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.