Still planning the mutagenesis project

OK, I'm abandoning the old mutagenized stocks and starting the mutagenesis project from scratch.

I want to take advantage of having selectable alleles linked to each of the genes where mutations produce hypercompetence.  That's 1) the StrR point mutation linked to sxy (about 50 kb; 50% cotransduction), 2) the CatR cassette linked to murE (about 4 kb; 90% cotransduction?), and the CatR cassette tightly linked to rpoD (~100 bp; > 90% cotransduction).

Last time I tried to do this by doing the EMS-mutagenesis in vitro (See blog posts: here and here). Call this strategy C.   I directly mixed EMS with DNA of three strains carrying the above selectable markers and wildtype alleles of the hypercompetence genes, and then transformed the mutagenized DNAs into competent wildtype cells, selecting for each of the marker strR and CatR alleles.  But this failed.  I didn't get any low-level novobiocin resistance mutations that would have indicted that the EMS caused mutations, and I didn't get any hypercompetent mutants.  I suspect that transforming cells with EMS-damaged DNA is a very inefficient way to create mutations.

This time I'm going back to doing the mutagenesis in vivo (call this strategy B), incubating the three marked strains with EMS and allowing them to grow for 1-2 hr to convert the damage into mutations.  Then I'll isolate the DNA from each mutagenized culture, and use this to transform wildtype cells to the three markers.  This will give me pools of cells that have experienced a high mutation rate in the neighbourhood of sxy, murE and rpoD.

But before I do this I need to do the math, to see if this is really any better than just mutagenizing wildtype cells and screening them all for hypercompetence (call this strategy A).  Having the linked markers will certainly be handy later, once I've found hypercompetence mutants and want to find out which gene they're in.  But will using them as described above really let me find more mutations in these specific genes?

OK, I've laid out a situation with realistic numbers, and I've run it by the PhD student and the summer student.  Bottom line:  Strategy B does not enrich for mutations in the desired region.  It's only strength is that it allows use of much higher concentrations of EMS than would be tolerated in Strategy A.

Here's the numbers analysis for strategy A:

1. Start with 10^9 cells.  (Below I'll consider whether fewer would be OK.)
2. Treat with EMS (0.08M for 30 min).  Previous work suggests that this creates about 1 mutation per surviving cell.
3. Grow cells for about 3 hr or more, keeping cell density below OD600= 0.2.  The goal is to allow enough time for the cells to recover from the DNA damage, undergo two rounds of DNA replication to convert some damage into G->A mutations, and express the mutant hypercompetent phenotype.  Assume that the cell numbers increase 10-fiold during this period.
4. How many mutations will we have?  The genome has about 4 x 10^5 Gs.  Mutations at some of these will be lethal or sub-lethal, so assume about 2 x 10^5 positions where mutants have normal or near-normal growth.  With 10^10 cells, we will have about 5 x 10^4 occurrences of each mutation, on average.
5. How many hypercompetence mtuations will we have? 10 of these are positions we have already found to be sites of hypercompetence mutations, so in our 10^10 cells we'll have at elast 5 x 10^5 hypercompetent cells.
6. To select for these hypercompetent cells, transform the OD600=0.2 cell population with the 8 kb NovR DNA fragment from plasmid pRRnov1.  This DNA transforms much better than the equivalent PCR fragment or than chromosomal DNA with the equivalent mutation.
7. The normal cells will transform at a frequency of about 10^-8 - 10^-7.  (I'm guessing here; with chromosomal DNA it's ~10^-9.) That would give about 100-1000 novR colonies from the 10^10 cells.
8. The hypercompetent cells will transform at higher frequencies:  rpoD: cells with the known rpoD mutations will probably have a TF of 10^-4 - 10^-3, so the 5 x 10^4 cells of each mutation would give about 5-50 novR colonies.  sxy:  cells with the known sxy mutations will probably have a TF of 10^-3 - 10^-2, so the 5 x 10^4 cells of each mutation would give about 50-500 novR colonies.  murE:  cells with the known murE mutations will probably have a TF of 10^-2 - 10^-1, so the 5 x 10^4 cells of each mutation would give about 500-5000 novR colonies.   
9. According to this analysis, most of the colonies will be hypercompetent mutantx, and most of the hypercompetence mutants we find will probably have their mutations in murE. Of coulrse this only considers the hypercompetence mutations we already know about.

Things to do first:  

1. Check that our EMS stock is still good, by mutagenizing wildtype cells and scoring low-level novobiocin resistance.  A resistance frequency of ~5 x 10^-6 is approximately one mutation per viable cell.
2. Make a big prep of pRRnov1.  The yield of this plasmid is often poor so this may take several attempts and large volumes.  Check the transformation frequencies and efficiencies this DNA gives.

Are the old mutagenized cells worth using?

Last month I wrote a post about an old experiment (What can I recover from an old failed experiment).  I concluded that some of the frozen mutagenized cells might be worth using, but that I would first need to test how effectively they had been mutagenized.  Now it's time to do that.

The cells were incubated with the mutagen ethane methylsulfonate (EMS), which alkylates G bases and causes G->A transition mutations.

I can test the efficiency of this mutagenesis by plating cells on novobiocin agar plates, using a lower-than-normal novobiocin concentration (1 µg/ml rather than 2.5 µg/ml).  I also need to test the overall viavbility of the frozen cells, by plating them on plain agar plates, but I'll need to do this anyway to test the mutagenesis. 

I have two tubes of each of six cultures (three strains at two EMS concentrations).  Each was grown for 90 min after the EMS treatment, to allow DNA replication to convert the DNA damage to mutations:

• B and C: KW20 (wildtype), 0.05 mM and 0.8 mM EMS
• D and E: RR514 (StrR, linked to sxy), 0.05 mM and 0.8 mM EMS
• F and G: (RR805 (CmR, linked to murE), 0.05 mM and 0.8 mM EMS

I'll thaw and test one tube of B and one of C; these should be representative of the others.

Rather than just discarding the remaining cells I thawed, I could at the same time put these cells through the next step, by growing them at low density and transforming them with either novR chromosomal DNA or a novR PCR fragment (better because higher transformation efficiency).  But I won't do this, because first I need to find the novR PCR fragment and test it.

I just checked my old notes.  In expt #1290 I had tested the PCR products (novR and nalR) and found that they gave only slightly higher transformation frequencies than MAP7 chromosomal DNA.  This is surprising, since the effective concentration of the resistance-conferring fragments is much higher in the PCR DNA prep (maybe 10-50-fold higher, depending on the concentration of the PCR DNA, which wasn't measured.  The MAP7 DNA was used at a concentration that's saturating for transformation.

BUT, on more carefully re-reading my old notes (as usual not as limpidly clear as I would desire), I can't be sure that the cells I have saved are the cells I need (they might instead be cells that have already been incubated with the wrong DNA).  I think I'd better abandon this mess and start fresh.

Wildtype strain weirdness

While looking over some of the RNA-seq analyses done by our summer student (former undergrad, someday grad student somewhere), I noticed something unexpected.

We know that cultures in the rich medium sBHI become moderately competent when they reach high density.  Their transformation frequencies reach about 10^-5 - 10^-4, which is 100-fold lower than fully induced cultures but 1000-fold higher than log phase cultures.  Consistent with this, in the microarray analyses we did about 12 years ago we saw modest induction of all the competence genes except ssb in this condition (we didn't publish the data but described it as 4-20-fold induction).

So I expected to see similar induction in in rich medium in the RNA-seq data.   But there's no consistent induction at all; most genes don't show any change at all, and a few go up or down a bit.  You can look at the complete data here, but unfortunately the individual graphs are very low resolution.  Here's a blowup of one pair of graphs, for comE, which encodes the secretin pore:

On the left are cells in the competence-inducing medium MIV.  We see very strong induction of comE in wildtype (KW20) cells (brown dots and line), and no induction when the Sxy regulator is knocked out (blue dots and line).  On the right are cells in rich medium.  Here we see strong induction in the presence of the sxy1 hypercompetence mutation (blue dots and line) but no induction at all in the wildtype KW20 cells.

So the summer student did more analyses.  First he did more work with the RNA-seq data:

1.  The baseline (log phase) levels of expression of each competence gene are the same in cultures grown for the rich-medium experiments and cultures grown for the MIV-induction experiments rich medium.  This suggests that there was nothing very wrong with the rich medium cultures.  (The rich medium experiments took their log phase samples from very dilute cultures (OD600 = 0.02), and the MIV-induction ones took theirs at OD600 = 0.2.  Later we plan to use this to test whether both densities are genuine log phase, by seeing if this density difference changes expression of any genes at all.)

2.  The sxy gene is slightly induced as culture density increases, though we don't really know how much it should be induced.

3. The genes required for induction of the competence regulon are intact in the reads of the rich-medium cultures: sxy, crp, cya.  That means we didn't accidentally use a strain carrying a knockout of one of these genes instead of wildtype cells.

4.  To check whether the supposedly wildtype strain might have a knockout of another gene, he looked for reads derived from antibiotic-resistance cassettes.  He found a few reads of a spectinomycin cassette (like the one we have used for many of our knockouts), but the numbers were so small they're probably just contaminants.

Then he managed to do comparisons between the RNA-seq data and the old microarray data:

The blue-line graph shows the microarray data.  The Y-axis is fold-change in expression of each competence gene, and the X-axis is time relative to when a separate sample was removed for competence induction.  The red-line graph shows the RNA-seq data, squished to make the spans of its axes roughly consistent with those of the other graph.  The Y-axis is again fold-change in expression, but now the X-axis is the density of the culture, measured as OD600.  

In the microarray data, some genes aren't induced at all but others are induced as much as 11-fold.  In the RNA-seq data, only one gene is induced even 2-fold, and many are down-regulated.  So we definitely do have a problem.

So then it became my turn to do some experiments to figure out what's going on.  Fortunately I had saved frozen samples of the cells used for every RNA sample we analyzed by RNA-seq.  

First I thawed out and transformed the three samples of supposedly wildtype cells at OD600 = 1.0. Their transformation frequencies were all a lot lower than they should have been ('FK': 3.6 x 10^-7; 'GK': 2.5 x 10^-7; 'HK': 7.6 x 1-^-7, rather than about 10^-5).  That's consistent with a genuine lack of induction of their competence genes (and not with my alternate hypothesis, that there was just some error in the analysis of this RNA-seq data).

I streaked the cells on spectinomycin plates, to check if they had an unexpected spcR cassette.  None grew, and they all grew on the control plates.  So we didn't use any of our spc-cassette knockout mutants by mistake.

Finally I inoculated two of the supposedly wildtype strains ('GK', from a plain-plate colony of the above transformation test, and 'FK', from its OD600 = 0.02 frozen sample) into rich medium, along with a wildtype control strain, and tested competence development under three conditions.  The left columns show the transformation frequency seen 60 min after adding 1mM cAMP to a log-phase sBHI culture - I did these in case we didn't get normal transformation in the other tests, since this would tell us if the strains were somehow unable to produce cAMP (e.g. if they had a phosphotransferase mutation).  The middle columns are cells at high density in sBHI; both of the suspect strains have near-normal transformation frequencies.  The right columns are cells transferred to the competence-induction medium MIV; one of the suspect strains has normal transformation and the other is down 10-fold.

(I must admit that I don't have high confidence in the numbers from this experiment, for several reasons.  First, the colony sizes and counts were very erratic (inconsistent from one dilution or plating volume to another), perhaps because I used mostly old novobiocin plates left by our now-gone Co-op tech. Second, the 'latelog' samples were allowed to grow longer than I intended, so their competence may have been decreasing, and this problem was slightly worse for the GK and FK cultures.  Third, I was testing a new competence protocol (see p.s. below).)

So what have we learned?  Mainly that there's nothing obviously wrong with the 'wildtype' cells used for the sBHI RNA-seq samples.

So what should we do next?  I don't know.  Maybe I should repeat the competence-induction tests with fresh plates, to get better numbers.

p.s.  The KW20 MIV transformation frequency is slightly lower than I usually see, probably because I was testing a new scaled-down protocol that doesn't use an expensive disposable filter funnel to collect and wash the cells.  Our usual protocol is to collect and wash 10 ml of cells using a Nalgene disposable filter funnel (0.2 µ size, designed for water sampling), and then resuspend them in 10 ml of MIV in a flask shaking in the waterbath for 100 min. But my new attention to economy has revealed that each funnel new costs nearly $7.  So this time I just pelleted 2 ml of cells in a microfuge tube, resuspended the cells in 1 ml MIV, pelleted them again, and resuspended them in 2 ml of MIV in a large glass culture tube, which I incubated on the roller wheel in our air incubator.  The transformation frequencies for KW 20 and GK are plenty high.  The lower transformation of the green 'FK' sample may be because I ran out of MIV and skipped its washing step.

Recommended Twitter accounts for neuroscience grad students to follow

Next week I'm running a small science-communication workshop for some neuroscience grad students from developing countries.  I don't know much about neuroscience, so yesterday I asked the Twitterverse to recommend accounts that neuroscience grad students should follow.

Here's the list (in no particular order because I couldn't persuade Word to 'sort' it):

@bradleyvoytek

@betenoire1

@pollyp1

@MayBrittMoser

@debivort

@Neuro_Skeptic

@GaryMarcus

@SfNtweets

@artcollisions also

@Dr_KarenLee

@SteveKerfoot

@KittyM79

@mripathology

@guthyjacksonfdn

@superkash

@CousinAmygdala

@neurobongo

@neuronJoy

@JohnBorghi

@nickwan

@AlquierThierry

@emilysinger

@BSDneuro

@jillumine

@neuromusic

@prerana123

@DennisEckmeier

@manes

@BenSaunders

@drugmonkeyblog

@nucAmbiguous

@doc_becca

@markgbaxter

@tollkuhn

@sheacshl

@schoppik

@neuroecology

@thefreemanlab

@Bashir9ist

@andpru

@jessicapolka

@BiophysicalFrog

@FORsymp

@EEGesus

@CSHLaboratory

@Parklifensci

@leon_summer

@bakermind

@K_dele

@SciTriGrrl

@sumscience

@Namnezia

@brembs

@DoctorZen

@Neuro_Skeptic

@MHendr1cks

@tatterededge
@Dr_KarenLee

Finally the final RNA-seq steps

Yesterday I did the Ribo-Zero treatment of the 24 samples for our final RNA-seq analysis.  It was a bit complicated to plan because I was using a kit designed for only 6 samples (because the kits are so expensive).  Luckily we had leftover reagents from the 72 samples we treated last summer, which let me set up reactions that were 3/4 of the standard volume.

 The kit removes the abundant ribosomal RNA from the sample, leaving only the desired mRNA and some small RNAs for sequencing.  It works by first annealing tagged oligos to the rRNA, and then removing the rRNAs with magnetic beads that bind to the oligo tags (probably biotin?).  We had quite a lot of the buffer and oligo solutions left from last time, but no beads.  A mixup had led to the beads in the new 6-sample kit being ruined by being stored at -80 °C rather than 4 °C, but Illumina very kindly provided us with replacement beads. 

The replacement beads came with a new tube of the solution used to resuspend the beads after their preliminary washing, so I was able to resuspend the beads in 3/4 of the volume needed for 24 samples rather than 1/4.  Using the larger volume was important, because the separation step depends on the right relationship between the liquid in the tube and its position in a special rack with magnets on one side.

The final step is to ethanol precipitate the depleted RNAs and resuspend them in a special buffer for the first library-prep step.  The former RA is going to do the library prep and sequencing in her new lab (she also provided the Ribo-Aero kit), and she's given us the buffer for the resuspension.  Right now the 24 ethanol precipitations are in the -20 °C freezer; this morning I'll spin them down (30 min, because the concentrations are so low), wash tyhe pellets carefully with 70% ethanol, dry them carefully, resuspend them, and deliver them to her.

Then I'll give a big sigh of relief and move on to other projects.

Today's work on the RNA-seq samples

The Co-op tech has pressed half the samples through the RNeasy kit spin columns, and will probably get the rest done today.

But she also is still working on the PCR checks (problems getting amplification from the colony-DNA material) and redoing some of the transformation assays (using the frozen cells I'd saved for each sample) because two of her original tests gave surprisingly low transformation frequencies.

I'm hoping we can also get started on the next steps today - checking the RNA concentrations using the Nanodrop and running aliquots in a gel to check that the rRNA bands are intact.  First step for this is to clean up a gel box and comb and see if we have any RNA-loading dye made up.

Other tasks for the soon-to-be-departing Co-op tech

The Co-op tech will be leaving us at the end of the month.  Last week she gave an excellent bab meeting presentation, and this revealed a couple of loose ends and interesting possibilities that should be cleared up before she leaves.

First, she and the sabbatical visitor (now back home in Regina) isolated a new mutation in rpoD that causes hypercompetence.  But this strain hasn't yet been added to our formal collection of frozen strains and the associated 'Strain List' database.  This is essential and urgent.

Second, the mutant hunt turned up a couple of other 'possibly hypercompetent' mutants whose phenotypes haven't been checked out.  These need to be checked in competence time courses, and added to the Strain List collection if they turn out to be genuinely hypercompetent.

A weird result that I'd forgotten about is the finding that cells transformed with a mixture of NovR and KanR DNA fragments (generated by PCR) showed a much LOWER cotransformation frequency than expected.  We suspect this reflects some chromosome-level interaction between these linked segments, but we'd first need to reproduce the result.

RNA-seq progress

I've collected and frozen all the 24 samples for our make-up RNA-seq run. (not the Trizol-prep ones - they've been deep-sixed).  And this morning the co-op tech learned to prep RNA from each sample. She's done the first 4, and will do the rest over the next couple of days.

The next steps are:

1. Complete the PCR tests of strain genotypes and the analysis of transformation frequency data. She's still working on the PCR tests, but so far everything looks OK.
2. Check the RNA concentration using the Nanodrop
3. Run aliquots of the samples in a gel to check integrity of the rRNA bands (surrogate for integrity of the mRNA).
4. Treat 5 µg of each sample with DNA-free.  We found our stock from last year, and there's still enough to treat all our samples.
5. The former RA says we can take the DNA-free-treated samples directly to the RiboXero ste; we don't need to first do a 'clean-up' step with the RNeasy Minelute kit spin-columns.
6. Treat an aliquot of each sample with RiboZero.  We will use only half as much RNA as recommended, and only 1/4 as much of the other reagents (in 1/4 of the recommended volume, of course). This will let us treat 24 samples with a 6-treatment RiboZero kit.
7. Give the samples to the former RA in her new lab for library preparation and sequencing.

What can I recover from an old failed experiment

About 18 months ago I did a big mutagenesis experiment, intending to isolate new hypercompetent mutations.  I made several mistakes and the experiment was a failure, but I did freeze stocks of intermediate cultures.   At the time I thought that some of these could be used in a future attempt, because they came from stages before the mistakes were made.

I still want to repeat this experiment, and I just found the stocks in the -80 °C freezer.  Now I need to decide which are potentially useful, and throw out the rest.  Here's photos of what I found:

  

The letters A-G refer to different strains, each with a wildtype version of a gene known to give rise to hypercompetence-causing mutations, and to different levels of mutagenesis

• A, B & C: wildtype cells, incubated in 0 (A), 0.05 (B) and 0.08 (C) M solutions of the mutagen EMS.
• D & E: strain RR514, which has a Streptomycin-resistance mutation (StrR) close to the wildtype sxy gene, incubated in 0.05 (D) and 0.08 (E) M solutions of EMS.
• F & G: strain RR805, which has a chloramphenicol cassette (CmR) inserted within a few kb of (= closely linked to) the wildtype murE gene, incubated in 0.05 (F) and 0.08 (G) M solutions of EMS.

The big tubes turn out to be useless, since they contain cells that were incubated with the wrong DNA after the mutagenesis.  Most of the small tubes also are from stages that have been incubated with DNA, (e.g. label 'F DNA'), but others (the ones labeled '90') were frozen after 90 min of post-mutagenesis growth, before the DNA addition step.  These ones I can use.

The first step now is to do a test I didn't do in the original experiment, to check that the EMS mutagenesis did indeed cause mutations by plate some of the cells on low-concentration novobiocin. I'll do this test on the wildtype cells (B & C), so not to unnecessarily use up the more valuable cells in the marked strains (D-F).  I don't have tubes of the control A culture, so I'll just use normal wildtype cells.

If this test shows that the mutagenesis worked, I have two alternatives.  1.  I could isolate DNA from the mutagenized marked cultures and use it to transform wildtype cells to StrR (D & E, to enrich for cells with sxy mutations) or CmR (F & G, to enrich for cells with murE mutations).  Then I'd enrich these transformants for hypercompetent mutants by transforming them with the PCR'd NovR fragment (after first testing that this works well).  2.  I could do the hypercompetence-selection transformation first, and then isolate DNA and transform wildtype cells with selection for the linked marker.  The advantage of 1 is that I can pool many thousands (millions?) of transformants, maintaining whatever genetic diversity my mutagenesis has created in the gene of interest.

New RNA-seq work

(OK, I just checked, RNA-seq should be hyphenated.)

I've made a couple of posts about plans for new RNA-seq work on the Sense Strand blog: http://sensestrand.blogspot.ca/2015/03/planning-more-rnaseq-runs.html and http://sensestrand.blogspot.ca/2015/04/back-to-planning-new-rna-seq-samples.html

Now it's time to get down to work.

Here's the planned samples.  We have 26 on the list, but a standard run will only be 24, so two need to be dropped.  Conveniently, the two KW20-in-Trizol samples might not be needed, depending on the available small-RNA data for H. influenzae, so I won't consider those right now.

For the rest, we have 6 mutant strains.  Given the mixups that have occurred so far, it would be prudent to check these every way we can.

We are checking antibiotic resistances and will transform each strain when we grow its culture and collect the samples for the RNA preps. One of the Honours Zoology undergrads has already checked the toxin and antitoxin strains by PCR (she's the one who suffered most from the mixup), and we'll use the same primers to check the cultures we're sampling from.  We need the former RA's help to find the primers for the ∆hfq mutant, but luckily its phenotype is quite distinctive - in our whole collection I can only think of one other mutant that has its competence down 10-fold.  RR753's phenotype is also distinctive, hypercompetent, but not very.  The crp and sxy mutants have the same phenotypes (same drug resistance, same complete lack of competence.  We have lots of sxy PCR primers, but we'll have to check to see which ones will work with this insertion mutant.  And, for both crp and sxy there's the additional problem that miniTn10 insertions do not amplify well because of their end repeats.  I wonder if we have an internal primer for miniTn10kan.

The co-op tech has checked antibiotic resistances and frozen fresh stocks of all the strains.  She's inoculated the 4 strains for the MIV-competence preps, and tomorrow we'll toy to collect all those samples.  (No, I haven't done anything in preparation yet.)