Difference between revisions of "Team:Humboldt Berlin/Experiments"

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                                 <p>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.</br></br>
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                                 <p>To check if a construct was successfully transformed into <i>C. reinhardtii</i>, 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.</br></br>
 
<p><b>Preparation of DNA:</b></p>
 
<p><b>Preparation of DNA:</b></p>
 
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                               <p>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. </br></br>
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                               <p>After having obtained successful results of the integration of YFP into a <i>C. reinhardtii</i> clone from the colony PCR, it can be screened for fluorescence. If the YFP protein was expressed correctly, the clone should display a signal. </br></br>
 
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<li>For each clone to be screened, place 160 µl of resuspended cell culture in a 96-well plate</li>
 
<li>For each clone to be screened, place 160 µl of resuspended cell culture in a 96-well plate</li>

Revision as of 10:06, 13 October 2019

Experiments

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 of 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 10^7 . 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 was 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, under a biosafty cabinet, into the cultivation tubes and measurments were made. 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 unter 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% +/-

  6. 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
    4. 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.
    5. 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.
    6. Seal recovery plate with parafilm and incubate overnight with gentle shaking (110 rpm) under continuous light (50 µmol m-2 s-1).
  7. 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 cycling
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.

Fluoreszenz 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

DNA-Isolation

TODO

Synthesis Protocols

Sequence & Primer Design

TODO

PCR

TODO

Digestion

TODO

Ligation

TODO

General

Gel Electrophoresis

TODO

Protein Expression: SDS-page

TODO

Workflow

Plasmid assembly

TODO