Team:Humboldt Berlin/Experiments

Experiments

Experiments

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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.

    5. 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).
      3. 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 BpiI and BsaI. 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 BpiI recognition sites are positioned on the outsides of the construct surrounded by two random bases which are represented as “n”. The BpiI 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 BpiI cutting site (blue) is similar to the BpiI 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

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


    6. Component Amount [µl] Amount [µl]
    10x FD (Fast Digest) 6 µl 2 µl
    Backbone 2,5 µg = x µl 1 µg = x µl
    BamHI 2,5 µl 1 µl
    XhoI 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 BamHI & XhoI without purification, directly with PCR product

      Component Amount [µl]
      10x FD (Fast Digest) 10 µl
      PCR Produkt (Insert) 50 µl
      BamHI 5 µl
      XhoI 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
    2. 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
BpiI 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 BsaI and BpiI, 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 BpiI.

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
    BpiI 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” 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 BpiI 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 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.
  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 of overnight cultures, labeled with clone number and store at -80°C.
  6. Plasmid prep 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 BsaI and BpiI 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 BsaI, BpiI, BamHI and SpeI 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 BamHI, XhoI and XbaI 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 BpiI 0.5 µl
    Thermo FastDigest Buffer (10x) BpiI 2 µl
    Thermo T4 DNA Ligase buffer (10x) 10x 2 µl
    Thermo T4 DNA Ligase (5 U/µL) 1 µl
    H2O 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 is our structured overview of importent experiments and their order. It's sorted by workflow topic (Synthesis, E. coli, Chlamydomonas and general)

Learn more...

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Contribution

To get a better understanding of the protocols followed to measure absorbance and fluorescence spectra with a plate reader, we conducted some measurements with fluorescent and non-fluorescent chromoproteins expressed in E. coli.

experiments preview

Demonstrate

Following the iGEM guidelines we demostrated that our engineered system works.

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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