Inspiring the next generation of NMR scientists during British Science Week

On Friday 15th March 10 chemistry A-Level students from Range High School visited the Institute for the annual Analytics Day held in the NMR Centre for Structural Biology organised by Dr Jill Madine and Dr Marie Phelan. This visit has been an annual event for the past several years which the students look forward to in order to gain enhanced understanding of NMR to help with their A-level courses and also provide an opportunity to chat with PhD students about what is involved in University life and academic research.  The students were given lectures on the basic applications of mass spectrometry and NMR from Stephen Moss (School of Physical Sciences) and Dr Marie Phelan. This was the followed by practical workshops where the students carried out chromatography and learnt to prepare and run NMR samples along with how to interpret the data.  Prior to their visit, as part of a school practical, they have made salicylic acid – a precursor for aspirin. We obtained these samples and collected NMR spectra of their products ready for analysis on the day.  This enabled them to establish how successful their synthesis had been and compare their results across the class, with previous years’ students (and to the teacher!). The pupils response at the end of the day was that they had learnt a lot and they can now ‘do’ NMR. Watch out for future budding NMR Nobel Prize Winners inspired during British Science Week in IIB!

Pupils were assisted on the day by Michelle Tan, Adika Sen (visiting interns in the NMR Centre), Zain Ghanameh (IACD), Jeremy Chazot (IACD) and James Torpey (IIB).

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Plant Power at the VGM

On Thursday 21 February 2019, during school half term the Walley group were at the Victoria Gallery and Museum (VGM).  We took with us a display of cultivated brassica crops, and their crop wild relatives to demonstrate how over many years the weedy Brassica oleracea crop wild relatives have been domesticated and bred into so many different vegetables through selection.

Visitors had a go at performing crosses between different brassica crops by transferring pollen between flowers using paint brushes and, inspired by how new vegetables can created, such as flower sprouts or ‘Kalettes’ (crossing a sprout with a kale plant), designed their ideal or fantasy brassica plants for us to display.

The methods of modern plant breeding that we are using within the BRESOV project (Breeding for Resilient, Efficient and Sustainable Organic Vegetable production) were discussed and compared to conventional plant breeding.

Visitors had the opportunity to taste many of the different vegetables derived from wild Brassica oleracea, including cauliflower, sprouts, kale, kalettes, red cabbage, pak choi, chinese cabbage and broccoli- they were very popular!

 

Learning synthetic biology techniques in Denmark – Johnston Post Doc Fund report

Learning synthetic biology techniques in Denmark – Johnston Post Doc Fund report

Guest post by Dr Hannah McCue, postdoctoral researcher at the Institute of Integrative Biology

With the help of IIB’s Johnston Postdoctoral Development Fund, I was able to visit a world-leading lab in Denmark in order to enhance my knowledge of advanced synthetic biology techniques. Prof Mortensen’s lab is situated at the technical University of Denmark (DTU) located in Lyngby, just outside central Copenhagen. The Johnston Fund kindly covered costs for my travel and AirBnB accommodation close to the DTU, giving me almost two weeks to experience life working at the DTU and learning novel molecular biology techniques.

The key aim of my trip was to learn the ‘tricks of the trade’ of Uracil-Specific Excision Regent (USER) cloning, a technique which multiple scientists at the university have struggled to utilise. In principle, USER cloning should be a straight forward one-pot cloning reaction which holds several advantages over other traditional and more modern cloning methods. Specifically, USER cloning utilises a ligation-free protocol, generates highly specific sticky ends and does not rely on the presence of restriction enzyme recognition sequences. The premise of USER cloning is that by incorporating a single deoxyuracil around 8-12 bases from the 5’ end of each primer, highly specific and long sticky ends can be created on the resulting PCR product with the USER enzyme mix. USER enzyme contains uracil DNA glycosidase (UNG) which excises uracil nucleotides from PCR products and DNA glycosylase-lyase endo VIII which releases the sequence upstream of the uracil nucleotide. The overhangs created are sufficiently long that DNA assembled into a circular plasmid is suitably stable to be transformed into bacteria without prior ligation.

My visit to Prof Mortensen’s lab gave me hands on experience of USER cloning alongside established experts in the field of cell factory construction and engineering. Whereas my expertise lies mainly with the use of bacteria for the production of heterologous proteins and secondary metabolite pathways, Prof Mortensen’s lab focuses on yeast and fungi such as Aspergillus. The main focus of the lab is the discovery of valuable products from fungi and the development of optimal cell factories for their production. To this end, they use CRISPR technology both to insert gene pathways into the organism of interest and to regulate the pathway to give optimal output of the desired molecule.

I was lucky enough to work alongside Dr Katherina Vanegas Garcia who developed “SWITCH” and “TAPE” techniques to help speed up strain construction when developing yeast cell factories. Using these techniques strains can be generated that can iteratively switch between a genetic engineering and a pathway control state. For instance a multi-gene pathway can be inserted into an innocuous location in the genome of the desired strain using Cas9 nuclease in genetic engineering mode. Subsequently the cell factory can be switched into the pathway control state using a dCas9 mutant to up or down regulate different genes in the pathway and monitor the effects to optimise final product yield. She also helped developed a Technique to Assess Protospacer Efficiency (TAPE) whereby the efficiency of particular sgRNA protospacer sequences are assessed for their efficiency to target Cas9 to genomic DNA and cause double strand breaks. The principle is that double strand breaks are lethal in yeast and therefore the efficiency of a protospacer sequence should be reflected in the survival rate of transformants in the absence of a repair template. This technique is also applicable in Aspergillus nidulans NID1 strain which is deficient for non-homologous end joining and hence double strand breaks will also be lethal in this strain.

I designed two experiments to test the application of USER cloning for future use in GeneMill. The first was to assemble 5 stretches of DNA encoding an operon of 13 genes and spanning almost 14 kilobases. USER overhangs were designed to assemble these genes into a USER backbone developed by Dr Vanegas Garcia. Unfortunately, a plasmid encoding all 13 genes was not obtained from these experiments, however, staff and students at the DTU have succeeded in cloning large gene constructs in this manner. Presumably there is an issue with the specific DNA sequence used in this construct which has also proved problematic when using other cloning techniques in the past.

The second experiment was to clone three sgRNA protospacer sequences into a USER backbone designed for CRISPR in Aspergillus nidulans. This cloning was successful on the first attempt and subsequently I was able to carry out CRISPR TAPE experiments to assess the efficiency of targeting of the protospacer sequences to my gene of interest in A. nidulans. All three sgRNA constructs were lethal in NID1 strain when compared to the control transformation showing that all three protospacer sequences were highly efficient. In parallel, I also transformed each sgRNA along with a repair oligo to insert single amino acid changes in my gene of interest. Unfortunately, all three transformants were extremely sick with only one colony from one sgRNA proving viable. This could indicate either that the mutations encoded by the rescue oligos were also lethal or repair using the rescue oligo was not achieved. Without viable transformants to PCR from this is difficult to check. Instead I plan to design oligos encoding silent mutations in the hope that I will then obtain viable transformants.

In summary, my visit to the DTU gave me the opportunity to test USER cloning in both challenging and simple applications. I was also able to conduct a series of CRISPR experiments in A. nidulans, an organism with which I had no prior experience. In addition to receiving hands-on training in the lab, I was given the opportunity to speak to members of different research groups and attend a number of research seminars during my stay. Research areas ranged from discovery of novel antibiotics in fungi to pleasant smelling moss that can be used as an alternative to air freshener! Of particular interest was the Diversify project which is a huge collaboration between many different researchers at the DTU and industrial partners Novozymes and Novo Nordisk. This project aims to take hundreds of yeast and fungal strains and adapt them for the aforementioned SWITCH technique by identifying innocuous sites for heterologous pathway integration. These strains can then be rapidly screened for optimal production of desired metabolites. Ambitious, high throughput, multi-partner, synthetic biology challenges such as this have the ability to change the wider approach to industrial biotechnology enabling sufficient production of useful or valuable compounds that would otherwise be ignored due to underperforming host strains.

I have been extremely privileged to have been selected for receipt of the Johnston Fund and as a consequence I have obtained invaluable experience of how another synthetic biology-focused research lab works. I have renewed enthusiasm that synthetic biology can revolutionise biological research and has the potential to have a significant impact on how we think about the future of industrial biotechnology. Not only am I now equipped to teach and supervise students and colleagues about how to utilise USER cloning, the visit to Denmark has given me a wider perspective on how to approach various industrial projects with which I am involved. I therefore believe that the experience has greatly enhanced my professional development and will aid my productivity across all aspects of my work.

Pint of Science

The Pint of Science events around Liverpool ran for three nights (14-16 May 2018), all evenings were sold out!

The Centre for Cell Imaging’s technician, Jen Adcott, took to the stage at LEAF on Bold Street on the first evening of #Pint18 to present a two minute shot talk about ‘The science of imaging, a technician’s perspective’.

Jen took on the challenge, initially not realising that PowerPoint slides were not permitted for the shot talks, only small props that could be carried on stage… adding to the challenge of giving her first public talk, by discussing microscopy without being able to show any images!

The training provided by Steve Cross, organised by the University of Liverpool, was a huge help, and it was great to see how everyone’s talks progressed following the training.

Jen talked about how technicians support scientists in imaging. She demonstrated the use of different microscopes using a laser pointer to show the principle of the laser scanning confocal microscope, with the addition of a cylindrical lens (glass stirrer borrowed from lab B!) to turn the laser beam into a Lightsheet, as used in Lightsheet imaging.

 

Unfortunately due to broadcasting issues Jen’s talk was just missed off the live Facebook feed. The recording from the night started from the third talk in, and can be viewed here. Feedback from the event was very positive, with many people stating that ‘the shot talks were the highlight of the night!’

Jens advice for anyone thinking about taking part in the Pint of Science events is

“Do it! It’s great fun, although slightly nerve racking. It’s great to be a part of an event like Pint of Science, meeting new people, learning new things, and talking science. The support and training was fantastic! I was nervous that a technician giving a talk may not go down well with a room full of people wanting to hear science, but later at the bar I had people coming up to me wanting to learn more about the microscopes – it was great! And made me realise that anyone working in science, no matter at what level can take part in events like this.”

 

Meet the Scientists

On Saturday 17th March IIB led the Meet the Scientists Event at the World Museum. Activities included stands led by the CCI and Madine group from IIB along with other stands from Life Sciences, ITM and IGH.

The CCI had a large team, and all worked together brilliantly on the Seeing is Believing stand! The team included:

Violaine See (CCI staff): Preparation of samples for imaging, and assistance at the event.

Dave Mason (CCI staff): Preparation of samples, imaging of samples, produced posters for the event, and assistance at the event.

Marco Marcello (CCI staff): Organisation of virtual reality tours of microscopy images, with Virtual Arcade

Daimark Bennett (CCI staff): Preparation of samples for imaging, and assistance at the event.

Raphael Levy (CCI staff): Preparation of samples for imaging, and assistance at the event.

Anne Herrmann (Postdoctoral researcher): Imaging of samples, preparation of printed materials for the drawing microscopy station.

Sophie Cowman (PhD student): Filmed and produced a tour of the CCI facility, which was on display during the event.

Rebecca Kelly (PhD student): Preparation of CCI postcards, set up and take down of stand, and assistance at event especially for the match the picture quiz.

Claire Kelly (PhD student): Set up and take down of stand, and assistance at event especially for the virtual reality microscopy tour.

Hammed Badmos (PhD student): Preparation of samples, and assistance at event especially for the microscope demonstrations.

Jen Francis (PhD student): Assistance at event especially for the microscope demonstrations.

Sumaira Ashraf (Postdoctoral researcher): Set up and take down of stand, and assistance at event especially for the microscope demonstrations.

Jen Adcott (CCI Staff): Organisation of the Seeing is Believing stand and co-ordinator of activities, imaging of samples, designed and produced the match the picture quiz and microscopy stickers, and assistance at the event.

Feedback from the CCI stand, seeing is believing:

Violaine See – “It was great, and the activities were all very popular. What I really liked about our exhibit is that it was real science. Well done Jen A for leading this, the result was absolutely awesome. Well done Jen F, Hammed, and Sumaira for guiding the kids with the microscopes with so much patience and enthusiasm. Dave has been an absolute star with the colouring sheets and at explaining what we do with microscopes. Rebecca and Claire have been fantastic with the quiz and virtual reality. An amazing team effort. I feel very fortunate to have you all around, you are amazing.”

Daimark Bennett – “Fantastic effort by everyone and great activities – it was great to see how busy it was even later on. The VR clearly went down a storm and everything from the stickers to the CCI movie looked really professional and well put together. It really is hard to convey the science when it’s so chaotic but I think the exhibit was pitched at the right level. In any case, my daughter, who is not easy to impress, gave the thumbs up 🙂 Well done everyone!”

Jen Adcott – “It’s great to work with such a fantastic team of people! The day was busy, and the CCI stand seeing is believing was hugely popular with many repeat visitors. I am looking forward to meeting more future scientists at the next events.”

The Madine group ran 2 activities ‘How does the heart work?’ and return of the popular ‘A lego treasure hunt for new medicines!’ with the help of PhD students James Torpey and Nathan Cumberbatch, MRes student Kiani Jeacock and undergraduate volunteers.  Visitors enjoyed learning how blood is transported around the body by watching blood cells flow around the giant circulatory system (borrowed from IACD created with a Wellcome Trust Public Engagement award, granted to Dr Valentina Barrera). Children of all ages were keen to take part in the Lego treasure hunt around the museum to find the correct drug that fit the Lego protein molecule, and be rewarded with a Lego Scientist to take home. Thanks to members of the group for their help and enthusiasm when describing the drug development process through the use of Lego.

Range High School Students annual visit to NMR Centre

On Friday 9th March 15 chemistry A-Level students from Range High School visited the Institute for a workshop in the NMR Centre for Structural Biology organised by Dr Jill Madine and Dr Marie Phelan. This visit has been an annual event for the past several years which the students look forward to in order to gain enhanced understanding of NMR to help with their A-level courses and also gain an insight into what goes on in an academic research environment.  The students were given lectures on the basic applications of mass spectrometry and NMR from Stephen Moss (School of Physical Sciences) and Dr Marie Phelan. This was the followed by practical workshops where the students carried out chromatography and learnt to prepare and run NMR samples along with how to interpret the data.  Prior to their visit, as part of a school practical, they have made salicylic acid – a precursor for aspirin. We obtained these samples and collected NMR spectra of their products ready for analysis on the day.  This enabled them to establish how successful their synthesis had been and compare their results across the class, with previous years’ students (and to the teacher!). This final part of the day is always the most exciting for the students where there is no hiding that they actually dropped their sample and scraped it off the desk!

PhD student James Torpey along with internship students Daniel Thomas and Raven Chandramohan  helped with the day providing practical and theoretical advice.

Lancashire Science festival

On Thursday 29th of June, Marie Phelan of the Technology Directorate (NMR Metabolomics) and member of the Biochemical Society helped out the Society at the Lancashire Science Festival held in the Halls of University of Central Lancaster, Preston. The event was attended by primary and secondary schools from Cheshire, Merseyside, Greater Manchester and Lancashire with the aim to engage and inspire the next generation in science technology engineering and mathematics (STEM). The Biochemical Society ran several activities in order to inform and debate genome editing including recent advances such as CRISPR-Cas9 technology with pupils as young as 6 engaging in DNA editing methodology and ethics.

Figures: Some of the Biochemical Society volunteers speaking to local pupils at the Lancashire Science festival. Pupils learn about “Scientific Scissors”. Science Festival Exhibitors Main Hall