If I have lots of KanR colonies from yesterday's hypercompetence-enrichment step...

Today:
  1. Pool the colonies (from each of 6 original mutagenized cultures).
  2. Freeze part.
  3. Make chromosomal DNA preps from the rest.  These will be used for sequencing and for the backcrosses (steps 5 & 6)
  4. As controls also make DNA from the pooled NovR colonies from yesterday.
  5. Use the 2 DNAs from the StrR parent and the 2 DNAs from the CmR parent to transform KW20 to StrR and to CmR.
  6. Maybe use the DNAs from the wildtype parent to transform KW20 to KanR.
  7. (Incidentals: make lots more BHI agar, pour plates, use frozen competent KW20, test transformation by NalR fragment, wash and autoclave flasks again.)
Tomorrow (one final round of hypercompetence selection):
  1. Pool the backcross colonies derived from each of 6 original mutagenized cultures.
  2. Freeze part.
  3. Grow part in log phase for at least 2 hr (like yesterday)
  4. Transform with a different marker (NalR fragment, or MAP7 DNA with selection for NalR or SpcR).
Monday:
  1. Pool the transformant colonies (from each of 6 original mutagenized cultures).
  2. Make chromosomal DNA preps from them for sequencing.

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Yet more on-the-fly experiment planning

Hm, yesterday's attempt to do two rounds of enrichment for hypercompetent mutants yielded very few colonies (about 10 total).  That's probably because I didn't let the NovR cells grow to a high enough density before transforming them with KanR DNA.  There were so few cells present with the KanR DNA was added, 5 x 10^5/ml - 6 x 10^6/ml based on the numbers of colonies that grew on the plain plates after the second round transformation).

But I can do the second round enrichment again, by pooling all the NovR colonies that grew on these plain plates.  I have between 1000 and 60,000 colonies to pool, depending on the culture, and the cultures were diluted so much that I don't have to worry that any of these colonies might be KanR. And the colonies are all NovR transformants from the previous round, though many of them are identical descendants of the original mutants.

Plan:  Put some BHI on the plates and scrape them to resuspend all the colonies.  Dilute the pooled cells in sBHI to OD600 = 0.1, then dilute 50-fold in more sBHI.  Grow (37°C, shaking) for 2-3 hr, until OD600 reaches 0.05.  Add KanR chromosomal DNA and let continue growing for another hr.  Dilute and plate on plain, Nov and Kan plates.

Progress:  OK, I've counted the colonies (previously had just estimated them), pooled them (excluding one contaminated plate), and now they're growing in 10 ml sBHI at a starting OD600 of 0.002.  Now I need to pour lots of plates.

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AAACCCKKK!!! - I screwed up the selection

I did Day 1 of the big experiment yesterday.  But because the NovR DNA fragment the postdoc gave m hadn't transformed very well in the test, and I didn't have enough of it for the large volumes I was transforming, I used MAP7 DNA for the transformations that select for hypercompetent mutants.  At the time I thought the only disadvantages were a slightly lower overall transformation frequency and the 1-2% risk (for each new mutant) that transformation would also replace the mutation itself.

I forgot that, because the MAP7 DNA carries resistance to all the antibiotics we commonly select for, the pool of NovR cells I'd get would contain 1-2% cells resistant to teach of the other antibiotics.  This means that I can't just reselect the cells in the NovR pool for hypercompetence using DNA with another mutation, because this will just select those cells in the pool that got that mutation in the first round of selection for hypercompetence.

I see several options:

Option 1:  Yesterday I froze lots of mutagenized cells that hadn't yet been incubated with any DNA.  I can thaw these out, grow them for an hour at low density, and transform them to NovR with either the NovR DNA fragment or chromosomal NovR DNA that doesn't carry any other antibiotic resistances.  (I may have an old stock of this DNA.)   Then I can grow the pooled cells in medium with novobiocin for 6 hr, dilute them, and do the second-round transformation (NalR or KanR DNA)) and plate.  If I'm using the NovR fragment I may need to do this transformation on cells that have been concentrated - this will increase background, but the next round of hypercompetence-selection should take care of this.

Option 2 (BAD): I can plate the NovR transformants and manually check single colonies for resistance to the antibiotic I want to use next, by toothpicking them onto other antibiotic plates.  Then I could either test them individually for hypercompetence or pool the ones that are sensitive to whatever antibiotic I plan to use next.  Testing them individually would make the planned pool-sequencing unnecessary, and pooling them would be a lot of work.  But I might as well freeze my pools of NovR cells for later manual screening, just in case we  want to do this.

Option 3 (BAD):  I can make DNA from the pooled NovR colonies from the strain carrying the StrR mutation linked to sxy and the strain carrying the CmR mutation lnked to murE, and use this DNA to transform wildtype cells to StrR and CmR. Then I can do another round of hypercompetence selection, transforming the pooled cells with a marker that's not linked to the one I selected for.  BUT, these new pools will probably (maybe) include cells with the unselected antibiotic resistances from the MAP7 DNA.

 Expanded plans for Option 1: 
  1. Thaw 1 vial of each of the 7 cultures (wildtype treated with 0, 0.05 and 0.08 mM EMS, StrR and CmR treated with 0.05 and 0.08 mM EMS).  
  2. Dilute way down in sBHI, to a density equivalaent to that used yesterday.  Grow 1 hr or more at low density.  
  3. Look for NovR chromosomal DNA.  If using the PCR fragment, concentrate the cells by filtration just before adding the DNA.
  4. Incubate cells with DNA for 15 or 30 min, then DNase-I-treat, filter, wash, and resuspend in sBHI + Novobiocin.  
  5. Grow 6 hr or more, keeping OD600 below 0.1.  
  6. Transform a fraction of this culture (no benefit from using it all).  Add KanR chromosomal DNA or NalR PCR fragment.  Incubate 15 min, DNase-treat and plate (if KanR) or grow for 90 min before plating (if NalR).
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Timing for the big mutagenesis experiment

In the previous planning post I ended with the following breakdown:
Day -1: Streak out the cells.  (3 strains)
Day 0:  Inoculate single colonies overnight.  (3 strains)
Day 1: Dilute and grow, mutagenize, wash, dilute and grow, 
            freeze, dilute and grow, transform with NovR, wash, 
            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)
Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))
Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)
Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)
Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)
- See more at: http://rrresearch.fieldofscience.com/#sthash.0AuGwsEC.dpuf

Day -1: Streak out the cells.  (3 strains)
Day 0:  Inoculate single colonies overnight.  (3 strains)
Day 1: Dilute and grow, mutagenize, wash, dilute and grow,
            freeze, dilute and grow, transform with NovR, wash,
            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)
Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))
Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)
Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)
Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)

 I'd like to get the whole thing one as quickly as possible, because next week may be our last chance until next April to get the DNAs in for sequencing.

I have the EMS, and I've streaked out the strains, but I didn't inoculate single colonies last night.  Could I still do the big Day 1 mutagenesis, transformation and selection today, or will there not be enough time to have the cells grow up first from single colonies?

What's the actual time commitment for day 1?
  1. Inoculate cells from single colonies into sBHI, grow to OD600 = 0.1.  (time = several hr)
  2. Add equal volume of sBHI containing 2X the desired concentrations of EMS.  Incubate 30 min at 37°C.  Cool cells down quickly. (time = 30 min)
  3. Filter-wash cultures (7 cultures) and resuspend in larger volumes of sBHI. (time = 30 min?)
  4. Grow washed cells for 90 min or until OD is back to 0.1. (time = 90 min)
  5. Dilute part of each culture and grow for 1 hr longer. (time = 1 hr)
  6. While cells are growing, filter-concentrate and freeze the rest of the cultures.
  7. Add NovR DNA fragment to cultures. Incubate 30 min, then DNase I for 5 min. (time = 40 min)
  8. Filter cultures, resuspend in sBHI + novobiocin. (time = 30 min)
  9. Grow (at least 6 hr or) overnight.
  10. The next morning, plate some cells on Nov5 plates, freeze some, and grow some back into log phase for KanR transformation with MAP7 DNA.
OK, I think I can do steps 1-8 today.

How much EMS to use:

In #181 I calculated that adding 79.5 µl of EMS to 15 ml culture gave 0.05 mM, and 127.3 µl gave 0.08 mM.  So for my 10 ml of cells I would use 53 µl and 85 µl of EMS.

What about the volumes of culture to use?
  1. Mutagenize 10 ml at OD600 = 0.1
  2. Resuspend in 80 ml.
  3. Freeze 70 ml after 90 min (concentrate cells first) and dilute 10 ml to 40 ml in fresh sBHI
  4. Grow 1 hr, add DNA etc, filter.
  5. Freeze half the cells.
  6. Resuspend the rest in 100 ml + novobiocin2.5.  Grow 6 hr - overnight
Should I mutagenize a larger volume?  10 ml will be only about 3 x 10^9 cells.  In #181 (the original EMS experiment) I used 15 ml at OD600 of 0.33, which is about 5 times as many cells.

How much EMS do I have?  OK, 5 ml (after brief "Where on earth did I put it???").  It doesn't keep well once opened ("Store under inert gas") so maybe I should double the volumes for the first two steps and freeze more of the mutagenized cells for possible later analysis.  So use 106 µl and 170 µl EMS for 20ml cultures, and dilute to 160 ml.

What about the EMS-contaminated waste culture and tips?

EMS is inactivated in 1.0 M NaOH.  My filter flask will contain about 400 ml of EMS waste (including washes), so I'll add 16 g of solid NaOH to that, and let it sit for an hour before neutralization and disposal.  I'll put the contaminated tips in there too.





Day -1: Streak out the cells.  (3 strains)
Day 0:  Inoculate single colonies overnight.  (3 strains)
Day 1: Dilute and grow, mutagenize, wash, dilute and grow, 
            freeze, dilute and grow, transform with NovR, wash, 
            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)
Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))
Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)
Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)
Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)
- See more at: http://rrresearch.fieldofscience.com/#sthash.dxAGHC0t.dpuf
Day -1: Streak out the cells.  (3 strains)
Day 0:  Inoculate single colonies overnight.  (3 strains)
Day 1: Dilute and grow, mutagenize, wash, dilute and grow, 
            freeze, dilute and grow, transform with NovR, wash, 
            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)
Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))
Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)
Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)
Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)
- See more at: http://rrresearch.fieldofscience.com/#sthash.0AuGwsEC.dpuf
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