Oct 27, 2022
Version 1
High-throughput workflow for the genotypic characterization of transposon library variants V.1
- 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
- Ángel Goñi-Moreno: angel.goni@upm.es
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
Manuscript citation:
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: September 19, 2022
Last Modified: October 27, 2022
Protocol Integer ID: 70220
Keywords: High-throughput, Transposon library, marC9, Tn5, SEVA, OT-2, Opentrons, genotypic characterization, genotyping, bacterial genome, 96-well
Funders Acknowledgement:
Comunidad de Madrid
Grant ID: Y2020/TCS-6555, 2019-T1/BIO-14053
MCIN/AEI
Grant ID: CEX2020-000999-S, PID2020-117205GA-I00
European Research Council
Grant ID: 101044360
Abstract
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.
Materials
Equipment:
Equipment
Incubating mini-shaker
NAME
Incubating shaker
TYPE
Fisherbrand
BRAND
15554070
SKU
LINK
Equipment
OT-2
NAME
Liquid handler
TYPE
Opentrons
BRAND
OT-2
SKU
Equipment
SPECTROstar Nano
NAME
plate reader
TYPE
BMG
BRAND
SPECTROstar Nano
SKU
LINK
Equipment
Mastercycler® nexus - PCR Thermal Cycler
NAME
Thermocycler
TYPE
Eppendorf
BRAND
12304943
SKU
LINK
Equipment
Centrifuge Tube Mini-Cooler
NAME
Cold Block
TYPE
BRAND
BRAND
10141921
SKU
LINK
Electrophoresis machine
Wet-lab requirements:
Material
- Non-treated flat bottom sterile 96-well plates (i.e 96-well plates flat bottom non-treatedVWR AvantorCatalog #734-2781 )
- Sterile breathable membrane for 96-well plate (i.e Greiner Bio-One Sellador BREATHseal™Fisher ScientificCatalog #11920667 )
- 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 )
- Agarose
Oligonucleotides:
- Spurious integration control: PS3, PS4, PS5, PS6 (
- Arbitrary PCR: ARB2, ME-O-Km-Ext-F, ME-O-Km-Int-F, ME-O-Sm-Ext-F, ME-O-Sm-Int-F, ME-O-Gm-Ext-F, ME-O-Gm-Ext-R (https://doi.org/10.3389/fbioe.2014.00046)
- Optional insert control: PSMCS
Dry-lab requirements:
- Python 3
- Command-line BLASTN
Colony picking in selective media
Colony picking in selective media
1d
1d
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 protocolCREATED 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-SelectionCREATED 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 µL of 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 µL of 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 BLASTNCREATED 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-PCRs-OT.csv Variables for source plate 2 arbitrary PCR 1: Variables-PCRs-OT.csv
Variables-PCRs-OT.csv Variables for source plate 3 arbitrary PCR 1: Variables-PCRs-OT.csv
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-PCRs-OT.csv Variables for source plate 2 arbitrary PCR 2: Variables-PCRs-OT.csv
Variables-PCRs-OT.csv Variables for source plate 3 arbitrary PCR 2: Variables-PCRs-OT.csv
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
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 Note
We only sent 61 samples, that is why sequencing_results.zip has 61 files