Adventures around the water bath

The expression of proteins in cellular models or bacteria is quite common in order to study their function and structure or for large scale production.

To make a bacteria express a certain protein, the cells must take up the DNA sequence that encodes for that protein. Because proteins can be modified after production by some cell-specific mechanisms, the final protein produced by a bacteria is not necessarily exactly the same as we would find in the original environment.

Not all bacteria can take up DNA, the bacteria who can do this are called competent bacteria, and to the process of inserting foreign DNA into a bacteria, we call transformation.

About a year ago, I had to do transformation of some competent bacteria. I did not want to express the protein, I needed to be able to distinguish a sequence of DNA that had been mutated from its normal version. We were hoping that in every four bacteria: one would have taken up the mutated version, one would have the control version and the other two would have  a mix of these two sequences. Because bacteria grow very fast, overnight I would have more than 100 colonies, from which I could select 10-20 and hoping “Mendel was on my side” send this for sequencing and voilà: I would know the exact mutation I had introduced in that DNA.

I have discovered the hard-way that even competent bacteria are not so competent if the scientist is not bright. After two months of failing to see bacterial colonies, asking for help from everyone I could find in the lab, making them read my protocol, comparing it to their protocol to no avail, the issue started bugging the professor. On a hallway conversation he says “Are you sure the water bath is at 42 C?” To which I reply “which water bath? I am using the thermo-block.”. It so seems that there is a preference for water baths over heating-blocks among the older generation of scientists. Is this well supported? The short answer is: of course not!

But there are differences between water-baths and heating blocks

Water baths need a very big amount of water to function, thus once they reach a certain temperature they will be very stable in maintaining it constant throughout the entire surface. This means that every single sample will be exposed to the same temperature as the one next to it and that every single particle of a given sample will be at that temperature.

Heating blocks on the other hand are constantly giving away heat, the temperature along its surface varies a bit (I never measured but I wouldn’t expect it to be much more than a few tenths of degree), also the temperature from the bottom of the tube to the upper part should vary a bit.

Is this sufficient to make a difference in getting colonies of bacteria growing?

In our lab, we will never know: people that used to use water bath still use them, people who used heating blocks still use them. And I, I have converted! Working experiments are better than non-working ones!

In any case, how does putting bacteria at 42 C help them taking up DNA?

Basically, we “heat-shock” them: the bacteria are on ice for 30 minutes, all cold and stiff. Then they go directly into 42 C, which makes them more comfortable. Suddenly, after 50 seconds, they go back to ice. As bacteria membranes are made of lipids, and lipids are very fluid, the changes in temperature allow for pores to be open in the cell membrane, through which DNA can move in without resistant.

Os porquês da minha vida

Madeirense de raíz, mas já sem sotaque (a não ser quando falo ao telefone com alguém da ilha), sou catalogada como nunca tendo saído da idade dos porquês – assumo que seja por esta minha “irreverência” que acabei diretamente ligada à investigação científica. Fascina-me não só aprender, mas também ensinar. Sou licenciada em Biologia Celular e Molecular (BCM) pela FCT/UNL. Como as células animais eram um mistério, decidi fazer o mestrado em Biologia Celular e Molecular com especialidade em Neurobiologia pela Universidade de Coimbra, onde estudei o papel de uma molécula na morte neuronal, tentando fazer o paralelo com o que acontece após a lesão da medula espinhal. Já mais recentemente, acabei o doutoramento em Biologia Celular, Molecular e do Desenvolvimento. Durante o doutoramento vivi dois anos e meio nos Estados Unidos (colaboração com um laboratório da Johns Hopkins University) e estudei como diferentes estímulos influenciam as células estaminais embrionárias. Enquanto acabava o doutoramento fui à aventura e trabalhei como técnica de microbiologia numa CRO (Instituição de Desenvolvimento de Produtos, Desenvolvimento e Validação de Métodos Analíticos) e CMO (Produção sob contrato e Prestação de Serviços de Análises de Controlo de Qualidade) onde descobri que adoro o laboratório, mas adoro ainda mais desafios! Assim sendo, estou neste momento a transitar para uma nova etapa… aguardam-se agora desenvolvimentos…

What could have been and what indeed was

Someone once though he wanted to work in a lab; with some luck he managed to spend a summer during high school wearing a white coat following SOPs in one of those wonderful places. Therefore he decided he had to do a Ph.D. so that he didn’t need to follow them anymore. After a bachelor in Molecular and Cell Biology and motivated by another summer in a lab (in this case a pharma lab, so that he could confirm that not being accepted in this course was positive) he started turning his back to fundamental science and to the wet lab for something his colleagues didn’t like and he found interesting (data analysis, modeling and programing). With a master and Ph. D. more applied (Biotech and Bioengineering) and chasing the opportunities that came across his path he jumped between metabolic networks, systems and synthetic biology, stem cells, bioreactors, Portugal and Austria with (for now) his last jump being the vaccine production in Belgium… (to be continued)

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Alguém uma vez pensou que queria trabalhar no laboratório; com alguma sorte conseguiu passar um Verão durante o secundário de bata branca a seguir SOPs num desses maravilhosos sítios. Assim, esse alguém decidiu que queria fazer um Ph.D. para não as ter que seguir nunca mais. Após uma licenciatura em Biologia Celular e Molecular e motivado por um outro Verão num laboratório (no caso de farmácia, para confirmar que não ter entrado nesse curso foi algo positivo) foi virando as costas à ciência fundamental e ao laboratório propriamente dito por algo que os colegas não gostavam e que ele achava interessante (análise de dados, modelação e programação). Com um mestrado e Ph. D. mais aplicados (Biotec e Bioengenharia) e aproveitando as oportunidades que foram surgindo, esse alguém acabou por saltar entre redes metabólicas, biologia sintética e de sistemas, células estaminais, biorreatores, Portugal e Áustria sendo (por agora) o último salto para a área de produção de vacinas na Bélgica… (em actualização)

Era uma vez…Episódios da vida académica e científica

Nascido e criado nesta terra de sonhos que é a Margem Sul, cedo senti a necessidade de explorar o vasto mundo que nos rodeia. Foi assim natural, após demorada ponderação, a minha escolha em 2006 por uma faculdade que me permitia essa mesma exploração: afinal teria que mudar de freguesia todos os dias o que nunca tinha acontecido na minha (ainda) jovem vida. Começou assim um já razoavelmente longo percurso que incluiu uma licenciatura (Biologia Celular e Molecular), um mestrado (Biotecnologia) e um Doutoramento (Bioquímica Estrutural) na instituição de referência que dá pelo nome de Faculdade de Ciências e Tecnologias da Universidade Nova de Lisboa (no meu tempo FCT/UNL, nos dias de hoje FCT/NOVA).

Os meus actuais interesses de investigação passam pela aplicação de técnicas de Biologia Estrutural ao processo de desenvolvimento de novos fármacos (vulgo drug design) e espero, assim haja financiamento e saúde, continuar focado neste tópico nos próximos tempos.  

Quem me conhece sabe que nunca resisto a opinar, intervir em discussões e manifestar a minha opinião pelo que faz todo o sentido começar a colaborar neste projecto. Quem me conhece também, saberá que a frase anterior não corresponde exactamente à verdade o que torna a minha participação ainda mais meritória.

Veremos o que resulta daqui, mas desconfio que a maioria dos textos irão abordar aspectos e curiosidades na interface ciência-humanidades que, tantas vezes ignorada ou desvalorizada, possui um interessante potencial para ser explorado.

The one that dances

I am the one who dances.

I have been interested in life sciences research since I have first seen movies about Ebola epidemics. I dreamt of being the one to be called and save the world. I grew up and aware of the difficulty behind saving the world. I haven’t quite given up yet. After doing my bachelor studies in Cellular and Molecular Biology, from FCT/UNL, I went on for a masters in Neuroscience. My stay in Rotterdam (Erasmus MC) made me realise people around the world are more similar than different. I also start taking what is good and I like from the places where I live and try to ignore what is missing. Since then I have also lived in the UK, where I worked in a pharmaceutical company. The shock of moving from academia to industry was only surpassed by the shock of moving back to academia. I have been doing my PhD in Immunology for two years now, studying Multiple Sclerosis in Zurich. I have deleted the genes coding for pattern recognition receptors from mice and now study the effect of this deletion in the onset of experimental autoimmune encephalomyelitis (the mouse equivalent of MS).

The last two years have been a journey of discovery: a PhD project takes time to materialise, research is slow, life is stressful and results are often frustrating. We have a team of about 15 people, they are all working in slightly (or completely) different topics, I learn new thing everyday just by having coffee with my colleagues, and this really keeps me going. I have discovered I like the inter-phases: when talking with someone from a different area, we need to ask more questions, be more precise, adapt vocabulary, stop assuming everyone has the same level of understanding or access to the same information.