Complementation of menC- KO E. coli

menC (the gene that encodes OSBS) has been replaced by a Kanamycin
resistance (KanR) gene, so it cannot synthesize menaquinone

menaquinone is required for facultative respiration under anaerobic conditions
when an appropriate electron acceptor is supplied (we use trimethylamine Noxide)

If you are expressing the protein from a pET vector or any other vector with a T7
promoter, use the menC- (DE3) strain. The gene for T7 RNA polymerase has
been integrated into the genome in this strain.

Wear gloves; wash with 70% EtOH and/or change whenever you think you have
splashed your hands.
1. Transform plasmids into menC- cells
empty vector (negative control), WT (positive control), and all samples
2. start 1 ml o/n cultures with single colonies in LB + antibiotics (Kan for the cells, Carb
(or whatever is required) for plasmids)
3. Make a 1:20 dilution of each saturated o/n culture in liquid menC minimal media and
place on ice.
4. Prepare menC media with antibiotic. Add enough menC minimal media to a flask for
all samples and add the appropriate antibiotics. (13mm screw top tubes hold ~ 9?
ml). KEEP THE FLASK COVERED WITH FOIL UNLESS YOU ARE PIPETTING
FROM IT.
5. Aliquot menC minimal media + antibiotics into each tube. Fill to a little below the
neck. You do not need to be exact because you will fill it to the top later. Use a 25 ml
pipet to minimize the number of times you have to refill. KEEP THE CAP LOOSELY
ON THE TUBE UNLESS YOU ARE FILLING IT.
6. Inoculate with 2 l of the dilution from #3. (See below for setting up quantitative
growth curves). Inoculate each tube and replace cap loosely. If you splash or drip
media, immediately clean the sides of the tube and your gloves with 70% EtOH.
Change gloves if necessary.
7. Filling and sealing tubes:
a. Do one tube at a time. Do in this order:
i. mutant 1 (the one you think is likely to grow slowest)
ii. mutant 2
.
.
.
n-2. last mutant (the one you think is likely to grow best)
n-1. WT (positive control)
n. empty vector (negative control)
b. Use a 1 ml micropipette to fill the tube to the top with menC minimal media. I
fill it up until it forms a dome at the top.
c. Screw the cap on all the way.
d. Rinse the outside of the tube and your gloves with 70% EtOH. Dry with paper
towel or Kimwipe.
e. Seal with a strip of parafilm. Invert to make sure there are no bubbles.
f. Wash your gloves with 70% EtOH and dry with a clean paper towel.
g. Repeat for each sample. Do the negative control last.
8. Place in 37o C incubator (NOT the shaker. You don’t need to aerate these cultures).
9. Check OD at 600 nm. Put the single cuvette holder in the spectrophotometer (tubes
will not be stable in the multicuvette holder). Blank with MenC minimal media (I
usually just pour some into a clean tube).
Cells will settle to the bottom, so I invert the tube a few times and wipe it off
before reading the OD. You will notice that there is usually a bubble after a day
or so in cultures that grow, even if there wasn’t one when you set up the culture.
10. Verify that cell growth is dependent on the plasmid.
Do a mini-prep of cultures that grow and sequence the insert.
(In some cases, we may also want to check protein expression levels using the
mini-purification protocol or a Western Blot.)
Non-quantitative complementation:
If you just need to determine whether there is growth or no growth, inoculate as
discussed above and check OD about 1X per day for several days.
Quantitative complementation:


To determine relative levels of activity between mutants and WT protein,
you need to do a quantitative growth curve. For publication, do them in
triplicate.
To be quantitative, you need to start each culture with approximately the
same number of cells.
1. Check the OD of each 1:20 dilution from #3 above. (OD should be between
0.1 - 1 to be accurate)
2. Normalize the inoculation volume for each sample so that you add the same
number of cells to each culture.
3. Keep the 1:20 dilutions on ice while you set up the cultures so the OD doesn’t
change as you set up the cultures. Put each anaerobic culture on ice after you
seal it.
4. If you start the culture with the right amount of cells at the right time of day, the
density will not be high enough to read until morning. If you time it wrong, you’ll
have to measure ODs overnight.
5. Measure ODs every 2-4 hours (or more ore less often, as needed).
6. Graph your results and calculate the doubling time during log phase.