TITLE: Singleplex Assay for Fitness Measurements
AUTHORS: David Ross

[DESCRIPTION]
This protocol outlines an assay for the singleplex measurement the fitness of plasmid variants in E.coli. 

This protocol is used during onboarding of a new protein function for the Pooled, Growth-Based Assay. In this protocol, clonal bacterial stocks are measured instead of a pooled mixture, and the end-point OD measured after each growth cycle is used as a proxy for the DNA barcode count data that is produced by the Pooled, Growth-Based Assay. The fitness estimates from the end-point OD measurements are used to fine-tune the plasmid circuit and the assay conditions in advance of running the Pooled, Growth-Based Assay.

The inputs include separate E. coli glycerol stocks for each variant. The protocol begins with several growths which convert the separate glycerol stocks into cultures that have reach stationary phase in a 96-well plate. The glycerol stocks are first grown overnight in separate tubes. The next morning, the optical density (OD) of each culture is measured, and then each culture is distributed into a 96-well growth plate. This plate is placed in a plate reader/incubator to grow to stationary phase (~12 hours) without antibiotics or additives (except those required for plasmid maintenance), with optical density (OD) and fluorescent measurements being taken every 5 minutes and at the end of each growth plate's incubation. After this point, the cultures are ready to act as a inputs for subsequent growth cycles. 

The four subsequent growth cycles (i.e., timepoints 1-4) are all ~3 hours long, so that cells stay in mid-log phase. Small amount of the plated cultures at the end of each cycle acting as input for the subsequent growth cycle. The OD measured at the end of each growth cycle (i.e., the end-point OD) will be used in the downstream fitness calculation. 

Contents of each growth plate for timepoints 1-4 (growth plates 2-5):
Timepoint 1: This growth plate contains additives to initiate gene expression, but no selection antibiotic.
Timepoint 2: This growth plate contains additives to initiate gene expression and the selection antibiotic.
Timepoint 3: This growth plate contains additives to initiate gene expression and the selection antibiotic.
Timepoint 4: This growth plate contains additives to initiate gene expression and the selection antibiotic.


[STEPS]
SECTION: Culture Preparation
1. Use a scraping from the glycerol stock for each clonal variant to start 5 mL cultures in M9 media, each in a 15 mL snap-cap culture tube. 

SECTION: Prepare the automation system or liquid handler
4. Load required reagents (M9 media, additives, antibiotics, etc.) and growth-plates into automation system or liquid handler.
SECTION: First Growth Plate: cells reach stationary phase
5. Prepare growth plate by pipetting 450 μL M9 media and 50 μL cell culture into each well.
SECTION: First Growth Plate: cells reach stationary phase
6. Apply gas-permeable seal to the plate.
SECTION: First Growth Plate: cells reach stationary phase
7. Incubate for 12 hours at 37°C, with fastest shaking possible in the plate reader (e.g., in Biotek Neo2SM reader: double orbital shaking at 807 cpm and 1 mm shaking diameter).

SECTION: First Growth Plate: cells reach stationary phase
7.3. Approximately ten minutes before the end of the 12-hour incubation, pre-warm the second growth plate. Adjust pre-warming temperature and timing so that the media temperature in the plate is 37C at the end of the pre-warming, and so that the pre-warming step ends at the same time as the 12-hour incubation.
SECTION: Second Growth Plate (timepoint 1)
8. After 12-hour incubation, remove gas-permeable seal. 
SECTION: Second Growth Plate (timepoint 1)
9. Transfer 10 uL from each well in the first growth plate to the corresponding well in the second growth plate. 
There are some subtleties in the 96-channel pipetting required to get a reproducible transfer, details are described in the SI of this paper: https://academic.oup.com/synbio/article/7/1/ysac013/6659220 
SECTION: Second Growth Plate (timepoint 1)
10. Apply gas-permeable seal to second growth plate.
SECTION: Second Growth Plate (timepoint 1)
11. Incubate the second growth plate for approximately 3 hours,  with fastest shaking possible in the plate reader (e.g., in Biotek Neo2SM reader: double orbital shaking at 807 cpm and 1 mm shaking diameter).
The exact incubation time for this step needs to be worked out during the testing phase. The time needs to be adjusted so that in the wells with the fastest growing cultures (e.g., with zero antibiotic):
The cell density always remains in mid-log phase or lower.
The cell density at the end of each subsequent incubation step is constant or slightly decreasing. 
SECTION: Second Growth Plate (timepoint 1)
11.2. During incubation, prepare the third growth plate. 
Total volume should be 450 uL per well. Each well contains the same additives as in the second growth plate, plus the addition of the selection antibiotic in some of the wells.
SECTION: Second Growth Plate (timepoint 1)
12. After ~3-hour incubation, remove the gas-permeable seal and take an end-point OD600 measurement of the second growth plate.
SECTION: Third Growth Plate (timepoint 2)
13. Transfer 50 uL from each well in the second growth plate to the corresponding well in the third growth plate.
SECTION: Third Growth Plate (timepoint 2)
14. Apply gas-permeable seal to third growth plate.
SECTION: Third Growth Plate (timepoint 2)
15. Incubate the third growth plate for approximately 3 hours (same incubation time as second growth plate).
with fastest shaking possible in the plate reader (e.g., in Biotek Neo2SM reader: double orbital shaking at 807 cpm and 1 mm shaking diameter).
SECTION: Fifth Growth Plate (timepoint 4)
24. After the 3-hour incubation of the 5th growth plate, remove the gas-permeable seal and take an end-point OD600 measurements. 
SECTION: Fifth Growth Plate (timepoint 4)
22. Apply gas-permeable seal to fifth growth plate.
SECTION: Fifth Growth Plate (timepoint 4)
21. Transfer 50 uL from each well in the fourth growth plate to the corresponding well in the fifth growth plate (automated liquid handler).
SECTION: Fourth Growth Plate (timepoint 3)
20. After ~3-hour incubation, remove the gas-permeable seal and take an end-point OD600 measurement of the fourth growth plate.
SECTION: Fourth Growth Plate (timepoint 3)
19.2. During incubation, prepare the fifth growth plate. 
Total volume should be 450 uL per well. Each well contains the same additives and selection antibiotic as in the third and forth growth plates.
SECTION: Fourth Growth Plate (timepoint 3)
19. Incubate the fourth growth plate for approximately 3 hours (same incubation time as third growth plate).
with fastest shaking possible in the plate reader (e.g., in Biotek Neo2SM reader: double orbital shaking at 807 cpm and 1 mm shaking diameter).
SECTION: Fourth Growth Plate (timepoint 3)
18. Apply gas-permeable seal to fourth growth plate.
SECTION: Fourth Growth Plate (timepoint 3)
17. Transfer 50 uL from each well in the third growth plate to the corresponding well in the fourth growth plate.
SECTION: Third Growth Plate (timepoint 2)
16. After ~3-hour incubation, remove the gas-permeable seal and take an end-point OD600 measurement of the third growth plate.
SECTION: Third Growth Plate (timepoint 2)
15.2. During incubation, prepare the fourth growth plate. 
Total volume should be 450 uL per well. Each well contains the same additives and selection antibiotic as in the third growth plate.
SECTION: Fifth Growth Plate (timepoint 4)
23. Incubate the fifth growth plate for approximately 3 hours (same incubation time as third and fourth growth plate).
with fastest shaking possible in the plate reader (e.g., in Biotek Neo2SM reader: double orbital shaking at 807 cpm and 1 mm shaking diameter).
SECTION: Quality control check the overnight culture
3. After incubation, measure the OD600 of each overnight culture as a growth check.
SECTION: Culture Preparation
2. Incubate cultures overnight (18-24 hours) at 37°C with shaking at 300 rpm.
SECTION: First Growth Plate: cells reach stationary phase
7.2. During incubation, prepare the second growth plate by adding 490 µL mixed media (media, additives  like inducers, maintenance antibiotics, etc but no selection antibiotic) per well to the second growth plate. 
SECTION: Second Growth Plate (timepoint 1)
11.3. Approximately ten minutes before the end of the 3-hour incubation, pre-warm the third growth plate. Adjust the timing here so that the media in the plate is pre-warmed to 37C at the same time as the end of the end-point OD600 measurement (next step).
SECTION: Third Growth Plate (timepoint 2)
15.3. Approximately ten minutes before the end of the 3-hour incubation, pre-warm the fourth growth plate. Adjust the timing here so that the media in the plate is pre-warmed to 37C at the same time as the end of the end-point OD600 measurement (next step).
SECTION: Fourth Growth Plate (timepoint 3)
19.3. Approximately ten minutes before the end of the 3-hour incubation, pre-warm the fifth growth plate. Adjust the timing here so that the media in the plate is pre-warmed to 37C at the same time as the end of the end-point OD600 measurement (next step).
SECTION: Data Analysis- Calculating end-point density Fitness
25. At the end of the protocol, you will have end-point OD measurements, corresponding to the density of cells, for plates 2-5 (i.e., timepoints 1-4).
SECTION: Data Analysis- Calculating end-point density Fitness
26. Plot log(end-point density) vs. timepoint
SECTION: Data Analysis- Calculating end-point density Fitness
27. Use linear fits from growth plates 3-5 to get Fitness. 
Fitness = slope of fit line + log(10), where 10 is the dilution factor used to passage cells from one timepoint to the next. 
SECTION: Data Analysis- Plotting Fitness vs Function (along with Function measurements)
28. Plot a separate curve for each antibiotic concentration (and zero antibiotic).
SECTION: Data Analysis- Plotting Fitness vs Function (along with Function measurements)
29. The measurable range of function typically needs to span multiple orders of magnitude, so make the x-axis logarithmic (i.e., log(function))
SECTION: Data Analysis- Plotting Fitness vs Function (along with Function measurements)
30. Look at the difference between Fitness with and without antibiotic to choose the best selection plasmid for the assay (which combination of resistance gene and RBS strength) and the best concentration(s) to use for the selection antibiotic.
If this Fitness difference has non-zero slope over the relevant range of function for one or more antibiotic concentrations and the zero antibiotic concentration is approximately constant across the functional range of the protein, then the selection plasmid is good. 
SECTION: Data Analysis- Calculating end-point density Fitness
25.1. The data for log(end-point density) vs. growth plate number is also used to adjust the length of incubation periods for growth plates 2-5 (i.e., timepoints 1-4). Ideally, the end-point density of the fastest growing cultures should remain constant or decrease slightly for each subsequent growth plate. 
If there is a slight increase in end-point density from timepoint 1 to 4, the incubation period should be reduced slightly. 
If the end-point density for the fastest growing culture decreases more than 2-fold from timepoint 1 to timepoint 4, the incubation period should be increased. 
Once the incubation period is adjusted to give constant or slightly decreasing end-point density for the fastest growing cultures, that incubation period should be used for all subsequent growth protocols (for the singleplex fitness, singleplex function, and pooled assays).
SECTION: Second Growth Plate (timepoint 1)
11.1. Measure OD600 every 5 minutes throughout the incubation. 
Optionally, measure fluorescence if appropriate for the plasmid circuit.
SECTION: Third Growth Plate (timepoint 2)
15.1. Measure OD600 every 5 minutes throughout the incubation. 
Optionally, measure fluorescence if appropriate for the plasmid circuit.
SECTION: Fourth Growth Plate (timepoint 3)
19.1. Measure OD600 every 5 minutes throughout the incubation. 
Optionally, measure fluorescence if appropriate for the plasmid circuit.
SECTION: Fifth Growth Plate (timepoint 4)
23.1. Measure OD600 every 5 minutes throughout the incubation. 
Optionally, measure fluorescence if appropriate for the plasmid circuit.
SECTION: First Growth Plate: cells reach stationary phase
7.1. Measure OD600 every 5 minutes throughout the incubation. 
Optionally, measure fluorescence if appropriate for the plasmid circuit.