Both my gels gave OK results. But I didn't really use enough DNA in the conventional gel, and the CHEF gel ran faster (or longer) than would have been ideal. But the conclusion is that both DNA preps consist almost entirely of fragments bigger than 40kb and smaller than 150kb. This is fine for my initial experiments.
The next step is to tag the ends of the DNA molecules with biotin. At most ends the two strands are likely to be unevenly broken (i.e. either the 5' end or the 3' end overhanging), so I can use Klenow polymerase to fill in the ends that have 3' overhangs. I'll use a biotinylated deoxynucleotide in place of done of the normal nucleotides (e.g. biotin-dUTP), so those ends whose 3' overhangs include an A will have a biotin. Ends lacking an A won't be labeled, nor will blunt ends and those with 5' overhangs. But this shouldn't be a problem as the presence of fragments with one or two unlabeled ends won't interfere with attachment of the biotin fragments to the streptavidin beads. I expect 3' overhangs and 5' overhangs to be equally common, and both to bed much more common than blunt ends. H. influenzae DNA us rich in As and Ts so, if all goes well, 30-50% of the ends will be labeled.
I can't do this until we get some biotin-dUTP; I'll order it first thing Monday morning.
To check whether the labeling reaction works, I'll do a test reaction that also includes a small amount of a radioactive nucleotide; we have some in the fridge that should still be good as it is labeled with 33-P which has a 30-day half-life. I'll both check for incorporation of 33-P into the DNA directly, and run the DNA in a gel and do an autoradiogram (expose it to a radiation-detecting screen) to check that most of the label is in big fragments. This is important because, although small fragments are less conspicuous in gels (longer fragment shave more DNA in them so they fluorescence brighter). their ends label just as well as those of long fragments fragments.
On Monday I'll also order the streptavidin-coated polystyrene beads needed for the next step.
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Running gels!
I've got two gels running while I type. Both will help me decide if the MAP7 DNA preps we have are suitable for my tweezers experiments. Both gels contain high and low concentrations of two different MAP7 DNA preps, along with a size standard consisting of intact phage lambda DNA (48.5kb) and a HindIII digest of the same DNA.
The first gel is a conventional agarose gel - the voltage is created by a pair of simple wire electrodes, one running across each end of the gel box. To increase the resolution (i.e. separation) of DNA fragments bigger than 15-20kb, the gel has a lower concentration of agarose than is usually used (0.6% rather then 0.8-1.0%). This makes it more fragile, so I'll need to handle it very carefully tomorrow when I'm photographing it. I'm also using a much lower voltage, which will also help spread out the big fragments and squeeze together the little ones I don't need to resolve.
The second gel is a "CHEF" pulsed field gel. This uses pulsing electric fields in different directions (120 degrees to each other) to jiggle the DNA fragments back and forth as they move through the gel. This forward-left then forward-right pushing has little effect on the separation of small fragments (less than about 20kb) but it greatly improves the separation of the big fragments. The new apparatus (belonging to the new lab next door) is much more sophisticated than the old one that's collecting dust on our top shelf, and I had to call technical support to learn how to dumb it down enough that I could control what it was doing.
In admiring this new apparatus we developed a new rule of thumb relating the cost of a piece of scientific equipment to the number of buttons it has. This one has more than 50 buttons, and cost about $40,000; our old one had about 7 buttons and cost $4500. This only applies to equipment that doesn't come with its own computer. The real-time PCR machine we share with other labs cost $70,000; it gets away with having only a single button because it's controlled by very complicated software.
The first gel is a conventional agarose gel - the voltage is created by a pair of simple wire electrodes, one running across each end of the gel box. To increase the resolution (i.e. separation) of DNA fragments bigger than 15-20kb, the gel has a lower concentration of agarose than is usually used (0.6% rather then 0.8-1.0%). This makes it more fragile, so I'll need to handle it very carefully tomorrow when I'm photographing it. I'm also using a much lower voltage, which will also help spread out the big fragments and squeeze together the little ones I don't need to resolve.
The second gel is a "CHEF" pulsed field gel. This uses pulsing electric fields in different directions (120 degrees to each other) to jiggle the DNA fragments back and forth as they move through the gel. This forward-left then forward-right pushing has little effect on the separation of small fragments (less than about 20kb) but it greatly improves the separation of the big fragments. The new apparatus (belonging to the new lab next door) is much more sophisticated than the old one that's collecting dust on our top shelf, and I had to call technical support to learn how to dumb it down enough that I could control what it was doing.
In admiring this new apparatus we developed a new rule of thumb relating the cost of a piece of scientific equipment to the number of buttons it has. This one has more than 50 buttons, and cost about $40,000; our old one had about 7 buttons and cost $4500. This only applies to equipment that doesn't come with its own computer. The real-time PCR machine we share with other labs cost $70,000; it gets away with having only a single button because it's controlled by very complicated software.
Not beads-on-a-string but strings of DNA on a bead
The other project I'm taking on is the controls and preparation for our laser tweezers analysis of DNA uptake. Our previous work (really the physics grad student's work) was trying to get cells to attach to and take up a specific bead-attached DNA fragment with a single USS. If this had worked we hoped it would let us study how DNA uptake depends on the orientation of the USS. We now realize that this was too ambitious an initial goal.
Our new experimental goals are to study the forces that act on the DNA during uptake, in wildtype cells and in cells with mutations in specific uptake proteins. This doesn't require use of a defined DNA molecule with only one USS, so we can change the setup to optimize our chances of success.
We also belatedly realized that we can use Bacillus subtilis as a positive control. Laser tweezers have already been used to study forces generated during B. subtilis DNA uptake. (Because the B. subtilis uptake mechanism is quite different than that of H. influenzae this serves as proof-of-concept for our experiments but doesn't make them redundant.) B. subtilis cells are larger and more robust than H. influenzae cells, and their DNA uptake does not depend on a particular sequence. Because the conditions for B. subtilis tweezer assays have already been worked out, we will make sure we can demonstrate uptake by it before moving on to uptake by H. influenzae.
The new plan is to first make sure we have lots of DNA fragments attached to each polystyrene bead, before trying to detect uptake by either B. subtilis or H. influenzae. My first step is to get a preparation of randomly broken H. influenzae DNA that consists mainly of fragments 50-100kb long, and then to attach these fragments to the beads by sticking biotin on the ends of the DNA, and using beads pre-coated with streptavidin, which binds tightly to biotin.
I've decided on 50-100kb for several reasons. First, it's hard to work with fragments longer than this, because longer fragments break unless they're handled very gently. Second, it's easy to get fragments about this size simply by not being gentle with the DNA prep. Third, this will let me attach a LOT of DNA to each bead. Fourth, the individual fragments will be substantially longer than the cells and the beads: the beads are 1 micrometer in diameter, the cells are 1-3 micrometers long, and a 60kb DNA fragment is about 20 micrometers. But they aren't so long that they extend a long way away from the bead they're attached to.
I'll use DNA from strain MAP7, partly because we already have lots of it (made by one of the post-docs) and partly because it carries antibiotic-resistance alleles that we can use to detect whether cells have taken it up. This will be important in checking whether the beads really do have DNA attached to them.
The DNA in the prep we have may already be broken into appropriately-sized fragments. I can check for small pieces by running this DNA in a normal agarose gel; fragments bigger than about 25kb will all jam up at the top of the gel, and only smaller fragments will spread out in the gel. A better check for DNA size will be to run the DNA in a pulsed-field agarose gel, whose rapidly reversing electrical current allows even very big fragments to spread out in the gel. Luckily our neighbours in the lab have a new apparatus to run these gels. (We have a 15-year old system which I suspect no longer works.)
If this analysis says the DNA fragments are too small, I'll make a fresh prep, handling it more gently. If the analysis shows that the DNA fragments are too big, I'll just rough up the DNA prep by whirling it in the vortex mixer, or by forcing it through a narrow syringe needle.
The next step will be adding the biotin to the ends....
(Hmm, the spell-checker thinks that tweezers must be plural; it doesn't approve of "laser tweezer analysis".)
Our new experimental goals are to study the forces that act on the DNA during uptake, in wildtype cells and in cells with mutations in specific uptake proteins. This doesn't require use of a defined DNA molecule with only one USS, so we can change the setup to optimize our chances of success.
We also belatedly realized that we can use Bacillus subtilis as a positive control. Laser tweezers have already been used to study forces generated during B. subtilis DNA uptake. (Because the B. subtilis uptake mechanism is quite different than that of H. influenzae this serves as proof-of-concept for our experiments but doesn't make them redundant.) B. subtilis cells are larger and more robust than H. influenzae cells, and their DNA uptake does not depend on a particular sequence. Because the conditions for B. subtilis tweezer assays have already been worked out, we will make sure we can demonstrate uptake by it before moving on to uptake by H. influenzae.
The new plan is to first make sure we have lots of DNA fragments attached to each polystyrene bead, before trying to detect uptake by either B. subtilis or H. influenzae. My first step is to get a preparation of randomly broken H. influenzae DNA that consists mainly of fragments 50-100kb long, and then to attach these fragments to the beads by sticking biotin on the ends of the DNA, and using beads pre-coated with streptavidin, which binds tightly to biotin.
I've decided on 50-100kb for several reasons. First, it's hard to work with fragments longer than this, because longer fragments break unless they're handled very gently. Second, it's easy to get fragments about this size simply by not being gentle with the DNA prep. Third, this will let me attach a LOT of DNA to each bead. Fourth, the individual fragments will be substantially longer than the cells and the beads: the beads are 1 micrometer in diameter, the cells are 1-3 micrometers long, and a 60kb DNA fragment is about 20 micrometers. But they aren't so long that they extend a long way away from the bead they're attached to.
I'll use DNA from strain MAP7, partly because we already have lots of it (made by one of the post-docs) and partly because it carries antibiotic-resistance alleles that we can use to detect whether cells have taken it up. This will be important in checking whether the beads really do have DNA attached to them.
The DNA in the prep we have may already be broken into appropriately-sized fragments. I can check for small pieces by running this DNA in a normal agarose gel; fragments bigger than about 25kb will all jam up at the top of the gel, and only smaller fragments will spread out in the gel. A better check for DNA size will be to run the DNA in a pulsed-field agarose gel, whose rapidly reversing electrical current allows even very big fragments to spread out in the gel. Luckily our neighbours in the lab have a new apparatus to run these gels. (We have a 15-year old system which I suspect no longer works.)
If this analysis says the DNA fragments are too small, I'll make a fresh prep, handling it more gently. If the analysis shows that the DNA fragments are too big, I'll just rough up the DNA prep by whirling it in the vortex mixer, or by forcing it through a narrow syringe needle.
The next step will be adding the biotin to the ends....
(Hmm, the spell-checker thinks that tweezers must be plural; it doesn't approve of "laser tweezer analysis".)
E. coli strain construction the old-fashioned way
I haven't done a P1 transduction (or any E. coli genetics) for 20 years. But I'm being sent E. coli strains carrying chromosomal lacZ fusions and will need to move these fusions into different genetic backgrounds. This is stuff I did in grad school and I'm delighted to be doing it again.
P1 is a phage (bacteria virus). Particles of phage inject their DNA into E. coli cells, where the DNA is replicated and instructs the cell to make lots of phage proteins. Then the phage proteins self-assemble into phage particles that each fill themselves with a phage DNA, and other phage-encoded proteins cause the cell to lyse (burst open), freeing the new phage to go infect more cells.
What makes P1 useful for strain construction is its tendency to make the mistake of filling itself with a fragment of E. coli DNA rather than P1 DNA. The particles that do this still go on to inject their DNA into new cells, but this DNA (of course) doesn't make new phage or burst the cell. Instead it often recombines with the chromosome of this cell. If the original cell (the 'donor') and the new cell (the 'recipient') had different versions (alleles) of a gene, the donor cell's allele will sometimes get recombined into the recipient cell. The fragment of DNA that's transferred is about 100 genes long, so even big differences (presence or absence of whole genes) can be transferred.
Say strain A has an gene that I want to put into strain B. I first replicate P1 with strain A as host. This produces about 5 ml of a phage 'lysate', usually containing about 10^10 Pi phage per ml. Most of these are normal but some have DNA from strain A. I then infect strain B with these phage, using fewer phage than I have bacteria (I think), so most bacteria get infected by only a single phage. Most of these are infected by normal phage, and go on to lyse and release new phage. To prevent these phage from killing other cell, after the first cells have had time to inject their DNA into their hosts (10 minutes?) I add some citrate, which binds up the calcium the phage need to attach to new cells. Then I put the strain B cells onto agar plates with medium that only cells with the strain A gene can grow on, and I leave them in the incubator overnight. If I haven't messed up, the next day the plates will have lots of strain B colonies with the desired gene.
Lovely pure genetics. No enzymes, no PCR, no kits, and the cells do all the work.
P1 is a phage (bacteria virus). Particles of phage inject their DNA into E. coli cells, where the DNA is replicated and instructs the cell to make lots of phage proteins. Then the phage proteins self-assemble into phage particles that each fill themselves with a phage DNA, and other phage-encoded proteins cause the cell to lyse (burst open), freeing the new phage to go infect more cells.
What makes P1 useful for strain construction is its tendency to make the mistake of filling itself with a fragment of E. coli DNA rather than P1 DNA. The particles that do this still go on to inject their DNA into new cells, but this DNA (of course) doesn't make new phage or burst the cell. Instead it often recombines with the chromosome of this cell. If the original cell (the 'donor') and the new cell (the 'recipient') had different versions (alleles) of a gene, the donor cell's allele will sometimes get recombined into the recipient cell. The fragment of DNA that's transferred is about 100 genes long, so even big differences (presence or absence of whole genes) can be transferred.
Say strain A has an gene that I want to put into strain B. I first replicate P1 with strain A as host. This produces about 5 ml of a phage 'lysate', usually containing about 10^10 Pi phage per ml. Most of these are normal but some have DNA from strain A. I then infect strain B with these phage, using fewer phage than I have bacteria (I think), so most bacteria get infected by only a single phage. Most of these are infected by normal phage, and go on to lyse and release new phage. To prevent these phage from killing other cell, after the first cells have had time to inject their DNA into their hosts (10 minutes?) I add some citrate, which binds up the calcium the phage need to attach to new cells. Then I put the strain B cells onto agar plates with medium that only cells with the strain A gene can grow on, and I leave them in the incubator overnight. If I haven't messed up, the next day the plates will have lots of strain B colonies with the desired gene.
Lovely pure genetics. No enzymes, no PCR, no kits, and the cells do all the work.
HOW to turn on sxy in E. coli
We think that the previous attempts at inducing E. coli's ppdD were unsuccessful because the researchers didn't know that this gene has a CRP-S promoter that requires Sxy, and didn't know what we know about regulation of sxy expression and of competence genes in H. influenzae.
We know that competence is induced by an abrupt nutritional downshift, so I'll try to induce E. coli sxy the same way. The previous researchers tested nutritionally-limiting conditions, but only in steady state (log phase) culture. So I'll grow E. coli in rich medium (LB or sBHI) and transfer it to either MIV or the simple salts used in various E. coli defined media. I'll also test the effect on ppdD of abruptly cutting off the culture's oxygen supply, and of the onset of stationary phase.
Because we have evidence that in H. influenzae sxy induction depends on depletion of nucleotide pools, I'll try various genetic tricks that may cause E. coli to suddenly run out of nucleotides for DNA or RNA synthesis. The most straightforward is culturing purine auxotrophs in rich medium, and then abruptly transferring them to a defined medium lacking purines.
cAMP induces sxy transcription in H. influenzae, but I don't think the E. coli sxy gene has any obvious CRP site. However I'll probably add cAMP to the various test media, both because we know the CRP-S promoters need it and just in case sxy transcription does too.
We know that competence is induced by an abrupt nutritional downshift, so I'll try to induce E. coli sxy the same way. The previous researchers tested nutritionally-limiting conditions, but only in steady state (log phase) culture. So I'll grow E. coli in rich medium (LB or sBHI) and transfer it to either MIV or the simple salts used in various E. coli defined media. I'll also test the effect on ppdD of abruptly cutting off the culture's oxygen supply, and of the onset of stationary phase.
Because we have evidence that in H. influenzae sxy induction depends on depletion of nucleotide pools, I'll try various genetic tricks that may cause E. coli to suddenly run out of nucleotides for DNA or RNA synthesis. The most straightforward is culturing purine auxotrophs in rich medium, and then abruptly transferring them to a defined medium lacking purines.
cAMP induces sxy transcription in H. influenzae, but I don't think the E. coli sxy gene has any obvious CRP site. However I'll probably add cAMP to the various test media, both because we know the CRP-S promoters need it and just in case sxy transcription does too.
Turning on sxy in E. coli
(Sorry for the infrequent posts. Teaching biology to 400 first-year students, and the associated administrative crap, has been sucking all the oxygen out of my brain.)
Once classes end (only a couple more weeks...) I'm planning research in two directions. One is the preliminary work for the laser tweezers measurements of DNA uptake. I'll post about that later. The other is searching for conditions that induce expression of the E. coli sxy gene.
In Haemophilus influenzae expression of sxy induces the genes of the CRP-S operon, and these in turn enable the cell to take up DNA. E. coli has most of the same CRP-S genes, and it has a sxy homolog. The grad student (we're down to one; the other one finished) has shown that artificial expression of E. coli sxy does induce expression of its CRP-S genes, but nobody has found conditions that naturally induce these genes. Such conditions would, we expect, be conditions that induce expression of sxy. Understanding how sxy is regulated, in both E. coli and H. influenzae, is key to understanding the evolutionary function(s) of the CRP-S regulon.
So my plan is to subject E. coli to the same kinds of conditions that induce sxy in H. influenzae. To tell when sxy has been induced, I can either use a fusion of the E. coli lacZ gene to the sxy gene, or use a fusion of the lacZ gene to the ppdD gene, which is one of the CRP-S genes that we know to be strongly induced by Sxy. Then I can easily detectg expression of lacZ, either in broth by incubation with the lactose analog ONPG (turns yellow), or on plates with the lactose analog X-GAL (turns colonies blue). The grad student has emailed the French researchers who studied ppdD induction, asking if they will send us their ppdD fusion.
Once classes end (only a couple more weeks...) I'm planning research in two directions. One is the preliminary work for the laser tweezers measurements of DNA uptake. I'll post about that later. The other is searching for conditions that induce expression of the E. coli sxy gene.
In Haemophilus influenzae expression of sxy induces the genes of the CRP-S operon, and these in turn enable the cell to take up DNA. E. coli has most of the same CRP-S genes, and it has a sxy homolog. The grad student (we're down to one; the other one finished) has shown that artificial expression of E. coli sxy does induce expression of its CRP-S genes, but nobody has found conditions that naturally induce these genes. Such conditions would, we expect, be conditions that induce expression of sxy. Understanding how sxy is regulated, in both E. coli and H. influenzae, is key to understanding the evolutionary function(s) of the CRP-S regulon.
So my plan is to subject E. coli to the same kinds of conditions that induce sxy in H. influenzae. To tell when sxy has been induced, I can either use a fusion of the E. coli lacZ gene to the sxy gene, or use a fusion of the lacZ gene to the ppdD gene, which is one of the CRP-S genes that we know to be strongly induced by Sxy. Then I can easily detectg expression of lacZ, either in broth by incubation with the lactose analog ONPG (turns yellow), or on plates with the lactose analog X-GAL (turns colonies blue). The grad student has emailed the French researchers who studied ppdD induction, asking if they will send us their ppdD fusion.
Long-term plans inspire short-term plans
Now that the rush of grant-proposal preparation is over it's time to consider the immediate implications for the experiments we're doing. Because the granting agency's funds are especially tight right now, our plans need to include the possibility that one or both proposals won't be funded in this round, so that we'll need to reapply in September.
(Summaries of both proposals are available on the "What we're planning" page of our lab's web pages (link in sidebar). The complete Sxy proposal is also available there, but the DNA uptake proposal is not because I left one of the figure files at home; I'll post it tomorrow.)
So this afternoon at lab meeting we discussed the proposed DNA uptake experiments, with a particular focus on identifying the aspects of those experiments that will be seen as strong preliminary work in support of a resubmitted application. This was all too easy, because we haven't yet done nearly as much preliminary work as I'd like.
Luckily the post-doc working on uptake is happy to consider new directions (we submitted her paper on Tuesday), and I'm eager to get my hands wet again after so many months of paper and proposal writing (and teaching, though that's not done for another month).
(Summaries of both proposals are available on the "What we're planning" page of our lab's web pages (link in sidebar). The complete Sxy proposal is also available there, but the DNA uptake proposal is not because I left one of the figure files at home; I'll post it tomorrow.)
So this afternoon at lab meeting we discussed the proposed DNA uptake experiments, with a particular focus on identifying the aspects of those experiments that will be seen as strong preliminary work in support of a resubmitted application. This was all too easy, because we haven't yet done nearly as much preliminary work as I'd like.
Luckily the post-doc working on uptake is happy to consider new directions (we submitted her paper on Tuesday), and I'm eager to get my hands wet again after so many months of paper and proposal writing (and teaching, though that's not done for another month).
Grant proposals done!
Both my grant proposals are done. Competition is very tight this year, and I won't learn the outcomes until sometime in July.
I think the problems we propose to address are very important, the questions we pose get to the heart of the problems, and the experiments we're proposing are excellent ways to answer them. The proposals themselves are very clearly written and nicely presented; the text is well spaced and has little colour illustrations embedded in it, and is supplemented with very clear and carefully drawn illustrations. Much of the credit goes to the grad students and post-docs, whose insights and work were critical in getting the proposals done.
The proposals would have been even better if I had been able to include more preliminary data for some of the experiments, to reassure the reviewers that the planned experiments will actually work. A related problem is the need for more discussion of what problems could arise with the proposed experiments and how such problems will be dealt with.
Now we have these great research programs worked out, we need to adjust our immediate research priorities a bit. This will have the added benefit of generating some preliminary data and a better understanding of possible problems, so if the proposals don't get funded we'll be ready to submit stronger ones in September.
I think the problems we propose to address are very important, the questions we pose get to the heart of the problems, and the experiments we're proposing are excellent ways to answer them. The proposals themselves are very clearly written and nicely presented; the text is well spaced and has little colour illustrations embedded in it, and is supplemented with very clear and carefully drawn illustrations. Much of the credit goes to the grad students and post-docs, whose insights and work were critical in getting the proposals done.
The proposals would have been even better if I had been able to include more preliminary data for some of the experiments, to reassure the reviewers that the planned experiments will actually work. A related problem is the need for more discussion of what problems could arise with the proposed experiments and how such problems will be dealt with.
Now we have these great research programs worked out, we need to adjust our immediate research priorities a bit. This will have the added benefit of generating some preliminary data and a better understanding of possible problems, so if the proposals don't get funded we'll be ready to submit stronger ones in September.
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