Janet Matsen's protocols for metabolic engineering.

ACS Library Screening with Nash Reagent

20 Dec 2014

Things that can be done at “waiting steps”:

  • Prepare labeled plates:
    • assay plates: non-UV 1/2 area plates. Label non-UV on the left skinny side, and label the date and description on the front wide side.
  • add data from the plate reader software to the gSpreadsheet.
    • Include a title like 141220 plate4 because the R script will parse out the plate number and use it to match to the well description.
  • label calculated uM values on ADH tubes for later freezing.

Have ready:

  • Purified ADH.
    • Takes a while to thaw from -80oC
    • 150204 idea: move right amount to -20oC the previous night so it goes faster in the AM
  • Fully grown-out autoinduced ACS enzyme variants with controls present on the plate.
    • Include a diagonal stripe of the + control ACS L641P, and some P641L and K609A controls elsewhere
    • Was selective antibiotic included?
  • Plenty of NADH, ATP, CoA (keep an eye for stock running anywhere near low).
  • Assay buffer. Record date of prep in notebook & spreadsheet.
    • currently using 50mM PO4 buffer, pH ~7.4

Weigh chemicals

  • Keep buffer at room temp; want enzyme reaction to happen at RT.
  • Use spreadsheet to calculate amount of each ingredient to weigh out.
  • Prepare eppendorf tubes for cofactors: NADH, CoA, Mg++. Name & date on top.
    • Don’t put these empty tubes on ice or they will attract water precipitation.
  • Label a 15mL tub for formate: date, formate, concentration,
  • Item locations:
    • NADH, ATP, CoA: -20oC freezer closest to Amanda’s bench. NADH & CoA are in a plastic baggie at the very bottom, in the cl
      ear bin. ATP is in a bigger bottle a few shelves up, and is shared with Amanda.
    • formate is in the Na (sodium bin), and MgCl2 is in the M bin.
  • Put temperature sensitive reagents on ice. (NADH, ATP, CoA)
  • Weigh on most precise balance, being careful not to get water on the tubes (will effect measurement)
    • CoA is expensive ($231/100mg bottle); don’t get much more than is required. NADH is medium-expensive, so don’t go crazy with that either.
    • Watch out for static.
  • Record weights measured on tube, in notebook, and in spreadsheet.
  • Add the appropriate amount of buffer based on spreadsheet calculations.
  • Make sure we don’t run out of reagents:
    • keep an eye on CoA reserves. If you open the last bottle, order 2-3 more.
    • If the NADH bottle gets low, order another. (We don’t use this one as fast, so we don’t need to keep an unopened bottle around.)

Move the following items to the plate reader

  • a 50mL tube with some assay buffer
  • all the electronic and non-electronic pipettes (in a bucket)
  • pneumatic pipette
  • stopwatch
  • BugBuster
  • purified ADH
  • lab notebook
  • leave the ACS cells on the shaker so they don’t settle.
  • Nash reagent. Ideally keep on ice in a dark bucket. Make sure it isn’t “to yellow”. Record prep date.
  • falcon tube holder (for buffer & formate stock)
  • headphones
  • timer

Prepare the plate reader (only if doing TCA precip & scanning spectrum)

  • Establish a connection between the computer & plate reader. May require restarting the computer.
  • Make or copy an old PDA file into a new folder with the expriment date
    • set spectra to scan from 350nm to 550nm with step size of 2nm
  • Note that when you save the PDA files, do save as and change the filename a little bit. This is because saving is WAY slower than save as. Eg. filename–v5.pda is saved as filename–v6.pda
  • Naming of the scan is crucial: the name is parsed by the R file, and the plate name is crucial for matching data to the descriptive well information in a different spreadsheet.

Screening a whole plate:

  • Label plates
    • Plate = 1/2-area polystyrene (non-UV).
    • Put the date on the A1-H1 side of the plate. Write non-UV if plate is polystyrene.
  • Pipette autoinduced cells into 96-well plate.
    • Do at bench with flame and cheap non-filtered but sterile tips.
    • Make sure source cells just came off the shaker so they will not have settled out of solution.
    • Eyeball that each tip does in fact have 4uL of cell suspension. (Loose fitting pipette tips don’t suck up liquid).
    • Put cells at the bottom of the wells so they will be sure to be in the reaction.
  • Make a master mix of ADH (7uM final), NADH (6mM final), CoA (1mM final), ATP (4mM final), Mg2+ (5mM final), and formate (700+mM final) according to gSpreadsheet
    • Make sure:
      • the uM of the ADH stock represents what you are going to use (different batches have different final uM values)
      • the number of plates is set correctly (usually 4)
      • you are making an appopriate amount of formate
    • Excess factor should be ~1.2
      • 1.1 is definitely not enough 141215 JM. 1.2x was too much 150204. 1.15 was too little 150409. Settle for 1.18

-OR-Current recipe when NOT using a master mix:

Current recipe as of 141220:
* 4uL of autoinduced ACS cells
* 2uL of 10X BugBuster (fairly arbitrary given the rxn volume is 40uL)
* 3uL of NADH (3uL of 80mM in 40 uL –> 6mM)
* 3uL of ATP/CoA/Mg2+ mix, which includes:
* stock concentrations: 15.15 mM CoA, 75.72 mM MgCl2, 60.6mM ATP
* final concentrations: 1mM CoA, 5mM MgCl2, 4mM ATP.
* _X_uL of purified ADH. Want ~7uM for 4uL of autoinduced ACS cell extract.

Make a stock right before prepping a plate:

  • Use a rinsed (DI water) or new pipette boat to mix in.
  • Make excess of the mix. ~20% extra is appropriate.
  • Load ACS cells first, using sterile technique at the bench

Start the formaldehyde producing reaction

  • First make sure the plate reader file is ready:
    • Scan from ~350 to 500 nm, with 2 nm steps. Scanning a whole plate this way takes ~10 minutes!
      • Can use less steps if it is a less important read.
  • Decide how long the reaction will be run. Include this in the file name.
    • Assume 15 minutes for now.
      • 10 min not enough JM 150129 <– ??
    • 7 minutes has been used since 2/4/2014 for 1/2 mL autoinduced cultures
    • May need to change the time as the autoinduction media recipe and culture volume changes.
  • Start reaction by adding 40-x uL of the reaction cocktail that includes formate, ATP, CoA, Mg++, purified ADH, BugBuster, and make-up buffer.
    • 37uL if 4uL of cells per well
      • Set pipette to 4*36uL dispensing. Don’t want “soapy” solution sucked into the pipette itself.
    • Move tips & waste close together so loading time is quick and wells have comparable reaction times.
    • ** Use new tips for each pipetting up** because BugBuster makes sucking bubbles into the pipette likely.
  • Put a cover on the plate to help prevent formaldehyde evaporation (this may not actually help; to be tested).
  • Start a timer when the first plate’s loading is complete.

Add Nash reagent

  • Usually an equal volume of Nash reagent is added. The reaction essentially(?) stops when the Nash reagent is added.
  • Move plate to 55oC or 61oC box for color development. ~30 minutes is appropriate.
    • Note the temperature of the box in a notebook.
  • Remove plate to scan spectrum.

If you want to scan the plate to plot the data, TCA precipitation is necessary

  • See safety notes below
  • This removes flecks of precipitated protein
  • Add 40uL to a 40uL rxn with 40uL Nash?
    • LOOK UP OLD DATA!
  • Centrifugation is necessary to push down particulates.

Copy data into electronic spreadsheet/notebook

  • Export data from .pda to a text file.
    • Select the plate reads you want to include. You can only do within one experiment at a time
    • Verify that the assay plate number is included in the file names
    • Save to same folder as the PDA files, or a sub-folder
    • Open .txt files with Vim, and copy to the google spreadsheet.
  • Optional: go through plate and assign approximate yellowness values to each well.
    • 2 = comparable to average + control yellowness
    • 1 = less yellow than control
    • 3 = noticeably more yellow than + control.

Archive cells

  • Record in lab notebook
  • Restreak on an LB plate with the appropriate antibiotic.
    • Are you restreaking for single colonies or just putting blobs on agarose?
    • For single colonies, store up to 5 strains on a plate.
    • Can squeeze 20+ on a plate if you just want blobs.
  • Put cell-filled 96-well plates in the fridge with aluminum seals.

Deposit summary here:

Clean up

  • Charge electronic pipettes that are low in battery.
  • Make sure the plate shaker is turned off.
  • Return pipettes to appropriate places
  • Discard used 96-well plates.

Trichloroacetic Acid Safety

  • Trichloroacetic acid is a strong acid that will burn your skin and eyes very badly.
    • In 2/2015 Janet got a tiny bit (smaller than a “drop”) on her arm and it left a brown spot where it burned.
  • Proper handling:
    • gloves and closed-toe, skin-covering shoes are mandatory
    • goggles and a lab coat are encouraged.
    • do not wear shoes that fail to protect your feet (e.g. mary jane shoes or sandals)
  • If you do get some on you, wash it off immediately.
    • It probably won’t cause cancer, but will definitely leave a mark.
  • See the [EPA notes[(http://www.epa.gov/iris/toxreviews/0655tr.pdf) about toxicology to better understand the health hazards.

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