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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