TITLE: CRISPR-Cas9 episome conjugation into Phaeodactylum tricornutum
AUTHORS: Mark Moosburner, Andrew Allen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Bacterial-conjugation methodology used to introduce the CRISPR-Cas9 episome generated using the GG2 assembly protocol.</div><div class = "text-block">This method, though, can be adapted for any episomal transfromation from an E. coli culture that contains a cargo plasmid (episome) and conjugation plasmid (pta-MOB) to Phaeodactylum. </div></div>

[STEPS]
?. [Prepare conjugation transformation plate]
Prepare conjugation plate
?. [Prepare conjugation transformation plate]
Autoclave   of 1.6% agarose solution (in MQ-H2O)Incubate autoclaved bottle/flask in  waterbath
45 ml
50 °C
?. [Prepare conjugation transformation plate]
Pipette  mL of N-free ASW (Artificial Sea Water) into 250 mL flaskAdd  LB mediaAdd  NaNO3 (880uM)Ad  NH4Cl (880uM)Incubate ASW+LB in  waterbath(or until agarose has acclimated to 50oC)
50 ml
5 ml
25 µl
25 µl
50 °C
?. [Prepare conjugation transformation plate]
Once both solutions have reached , pour the agarose into the ASW flask in the PCR hood to maintain sterilityContinuually mix in flask until mixed thoroughly
50 °C
?. [Prepare conjugation transformation plate]
Pipette  of ASW (0.8% agarose, 5% LB) mix into each wellAvoid bubbles on plate by pipetting up 10 mL while only dispensing  8 mL
8 ml
?. [Prepare conjugation transformation plate]
During incubation, place 2 6-well culture plates (flat bottom) in a PCR hood
?. [Prepare conjugation transformation plate]
Solidify and dry plates in PCR hood During, prepare P. tricornutum cells
?. [Prepare Phaeodactylum tricornutum cells]
Obtain an axenic culture of Phaeodactylum tricornutum (CCAP1055/1) growing in exponential phase
?. [Prepare Phaeodactylum tricornutum cells]
Determine culture health by Fv/Fm measurement using Dual-PAM fluorometer    (Exponentially growing culture typically has a Fv/Fm value of 0.6 to 0.7)
?. [Prepare Phaeodactylum tricornutum cells]
Collect enough culture volume for  6e8 cells (total)    Calculation:  P. tricornutum at concentration of 5e8 cells/mL per well                            12 wells X  =  (5e8 cells/mL) --> 6e8 cells    Ex:                            P.t. concentration = 1.1e6 cells/mL                            6e8 (cells) / 1.1e6 (cells/mL) =                             *** I round up to  ***
100 µl
100 µl
1.2 ml
545.5 ml
600 ml
?. [Prepare Phaeodactylum tricornutum cells]
Measure cell concentration    (Ideally by flow cytometry side scatter (SSC) vs. FL3 (longpass filter, 570nm)
?. [Prepare Phaeodactylum tricornutum cells]
Pellet cells
3000
?. [Prepare Phaeodactylum tricornutum cells]
Remove supernatent with graduated pipette-man so to not disturb pelletAdd cells  to 2 2mL tubes
?. [Prepare Phaeodactylum tricornutum cells]
Pellet cells again
2000
?. [Prepare Phaeodactylum tricornutum cells]
Remove supernant with pipette
?. [Prepare Phaeodactylum tricornutum cells]
Add 100-200 uL NO3-ASW to one 2 mLtube and mix cells with pipetteMix all cells together in 1 tube
?. [Prepare Phaeodactylum tricornutum cells]
Add appropriate volume of NO3-ASW to cells to bring final volume to
1.2 ml
?. [Plate P. tricornutum cells onto conjugation plates]
?. [Plate P. tricornutum cells onto conjugation plates]
Pipette  P. tricornutum cells on each well, directly in the middle of the well
100 µl
?. [Plate P. tricornutum cells onto conjugation plates]
Spread cells by rotating plate in an orbital motion with hands
?. [Plate P. tricornutum cells onto conjugation plates]
Dry cells after spreading under PCR hood
?. [Plate P. tricornutum cells onto conjugation plates]
Cover 6-well plates and seal lid to plate with breath-easy culture tape
?. [Plate P. tricornutum cells onto conjugation plates]
Incubate cells at  and under medium-intensity light )
18 °C
?. [Prepare E. coli for conjugation transformation]
The  night before, grow E. coli cultures in preparation for conjugation
?. [Prepare E. coli for conjugation transformation]
Inocculate  LB media cultures with pipette stab of conjugation-ready E.coliAmpicillin Tetricyclin Gentimycin  shaking
2 ml
37 °C
?. [Prepare E. coli for conjugation transformation]
Ensure to include positive and negative controlsPositive (conjugation episome containing a RFP bacterial expression cassette)Negative (conjugation episome that confers resistance to nourseothricin antibiotic, in contrast to resistance to phleomycin)
?. [Prepare E. coli for conjugation transformation]
Next day, remove E. coli cultures from
37 °C
?. [Prepare E. coli for conjugation transformation]
Prepare out-growth E. coli culture mediaPrepare  LB media (Amp-50, Tet-5, Gent-20) for each conjugation and pipette into 50 mL conical tube (one per conjugation reaction)
12.5 ml
?. [Prepare E. coli for conjugation transformation]
Pipette  overnight E.coli culture to each 50 mL out-growth tubeDo not seal tube with cap. Rather, place cap on tube loosely and secure with tape
250 µl
?. [Prepare E. coli for conjugation transformation]
Shake out-growth cultures a
37 °C
?. [Prepare E. coli for conjugation transformation]
Measure optical density of out-growth culturesIdeal absorbance reading: OD600 = 0.9-1.0
?. [Prepare E. coli for conjugation transformation]
Pellet out-growth cultures
3000
?. [Prepare E. coli for conjugation transformation]
During pelleting, dry 6-well conjugatin plates under a PCR hood and label each well accordingly
?. [Prepare E. coli for conjugation transformation]
After pelleting, remove media using vacuum manifold
?. [Prepare E. coli for conjugation transformation]
Resuspend E.coli pellet
?. [Prepare E. coli for conjugation transformation]
Pipette   S.O.C. media into each tube
100 µl
?. [Prepare E. coli for conjugation transformation]
Mix well by vortexing
?. [Prepare E. coli for conjugation transformation]
Add E. coli to P. tricornutum cells on conjugation plates
?. [Prepare E. coli for conjugation transformation]
For each conjugation reaction, pipette  E. coli culture on top of P. tricornutum cells in the middle of the well. Mix the E. coli culture very well with a pipette prior to dispensing in well. Spread E. coli in an orbital motion (refer to video) for each well (do not wait until all 6 wells have E. coli to spread, spread after every dispensing)
100 µl
?. [Prepare E. coli for conjugation transformation]
Dry E. coli on top of P. tricornutum cells (10-20 minutes)
?. [Conjugation Transformation]
The conjugation mechanistically operates when the E. coli are exposed to an optimal growth temperature and for a certain duration.P. tricornutum is most competent  to conjugation at
30 °C
?. [Conjugation Transformation]
After all E. coli have dried, wrap each plate with breath-easy tape
?. [Conjugation Transformation]
Incubate 6-well plate at   and in the dark
30 °C
?. [Conjugation Transformation]
After incubation, place conjugation plates at   for
18 °C
?. [Scrape Phaeodactylum cells off conjugation plates]
After 48 hrs, collect P. tricorntum cells in 2 mL tubesRemove conjugation plate(s) from incubation and put them under a PCR hood, take off lid to start drying plate(s)
?. [Scrape Phaeodactylum cells off conjugation plates]
Obtain ASW media supplemented with both NO3 and NH4Prepare 2mL tubes to collect scraped cellsI recommend using a hockey stick styled cell scraper. It allows more control of scraping in a small well area
?. [Scrape Phaeodactylum cells off conjugation plates]
For each well...Pipette   media and gently scrape off cells with a hockey-stick shaped cell spreader *** DO NOT USE A LOOP ***Collect cells with a P1000 pipetteRepeat once
500 µl
?. [Scrape Phaeodactylum cells off conjugation plates]
Mix collected cells very well with pipettePlace tubes at   for
18 °C
?. [Select transformed Phaeodactylum tricornutum cells]
During 1 hr incubation, dry P. tricornutum selection plates
?. [Select transformed Phaeodactylum tricornutum cells]
Selection Plates:For each conjugation reaction, prepare 3 selection plates (100mm x 15mm culture plates) with 35 mL media eachEx: Prepare   of plate selection media (enough for 1 6-well conjugation plate, 18 plates)        1. Autoclave 325 mL 1.6% agarose        2. Incubate at  after sterilization        3. Incubate  ASW supplemented with  NaNO3 and  NH4Cl, also at         4. Thaw Phleomycin stock (100mg/mL) and chloramphenicol (20 mg/mL)        5. Pour agarose into ASW flask and mix        6. Add  Phleomycin (50 ug/mL final concentration) and  Chloramphenicol (10 ug/mL final concentration) when temperature of ASW/agarose falls below         7. Pipette  selection media into each culture plate        8. Dry under PCR hood for         9. Store at  until needed
650 ml
50 °C
325 ml
163 µl
163 µl
50 °C
325 µl
325 µl
50 °C
35 ml
4 °C
?. [Select transformed Phaeodactylum tricornutum cells]
Dry selection plates under a PCR hood for at least
?. [Select transformed Phaeodactylum tricornutum cells]
Pipette 300 uL of  collected P. tricornutum cells per plates
?. [Select transformed Phaeodactylum tricornutum cells]
Spread on plate with cell spreader (or loop is fine here)
?. [Select transformed Phaeodactylum tricornutum cells]
Dry cells on selection plates and seal plates with breath-easy tape
?. [Select transformed Phaeodactylum tricornutum cells]
Incubate selected P. tricornutum at   for at least 10 days or until dark circular colonies begin forming.
18 °C