High-throughput workflow for the genotypic characterization of transposon library variants

Dispense 100µL of selective media (M9-citrate for P. putida or Luria-Bertani plus 20 ng/µL nalidixic acid for DH5 α E. coli ) plus transposon cassette antiobiotic in a 96-well plate

Pick individual colonies into a 96-well plate with selective media

Tip: Keep tips inside of wells to keep track

Cover with a sterile breathable membrane

Grow at 30°C ( P. putida ) or 37°C ( E. coli ) / 500rpm

Measure OD_600nm of overnight culture grown in selective media from plus transposon cassette antibiotic in a plate reader

Inoculation of counter-selection plate in selective media:

• Dispense 100µL of selective media (M9-citrate for P. putida or Luria-Bertani plus 20 ng/µL nalidixic acid for DH5α E. coli ) plus ampicillin (backbone antibiotic) to select against spurious integration events.
• Transfer 5µL of overnight culture from to counter-selection (ampicillin) plate
• Cover with a sterile breathable membrane
  Note

  For steps 6 and 7, if two or more 96-well plates are used as input it is advised to use the OT-2 protocol below to minimize human error. Dispensed volume and culture volume inoculated should be that described in these steps. Note that steps 6 and 7 could be completed together in a single run depending on the number of initial plates.

OT-2 Media dispensing and culture inoculation protocol

Inoculation of precultures for glycerol stock in rich media:

• Dispense 100µL of Luria-Bertani media plus transposon cassette antiobiotic in a 96-well plate
• Transfer 5µL of overnight culture from to counter-selection (ampicillin) plate
• Cover with a sterile breathable membrane

Grow counter-selection and glycerol stock pre-culture plates at 30°C ( P. putida ) or 37°C ( E. coli ) / 500rpm

Measure OD_600nm of overnight culture grown in selective media plus ampicillin in a plate reader

Selection of colonies to store as glycerol stocks and do further PCR reactions by running the following OT-2 protocol with its corresponding template.csv

OT-2 Counter-Selection

Cover glycerol stock plates with a storage membrane and store at -80ºC

If not proceeding to the next step right away: Store "PCR plate" at 4ºC for a few days or cover with an storage membrane and store at -20ºC for longer term

Transfer 50µL of selected colonies from one or more libraries to a 96-well plate with the following OT-2 protocol:

OT-2 Protocol to transfer volume from several plates to a single plate 

Spurious integration control with SEVA primers pairs PS3/PS4 and PS5/PS6

OT-2 PCR sample preparation protocol  

Optional: Cargo integration control with primers PSMCS and either ME-O-Km-R/ME-O-Sm-R or ME-O-Gm-R (depending on transposon cassette antibiotic )

Arbitrary PCR#1 using primer pairs ARB6 and ME-O-Km-Ext-F/ME-O-Sm-Ext-F or ME-O-Gm-Ext-F depending on transposon antibiotic cassette

OT-2 PCR sample preparation protocol  

Seal 96-well plate, place it in thermocycler and run the following PCR program:

A	B	C
98ºC	5 min
98ºC	10 s	x6 cycles
30ºC	30 s
72ºC	1 min 30 s
98ºC	10 s	x30 cycles
45ºC	30 s
72ºC	1 min 30 s
72 ºC	5 min
4ºC	hold

Select 8-12 Arbitrary PCR#1 reactions from the 96-well plate and run them on a 1% agarose gel to verify amplification.

Arbitrary PCR#2 using primers pairs ARB2 and ME-O-Km-Int-F/ME-O-Sm-Int-F or ME-O-Gm-Int-F depending on transposon antibiotic cassette

OT-2 PCR sample preparation protocol  

Seal 96-well plate, place it in thermocycler and run the following PCR program:

A	B	C
98ºC	30 s
98ªC	10 s	x30 cycles
52ºC	30 s
72ºC	1 min 30 s
72ºC	5 min
4ºC	hold

Select 8-12 Arbitrary PCR#2 reactions from the 96-well plate and run them on a 1% agarose gel to verify amplification

Prepare a PCR plate to send to sequencing by mixing 10µL of unpurified Arbitrary PCR#2 reaction and 10µLof 10 µM sequencing primer (ME-O-Km-Ext-F/ME-O-Sm-Ext-F or ME-O-Gm-Ext-F depending on the transposon antibiotic cassette)

Annotate sequencing results by running the following protocol:

Bacterial genome annotation script using BLASTN

In this section we show a specific example on how to run the workflow with the OT-2 liquid handler using a starting point three 96-well plates with P. putida KT2440 colonies picked from matings using cargos with either a kanamycin (pBLAM1-2), streptomycin (pBLAM1-4) or gentamicin (pBLAM1-6) resistance gene.

Counter-selection and glycerol stocks pre-cultures s using the following OT-2 protocol in in two runs

The following template. csv will dispense M9-citrate media containing ampicillin to three plates. Subsequently, each plate will be inoculated from each of the three starting plates.

Variables-AntibioticPlatesCreation-OT.csv

The following template. csv will dispense LB media with either gentamicin, kanamycin or streptomycin to each plates. Subsequently, each plate will be inoculated from each of the three starting plates.

Variables-AntibioticPlatesCreation-OT.csv

Colony selection in OT-2 liquid handler robot.

Each LB plus cargo antibiotic plate will be run separately with the script in . For each library there is an input .csv file for the absorbance at 600nm in M9-citrate with either kanamycin, gentamicin or streptomycin and another .csv file for the absorbance at 600nm in M9-citrate ampicillin. The LB plate from step 25.2 will be used to inoculate the two glycerol stock plates and PCR plate.

pBLAM1-2: .csv files and template .csv to run script

Variables-ColonieScreening-OT.csv

220920_M9Cit_ant_LibpBLAM12.csv

220921_M9Cit_amp_LibpBLAM12.csv

Output from the run (map with identifiers)

map_220921_LibpBLAM12.csv

pBLAM1-4: .csv files and template .csv to run script

Variables-ColonieScreening-OT.csv

220920_M9Cit_ant_LibpBLAM14.csv

220921_M9Cit_amp_LibpBLAM14.csv

Output from the run (map with identifiers)

map_220921_LibpBLAM14.csv

pBLAM1-6: .csv files and template .csv to run script

Variables-ColonieScreening-OT.csv

220920_M9Cit_ant_LibpBLAM16.csv

220921_M9Cit_amp_LibpBLAM16.csv

Output from the run (map with identifiers)

map_220921_LibpBLAM16.csv

Master 96-well plate for PCR steps to combine cultures from different transposon libraries in a single 96-well plate

The following template.csv is to be run with the OT-2 script in

Variables-SamplesMerging-OT.csv

Output from the run (map with identifiers)

maps_master_pcr-220923_5.csv

Control PCRs to account for spurious integrations and the correct integration of the cargo

Spurious integration control with PS3/PS4 and PS5/PS6 SEVA oligonucleotides

The following template.csv was used for the OT-2 script in to prepare each mastermix with either PS3/PS4 or PS5/PS6 primer pairs and transfer cultures from Master 96-well plate

Variables-PCRs-OT.csv

Gels after PCR protocol in thermocycler

Cargo integration control with PSMCS and either ME-O-Km-R/ME-O-Sm-R or ME-O-Gm-R

The following template.csv was used for the OT-2 script in to prepare the mastermix and transfer cultures from Master 96-well plate

We have discarted the first 3 columns because they came as negative (without integration) in the spurious PCRs.

Gels after PCR protocol in thermocycler

Arbitrary PCRs

Arbitrary PCR#1

The following three different template.csv were used (one for each primer pair) in three independent runs with the OT-2 script in using the same input and output plate to amplify the genomic context around each transposon cassette

Variables for source plate 1 arbitrary PCR 1: Variables-PCRs-OT.csv

Variables for source plate 2 arbitrary PCR 1: Variables-PCRs-OT.csv

Variables for source plate 3 arbitrary PCR 1: Variables-PCRs-OT.csv

Arbitrary PCR#2

The following three different template.csv were used (one for each primer pair) in three independent runs with the OT-2 script in using the Arbitrary PCR#1 plate as input and the same output plate to amplify the genomic context around each transposon cassette

Variables for source plate 1 arbitrary PCR 2: Variables-PCRs-OT.csv

Variables for source plate 2 arbitrary PCR 2: Variables-PCRs-OT.csv

Variables for source plate 3 arbitrary PCR 2: Variables-PCRs-OT.csv

Gel results for arbitrary PCRs

Sequencing and annotation of arbitrary PCR#2

The following files were used:

alignment_and_annotation_blastn.py Pseudomonas_putida_KT2440_110.fna Pseudomonas_putida_KT2440_110.csv sequencing_results.zip