TITLE: High-throughput workflow for the genotypic characterization of transposon library variants
AUTHORS: Lorea Alejaldre, Ana Mariya Anhel, Ángel Goñi-Moreno

[DESCRIPTION]
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.

[STEPS]
SECTION: Colony picking in selective media
1. 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 



SECTION: Counter-selection and glycerol stocks pre-cultures
6. 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   to counter-selection (ampicillin) plate
Cover with a sterile breathable membrane
 

 

SECTION: Counter-selection and glycerol stocks pre-cultures
7. 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    to counter-selection (ampicillin) plate
Cover with a sterile breathable membrane



SECTION: Control PCRs
14. 
 

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

 
 

 

SECTION: Arbitrary PCRs
16. 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 


 

 


 

SECTION: Sequencing and annotation
23. Annotate sequencing results by running the following protocol:

 

 


SECTION: Colony picking in selective media
2. Pick individual colonies into a 96-well plate with selective media
Tip: Keep tips inside of wells to keep track

SECTION: Colony picking in selective media
4. Grow   at 30 °C (P. putida) or 37 °C (E. coli) /    



SECTION: Colony picking in selective media
3. Cover with a sterile breathable membrane
SECTION: Counter-selection and glycerol stocks pre-cultures
8. Grow counter-selection and  glycerol stock pre-culture plates   at 30 °C (P. putida) or 37 °C (E. coli) /    
SECTION: Master 96-well plate for PCR steps
13. Transfer 50 µL of selected colonies from one or more libraries to a 96-well plate with the following OT-2 protocol: 

 

SECTION: Colony selection in OT-2 liquid handler robot
10. 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


 

 

SECTION: Counter-selection and glycerol stocks pre-cultures
5. Measure OD600nm of overnight culture grown in selective media from    plus transposon cassette antibiotic in a plate reader

SECTION: Counter-selection and glycerol stocks pre-cultures
9. Measure OD600nm of overnight culture grown in selective media plus ampicillin in a plate reader
SECTION: Control PCRs
15. 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)
SECTION: Colony selection in OT-2 liquid handler robot
11.  Cover glycerol stock plates with a storage membrane and store at -80ºC 
SECTION: Colony selection in OT-2 liquid handler robot
12. 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
SECTION: Arbitrary PCRs
19. 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 


 

 

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


 

SECTION: Arbitrary PCRs
20. Seal 96-well plate, place it in thermocycler and run the following PCR program:

 



SECTION: Arbitrary PCRs
17. Seal 96-well plate, place it in thermocycler and run the following PCR program:

 

SECTION: Sequencing and annotation
22. 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)

 

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

SECTION: Example
24. 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.
SECTION: Example
25. 
Counter-selection and glycerol stocks pre-cultures  using the following OT-2 protocol in   in two runs
SECTION: Example
26. 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.
SECTION: Example
27. 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   

  

Output from the run (map with identifiers)

  

SECTION: Example
28. Control PCRs to account for spurious integrations and the correct integration of the cargo
SECTION: Example
29. Arbitrary PCRs

SECTION: Example
30. Sequencing and annotation of arbitrary PCR#2
The following files were used:
        

 


SECTION: Example
25.1. 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.

  

SECTION: Example
25.2. 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.

  

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

  

 
  

Output from the run (map with identifiers)

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

  

  
  

Output from the run (map with identifiers)

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

  

  
 

Output from the run (map with identifiers)

  
SECTION: Example
28.1. 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

  

Gels after PCR protocol in thermocycler

  

  
  

  
SECTION: Example
28.2. 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

  
  
  
SECTION: Example
29.1. 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 for source plate 2 arbitrary PCR 1:   
Variables for source plate 3 arbitrary PCR 1:   

SECTION: Example
29.2. 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 for source plate 2 arbitrary PCR 2:   
Variables for source plate 3 arbitrary PCR 2:   

Gel results for arbitrary PCRs