1. Culture Vt.

1.1 Spread plate with Vt liquid culture (what kind of media is in the plate?
How to make plates?)

1.2 Put the plate in RT for 2 days.

1.3 Add 5ml of 1% LB broth (with 1ul/ml ampicillin) to each glass tubes
(several Vt tubes and a blank).

1.4 Use tips to scratch a colony in the plate and throw the tip in the tube
(except for the blank).

1.5 Put the tubes in a roller drum at RT or 37C for 2 days.

1.6 Read OD420.

2. Culture E. coli.

2.1 Spread plate with E. coli liquid culture (what kind of media is in the plate?
How to make plates?)

2.2 Put the plate in 37C? for 1 day?.

2.3 Add 5ml of 1% LB ? (with 1ul/ml strep) to each glass tubes (several E. coli
tubes and a blank).

2.4 Use tips to scratch a colony in the plate and throw the tip in the tube
(except for the blank).

2.5 Put the tubes in a roller drum at 37C overnight.

2.6 Read OD420.

3. Azocasein Assay (test proteinase activity)

3.1 Centrifuge at 13,000 rpm for 10 min.

3.2 Transfer 100ul of supernant to a new tube (keep the rest just in case).

3.3 Add 400ul Azocasein to each tube, vortex, incubate at 37C for 30 min.

3.4 Add 600ul TCA to each tube, vortex, incubate on ice for 40 min.

3.5 Centrifuge at 13,000 rpm for 10 min.

3.6 Transfer 800ul of supernant to a new tube, add 200ul NaOH to each tube.

3.7 Read OD420.

4. Wash cells.
4.1 Centrifuge at 13,000 rpm for 10 min to harvest cells.

4.2 Discard supernant, add 1 ml seawater to each tube. Vortex well to

resuspend cells.

4.3 Centrifuge at 3,000 rpm for 5 min.

4.4 Discard supernant, add 1ml seawater to each tube. Pipet to resuspend
cells.

4.5 Read OD420 to determine cell concentration.

Things needed:

Tube, tip, plate, “the stick to spread plates”

Azocasein, 1% LB, 1% LB broth, TCA, ampicillin, strep

Bacteria hood, spectrophotometer, incubator

Azocasein (light sensitive): dissolve 1g in 100ml water (gentle heating and

stirring may be needed), filter with ?. Store solution at 4C. Always cover with
foil.

1% LB broth ?

1% LB ?

TCA ?

LB Broth Media Recipe

1. Add the following to 800ml H2O
 10g Bacto-tryptone.
 5g yeast extract.
 10g NaCl.
2. Adjust pH to 7.5 with NaOH.
3. Adjust volume to 1L with dH2O
4. Sterilize by autoclaving
LB Agar Recipe
1. Add the following to 800ml H2O
  10g Bacto-tryptone
 5g yeast extract
 10g NaCl
2. Adjust pH to 7.5 with NaOH
3. Add 15g agar
4. Melt agar into solution in the microwave
5. Adjust volume to 1L with dH2O
6. Sterilize by autoclaving
Don't forget to put a stir bar inside the bottle before autoclaving when
making LB agar.

We culture Vt in LB with 1% sodium chloride (plates or broth). E.coli

grows better in LB only (no NaCl), but both Vt and E.coli can be grown
LB and 1% LB solid (plate) or liquid (broth). To prevent contamination
you can add, as we do here, Ampicilin for Vt and Strep for E.coli (plates
and liquid).