Sep 10, 2021

Public workspaceChlamydomonas reinhardtii nuclear transformation by electroporation. V.3

PLOS One
DOI
dx.doi.org/10.17504/protocols.io.bx5cpq2w
  • João Vitor Molino1
  • 1University of Zurich
  • Lab
Joao Vitor Molino
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DOI: dx.doi.org/10.17504/protocols.io.bx5cpq2w
External link: https://doi.org/10.1371/journal.pone.0192433
Protocol Citation: João Vitor Molino 2021. Chlamydomonas reinhardtii nuclear transformation by electroporation.. protocols.io https://dx.doi.org/10.17504/protocols.io.bx5cpq2wVersion created by Joao Vitor Molino
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: September 10, 2021
Last Modified: September 10, 2021
Protocol Integer ID: 53124
Keywords: Microalgae, Recombinant, electroporation, plasmid, Chlamydomonas,
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Abstract
This protocols describe the steps required for nuclear transformation of Chlamydomonas reinhardtii by electroporation.

Here you can find a video following the protocol.
Guidelines
Cell density for harvesting is important to overall transformant yields. It should be aimed to mid-log phase cells. 
*Transformation tested from 1-6 x 10^6 cells/mL - Worked.
Materials
MATERIALS
ReagentMAX Efficiency™ Transformation Reagent for AlgaeThermo FisherCatalog #A24229
Protocol materials
ReagentMAX Efficiency™ Transformation Reagent for AlgaeThermo FisherCatalog #A24229
ReagentMAX Efficiency™ Transformation Reagent for AlgaeThermo FisherCatalog #A24229
Safety warnings
  • High voltage is used in the electroporation, use EPIs and avoid contact with electrodes on the electroporator.
Before start
  • Prepare a ice bucket
  • Separate cuvettes, keep them on ICE
  • Allow linearized vectors to melt 
  • Keep transformation buffer on ICE/Fridge
  • Prepare 50 mL centrifugal tubes with 10 mL TAP medium for recover stage
DNA Preparation
DNA Preparation
6h 30m
6h 30m
Digest a large enough amount of plasmid. The goal is to have a concentrated digested sample in the range of 250-700 ng/uL.

  1. Select the appropiate enzymes for linearization. Usually, restrictions sites in flanking position to the expression cassete.
  2. Mix all components for digestion Amount40 µg uncut vector . Digest for Duration06:00:00 at Temperature37 °C .
  3. Column purify digestion (Avoid gel purify, since vector backbone may helps to prevent intracelular DNAses action). *Use a PCR purification kit to purify the digestion reaction.
  4. Quantitate by absorbance measurement (i.e. Nanodrop).


ComponentAmount
10X Cutsmart NEB6.0 uL
XbaI | NEB 20 U/uL3.0 uL
KpnI HF | NEB 20 U/uL3.0 uL
Plasmid 1219.9 ng/uL40 uL
ddH20, Molecular grade8.0 uL
Typical reaction setup

Expected result
Result example
Concentration0.641 µg/µL Cutted vector
Amount30 µL Final elution volume
Amount19.230 µg total mass



6h 30m
Cells preparation
Cells preparation
  1. Aseptically inoculate Amount250 mL Tap media with wild type cells. Either by scrappeing cells of a plate with a inocculating loop or from a previous cultured cells.
  2. Incubate at Temperature25 °C , under constant shaking (~150-180 RPM) and light (60-80 µmols de photons/m2s) until a cell density from Amount3 x 10^6 cells/mL to Amount6 x 10^6 cells/mL is reached.
Pellet cells in centrifuge tubes. Separate culture in sufficient amount of sterile 50mL centrifuge tubes or larger volume tubes, and centrifuge for Centrifigation2000 x g, 25°C, 00:10:00 .  
Pellet Cells
  1. Genttly ressupend cells at 3-6-108 cells/mL in Transformation Buffer.
ReagentMAX Efficiency™ Transformation Reagent for AlgaeBio-rad LaboratoriesCatalog #A24229

Expected result
Culture at 3x106 cells/mL usually yield 12-13 transformations.






Transformation
Transformation
  1. Add cutted vector to the bottom of the electroporation cuvette. Typically from Amount250 ng cutted vector to Amount1000 ng cutted vector
Add Amount250 µL ressuspended cells (at approximatelly Concentration3 x 10^8 cells/mL ) to each cuvette. Pippet up and down on DNA sample. Flick cuvette to mix DNA and cells. Shake cells to the bottom of the cuvette. Also add no DNA control (Elution buffer or water).
Cell suspension ready for electroporation
  1. Incubate cells with DNA TemperatureOn ice for Duration00:10:00
  2. Wipe cuvette (to remove condensated water) and electroporate (Table Electroporation).
  3. Let it recover for Duration00:10:00 on the cuvette
  4. Add cells to Amount10 mL TAP/40mM sucrose, pH 7.0 inside sterile 50mL centrifuge tubes. Gently transfer cells from cuvette to TAP/40 mM sucrose. Rinse cubette with TAP/40 mM sucrose to transfer any remaining cells.
Incubate at TemperatureRoom temperature on rocker or shaker at 50 rpm DurationOvernight ambient light.
Recover step of cells in shaker, low mixing.
  1. Pellet cells by centrifuging for Centrifigation2000 x g, 25°C, 00:10:00
  2. Aseptically poor off supernatant. Add Amount300 µL TAP/40mM sucrose to pelet. Gently re-suspend cells and pipette onto 2 plates with appropriate antibiotics. ie. Amount200 µL cells ressuspended  per plate, and let it dry a,septically without plate cover.
  3. Spread cells evenly over the plate with a innoculation loop. Avoid spreading to the borders.
  4. Use parafilm to block evaporation and place plates under constant light (60 µmols de photons/m2s), Temperature25 °C . Colonies should be visible in 5-7 days. 
Table Electroporation - Settings
Voltage800 V
Time Constant20 ms
Cuvette gap4 mm

Equipment
Gene Pulser Xcell Electroporation Systems
NAME
Electroporator
TYPE
Biorad
BRAND
1652660
SKU
https://www.bio-rad.com/en-ca/sku/1652660-gene-pulser-xcell-total-system?ID=1652660
LINK


Typical output after electroporation
Time constant (ms)Voltage (V)Capacitance (uF)Resistance ( Ohms)
20.178850650
20.478950600
19.878950550


Expected result
Green colonies should appear in the plate as in the pictures below.

Positive result
Negative control