The meaning of #Chem4Life: Chemistry for Life and Life in Chemistry

Ladies and Gentlemen,

Welcome to the 2018 #RealTimeChem week! My suggestion for this year’s theme was the most voted on Twitter and it’s now time to explain to all of you why I proposed this hashtag.

Chem4Life

First of all, my name is Gabriele Laudadio, and I am currently pursuing my PhD at the Eindhoven University of Technology in the group of Dr. Timothy Noël. Since I was a child, I have been fascinated by chemistry and how the interaction between different molecules originated life as we know it. For this reason, as soon as I graduated high school student, I decided to go for it: I wanted to be a chemist!

From the very beginning, I realized how tough studying chemistry can be. So many different disciplines, so many different courses to attend. None of this scared me though, quite the opposite happened: the more I got into the field, the more I knew that it was worth all my time.

Learning the basics of chemistry felt like learning a new language. Atoms make up the alphabet, molecules can be used to form sentences and drawing reaction mechanisms pretty much compares to grammar: the rules are clear but there’s always an exception! The amazing thing about the chemical language? It’s universal! Chemistry can be used to communicate with any other fellow chemist around the world and to unravel the mystery of our universe.

Looking back to the last couple centuries, it’s so clear that chemistry not only serves the purposes of the chemical community, but affects society as a whole. From the understanding of microbiology to new approaches in the synthesis of bioactive compounds, from the production of polymers to the relevance of transition metal complexes in drug discovery…this list could be so long! What’s certain is that the Chemistry in all these discoveries radically changed the Life of every human being on this planet.

And that’s where the theme I suggested comes from: a reminder that Chemistry and Life are indissolubly linked together. What’s left to us chemists, scientists, and curious tweeps around the world is the task to learn the language of chemistry to interact with Nature. Have you had the chance in your career to glimpse into the bond between Chemistry and Nature? How did your research contribute to bring us one step further as society? Tell us all about it this week! This will be an awesome discussion I am certainly looking forward to!

But there’s also a second meaning for this #Chem4Life RealTimeChem week, one for all the insiders of the chemistry world. Nothing in life binds people together like common passions and interests, so this week we want to know how Chemistry impacted your own life. Which people/places/adventures changed your life as a chemist? Myself, I had the chance to meet fantastic people during my studies: many of my lab mates are among my best friends and I even had the luck to find love over chemistry books (My wife probably knew I liked her the moment I enthusiastically offered her my jealously guarded Org Chem notes during our masters )

So let’s use the great platform of Twitter to talk about Life in Chemistry!

And don’t get me wrong: I know life as a chemists is not all sunshine and rainbows. There’s many challenges all of us face when devoting our time to Chemistry, both as scientists and as human beings. So don’t hold back and feel free to start any discussions on “the dark side” of chemistry as well… (what about working-life balance, funding situation, preprint papers, issues with the peer-reviewing systems, diversity in science, intercultural awareness in international labs…)

I certainly hope that this #Chem4Life will be a great opportunity for all chemists to remind ourselves of how lucky we are to work in this great field, to inspire and support each other through real-life examples and to reflect as a community on what we hope to achieve/improve in the future.

 

Chemistry for Life and Life in Chemistry, one for all, all for one!

 

Enjoy this lovely week, and don’t’ forget the hashtag! #Chem4Life.

 

–Gabriele–

Gabriele_Laudadio_ST_PO_VH_3555_PhD

Author Biography: 

Gabriele Laudadio received his masters degree in Organic Chemistry from the University of Pisa in 2016. 
He is currently pursuing his Ph.D. in the group of Dr Timothy Noël at the Eindhoven University of Technology. 
His research focuses on the application of Green Chemistry to improve Organic Chemistry methodologies, 
combining continuous-flow microreactor technology with electrochemistry and photochemistry.

 

 

Advertisements

#RealTimeChem Week 2016 – FAQ

rtc-week-2016

Hello everybody,

It’s almost that time again, time for #RealTimeChem Week! For those not in the know, #RealTimeChem Week is a 7 day event to help raise awareness of the #RealTimeChem chemistry community on Twitter and encourage as many chemists to tweet about their chemistry as possible. During the week various events, competitions and prizes are on offer, just to make it all the more fun and exciting.

If you are completely new and want to know more about #RealTimeChem in general, then following this link to the regular FAQ, where you can learn all about the project and the community.

If you don’t use #RealTimeChem all that regularly, this is the week to give it a go. Why not take some time during #RealTimeChem to share some chemistry and connect to other chemists in the world and have some fun while you’re at it?

 

When is it?

31st October-6th November. It runs all day for all 7 days.

 

How do I take part again?

Just tweet about chemistry using the hashtag #RealTimeChem. Simple as that.

 

So whats going on during this years event?

This year has seen the proposed addition of four brand new elements to the periodic table: nihonium, moscovium, tennessine and oganesson (due to be ratified in November). And with these additions the periodic table is now full. Row 7 is done folks! Such a big change doesn’t happen all that often and it’s been just one of a number of big changes that have occurred, and not just in chemistry, in chemistry during 2016.

From a personal perspective, I became a father for the first time this year and while asking about the community for ideas for #RealTimeChem Week this year, a theme that resonated with me was a suggestion from @nadineborduas:

As such, this year’s overall event theme is “New Elements in Chemistry” i.e. #NewElemChem – but this is not just about the periodic table’s new additions, it’s all about you. What new elements have been introduced in your life as a chemist or to your chemistry this year? Perhaps you’ve just started your life as a chemist? Maybe you’ve made some new, ground breaking discoveries? Had to learn new skills? Got new equipment or glass ware?  All the new things.

Of course the event isn’t limited to the theme, but this is just a few suggestions to think upon and will also be the subject of all #RealTimeChem Blog Carnival posts (more on that below).

 

#RealTimeChem Week Advert

Below you can find some banners to help you share the word about #RealTimeChem Week 2016. Designed again by the awesome Andy Brunning of @compoundinterest (www.compoundchem.com)

rtcw-about-poster

Plus in a few different colours:

rtcw-about-poster_bluertcw-about-poster_redrtcw-about-poster_purple

#RealTimeChem Awards 2016 (31st October – 4th November)

Just like previous years, I will be offering awards for the best tweets during #RealTimeChem Week. The format has, again, changed a little due to circumstances and budget. This year, after noting that tweets are often quite slim on the weekend portion of #RealTimeChem week, I’ve decided to focus the awards on the 5 days of the working week, with The Great #RealTimeChem Cook off taking up the weekend slot.

So, all you have to do to potentially win a prize in the awards is tweet using #RealTimeChem on the Monday-Friday. There are three different awards available: Ag, Au and Pt. 10 awards will be given each of the five days (6 Ag, 3 Au, 1 Pt). Those winning the coveted Platinum award (5 total) will each win a prize, which is, as usual, a #RealTimeChem mug with this year’s snazzy logo emblazoned on it:

mug-preview

This years mug prize.

Sadly, due to severe budget restrictions this year, Gold and Silver award winners don’t get a prize unfortunately, except recognition that you are, completely awesome.

 

The Great #RealTimeChem Cook Off (5th– 6th November) 

 

It’s back! Introduced last year, The Great #RealTimeChem Cook Off celebrates the perfect combination that is chemistry and cooking.

This year’s contest is sponsored by @WileyVCH‘s society chemistry journals.* Five winners will receive a ChemPubSoc Europe package containing

A copy of What’s Cooking in Chemistry: How Leading Chemists Succeed in the Kitchen
-An exclusive #chemquackers scientist rubber duck (which you can use for all your #RealTimeChem posts!)
-Other Wiley-VCH goodies!

cpse-realtimechem-whatscooking-2016-prizes

This year’s cook off prizes

The competition only takes place on the weekend of #RealTimeChem Week. Feel free to cook something during the week, but the tweet must be shared on the weekend of November 5th-6th to count.

All you have to do to enter the competition is to tweet your culinary creation (anything cooking, baking or food related) and include #RealTimeChem #whatscooking & @ChemPubSoc_Euro at the end of your tweet. Your tweet should include a picture or video of your creation and ideally have a short description (the description can even talk about the chemistry in your cooking! It’s up to you). Alternatively, you can write/link a recipe for others to try.

Everyone who tweets a cooking-related post using these hashtags will be entered into the competition, and 5 favourites will win a prize.

Hopefully with a bit more notice this time, you’ll all have time to get some ingredients in and post a tweet. I look forward to seeing what you all come up with!


*@ChemEurJ, @ChemistrySelect, @ChemistryOpen, @ChemBioChem, @ChemCatChem, @ChemMedChem@ChemElectroChem, @ChemPhysChem, @ChemPlusChem, @ChemSusChem,@ChemPhotoChem, @EurJIC, and Eur. J. Org. Chem (all journals of @ChemPubSoc_Euro); @ChemAsianJ, @AsianJOrgChem, and @ChemNanoMat (all journals of the Asian Chemical Editorial Society); and @angew_chem (a journal of @GDCh_aktuell).

 

 

The #RealTimeChem Week Blog Carnival – #NewElemChem (31st October-6th November)

rtcw-new-elements

While the primary action for #RealTimeChem Week takes place on Twitter, there is also a blog carnival that runs alongside it. Chemistry bloggers are part of a thriving community and there are some excellent writers out there just waiting for readers.

Last year, #RealTimeChem Week had a “Back to the Future” theme, resulting in some fantastic posts on #OldTimeChem and #FutureTimeChem (highlights from last year are available here at SciTechConnect).

This year the theme is “New elements in chemistry” (#NewElemChem) and here’s the brief:

Write a blog post about the new chemistry in your life or the new life in your chemistry. The key is the “new” part. Our lives in chemistry are made up of many elements, both chemically and non-chemically speaking, and this is your chance to tell the community all about it. What new reactions have you run this year? Have you had fun with new chemicals? Did you learn something mind-bogglingly for the first time? Are you adapting to a new life situation that’s affecting your chemistry? Have you just started your life as a chemist? You can answer any of these questions and more. Write as little or as much as you like and share it during #RealTimeChem Week with #NewElemChem to be part of the blog carnival.

So, if you are a blogger, write a post and share it during the Week on Twitter using the hastag #NewElemChem. The carnival this year is being kindly hosted again by Elseviers SciTechConnect (thanks Katey Birtcher!). They will be looking out for this hashtag and will collect your blog post into a round up each day so they are all in one place for easy access.

Please note, if you don’t use the hashtag, then your post won’t be included in the carnival, so please make sure you remember to add it. I’ll also be retweeting these via @RealTimeChem to draw attention to them during the week. Happy writing!

 

Compound Interest competition

rtcw-chemunicate-competition

Want a graphic made based on your research? For this year’s #RealTimeChem Week, we’re once again after chemistry researchers who want to explain their research in easy-to-understand terms. To enter, all you have to do is write a piece no longer than 500 words, detailing your work and its potential applications. Note that it should be written so it’s understandable for an audience of non-scientists!

From the submitted pieces, three will be chosen to have graphics made based on them, and these graphics will then be featured alongside your written piece on the Compound Interest site during #RealTimeChem Week, which this year runs from 31 October until 6th November.

You can find details on how to enter here.

 

Other events/competitions

There is always room for more chemistry-based fun. If you would like to run an event or competition during #RealTimeChem Week or to sponsor one of the above events, then please get in touch with me via realtimechem@gmail.com and I’ll be happy to chat about the possibilities.

 

-Doctor Galactic-

 

 

  

It’s #Time4Chem

Hello everybody,

First off, apologies for the lack of updates recently. I’ve been rather busy so far this year both at work and at home so haven’t had the time to do much on the blog. I’m hoping to relaunch the major features like #RealTimeChemInFocus soon.

In the meantime, it’s no secret that I work for the Royal Society of Chemistry as a publishing editor. Generally, I’ve kept #RealTimeChem and the RSC apart, but this year I’ll be making a bit of an exception.

175years

The RSC is the world’s oldest chemical society and is celebrating is 175th anniversary in 2016. As such, it’s going all out this year to recognise it’s history and the chemical community.

It’s hoping that everyone will take some time this year to dedicate 175 minutes to chemistry and then share your story with the rest of the community. Obviously, I know a lot of your spend near 24/7 dedicating yourself to chemistry, but this is a good opportunity to try something different that you may not have considered before.

How does this link into #RealTimeChem and social media? Well, you can share your stories via the dedicated hashtag #Time4Chem, which I’ll be keeping an eye out for this year too.

I’d greatly encourage everyone in the #RealTimeChem community to have some fun with this if you have the time this year. It’s only 175 mins (that’s, like, less than 3 hours) The possibilities of what you can do for your 175 minutes are pretty much endless, but here’s a few suggestions:

  • Take part in some education outreach – a lot of Universities have outreach departments
  • Start a chemistry podcast/youtube channel – chat about chemistry, show off some reactions or chemistry concepts, have some fun.
  • Spend some time editing wikipedia – chemistry articles can always be updated and your knowledge may be just what the world’s biggest free encyclopedia needs.
  • Start a chemistry blog – enjoy writing? Enjoy chemistry? Why not combine both together?
  • Contribute to ChemSpider Synthetic Pages (http://cssp.chemspider.com/)

 

There are many more examples on the RSC website:

http://www.rsc.org/about-us/our-history/175-anniversary/

Just don’t forget to let the RSC know by keeping them up-to-date with #Time4Chem.

Now in particular is a great time to start, as this week is the anniversary week and sees the start of the RSC’s 175 faces of Chemistry exhibition at Burlington House in London – so why not spend some of your 175 minutes celebrating diversity in science? It runs from 22nd February to 4th March.

More information at the link:

http://www.rsc.org/diversity/175-faces/

http://www.rsc.org/events/detail/21557/175-faces-of-chemistry-exhibition

 

Ciao for now,

-Doctor Galactic and The Labcoat Cowboy-

#RealTimeChem Week 2015 Awards and Cook Off prize winners

Hi RealTimeChemists,

Once again I have to say a massive thank you every single chemist out there who took part in #RealTimeChem week. This community only exists because of you and it’s always great fun to witness the massive variety of chemists taking part from all over the world. It was nice to see tweet not just from regulars, but also a lot of newcomers. Welcome to #RealTimeChem I hope you enjoy your stay.

While the week event is over for another year, don’t forget that #RealTimeChem is a 24/7 project, so feel free to keep sharing chemistry whenever you want and engage with your fellow chemists around the world.

I have some very special thank yous to give out this year to the following folks:

  • Andy Brunning of @compoundchem fame. Not only did Andy design all of the graphics for this year, he also provided a really cool infographic competition of his own to celebrate #RealTimeChem Week. I look forward to working with Andy again in the future.
  • Katey Birtcher and all the folks at Elsevier’s SciTechConnect who ran the Blog Carnival this year and promoted the week. Your enthusiasm for the project has really kept me going and I loved the round ups.
  • Nicola Burton, formerly @Elements_UD now @SpaceBambee, thanks greatly for the #RealTimeChem Award badges
  • Kudos to the Royal Society of Chemistry and all my friends at work for supporting the project and also getting on board from time to time.
  • Finally, thanks to Guido Kemeling, Editor-in-Chief of @ChemSusChem, who was kind enough to organise the prizes for the The Great #RealTimeChem Cook Off. Thanks to all the other ladies and gentlemen at WileyVCH for the support as well.

With all of these out of the way, it’s time to get onto this years awards. This was, as ever, ridiculously tough because there were many excellent tweets all deserving a prize. Thankfully, I have a few more prizes to give out this time around!

Below you will find out the results. Drum roll please?


 

RTCWTOW

TOP PRIZE WINNERS

The three tweeters below all win a #RealTimeChem Week 2015 mug of their very own like the below:

Everyone loves a mug right? You can do all kinds of things with them!

BrianWagner

From Wednesday. Brian Wagner (@drummerboy2112) is one of THE chemists to follow on Twitter in my humble opinion and this simple demonstration of Boyle’s Law using a marshmallow, a flask and some suction was arguably the most popular tweet of the whole week in terms of retweets and favourites. Science can be fun and informative both at the same time.

Lauravanlaeren

From Wednesday. Laura van Laeren (@lauravlaeren) strikes again, sharing a lot of great tweets throughout the week, including several pictures of her beautifully painted finger nails. However, it was her starry night flask that caught a lot of attention this year and I couldn’t ignore it’s awesomeness. Pretty colours for the win!

emilyhardy

From Saturday. Speaking of pretty colours, it was National Chemistry Week in the US at the same time, with colours being the big theme. Emily Hardy (@EmilyEHardy) snuck in on the Saturday to show off some fantastic chemiluminescence. Really spectacular.

Don’t forget to DM me your address so I can post your prize to you.

RUNNERS UP

The remaining 9 Pt Awards winners will all get a #RealTimeChem keyring like the one below:

keyring

All winners, DM me your address & I’ll post your key ring to you.


NOTE: Au/Ag award winners. Unfortunately, you don’t get a prize (except the kudos),  but thank you for taking part!


GRTCCO

I was lucky this year to be given some extra prizes for a new competition. The first #RealTimeChem Cook off! There were quite a few entries and below you can find the 6 chosen winners.

The Grumpy Chemist (@Chemistry_Kat) 

Henrik Pedersen (@hacp81)

Victoria Stafford (@ToriaStafford)

Massimo Grillo (@MassimoGrillo63)

Tom Kuntzleman (@pchemstud)

Debbie Mitchell (@heydebigale)

 

All 6 of these tweeters win a copy of “What’s Cooking in Chemistry?: How Leading Chemists Succeed in the Kitchen“.

Send me your address details via DM and the folks at Wiley will send you your prize*!


 

*Book prizes sponsored by @WileyVCH’s society chemistry journals. These are: @ChemEurJ., @ChemistryOpen, @ChemBioChem, @ChemCatChem, @ChemMedChem@ChemElectroChem, @ChemPhysChem, @ChemPlusChem, @ChemSusChem, @EurJIC, and Eur. J. Org. Chem (all journals of ChemPubSoc Europe); @ChemAsianJ, @AsianJOrgChem, and @ChemNanoMat (all journals of the Asian Chemical Editorial Society); and @angew_chem (a journal of @GDCh_aktuell)


 

 

Well, that’s all folks. All awards are now given and #RealTimeChem Week is officially over for this year. Once more, thank you to everyone who took part. May all your chemistry dreams come true.

mischief managed

-Doctor Galactic & The Lab Coat Cowboy-

Announcing the host of the #RealTimeChem Week 2015 Blog Carnival

Hi #RealTimeChemists,

 

A brief update on #RealTimeChem Week 2015 (more to come). In previous years the Blog Carnival has been hosted by @JessTheChemist on her blog “The Organic Solution”. Unfortunately, Jess has moved on to pastures new and her blog is no longer that active, so I’ve sought out a new blog to host the carnival this year.

I am pleased to announce that the folks at Elsevier’s SciTech Connect have kindly volunteered to host this the #RealTimeChem Week 2015 blog carnival. You can find a preview post on that SciTech Connect have put up on the subject here, which contains all you need to know about the blog carnival.

I am really hoping that many of you chemistry bloggers out there will join in to write a blog post for #RealTimeChem Week 2015, which starts on 19th October. Once you’ve written your post don’t forget to share it during #RealTimeChem Week with one of the two topic hashtags: #OldTimeChem and #FutureTimeChem. The nice folks at SciTech Connect will be looking out for these hashtags and collecting your blog posts together in daily round ups.

 

If you need a reminder of the two topics this year:OldTimeChem-PosterFutureTimeChem-Poster

Happy writing!

-Doctor Galactic-

Joining the dark side of the Force for a week

Hello! I am Clemens, a postdoc at the University of Cambridge, and in this #RealTimeChemInFocus blog post you will follow me, a chemist, doing some “biology” a.k.a. the dark side of the Force.

 

I know it’s weird. Why would a chemist venture into the world of biology in the first place? Honestly, it just happened! Organic chemistry was my first love as an undergrad, even after my final product of a 9-step carbohydrate synthesis decided to spontaneously decompose! I still try holding on to my first love by attempting to solve the Denksport problems of Dirk Trauner’s group and I still admire elegant total syntheses. However, the dark side of the Force has always been strong in me and over the course of my PhD and postdoc, I gradually moved towards chemical biology. I can’t help it; I am simply fascinated how chemistry can give answers to complex problems by probing or perturbing cellular systems. So, without further ado, this is a typical week in my life.

Monday

Although I am not a particular fan of Monday mornings, this Monday morning is one of the toughest of the year! I just came back from an exciting week featuring the ISACS16 conference in Zurich and a 3-day music festival in Austria (Figure 1). It made me realize how similar festivals and conferences are. Long days, short nights, meeting new people and listening to some raw talent all day long.

Figure 1: A tough start to this week after a conference & festival double feature last week.

Figure 1: A tough start to this week after a conference & festival double feature last week.

Don’t get me wrong, I enjoyed it a lot, but it took a lot of energy out of me and getting up for the obligatory Monday morning group meeting at 9 a.m. is tough. I get coffee and arrive on time, a miracle! After the meeting, which is held in the Chemistry department, I postpone my plans to go to the Cancer Research UK (CRUK) Cambridge Institute, where the biology projects of our group happen, for one day and take some time to recover. After all, I missed a lot of science in the last week, as my RSS feed and email client tell me (Figure 2). Even better, I have to analyze some exciting sequencing data, which were generated in my absence. Having multiple, diverse projects running in parallel is one of the great things about being a chemical biologist. As my computer does all the hard work, aligning millions of reads to a reference genome, all I need to do is drink coffee and use the software correctly. The latter is something I am still struggling with (Figure 2). Nevertheless, at the end of the day I get everything analyzed and the results tell me that I am all set for writing my first manuscript as a postdoc! I also managed to catch up with my emails and the RSS feed so it’s time to cycle home and get some much-needed rest!

Figure 2: Clearly, I haven't figured out how to take screenshots on a mac…

Figure 2: Clearly, I haven’t figured out how to take screenshots on a mac…

Tuesday

Another morning and it is time to head to the CRUK Cambridge Institute (Figure 3), where I will spend the rest of my working week.

Figure 3: The CRUK Cambridge institute and yes, we do have the occasional sunshine here in the UK.

Figure 3: The CRUK Cambridge institute and yes, we do have the occasional sunshine here in the UK.

I am not sure how many of you fellow chemists have ever set food in a hardcore biology working environment, so let me give you a short tour. Things here are a lot cleaner and the number of fume hoods is sadly kept to a bare minimum. They are mostly used for “dangerous” phenol-chloroform extractions of nucleic acids. Being thrown into a new working environment, I always look out for things I recognize or can relate to. Lab coats are mandatory and even wearing eye protection is reinforced (Figure 4). You can also spot the occasional TLC chamber (everybody loves TLC chambers), although they are often used for a completely different purpose than analyzing your reactions.

Figure 4: Familiar sights for a chemist in a biology lab.

Figure 4: Familiar sights for a chemist in a biology lab.

Once you are feeling more comfortable in the world of biology, you might even find more similarities to your familiar chemistry lab. For working with tissue cultures, we have special hoods that remind me a lot of glove boxes. Instead of using an airlock you are using ethanol to decontaminate everything before placing it inside the hood. Of course, once you put your thoroughly washed hands inside, your nose starts to itch. Another similarity to the familiar glove box; you have to keep the place spotless as contamination with evil bacteria or yeast will spoil not only your cells, but could affect the cultures of a whole lot of other people (Figure 5). This scenario is especially bothersome, when you have worked for months creating a cell line for a particular disease you’re studying, only to find it contaminated and yourself right back at the start of your project.

Figure 5: Things you do not want find in your mammalian cell cultures! (https://www.microscopyu.com/articles/livecellimaging/livecellmaintenance.html)

Figure 5: Things you do not want find in your mammalian cell cultures! (https://www.microscopyu.com/articles/livecellimaging/livecellmaintenance.html)

Unfortunately for you, I won’t culture any cells this week, so I can’t show of my recently acquired and still embarrassingly clumsy skills, but I encourage anyone who’s curious to give it a go. It is surprisingly simple to culture mammalian cells like HeLa or HEK293 and as a chemist you have enough skills in your repertoire to learn it quickly. Because you have to work carefully and be gentle with the cells, I always picture myself handling tert-BuLi, which freaks me out, but my cells seem to appreciate the gentle treatment.

The plan for the week is to continue with a project I stopped working on before my week abroad. To cut a rather long story short, we identified some potential protein targets in a screen and are now keen on validating these hits. To get an independent confirmation, we need to clone all  28 proteins of interest (P.O.I.) into a transfection vector and express them inside the cell as a tagged version, in order to confirm the interaction by Western blot. That should suffice to give you a rough idea, and the rest of my day is spent planning everything and diluting 56 primers to the right concentrations. By the time night falls, my pipetting thumb has had a good workout!

Wednesday

I spent my PhD in an enzyme-engineering lab, so I did my fair share of cloning and from my experience I can tell you everything starts approximately like this:

Figure 6: Every good cloning starts with a successful PCR. The tricky thing is where to go from there.

Figure 6: Every good cloning starts with a successful PCR. The tricky thing is where to go from there.

For the current task at hand it is a bit trickier. For half of our P.O.I.s we were lucky and could obtain the cDNA – that is the complementary DNA synthesized from the corresponding messenger RNA – in the form of E. coli glycerol stocks that carry a vector containing the cDNA. For these proteins, cloning is easy: isolate the plasmid from the E. coli precultures and simply amplify the cDNA with the correct primers. We use primers that have 5’ and 3’ overhangs, which allow us to subclone the amplified cDNAs into the Gateway cloning system (Figure 7, http://scienceftw.wikia.com/wiki/Gateway_cloning).

Figure 7: Step-by-step workflow of Gateway cloning.

Figure 7: Step-by-step workflow of Gateway cloning.

This method is neat, because it uses a recombinase instead of restriction enzymes and the main objective is to bring your insert into the entry vector for the Gateway system. From there, you can use another recombinase and insert your cDNA into a whole bunch of different vectors that carry appropriate tags and also allows transfecting mammalian cells! Compared to 10 years ago, when I first tackled a cloning problem, this protocol is a piece of cake.

As I started the E. coli precultures from the glycerol stocks before I left yesterday, my day consists of isolating the plasmid, doing the PCR reactions, and purifying the inserts. Sounds like a walk in the park, but it takes time (a lot of pipetting again). By the end of the day, I got 12 out of 28 cDNAs ready to insert them into the entry vector. It was clearly a successful day and I leave the lab happy for my weekly basketball game!

Thursday

Today, I start the cloning of the P.O.I.s we couldn’t obtain from the cDNA in the convenient E. coli glycerol stock form. This cloning is a lot trickier, as we have to prepare our own cDNA. For this, we isolate the total RNA from HeLA cells, which involves a phenol-chloroform extraction (so dangerous!) in a real hood (so happy!). Next, we use a poly dT primer that – at least in theory – is expected to hybridize with all mRNAs in the cell as they carry a complementary poly A tail. Creating an RNA-DNA duplex allows us to reverse transcribe the whole transcriptome and generate our sought-after cDNAs. In principle, we should have our P.O.I. cDNAs in there as well, however there is no easy way of determining the concentration and whether they were fully reverse transcribed in the first place. Nevertheless, we take the crude reaction as a template for some PCR reactions. Given that we are working with a complex mixture, I start a gradient PCR – which probes annealing temperatures between 50 and 70 degrees – and hope for the best. A few hours later, I load the first 48 out of 96 PCR reactions onto an agarose gel (Figure 8), and after size separation take a look at the gel under UV light! Hurray, for 4 of 6 targets we amplified something (Figure 8). I check, whether they have the right size, which they all do, and purify them. The second batch looks equally good, which means that combined with the inserts I amplified yesterday, I got 25 out of 28 constructs ready for the recombination reaction. That’s pretty awesome and I call it a day!

Figure 8: Loading of my agarose gel on the left (hoping), results on the right (celebrating).

Figure 8: Loading of my agarose gel on the left (hoping), results on the right (celebrating).

 

Friday

It’s Friday! And it’s a special Friday, as we are invited to our bosses place for a British “summer” BBQ. I prepared some salmon-spinach roles yesterday night, but they really looked ugly so I didn’t dare taking a picture. With the BBQ coming up later in the afternoon, it will also be a short day in the lab, which suits me, as all I need to do is finishing the first stage of my cloning efforts.

To insert my cDNAs into the Gateway entry vector, all I need to do is mix the vector with my PCR products. I then add the recombinase enzyme that swaps the standard insert – a gene encoding for a toxic protein that prevents growth of false positives – with my cDNAs. After incubating the reaction for an hour at room temperature, I thaw some chemically competent E. coli cells, which I will use for the upcoming transformation. These bacteria are suspended in a buffer-DMSO mixture, conditions that promote plasmid uptake through the cell membrane when heated to 42 degrees Celsius for a short time (about a minute). This procedure is a bit cruel as it kills most of the bacteria; after all they don’t like the DMSO too much. However, some bacteria that took up the plasmid during the heat shock survive and they are allowed to recover at 37 degrees Celsius in a rich medium for half an hour. Next, they are pelleted by centrifugation and resuspended in 100 μL of medium. The E. coli suspension is finally spread onto LB agar plates that have the right antibiotic (kanamycin) in them, which ensures the plasmid is amplified while the bacteria happily divide. Normally, you would incubate at 37 degrees Celsius overnight, which will give you good-sized colonies; however, with the BBQ and the weekend coming up, I just place them on my bench, where they will incubate at room temperature over the weekend, delaying the growth by about two days (Figure 9).

Figure 9: My prize after a week of cloning! Grow E. coli, grow!

Figure 9: My prize after a week of cloning! Grow E. coli, grow!

All there’s left to do: Head home, get the ugly salmon-spinach roles out of the fridge, cycle to my bosses place and enjoy a burger and some beer at the BBQ. Obviously, rain begins to fall as soon as the BBQ starts, but hey, that’s life in the UK after all.

 Figure 10: A proper British “Summer” BBQ in the rain.


Figure 10: A proper British “Summer” BBQ in the rain.

Author biography:

ClemensClemens Mayer is a postdoc, working at the University of Cambridge under the supervision of Prof. Shankar Balasubramanian. He was born in Graz, where he completed his undergrad in 2008. Subsequently, he moved to Zurich to pursue his Ph.D. in the field of enzyme engineering. In 2014 he joined the University of Cambridge, where he is currently investigating the role of RNA structures in biological processes. Passions include coffee, basketball, the accumulation of useless knowledge, being a geek, and dreading the English summer.


If you are interested in writing a guest post for #RealTimeChemInFocus, please get in touch with @RealTimeChem on Twitter.

A week in my life

Hello! My name is Laura Jane, and I’m a PhD candidate hailing from Stellenbosch, South Africa, here to show you what a week in my #RealTimeChem life entails!

 

One of the things our group is working on is a class of molecules called dithiadiazolyls (see this paper for more). Dithiadiazolyls (or DTDAs) are sulphur- and nitrogen-containing heterocycles that exist as neutral radicals. (It is interesting to note that the SOMO, in which the unpaired electronDTDA resides, is nodal at the carbon of the DTDA ring, so it is possible to alter the nature of the R-group without significantly altering the nature of the DTDA ring.) Thiazyl radicals have been investigated as potential building blocks for the design of molecular materials with interesting and desirable physical properties, such as conductivity and magnetism. Their magnetic and electrical conducting properties relate directly to their solid state structure. Unfortunately, many DTDAs tend to diamerise in the solid state, which results in spin pairing and, consequently, loss of any magnetic or conductive properties. We therefore look into ways to override this diamerisation and direct the structure of these materials in the solid state. My project involves the use of porphyrins as supramolecular scaffolds to create novel materials.

Monday

Monday morning starts like any other, with a cup of tea and `n Ouma beskuit while I read the news, then a breakfast of fresh fruit while I check up on what’s new in the Chemistry world. After checking my email, it’s off to my supervisor’s office, to discuss my plans for the week, but more importantly – to discuss our group’s plans regarding data backups (and storing data off-campus), spurred on by the previous day’s fire at one of our neighbouring buildings. Today ended up being an office day, not a lab day. First, backing up my data. While that’s running (my laptop tends to crash if you try giving it two things to do at once), I head off on a library run. When I return, it’s time to go play catch-up by going through some data from the last two weeks that I collected, but didn’t process, as I had fallen ill.

On Tuesday afternoons I have to demonstrate (“demi”) for an undergraduate practical session. First though, marking a stack of my class’s lab reports (nothing like leaving your marking to the last moment!). By the time that is finally done, there’s only an hour or two to spend in the lab, so I catch up on the always-fun tasks such as cleaning the never-ending pile of dirty glassware, sweeping the floor, taking inventory and so on. After a quick lunch at my desk while I catch up with what’s happening on Twitter, I haul myself and my giant stack of books across the road and around the block to one of the other Chemistry buildings for my demi duty. (The Department of Chemistry and Polymer Science at SU is spread over five buildings). This semester I’m involved in second year Inorganic Chemistry, a fun course to demi for as the pracs involve fundamental concepts and lots of pretty colours! Today’s practical involves introducing the students to the concept of qualitative analysis. South Africa has a very diverse population and consequently has 11 official languages – so language policy is a very important topic. While SU has traditionally been an Afrikaans university, undergraduate programs are now mostly bilingual (with postgraduate programs typically run only in English), so it’s quite a challenge constantly switching between the two languages when explaining to the students if your brain isn’t fully engaged.

Tuesday 

Wednesday arrives and it’s time to hit the lab for some DTDA synthesis! DTDAs are very moisture sensitive, so it’s all about the Schlenk line. I work in a tiny little synthesis lab, where currently only myself and a MSc student are working in the fume hoods.  Today it’s just step one of the DTDA synthesis, first creating LiHMDS in situ (it arrives in an unusable state when purchased as-is), then – no, wait, load shedding has kicked in again. Luckily, our building can get power from back-up generators (otherwise it’s 2.5 hours without power each time), but it’s still a minute of standing around in the dark waiting for electricity to return. Once the lights are back on and the stirrer plate is working again, it’s on adding the desired aromatic nitrile to form a silylated amidine.  While those reactions are stirring away until completed, I turn my attention to my DTDA – metalloporphyrin complexations. These tend to take (what seems like) forever to form diffraction-quality crystals, so there are normally lots of these running in the background. Because of the moisture-sensitive nature of the DTDA radicals, I tend to set up these crystallizations in skinny Schlenk tubes rather than crystallization vials – it turns out that old-school test tube racks are perfect for holding these flasks when there’s only so much room to clamp flasks in your fume hood!

Wednesday

Thursday brings step two, condensation of the silylated amidine with SCl2 to form a dithiadiazolylium chloride salt. SCl2 is another reagent that we have to synthesise ourselves (from powdered sulphur and chlorine gas), and smells just about as lovely as you can guess, so luckily I don’t have many lab-mates to irritate! Once the product has formed, it’s time to filter and wash it – inertly of course. After drying in vacuo, the dithiadiazolylium chloride salt is obtained as a yellow powder. Halfway through the day, there’s a short break from the lab for group meeting. Typically, our group meetings involve one student presenting their current research and another presenting a paper in a relevant field. This week, however, was something a little bit different as our group was hosting Prof. Wais Hosseini (University of Strasbourg), who was given the opportunity to discuss some of his group’s work in molecular tectonics.

Thursday

The last thing to do for Friday is reduce the dithiadiazolylium chloride salt to the dithiadiazolyl radical. There are several ways to do this, but my favourite is a solid-state reduction using triphenylantimony. (Zinc-Copper couple in THF is another option.) If the reaction is successful, a drastic colour change from yellow to purple is observed. Purification is then achieved by means of sublimation to get shiny dark purple crystals, all ready to meet up with some porphyrins next week.

Friday

Finally, the week comes to an end and it’s time to enjoy the late afternoon sun with a glass of cold Sauvignon Blanc on the lawns of a wine farm just up the road! Life in Stellenbosch isn’t all too bad!

wine

Author biography:

 

LauraJane

Laura van Laeren is a PhD candidate at Stellenbosch University in South Africa. She is currently investigating novel thiazyl radical – metalloporphyrin complexes under the supervision of Prof Delia Haynes and Dr Katherine de Villiers. Her passions include the written word, scientific education and the Cape Winelands.

Blogs at Whimsical Science (http://www.whimsicalscience.com/) & Whimsy Is Forever (http://www.whimsyisforever.com/)