VECTORs

Riley Williamson; Nicholas Dusek; Eglantina Lopez Echartea; Barney Geddes

Mar 14, 2025

VECTOR^s

DOI

dx.doi.org/10.17504/protocols.io.rm7vzkwr8vx1/v1

¹North Dakota State University

Riley Williamson

North Dakota State University

DOI: dx.doi.org/10.17504/protocols.io.rm7vzkwr8vx1/v1

Protocol Citation: Riley Williamson, Nicholas Dusek, Eglantina Lopez Echartea, Barney Geddes 2025. VECTORs. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzkwr8vx1/v1

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: March 12, 2025

Last Modified: March 14, 2025

Protocol Integer ID: 124274

Funders Acknowledgements:

FFAR

Grant ID: FF-NIA21-0000000061m

Abstract

Our protocol lays out a high-throughput microbial engineering platform for the isolation of desired engineering events. Using fluorescence monitoring, one would be able to identify single colonies of their desired strain containing their desired plasmid construct. This protocol may be used to explore two different engineering approaches. This first approach involves the delivery of a single plasmid variant to multiple bacterial
strains. This may be used to introduce specific cargo to multiple community members, offering a method for tracking mutations or genome integration. The second approach involves the delivery of multiple plasmids to a single strain. This process is ideal for providing a comprehensive view of a strain's adaptability to genetic modification and the vector components needed to do so. 
The expected result of this protocol is to end up with isolated colonies containing the user's ideal engineered microorganism.

Attachments

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Guidelines

Troubleshooting Guide

Cultures Splashing on Cover of Plate (Days 3-8)
To avoid splashing during the agitation of culture plates, gently shake the plates back and forth on a at surface in a fluid motion. Do not stop and start abruptly. If the liquid cultures are too disturbed and are found on the lid of the plate, carefully remove the cover and replace it. Failure to replace the cover may result in contamination between wells.


No Bacterial Growth in Wells After Incubation of Recipient Cells (Prep, Day 1)
If no bacterial growth is observed after incubation, this may be due to improper transfer during inoculation. Be sure that enough freezer stock is on loop/needle or liquid culture is being extracted in each pipette tip when inoculating.


No Bacterial Growth in Wells After Incubation of Donor Cells (Day 1)
If no bacterial growth is observed after incubation, this is most likely due to the concentration of antibiotic being too high, the wrong antibiotic being added to the media, or an absence of the necessary selection supplement. Fix by restarting the process, making sure all additives are present and proper antibiotic is added at the correct concentration. Be sure that enough freezer stock is on loop/needle or liquid culture is being extracted in each pipette tip when inoculating.


Para film Degradation (Prep-Day 6)
Para film on the edges of plates are likely to
tear and fall apart as they incubate. It is important to replace these wraps as
exposure to air can lead to evaporation of bacterial cultures.


Echo 525 Exceptions Report Showing Errors (Prep, Day 2)
Should there be exceptions during the acoustic handling process, indicating that the proper amount of liquid was transferred to the destination plate, the cause is often that the volume in the source plate well was too high or that the well ran out of sufficient stock. To remedy this, add or remove the desired liquid to the source plate well. The Echo source plates may have an issue transferring any liquid should the volume in the well fall below 12 µL or above 48 µL. Another issue may be the presence of bubbles inside the source plate wells. Use sterile 20µL pipette tips to manually move or "pop" bubbles.

Materials

Biologic materials
Donor E.coli (Frozen stock)
Recipient cells (Frozen stock)

Reagents
Select yeast extract (Sigma-Aldrich, product no. Y0875)
Tryptone (Sigma-Aldrich, product no. T2559)
Luria Bertani broth (LB) (Sigma-Aldrich, product no. L3022)
Gentamicin sulfate salt (Sigma-Aldrich, product no. G1264)
Neomycin trisulfate salt hydrate (Sigma-Aldrich, product no. N6386)
Tetracycline hydrochloride (Sigma-Aldrich, product no. T7660)
Magnesium chloride hexahydrate (MgCl2·6H2O, Sigma-Aldrich, cat. no. M2670-100G)
Sodium chloride (NaCl, VWR cat. no. X190-1kg)
Sodium phosphate dibasic dihydrate (Na2HPO4·2H2O, Sigma-Aldrich, cat. no. 10028-24-7)
Sodium phosphate monobasic monohydrate (NaH2PO4·H2O, Sigma-Aldrich, cat. no. 71504-250G)
Sodium hydroxide (NaOH, Sigma-Aldrich, cat. no. S8045-500G) 
Na2-EDTA·2H2O (JT Baker cat. no.4040-01)
Tris-HCl (IBI, cat. no. IB70162)
Glycerol (Thermo Scientific, cat. no. A16205.0F)
Agar (Thermo Scientific Agar powder cat. no. AAA107520E)
Ethanol (Fisher cat. no. BP2818-500)
Nuclease-free water (Qiagen, cat. no. 129114)
Sodium Chloride (Sigma-Aldrich, product no. S9625)

Equipment
Single well, rectangular plate (Thermo Fisher, cat. no. 165218)
White reservoirs (Southern Labware cat. no. P8050-5)
Reagent reservoir PP (Sigma-Aldrich, product no. BR703409)
Standard microcentrifuge tubes (1.5 and 5 ml; Eppendorf, cat. no. 0030125.150, 30119401)
96 U-bottom deep well plate (VWR, cat. no. 7323803)
96-well cell culture plate (Amazon, NEST, cat. no. 701001)
96-well PCR plate (Hard-Shell, BIORAD, cat. no. HSS9601)
96-well EIA/PIA plate (Costar, cat. no. 3590)
Echo Mass Spectrometry (MS) Qualified 384-well Polypropylene (PP) Microplate (Beckman Coulter, product no. C74290)
2.0-ml cryogenic vial (NEST, cat. no. 607001)
Multi-channel pipettes (Rainin cat. no. 17013803, 17013805, 17014496
Single channel pipettes (Rainin cat. no. 17014393, 17014388, 17014384 and 17014382)
Pipette tips (nuclease-free, sterile filter tips; 20, 200 and 1,000 µl; Rainin cat. no.  17014961, 30389239, and 30389213)
Dry bath metal beads (Thermo Fisher, cat. no. A1254301)
Dry bath (Sigma-Aldrich, product no. Z742509)
Inoculating loops and needles (10µl capacity, VWR cat. no. 612-9358)
Breathe-Easy sealing membrane (Sigma-Aldrich, product no. Z380059)
Parafilm (VWR, cat. no. 291-1213))
Plate seals (VWR cat. no. 732-3212)
Adhesive foil seals (VWR, cat. no. 60941-074)
Class II biological safety cabinet (Nu-Aire model NU425-400 series 30)
Vortex mixer (VWR cat. no. 10027-194)
Electronic balance (Mettler Toledo, cat. no. AL104)
Incubator-shaker (Eppendorf, cat. no. M1299-0090)
-80 °C Upright Ultra-Low Temperature Freezers (Thermo Fisher Scientific, model no. 907)
-20 °C freezer (VWR Lab HC Manual Def -20 CF, cat. no. 75836-238) 
Echo 525 Acoustic Handler (Beckman Coulter, product no. 001-10080)
BioTek Cytation 5 Cell Imaging Multimode Reader (Agilent)

Computer requirements
Operating system requirements: The bioinformatic pipeline was developed and tested on a 64-bit RedHat Enterprise Linux version 8+ system, but any operating system capable of running a modern version of the R language – Linux, MacOS, or Windows – should work.
Hardware requirements: 16+ GB of RAM is recommended for most datasets. RAM requirements increase with number of plates per analysis. If analyzing only a few plates, 4-8 GB of RAM may be sufficient.

Software Requirements:
Echo Plate Reformat Software (Beckman Coulter)
Echo Cherry Pick Software (Beckman Coulter)

Safety warnings

1.  Risk of Contamination
Cross-contamination between wells and plates can affect the accuracy of microbial isolation and
plate readings. Work in a laminar ow hood when handling cultures. Always use sterile pipette tips, nuclease-free water, and reagents to minimize contamination risks.
2.  Handling of Microbial Cultures
Some environmental bacteria may pose biosafety risks, especially opportunistic pathogens. Always wear appropriate personal protective equipment (PPE) (lab coat, gloves, eye protection) when handling cultures. Dispose of biohazardous waste properly according to institutional guidelines.

3.  Storage and Freezing Precautions
Bacterial glycerol stocks at -80°C must be handled carefully to prevent repeated freeze-thaw cycles, which can reduce viability. Ensure proper mixing of glycerol and bacterial culture before freezing to maintain even distribution.

Start Recipient Cultures (Prep)

Inoculate TY Broth:
Add 150 µL of TY broth to each well of a 96-well at-bottom plate.
Using a loop, inoculate one of the wells directly from recipient freezer stock. Repeat for each recipient strain.
Include replicates of inoculations to ensure successful growth.    

Plate Layout:
Use a pattern that facilitates easy transfer to a 384-wellsource plate.
Include replicates to ensure successful growth.

Control Wells:
Add at least one blank well containing uninoculated TY broth to act as a control.

Storage:
Place the lid on the plate and seal the edges with parafilm.
Store the plate in a drawer at room temperature.

Check Growth:
Monitor the plate daily for successful growth (3-7 days).

Prepare Conjugation Mating Spot Plates and Selection Plates (Prep)

Prepare TY Agar:
Make TY agar and autoclave as required.
Allow agar to cool slightly before proceeding.
Add the appropriate volumes of 0.5M CaCl2  and other nutrients to the TY agar.

Heat Bead Bath:
Set the bead bath to "high".
Allow time for the bead bath to heat up, adjusting as needed.

Dispense Agar into Wells:
Place the reservoir containing the media into the beads to keep the agar warm.
Place the 96-wellat-bottom plate on the heating block, moving it occasionally to prevent plastic melting.
Add 100 µL of TY + ALA agar to each desired well in the plate.
While still on the heating block, use pipette tips to carefully remove any bubbles from the agar.
Remove the plate from the heating block to allow agar to cool and solidify inside the hood.

Prepare Selection Plates:
Use rectangular single-well plates.
Add approximately 35 mL of TY agar per plate.

Develop Echo Protocol (Prep)

Create Mating Spots Protocol:
Use Echo plate reformat software to develop a protocol for creating mating spots with donor and recipient cell cultures.

Test Protocol:
Run a test using water to verify the protocol is successful.

Save and Export:
Save the transfer report and convert it to a .xlsx file.
In the .xlsx file, add commands to calculate the required transfer volume from each source well based on cell counts (to be measured later).
Include calculations to ensure the final volume in each mating spot is 5 µL.

Start Overnight Cultures (Day 1)

Recipient Cultures:
Add 15 µL of each recipient culture to 135 µL of TY broth in a new 96-well flat-bottom plate. Follow the same inoculation pattern as before.
Cover the plate with Breath-Easy film.
Incubate overnight at 28°C in a shaking incubator at 225 rpm.

Donor Cultures:
Inoculate a 96-well at-bottom plate with E. coli donor cells, directly from freezer stock, in a similar pattern to the recipient cells.
Each well should contain: LB broth + required nutrients and antibiotic corresponding to the resistance module in the donor cells. Cover the plate with Breath-Easy film.

Echo Setup and Conjugation (Day 2)

Prepare for Measurement:
Replace the Breath-Easy film with clear plastic film.
Measure OD600 using a Cytation5 instrument.

Verify Cell Growth:
Remove the plates from the incubators and confirm the cultures are normalized in density. 
If needed, return the plates to the incubators and recheck later that day.

Input Cell Counts:
Use the previously saved Excel protocol le to input OD600-derived cell counts for donor and recipient cultures.
Assume OD600 = 1.0 ≈ 1 million cells/mL.

Adjust Volume:
Calculate the required culture dilutions to ensure that total volume in each mating spot is ≤ 5 µL.
Prepare donor and recipient cultures accordingly

Prepare Source Plate:
Ensure volumes in each source well are between 12 µL and 48 µL.

Convert File:
Convert the updated .xlsx file to .csv.

Run Cherry-Pick Protocol:
Add the required culture volumes and media to the source plate.
Load the cherry-pick program on the Echo-525 instrument.
Select the appropriate source and destination plates and input the .csv file.

Handle Exceptions:
Review the exceptions report.
Rerun the protocol with the exceptions report or adjust volumes manually as needed.

Conjugation Incubation:
Allow the conjugation plate to dry inside sterile hood.
Seal the edges with para film.
Place the plate upside down and incubate overnight at 28°C.

Resuspension and Plating (Day 3)

Resuspend mating spots in 150 µL of TY broth:
Use a micropipette to wash the bottom of each well carefully.

Transfer resuspended cells to a new 96-well at-bottom plate.

Prepare serial dilution plates:
Fill each well with 90 µL of saline.
Leave unnecessary wells empty.

Spot plate dilutions:
Transfer 10 µL from the first row of the conjugation plate to the first row of a dilution plate (10⁻¹ dilution).
Transfer 10 µL from the conjugation plate to solid media selection plates.
Repeat the process for all antibiotic selection conditions and control plates.
Mix the first row of the dilution plate and transfer 10 µL to the row below (10⁻² dilution).
Repeat serial dilutions to 10⁻⁷.

Leave spot plates uncovered in the hood to dry (~10 minutes).

Cover each selection plate with caps, seal edges with para film, and store plates upside down in a dark drawer.

Monitor and Image (Day 4-9)

Monitor plates daily for contamination and growth:
Replace parafilm as needed.
If overgrowth occurs, store plates in a 4°C refrigerator.

On the final day, image plates to assess GFP expression using Cytation 5 with the following settings:
Magnification: 1.25X PL APO
Wide FOV
Color: GFP (469/525)
LED: 10
Integration Time: 5 ms
Gain: 17.6
Offsets: Horizontal (-3611 µm), Vertical (32684 µm) Montage: Yes (9x13 rows x columns) 
Tile Overlap: Auto for stitching.

Prepare PCR Samples:
Fill 96-well PCR plate with 25 µL of saline.
Inoculate each well with a single colony from one of the transconjugant samples.

Para film edges of all plates and store in a 4°C refrigerator.

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