LETTER TO THE EDITOR: GENETIC EDITING OF SECRETORY PATHWAY OF PENICILLIUM CHRYSOGENUM AFTER OBSERVATION OF INCREASED SECRETORY RATES IN AN INCREASED STRESS ENVIRONMENT (MICROGRAVITY), A RESEARCH PROPOSAL BY HIGH SCHOOL STUDENTS IN DUBAI AKIO SHIRALI 1 ,* , WILSON MINTER HUIJSMANS 2 ,* AND MATTEO SOTTOCORNOLA 3 ,* 1 THE CRANLEIGH SCHOOL, ABU DHABI, UNITED ARAB EMIRATES * AUTHORS HAVE CONTRIBUTED EQUALLY TO THIS WORK Copyright 2018, Fine Focus all rights reserved MANUSCRIPT RECEIVED 12 DECEMBER 2017 This piece was received as an unsolicited contribution to XX at ASM from a group of high school students in Dubai UAE. The form is that of a generally written research proposal. We opted to take it and publish it as a Letter to the Editor here, in order to serve as an example of the type of research-based as- signments in STEM curricula internationally. Please direct all queries or ques- tions to Ms. Sarah Groves, teacher using email provided. 163 In this study, we aim to amplify the secretory pathway of Penicillium Chrysogenum within the ISS or similar simulated microgravity using the miniPCR and/or RTQ-PCR and then optimizing Penicillium Chrysogenum function using CRISPR cas-9 (Clustered Regularly Interspaced Short Palindromic Repeats), a new technology in the genetics which can help in gene alteration for better drug production. The secretory pathway of Penicillium Chrysogenum is controlled by genes pcbAB , pcbC and penDE ABSTRACT CORRESPONDING AUTHORS Akio Shirali Student, Cranleigh AD, Abu Dhabi, UAE Email: shiraliakio@gmail.com Ms. Sarah Groves Science Teacher, the Cranleigh AD, PO BOX 126888 Abu Dhabi, UAE Email: sgroves@cranleigh.ae Wilson Minter Huijsmans Student, Cranleigh AD, Abu Dhabi, UAE Email: wilbuoy@gmail.com Matteo Sottocornola Student, Cranleigh AD, Abu Dhabi, UAE Email: m.sottocornola@icloud.com INTRODUCTION 164 • FINE FOCUS, VOL. 4 (2) KEY WORDS - Penicillium Chrysogenum - CRISPR cas-9 - miniPCR - RTQ-PCR - microgravity - beta – lactam antibiotics - pcbAB - pcbC - penDE The secretory pathway of microorganisms have shown promising results in regard to increased secretory rates when simulated by microgravity. Therefore, in microgravity, this microorganisms can be used to obtain higher amount of microorganisms’ secon`dary metabolites. Beta lactam bacteria are of great biotechnological interest due to their ability to secrete bioactive antibiotica as secondary metabolites. Secondary metabolites are organic compounds produced through the modification of primary metabolite synthesis. Secondary metabolites do not play a role in growth, development, and reproduction like primary metabolites do, because they are typically formed during the end of log phase or near the stationary phase of growth. The secretory pathway of Penicillium Chrysogenum is controlled by genes pcbAB, pcbC and penDE. These genes control the production of penem, a type of beta lactam antibiotics. Space flight experiments have been suggested to affect cellular processes in microorganisms. For instance, preliminary reports on the effects of spaceflight on secondary LETTER TO THE EDITOR • 165 metabolism. In our study, we plan to fill the gap between same quality higher yields versus same quality lower yields of secondary metabolites during space flights. Furthermore, Penicillium Griseofulvum is found to have a penicillin gene cluster similar to that of Penicillium Chrysogenum. No other species among the studied fungi were found to produce penicillin or to possess the penicillin biosynthetic genes, except P. verrucosum, which contains the pcbAB gene but lacks pcbC and penDE. Hence Penicillium Chrysogenum is our fungi of choice. The process of regulation of penicillin biosynthesis has been studied for many years. Specifically, the improvement of P. chrysogenum strains to obtain higher penicillin yields is a main intense objective in industrial research. To simulate the microgravity environment on earth, several models have been developed and applied to examine the effect of microgravity on secondary metabolism. The purpose of our study is to use Penicillium Chrysogenum for production of higher penicillin yields in microgravity environment. METHODS We divided the method of the experiments into two parts: 1- Freeze and fly: A simple experiment where the Penicillium Chrysogenum is monitored in a low gravity environment on earth. This acts as a proof of concept experiment. 2- Further plan: Use of data obtained by the freeze and fly experiment to edit the microbe gene structure using CRISPR technology in order to produce a permanent artificially induced stress condition to increase penicillin production rate by Penicillium Chrysogenum. 1- FREEZE AND FLY EXPERIMENT: The “Freeze and fly experiment” will be a proof of concept and a chance to gather information on how the expression rate of genes is different to the ones on plain gravity. Genes for the biosynthesis of secondary metabolites are arranged in clusters together with genes for resistance to the toxic action of secondary metabolites on the producer organisms. Likewise, mRNA templates of the Penicillin biosynthesis cluster of P. Chrysogenum - pcbAB, pcbC , and penDE will be used on the miniPCR. These templates will be prepared and purified on earth. The frozen mRNA templates will be sent on the ISS to run the miniPCR. The freeze and fly will be used with the three genes (pcbAB , pcbC and penDE) and their primers (from Ref 15): pcbAB: FWD: GAA GAC GTC ATA CTT ATT CTC TG REV: CGG CAT CGG ATA AAG AGA TCT GG pcbC: FWD: GAT TGG CGC TCC TCG TTC ACC REV: CCA TTA TTT TTC TAG TCG ACA TGG CAT CGA TTC CCA AGG CCA ATG TCC CC penDE: FWD: CCC GCA GCA CAT ATG CTT CAC ATC CTC TGT CAA GGC REV: ATG ACA AAC ATC TCA TCA GGG How can this help the world? Our Idea is there to fill any of two voids: 1. The idea can be used to increase the production of stock which then can help 166 • FINE FOCUS, VOL. 4 (2) Sample 1 - Control: no polymerase Sample 5 - Primers pair: pcbC Sample 2 - Control: no template Sample 6 - Primers pair: pcbC Sample 3 - Primers pair: pcbAB Sample 7 - Primers pair: penDE Table 1: PCR experiment will be run on the genes pcbAB, pcbC and penDE from Penicillium Chrysogenum The experiment will be done in a miniPCR in duplicate, with 8 sample reac- tions as following: While this is being done in microgravity there would be another batch from the same strain of Penicillium chrysogenum would be monitored on normal gravitational conditions. decrease the problem of a low inventory. This will make sure that in a financial sense, the constant fear of not being able to accommodate the high demand with a low supply can be cancelled or at the least reduced. 2. The increase in the stock of medicine can be help full as any charity buys the medicines at either a lower cost or in a greater quantity at the same cost which will help the underprivileged to help procure a medicinal cure to ailments that they may suffer from. We hope that our work can have a massive impact on the lives of others, as the blessing and happiness of a person is far greater than any sort monetary gain that anyone or we could gain. Considering the method that we have tried to introduce here we believe that this will have many implications on the pharmaceutical business as there are many types of medicines procurable from bacterial secondary metabolites. Here are some examples: 1. Cancers- the chemotherapy drugs come from secondary metabolites of plant. 2. Diabetes Type 1 - Humulin, the most successful medicine to treat type 1 diabetes is the recombinant insulin produced in bacteria Escherichia coli and yeast. 2- FURTHER PLAN EXPERIMENT: The expression rates of the three genes on earth would be compared to their expression rates in microgravity. This would be done using a reverse transcription method form their RNA to create a cDNA, a technique known as RT PCR. When this is done, we plan to create a draft plasmid containing an edited version of the DNA to account for the changes that have been made in the expression rates in microgravity. This plasmid would then be implanted into a cell which can express the plasmid; like F+ cell. Once F+ cell is placed with a group of F- cells, the DNA as a plasmid can move from the F+ cell to the F- cell via the help of the protein relaxosome and relaxase. This is the process of conjugation. Once complete the F+ cell could be placed in a colony with massive amounts of F- cells. This would then allow the F- cells to procure the same traits of the F+ cell, which is an increased secretory rate. LETTER TO THE EDITOR • 167 Graph 1: The Bacterial growth curve 1 4 This is to ensure that the bacterium does not create offspring that are resistant to the stress because they die at the end of the graph due to lack of nutrients and food. Why the stationary phase is important for secondary metabolites production? First, we would like to reemphasize the objective of the experiment “Genetic editing of secretory pathway of Penicillium chrysogenum , after observation of secretory rates in increased stress environment”. This idea is based on multiple facts: 1. Stress that is induced on micro bacteria increases the rate of secondary metabolite production 1 . 2. Genetic editing is now quite widely used. 3. Other studies done on bacterium with similar genomic structures have shown promising results 2 , 4 . To understand the process of secondary metabolite production, it is important to take into account that secondary metabolite production is only temporary as a results of adaptation to stress by bacteria and fungi. This best to compare to puts of Charles Darwin, one of history’s best biologist, “it is not the strongest that survives, but the species that survives is the one that is able to adapt to and to adjust best to the changing environment in which it finds itself”. To ensure that the fungi cannot adapt to the stress we propose that the edited stress is added to the micro bacteria in their stationary phase ( see graph 1 ). DISCUSSION How can this help the world?? Our Idea is there to fill any of two voids: 1- The idea can be used to increase the production of stock which then can help decrease the problem of a low invento- ry. This will make sure that in a financial sense, the constant fear of not being able to accommodate the high demand with a low supply can be cancelled or at the least reduced. 168 • FINE FOCUS, VOL. 4 (2) ACKNOWLEDGEMENTS • Dr. Wael Mohammed Osman (Khalifa University, Abu Dhabi, UAE) • Dr. Claudia Schafer (MIT, Boston, USA) • Mr. Matthew MacNaught (The Cranleigh School, Abu Dhabi, UAE) • Ms. Sarah Groves (The Cranleigh School, Abu Dhabi, UAE) • Mr. Damien Ward (The Cranleigh School, Abu Dhabi, UAE) • miniPCR (Amplyus 1770 Massachusetts Avenue Cambridge MA 02140 USA) ABOUT THE AUTHORS Akio Shirali Hello, my name is Akio Shirali, I am a 13 year old student at Cranleigh Abu Dhabi. I have always been interested in the sciences and have always excelled in mathematics. One of my main motivations is my Grandmother who was a microbiologist. My area of specialty is biology. Wilson Minter Huijsmans My name is Wilson Minter Huijsmans and I am 15 years old. I have always excelled at Mathematics and the Sciences, specifically Physics. I have won/ achieved multiple things in all these areas, with the most recent being a finalist prize in the Genes in Space UAE competition, the motivation for this paper, alongside my science obsessed teammates, the co-authors of this paper. 2- The increase in the stock of medicine can be help full as any charity buys the medicines at either a lower cost or in a greater quantity at the same cost which will help the underprivileged to help procure a medicinal cure to ailments that they may suffer from . We hope that our work can have a mas- sive impact on the lives of others, as the blessing and happiness of a person is far greater than any sort monetary gain that anyone or we could gain. Considering the method that we have tried to introduce here we believe that this will have many implications on the pharmaceutical business as there are many types of medicines procurable from bacterial secondary metabolites. Here are some examples: 1- Cancers- the chemotherapy drugs come from secondary metabolites of plant. 2- Diabetes Type 1 - Humulin, the most successful medicine to treat type 1 diabetes is the recombinant insulin produced in bac- teria Escherichia coli and yeast. LETTER TO THE EDITOR • 169 REFERENCES 1. Ana M. Calvo, Richard A. Wilson, Jin Woo Bok, and Nancy P. Keller. 2002. Relationship between Secondary Metabolism and Fungal Development. Microbiol Mol Biol Rev; 66(3): 447–459. 2. Hong Gao, Zhiheng Liu, Lixin Zhang. Secondary metabolism in simulated microgravity and spaceflight. Protein Cell 2011. 2(11): 858–861 DOI 10.1007/s13238- 011-1125-z 3. Microbiology Book Series number 2: Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology (Volume 1), Publisher: FORMATEX RESEARCH CENTER, Badajoz, Editors: A. Méndez-Villas, pp.577-588. 4. Nitin Kumar Singh, Adriana Blachowicz, Aleksandra Checinska, Clay Wang, Kasthuri Venkateswaran. Draft Genome Sequences of Two Aspergillus fumigatus Strains, Isolated from the International Space Station. Genome Announc . 2016 Jul-Aug; 4(4): e00553-16.Published online 2016 Jul 14. doi: 10.1128/ genomeA.00553-16 5. Efimov VP, Morris NR. The Lis1-Related Nudf Protein of Aspergillus nidulans Interacts with the Coiled-Coil Domain of the Nude/Ro11 Protein. J Cell Biol . 2000 Aug 7; 150(3): 681–688. 6. Marco A van den Berg, Richard Albang, Kaj Albermann, Jonathan H Badger et al. 2008. Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nature Biotechnology 26:1161-1168. 7. Eric Fourest, Catherine Canal and Jean-Claude Roux. 1994. Improvement of heavy metal biosorption by dead biomasses and Penicillium pH control and mycelial (Rhizopus arrhizus, Mucor miehei chrysogenum). FEMS Microbiol. Rev. 14(4):325-332. 8. David J. Smith, Martin K. R. Burnham, John H. Bull et al. B-Lactam antibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes. 9. . Henry F. Chambers, Delphine Moreau, David Yajko et al. 1990. Can Penicillins and Other -Lactam Antibiotics Be Used To Treat Tuberculosis? EMBO J. 9(3):741-747. Matteo Sottocornola Hello, my name is Matteo Sottocornola. I’m Italian and French and speak flu- ently three languages. I’m currently studying for my A levels as I am a 16 years old. My main interests lie in the fields of Physics and Mathematics although I also take interest in subjects such as biology and chemistry.