igem.org/mediawiki/2019/4/46/T--Humboldt_Berlin--results_L1_1.png) | ![](https://static.igem.org/mediawiki/2019/a/a3/T--Humboldt_Berlin--results_L1_2.png) | ![](https://static.igem.org/mediawiki/2019/2/25/T--Humboldt_Berlin--results_L1_3.png) |

#### Cloning Results of Backbones

| Part Name | Registry Name | Description | Successful Amplification | Successful Transformation into *E. coli* | Successful Sequencing |
| --- | --- | --- | --- | --- | --- |
| L1a | [BBa\_K2984003](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984003) | Level 1 backbone with ampicillin resistance and RFP flanked by GoldenGate restiction site | ✓ | ✓ | - |
| L1b | [BBa\_K2984005](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984005) | Level 1 backbone with ampicillin resistance and RFP flanked by GoldenGate restriction sites | ✓ | ✓ | ✓ |
| L1c | [BBa\_K2984002](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984002) | Level 1 backbone with ampicillin resistance and RFP flanked by GoldenGate restriction sites | ✓ | ✓ | ✓ |
| L2 | [BBa\_K29863](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984063) | Level 2 backbone with paromycin resistance flanked by GoldenGate restriction sites | - | - | - |

#### Examples for Successful Results of L1 Composite Parts

| Successful Transformation into *E. coli* | Successful Sequencing | Successful Transformation into *C. reinhardtii* |
| --- | --- | --- |
| ![](https://static.igem.org/mediawiki/2019/2/24/T--Humboldt_Berlin--results_L1compo_2.png) | ![](https://static.igem.org/mediawiki/2019/e/ee/T--Humboldt_Berlin--results_L1compo_1.png) | ![](https://static.igem.org/mediawiki/2019/0/01/T--Humboldt_Berlin--results_L1compo_3.png) |

#### Cloning Results of L1 Composite Parts

| Part Name | Registry Name | Description | Successful Amplification | Successful Transformation into *E. coli* | Successful Sequencing | Successful Transformation into *C. reinhardtii* |
| --- | --- | --- | --- | --- | --- | --- |
| L1c-PsaD-YFP-RbcS2 | [BBa\_K2984019](http://parts.igem.org/Part%3ABBa_K2984019) | Reference for fluorescence measurements | ✓ | ✓ | ✓ | ✓ |
| L1c-PsaD-ARS-YFP-Rbsc2 | [BBa\_K2984027](http://parts.igem.org/Part%3ABBa_K2984027) | Secretion of YFP | ✓ | ✓ | ✓ | - |
| L1-PsaD-ARS-PETase-YFP-SP20-RbcS2 | [BBa\_K2984028](http://parts.igem.org/Part%3ABBa_K2984028) | Secretion of PETase and expression measurement via YFP fluorescence | ✓ | ✓ | ✓ | ✓ |
| L1-PsaD-GLE-PETase-YFP-SP20-RbcS2 | [BBa\_K2984029](http://parts.igem.org/Part%3ABBa_K2984029) | Secretion of PETase and expression measurement via YFP fluorescence | ✓ | ✓ | ✓ | ✓ |
| L1c-PsaD-ARS-YFP-SP20-Rbcs2 | [BBa\_K2984030](http://parts.igem.org/Part%3ABBa_K2984030) | Secretion of YFP to test the ARS secretion signal and SP20 efficiency | ✓ | ✓ | ✓ | ✓ |
| L1-PsaD-ARS-PETase-YFP-RbcS2 | [BBa\_K2984031](http://parts.igem.org/Part%3ABBa_K2984031) | Secretion of PETase and expression measurement via YFP fluorescence | ✓ | ✓ | ✓ | - |
| L1-PsaD-ARS-PETase-3xHA-RbcS2 | [BBa\_K2984032](http://parts.igem.org/Part%3ABBa_K2984032) | Secretion of PETase and purification via HA-tag | ✓ | ✓ | ✓ | - |
| L1c-AR-ARS-YFP-Rbcs2 | [BBa\_K2984033](http://parts.igem.org/Part%3ABBa_K2984033) | Secretion of YFP | ✓ | ✓ | ✓ | ✓ |
| L1c-PsaD-PETase-YFP-His-RbcS2 | [BBa\_K2984035](http://parts.igem.org/Part%3ABBa_K2984035) | Expression of PETase and expression measurement via YFP fluorescence to be purified via His-Tag | ✓ | ✓ | ✓ | - |
| L1c-PsaD-PETase-YFP-Rbcs2 | [BBa\_K2984036](http://parts.igem.org/Part%3ABBa_K2984036) | Secretion of PETase and expression measurement via YFP fluorescence | ✓ | ✓ | ✓ | - |
| L1c-PsaD-PtxD-Rbsc2 | [BBa\_K2984037](http://parts.igem.org/Part%3ABBa_K2984037) | Evaluation of C. reinhardtii growth on phosphite-containing media | ✓ | ✓ | ✓ | ✓ |
| L1c-Psad-PETase-ptxD-Rbcs2 | [BBa\_K2984038](http://parts.igem.org/Part%3ABBa_K2984038) | Expression of PETase and evaluation of*C. reinhardtii*growth on phosphite-containing media | ✓ | ✓ | - | - |
| L1c-PsaD-PETase-3xHA-RBCS2 | [BBa\_K2984039](http://parts.igem.org/Part%3ABBa_K2984039) | Expression of PETase and purification via HA-Tag | ✓ | ✓ | ✓ | - |
| L1c-PsaD-ARS-PETase-3xHA-SP20-RbcS2 | [BBa\_K2984041](http://parts.igem.org/Part%3ABBa_K2984041) | Enhanced Secretion of PETase and SP20 efficiency and purification via HA-Tag | ✓ | ✓ | ✓ | ✓ |
| L1c-PsaD-MHETase-YFP-RbcS2 | [BBa\_K2984042](http://parts.igem.org/Part%3ABBa_K2984042) | Expression of MHETase and expression measurement via YFP fluorescence | ✓ | ✓ | ✓ | - |
| L1c-PsaD-MHETase-3xHA-RbcS2 | [BBa\_K2984043](http://parts.igem.org/Part%3ABBa_K2984043) | Expression of MHETase and purification via HA-Tag | ✓ | ✓ | ✓ | - |
| L1c-AR-PETase-YFP-RbcS2 | [BBa\_K2984047](http://parts.igem.org/Part%3ABBa_K2984047) | Expression of PETase and expression measurements via YFP fluorescence | ✓ | ✓ | - | - || L1c-PsaD-bleRscp-ARS-PETase-SP20-RbcS2 | [BBa\_K2984048](http://parts.igem.org/Part%3ABBa_K2984048) | Enhanced Secretion of PETase and SP20 efficiency and bleomycin resistance to  *C. reinhardtii* | ✓ | ✓ | - | - |
| L1-PsaD-Paro-RbcS2 | [BBa\_K2984055](http://parts.igem.org/Part%3ABBa_K2984055) | Construct lending paromomycin resistance to *C. reinhardtii* | ✓ | ✓ | ✓ | ✓ |
| L1-PsaD-Hyg-RbcS2 | [BBa\_K2984056](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984056) | Construct lending hygromycin resistance to *C. reinhardtii* | ✓ | ✓ | ✓ | ✓ |

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![](https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg)

### Expression of YFP-containing constructs

The yellow fluorescent protein YFP was used as a fluorescent tag in some of our constructs. The goal of using YFP as a tag was to be able to measure enzyme expression and secretion and to screen for successful mutants using YFP as a marker. Additionally, we wanted to use a YFP-expressing *C. reinhardtii* to analyse possible locus effects on expression. We were able to successfully transform a YFP-expressing *C. reinhardtii* with a [construct of our own design](http://parts.igem.org/Part%3ABBa_K2984019).

But measurements of YFP in *C. reinhardtii* turned out to be a great challenge, because of the strong interaction of the algae with light (by their photosystems, pigments, chlorophyll and light antennae). More information on our process of measuring YFP can be found on our [Measurement page](https://2019.igem.org/Team%3AHumboldt_Berlin/Measurement).

We were able to successfully measure YFP fluorescence intensity and fluorescence spectra of YFP-expressing *C. reinhardtii* clones in comparison to the wild type (WT). The results showed that our clone exhibited a higher fluorescence intensity at 528 nm than the WT (YFP emission peak) and the fluorescence spectrum of YFP confirmed the presence of the yellow fluorescent protein. This YFP-expressing clone also allowed us to characterize the light induction of the PsaD promoter by doing a time-resolved measurement of the fluorescence intensity.

During this measurement we exposed *C. reinhardtii* cultures which contained our YFP construct to different growth conditions. One was exposed only to the dark, the other to synchronized growth lighting conditions (10 hours dark, 14 hours illuminated). Then, we started a time-resolved fluorescence intensity measurement in the dark, with a WT control. After approximately four hours, we activated a light source and exposed the cultures to light, thus activating the light inducible PsaD promoter. Our results showed that for the dark and synchronized cultures containing the YFP construct a peak in fluorescence intensity could clearly be seen after the light induction. This proved the light induction of the PsaD promoter. If you are interested in this measurement, please visit the page of our YFP mVenus construct in the iGEM registry [here](http://parts.igem.org/Part%3ABBa_K2984019).

![fluorescence intensity](https://static.igem.org/mediawiki/2019/7/73/T--Humboldt_Berlin--YFP_fluorescence_intensity.png)
 ![fluorescence difference spectrum](https://static.igem.org/mediawiki/2019/5/50/T--Humboldt_Berlin--YFP_fluorescence_difference_spectrum.png)
 ![fluorescence intensity time resolved](https://static.igem.org/mediawiki/2019/d/d9/T--Humboldt_Berlin--YFP_Lichtinduziert_wt_Sync_21_10_19.png)

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![](https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg)

### Secretion of Enzymes

Secretion of enzymes is of high importance to solve complex problems with the help of cells like *C. reinhardtii*. For PET degradation specifically, the secretion is fundamental, so that the enzyme PETase can reach its substrate PET. We used several different secretion signal peptides and designed constructs specifically to test and quantify the secretion out of the cell. There were three level 1 contructs we designed to test secretion:

1. [L1c-Psad-ARS-YFP-RbcS2](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984027)
2. [L1c-Psad-ARS-YFP-SP20-RbcS2](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984030)
3. [L1c-AR-ARS-YFP-RbcS2](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984033)

The first and third construct were designed for measuring the influence of arylsulfatase secretion signal (ARS) on mVenus secretion, expressed by two different promoters. The first and second constructs could be compared to see how the SP20 glycomodule increased the secretion driven by the ARS peptide. The level 1 constructs were transformed into *C. reinhardtii* via electroporation. After two weeks of growth on TAP-agar plates containing paromomycin, the present clones were picked for colony PCR or sequencing.

To see if and how our different constructs worked, we conducted supernatant measurements.
For each positive clone 50 ml flasks were prepared 4 days in advance. Of each flask 150 µl were transferred to a black 96-well plate and sequentially diluted up to 1:128. These flasks were then centrifuged for 10 min with a speed of 2000 rpm at 4°C. The supernatant was separated from the pellet in a fresh falcon tube for measurement. Before starting another centrifugation process, 12 x 150 µl of each supernatant were transferred to a black 96-well plate. This step was repeated after the second centrifugation, creating a third well plate. All three well plates were then analyzed under the plate reader to measure for fluorescence emission.

Unfortunately, we were unable to measure fluorescence in any of the supernatants.
For every well in each plate, absorbance was measured at a wavelength of 680 nm to check the supernatants for possible cell debris or leftover Chlamys and absorbance scans were performed to scan for the absorption by YFP. Additionally, we measured the fluorescence with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The fluorescence intensity, plotted against the absorbance of the medium, can be seen in Fig. 4. Here we see that the fluorescence intensity correlates with the optical density of the medium, which explains the fluctuations in fluorescence intensity due to the presence of residual cells or debris in the medium. It was not possible to assess with certainty if there was YFP present in the medium.

Nevertheless, we were able to prove that YFP was present in vesicles near the membrane area of one of our YFP mVenus secretion clones ([BBa\_K2984030](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984030)). This was done by imaging *C. reinhardtii* cells using confocal microscopy (Fig. 4.1). Although we were not able to measure YFP in the medium surrounding the algae, which is difficult, as can be read in our [measurements page,](https://2019.igem.org/Team%3AHumboldt_Berlin/Measurement) it is possible to assume that secretion did occur if we observe the confocal micrsocopy images. The presence of the vesicle strongly suggests secretion of YFP mVenus.

Additionally, we can verify that the ARS secretion signal works in principle during the screening of one of our CRISPR/Cas9 transformations, where secretion is used to color positive clone blue (Fig. 5). By knocking out the SNRK gene, which regulates the expression of the arylsulfatase, uncontrolled secretion of this enzyme takes place. By adding a special pigment to the colonies, the knockout colonies become blue, demonstrating the function of the ARS secretion signal.

![secretion measurements](https://static.igem.org/mediawiki/2019/b/ba/T--Humboldt_Berlin--SecretionMeasurement.png)
 ![confocal microscopy - YFP](https://static.igem.org/mediawiki/2019/b/b2/T--Humboldt_Berlin--Microscopy-YFP.png)

 ![blue screening](https://static.igem.org/mediawiki/2019/f/f0/T--Humboldt_Berlin--WhenARSworks.jpeg)

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![](https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg)

### Phosphite decarboxylase Controlled Growth: PtxD as a selection advantage?

Utilizing an alga that can compete against other organisms in culture by using another phosphorus source, e.g. phosphite, can have great advantages in controlling contamination and competition in a cultivation. The phosphite decarboxylase (ptxD) enables *C. reinhardtii* to outcompete against other algae as Loreza-Quezada et.al. (2016) have shown. Thus, the combination of PtxD expression coupled with expression of PETase and MHETase would enable *C. reinhardtii* to degrade microplastic within a closed or open cultivation setup.

Furthermore, b< transforming such an enzyme into a *C. reinhardtii* strain these transformants gain a selection advantage in comparison to the wild type. Trough coupling of PtxD to PETase or MHETase we could avoid the problem of *C. reinhardtii* cells, which lost PETase/MHETase activity by expulsion of the plasmid prevailing.

Based on these advantages we want to transform the PtxD gene into *C. reinhardtii*. To determine the impact of phosphate and phosphite on growth of *C. reinhardtii*, we tested different strains under several media conditions. As a reference we used the TAP medium containing phosphate. We further tested growth in a medium without phosphate (TA medium) and in TAP medium supplemented with phosphite (TAPi).

The first graph shows the growth curve of the wildtype strain SAG11-32b.
The cells growing on TAP media show growth up to OD 2.3 [R.U.]. Cultures growing on phosphorus-deficient media (TA) grow similarly. This is probably due to the phosphate storage of *C. reinhardtii* in the cytosol (Vered Irihimovitch et al., 2008).

In comparison, the cultures growing on phosphite (TAPi) media show strongly reduced growth. This is explained by the fact that phosphonate acts as a potent enzyme inhibitor which competes with its structural analogues for binding the enzyme's active sites (White & Metcalf, 2007).
In conclusion, the metabolism of phosphate is inhibited by phosphite in *C. reinhardtii* wild type cells.

To show how the *C. reinhardtii* mutant strain UVM4 (mutant without cell wall) can handle phosphate starvation, cultures were grown in TA medium for 9 days and were then used to inoculate the cultures on TAPi medium.
The graph shows three replicates of UVM4 cultures that were cultivated in phosphate-depleted medium (blue, red, green). These cells were not able to grow at all but if these were rescued in TAP medium they were able to grow again. Cells that grew on TAP medium as a control displayed normal growth.
This experiment shows that *C. reinhardtii* cannot grow under phosphate starvation if the storages of phosphate were previously depleted.

![Sag11 growth TA and TAPi](https://static.igem.org/mediawiki/2019/9/97/T--Humboldt_Berlin--growth_of_sag11_ptxd.png)

![UVM4 growth TA and TAPi](https://static.igem.org/mediawiki/2019/a/a8/T--Humboldt_Berlin--growth_uvm4_ptxd.png)

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![](https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg)

### CRISPR/Cas9 Directed Transgene Insertion

Achieving high expression rates of recombinant proteins is often difficult in *C. reinhardtii*. Transgenes are randomly integrated into the genome and consequently their expression is influenced by the genetic surrounding of the insertion site. We hoped to [increase expression](/Team%3AHumboldt_Berlin/Design), by inserting our DNA construct into a defined region of the *C. reinhardtii* genome.
With CRISPR/Cas9, we targeted three different loci to insert our transgenic constructs.

To see if our Cas9 assays work in general, we first used the easy screenable SNF-related serine/threonine-protein kinase (*SNRK2.2*) locus. Inactivation of the *SNRK2.2* gene leads to the constitutive expression of arylsulfatase, which gets secreted into the medium. The presence of arylsulfatase can easily monitored with a color reaction. By cleaving the sulfate group from the colourless substrate 5-bromo-4-chloro-3-indolyl-sulfate (X-SO4) the dye indole blue is formed (Fig. 8). This blue-green screen poses as an efficient method of finding CRISPR/Cas9-directed insertion mutants.

The first construct we inserted into the *SNRK2.2* locus was the level 1 construct [L1c-Psad-YFP-Rbcs2](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984019). This experiment delivered us our first positive YFP-expressing clone exhibiting a robust expression of mVenus. Then, we performed a colony PCR with *SNRK2.2* locus-specific primers to amplify the insert. The *SNRK2.2* specific amplification without an insert would lead to bands with a length of approximately 200 bp. If the insert was successfully inserted into the *SNRK2.2* gene, we expected a band of approximately 2000 bp. The PCR results showed that the insertion of the YFP construct into the *SNRK2.2* locus was only partly successful (Fig. 9). We can see a bigger band at around 5000 bp. The YFP-expressing clone is labeled on Fig. 9 as A4. We sent the bands of a length of 5000 bp to sequencing but obtained only overlayed signals from a mix of PCR products. So the *SNRK2.2* target site was cut by CRISPR/Cas9 but we have no certainty if the YFP construct is inserted along with other sequences, inserted twice or not inserted at all.

After confirming that we can insert our YFP construct at targeted regions into the genome we now thought of loci that would boost the expression of our transgene. We chose two of the most highly expressed genes in *C. reinhardtii*, *PSAD* and *RBCS2* and designed guide RNAs for CRISPR/Cas9 to insert our YFP-constructs into the 3'UTR region of these genes. By inserting our construct into these loci we hoped for an increased expression of our YFP mVenus.

After the [transformation of our algae with CRISPR/Cas9 ribonucleoproteins](https://2019.igem.org/Team%3AHumboldt_Berlin/Experiments) we performed colony PCRs as a screening method to examine if the insertion into the loci was successful. For this, we designed primers that bind to the *PSAD* and *RBCS2* genes to investigate if the insertion worked as planned.

The PCR result, seen on Fig. 10, shows the analysis of 96 clones that were transformed to target a YFP construct into *PSAD* and *RBCS2*. Only for the wild type (WT) probe of RbcS2 a clear PCR band can be seen at the expected length. No clear bands can be observed in the other samples. This indicates that there was a problem with the PCR parameters and fragments were not amplified correctly. Due to the fact that we did this experiment at the end of our project we did not have enough time to do further screening or transformations to identify high expression loci in *C. reinhardtii*.

![blue screening](https://static.igem.org/mediawiki/2019/f/f0/T--Humboldt_Berlin--WhenARSworks.jpeg)

 ![YFP gel](https://static.igem.org/mediawiki/2019/thumb/d/d7/T--Humboldt_Berlin--A4-PCR_4.tif/372px-T--Humboldt_Berlin--A4-PCR_4.tif.jpeg)

 ![Crispr PCR](https://static.igem.org/mediawiki/2019/9/91/T--Humboldt_Berlin--CRISPR-GEL.jpg)

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![](https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg)

### RESDA PCR for Identification of Transgene Insertion Locus

The locus where a transgene is inserted is of great importance for the expression rate of the gene. Many conventional methods of transformation lead to random insertion of the transgene, so that there is no influence on the insertion locus. When a transformation is successful and a transgene is abundantly expressed, it might be of interest to know in which locus the gene was inserted. The RESDA-PCR method is a method that allows localization of the insertion locus.

We cultivated a *C. reinhardtii* clone which was successfully transformed with our [YFP mVenus construct](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2984019) and showed abundant expression of the protein. We wanted to investigate in which locus our transgene was inserted, so we planned a RESDA PCR.

The RESDA PCR method is a PCR method designed to identify the insertion locus of transgenes. The method was developed by González-Ballester and his colleagues and is based on the amplification of a transgene from the genome using primers that bind on recurrent restriction sites in the genome (González-Ballester et al. 2005). To amplify the transgene, one primer that binds to the insert is used and another primer that binds to one of the recurrent enzymes in the genome, in hope that the insert is close enough to a restriction site to be amplified. The amplification is done through two different rounds where the second round uses nested primers to narrow down the specificity of the amplification. After the amplification the fragment can be sequenced to determine its locus.

Our goal was to determine the locus of insertion of our YFP mVenus construct, to see if we could use this locus for future directed insertion of transgenes with CRISPR/Cas9. Additionally, we also performed the RESDA-PCR with a luciferase-expressing clone kindly provided by the research group of Prof. Dr. Hegemann of the Humboldt University of Berlin.

The PCR was made with primers for four different restriction sites. These are *AluI*, *PstI*, *SacII* and *TaqI*. We performed the PCR with a temperature gradient and with different primer concentrations to check for optimal conditions of amplification. Unfortunately, we were not able to observe any bands on the agarose gel after our RESDA-PCR (Fig. 1). We repeated the PCR twice, varying the conditions each time to optimize the formation of bands. Still, we were not able to reproduce the results described by González-Ballester et al. (2005). This means that we were unfortunately unsuccessful to determine the locus of insertion of our YFP expressing clone.

![](https://static.igem.org/mediawiki/2019/9/97/T--Humboldt_Berlin--RESDA-PCR.jpg)

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![](https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg)

![](https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg)

## 2. PET-degradation

### PET-degradation *in-silico*

The viability of PET degradation by *C. reinhardtii* at a larger scale is yet unknown. Models of biological systems allow us to design experiments *in silico* that are difficult to reproduce  *in vivo* and give us special insights into the role that parameters might play in the given biological system. Therefore, to assess the efficiency of PET degradation by *C. reinhardtii*, a mathematical model of PET degradation in a continuous culture of *C. reinhardtii* was designed.

The overall goal of the model is to determine the time needed to degrade 1 mg of PET. The model takes into account enzyme expression, secretion and kinetics and also the cultivation density of the algae as decisive factors for the PET degradation rate. It predicted that for a cultivation density of 1:10, a 40 g PET bottle would be degraded in approximately 10 years. For a cultivation density of 1:100, the predicted time to degrade a bottle was 100 years. Additionally, an improvement of the PETase enzyme by a factor 1000 was made. For a cultivation density of 1:10 and the improved enzyme, the time needed to degrade a bottle was 119 days. For a cultivation density of 1:100 and the improved enzyme, the time needed to degrade a bottle was 3,3 years. The results of the model led us to take several decisions regarding the improvement of the PET degradation. We chose to use the light-inducible PsaD promoter for our constructs, the secretion enhancing glycomodule SP20, the specialized *C. reinhardtii* strain UVM4 for increased transgene expression and the flat panel cultivation method to achieve higher cultivation densities. For more information please visit our [model page](/Team%3AHumboldt_Berlin/Model).

![PET degradation](https://static.igem.org/mediawiki/2019/6/6e/T--Humboldt_Berlin--PETase_Model_1_to_10.png)
 ![PET 1 to 100](https://static.igem.org/mediawiki/2019/7/7f/T--Humboldt_Berlin--PETase_Model_1_to_100.png)

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### PET degradation by *C. reinhardtii*

Expressing and secreting PETase and MHETase in *C. reinhardtii* was one of the main goals of our project. To be able to express the PETase, we designed a wide variety of constructs with functional parts from our [Chlamy-HUB Collection](/Team%3AHumboldt_Berlin/Part_Collection).

These constructs were systematically designed to experimentally test the expression and secretion of PETase. Additionally, we designed the constructs with different markers for detection or measurement of the enzyme, like the yellow fluorescent protein mVenus, HA-tags and His-tags. Construct [BBa\_K2984028](http://parts.igem.org/Part%3ABBa_K2984028)  was for example designed for enhanced secretion of PETase, marked with the YFP mVenus. Or construct [BBa\_K2984039](http://parts.igem.org/Part%3ABBa_K2984039), designed for expression of the enzyme, marked with a HA-tag for detection, isolation and purification. To see more constructs you can visit our [composite parts](/Team%3AHumboldt_Berlin/Composite_Part) page.

![PETase](https://static.igem.org/mediawiki/2019/3/39/T--Humboldt_Berlin--PETase.jpg)
 ![PETase Gel](https://static.igem.org/mediawiki/2019/1/1b/T--Humboldt_Berlin--PETase_Gel.jpg)

**Fig. 15 - Amplification of the PETase transgene of *C. reinhardtii* through a colony PCR shows successful transformation of the construct.** Expected band length: 885 bp.

![MHETase](https://static.igem.org/mediawiki/2019/1/12/T--Humboldt_Berlin--MHETase.jpg)

**Fig. 16 - Crystal structure of the MHETase enzyme.**

![MHETase Gel](https://static.igem.org/mediawiki/2019/0/0a/T--Humboldt_Berlin--MHETase_Gel_2.jpg)

**Fig. 17 - Amplification of the MHETase transgene out of *C. reinhardtii* through a colony PCR.** Due to problems with the primer we had issues with unspecific bands in the gel. The successful transformation of the construct could not be confirmed.

The constructs were all assembled using the MoClo standard and then transformed into *C. reinhardtii*. After the transformation, the clones had to be screened for positive transgene insertion. The screening was done through colony PCRs, where the inserted fragments were fully or partially amplified to verify transgene insertion. Unfortunately, our initial PETase level 0 part was missing one nucleotide, which led to a frameshift of all subsequent parts in the construct. Although we had done many transformations of PETase constructs it was not until two months into our transformation workflow that we discovered this mistake. This made our PETase impossible to screen, because all markers for detection were located after the PETase sequence and had the aforementioned frameshift.

After successfully correcting the mistake in the PETase sequence, we were able to successfully transform a PETase construct into *C. reinhardtii*. We transformed construct [BBa\_K2984032](%E2%80%9Dhttp%3A//parts.igem.org/wiki/index.php?title=Part:BBa_K2984032”) into *C. reinhardtii*, which contains the secretion signal ARS and an HA-tag to detect and isolate the enzyme. In Fig. 15, the successful amplification of the PETase enzyme of *C. reinhardtii* via colony PCR can be seen. The expected length of the fragment was 885 bp or approximately 900 bp. The samples were sent to sequencing and we could verify that the sequence of the PETase was intact after the insertion into *C. reinhardtii*. Unfortunately we did not have enough time to do further tests and experiments with the transformed clone.

Additionally to the PETase enzyme, we wanted to transform the MHETase enzyme into *C. reinhardtii*. For this enzyme we also designed constructs systematically, as can be seen in our [composite parts](/Team%3AHumboldt_Berlin/Composite_Part) overview. The MHETase we used for these constructs was kindly given to us by the  [TU Kaiserslautern](/Team%3ATU_Kaiserslautern) 2019 iGEM team. Nevertheless, after various attempts to transform the MHETase into *C. reinhardtii*, we were unable to find a successful clone in our screening. We had problems amplifying the MHETase through a colony PCR because we had difficulties with the primer specificity. The primers we designed first showed well defined bands at the wrong length in the gels. We sent our probes to sequencing and it turned out that our primers were amplifying parts of chromosome 16 of *C. reinhardtii*. After ordering new primers, we encountered another specificity issue: several unspecific bands were appearing in our gel (Fig. 17). Because of these problems with the MHETase screening we were not able to assess in time if one of our transformed clones was positive.

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### Growth Experiments: Toxicity of microplastic and its degradation products

####

When it comes to cultivating our transgenic alga in the bioreactor it is not only important to know about the toxicity of the PET degradation products. Since the alga should degrade PET, we also checked if the presence of substantial amounts of microplastic in the media affected the growth of *C. reinhardtii*.
To determine the range of possible applications for *C. reinhardtii* for plastic degradation we performed toxicity tests on different strains. We assessed whether the degradation products of MHET, terephthalic acid (TPA) and ethylene glycol (EG) are toxic for *C. reinhardtii* and showed that TPA is not toxic for the alga whereas EG is toxic in concentrations only above 7%. As shown by our model, even the most optimistic catalytic rates would not yield such high EG concentrations. Therefore, we can be confident that our algae can survive degrading PET in a bioreactor setup.

**Toxicity test setup**

We performed our toxicity tests in a Multi-cultivator “MC1000”. This cultivator enables measurement of growth in different media creating growth curves.

The growth of *C. reinhardtii* was observed by measuring the optical density at OD 680 nm. At this wavelength, photosystem II has its absorption maximum, so measuring at this wavelength is common when cultivating phototrophic organisms. To exclude a possible contamination the cultivator also measured the OD at 720 nm where every other organism absorbs. The ratio of both measurements shows the reproduction of either *C. reinhardtii* or a contamination (e.g. bacteria).

![Multi-cultivator MC1000](https://static.igem.org/mediawiki/2019/3/34/T--Humboldt_Berlin--results_reactor_set-up.png)

**Toxicity test for Terephthalic acid (TPA)**

To determine the toxicity of TPA growth experiments were performed with the wild type *C. reinhardtii* strain CC-125. The solubility of TPA is fairly low with ~15 µg/l. In these measurements different dilutions of TPA in the standard TAP medium were tested inside the Multi-cultivator 1000. Figure 19 shows the growth curve of *C. reinhardtii* determined at 680 nm (shown in green) by measuring the OD. TPA was dissolved in TAP medium. Even concentrations of more than 15 µg/l (where TPA precipitates) did not have an influence on the reproduction of *C. reinhardtii*. The red curve illustrates the OD at 720nm. Since these curves do not show an abnormal increase, the cultivator was not contaminated.
This shows that growth of *C. reinhardtii* is not influenced by TPA.

![fluorescence intensity](https://static.igem.org/mediawiki/2019/a/ac/T--Humboldt_Berlin--results_toxtest_tpa_cc125.png)

**Fig. 19 - Growth of *C. reinhardtii* wild type CC-125 at 680 nm and 720 nm under soluble and saturated (15 µg/l) TPA concentration in comparison to a TAP control.** Cells were cultivated inside the Multi-cultivator 100 under 150 µE light at 30°C.

**Toxicity test for ethylene glycol**

Ethylene glycol, Ethan-1,2-diol, (EG) is a colourless organic compound with the formula (CH2OH)2. It has a LD50 in rats (orally) 4700 mg·kg−1. The toxic effect relies on its byproducts glycolaldehyde, glyoxal and glyoxylic acid. To determine the toxic effect of EG on *C. reinhardtii* the growth of several strains at concentrations of 1 % to 10 % EG was tested.

**Figure 20 shows that *C. reinhardtii* wild type CC125 is able to tolerate EG concentrations up to 5 % without showing growth limitation.** A concentration of 4 % decreases the growth speed but nevertheless growth maximum is reached. Concentrations starting at 7 % are lethal to the alga.
But due to the turnover rate of the enzymes PETase and MHETase the concentration of EG will never reach a concentration more than 1-2 %.
This fact makes it possible for *C. reinhardtii* to survive in an environment where these products arise. Thus, *Chlamydomonas* represents a good model organism to tolerate the process of PET degradation.
We further did the same measurement using the *C. reinhardtii* mutant strain UVM4 that lacks a cell wall. The graph in figure 21 also demonstrates that already concentrations of 6 % inhibit the growth of this mutant strain.

![fluorescence intensity](https://static.igem.org/mediawiki/2019/6/6f/T--Humboldt_Berlin--results_toxtest_eg_uvm4.png)

Fig. 20 - Growth of *C. reinhardtii* wild type CC125 at 680 nm and 720 nm under soluble and saturated (15 µg/l) TPA concentration in comparison to a TAP control. Cells were cultivated inside the Multi Cultivator MC100 under 150 µE light at 30°C.

![fluorescence intensity](https://static.igem.org/mediawiki/2019/5/5f/T--Humboldt_Berlin--results_toxtest_eg_cc125.png)

Fig. 21 - Growth of *C. reinhardtii* mutant strain UVM4 at 680 nm with different EG concentrations between 0-7% in TAP media. Cells were cultivated inside the Multi Cultivator MC1000 under 150 µE light at 30°C.

**Toxicity test on the effect of microplastic**

When it comes to cultivating our transgenic alga in the bioreactor it is not only important to know about the toxicity of the PET degradation products. Since the alga should degrade PET, we also checked if the presence of substantial amounts of microplastic in the media affects the growth of *C. reinhardtii*. From our visit at the wastewater treatment facility we learned that the cleaned water that leaves the facility has an average microplastic concentration of 15 mg/L (A. Matzinger, 2018). We set up cultivation experiments with varying concentrations of microplastic particles (<40 µm).

![fluorescence intensity](https://static.igem.org/mediawiki/2019/5/54/T--Humboldt_Berlin--results_toxtest_microplastic_sag11-32b.png)

**Fig. 22 - For the amount of microplastic and the period of time that we tested we could not measure an effect on the alga growth.** For some measurements we observed deviations which could be due to microplastic particles interfering with the OD sensores of the multi-cultivator.

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## 3. *C. reinhardtii* for industrial use:

### *C. reinhardtii* in our use-case scenario

One of the main goals targeted by projects like ours is the development of a use-case scenario. We therefore visited a wastewater treatment facility in Berlin to test growth of *C. reinhardtii* under natural and industrial conditions. Our idea: The *C. reinhardtii* mutant strain UVM4 should degrade microplastic inside a wastewater treatment facility.

As UVM4 has no flagella it sediments. Together with the sinking terephthalic acid (TPA) both can be pumped out. This could be included into the existing cleaning process: Inside the secondary clarifier the sewage sludge settles on the ground of the pond and gets pumped out. The clean top layer of this water flows into the river network of Berlin. To test this if such a system would be feasible we took some water samples from the different cleaning stages of the wastewater treatment facility into our lab and performed growth tests with *C. reinhardtii* in it. We took samples from the secondary clarifier as well as water that had been cleaned by UV light in the facility.

Unfortunately Chlamy did not grow in any taken wastewater samples. We realized that cultivating Chlamy in wastewater requires many further examinations, experiments and modifications. Thus, different water conditions tolerable by *C. reinhardtii* must be examined.

![PET degradation](https://static.igem.org/mediawiki/2019/d/db/T--Humboldt_Berlin--secondary-clarifier.jpeg)

**Fig. 23 - Water received from wastewater facility in Berlin.**

![Chlamy growth in wastewater](https://static.igem.org/mediawiki/2019/8/86/T--Humboldt_Berlin--growth_uvm4_od680_wastewater.png)

**Fig. 24 - Growth of *C. reinhardtii* at OD 680 under 150 µE light.** Incubation of 1ml
UVM4 strain at OD 1,7 in 50 ml media (e.g freshwater from lab sink, secondary clarifier and the UV cleaned water of the wastewater facility).

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### Filtration of *C. reinhardtii* for selection inside the bioreactor

When using a bioreactor to degrade PET, Chlamy will mix with PET, MHET, EG and TPA. We wanted to find a way to separate *C. reinhardtii* from the other components and to extract the cells out of our bioreactor. Therefore we tested several microfilters with different pore widths.
For this test we used the *C. reinhardtii* cell-wall-deficient UVM4 mutant strain. By using vacuum filtration, UVM4 cells were filtered through different filters.

We tested three different pore sizes: 7 µM, 5 µM, 0.8 µM for filtration success. To determine the efficiency of the filtration process we measured the optical density of the culture and the weight of the filters. The table shows that with decreasing pore size the filter gains weight and the optical density of the filtrate decreases. By the second filtration through a 0.8 µM filter less cells stick to the filter.

| filter pore size | filter's weight increase | culture's decrease of OD |
| --- | --- | --- |
| 7 µM | 0.0007 g | 0.0007 |
| 5 µM | 0.0043 g | 0.159 |
| 0.8 µM (first filtration step) | 0.0061 g | 0.277 |
| 0.8 µM (secondary filtration step) | 0.0041 g | 0.224 |

The pictures taken of the filters after the filtration process are shown in Figure 25. The first filtration through a 0.8 µM filter yielded the greenest color and therefore shows the highest net filtration.
By this measurement we could show that only a pore size of 0.8 µM can filter *C. reinhardtii* cells sufficiently well. Since *C. reinhardtii* cells have a size of 10 µM this result is surprising. An explanation for the small required pore size could be the missing cell wall, as we used the cell-wall-deficient mutant strain.
With this knowledge we can build filter systems inside our bioreactor to sufficiently filter *C. reinhardtii* out of the bioreactor and to separate it from other components.

![vacuum filtration setup with different filter pads.](https://static.igem.org/mediawiki/2019/7/72/T--Humboldt_Berlin--filtration_set-up.png)

![Different pore sizes and their cut-off rate](https://static.igem.org/mediawiki/2019/2/23/T--Humboldt_Berlin--filters_green.png)

**Fig. 26 - *C. reinhardtii* cell-wall-deficient UVM4 mutant strain filtered through microfilters with different pore sizes.** Cells stick to the filter with 0.8 µM sized pores.

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### Learn more...

[![plant](https://static.igem.org/mediawiki/2019/2/29/T--Humboldt_Berlin--plants.jpg)](/Team%3AHumboldt_Berlin/Plant)
[![plant](https://static.igem.org/mediawiki/2019/2/21/T--Humboldt_Berlin--Achievements_bw.jpeg)

#### Achievements

Find out about our achievements and the goals we reached during our iGEM project.](/Team%3AHumboldt_Berlin/Achievements)

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González–Ballester, D., de Montaigu, A., Galván, A. and Fernández, E. (2005). Restriction enzyme site-directed amplification PCR: a tool to identify regions flanking a marker DNA. Anal. Biochem. 340, 330–335. doi:10.1016/j.ab.2005.01.031

Vered I., Shlomit Y., Phosphate and sulfur limitation responses in the chloroplast of Chlamydomonas reinhardtii, FEMS Microbiology Letters, Volume 283, Issue 1, June 2008, Pages 1–8, https://doi.org/10.1111/j.1574-6968.2008.01154.x

White, A. K., & Metcalf, W. W. (2007). Microbial metabolism of reduced phosphorus compounds. Annu Rev Microbiol, 61, 379-400. doi:10.1146/annurev.micro.61.080706.093357.

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