TITLE: LDM protocol for estimating plasmid conjugation rates
AUTHORS: Olivia    Kosterlitz, Claire  Wate, Adamaris  Muñiz Tirado, Benjamin  Kerr

[DESCRIPTION]
This is a general protocol to implement the LDM approach for estimating plasmid conjugation rates. This version of the protocol was originally submitted with the accompanying manuscript. The recommended version (i.e., the most up-to-date) of the LDM protocol will be linked in the corresponding GitHub repository. To get a conceptual overview of the approach, please read the accompanying manuscript.

The protocol is intentionally designed to facilitate proper implementation by explicitly verifying that the modeling assumptions are met. We provide 5 streamlined phases, where the last phase involves executing the LDM conjugation assay. The initial 4 phases are to gather the necessary information for the proper execution of the LDM conjugation assay (i.e., phase 5). Here, we provide a brief description and goals for the initial 4 phases. 

Phase 1: Minimum inhibitory concentration assay with the transconjugant-selecting medium. The goal is to find a concentration of selective agents (e.g., antibiotics) in the transconjugant-selecting medium that inhibits donors and recipients while simultaneously permitting the growth of transconjugants. 

Phase 2: Extinction probability assays for selective liquid and agar media. The goal is to check the assumption that the focal cell type will successfully establish a lineage under the appropriate selective conditions. 

Phase 3: Growth rate assay. The goal is to determine the donor- and recipient-specific incubation times that will ensure the cultures enter exponential growth before initiating the conjugation assay. 

Phase 4: Finding an incubation time and initial densities for executing the LDM conjugation assay. The goal is to find a combination of incubation time and initial densities such that the probability of a conjugation event occurring is between 0 and 1 (i.e., where there will be a mixture of turbid and non-turbid co-cultures after incubation with transconjugant-selecting medium). 

Phase 5: LDM conjugation assay. The goal is to gather experimental estimates for the variables in the LDM equation   to calculate an estimate of the conjugation rate. 



[STEPS]
SECTION: Phase 1: Minimum inhibitory concentration assay with the transconjugant-selecting medium.
1. Prepare bacterial cultures.

 Start cultures from freezer stocks for the donor, recipient, and transconjugant. Supplement the donor and transconjugant growth culture medium with the appropriate selective agents to maintain the plasmid. If using a microtiter plate format, surround the culture wells with the sterile growth medium to serve as contamination controls. Incubate  .
Transfer each turbid overnight culture from the previous step by diluting it 100-fold into growth medium initiating a second overnight culture (in order to acclimate the previously frozen strain to laboratory conditions). Prepare the growth medium and contamination controls as described in the previous step. Incubate  . 
SECTION: Phase 1: Minimum inhibitory concentration assay with the transconjugant-selecting medium.
2. Create a 2-fold gradient of the transconjugant-selecting medium.

Add 500 µL of growth medium to rows 2 through 8 (skip the first row) of a deep 96-well microtiter plate. 
Add 1 mL of growth medium supplemented with transconjugant-selecting antibiotics to row 1 (aiming for concentrations high enough to inhibit non-transconjugant strains). 
Create the 2-fold gradient by transferring 500 µL serially down the rows of the deep-well plate mixing thoroughly by pipetting up and down.
 

SECTION: Phase 1: Minimum inhibitory concentration assay with the transconjugant-selecting medium.
3. Innoculate the transconjugant-selecting medium gradient.

Dilute the donor, recipient, and transconjugant cultures 100-fold into growth medium.
Add 500 µL of the diluted cultures to each well in the appropriate columns in the deep-well plate prepared in the last step.
Incubate  .
SECTION: Phase 1: Minimum inhibitory concentration assay with the transconjugant-selecting medium.
4. Identify a viable transconjugant-selecting medium concentration.

Record the turbidity from the MIC plate.
Analyze the turbidity data to determine the strain-specific MIC for the transconjugant-selecting medium. Identify a concentration (or range of concentrations) above the donor and recipient MIC, but below the transconjugant MIC. 

SECTION: Phase 2: Extinction probability assays for selective liquid and agar medium.
5. Prepare bacterial cultures.

 
SECTION: Phase 2: Extinction probability assays for selective liquid and agar medium.
6. Prepare the selective liquid and agar mediums.

Prepare two deep-well plates to be inoculated with transconjugants: one with an antibiotic-free medium and one with the transconjugant-selecting medium. Add 950 µL of growth medium to each well in the deep-well plate. Supplement the transconjugant-selecting medium with the relevant antibiotics at the concentration identified in Phase 1.
Prepare two agar plates for each cell type (donor, recipient, and transconjugant): one with an antibiotic-free medium and one with the strain-specific selecting medium (donor-, recipient-, and transconjugant-selecting agar, respectively). Supplement the agar plates with the strain-specific selective agents (e.g., relevant antibiotics at relevant concentrations) that will be used throughout the assay. 

SECTION: Phase 2: Extinction probability assays for selective liquid and agar medium.
7. Dilute and innoculate the bacterial cultures.

Dilute the donor, recipient, and transconjugant cultures from step 5. 
Innoculate both deep-well plates (antibiotic-free and antibiotic-containing) by adding 50 µL of diluted transconjugant culture. Note: the transconjugant cultures should be diluted to add approximately 1 transconjugant cell per well. 
Innoculate both agar plates (antibiotic-free and antibiotic-containing) for each cell type (donor, recipient, and transconjugant) with 100 µL of the appropriate diluted culture. Note: each culture should be diluted to add approximately 30 to 300 cells per plate. 
Incubate the deep-well plates and the agar plates  .  
SECTION: Phase 2: Extinction probability assays for selective liquid and agar medium.
8. Determine the extinction probabilities. 

Record the turbidity from both deep-well plates.
Calculate the extinction probability   of the transconjugants in the transconjugant-selecting medium: 
 

Record the colonies from both agar plates for each cell type. 
Calculate the strain-specific extinction probabilities   for the strain-specific agar plate.
 

 

SECTION: Phase 3: Growth rate assay.
9. Prepare bacterial cultures

  
SECTION: Phase 3: Growth rate assay.
10. Dilute and innoculate bacterial cultures.

Dilute the donor and recipient cultures 10,000-fold into growth medium. (Note: if the experimenter has a non-zero extinction probability for the transconjugants in the liquid transconjugant-selecting medium 
Add 250 µL of the diluted cultures to each well in the appropriate columns in a standard microtiter plate prepared in the last step. Innoculate one row per time point. 
 
 
SECTION: Phase 3: Growth rate assay.
11. Dilute and plate bacterial cultures at 1-hour intervals.

Dilute and plate the donor and recipient cultures from the wells in the corresponding row. Incubate the agar plates  . 
Incubate the microtiter plate for 60 min. 
Repeat this step for each time point. 
SECTION: Phase 3: Growth rate assay.
12. Determine an appropriate incubation time for the donors and recipients to isolate the cultures in exponential growth by calculating the growth rate over time.

Record the colonies from the agar plates.
Calculate the growth rates   using the density information from neighboring time points.
 

 

SECTION: Phase 4: Finding an incubation time and initial densities for executing the LDM conjugation assay.
13. Prepare bacterial cultures

  
SECTION: Phase 4: Finding an incubation time and initial densities for executing the LDM conjugation assay.
14. Isolate bacterial cultures in exponential growth

Dilute the donor, recipient, and transconjugant cultures 10,000-fold into growth medium.
Incubate for strain-specific times determined in Phase 3 to ensure the cultures enter exponential growth.
SECTION: Phase 4: Finding an incubation time and initial densities for executing the LDM conjugation assay.
15. Dilute bacterial cultures for a full factorial treatment of initial densities and incubation times.
 

Dilute the exponentially growing donor and recipient cultures into pre-warmed growth medium to the relevant initial densities (e.g., 104, 105,  and 106-fold dilution).
Mix the diluted donor and recipient cultures at equal volumes to create the co-cultures.
Dispense 100 µL of diluted co-culture into each appropriate well in the corresponding columns in the experimental plate and the control plate according to the plate maps.
Dispense 100 µL of diluted donor, recipient, and transconjugant monocultures into each appropriate well in the corresponding columns in the control plate according to the plate map. 
SECTION: Phase 4: Finding an incubation time and initial densities for executing the LDM conjugation assay.
16. Add transconjugant-selecting medium, plate from the mating co-cultures for estimating densities, and create the relevant controls at 1-hour intervals.

Dilute and plate from the appropriate co-culture wells in the control plate onto donor- and recipient-selecting agar plates to uncover densities and determine whether donors and recipients maintain constant growth. Incubate the agar plates  . 
Mix 50 µL from the appropriate donor and recipient monocultures into the corresponding empty well in the control plate to create a co-culture control and verify that new transconjugant cells do not form via conjugation after transconjugant-selecting medium is added.
Add 900 µL of transconjugant-selecting medium into each appropriate well in the corresponding two rows in the experimental plate and the control plate according to the plate maps. 
Incubate the deep-well plates for 60 min. 
Repeat this step for each time point. Upon completion of the last time point, incubate the deep-well plates until the turbidity is stable which will most likely occur between 2880 min to 5760 min (this depends on the experimental system). 
 

SECTION: Phase 4: Finding an incubation time and initial densities for executing the LDM conjugation assay.
17. Find an incubation time and initial density where there is a mixture of turbid and non-turbid cultures and the bacterial cultures have a constant growth rate. 

Record the colonies from the agar plates.
Calculate the growth rates   for each time point.  
Record the turbidity from both deep-well plates.
Analyze the turbidity data (in conjunction with the growth rates) to determine an incubation time and initial density that satisfies the assay requirements. 
 

SECTION: Phase 5: LDM conjugation assay.
18. Prepare bacterial cultures. 

  and   

SECTION: Phase 5: LDM conjugation assay.
19. Dilute and innoculate bacterial cultures and plate from the mating cultures for estimating initial densities.

Dilute the exponentially growing donor and recipient cultures into a pre-warmed growth medium to the relevant initial densities identified in Phase 4. 
Mix the diluted donor and recipient cultures at equal volumes to create the co-cultures.
Dispense 100 µL of diluted co-culture into each appropriate well in the corresponding columns according to the plate map. An example plate map is available Figure 5 in the accompanying manuscript. 
Dilute and plate the diluted co-culture used in the previous step onto donor- and recipient-selecting agar plates for estimating the initial densities ( ). Incubate the agar plates  . 
Dispense 100 µL of diluted donor, recipient, and transconjugant monocultures into each appropriate well in the corresponding columns according to the plate map. 
Incubate the deep-well plate for the incubation time   identified in Phase 4.
SECTION: Phase 5: LDM conjugation assay.
20. At the chosen incubation time   , plate from the mating cultures for estimating final densities and add transconjugant-selecting medium to all relevant wells. 

Dilute and plate from the appropriate co-culture wells onto donor- and recipient-selecting agar plates for estimating the final densities ( ). Incubate the agar plates  . 
Mix 50 µL from the appropriate donor and recipient monocultures into the corresponding empty well(s) to create a co-culture control and verify that new transconjugant cells do not form via conjugation after the transconjugant-selecting medium is added.
Add 900 µL of the transconjugant-selecting medium into each appropriate well according to the plate map. 
Incubate the deep-well plate until the turbidity is stable which will most likely occur between 2880 min to 5760 min (this depends on the experimental system). 
SECTION: Phase 5: LDM conjugation assay.
21. Calculate the LDM estimate.

Record the colonies from the agar plates.
Record the turbidity from both deep-well plates.
Calculate the LDM estimate for the conjugation rate.