TITLE: Modified Bacterial Conjugation Protocol For Pseudo-nitzschia multiseries
AUTHORS: G Jason Smith, April Woods

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a modified version of  'Conjugation of Thalassiosira pseudonana', publishedby J. Turnsek </span><a href="http://dx.doi.org/10.17504/protocols.io.f55bq86" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">dx.doi.org/10.17504/protocols.io.f55bq86</span></a><span> .</span></div><div class = "text-block"><span>The protocol was modified to enhance viability in the diatom </span><span style = "font-style:italic;">Pseudo-nitzschia multiseries. </span><span style = "font-style:italic;"></span></div><div class = "text-block"><span>This version was used to transform </span><span style = "font-style:italic;">Pseudo-nitzschia multiseries</span><span> isolate 15091C3 with an episomal plasmid derived from pPtPUC3 to express a egfp gene under control of the </span><span style = "font-style:italic;">P. multiseries</span><span> actin promoter and termination domains. </span></div><div class = "text-block"><span style = "font-style:italic;">PLASMID INFO</span></div><div class = "text-block"><span style = "font-weight:bold;">pPmAGFPC10 EPI</span><span> : A P. multiseries derived expression cassette under control of P. multiseries ca 1000 bp of actin promoter and termination domains.   </span><span style = "font-style:italic;">Pm actPs::eGFP::actTs</span></div><div class = "text-block"><h2></h2></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/3e260d53a20cb2b3651419051fbb25e6ee617805b883dbb4e4e005e3869209bb/brxndnqn.png" /></div></div>

[STEPS]
?. [Growth and preparation of E. coli pTA-MOB host and episomal plasmid donor]
Innoculate G418+Kan plates with the appropriate host strain and grow overnight (use within 7 days). Pick isolated colonies and and inoculate into 10 mL LB medium. Grow overnight at 37oC, 200 rpm.
?. [Preparation of E. coli donor (cont)]
Centrifuge at 4,000 rpm, 10oC, for 10 min.Decant supernatant and resuspend bacterial cell pellet in 100 μL SOC*
?. [Preparation of E. coli donor (con]
Measure OD600 and start a 150 mL LB subculture (recommended starting OD600 either 0.05 or 0.1).
?. [Preparation of E. coli donor (cont)]
Grow at 37oC, 200 rpm until OD600 reaches 0.3-0.4
?. [Growth and preparation of Pseudo-nitzschia cells]
P. multiseries stock cultures are maintained in filtered (0.2μ) autoclaved seawater (FASW) + Guillard's Marine Enrichment f/2 medium.Subcultures are maintained in active mid-log phase growth, by serial batch culture in FASW+f/2Typical cell numbers at log phase harvest are in the 10-30 *10^3 cells/mL range.  Centrifugal concentration of 100mL of culture yields around 2*10^6 cells for conjugation.
- I've never tested if cell density before spinning cells down matters with respect to final conjugation outcome same as the Alverson Lab did for P. tricornutum. They observe ~8 x 106 cells/mL to be a sweet spot. In my hands spinning cells down at ~4-8 x 106 cells/mL seemed to work fine. According to my information harvesting T. pseudonana at ~0.8 x 105 cell/mL works best for biolistic experiments. 
- I counted cells with a Sedgewich-Rafter cell.
?. [Harvest Pseudo-nitzschia cells]
Spin down cells at 4000 rpm, 10oC, for 20 min in 50mL falcon tubes. Remove supernatant with vacuum pipette.Add additional 50mL culture and repeat centrifugation.  Pellet will contain cells from a total of 100mL culture. Re-suspend cell pellet in 1 mL of seawater enrichement media. Allow suspension to come to room temperature.
?. [Conjugation]
Mix 450 µL Pseudo-nitzschia cells and 50 µL E. coli cells in a 1.5 mL tube.Mix fully by gentle pipeting.Incubate mixture at 30 oC for 5 min.
This is the major modification forPseudo-nitzschia conjugation. A final concentration below 10% SOC greatly enhances Pseudo-nitzschia viability.
?. [Conjugation]
Gently spread mixture on pre-warmed 1% agar plates containing 5% (v/v) LB and 50% (v/v) filter sterilized seawater - L1 media.
Plates were incubated at 37° for an hour before plating cells.
?. [Conjugation]
Incubate in dark at 30oC for 90 minutes.
?. [Conjugation]
Move plates to standard Pseudo-nitzschia growth conditions - in my case 15oC and constant light - for 4 hours.
?. [Conjugation]
Add 1 mL reduced Nitrogen L1 medium*. Gently scrape agar surface with a cell scraper or L spreader to resuspend bacterial and diatom cells. Expect to recover ~500 µL co-culture suspension after scraping.
At tis point, we use L1 media enrichment and adjust the amount of Nitrogen so that Silica is not a limiting nutrient. Nitrogen:Silica ration is 1:3.
?. [Prepare for Solid Phase Growth in 1% (w/v) LGTA]
LGTA has been prepared following https://www.protocols.io/view/low-gel-temperature-agarose-lgta-media-gsibwceLGTA has been maintained at 20 oC. LGTA can also be microwaved to reliquify from solid storage, be sure to cool to 20 oC.Aliquot 4 mL, molten LGTA (20 oC) into sterile culture tubes (e.g. Falcon 2059).Add as much cell suspension as you are able to scrape off the conjugation plate (ca. 500uL) into LGTA.Gently vortex to mix.
The LGTA contains the same adjusted 1:3 NO3:Si ratio.
?. [PLATING CULTURES:]
Transfer 1 mL to well of Costar 12-well culture plate avoiding introducing bubbles.Repeat for 4 wells.For selection experiments may reduce volume to 500uL per well and include selection-free matched control wells.(LGTA will solidify during subsequent incubation at 15 oC.)
?. [Screening]
Observe plates under fluorescence microscopy for viability and growth (chlorophyll fluorescence). Screen for expression of egfp under fluorescence microscopy.As cells grow, some may emerge from the agarose and into the liquid overlay. These are available for isolate pics.Gel plugs may be removed and resuspended into liquid growth. Isolate cultures may be screened for presence of plasmid by PCR.DNA extraction of isolate cultures may be used to transform E. coli and selected for Kanamycin resistance.