Dilution-to-Extinction Experiment Protocol_DNA Extraction Pipeline

Defined Medium

Can be prepared in advance according to SJB Artificial Media Protocol DOI: dx.doi.org/10.17504/protocols.io.rm7vzy615lx1/v1 or using medium of choice.

Culturing Hardware Preparation

All culturing hardware (carboys, flasks, glassware, PTFE plates, sealing mats, stir bars) are washed first with dishwasher or lab-grade detergent; rinsed with hot tap water, cold tap water, and then DI water; and let dry.

They are then soaked in a 10% HCl acid bath overnight, rinsed six times with DI water and MilliQ water, lids applied or wrapped in foil, and autoclaved prior to storage or use.

Collect samples at any source of interest with sterile, acid-washed carboys appropriate to the required volumes, taking as many precautions as practical to avoid contamination.

Collectors should wear gloves.

Samples should be transported back to the laboratory with the shortest delay possible to reduce potential bottle effects that can alter the community prior to inoculation.

Filter sample(s) prior to enumeration using a (2.7um) Whatman GF/D glass fiber filter to exclude large aggregates/particles and increase the proportion of planktonic cells in the seawater.

Sample Preparation

Enumerate DTE inoculum sample prior to dilution with artificial medium and PTFE plate inoculation.

Total cells μL^-1 needs to be between 1,000 and 5*10^6 for a given sample to stay within the accurate counting range of the BDAccuri. If samples are higher, make appropriate dilutions in sterile medium.

Add two artificial medium controls to count (one stained and one unstained).

Controls. Unstained controls (artificial medium) are used to set a green fluorescence threshold for exclusion of autofluorescence and system noise, respectively. Stained controls ( one stained and one unstained artificial medium sample) are used for gating results around standard noise of the artificial media and identify the potential area for positive cell growth. Control samples are counted along with the experimental samples.

Transfer 198 μL of each sample, in triplicate, into a round- bottom 96-well plate.

Transfer 400 μL of each of the desired controls into a 0.5 mL microcentrifuge tube and add 198uL of each to 96-well plate. Typically, controls consist of stained and unstained medium and stained and unstained E. coli.

Stain samples and one control with 1x final concentration SYBR Green (2 μL 100x SYBR Green added to 198 μL sample).

Incubate samples for 30 minutes in the dark.

Run Settings and Worklist Setup

The following is used with BD Accuri C6 Plus Software for the BD Accuri C6 Plus Flow Cytometer systems from BD Biosciences (San Jose, California, United States).

Adjust Thresholds. Run control samples in manual collect tab using no thresholds and the general run settings: "Run with Limits" , volume: 10uL , flow rate: medium. The green fluorescence (FL1 or FITC) and forward scatter (FSC)/ side scatter (SSC) thresholds are set to exclude noise based on the unstained controls (Fig. 2). These settings are used for counting the samples in the auto collect tab.

Worklist need to be tailored by application and stain used. General run settings (w/SYBR Green): "Run with Limits"; volume: 10uL; flow rate: medium; Thresholds - FSC or SSC: 10, FL1 or FITC: 100 (thresholds may vary depending on media type and culture signature).

Plots. Forward scatter (log scale) vs. green fluorescence (log scale), side scatter (log scale) vs. green fluorescence (log scale), and green fluorescence (log scale) vs. yellow fluorescence (log scale) are used to evaluate the counts.

SIP Settings

SIP Rinse: 1 cycle per sample

SIP Clean after completed run

Analysis and Gating

Cell concentration is calculated using a gated region, which is selected based on the side scatter vs. green fluorescence plot. Forward scatter vs. green fluorescence can also be used but may reduce resolution depending on the sample.

Initially, a global gate is applied to plots using the stained artificial medium such that the gate excludes the stained medium signal.

However, especially in the case of isolates, but sometimes with whole seawater, individual samples must have the gate customized to account for variations in signal. In practice, this will be empirically defined, as different media will give different amounts of background noise.

All steps except incubation are performed inside a biosafety cabinet or laminar flow hood.

Based on enumeration (Sect. above), cells are diluted in medium such that they can be inoculated into deep 96-well plates using minimal volume. The average number of estimated cells per well should be 1–3 after inoculation.

Example. With a medium plus inoculum final volume of 900mL, an inoculum cell density of 5*10³ cells/mL and final concentration of 1-3 cells/1.7 mL after distribution ; the inoculation volume should be approximately 0.1062 mL or 106 µL.

Distribute with a P1200 multichannel pipette or the Eppendorf epMotion 5075 automated liquid handling system.

Manual Distribution

Inoculated medium is distributed into sterile, acid-washed deep 96-well plates (2.1 mL PTFE) at 1.7 mL per well in a biosafety cabinet using a P100-1200 µL multichannel pipette.

Swirl inoculated medium thoroughly and aliquot into sterile liquid reservoir for ease of distribution.

Top full plates with sterile silicon lids. Incubate plates at room temperature (~25C°) for 2 - 4 weeks.

Automated Distribution

Eppendorf epMotion 5075 can be set up following the manufacturer’s instructions.

Distribution and incubation protocol is consistent between distribution types.

Carried out in the same manner as enumeration for medium inoculation with the following adaptations for high-throughput counting with 96-well plates.

Aliquoting samples for enumeration can be done with the Eppendorf epMotion 5075 automated liquid handling system or P200 multichannel pipette.

In a biosafety cabinet or laminar flow hood, using a multichannel pipette, transfer 198 μL from the deep 96-well incubation plates to 96-well round-bottom counting plates.

Wells with cell densities of 10^4 and higher are considered positive and should be transferred.

The four corners of each plate containing a negative control should have very little signature apart from the media signal and a cell density of 10^3 or less. If a biotic signature is seen in a negative control, consider the plate contaminated and should not be used.

Transfer 200uL of each positive well to a 250mL flask filled with 50mL of medium of choice.

• transferring inoculum and culture volume can vary with preferred starting culture cell density.

Prepare medium before day of transferring.

Following the "Inoculum Sample Enumeration" section, count all newly transferred cultures.

Take daily cell counts and plot growth curves to get a visual of when to transfer and filter cultures. The general time for transferring and filtering is when a culture is in late log phase of its growth cycle. (cell density will vary depending on strain)

Transfer cultures prior to filtering.

When cultures are at late log phase create a new transfer group using 200uL of culture into 50mL of fresh medium.

Once transferred, filter 40-45mL of late log phase culture using a 0.1-0.2 Supor PES filter. Filter either by hand or with vacuum filtration.

Store filters with cells in the -20°C until ready for DNA extraction.

Extract DNA from filtered cells using the GenElute™ Bacterial Genomic DNA Kit.

Quantify the gDNA concentration after extraction using the Qubit Fluorometer.

Follow kits protocol.

Once DNA is extracted, prepare the PCR master mix for the number of samples

Volume of gDNA will vary depending on concentration.

Can use taq Polymerase kit (Invitrogen, OneTaq, etc) and dNTP's or premade master mix (GoTaq G2, etc).

To each PCR tube add:

Master Mix: 15uL

gDNA: 3uL

H20: 32uL

Run PCR's using the following Thermocycler conditions:

Lid Temp: 105C

Sample Volume:50uL

1. 94C, 3:00 mins

2. 94C, 0:30 mins

3. 50.8C, 0:30 mins

4. 72C, 2:00

5. GoTo step 2, 34x

6. 72C, 10:00

7. 4C, infinite hold

Store PCR products in 4°C or continue to gel and purification.

Run a 1.5% agarose gel to verify success of PCR amplification at the correct band length (usually 1500 bp).

Gel reagents in Materials section of protocol.

Set up gel casting tray in stabilizer and use leveler.

Add a 50mL solution of 1.5% agarose and 1x TAE Buffer to the designated gel pyrex bottle.

Microwave in 30 second intervals until all agarose is dissolved (swirl intermittently).

Add 30ul of 100x SYBR Green to molten agarose solution, swirl and pore into gel mold.

Remove any bubbles with a pipette tip by either sucking up bubble or moving it to the side.

Place gel mold in a dark place(SYBR Green is light sensitive) for at least an hour for gel to harden.

Purify PCR product with either GeneJet PCR purification kit or Ampure XP beads.

Follow GeneJet PCR purification kit protocol.

OR

Follow Ampure XP bead protocol (lab protocol).

Quantify purified PCR product using Qubit assay (following kit instructions) and set up sequencing samples as follows:

For each PCR product there will be two sequencing samples made (one for 16S rRNA 27F Forward and one for 1492R Reverse sequences).

Sequencing Samples (Total volume 15uL):

10uM 27F OR 1492R Primer: 5uL

PCR Product: need ~40 ng of DNA in 10uL

Molecular H20: remaining volume in the 10uL after PCR Product addition

Once samples are set up, submit sequencing request through Azenta (Genewiz) or other sequencing facility.