PROCEDURE:

• Take a colony from a plate and put in 20uL dH2O in PCR tube (at least one colony from each plate)
• Restreak on new plate using the same pipette tip (or wooden stick)
• Boil tube content in PCR at 98C for 10min (optional)
• Use that as template for colony PCR (25uL reaction)
• Mix in PCR tube:

• PCR and verify on gel
• PCR program: 98°C 30s → 35*[98°C 10s → 53°C 20s → 72°C 35s] → 72°C 5min → 4°C forever
• Primers come in little envelope (get paper spreadsheet due to stickers)
• Primers come in little blue tubes in a package
• Take one; see DNA dried up on the side of the tube
• Put little blue tubes in centrifuge (all of them) to spin DNA to bottom, and click double arrow for about 2 seconds to spin lightly
• Now, dissolve them to 100 uM concentration in nanopure water (ideally sterile)
• Amount of DNA on tube (ex 22.6 nm (nanomoles)) -> add 226 uL of water to get it to 100uM concentration
• 2 at a time, vortex them for 5 seconds to mix DNA and water
• Phusion DNA polymerase (not tac): phusion is expensive because it’s a high fidelity polymerase (few mistakes) so it’s better if you’re gonna use the DNA later
• Phusion is from the freezer, 2x master mix
• Add water, primer, and template to phusion
• Template: from mini prep set
  • PSB1T3: vector which has tetracycline resistance gene
  • pMQ30: plasmid for gene deletions
  • pMQ95: expression plasmid
  • pMQ97: expression plasmid
• amplify pMQ97, 30, 95 to amplify the backbone (will amplify everything except tetracycline resistance)
• 2 rxns of PSB1T3, because overhangs for 30&97 are the same so they share
• Ending in r=reverse, f=forward; starting with bb=backbone
• Mix primers that go together to concentration of 10 uM
• Put primers in freezer in pMQ vector box
• get 5 PCR tubes, in cupboard, with racks on the counter
• look online at phusion site (neb.com) and look up 50 ul reaction protocol
• 2.5 ul primer mix, .5 ul of template, 22 ul H20 = 25 total, then 25 ul phusion mastermix (from 2x phusion)
• put in pSB1T3 into the i’s, and the respective pMQ’s into each tube for the template
• for the primer mix, the bb97 and bb30 share the bb30 mix, all others get paired with their respective numbers
• 25 ul phusion into each tube
• put leftover phusion in buffers box in freezer
• largest backbone: 10 kb -> 2.5 minute extension time

Electrophoresis with 0.1% agarose gel

PREPARATION:

• 0.5 g agarose
• 0.5 ul cybersafe stain
• 50 mL TAE 1x buffer

PROCEDURE OF MAKING GEL:

• Mix the agarose and 50 mL of TAE buffer together
• Microwave mixture until mixture does not have floating particles (roughly a minute)
• Mix in the cybersafe stain until solution is homogeneous
• Pour into gel box mold (make sure the comb/teeth are in the gel box for the wells)
• Let solidify (about 30 minutes) at room temperature

PROCEDURE OF OF RUNNING GEL:

• Place gel in electrophoresis chamber where the wells are closer to the side that hooks up to the negative electrode
• Add enough TAE buffer such that the gel is just barely submerged in buffer
• Load samples (with dye)
• Hook up electrodes
• Run (~30 minutes)

PROCEDURE OF OF IMAGING GEL:

• Remove gel from electrophoresis box
• Put gel into UV-box (in the Murray Lab), make sure UV light is off when you open the box
• Turn on the UV light, make sure the bax is closed
• Open up the imaging program
• Change the settings until gel is in focus
• Save gel image in iGEM folder (file should be named dateYOUNAME, ex: 7_12Debbie)
• Print gel

Yeast transformation

PROCEDURE:

• Thaw competent yeast cells at RT (RT ok for all steps)
• Aliquot 50 uL into new tube
• Add 100 ng of plasmid to 50 uL cells
• For homologous recombination, add 100 ng of plasmid + 300-500 ng insert
• Vortex vigorously for 2 minutes
• Heat shock 42C water bath for 40 min (up to 180 min ok)
• Add 250 uL of sterile water, vortex vigorously for 2 minutes
• Plate 100 uL on selective plate

Plasmid Transfer by Conjugation

Plasmid with a transfer origin (oriT) can be shuttled between strains using conjugation, provided that the donor strain (or a helper strain) contains the code for the necessary transfer machinery. The standard donor strain for conjugation in the Newman lab is E. coli BW29427 (DKN164, formerly known as WM3064). BW29427 is a diaminopimelic acid (dap) auxotroph, i.e. dap (300 µM) has to be added to any medium before it can grow. Plates can be prepared by spreading 200 µl of a 60 mM dap stock.

Biparental conjugation:

• Mix 800 µl of an overnight culture (LB + dap + suitable antibiotic) of E. coli BW29427 harboring the plasmid to be transferred and 200 µl of the recipient strain (e.g. PA14, grown overnight in LB).
• Centrifuge the cells and wash the pellet in LB to remove the antibiotic.
• Centrifuge and resuspend the pellet in 30 µl LB and, spot onto the center of an LB+dap plate and incubate at 30°C overnight.
• Pipette ~2 ml of LB onto the mating plate and resuspend the cells by scraping, e.g. with a pipette tip.
• Collect the cell suspension with a pipette after tilting the plate, centrifuge, and resuspend the cells in a volume suitable for plating.
• Plate onto one or more plates without dap (so BW29427 cannot grow) and with a suitable antibiotic to select for the plasmid (this prevents growth of plasmidless recipient cells). Only recipients carrying the plasmid will form colonies.
• Presence of the plasmid (replicable, transposon carrier, or integrated suicide vector) in colonies can be verified by PCR.

PREPRARATION:

• Liquid cultures of donor strain (DKN1005) and receptor strain (Z. mobilis ZM4)
• DAP
• 0.9% salt solution

PROTOCOL FOR pLAFR5 CONJUGATION:

• Treat RM plates made for Z. mobilis with 100 uL of DAP
• Incubate membranes on plates for 30 minutes
• Mix 750 uL of the donor strain (DKN1005) and 250 uL of the receptor strain (Z. mobilis ZM4). If the receptor strain has low density, then mix the strains 50:50.
• Centrifuge for 1 minute and pour out the supernatant (This step removes the antibiotics)
• Resuspend in 1 mL of RM
• Repeat steps 4 and 5
• Then plate 50 uL of the mixture onto the filter membranes
• Incubate at 30 C
• Place the filter in a falcon tube containing the 0.9% salt solution (1 mL each)
• Vortex vigorously until solution is cloudy
• Plate 100 uL onto RM + tet plates (no DAP on these plates causes the DKN1005 donor to die)

Ethanol Assay

Ethanol Color Assay Reaction

Materials

• water
• 1.0M bicine (pH 8.0)
• 40 mM EDTA
• 10 mM NAD
• 4.2 mM thiazolyl blue
• 16.6 mM phenazine ethosulfate
• 1 mg/mL alcohol dehydrogenase enzyme
• ethanol

Method

Make a master mix consisting of:

• 3.6 mL water
• 1.2 mL bicine
• 1.2 mL EDTA
• 1.2 mL NAD
• 1.2 ml thiazolyl blue
• 2.4 mL phenazine ethosulfate

Before running the experiment, add 1.2 mL of alcohol dehydrogenase enzyme.

Pipette 5 uL of sample into the 96-well microtiter plate. Add 95 uL of master mix. Measure absorbance at 570 nm. We found that the maximum absorbance of each well occurred at a different time relative to the concentration, so it is best to run a series of measurements over a period of approximately 4 hours.

MOPS Buffer

We used the recipe from Teknova listed here but substituted 10 ml of 20% glucose for 8 ml of 50% glucose. Mix all the following ingredients together to get MOPS buffer:

• 100 ml 10X MOPS Mixture
• 10 ml 0.132 M K2HP04
• 100 ml 10X ACGU
• 200 ml 5X Supplement EZ
• 582 ml Sterile H20*
• 8 ml 20% Glucose