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.

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British Science Week – Starting Young

During British Science Week Jill Madine went into Orchard Day Nursery in Huyton to inspire budding young scientists with exciting experiments. Following a discussion on what a real researcher does and looking at models of the heart, brain and circulatory system the children were asked to think like a real researcher and predict and draw what they thought would happen. The scientific question was what will happen when skittles sweets are placed on a plate of water. The children cam up with 3 hypotheses: they would go soggy, the colours would run or they would go bang! The children then watched with awe as the skittles made a rainbow pattern and went white. This was then followed by much discussion as to whether this was what happening in their tummies when they ate skittles!  The second experiment was to make a lava lamp and watch the coloured bubbles float around in the oil to much amazement. Now that the children were real researchers the afternoon ended with them dressing up in a lab coat and safety goggles for photographs which was enjoyed by all. Whilst it was definitely a challenge to come up with experiments for 3-5 year olds it was a fun and rewarding afternoon which hopefully inspired some future scientists.

Merseyside Young Life Scientists ‘Becoming Scientists’ Day

The Merseyside Young Life Scientists scheme offers an exciting series of events for Year 12 students interested in learning more about a career in life sciences.
Whilst most students were enjoying their Easter Break, 30 Year 12 students who are part of Merseyside Young Life Scientists were up early making their way to the School of Life Sciences for a Becoming Scientists Taster Day.
Students were able to experience a first year lecture on DNA and spend time in the teaching labs. Lecturers, Postgraduate students and Undergraduate students were on hand to show these year 12 students what being an undergraduate in life sciences is really like.

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After a busy morning, of lectures and practical laboratory experiments students caught up with their PhD mentors over lunch. Choosing a topic to research for the academic poster presentation is the next challenge for the students.
Over the next few months, students on the scheme will produce posters on a scientific topic with the help of their PhD student mentors. The culmination of their hard work will be a Merseyside Young Life Scientists conference showcasing their posters in September.

Thanks to everyone who helped out with the Becoming Scientists Taster Day!

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To find out more about studying Life Sciences at Liverpool join us at an Open Day http://www.liv.ac.uk/study/undergraduate/open-days-and-visits/ or visit our course pages https://www.liverpool.ac.uk/life-sciences/undergraduate/

Knowledge Exchange – Advancing Food Security with Sensors and Analytical Instrumentation

chulalongkorn university march 2018

Dr Simon Maher (EEE) with Dr Iain Young (IIB) and Prof Joe Spencer (EEE) in Thailand last week as part of an Institutional Links Award with Chulalongkorn University where they delivered a workshop Advancing Food Security with Sensors and Analytical Instrumentation to staff and students from Chulalongkorn University and representatives from leading Thai biotechnology and food companies. They also provided their partners on the Award, Dr Thanit Praneenararat and Prof. Tirayut Vilaivan, a prototype bespoke portable Ion Mobility Spectrometer to test for antibiotics in food utilizing technologies developed jointly with the partners. While in Thailand, the group met with several companies in the global food industry including senior managers from the CP Group (seen in the picture), a global conglomerate with revenues > $45 billion USD. The plan is to expand the utility of the devices for the food and biotechnology sector and to expand its capability to include detection and quantification of antibiotics and other pharmaceuticals, food-borne pathogens and other contaminants.

 

Shrewsbury School visit

The Lower Sixth Biology visited the Institute for Integrative Biology.

With a world-class biological research facility only a couple of hours away, it would seem silly for us not to pay them a visit each year, and once again we were generously hosted by Professor Alan McCarthy – Head of Undergraduate Admissions for Liverpool’s School of Life Sciences (and Shrewsbury School Governor). In the morning we heard talks giving overviews of the key technologies we would later be seeing.

You can read the rest of their visit report here.

 

 

 

Pinfold Junior School day at the Millennium Wood

by Meriel Jones

Getting children out of the classroom to connect with the natural world should be a feature of primary education and is also an excellent way to introduce science.  This is why, towards the end of the summer term on July 5th, children from Pinfold Junior School in Scarisbrick near Southport found themselves in their local Millennium Wood for the day.

Along with building dens, hunting for treasure and making mini scarecrows with their teachers, they went on a bug hunt with Dr James Davies, a postdoctoral associate in the Institute of Integrative Biology.  Extracting creepy crawlies from the undergrowth and then admiring dragonflies and butterflies as they flew past kept the young hunters, and James, very busy.

In addition, Patrick Hamilton, Lois Ellison and Kelly Roper, undergraduate students from the School of Life Sciences Student Outreach Society, were on hand with activities in the local church hall that was the base for lunch. Kelly said ‘We all really enjoyed the day and it has sparked some new ideas for outreach activities we can develop further. Therefore it was a beneficial experience for us as well.’

‘I would say the main thing I took away from the day was how much fun the children had applying what we had told them about adaptations, to the creation of their own creatures which had a whole range of creative/imaginative features.’

This event is the most recent in the Institute of Integrative Biology’s relationship with Pinfold School that began in 2010 and has included a project that won the annual national Rolls-Royce Eden Award for the best implemented environmental project meeting the needs of a school in 2013.

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Visit to Cannon Sharples Primary School

On Tuesday 16th of May, Marie Phelan of the Technology Directorate (NMR Metabolomics) and two interns from the Ngee Ann Polytechnic in Singapore visited Cannon Sharples Year 6 pupils. The primary school in Wigan was holding a careers week, so as part of their #raising aspirations initiative Marie was invited to talk about higher education and scientific careers. In addition, interns Shina Teo and Xin Hui Er on their 4-month placement with the University of Liverpool spoke to the pupils about college life in Singapore and their experiences at the University. The 38 pupils in attendance gathered into groups to figure out what specialist skills various careers required and to play the celebrity education quiz.

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Marie talking about her career to year 6 pupils at Cannon Sharples Primary School in Wigan.

Interns Shina Teo and Xin Hui Er     Pupils pick skills for specific careers