Experiments

Experiments. Two team members in white coats and gloves pipette at a bench crowded with media bottles.

Abstract

Experiments collects the twenty-four protocol sheets behind the wet lab work, grouped into four stages, each as a downloadable PDF beside the procedure diagrams drawn for it.

The sheets follow four stages in the order one hands its product to the next: ten for cloning, seven for protein production, five plant assays and two for the bioreactor. All but one carry the full procedure as a downloadable PDF, down to the reagent volumes, the cycling conditions and the blank or untreated arm the readout is scored against. The newest is the DNPH assay for ACC deaminase activity, scaled down twenty-fold from the published method onto a 96-well plate; it read α-ketobutyrate in the reactor permeate on 7 and 8 October, after which DNPH could not be bought and the Berthelot assay replaced it. Three sheets produced no result on this wiki: the Bradford assay, large scale extraction, and purification and dialysis. Electroporation ran on both strains in the sheet: B. subtilis WB800 gave countable colonies at 2.1 to 2.5 kV, strain 168 only a handful.

Stages

Contents

On this page

Overview

ReLEAF makes plant protectants in Bacillus subtilis and tests them on Arabidopsis under salt and heat stress. The wet lab work runs in four stages, and each hands its product to the next: cloning builds the constructs, protein production gets the protectant out of the culture and measures its activity, the plant assays test it, and the bioreactor sheets calibrate the instruments that watch the culture grow.

Whole-project experiment map: cloning in E. coli, electroporation into B. subtilis, bioreactor culture, secreted protectants, plant model testing on agar, soil and hydroponics, and plant growth evaluation by chlorophyll, root length, leaf area and fresh weight.

Protocols

Molecular cloning

Culture agar preparationPrepare and make agar plates to cultivate bacteria.

PDFFull protocol sheetCulture agar preparation, 197 KB

Procedure diagram for culture agar preparation: weigh LB agar into a flask with water and glycerol, stir, autoclave, cool in the cabinet, add antibiotic, pour plates.
Culture agar preparation.
PCR amplificationA nucleic acid amplification technique to rapidly produce copies of DNA segments from a small initial sample.

PDFFull protocol sheetPCR amplification, 248 KB

Procedure diagram for PCR amplification: assemble the reaction on ice, then run the thermal cycle.
PCR amplification with Ex Taq DNA polymerase.
Restriction enzyme digestionLinearize purified plasmids with restriction enzymes to verify their sizes.

PDFFull protocol sheetRestriction enzyme digestion, 130 KB

Procedure diagram for restriction enzyme digestion: calculate the DNA volume, assemble buffer, enzyme, DNA and water on ice, incubate.
Restriction enzyme digestion.
DNA gel electrophoresisResolve specific DNA fragments to allow visual verification and confirmation of product band sizes.

PDFFull protocol sheetDNA gel electrophoresis, 285 KB

Procedure diagram for DNA gel electrophoresis: cast the agarose gel with stain, load ladder and samples, run at 100 volts, image.
DNA gel electrophoresis.
Golden Gate assemblyClone and assemble multiple core genetic parts of the light-controlled optogenetic circuit.

PDFFull protocol sheetGolden Gate assembly, 218 KB

Procedure diagram for Golden Gate assembly: combine vector, inserts, T4 ligase, buffer and type IIS enzyme, then cycle digestion and ligation.
Golden Gate assembly.
E. coli transformationIntroduce foreign plasmid DNA in competent cells in order to replicate plasmids in E. coli.

PDFFull protocol sheetE. coli transformation, 379 KB

Procedure diagram for heat-shock transformation: thaw competent cells on ice, add plasmid, heat shock at 42 degrees, recover in SOC, plate.
Chemical transformation into E. coli DH5α.
Electroporation into B. subtilisIntroduce foreign plasmid DNA in competent cells through exponential electrical flow in order to replicate plasmids in B. subtilis.

PDFFull protocol sheetElectroporation into B. subtilis, 294 KB

Procedure diagram for electroporation: grow and wash electrocompetent Bacillus subtilis, mix with plasmid in a chilled cuvette, pulse, recover in SOC, plate on selective agar.
Electroporation into B. subtilis.
Colony PCRRapidly screen individual bacterial colonies to verify the presence of the desired DNA insert or recombinant construct using the colony as the PCR template.

PDFFull protocol sheetColony PCR, 204 KB

Procedure diagram for colony PCR: grid a backup plate, pick each colony with a tip, streak it on the grid, drop the tip in a PCR tube with master mix, run the cycle.
Colony PCR with a gridded backup plate.
Liquid bacterial cultureGrow bacteria such as E. coli or B. subtilis in nutrient-rich broth, allowing rapid multiplication in a suspended, aerated environment.

PDFFull protocol sheetLiquid bacterial culture, 228 KB

Procedure diagram for liquid culture: label tubes, add TB medium and antibiotic, pick a colony with a tip, drop it in, incubate at a slant.
Overnight liquid culture.
DNA isolationIsolate plasmid DNA from bacterial cultures for concentration measurement and downstream applications.

PDFFull protocol sheetDNA isolation, 400 KB

Procedure diagram for plasmid miniprep: pellet the culture, resuspend, lyse, neutralise, bind to the column, wash, elute, measure on the BioDrop.
Plasmid DNA isolation, column to BioDrop.

References

  1. Arnon DI. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology 1949;24(1):1-15.
  2. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 1976;72:248-254.
  3. Viogene. Mini Plus Plasmid DNA Extraction System handbook.
  4. Thermo Fisher Scientific. HisPur Ni-NTA Spin Purification Kit and Slide-A-Lyzer G3 Dialysis Cassettes instructions.
  5. Sigma-Aldrich. CelLytic B Cell Lysis Reagent product information.
  6. Bio-Rad. Gene Pulser Xcell Electroporation System instruction manual.
  7. Penrose DM, Glick BR. Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiologia Plantarum 2003;118(1):10-15.
  8. Beal J, Farny NG, Haddock-Angelli T, et al. Robust estimation of bacterial cell count from optical density. Communications Biology 2020;3:512.
  9. van de Hulst HC. Light Scattering by Small Particles. New York: John Wiley & Sons; 1957.