1Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM)-Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA/CSIC), Madrid, Spain
Protocol Citation: Lorea Alejaldre, Ana Mariya Anhel, Ángel Goñi-Moreno 2022. High-throughput workflow for the genotypic characterization of transposon library variants. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg394jzg25/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
This is a workflow for the genotypic characterization of transposon library variants. It has been developed using an open-source Opentrons OT-2 robot, BLASTN for genomic annotations and modular sub-protocols (e.g., PCR sample preparation, OT-2 volume transfer, OT-2 counter selection, etc) that can be used for other tasks, thus providing a general-purpose pipeline.
All steps follow a 96-well plate format for high-throughput analysis. The protocol is described for the characterization of transposon library variants generated with SEVA-Sib pBAMD1-x and pBLAM1-x plasmid sets that follow Standard European Vector Architecture (SEVA, https://seva-plasmids.com) and can be amplified with the standard PS1-PS6 primers. After the description of the protocol we present the results of an example generated at our laboratory (https://biocomputationlab.com) using the soil bacterium Pseudomonas putida KT2440 as acceptor strain.
Guidelines
This workflow comprises the following sections: 1) Colony picking in selective media 2) Counter-selection and glycerol stocks pre-cultures 3) Colony selection in OT-2 liquid handler robot 4) Master 96-well plate for PCR steps 5) Control PCRs (spurious plasmid integration control and cargo insertion control) 5) Arbitrary PCRs 6) Sequencing and annotation. There is an additional section with an example on how to run the script.
We recommend the use of an OT-2 protocol specially if more than 2 libraries are to be analyzed. However, we recommend to do counter-selection in the OT-2 liquid handling robot even for one plate to avoid human errors.
Note that other pipettes can be used to run the workflow in the OT-2 but these were deemed the most appropriate for the overall workflow to minimize pipette changes.
Storage membrane for 96-well plate (i.e Thermo Scientific™ Láminas de papel de aluminio adhesivas para placas de PCRFisher ScientificCatalog #10130853 )
PCR plates (pink ones)
Lids for plates
Enzymes
DNA polymerase with green buffer
Phire Green Hot Start II PCR Master MixFisher ScientificCatalog #15391732
DNA polymerase (Thermo Scientific™ Phire Hot Start II PCR Master MixFisher ScientificCatalog #15361732)
Dispense 100 µL of selective media (M9-citrate for P. putida or Luria-Bertani plus 20 ng/µL nalidixic acid for DH5α 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 Overnight at 30 °C (P. putida) or 37 °C (E. coli) / 500 rpm
Counter-selection and glycerol stocks pre-cultures
Counter-selection and glycerol stocks pre-cultures
1d
1d
Measure OD600nm of overnight culture grown in selective media from go to step #4 plus transposon cassette antibiotic in a plate reader
Inoculation of counter-selection plate in selective media:
Dispense 100 µL 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 5 µL of overnight culture from go to step #4 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.
Protocol
NAME
OT-2 Media dispensing and culture inoculation protocol
CREATED BY
biocomp.cbgp Biocomputation Lab
10m
Inoculation of precultures for glycerol stock in rich media:
Dispense 100 µL of Luria-Bertani media plus transposon cassette antiobiotic in a 96-well plate
Transfer 5 µL of overnight culture from go to step #4 to counter-selection (ampicillin) plate
Cover with a sterile breathable membrane
10m
Grow counter-selection and glycerol stock pre-culture plates Overnight at 30 °C (P. putida) or 37 °C (E. coli) / 500 rpm
16h
Measure OD600nm of overnight culture grown in selective media plus ampicillin in a plate reader
Colony selection in OT-2 liquid handler robot
Colony selection in OT-2 liquid handler robot
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
Protocol
NAME
OT-2 Counter-Selection
CREATED BY
biocomp.cbgp Biocomputation Lab
Note
The OT-2 protocol will prepare three plates (2 glycerol stock plates and a "PCR plate") and perform the following:
Dispense 75 µLof PCR-grade water to "PCR plate"
Dispense 25 µL of 30% glycerol to two glycerol stock plates
Transfer 25 µL of grown pre culture in Luria-Bertani media plus transposon cassette antibiotic from go to step #7 to "PCR plate" and glycerol stock plates
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
Master 96-well plate for PCR steps
Master 96-well plate for PCR steps
Transfer 50 µL of selected colonies from one or more libraries to a 96-well plate with the following OT-2 protocol:
Protocol
NAME
OT-2 Protocol to transfer volume from several plates to a single plate
CREATED BY
biocomp.cbgp Biocomputation Lab
Control PCRs
Control PCRs
Safety information
Positive control (donor plasmid) and wild-type control (P. putida or E. coli) should be added to every reaction in this section.
Spurious integration control with SEVA primers pairs PS3/PS4 and PS5/PS6
Protocol
NAME
OT-2 PCR sample preparation protocol
CREATED BY
biocomp.cbgp Biocomputation Lab
Note
If <48 cfu are to be analyzed both PS3/PS4 and PS5/PS6 spurious integration controls can be done in a single 96-well plate
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 PCRs
Arbitrary PCRs
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
Protocol
NAME
OT-2 PCR sample preparation protocol
CREATED BY
biocomp.cbgp Biocomputation Lab
Note
The OT-2 protocol will perform the following steps:
Prepare a PCR master mix
Dispense 19 µL of PCR mastermix
Transfer 2 µL of pre-culture from go to step #12
Safety information
If different primer pairs are added to a single 96-well plate, the OT-2 script should be run separately for each primer pair
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.
Note
Several bands will appear and even DNA smears even when the reaction has worked perfectly.
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
Protocol
NAME
OT-2 PCR sample preparation protocol
CREATED BY
biocomp.cbgp Biocomputation Lab
Note
The OT-2 protocol will perform the following steps:
Prepare a PCR master mix
Transfer 1 µL of PCR product from Arbitrary PCR#1
Dispense 19 µL of PCR mastermix
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
Note
Several bands will appear and even DNA smears even when the reaction has worked perfectly.
Sequencing and annotation
Sequencing and annotation
1m
1m
Prepare a PCR plate to send to sequencing by mixing 10 µL of unpurified Arbitrary PCR#2 reaction and 10 µLof 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)
Note
These guidelines may vary depending on the sequencing service arranged for your laboratory.
Annotate sequencing results by running the following protocol:
Protocol
NAME
Bacterial genome annotation script using BLASTN
CREATED BY
biocomp.cbgp Biocomputation Lab
Note
The python script uses as input:
DNA sequencing results in .txt or .seq
Reference genome file in fasta format
Genome annotation file in fasta format
1m
Example
Example
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 using the following OT-2 protocol in go to step #6 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 go to step #10 . 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 go to step #13
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 go to step #14 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
Gel of the first 7 rows with the primers ps3 and ps4
Gel of the last 5 rows with the primers ps3 and ps4
Gel of the first 7 columns with the primers ps5 and ps6
Gel of the last 5 columns with the primers ps5 and ps6
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 go to step #14 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
Gel of the columns 4 to 9 of the control of integration primers
Gel of the columns 10 and half of the 11of the control of integration primers
Gel of the columns 11 and half of the 12 of the control of integration primers
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 go to step #16 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 go to step #19 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
Gel for several wells in the final plate of the arbitrary PCRs. .1 is result of the first PCR and .2 is the second PCR