Posted in Careers, General, General Lab, Ph D posts

Some reading material

Seeing as a lot of us are working from home, self-isolating, or getting bombarded with rather depressing news, I thought I’d compile a list of previous posts that might tickle your fancy.

For those that want to think about a career in research:

Want to know whether you’re cut out for it?

Who works in a research lab, and how is it structured?

Not sure which lab to pick?

How does a Ph. D work?

What’s a typical day like for a Ph. D student?

Things I wish I knew before I started a Ph. D

How to write a Ph. D thesis

What is a scientific conference?

How do you write a scientific paper/manuscript?

On the long road to scientific publication

Not sure what to do after graduating from a science degree?

For those that want to know more about me and my career journey:

Part 1: High school

Part 2: First to second year Undergraduate student

Part 3: Third year Undergraduate student to Ph. D

The stuff I worked on

Moth stuff

Friends appreciation post

Trying to sort out where to live

A general post at the end of 2019 on my progress

Experimental procedures:

DNA: A general protocol of cloning (with ongoing follow up posts), plus how to look at DNA

Proteins: Protein gels and Western blots (Part 1 and Part 2)

How to study Bacterial effector translocation (i.e. transport of teeny Bacterial proteins into the host cell)

Other fun, bits and bobs:

How to say ‘I love you’ as a scientist

Teeny lab stuff

Hilarious science comics

Posted in General Lab, Ph D posts

A miniature light show in the palm of your hand

I posted about western blots and protein gels recently, but they’re actually more like a validation for some big experiments I did last week. These are the big experiments I was referring to in some previous posts. I needed some Coxiella strains that make different proteins for me (I’ve posted quite a bit on this), so that I could use them to infect some human cells.

So, when I say human cells, they’re actually cervical carinoma/cancer cells. We use cancer cells all the time in the lab because, well, they’re very hardy. Cells can divide and replicate, but every cell has a limit on how many times they can do this. Most cells replicate a few times before they close the curtain, but cancer cells… they’re practically immortal. These cells in particular are from a woman named Henrietta Lacks. There’s a whole story on how she never gave permission for her cells to be harvested, but now these cells can be found worldwide. They’re actually one of the most commonly used epithelial cell lines (epithelium is the outer layer of tissue, so these cells form a ‘skin’ like barrier around different structures) in the world.

Anyway, we have some HeLa cells (that’s what these cells are called), and we grow them in liquid culture media called DMEM (Dulbecco’s modified Eagle’s medium- weird name), with a bit of serum to boost the nutrient levels.

Image result for dmem
A typical bottle of DMEM that we use to grow our HeLa cells.

HeLa cells are epithelial cells, meaning they self-adhere to the surface of their container. When we grow them, they grow on the bottom surface of our flasks, our plates, etc. It takes about 8 hours at 37 degrees, 5% CO2 for them to gentle settle and attach.

Image result for mammalian tissue culture
A typical tissue culture incubator looks something like this.

So for my experiments, I can get these cells, count them, and ‘seed’ them into an assay plate that looks something like this:

A typical 96 well black assay plate

We set up conditions in triplicate, so there’s three wells assigned for each condition in an experiment. If you look at it from the top, you’ll see wells that are pink, that have cells/samples in them, and clear wells that don’t have anything in them. You have 96 wells, so you can test many different conditions on the one plate.

Image result for 96 well assay plate with media
Here’s an example with a clear, see through plate. You can add cells quickly by using a multi-channel pipette like the one in the image.

Once I add my cells and leave them to incubate and self-attach, I can add my relevant Coxiella strains. Some strains make an artificial protein that I’m basically forcing the bacteria to make, so that I can study its activity. One portion of the protein is a Coxiella one, but the front portion of it is a foreign protein from another bacterial species (completely unrelated to Coxiella).

The red and blue in these ‘fusion’ proteins are from Coxiella, but the yellow isn’t.

The yellow portion of these proteins is actually a bacterial enzyme that cuts/cleaves things called ‘beta-lactam’ rings. If you know your antibiotics, you may have heard of beta-lactam drugs. They’re your penicillins, amoxycillins, ampicillins… anything that ends in ‘cillin’, really. The yellow protein is called beta-lactamase, and anything that ends in ‘ase’ is a protein enzyme that can modify its target in someway. In this instance, beta-lactamase can cleave beta-lactam rings found in certain compounds. It gives rise to antibiotic resistances in certain bacteria, and makes beta-lactam drugs completely useless against them.

So why am I playing with beta-lactamase? Well, I’m not working on antibiotics, for starters, but we can use this enzyme to our advantage.

There’s a kit that we use, and this kit comes with a special chemical compound. This compound, called CCF2-AM, contains a beta-lactam ring. Under normal circumstances, when you bombard this compound with a specific wavelength of light (410 nanometres), it emits a green colour (or 520 nanometre wavelength of light). But! In the presence of beta-lactamase, this enzyme can cleave the beta-lactam ring in this CCF2-AM, and the structure changes (because it gets cut almost in half). When CCF2-AM is cleaved by beta-lactamase, the light emitted turns into blue (or 450 nanometres). This colour difference is key.

The big honey comb looking structure is the CCF2-AM compound. Beta-lactamase can basically cleave this compound and this changes the colour of light it emits.
Newton P et. al. 2016. J Vis Exp

Lets add another layer of complexity (because, why not?)

I’ve got these infected HeLa cells. They contain lots of Coxiella. Healthy, normal Coxiella have this really cool mechanism of injecting (‘translocating’) bacterial proteins into the host cell, essentially by stabbing through membranes.

It’s that structure in this diagram that’s labelled, T4SS, which stands for Type 4 secretion system. You can see how during replication inside cells, these structures can pierce membranes and inject bacteria-derived proteins into the host cell.

The crux of this experiment is to figure out whether a particular protein of interest (the red or blue protein in the ‘Plasmids’ diagram) is getting translocated (injected) through the T4SS, into the host cell.

Because I’ve physically fused my proteins of interest with beta-lactamase/yellow protein, if the protein gets translocated through the T4SS, then beta-lactamase will also be found in the host cell (outside of the bacterial cell).

SO (still with me?)- putting all of this together:

Beta-lactamase, as I said, can cleave CCF2-AM. Uncleaved CCF2-AM emits a green colour, but cleaved CCF2-AM emits blue. Cleavage/blue signal can only occur if beta-lactamse is present in the host cell, outside of the bacteria.

So what I can do is basically add CCF2-AM dye to my infections, wait a couple hours (for the dye to be absorbed by the HeLa cells and get cleaved/not cleaved by any beta-lactamase present), and then put it in a special machine and read how much blue vs green signal is getting emitted by the cells.

Image result for clariostar
The machines look like this. You pop the plate in and it will bombard the samples with whatever wavelength of light you set it to (in our case it’ll be 410 nanometres), and it will detect and quantify how much green (520 nanometres) and blue light (450 nanometres) is being emitted at the same time. Pretty nifty!

All in all it looks something like this

Technically the T4SS should be coming out of the longitudinal ends of Coxiella.
Newton P et. al. 2016. J Vis Exp

CBU0077/MceA is a specific Coxiella protein we know is translocated via the T4SS. When fused to beta-lactamase (BlaM), and is being produced by Coxiella, it will translocate out into the host cell, dragging BlaM with it. Out in the host cell, the BlaM enzyme will cleave any CCF2-AM present, and cause a shift in colour emission so that there’s more blue over green. The machine we use to read the plates spits out numbers, and we can calculate them to get a blue over green (450:520 nanometre) ratio.

You can look at it visually, too (rather than just as numbers).

You can count how many cells there are in your field of vision/image, then calculate what percentage of those are blue. The little ‘p’ means ‘Plasmid’.
Carey and Newton et. al. 2011. PLoS Pathog

The specific experiments I was doing last week used this technique, but at a more complex level involving infecting the same HeLa cell with different strains of Coxiella at the same time. It’s a little too hard to explain right now so I’ll leave it at that!

Hopefully this made some sense- it’s more just to illustrate how many layers an experiment can have (human cells, bacterial cells, plasmids, proteins, chemicals, light…), and how we can use natural systems (antimicrobial resistance) to our advantage.

Posted in General Lab, Ph D posts

No, not the beer

2020 has seriously been a weird year so far! First it’s been bushfires, and now it’s this weird, novel Coronavirus (2019-nCoV)… Aside from political issues, the world is seriously falling apart (*coughs ‘also climate change’*)!

The institute I work at has recently been featured heavily on the news, as one of the labs within has become the first one outside of China to actually grow the virus! This is fantastic news, as they can provide live virus to study it’s infectivity, and test antivirals and vaccines to cure the infection (and maybe prevent further spread). I’m very proud to be able to work in such a fantastic place, although disclaimer- I have no relationship with the lab which grew the virus. They’re just in the same building.

Do check this video out. It’s super cool. 🦠

I did read that they’ve transported the virus to the CSIRO’s Australian Animal Health Laboratory (AHHL) in Geelong. A friend of mine works there, in that his Ph. D project (on influenza) takes him out there from time to time (hey Will). I’d jokingly suggested that he stay away from us from now on, but I’ve also got a medical doctor coming and going from my house who works at the Monash Medical Centre, where at least two patients are being treated (hey Dan). My housemate didn’t like the idea of autoclaving her boyfriend before he could enter our household. Dunno why…?

But while that’s somewhat on the giggly side, I do want to stress that the quarantining jokes are jokes. Because there have also been numerous reported incidents of racist comments being made against those of East Asian ethnicities. If an individual has recently returned from a heavily affected area, with lots of confirmed cases and the risk of having been infected is high, then it makes absolute sense to exercise precaution and quarantine them for the recommended 14 days. Stay home, rest up, keep an eye on people you’ve come into contact with, and if feeling unwell- call the GP or hospital and explain the situation before you rock up.

But- if you are just walking down the street and see someone of Asian decent- perhaps they may even be wearing a mask (I’ve encountered this all week), but you then feel negative towards them in any way… that’s not okay. Maybe they’re afraid of being infected, so they’re wearing a mask as a precaution- that’s a very common reason for wearing a surgical mask. Don’t tell them to ‘go back to China’. Don’t make any references to this outbreak as ‘yellow’ or make any additional poor puns. It is totally unnecessary and coming from a place of fear and ignorance. It’s okay to feel uncomfortable as the initial, reflex reaction. Just don’t act on it. You can override that primal fear, and choose not to make derogatory comments. It’s really not that hard.

For more information on the virus, I highly recommend sticking to official websites (for Victoria, the Better Health Channel, or anything that’s government related). Misinformation is rife, and is easily spread when people are scared.

If you really want to wear a mask while going about your day, apparently a P2 mask is better than the surgical masks, although it has to be skin tight with no gaps that allow in external air. We’re not at a stage yet when it’s necessary to wear masks, but the good news for me is that I have quite a few of those lying around from when there were tonnes of bushfire smoke. Hooray…? 😥

Posted in Careers, General Lab

This is how science funding works in Australia

Twitter is currently blowing up with the above image.

Just as an FYI to my non-science friends. The National Health and Medical Research Council funds a large number of grants that researchers absolutely rely on. Early career researchers need funding to establish themselves in their fields, yet the below success rate shows how skewed the funding is toward those who are at the top of their careers, who have already established themselves in their areas of expertise. This is how science works. It’s one of the reasons why I’m choosing to leave academic research as a career. This is ridiculous.

Posted in General Lab

Lab Hierarchy

If asked, I will say I “work” in a lab, even though I’m just a student. I’m “studying”, but I do experiments and try to publish papers. It’s like an apprenticeship. You’re still a wee baby, but you’re also working at the same time. But, while I’m not actually employed by my lab to “work” in the lab, there are staff members who are actually employees.

Labs are a rather unique, dynamic environment with staff and students interacting with one another as part of a team. But there is a heirarchy, and it’s mostly based on qualifications.

Top Tier: “The Boss” (Lab Head)

The Lab Head is the official “Boss” of the lab. The lab is generally named after them, they are the face of the lab, and they’re usually the most qualified of them all. They have the most power and make the final decisions on everything that goes on in the lab. If you were applying for any position (whether it be empolyee or student), you would approach them (or their PA) first. Managing everyone’s individual projects in the lab while also making sure you’re performing more and more administrative/political roles (constantly applying for grants, teaching within the faculty, attending board meetings, etc) can be very draining if someone really enjoys the hands on, experimental side of research.

Generally speaking they will at the very least have obtained a Ph. D in their relevant field (so they’re Doctor “something”), and may even be Associate Professors or Professors (ie. further and further into the clouds).

Depending on their availabilities, they may even be student Supervisors (for instance, mine is my Lab Head), but sometimes it can be a primarily academic title, with a more available person acting in their stead.

Next level down: “Post-Docs” (Post-Doctoral Researchers)

Post-Doctoral researchers, or “Post-Docs” are in the employee category. They may be self-funded (they have a grant for themselves, so the lab doesn’t pay for their salary directly), or lab-funded (the lab’s research grant/s pay their salary). They have a Ph. D under their belt, so they are fully fledged, independent researchers of their own. They should be able to form their own ideas and map out the direction of their research, and only touch base with the Lab Head to inform them of their decisions and show results. Obviously the Lab Head can still have the final say on what Post-Docs get to do (especially if the lab is paying their salary, but also because lab consumables may still be lab funded), but they certainly have more independence than a student would. Generally they’ll have a particular research project they are working on, much like a Ph. D student would.

If the Lab Head is busy, the Post-Doc may be required to supervise students directly as they are always on the ground, so to speak. They may also cover more administrative tasks to keep the lab running in a Lab Manager type role, whether it be to order consumables, attend safety meetings, etc. Some Post-Docs may be aiming to become Lab Heads one day, and may be seeking collaborations or teaching positions to solidify their status within their research institution.

League of their own: “RAs and Lab Techs” (Research Assistants and Laboratory Technicians)

Technically they’re below Post-Docs, due to them not having completed a Ph. D (generally speaking), but may be more experienced. Also in the employee category, their primary role is to keep the lab running in every way possible. Whether it be administrative tasks, replenishing lab consumables (ordering items, making reagents to meet demand) or actually running experiments to help overall lab research, they are generally the Jack of all trades. They may have their fingers in everyone’s pies, in that they are helping with multiple different experimental projects within the lab. An experienced and efficient RA can be like the keystone in the arch that is the lab. When they leave (or go on holiday), everything collapses! Usually holds a wealth of knowledge about how to run a lab, they are certainly highly valued for their skillsets. They may also be asked to supervise students at the technical level, but predominantly lower tier students like undergraduate students, or Ph. D students just starting out in the lab.

Slave Student categories: Ph. D students

We now enter the student category. Ph. D students are postgraduate students who, especially if the degree is “by research”, will be trying to complete a Ph. D thesis by performing experiments to obtain data, and writing it all up in a scientific format. Essentially a Ph. D should be teaching you how to become an independent researcher (ie. Post-Doc), so you’ll slowly learn how to direct your project. You’ll still need to meet up with you supervisor/s to discuss your progress, and they will initially guide you along with your project. Over time (hopefully), you’ll learn to become more and more like Post-Docs, and end up going to your supervisor to tell them what you would like to do (and simply ask for the go ahead to do so). While you have your own project, later down the line you may end up supervising undergraduate students (usually pre-Honours), and collaborating with other researchers based on gained skilllsets, which, again, leads you to progress further toward a Post-Doc mentality.
A Ph. D student may be supported by a research scholarship (whether it be government, industry, or university funded), but could also be supported financially by the lab itself. For us in Australia, depending on the type of scholarship, our course fees may be subsidised or covered… which is why, for a lab, a student is beneficial- because it’s essentially free labour.

Next level down: Masters students

Also in the student category, these students are also postgraduate students who generally only have two years in their degree. They may be Masters by coursework students with a short stint in the lab, or Masters by research students who spend the majority of their degree doing a research project (with a little bit of coursework alongside). If I decided that I didn’t want to stay on to do a full Doctoral degree, I could convert it into a Masters degree to finish earlier. Admittedly the qualification will be lower, but if something happened and I just went- I can’t do this anymore, I could techically convert degrees. The opposite holds true if someone decides they’d rather do a Ph. D, provided they are already qualified to start the degree (e.g. has completed Honours or a previous Masters degree).

Peasant level: Honours students (which I think is unique to Australia)

Of the permanent members of the lab, this is the lowest tier (sorry Honours students). You’re (generally) fresh out of your Bachelors degree, but have not had much experience being in a laboratory setting. Being a 9 month degree and generally with less experience than the rest of the lab, everyone ends up treating you like the baby of the lab. But, if you enjoy your time, you could apply for Masters or a Ph. D. For a lot of students, this is the first time working full time in an actual work place, so the step from simple undergrad student can be quite big. Those that used to complain about three hour weekly pracs during their Bachelors degree are in for a nasty surprise when they end up working 10-12 hour days with no weekends.

… and trailing in at the bottom: undergraduate/high school students

Whether it be work experience, a research subject, an internship, or an award, you can get temporary students popping in who are looking to gain an insight into what life in the lab may be like. It could be a day, week, month/s, but they’ll generally be under constant supervision while they’re inside the lab.

***

Personally, I like labs where this hierarchy exists but isn’t strictly adhered to. Scheduled meetings should be respected, and if someone asks me to do something, I will do my best to do it (unless it’s to fill their tip boxes or such like- that’s just too much). There are always professional, respectful interactions which should always exist in the workplace. But, I like labs where the Lab Head takes us out for drinks and banters with us (even better if they buy us drinks). Labs where Post-Docs and students can joke and laugh with each other without feeling that status difference. It’s definitely a cultural thing, but I do like the relatively relaxed nature of the lab I’m in. It does depend entirely on the Lab and its people, though, and your own personal preferences.