Flasks, smells, and mess!

Right, I promised to try and not start my posts with so, and the last one was.  This one I'm struggling already.
In the past two days, I've been preparing cells.  After getting my plasmid to stay in my E. Coli (well I hope anyway), I grew the cells on some agar with antibiotic on overnight, which I did by spreading the mixture of cells over some agar.  You make agar by mixing growth media (broth) with agar, which is a gelling agent.   You can put pretty much what you want in the growth media according to what your cells need.
The next day, I nicked 4 flasks off the shelf that had LB in (this is the growth media that we use, it smells a bit like marmite diluted down, and looks like a urine sample), but apparently E coli love it!!! When I say nicked, they are there to use, we have a make some more if you take the last ones, and as I made some that day as well that was alright.
In these flasks I added more antibiotic, and some of my cells.  You just scrape a few colonies off your plate and ping the pipette tip into the liquid.  Gilson pipettes work by displacing air, and you use a new tip that fits on the end for each measurement, and they have an ejector mechanism, for each tip, so the tips really do ping off.  The advantage of this is that the cells are sticky, so aren't very easy to get off anything the attach to, so it's easiest to just put the whole thing in!
I also made 16 big flasks.  These each hold 500ml, and were double strength LB.  That's a LOT of flasks!
The little flasks I grew overnight, then added a bit from them to each of the 16 big flasks, and grew those up (in an incubator that shakes them to get air in and keeps them at 37 degrees) for two hours before adding 20% arabinose, which apparently gets the cells to start making the proteins.
Then I spun them all down, hence most of the smells and the mess.  You have to pour the cells into bottles, then balance the bottles, so the centrifuge doesn't go walkabout (like your washing machine does, but the middle goes at about 8000 rpm, so you can imagine how bad it would be!), and then tip the liquid off after spinning, and add more cells until you've got through all the flasks.  The cells smell, and are really difficult to keep all in one place.  I used plenty of sodium hypochlorite (the active ingredient in bleach) today to get everything clean.
Prepared loads of pipette tips for autoclaving (the big ones go in coffee jars, a good gauge of how much caffeine you need to be a scientist!) whilst I was waiting for my cells to grow, along with random helping people whilst they dashed off to do all sorts of other things.
Will update on if I actually get any protein from all this!!! :D

Precipitation, and new discoveries!

So basically I got really excited about the ITC.  It came out with some amazing results, which I really wanted to prove.  So, I was on a roll, the machine was working, and I'd done everything I could with the various bits of my proteins.  I decided to (well it was my supervisor's idea really) try full length protein, and a mutation that had been made for something else.  I could work out why when I ran these proteins I ended up with no heat exchanges (NOT good) and a really weird curve, that no model could fit.
So anyway, when I took the liquid out at the end of the experiment, it wasn't clear (as it should be), it was cloudy- the precipitation.  That explains everything.
So after some discussion, it has been suggested that I change the salt concentration.  Unfortunately, I've run out of these proteins (loads of the other ones)  So I have to make some more.  So, this week, I'm going to be learning how to do protein extractions.  So far, I've just transformed the plasmids into the cells, using heat shock.
(Jargon here is...)  Plasmid:  a piece of circular DNA found in bacteria that are not part of the nucleus.
Transform: get the plasmid to get in the cells.  The cells are E. Coli cells.  To heat shock them, you leave them on ice for a while, then quickly heat them up to 42 degrees, then cool them again, before allowing them to grow up.  So you know you've only got the cells with the plasmid in (that contains the DNA that codes for the protein) the plasmid has an antibiotic resistance gene in it, so that when you grow them in some broth with antibiotics in them, only the cells you want survive, and the rest die because they can't grow in the antibiotic.
I will write with what else I'm doing later in the week.  For now, I'm happy to be doing something new, great as the results are from the ITC, you're relying on a computer, which is an amazing piece of technology, but I LOVE being in the lab.  It's one of the things I'm enjoying actually.  I really like the diversity of things you get to do every day!

Rain, ITC, Rain, Swing Dance and Rain

So, it's been, uh, raining, in Newcastle.  Not that I'm surprised by this fact, though I was hoping for slightly less wet weather in July.  Sorry for the lack of blogging.  It's not for lack of results I assure you, but more from the catching up needed when moving house as I have done.  Not quite managed to fully settle in, but that's another story.

So, what I've been doing for the past two weeks, is testing the interaction of my protein with a bit of E. Coli.  We figured out that my assays (where you grow up the cells and add the protein and end up with a pretty blue gel) might be binding to a different part of E. Coli (OmpF) to the bit I want to look at.   Because of this, I did my experiment again, with the different cell mutants, and then added a mutated version of my protein that doesn't bind OmpF to see what happened.  What happened was, nothing at all.  I don't know why, but either that protein isn't concentrated enough, or something else happens, because it doesn't show up on my gel at all.
I may try remaking the gels with a different stacking buffer, as the new stacking buffer I made I tried to pH with diluted acid.  Having forgotten one of the fundamentals of A level chemistry, my buffer, well, buffered the acid.  I eventually found the glacial HCl under the fume cupboard, but that was about 70ml too late, and in a 300ml solution, I think that really didn't help.  Anyway, I tried the whole thing twice and it didn't work, so if I get a chance to make up some new gels, I have my samples in the freezer, so that should be ok.

The main thing I've been doing is ITC.  This stands for Isothermal Titration Calorimetry.  Basically, you put the two things you want to test the interactions of in the same buffer.  You do this by dialysing one protein, then making up the other sample in the solution that you dialysed the protein in.  You then put one thing in the syringe of the machine and the other in the cell.  The machine then injects the contents of the syringe into the cell a bit at a time, and measures the heat exchange, and you end up with a nice peak on a graph.  The heat exchanges are supposed to start big and get smaller as the number of possible interaction sites gets smaller.  The heat exchanges can either go up (exothermic) or down (endothermic).  Mine appear to be going up, which apparently means that my interaction A. takes place (YAY!) and B. that it through hydrophobic binding, which is also good.

I tested my full length protein then different combinations of domains.  There are three domains.  Between two sets of results I found out that one part of my protein binds what I'm looking at.

The first few rounds I did this,  I only got some very little peaks on my graph, so it was suggested that I try the interactions at different temperatures.  I got some very good heat exchanges at 38 degrees (C), but my protein is unstable at that temperature and seems to precipitate out half way through the cycle, and give slightly strange results at the end, so that was out.  25 degrees, which is what I started with gave better results the second time round, and my protein is stable at that temperature. I think I might have made one of the solutions up more concentrated, which will have helped my results.  At 12 degrees, I got some good results, small, but not insignificant peaks on my graph.  However, when putting protein into buffer, as a control, my peaks started off small and got bigger.  I have a feeling that my protein interacts with itself at 12 degrees.  The graph looks mightily strange compared to the others!

I'm now testing different lengths of what I'm adding my protein to to see what happens there.  The results are looking nice, though I panicked this morning when one of my graphs decided to have no heat exchanges at all.  Either there was something wrong with the experiment, or I accidentally put the wrong thing in the wrong bit of the machine.  Likelihood is the latter, but the other two runs turned out the same, so I will put that result down as an anomaly.

I've been surprised how excited I've managed to get over what are essentially graphs of two solutions getting minutely hotter or colder, but when you start getting results that you hypothesised were going to happen (seem to be reliving A Level statistics today), it's hard not to get excited.  Trying to resist the urge to tell everyone and distract them from their research is quite hard, but there are a few people who are doing research directly related to what I'm doing, so they have a vested interest in what I'm doing.  The lab is quite friendly though, so people will usually chip in on conversations you're having and make useful suggestions or want to know what you're doing.  I think I've been blessed with being in a particularly friendly lab, and I'm very glad about it!

Between all of this, I've been enjoying the sites of Newcastle, whilst listening to swing music, and managing the odd spot of social swing dancing, which has been lovely.  The quayside in Newcastle, looking over to Gateshead is utterly idyllic.  The combination of the regeneration, which is very smart and modern, with the old bridges and pubs and art studios in old buildings gives an amazing atmosphere, and despite the rain, there have been a few beautiful evenings perfect for the odd dance down by the river.

One last thing.  In the lab.  Never bother to wear anything less comfortable on your feet than your MOST comfortable pair of shoes.  The pain in your feet from standing up all day genuinely is not worth it.  Lab coats and trainers all the way.  :D

Third time lucky!

Yesterday, for the first time I managed to get my blot to work!  I quite literally jumped around the lab and showed everyone.  I think they were a little confused by my enthusiasm, but considering as this blot actually has bands I felt like it was a massive achievement!  Have a picture to post if I can manage it.

This time, instead of having to soak the blot in one antibody, then another one, this blot I only had to soak in one antibody, as they've manage to get the marker to attach to the first antibody.  This means that instead of having an antibody to your protein, (primary antibody) followed another antibody to your primary antibody (secondary antibody), you only need one, which cuts out half the steps.

This new one seems to have worked very well, now I've made up the various new solutions, because the method is different.  I did have to put in 5 times as much developing solution as it said, but I think the solution I had was not quite as strong as the one on the sheet.

Image is of my blot.  First column is a marker of proteins of known weight.  Next two you can't see, as they are the negative control.  Next two are pellet and supernatant from normal cells, next four are mutated cells (pellet, supernatant, P, S) and the last one is my protein on it's own.  

This shows that my protein has stuck to the cell, but not all of it.  I can tell that this is my protein, and not other proteins from the cell because my protein has a special marker on the end, called a His-tag (lots of the amino acid Histidine in a chain on the end), which my antibody can bind to.  

This is the equivalent gel that I ran my proteins on.  I did two gels with the same samples, one to blot and one to look at like this.  These are with salt added to prevent charge reactions.  You can see the marker on the left then the normal cells without my protein.  You can see on the third column, there isn't a line, where there is on the 5th, 7th, 9th and 10th columns.  The spread of bands on the other columns is the cell pellet, so this is all the contents from the cell.  The other ones are the supernatant, which should just have my protein in.  The very far right is just my protein on its own.  

So, after impatiently waiting for my blot to develop, and then jumping around the lab because it had worked, I managed to calm down enough to learn how to do ITC.  This stands for Isothermal Titration Calorimetry.  Basically, you have two solutions of molecules, both in the same buffer, and you drop one into the other and see if there is any heat given off or taken in (heat exchange).  If there is (a significant amount of) heat exchange then your two molecules bind.

There are a lot of steps to prepare the machine, but once you've done it, you can leave it to run, and get on with something else.  The main thing (apparently) is to not have any bubbles or bits floating in your solution, both of which happen on a surprisingly regular basis.  Managed to prepare my solutions, which took a bit of time, due to various different steps and needing to concentrate some of the proteins down.
Forgot how to use the nanodrop, which is a mini spectrophotometer (so it measures how much stuff you have in solution usually in grams per millilitre, by passing light through it and looking at scattering), but am now a pro, as I remembered the method I had of holding the end of the pipette like a pen, which works really well.

Whilst waiting for this to run, I grew some more cells up, as my supervisor realised that my proteins probably weren't binding to the bit of the cell I'm looking at, but rather an outer membrane protein (OmpF), already known to bind my protein.  So for this set of cells, I have added the full length protein, mutated to not bind to OmpF, and a shortened protein that also can't bind OmpF.

Tomorrow, I will boil my samples to break up all the proteins so that they run properly on a gel; make the gels that I need; run the gels; run the blot; block the blot; wash the blot; add the antibody to the blot; wash the blot again and hopefully manage to develop the blot as well.  I say hopefully, I have to do this, as I can't leave the blot at any stage for the whole weekend, otherwise it just won't work.

Will update on how all this goes next week.  Need to start writing it up before long!  

Spot tests and pipette tips

Today I came in early to start my western blot from my gel that I ran yesterday.  I arrived to find I was the first to get in.  Think I might be a little too enthusiastic about this whole placement!  

Having located the nitrocellulose membrane and blot paper, which I was pretty chuffed to find, I set my blot to run, and panicked because my protocol and the top of the blot machine told me I needed different voltages.  Fortunately my memory served me correctly and it seems to have transferred well, from staining up the gel.  

I practically skipped to the incubator where my spot tests were, hoping that they had grown and worked and looked good.  For those who don't know what a spot test is: basically you pour some agar and let it set, then mix more agar (but more wobbly agar) and growth media (essentially like a very weak liquid version of marmite (well it contains yeast extract in any case)) with the cells you want to test, and pour that on top of your agar.  You then let the layer set and drop your bacteria killing compound on in different concentrations- mine were diluted by ten each time.  Once you've left it in a warm place overnight, the cells should have grown, but hopefully not where you've put your antibiotic, where there should be a clear spot.  

A revelation for me this week was finding out that antibiotic doesn't necessarily mean the type of thing you get at the doctors, but more anything that is anti something biological, so in  this case, my protein was the antibiotic.  

Anyway, my spot tests were pretty cool.  They seem to show that with my protein in a high enough concentration it can kill mutants we weren't expecting it to.  I tested the whole molecule, then 2/3 of it and then the last third on its own, so 12 spots on each agar plate.  I will try and put a picture up of the plates at some point.  I'm really pleased with this, and am testing the other two mutants, which theoretically should be partially killed, but not fully, tomorrow.  

It's nice to have something visual to look at, it shows what you know is happening in a way that you can immediately understand.  Not that I don't love gels of course, I don't think protein analysis could happen without gels.  Also they go really strange and solid if you let them dry by themselves!

I mentioned pipette tips.  We get through them at a rate of knots, as you have you use a clean one every time you pipette a different solution.  Spent this afternoon, whilst waiting for my agar to set, putting fresh ones in coffee jars (every lab seems to use coffee jars, I can't fathom getting through that much caffeine!) ready to be autoclaved.  Might have gone a bit overboard.  I reckon we've got enough for about the next month now!

 

Tomorrow I will finish my blot and spot tests, and move house, so it's going to be busy!  Not sure what my blot is going to show, but hopefully it might flag up some differences in concentration of the protein that I'm looking at.

New week, new conditions.

So, it's already Tuesday of my second week on my summer placement.  The days have gone so quickly!  Having had an exhausting but amazing weekend swing dancing, I was ready to see if my cells wanted to cooperate this week.

As you may remember, on Friday, I developed my blot, to find........  absolutely nothing at all.  The marker had just about transferred, but that was it, despite having looked at the gel, the bands I wanted had transferred onto the nitrocellulose sheet.

So, this week, like the weather here in Newcastle, I'm changing the conditions.  I'm adding salt, to make sure my reaction isn't electrostatic, as it looked like it could be that from staining the SDS gel.  I'm concentrating the samples more, and I'm using 10 well gels instead of 15, as you can get more sample in each well, which should give a clearer band.  

And today.......  I HAVE RESULTS!!!  This was a very pleasing finish to my longest day on my placement yet.  The bands are a lot clearer than the last, (though I think I made my gel a bit strangely, as the bands are a little wavy (I blame the acrylamide)) and the cells/proteins are doing more of what we thought they might do.  Overall a good day.

Even better, I'm looking forward to the results of my spot tests tomorrow, which I hunched myself over this afternoon, in order to make the perfect circle of liquid on my lovely smooth agar.  I've also found a use for my very useless skill of being able to write in mirror handwriting:  labelling the bottom of plates, so that I can see what it is when the plates are the right way round.

I also seem to have (finally after 4 years) mastered using moles.  For some reason I've always freaked out the minute I look at them.  Today's calculations were a success, without major panic.  Must find my own calculator though, Casio calculators just aren't my thing, thanks to a very determined statistics teacher that insisted on us using Sharps.

Will update on how the spot test and tomorrow's blot go soon.  Could be a while as I'm moving house this week, but I will try and post tomorrow.  

Week one. Proteins, mutants and teenagers...

So this week has been the start of my placement.  As I already know my way round the lab (I'm am so grateful for this, it usually takes weeks) I have been able to get straight on with doing things!  I've been growing up cells, mainly E. coli mutants, and adding proteins to them.  My agar even worked.  I have a history of agar hating me.  I can make it fine, and if it has come straight from autoclave it's great, but that rarely happens, so the 'melt in the microwave' technique is the best option, and either I don't heat it enough and it's lumpy, or I heat it too much, then panic that I'll denature the antibiotic and then leave it to get too cold, and it's still lumpy.  However, thanks to being made to practice earlier this year, which I'm very glad about, this week's agar worked really well.

This brings me on to word of the week, which is Kanamycin.  I know it's only an antibiotic, but it sounds great!!!

Panicked over my SDS page gel yesterday, which unusually decided it was going to take twice as long as usual to set.  The gels ran really well today (well after I was reminded to boil the samples anyway).  For anyone who doesn't know what the gel is, it allows you to separate proteins of different molecular weights using an electrical current.  The smallest proteins travel faster so end up at the bottom.  You can stain the gel and compare the bands that appear with a marker that has known molecular masses in it.  This helps you to identify if the protein you're looking for is there or not.

Today I was particularly happy as I started my first western blot ever.  This is where after running proteins on an SDS page gel, you then use electricity to drag the proteins onto a sheet, to which you can add antibodies to probe for the protein you want.  I was surprised to find that the process involves milk powder!

Feeling a bit heady due to pouring over my gel to see if we could find the protein, and breathing in a bit too many gel destain vapours!

I've also done loads of reading, and feel a bit more clued up on the subject of my project, there are so many things to know about just one protein, you'd be surprised.

I included teenagers in the title, as today we had a school visit, which the whole lab helped out in.  It was a microbiology based set of practicals.  I took great delight in telling them they were looking at the plague, anthrax, MRSA, TB, and various other lovely diseases.  Not so sure they appreciated it so much...  Also got the opportunity to judge the 'design a microorganism' competition along with Alex, a masters student in the lab.  There were some amazing ones, including ones that give you a six pac; clean your intestines; turn your hair into cheese; made you happy;  and made your eyeballs explode, but most of all, made you die a slow and painful death.  We ended up choosing the two that didn't actually kill you.  Had a really fun day and managed to do quite a lot of lab work in between.

Looking forward to finishing my western blot tomorrow.  I will find out if my first set of experiments have worked.  So far the only thing I really know is that my basic lab skills haven't been terrible so far!  I'll write another post, hopefully early next week about how it's all  going, and perhaps make it a little shorter!

First Post!

So, not much to say, other than that my studentship starts on Monday.  Really excited to be back in the lab, especially for this studentship, but also because I know that everyone there is lovely!!!  Promised I'd take in home-made cookies on Monday, so must not forget... that and my sharpies and calculator!