FIVE MINUTES IN L.I.T.

Posted in Science by sallybr

L.I.T., you ask? Well, that stands for Laboratory of Iron Transport, and it’s where I spend most of my waking hours. In brief, we study how bacteria such as Escherichia coli and Listeria monocytogenes get iron from the environment and “swallow it up.” The metal is indispensable for bacteria as well as all other living organisms to survive, but it is very tricky to obtain. Iron can be compared to money in the sense that everyone who has it tries to protect it from being taken away. However, bacteria developed sophisticated systems to do just that: steal the iron from you and use it to survive. Since all pathogenic bacteria need to obtain iron to cause disease, we hope that our research will lead to the discovery of new weapons to fight infections.

We thought – actually, let me give full credit to Phil for that – he thought it would be fun to walk through the lab and ask what each person was doing, in an informal and fun way. Unfortunately, two of our undergrad students were not available when the video was made: Noah Long and Dallas Hyder. It was tricky enough to get everyone else there at the same time. I think the video turned out pretty good, as it was totally improvised.

Check it out here.

It makes me very sad to see Brittany Nairn, our super star Research Professor, getting ready to leave our group. She is the person who shows up in the video once Phil goes through a closed-door to our darkened “Fluorescence Spectroscopy Room.” We wish she could stay longer as a member of our team, but unfortunately that’s not possible. She will go on to write her own story, and we know she will be successful and happy no matter the path life takes her. Our whole lab will miss her terribly…

CREDITS IN ORDER OF APPEARANCE

Yan Shipelskiy – Graduate student working super hard to develop a full system of evaluation of iron uptake in Gram-positive bacteria

Olivia Eliasson – Undergrad student working with Dr. Nairn to evaluate compounds with anti-bacterial activity linked to blocking of iron uptake

Dr. Somnath Chakravorty – Post-doc who recently joined our lab, working on iron uptake genes from Klebsiella pneumoniae

Aritri Majumdar – Graduate student performing very tricky experiments to evaluate the effect of bacterial membrane integrity on iron uptake

Ashish Somvanshi – Graduate student who just joined our lab, also working on iron uptake by Klebsiella pneumoniae

Dr. Brittany Nairn – Research Professor who joined our lab in August last year, and developed from scratch a whole system to evaluate iron uptake in a pathogen called Acinetobacter baumannii

Dr. Sally Newton – yours truly, molecular biologist by day, food blogger by night. Performing her favorite type of experiment using radioactive iron. Because, you know… some like it hot!

Director, Producer and Narrator, Dr. Phillip Klebba… My favorite scientist in the whole wide world!

Our undergrad student Noah Long, missing from the video, but proudly showing the results of his labor: the best batch of enterobactin ever purified in our lab! How cool is that look?

😉

Before I leave, I invite you to watch this video of Dr. Phil Klebba explaining in more detail the research of our group. Sorry, ladies, he is already taken.

15Oct2013

PCR AND A DANCE IN THE MIND FIELD

Posted in Articles, Science by sallybr

PCR stands for “Polymerase Chain Reaction”, but it could just as well be “Polymerase Chain Revolution”. I know that even those who do not work on DNA or molecular biology are aware that PCR is a tremendously powerful tool that influences many areas of our life. Forensics is a classic example, when PCR is used not only to help a prosecutor’s case, but what I find even more fascinating, to prevent innocent people from paying for a crime they did not commit. Many people on death row have been released from prison thanks to one of the most elegant and surprisingly simple techniques in molecular biology. Through PCR, a specific segment of DNA is replicated over and over and over inside a tiny plastic tube. The ability to make a lot of DNA starting with a few molecules opened the doors to countless types of studies, from evolution to detection of genetic and infectious diseases. For biochemists, it is actually impossible to do research without PCR. Taking our lab as an example, we use it almost on a daily basis, either to make precise alterations in bacterial genes, or to delete bacterial genes from the chromosome. Without this technology, many of our experiments could not be performed, whereas others would take months instead of days, or even hours.

The genius behind the invention of PCR is Dr. Kary Mullis, who won the Chemistry Nobel Prize exactly twenty years ago, in 1993. His own recollection of his scientific journey can be found in the fascinating (and at times controversial) book “Dancing Naked in the Mind Field”.

“We were at mile marker 46.58 on Highway 128, and we were at the very edge of the dawn of the age of PCR. I could feel it”. (Dancing Naked in the Mind Field, page 7)

Phil and I happened to travel right through that highway several times last week, and we made sure to take the book with us so we could read it under the spell of that beautiful setting.

“When you get the hang of it, science, like everything else people do for a living, is pretty straightforward. You are in the business of solving puzzles. The way to approach a puzzle is to think about it for a while, look at all the facts you can find out about it, and then take a guess. Propose a solution. The next step is to try your best to disprove your solution. Show that the pieces don’t fit together in the way that you have proposed. If you can do that, then propose another solution. And then do the same thing. Reality is a tricky little puzzle”. (K. Mullis, Dancing Naked in the Mind Field, page 50).

Through my work, I had the chance to meet incredibly impressive people. One such person was Joe Neilands, Phil’s PhD advisor from Berkeley. The impact of Neilands on Phil’s scientific career and political views was huge. Even though Joe passed away many years ago, Phil always includes a picture of him in his talks, a well-deserved tribute to the man who discovered siderophores. Siderophores (as I mentioned in the blog before) are molecules that allow bacteria and other microorganisms to survive in a world where iron is virtually unavailable. I knew that Kary Mullis was a PhD student in Neilands’ lab, in fact he was still around for a while when Phil joined the lab. I was thrilled to find out several references to his great mentor in the book.

“The lab in which I learned the most about life was presided over by Joe Neilands. (…) Joe Neilands made me aware of the present-day planet. I already knew about the universe but had spent little time thinking about today and the people around me”. (Dancing Naked in the Mind Field, page 35).

Tomorrow will be a special day for us. Kary Mullis will be in town to present a conference at our department, invited by Phil. He will have lunch with graduate students, talk to faculty, and certainly fascinate us with his recollections of the discovery of PCR. The talk is open to the public, so if you find yourself “in the neighborhood”, consider dropping by… 😉

NOTE ADDED AFTER PUBLICATION OF THIS POST: Conference will be streamed live and open to the public, so if you want to listen to him, join us by clicking here (you can also watch it later, it will be saved on the site).

I am so excited to finally meet him! I know it will be a great event for our department and a unique opportunity for graduate students to interact with someone who is not only brilliant, but is also not afraid to speak his own mind and to swim against the current, no matter how strong a current it is.

“The laws of science are demonstrable. They are not beliefs. When experiments in our century showed that Newton’s gravitational laws were not quite accurate, we changed the laws – despite Newton’s good name and holy grave in Cambridge. Relativity fits the facts better. This is the way science has been done now for almost four centuries, and because of science – not religion or politics – even people like you and me can have possessions that only a hundred years ago kings would have gone to war to own. Scientific method should not be taken lightly”. (Dancing Naked in the Mind Field, page 112).

ONE YEAR AGO: October 16: World Bread Day

TWO YEARS AGO: The US Listeria Outbreak 2011

THREE YEARS AGO: 36 Hour Sourdough Baguettes

FOUR YEARS AGO: October 16 is World Bread Day

14Nov2012

YOU ARE ALL INVITED!

Posted in General by sallybr

Cookies and coffee will be available at 3:45pm…

THE PUZZLES OF FERRIC ENTEROBACTIN TRANSPORT THROUGH FepA

When Gram-negative bacteria acquire iron, the metal crosses both the outer membrane (OM) and the inner membrane (IM). But, existing radioisotopic uptake assays only measure iron passage into the cell as the accumulation of the radionuclide in the cytoplasm. We devised a novel methodology that exclusively observes the OM transport reaction of ferric enterobactin (FeEnt) by Escherichia coli FepA. This technique, called postuptake binding, revealed previously unknown aspects of TonB-dependent ferric siderophore transport reactions. The experiments showed, for the first time, that despite the discrepancy in cell envelope concentrations of FepA and TonB (approximately 35:1), all FepA proteins were active and equivalent in FeEnt uptake, with a maximum turnover number of approximately 5/min. The accumulation of FeEnt in the periplasm required the binding protein and inner membrane permease components of its overall transport system; postuptake binding assays on strains devoid of FepB, FepD, or FepG did not show uptake of FeEnt through the OM. However, fluorescence labeling data implied that FepA was active in the fepB-minus strain, suggesting that FeEnt entered the periplasm but then leaked out. Further experiments confirmed this futile cycle; cells without FepB transported FeEnt across the OM, but it immediately escaped through TolC. These ferric siderophore acquisition systems are crucial to the pathogenesis of Gram-negative bacteria, and our results show that cathecolate siderophores, which are transported by OM receptors such as FepA, CirA, FecA and Fiu, play a defining role in colonization of the gut by E.coli.

Anyone who falls asleep will hurt my feelings! 😉

6Nov2012

THE LAB MOVE AND NEW BEGINNINGS

Posted in General by sallybr

Almost three months have gone by since we moved our lab from OU to KSU. I wish I had been able to compose a post about it sooner, but let’s just say that life has been tremendously busy ever since. Those who follow my blog might remember that our home move was a saga of epic proportions. As a consequence, I lost many nights of sleep worrying about the lab move because it’s a lot more complex, and too many things can go wrong. One of the major differences between a home move and a lab move, is a higher sense of urgency in the latter. When you move your home, you can always leave boxes hanging around, and set up just the basics: a place to sleep, a place to cook, a place to sit down and collapse at the end of the day. But, a lab is a full entity of sorts. The whole thing needs to be put in place as quickly as possible because: no lab, no work. No work, no progress. We anticipated a lag of a couple of weeks, and that’s more or less what happened. A big thanks goes to a company called TLM (Transportation Logistic Management), that moved all our stuff without breaking or losing anything! Plus, their whole crew was a pleasure to interact with, very nice group of men and women working hard and in great spirits. If you can believe it, they went through the trouble of wrapping up our large test tubes individually. And we have more than 500 of these!
One of the most stressful things about a lab move is that quick decisions need to be made on the spot. For instance, this “little” piece of equipment, called a French press, weighs a ton (well, almost a ton) and obviously once it sits on a spot, it better be its permanent home! 😉

Of all things that were moved, perhaps the most important was our – 70 C freezer. It holds all our bacterial strains, as well as very sensitive reagents. TLM had a nice strategy to deal with it: they packed the whole lab over a couple of days, and closed the truck. Next morning, at 5am they came to get the freezer, loading it just before starting the 312-mile drive. Six hours later, the freezer would be unloaded first and quickly plugged. If you don’t open the door, the temperature will stay cold enough to preserve its precious contents. Well, to add a little excitement to the adventure, once the freezer was unloaded, we realized that the plug and the outlet did not match! Frantic calls were made to electricians on campus, and they came to the rescue. Thanks to their efficiency, our freezer was plugged back when the temperature had risen only to – 44 C. Big sigh of relief!
Another very tricky piece of equipment to move was our double water-distiller. All parts are made of glass, connections are delicate and convoluted. I bet our grad student was wondering if it would ever be back in working condition…;-)

But it had a happy ending!

The huge baby shown below is a cell culture hood. We use it to work with mammalian cells, much more fragile and prone to contamination than bacteria. This machine has a special laminar flow of air that isolates the environment inside the hood. Plus, it also holds a germicidal UV lamp to sterilize that environment. It weighs a lot more than a ton, and it required a special lift to have its legs installed in place.

But once it is all said and done, setting up a new lab is a great experience!

Drawers are labeled… and stuff is stored inside….

New, clean bottles are labeled, solutions prepared, sterilized and stored, ready for use…

Chemicals are organized, benches get ready for experiments….

And we are finally back, cranking up some data! Here is a shot of our SLM Fluorometer 8100, a Rolls Royce of a machine, that needs to be operated in a dark room. It got a nice upgrade before our move, and it will be a major player in our projects in the near and not so near future.

I hope you enjoyed the glimpse on what a lab move involves. I can tell that we intend to retire here (not anytime soon, mind you!), because another move would just about kill us! 😉 Seriously, though, we are thrilled to join KSU and feeling energized and ready for this next phase in our professional life.

If you want to know a little more about our research, visit our webpage at KSU by clicking here….

ONE YEAR AGO: Honey-Oat Pain de Mie

TWO YEARS AGO: Carrot and Leek Soup

THREE YEARS AGO: Chicken Parmiggiana 101