Tuesday, August 25, 2009

Now I'm trying to wean myself off experiments and do lab tasks only. The final thing I want to finish is growing up new(!) dpra::spec cells and putting them in the strain collection. I've saved up most of the lab tasks for tomorrow, when the new(!) workstudy student will be coming in to learn the ropes.

Friday, August 21, 2009

I collected the results from the transformation assay on the double mutant (RecJ-ExoI-), but the KW20 (dprA::spec) was, unfortunately, contaminated. I'll have to go back and check the aliquots next week.

Last presentation on Monday!!! :0

Thursday, August 20, 2009

I mostly tried to recollect what I had been doing before I'd left.

-I was cloning Hi comM, ligA, and recBD genes using pTOPO and transforming into Ec.
I wasn't getting any plasmids out of the clones, though, and SS didn't get any either, AND we ran out of pTOPO, so this project's benched for now.

-I had done a transformation assay on KW20 str-R cells (using MAP7 DNA).

-I was going to test the transformation frequency of KW20 cells containing a spec-R cassette in the dprA gene.
If transformation frequency was low, this test would confirm that the KW20 cells were indeed unable to express the dprA gene.

-I was also going to retest the transformation frequency of my RecJ-ExoI- double mutants.
I need to do more replicates to check my previous results (they had differed significantly from what Kumar et al. had reported).

So I did the latter two today.

Also, good luck to anyone who applied for the Lab Assistant position! I'd say the job requires responsibility, ability to keep yourself on task, and probably some degree of neatness would help, too. :)

Friday, August 14, 2009

Today I did a plasmid miniprep on E.coli DH5-alpha cells that I had transformed with a cloning vector containing recBD genes. However, I must have done something wrong earlier, because when I ran the product on a gel, I didn't get any bands (I was expecting a supercoiled plasmid). So that was a little sad. I'll have to start back from the top, ligating genes into pTOPO then them into RbCl-competent E.coli DH5-alpha...

I also did a transformation assay on the H.in KW20 strR cells that SS asked me to grow up and make competent earlier this week. Since it's friday (and I won't be in tomorrow), I'll have to ask RR to kindly take them out of the incubator for me.
Publish Post

Between experiments, I read a paper titled "A Role for Single-Stranded Exonucleases in the Use of DNA as a Nutrient" by Palchevskiy and Finkel (2009). They also used exonuclease knockouts (RecJ and ExoI sound familiar?), but to study viability, not transformation. When exonucleases are not present, DNA is not degraded, so it can't be used as food to nourish the cell, so the cell (eventually) dies.

Lastly, I'm currently making competent the H.in KW20 that was given dprA::spec DNA earlier this week. I'll check the transformation frequencies of this strain to check that they have indeed recombined dprA- DNA into the chromosomal DNA, then I'll probably extract this strain's chromosomal DNA and feed it to my double mutant RecJ-ExoI- cells in order to make a triple mutant.

Wednesday, August 12, 2009

Restreaked KW20 (transformed with dprA::spec cassette).
Did a plasmid miniprep of strains (ligA cloning vector and comM cloning vector in E.coli) I had streaked out yesterday. Then I digested it with EcoR1 restriction endonuclease and ran the products on a gel.

Tuesday, August 11, 2009

Sooo...
Yesterday's transformation of cloning vector pTOPO (carrying comM) into DH5-alpha was supposed to yield blue and white colonies, depending on whether the correct gene was inserted into the plasmid but...
I got very nice pearly white colonies (OK, not exactly pearly, but white nonetheless) for the comM and ligA vectors, but recBD didn't grow much at all, so I replated the recBD cells from yesterday (E.coli magic; you can't "replate yesterday's cells" with H.in).

There are, of course, a number of things that could have gone wrong:

1. Ligation didn't work properly.
I have the ligation products, so maybe I could run them on a gel after cutting with a restriction enzyme?

2. Transformation didn't work properly.
Try it again?

3. Plating didn't go over so well.
I've checked my calculations for ampicillin and X-gal.
Amp stock = 50mg/mL
X-gal stock = 40mg/mL
Want final amp concentration of 100ug/mL and final x-gal concentration of 80ug/mL.
So, to 50mLs LB agar, I added 100uL amp and 100 uL x-gal.
That works out...



Also transformed KW20 strain with dprA::spec construct that SS made last week. Later, I'll use this new dprA::spec strain to test the transformation frequency of the RecJ-ExoI-dprA- triple mutant.

Monday, August 10, 2009

Today I ligated Hin's comM gene into pTOPO cloning vector. Last week, I amplified comM through PCR and purified it (with a kit hehehehe). The ligation was very very quick; probably didn't even take 30 minutes! I also transformed this cloning vector into DH5-alpha (does blogger speak Greek?), a strain of E.coli, then plated the cells on LB again containing ampicillin and X-gal. It was ... funny working with something other than Hin (E.coli even smells different), but it's cool to actually do something that I've studied in class! I remember learning about X-gal and the colonies appearing white if the cloning vectors have ligated with a gene (blue if they haven't).

Thursday, August 6, 2009

Did PCR to amplify comM gene from KW20 x-somal DNA, then ran a gel to check the product. There were two bands, not one, so I increased the annealing temperature (from 50 to 55 degrees Celsius) and am running another PCR.

The plates from yesterday's dprA- survival curve were ready to count, but sadly, all of the plain plates got contaminated (turns out it was the BHI!!! gah!!). The kan plates were fine, and I did count them, but since the growth of dprA- Hin cells could have been affected by the contamination, I don't trust the numbers very much.

Tuesday, August 4, 2009

As RR suggested in this comment, I excluded the first two points of the curve because the cell were not growing exponentially at that point, or there were too few of them doing so to trust the measurements.

The new graph (above) shows that the dprA- growth curve has a slightly lower slope, rather than a drastically lower one.

Today I transformed KW20, RecJ-, ExoI-, and RecJ-ExoI- competent cells with MAP7 DNA and selected for novobiocin. Tomorrow, we'll see how well the double mutant transforms.

Also tomorrow, I'll be doing a dprA- survival curve and will also learn about the dprA::spec plasmid that SS made. I'll use this DNA to make triple mutants (RecJ-ExoI-dprA-!) and test their transformation frequencies.

Friday, July 31, 2009

Grew up KW20, RecJ-, ExoI-, and RecJ-ExoI- cells in MIV to make them competent. Next week, I'll test the transformation frequencies of these strains.

I also counted colonies to determine the cfu/mL counts for the KW20, ExoI-, dprA-, ExoI-dprA- growth curves. I know this sounds trivial, but I get so happy when I check the incubator in the morning and the colonies look easy to count. By this I mean that they aren't bunched together on one side of the plate, they aren't only near the rim of the plate, there aren't too many bubbles in the agar, and that the colonies are nice and large (well, for Hin, anyways). Anyways, these plates were good to count.

Hm. More thinking will have to follow... but for now, happy long weekend! See you on Tuesday~

Thursday, July 30, 2009

Today's growth curve with KW20, ExoI-, dprA-, and ExoI-dprA- turned out pretty well in terms of OD values.

Here's the first graph. It's the OD values taken every hour, plotted on a log scale.

I fit exponential trendlines to each of the plots. When an exponential graph is plotted on a log scale, it'll turn out as a straight line. The slope of the line shows the growth rate of the culture; the larger the slope, the faster the cells are growing.
It really does look like the dprA- strain lags behind in growth, or maybe it dies easily overnight. Or maybe both.

Tomorrow, I'll see how the cfus turn out.

Wednesday, July 29, 2009

Today I ran a gel on the products of PCR on my kit-extracted RecJ-, ExoI-, and RecJ-ExoI- DNAs with ExoI primers. I have done this experiment before, but the last lane didn't show up, so I decided to do a replicate.
However, this time, the last two lanes didn't show up! Plus I got many more additional bands, the most noticeable one being around 700bp in length. It shows up in all lanes, so I'm guessing it could be the mastermix. Would it be worth adding the PCR ingredients to the individual reaction tubes, rather than making a mastermix? I've been using used buffer for my gels, maybe I should go with some fresh buffer next time. I hope my PCR skills aren't slowly fading away... :S I'll be repeating this experiment this week or early next week.

Tomorrow, I plan on doing a growth curve with these four strains: KW20, ExoI-, dprA-, ExoI-dprA-. I'll be measuring OD600 values and plating to determine the number of cfus (colony-forming units) in a mL. I'm doing this experiment to see how well the strains grow. If they don't grow very well, it might be a sign that the strain's mutation serves a (somewhat) vital function. Also, I've done this experiment with RecJ- and RecJ-dprA-, so the ExoI- set will be for completeness, too.

On Friday, I'll be making RecJ-ExoI- cells competent, as well as KW20, RecJ-, and ExoI- cells, too, since they'll be used as controls. Next week I want to use these cells to test their transformation frequency. Without two exonucleases, I'm expecting the transformation frequency to be high. Also next week, I plan to do a survival curve for the dprA- strain. This is to see how many cells will stay alive in MIV solution. It involves incubating dprA- cells in sBHI until they reach an OD600 of 0.2-0.225, then filtering and transferring them into MIV solution, and plating them (on plain sBHI plates? on kan plates?) at intervals (I'm thinking 0', 50', 100', and 150').

Also hoping the weather cools down next week!

Friday, July 24, 2009

On Thursday, I ran a PCR and a gel:
The top 5 lanes are the 1kbp ladder, the PCR products of MAP7, extracted RecJ-, extracted ExoI-, and extracted RecJ-ExoI- DNAs with ExoI primers. The bottom-right 5 lanes are the 1kbp ladder and x-somal DNAs of the same samples (so basically just the DNA, not a PCR). The bottom was to check that the x-somal DNAs had extracted nicely (which they had). The top lanes should look like this gel, which all of them, except the last, did. I don't know what's up with the last one. More on this later.

Today, I ran a gel on another PCR:
Lane 1: 1kbp ladder
Lane 2: MAP7 DNA + RecJ primers
Lane 3: kit-extracted RecJ- DNA + RecJ primers
Lane 4: kit-extracted ExoI- DNA + RecJ primers
Lane 5: kit-extracted RecJ-ExoI- DNA + RecJ primers
These guys should look like the gel in this post, which they do. I thought I didn't have any of the products of PCR with ExoI primers left, but I checked again, and I did! So I ran the PCR product of RecJ-ExoI- DNA + ExoI primers, the one that didn't show up last time. I was hoping last time had been an anomaly with the gel, but as you can (or can't, rather), see in the very last lane, nothing showed up again. I might have to repeat PCR with ExoI primers again.

Lastly, today I counted (:|) colonies from my transformation of KW20, ExoI-, dprA-, ExoI-dprA-, and pilA- strains with MAP7 DNA. Here are the transformation frequencies:

KW20: 3.8E-4
ExoI-: 4.5E-4
dprA-: 2.2E-7
ExoI-dprA-: 1.0E-7
pilA-: 1.2E-8

So the double mutant strain (ExoI-dprA-) can't transform very well. The pilA- strains were confirmed for SS. She mentions in this blog post that the strain is not naturally transformable.

Happy Friday!

Wednesday, July 22, 2009

I ran a gel on yesterday's PCR products, which were MAP7, RecJ-, ExoI-, and RecJ-ExoI- DNAs from the Oxford lab with ExoI primers.
The MAP7 and RecJ- lanes are aligned with the 2kbp rung. This shows that the ExoI gene in this samples is uninterrupted. The ExoI- lane is a little higher than the RecJ-ExoI- lane because the ExoI gene in the former is interrupted by a tetracycline resistance cassette, whereas the ExoI gene in the latter is interrupted by a kanamycin resistance cassette. The ladder's not too clear, but I'd estimate that the tet cassette is ~3kbp long and the kan cassette is ~1kbp long.

Well, now we know what the RecJ-, ExoI-, and RecJ-ExoI- DNAs are supposed to look like with RecJ and ExoI primers.

Today I also extracted DNAs from my RecJ-, ExoI-, and RecJ-ExoI- strains. I'll be running PCR on these to check that they look like the Oxford DNAs. The kit was a lot faster and easier to use than the manual extraction method. No phenol-chloroform, too.

Lastly, I counted colonies from a transformation I did yesterday with KW20, RecJ-, dprA-, and RecJ-dprA- strains and MAP7 DNA. The cells from this experiment are a different batch from my previous transformations, so I got a good biological replicate. This set also showed RecJ-dprA- cells are not great transformers.

Monday, July 20, 2009

I started off the day with some PCR. I ran RecJ primers with KW20, RecJ-, ExoI-, RecJ-ExoI- DNAs (the last three are from the Oxford lab). I did this experiment as a control. The results will show what we expect from the genotypes from the Oxford lab. Eventually I'll run PCR with the same types of DNA, but extracted from cells that I've transformed using the Oxford DNA.

It basically looks like this:

1. Oxford DNA => test with PCR
|
v
2. I use it to transform wildtype cells into RecJ-, ExoI-, and RecJ-ExoI- cells
|
v
3. I extract chromosomal DNA from these cells => test with PCR

I want to check that the DNA from step 3 is the same as in step 1.
The gel came out as expected. KW20 and ExoI- bands are lower because they're shorter; they're not interrupted by a tetracycline resistance cassette. RecJ- and RecJ-ExoI- bands are higher up on the gel because they're longer. The gene coding for RecJ has been interrupted by a tetracycline resistance cassette. They're quite faint, though. I suspect elongation time needs to be increased. So the gene for RecJ is about 2kbp long, and the tet cassette is about 2 or 3 kbp long (I'll have to check that somewhere).

Friday, July 17, 2009

Long live PCR!!!
Lane 1: 1kb DNA ladder
Lanes 2-4: SS's KW20 DNA with RecJ, ExoI, and Hi crp primers, in that order
Lanes 5-7: my KW20 DNA with RecJ, ExoI, and Hi crp primers, also in that order
I ran this gel for 45 minutes at 80V.

The bands are quite gloppy. Is it the new Taq?

The RecJ gene (HI_1214) is 1728 bp long, and the ExoI gene (HI_1377) is 1422 bp long. Both are interrupted by a tetracycline-resistance cassette (how long is it?). Hi crp is 675 bp long, so it's much smaller than what we expect for RecJ and ExoI.

I think the second band in the ExoI samples is due to annealing temperature (which was 45 degrees C for this reaction). SS also got a smaller second band in one of her PCRs, and she was going to lower the annealing temperature. The Tm (I forgot what the "m" stands for, but I think it means the temperature at which half of the primer is attached to the template DNA) of my ExoI_F primer is 57.2 degrees C, whereas my other primers (RecJ_F, RecJ_R, and ExoI_R) have Tms in the 47-50 range. So I guess the low annealing temperature made the ExoI_F primer bind with parts of the template that it was not specific to.

Happy weekend!

Thursday, July 16, 2009

Planning to make some more MIV-competent cells ASAP, as I've used them all in transformations. Actually, I did a transformation today. It's a replicate with KW20, ExoI-, dprA-, and ExoI-dprA- taking up MAP7 DNA and un/successfully expressing novobiocin resistance.

Also, we got new Taq today and I ran a PCR right alongside SS. If nothing shows up, woe is me. If I get bands, I'll be so relieved I might consider supporting Radio Paradise. (JK; that only came to mind because I hear it many, many times daily.) I'll put the samples in the fridge and run them on a gel tomorrow.

Tuesday, July 14, 2009

Through transformations with MAP7 DNA, it's starting to look like neither RecJ or ExoI are specifically blocked from degrading DNA by dprA's product.

Thursday, July 9, 2009

KW20, RecJ-, dprA-, and RecJdprA-- strains grew up quite nicely today. They were pretty much on par with each other in terms of OD, and tomorrow I'll see how the viable counts did.

I also counted colonies for the transformation of the four strains above with MAP7 DNA. Two transformations have shown that RecJdprA-- transforms quite poorly, comparable to the dprA- mutant.

Wednesday, July 8, 2009

I transformed KW20, RecJ-, dprA-, and RecJdprA-- strains with MAP7 DNA. I'm changing the concentrations that I plate, because last time, I got a lot of "film"s and "TMTC"s (too many to count).

Made KW20, ExoI-, dprA-, and ExoIdprA-- strains MIV-competent, which didn't take too long to grow up, thankfully.

It feels like today was a very busy day, and the sentences above probably don't do justice to how pooped I am from monitoring the MIV-competent cells, to diluting and plating the transforming cells, and getting stuff ready for a growth curve tomorrow. It's probably because I did it all simultaneously.

Monday, July 6, 2009

Today, to test how well RecJdprA-- mutants transform, I fed MAP7 DNA to KW20, RecJ-, dprA-, and RecJdprA-- strains. We already know how well the first three strains transform, but I'm doing them together so that I can compare across strains.

I also did a similar transformation with KW20 and pilA- strains for SS.

Lab meeting today was pretty cool, especially the stuff about "polonies". It really felt like JC was showing us a magic trick. George Church is my hero.

Sadly, it's raining outside and I think I'm coming down with a cold. :|

Friday, July 3, 2009

Above is a diagram I found to explain my last post. By "blob" I mean droplet B and by "lake" I mean droplet C. SS's samples resemble high surface tension droplet B, but my PCR products look like droplet C, which has low surface tension.

I wondered if:

1) PCR samples that show up on gels have high surface tension and samples that do not show up on gels have low surface tension. ==> No correlation. Although the MAP7 DNA + ExoI primer that SS ran did not show up on the gel, it was still a B droplet type.

2) The heat of the thermocycler had changed the surface tension. ==> Considering that I ran the PCRs on two different machines (ours and the Thompsons'), and SS and I both ran the same cycle, doesn't seem likely.

It seems SS and I are using different ingredients! :0

I have been mixing the PCR ingredients together (but not actually running PCR), but I've only been able to get droplet Cs.

Tune in next week for developments!
The PCR I ran yesterday was a replicate of one SS did last week. We used the same ingredients and thermocycler. Which means nothing's wrong with the stuff, but I'm using them improperly.

PCR variables and controls:

1. Used approximately 165ngs of lab stock MAP7 DNA. I've used this to transform competent cells before, so it shouldn't be weird. I could try some dprA::kan chromosomal DNA I have, but SS got a nice band with MAP7 last time.

2. Used Hi crp primers, which are SS's. She used them with MAP7 and got a product, so there's nothing wrong with the primers. Well, actually, maybe there is, since MAP7 and RecJ primers only showed up faintly and MAP7 and ExoI primers didn't show up at all when SS ran the PCR.

3. The gel electrophoresis equipment is fine, because the 1kb ladder runs nicely.

4. Is it the thermocycler, somehow? But the first time I tried this, I used the Thompsons', and that didn't work. Plus, as I've already said, SS used the same cycle on the same machine and got results.

5. Is there a specific way that I should be adding the ingredients? I always Taq last, but I don't have a certain order for the other ingredients. Should I be vortexing or centrifuging them? We don't have a lot of Taq left; maybe it's really old?

I've noticed that my products always seem to be very watery when I mix them with the gel dye. I can't explain this very well, but when I mix chromosomal DNA with the dye, the whole blob of liquid turns blue (and it remains a blob), but when I mix the PCR product with dye, the dye stays at the bottom and the blob turns into a flat lake, almost. I don't quite remember, but I ran the gel for SS's PCR, and her products seemed to remain a blob. I know that doesn't really confirm anything (yes, we know PCR's not working for you, Rhena).

Hm.

Thursday, July 2, 2009

Finally made the four strains MIV-competent today.

I still think the overnight method is faster, though, because you don't have the additional step of growing cells to OD600=0.1, which takes an additional day.

I also ran another PCR today, and not even the Good Luck Crystal could help. What is the problem? I added all the ingredients (I even checked them off as I went) in the correct amounts, and the cycler was set properly...

I found an online PCR troubleshooting guide. Hypothesis A ("Pilot Error") and I ("Bad Karma") look especially promising. Will read the page and try again later.

Tuesday, June 30, 2009

Grew up KW20, RecJ-, dprA-, and RecJdprA-- strains to an OD600 of 0.1ish today, then froze them. I'll grow these up to an OD600 of 0.2-0.225, then filter and resuspend them in MIV solution to make them competent.

Canada Day tomorrow! :D

Monday, June 29, 2009

You'd be surprised at how time flies when you're watching ice melt (and preventing it from spreading on the floor).

I spent most of today defrosting our -20C freezer, and the to-do list for this week includes:

1. Make KW20, dprA-, RecJ-, ExoI-, dprARecJ--, and dprAExoI-- cells MIV-competent in order to...

2. Transform these strains with MAP7 DNA to determine how well (or how badly) they take up DNA.

3. Run a successful PCR with wildtype DNA (probably MAP7) and RecJ, ExoI, and crp primers in order to practise PCR-ing and actually get it to work.

I'll be doing steps 1 and 2 in sets: [KW20, RecJ-, dprA-, RecJdprA--] is one and [KW20, ExoI-, dprA-, and ExoIdprA--] is another. This is so that comparisons can be made between strains without having to worry too much about differences in biology that occur due to experimentation on different days.

Thursday, June 25, 2009

About a week ago I did an experiment in which I fed KW20 wildtype and RecJ- strains some RecJExoI-- DNA. The experiment was done to test linkage of RecJ and ExoI genes.

The RecJExoI-- DNA is made so that a TetR gene interrupts the RecJ gene, and a KanR gene interrupts the ExoI gene. If genes were words, it'd look something like this:
...Re-[TetR]-cJ...
and
...Ex-[KanR]-oI...
These two genes are about 190-thousand base pairs apart on the H.influenzae chromosome.

For the experiment, I plated cells onto plain, Kan, Tet, and Kan+Tet plates. Why? Because
-Wildtype cells that grew on Kan must have taken up ExoI DNA (or both ExoI and RecJ DNAs),
-WT cells growing on Tet must have taken up RecJ DNA (or both ExoI and RecJ DNAs),
-And WTs growing on Kan+Tet means they must have taken up both DNAs.

If the KanR and TetR transformations were independent (i.e. they were never on the same strand of DNA and all cells in the culture were competent), then:
T.Freq. (Kan) x T.Freq. (Tet) = T.Freq. (Kan and Tet)
(Simple probability here, folks. The probability of two events occuring is the product of the probability of one and the probability of the other.) This was not the case, since (5.5E-4) x (5.0E-4) doesn't equal (1.0E-6). I saw higher numbers than expected.

Explanation:
Are all the cells in the culture equally competent?
YES: recJ and exoI genes are linked.
NO: congression is at work.

The first one is basic. If the two genes can be found on one intact strand of DNA, then the chances of gaining KanR and TetR are higher than if one cell had to take up two seperate strands of DNA. But this explanation depends on all of the cells in the culture being fully competent.

The second slightly more twisted.
Let's say you observe that 20% of all cells in the culture transform to become KanR and 20% become TetR. 8% become double mutants.
Through calculations, you'd expect (20%)(20%) = 4% of the culture to become double mutants.
Let's say we know that only half the cells in my culture are actually competent. Since transformation frequency is the number of cells out of all competent cells that transform,
instead of 20 KanRs out of 100 total cells (for example), there are actually 20 KanRs out of 50 competent cells, making the actual KanR transformation frequency 40%.

So we now have KanR and TetR transformation frequencies of 40%. We expect that out of 50 competent cells, we'll have (40%)(40%) = 16% KanR+TetR double mutants, or 8 double mutants out of 50 competent cells. Including the other 50 cells that aren't competent, we have a total of 100 cells, but we still have 8 double mutants. So it looks like our "total" transformation frequency for double mutants is 8%.

Using a 48-year old formula by Goodgal and Herriot, the fraction of competent cells for this example is:
T.Freq. (Kan) x T.Freq. (Tet) / T.Freq (Kan and Tet)
=(20%) x (20%) / (8%)
=50%
(Yes, I engineered the numbers to work out.)
Since the RecJ and ExoI genes are "just on the brink" of being linked, I'm probably seeing congression. For my experiment:
T.Freq. (Kan) x T.Freq. (Tet) / T.Freq (Kan and Tet)
=(5.5E-4) x (5.0E-4) / (1.0E-6)
=0.275
=27.5% of my cells were competent!
I don't know what I'm supposed to think, but that seems pretty low.

Thanks to Josh for helping me understand this!

Wednesday, June 24, 2009

I have been trying to befriend PCR today. It did not go over well.
Tomorrow will be a transformation. More details to follow.

Tuesday, June 23, 2009

LinkToday I followed a growth curve for these strains:

KW20,
ExoI-,
dprA-,
and ExoIdprA--.

ExoI- grew like KW20 for the most part, and I'll have to check the growth of dprA- mutants.

I've also had some think-time for congression and linkage a la Josh's blog post. I... think I'm going to need another run through. I'll go ask the post-docs tomorrow.

Look forward to a PCR repeat this week!

Friday, June 19, 2009

Big news!!!!
I ran my PCR to check my KW20, RecJ-, dprA-, and RecJdprA-- DNAs, and this is what I got:






That's right, nothing.
Will mull over this on Monday. For now, weekend!

Thursday, June 18, 2009

It turns out that the cells I was making MIV-competent were growing immensely slowly. RR's last comment said if all my optical density (OD) points went through an exponential line, I had started with a small number of viable cells. Alas, this was not the case. Bring out the graphs!

As you can see, the points aren't quite exponential. :S Why? Well, I am indeed running growth curves today for KW20, RecJ-, dprA-, and RecJdprA-- strains. It's been about 3 hours, and growth does seem quite slow. I'm also planning to run a gel on some DNA I made a few days ago. I extracted these chromosomal DNAs from the RecJ-, ExoI-, and RecJExoI-- strains I made from the Oxford DNA. We're going to check them with PCR (my primers are here!). I also read over Josh's blog post on linkage. I'll have to reread it to understand it better (it's not your writing, it's me :]).

Wednesday, June 17, 2009

I have been scratching my head at RR's comment to my last post. Why did they take forever to grow? How can I tell if there were only a few live cells, or if the strains just grow really slowly, or even both?!

I had transformed RecJ- and ExoI- MIV-competent cells with dprA- DNA,
then chosen a colony from these transformation plates and restreaked them on kan-tet plates,
then I chose colonies from the restreaks and grew them up in sBHI+KT overnight. They were cloudy in the morning, indicating they had grown.
That's where my cells came from.

I plotted a graph on Excel to help wrap my head around the information:
Sorry about the points not connecting... I'll figure that out.

Anyways, the cultures did reach their exponential phase between hours 5 and 6, but didn't feel inspired before that time. If they were growing slowly, wouldn't you expect a uniform slow growth for all hours after the start? So the growth curve would be more like a line than an exponential curve. But since the cultures did start growing rapidly in a short amount of time between hours 5 and 6, I think I may have started with a small number of viable cells. It's difficult to tell because optical density is a measure of all cells, alive or dead.

I'll be doing growth curve experiments tomorrow and Friday. Tomorrow I'll be monitoring the optical density of KW20, dprA-, RecJ-, and RecJdprA-- strains and Friday shall be KW20, dprA-, ExoI-, and ExoIdprA-- strains. I'm looking forward to learning more about the ExoI- growth curve, since I've noticed that it's always a log behind KW20 or RecJ-, for some reason.

Friday, June 12, 2009

I grew competent cells all day. They took forever (i.e. 5-6 hours) to get to an OD600 of ~0.2. Glehhhh. I'm still waiting for them to finish their "growing for 100 minutes in MIV solution" stage. Hopefully more exciting things to come next week. Stay tuned.

Thursday, June 11, 2009

This morning I read and took notes on the Kumar et al. paper. Just to show that I put some effort in, here are my notes:
I learned two sweet things from reading the paper (and doing some google-searching):

1) The proteins responsible for H.influenzae's virulence (i.e. ability to make its host sick) are controlled by a string of repeating DNA nucleotides, such as ATTGCATTGCATTGC... A change in the length of this string can change activate or deactivate certain genes, resulting in new phenotypes and new populations of cells that could survive an antibiotic attack. This is called "phase variation".

2) (The following information is from this site.) Mismatch repair (MMR) is so intense! MMR is a mechanism bacteria (like E.coli) use to fix incorrect base pairing after DNA replication. Basically, MutS binds to a mismatch, it calls over buddy MutL, which gives MutH a kick to get started, which binds to a GATC sequence near the mismatch and cuts the DNA strand there. From this cut to the mismatch, an exonuclease breaks down the DNA, and then DNA polymerase III and DNA ligase fill in the gap.

What is strange is that my ExoI- mutants' transformation frequencies were mucho lower than the published figures. Kumar et al said relative to a value of 1.0 for the transformation frequency of the wildtype, ExoI- got 0.9. I tried this with my figures, and got 0.2 for ExoI-! :\ (My RecJ- number was pretty close, though.)

I'm going to go regrow my streaked double mutants.

Wednesday, June 10, 2009

I counted my dprARecJ-- and dprAExoI-- colonies today, and they're looking OK. I picked out easily choose-able colonies and streaked them on kan-tet plates.

I tried to re-read the paper by Kumar et al. to glean more information from it, but... I didn't get much. I'll give it another go tomorrow, and I'll take notes as I read. That ought to help.

I've also been reading Mr. Tufte's The Visual Display of Quantitative Information, and it has some of the most interesting graphs I have ever seen, I kid you not. I was amazed at this one in particular:It's on the front cover, so I thought it was some complex Physics stuff, but it's a train schedule! The horizontal lines are stations and the diagonal lines show when a train left a station and when it will arrive at another.

Sometime later, we're going to order primers for PCR to check that my cells have the genotype I want them to have. Man, there were so many PCR questions in Biol 112. Hope it comes in handy.

Tuesday, June 9, 2009

Learned a lot of stuff from my turn at lab meeting yesterday. I've done many presentations before, thanks to IB, but they were mostly about Ethics or How Education Can Break the Cycle of Poverty, etc, etc... I'm new at talking about data. I have 17 points of advice written in my lab book, such as:

Always state the purpose/aim/goal of an experiment before going into procedural details.

Think about controls: negative and positive.

Group the experiments/data according to topic to keep thoughtflow.

And much much more. I hope to keep these points in mind and really improve in my next presentation! :]

Today I transformed some cells to create dprARecJ-- and dprAExoI-- double mutants. I have 7 cultures:
1. RecJ- cells without any DNA given
2. RecJ- cells given ~1ug of dprA::kanR DNA
3. ExoI- cells without any DNA given
4. ExoI- cells given ~1ug of dprA::kanR DNA
5. dprA- cells without any DNA given
6. dprA- cells given ~1ug of RecJ::tetR DNA
7. dprA- cells given ~1ug of ExoI::tetR DNA.

Hold on, yes, I know the dprA- cells don't transform well. I'm transforming them as a double-check. These guys shouldn't transform very well.

Also, the dprA::kanR DNA is some that I extracted, using the old-school non-kit method, from some stock, and it still has a lot of RNA left in it. Rosie said only about a tenth of the mass would probably be actual DNA, so I used quite a lot. 10 times more a lot, actually. :| Hope this works. Would having extra DNA floating around to take up be worse than having too little DNA to take up? Hm.

Thursday, June 4, 2009

Results came out mostly on par with the others. Very inbetween-ish.

I tried to make more MIV-competent cells, but they wouldn't grow :|. Tomorrow!

Mostly I'm looking at my data, trying to organize it for Monday's meeting!

Wednesday, June 3, 2009

Instead of just saying "Today, I did another MIV...", I'll explain why I did it.

I have done this experiment 3 times with good results each time. I'm getting clean controls, ExoI- and RecJ- strains are transforming as well as (sometimes even better than) the wildtype, and the dprA- strain is transforming poorly (which goes with the hypothesis).

Here are some sample transformation frequencies for the RecJ- strain (just as an example):
For experiment E09: 4.1E-4
For experiment E10: 6.6E-3
For experiment E11: 7.6E-4

I'm leaving out a lot of information by not including other numbers, but just know that for each experiment, the wildtype's transformation frequency was in the same log (i.e. "E-4" or "E-3") as RecJ-'s. Also, RecJ-'s no DNA control (they weren't given MAP7) had very low transformation frequencies, like 1E-8 or 1E-9.

Sounds good? Well, the funny thing is that E09 used MIV-competent cells that were made on the same day as E10. (E11 used cells made at a later date.) This means the biological aspect of E09 and E10 were the same, and something in the technique caused 41 out of 100000 cells to transform the first time, then 660 out of 100000 cells to do so the second time around!

Also, I do 1 in 10 dilutions of the cultures when plating, but sometimes I don't get 1 in 10 numbers. For example, if I count 100 colonies on a 10^-1 plate, I should get approximately 10 on the 10^-2, then around 1-ish on the 10^-3, but sometimes I get things like 667, 8, then 3. Huh??? It's a head-scratcher.

Since I couldn't think of anything that I could have done differently the second time (I even rechecked the calculations), today I did another replicate. This one was with cells that were made on the same day as the E11 ones.

So look forward to more results tomorrow!

Monday, June 1, 2009

You might get tired of hearing this, but I did another KREd + MAP7 transformation today.

I'll be making RecJdprA-- and ExoIdprA-- double mutants this week.

I'll also attempt to read through papers on ExoI, RecJ, and DprA and their functions in recombination. I've been through one paper so far, and the most I got out of it was something about UV radiation and maintaining something or other and ... I guess I'll try to read it again tomorrow.

Friday, May 29, 2009

Yesterday, I finished making more MIV-competent cells. I also froze some of my RecJExoI-- mutants. I grew up a sample in LB just in case, and it didn't grow, so it was H.influenzae. I checked under the microscope, too.

The results from my most recent transformation shows that the RecJ- and ExoI- mutants do transform just as well as the wildtype. The dprA- mutants, however, don't. So I've confirmed what was published in the paper. I don't actually know which paper. Maybe I ought to read it.

I'm going to do some more replicates next week using my new MIV-competent cells. So this time they'll be both technical and biological replicates.

Wednesday, May 27, 2009

Woah!

OK, so I tried to make some more MIV-competent cells, but 2 strains refused to grow, 1 grew too fast, and only 1 grew just right. So the 3 strains I couldn't/wouldn't do... I will do tomorrow.

I also did a replicate of my transformations with MAP7 DNA (with the usual suspects: KW20, RecJ-, ExoI-, and dprA-). I only had one epp of MIV-competent KW20 left, so I defrosted it, transferred half the volume to a new epp, spun them both down, resuspended in half the usual volume of MIV, and added half the usual volume of DNA to one tube. Should work, right?

Thirdly, I took my plates of RecJExoI-- double mutants, picked off 4 colonies with sterile toothpicks, and dropped the toothpicks into sBHI with special guests Kan (kanamycin) and Tet (tetracycline).

Lastly, I am slowly becoming familiar with Excel (spreadsheets and graphs galore). Data analysis is a large part of science that is not really shown to the public eye. I mean, if someone asked me to imagine a scientist at work, I'd think of someone at the bench, in the lab coat (ironically, only about 5% of people here wear lab coats), swirling colourful flasks of chemicals. I probably wouldn't think about someone in front of a giant spreadsheet, analyzing numbers. Basically, research involves a lot of skills other than just lab work. (Writing! Oh my! The other Redfields do a lot of penning reviews or editing manuscripts or getting those grants in.)

No new chicken today; I'm pooped and going home. Ciao.

Tuesday, May 26, 2009

I regrew my RecJExoI-- double mutants from Friday, but got lawns (this is when there are so many bacteria on a plate that they form a film instead of separate colonies). So today I diluted them down further and plated them.

Yesterday I also transformed my four lovely strains (KW20, RecJ-, ExoI-, dprA-) with my favourite DNA (MAP7). This is the fifth time I've done this. Which is good, because now I am more confident in the techniques and I feel like I know what I'm doing at each step. I'd better not get complacent, though.

In first-year Chemistry labs, we don't get to do labs twice, so we never get to follow through with the "improvements" and "changes in procedure" that we suggest in our write-ups. I think this sort of hinders us from developing a scientific approach to things, since we only write what we would do if we could. Not too helpful. Doing my transformations multiple times has allowed me to try out those woulds. :D

Friday, May 22, 2009

My cells are growing nicely, even on the kan-tet plates this time! This means I'll have some double mutants (RecJExoI--) soon. Mucho grande excitement, si.

I learned about biological and technical replicates today. In a biological replicate, you do the same experiment on different samples of cells at different times. The point is that your experiment will give the same results in batches of cells that are slightly different, making your conclusion more valid.

In a technical replicate, you do the same experiment on one batch of cells many times. This checks your technique (ie. protocols, diluting, plating, etc). This type of replicate can help point out flaws in technique, but since you're only testing on the same sample of cells, your conclusion is not as solid as it would be if you'd tested lots of different batches.

Definitely feels like a Friday today. Even the med students downstairs are slacking off a bit. Tsk tsk.

Thursday, May 21, 2009

Using the MIV-competent KW20 strain made yesterday, I did a transformation today.

Basically, you thaw the cells, spin them down, resuspend them in fresh MIV, add MAP7 DNA to the "+" DNA tube, incubate the tubes for 15 minutes, take them out, resuspend them in sBHI and incubate for 90 minutes (for them to be able to express the gene for tetracycline resistance), take them out, dilute, plate, shake, incubate!

I wonder if there's another word for "dilute" that ends in -ate. Hmmm... although not a real word, I think "unconcentrate" is the best.

I've also been organizing my lab book. It's started to fill up since the beginning of May, and I'm starting to get confused between "2nd attempt at..." and "3rd time transforming...". I've labeled my pages and filled out the table of contents. Today was the 8th experiment (E08) into my project! Maybe every 10th experiment I should eat something fantastic.

P.S. What are the Hallams doing? A camera guy has been following their lab members around all day.

Wednesday, May 20, 2009

Today we're going to make some more MIV-competent cells. These guys are grown up in supplemented BHI medium (sBHI) (brain-heart infusion of cows! Sounds like the witches from Macbeth :S) until they reach a certain density, then they're put in MIV starvation medium (not as nutritious as the sBHI) for 100 minutes. After that time, they're considered "competent", or able to take up DNA by themselves. Then they're put into the freezer for later use.

Friday, May 15, 2009

Results:

A) KW20, RecJ-, ExoI-, dprA- transformations with MAP7 DNA
Got unexpected numbers for the dilutions. Since the dilutions are 1 in 10, you'd expect 10 times less colonies as you move down. Not the case. Most plates decreased cell count by 2 times. Weird. Again, we found that transformation frequency was not as high as usual (for the WT). Plan is to make MIV-competent cells next week and start from the top.

B) Remaking RecJ-&ExoI- double mutants
Numbers on the plain plates were good (i.e. decreasing by 10X), but overall transformation frequency was low because I didn't get any colonies on the kan-tet plates! Which means the cells didn't transform well, or there weren't enough cells that survived to transform. Redo!

C) Getting to know H.influenzae
This was just an experiment to get myself acquainted with how H.influenzae grows, because some bacteria grow slowly and others very quickly. The aim was to give me a better background knowledge of this little guy. Every hour, I'd take a sample from a growing culture of cells and check it's optical density (OD) with a spectrophotometer. Optical density (cloudiness) is an indicator of how many cells are in the medium because the medium gets foggier as the cells grow. Then I took the same sample from the culture and diluted and plated it. Why? Just because the culture is at a certain OD, doesn't mean that all the cells are alive. Plating lets us determine the cfu (colony-forming units) per mL, which is basically the number of cells in 1 mL that are healthy enough to form colonies. I've collected my data and will learn how to make proper graphs next week.

(Unrelated but kind of related to point C: When we went down to the Invitrogen event yesterday, we saw an electronic cell-counter. You just put a sample on a slide, put it in, and it counts the cells. :O)

Long weekend!

Wednesday, May 13, 2009

Regrowing the ExoI-&RecJ- double mutant strain today. Plus, we'll be making more MIV-competent ExoI- and RecJ- cells tomorrow.

The transformants from yesterday (RecJ-, ExoI-, KW20, dprA-) are growing wee colonies, so I'll count them later today.

Quote of the day:
Invitrogen man: "Do you do DNA purifications?"
Me: "Ummm... maybe."

Tuesday, May 12, 2009

My MIV transformation turned out a little wonky. The hypothesis was that RecJ- and ExoI- would transform just as well as KW20 (the wildtype strain I'm using; it has genes of the bacterium as found in nature).

However, wildtype plates showed many more colonies than RecJ- and ExoI- plates. For example, KW20 with novobiocin resistance (by giving it MAP7 DNA) had 46 colonies (on a E-2 plate) whereas RecJ- had none!

I guess that doesn't mean anything without looking at the plain plates. The wildtype plates had around 3-4 times as many colonies as the RecJ- and ExoI- plates. Huh.

But when I repeated the experiment today, I noticed that some tubes of MIV-competent cells looked like they contained more cells than others. For example, the KW20 eppendorfs had a little brown mound at the bottom of the tube after centrifugation, whereas RecJ- and ExoI- were juuuust visible. Could it be that there's just a higher concentration of KW20 cells than the other strains? Hm.

If some RecJ- and ExoI- cells shown up on the novobiocin, I could have found transformation frequencies and compared them across strains. This is more important than simply the numbers of colonies, I reckon.

In other news:

1. I found out why I see green or blue backpacks all the time in LSC (the building in which our lab is located). The med students get free backpacks, and they get different colours according to their graduating year.

2. I haven't been much of a hockey fan since we lost television, but listening to last night's game was a gut-wrenching, hand-wringing, hair-pulling-outing, and teeth-grinding sort of experience.

3. Today's election day! I'll admit, even though I've been old enough to vote since... the last election (?), I am a first-time voter. :S Democracy! Huzzah! Viva la revolucion!

Monday, May 11, 2009

5 minutes before lab meeting!!!

I've counted plate after plate after plate today and have the data for

1) My KW20, ExoI-, and RecJ- transformed with MAP7 DNA!
2) My double mutants ExoI-&RecJ-!
3) The dilution and plating experiment!

More details to come!

Also found out that 3 bottles of BHI agar became contaminated!

Alas, 5 minutes have passed!

(What an exciting post...)

Friday, May 8, 2009

Finished the "Dilution and Plating Techniques" assay a while ago. We were all given 1mL of E.coli cells to dilute and plate out at our whim. Plate, shake, and incubate. If the results are the same, then the differences in techniques are not significant and our experiments can continue as usual (I assume). If we all get beserko-different numbers, we'll have to do more "Technique" assays. At least I'll get plenty of practise.

I guess I'll explain my project in more detail. Our lab is interested in transformation of bacteria. If you took Biol 112, as I did, you'll remember this (or you should). Certain bacteria can take up free-floating DNA from their environment and make it a part of their own chromosomal DNA (the new DNA has to be very similar to the old DNA). This can lead to the bacteria acquiring new abilities, such as being able to grow on an antibiotic.

A whole bunch of genes and proteins are needed for transformation to happen. Sometimes, the cell doesn't even get transformed because nucleases break down the incoming DNA before it can reach the chromosome. RecJ and ExoI are two nucleases that my project focuses on. dprA is a gene that is required for transformation. It's gene product probably acts as a sort of shield for the incoming DNA against nucleases. I want to test if dprA's action is specific to either RecJ or ExoI.

So let's say we have four H.influenzae cells.
Cell #1 is a wildtype cell. It has both the dprA (blocker) gene and the one that encodes, say, RecJ (cutter). We know (from experiments, I guess) that 1 in 1000 of these cells will transform.
Cell #2 is a mutant. We've taken out the dprA (blocker) gene, but RecJ (cutter) remains. We expect about 1 in 100 000 000 of these cell to transform because dprA isn't present to protect from RecJ's action. That's way down from cell #1.
Cell #3 is also a mutant. We took out RecJ's gene (cutter), and dprA (blocker) remains. We expect about 1 in 10 000 bacteria to transform because RecJ doesn't chop up the incoming DNA.
Cell #4 is a double mutant. That means it's missing two genes; we took out both dprA (blocker) and RecJ (cutter). This one's the real test. There are two things that could happen:
A) Bacteria transform juts as well as the wildtype. dprA is specific to RecJ and not another nuclease.
B) Not a lot of bacteria transform. dprA is specific to another nuclease.

I'll have to think about a clearer way to think about cell #4...

Thursday, May 7, 2009

Forgot to blog while at the lab, again.  D'oh!

Today I plated millions of bacteria!!!

Three strains, KW20, recJ-, exoI- had previously been made competent in MIV starvation medium.  I gave them some MAP7 DNA to take up, diluted them, then plated them on novobiocin plates to select for transformants.

The second thing I did today was make DNA from dprA::kan cells (grown last night).  This required adding chemicals, extracting twice, adding more chemicals, then scooping out the glump (read: gluey clump) of chromosomal DNA.  Most exciting part was when I had to make phenol/chloroform (one of the added chemicals).  Looking into the shelves underneath the fumehood was like looking into ... a prison.  For chemicals.

I then took this DNA, gave it to competent KW20 cells, let them express the gene, then plated them.  Hopefully, results will show tomorrow.

Wednesday, May 6, 2009

Tomorrow will be a happenin' day.  The plan is to test competences of all the strains I have accumulated so far (recJ-, exoI-, and wildtype KW20) with MAP7 DNA and select for novobiocin.  Since the recJ- and exoI- are missing nucleases (enzymes that chop up DNA), they should have similar/same transformation frequencies as the wildtype.  dprA- will be tested as well... if only I could find the DNA in the freezer.  0_0

I should've blogged while I was in the lab... now I can't remember squat.

Tuesday, May 5, 2009

Hi everyone,

I'm the new research assistant at the Redfield lab. OK, actually, I was a work-study student from September to early April, but I took a break in May (for exams and such) and now I've been re-hired with a better job title :).

I've only just finished first-year Science here at UBC, so most of the work I'll be doing is pretty basic and I'll try to explain things as clear as I can. I still have a few more baskets (!!!) of glassware to wash and autoclave, so more research-y stuff will come later.

Bye.