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.