Team:HK SSC/Description

Microcystis aeruginosa is one of the most common cyanobacteria responsible for harmful algal blooms. This cyanobacterium produces microcystin, a hepatotoxin that damages the liver. However, direct lysis of Microcystis aeruginosa may not best for the environment as it holds ecological values of heavy metal sorption and oxygen synthesis. In this project, we hope to silence the microcystin biosynthesis cluster(mcy) using a catalytically dead Cas9 (dCas9) enzyme lacking endonuclease activity. When the dCas9 enzyme is co-expressed with a guide RNA(sgRNA), the dCas9-sgRNA complex specifically binds to the McyB gene and blocks transcript elongation, leading to the repression of the McyB gene without altering the chromosome of the Microcystis. Here we provide the design of a dCas9-sgRNA expression gene in a shuttle vector that can replicate in both E.coli and cyanobacteria. We will also be conducting downstream analysis to see how our dCas9-sgRNA expression plasmid affects the microcystin-production rate and oxygen synthesis rate of Microcystis.

Project Inspiration

Our project is inspired from various aspects, including school lessons, books, news articles hiking trips, and even previous iGEM teams.

i. School lessons:

Chemistry:

We’ve learnt that certain species of cyanobacteria is capable of nitrogen fixation. We understand the ecological importance of nitrogen fixation. Microcystis aeruginosa can carry out metal sorption. However, they also produce neurotoxins and hepatotoxins, microcystin. These cyclic heptapeptides can cause lethal consequences.

Fig. 1 Chemical structure of Microcystin-LR

Biology:

We have learnt the cellular structure of cyanobacteria. From teachers and text books, we have had a brief understanding in how photosynthesis is carried out in cyanobacteria. More than half of the world’s oxygen is produced via phytoplankton (including cyanobacteria). On the contrary, we are aware that the bloom of cyanobacteria will blocked sunlight and slow down the photosynthesis rate of other water plants. Certain cyanobacteria (including Microcystis) may even produce toxins that inhibit certain enzymes in animals.

Fig 2. Microcystis Aeruginosa UTEX 2388

Geography:

Through studies in soil and water sources, we learnt how excessive use of fertilizers create eutrophic waters, which gives rise to cyanobacterial blooms. Teachers also taught us how global warming raises the temperature, which favours the rapid growth of cyanobacteria (especially Microcystis Aeruginosa).

ii. News Articles

Cyanobacterial bloom has always been a global concern. Countries from all around the world, especially countries with agricultural industries. New articles from around the world have shown economic losses and environmental damages caused by algal blooms. Severe cases of blue-green algal blooms resulted in the contamination of fresh water sources. Recreational activities in nearby areas have to be banned. Inspired by these articles, we hope to solve the problem of the accumulation of toxic substances produced by harmful algal blooms.

UK
China
US Texas
US Laowa
Lake Erie
Canada

iii. Trips

T-PARK

We visited T-PARK, at Tuen Mun Hong Kong, near Deep Bay. This picture is taken facing Shen Zhen. Oyster fisheries are common there. As seen from the photo, certain areas were found to be green. We were unsure whether the green patches are algae or not, and decided to conduct further investigation.

Fig 3. Photo taken in T-Park, Hong Kong

Hiking

Some team members discovered unusual green patches, which were thought to be algae. We were interested in knowing how these organisms impact our daily lives. As we dwell into this topic, we realized that cyanobacteria can bring both benefits and disadvantages to the ecological system. Therefore, we decided to design a project that minimizes the harms that cyanobacteria may bring.

Fig 4. Photo taken at Yuk Kwai Shan, Hong Kong by one of our members

Goals of project

The goal of our project is to remove Microcystin production from cyanobacteria Microcystis, without lysing or changing other characteristics in the cell. This is because Microcystis has its benefits in heavy metal sorption, supplying us with oxygen and many other more. We aim to maintain these abilities in Microcystis, while silencing its toxin producing gene.

Usage of synthetic biology

We used synthetic biology as the in vivo expression system in our design will not affect other species in the aquatic environment. This will pose minimal effects on the environment, while successfully silencing the toxin producing gene.

What is Cyanobacteria?

Cyanobacteria (also known as blue-green algae) are photosynthetic bacteria. They grow in warm, eutrophic freshwaters with sunlight, and obtain their energy through photosynthesis, making them the only photosynthetic prokaryotes. These cells can produce large surface blooms through rapid cell division. They are capable of nitrogen fixation, oxygen synthesis e.t.c. They also act as a food source for animals and insects.

Fig 5. Cyanobacteria bloom in Toledo, Ohio (https://waterandhealth.org/safe-drinking-water/treatment/harmful-algal-blooms-cyanobacteria-and-safe-drinking-water/)

What is Microcystis Aeruginosa?

Microcystis Aeruginosa is one of the most common cyanobacteria, well-known for producing harmful algal blooms by rapid cell division and producing toxins like Microcystin (see section below). However, it has its ecological value of heavy metal ion sorption (including Cadmium, Zinc, Antimony). It also acts as a food source for other species. Besides, it is capable of oxygen synthesis via photosynthesis.

What is Microcystin?

Microcystin are cyclic non-ribosomal peptides synthesized by most strains of Microcystis. With the cell division of Microcystis and sufficient sunlight, large amounts of Microcystin can be produced within a few weeks. It can pollute a whole water source, and eventually affect drinking water. Currently, over 50 different Microcystin peptides have been discovered. In this project, we will be targeting the most common Microcystin, Microcystin-LR. These peptides are a kind of hepatotoxin (toxin that damages the liver). They specifically and irreversibly inhibit protein phosphatase PP1 and PP2A in animals and human body, which are responsible controlling muscle contractions, glycogen metabolisms etc. Human illnesses caused by Microcystin ingestion has been recorded, with symptoms of stomach cramps, vomiting, diarrhoea, and pains in muscles and joints. In fact, there was a massive outbreak of acute liver failure in a dialysis centre in Caruaru, Brazil, which was later found to be due to Microcystin contamination. 100 patients developed acute liver failure and 52 died. Microcystin toxin is hard to destroy. It cannot be destroyed by boiling. They are highly soluble in water, and can persist for 21 days to 2-3 months in solution and up to 6 months in dry scum in dark. Guidelines and regulations have been imposed by the United States Environmental Protection Agency and the World Health organization in controlling the concentrations of Microcystin in drinking water.

Microcystin Biosynthesis Gene Cluster

The Microcystin Biosynthesis Gene Cluster (mcy) is responsible for Microcystin production. The clusters contain enzymes like peptide synthetase and polyketide synthase modules encoded by 10 mcy genes. These enzymes catalyse the formation of the Microcystin peptide. From previous researches, it was found Microcystin could not be produced without the McyB gene.

Fig.6 The Microcystin Biosynthesis Gene Cluster (Credits to Kurmayer, Rainer & Christiansen, Guntram)

Killing Microcystis Aeruginosa?

Theoretically, the lysis of Microcystis would lead to no production of Microcystin. However, Microcystis Aeruginosa has its ecological value of heavy metal ion sorption (including Cadmium, Zinc, Antimony) and nitrogen fixation. It also acts as a food source for other species. Besides, it is capable of oxygen synthesis via photosynthesis, supplying us with plenty of oxygen. Without these, the balance in nature could be disrupted and may lead to irreversible effects.

How should we solve the toxin producing problem without lysing Microcystis?

We decided to use utilize the in vivo CRISPRi system, involving the a dCas9 enzyme and a sgRNA. dCas9 or ‘dead’ Cas9 is a mutated version of Cas9 that has lost its endonuclease activity - it no longer cleaves double-stranded DNA, instead dCas9 simply binds to it. With the help of a guide RNA, it specifically binds to the target, usually 20 -30 bp , and blocks transcript elongation by RNA polymerase. This silences the gene without altering the cells chromosomes. In this project, we used dCas9-sgRNA to bind to McyB gene. dCas9 enzyme will then serve as a repressor to repress the expression McyB gene.

Fig.7 Comparison of functions of Cas9 enzyme and dCas9 enzyme. (Credits to Qi, Lei S)

Our Aims

Refrececes

ZENG Jin, ZHAO DaYong, JI YongBan & WU QingLong . “Comparison of heavy metal accumulation by a bloom-forming cyanobacterium, Microcystis aeruginosa .” Chinese Science Bulletin (2012): 3970-3797. Article.

Fengchang Wu, Fuhong Sun, Shan Wu, Yuanbo Yan, Baoshan Xing. “Removal of antimony (III) from aqueous solution by freshwater cyanobacteria Microcystis biomass.” Chemical Engineer Journal (2011): 172-179. Journal.

Runnegar M, Berndt N, Kong SM, Lee EYC, Zhang LF. 1995. In vivo and in vitro binding of microcystin to protein phosphatase 1 and phosphatase 2A. Biochem Biophys Res Commun 216:162–169. doi:10.1006/bbrc.1995.2605

Jing Liang, Tan Li, Ya-li Zhang, Zong-lou Guo, Li-hong Xu. “Effect of microcystin-LR on protein phosphatase 2A and its function in human amniotic epithelial cells.” Journal of Zhejiang University Science B (2010): 951-960. Journal.

National Center for Biotechnology Information. PubChem Database. Microcystin-LR, CID=445434, https://pubchem.ncbi.nlm.nih.gov/compound/Microcystin-LR#datasheet=LCSS (accessed on Oct. 13, 2019)

Azevedo S. M. F. O., Carmichael W. W., Jochimsen E. M., Rinehart K. L., Lau S., Shaw G. R., et al. (2002). Human intoxication by microcystins during renal dialysis treatment in Caruaru-Brazil. Toxicology 181 441–446. 10.1016/S0300-483x(02)00491-2

Rao, P. V. L., Gupta, N., Bhaskar, A. S. B., and Jayaraj, R. (2002). Toxins and bioactive compounds from cyanobacteria and their implications on human health. Journal of Environmental Biology, 3: 215-224.

Rapala, J., Niemela, M., Berg, K., et al.(2006). Removal of cyanobacteria, cyanotoxins, heterotrophic bacteria and endotoxins at an operating surface water treatment plant. Water Science and Technology, 54(3): 23-28.

Kurmayer, Rainer & Christiansen, Guntram. (2009). The Genetic Basis of Toxin Production in Cyanobacteria. Freshwater Reviews. 2. 31-50. 10.1608/FRJ-2.1.2. Dittmann, Elke. “Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis Aeruginosa PCC 7806.” Molecular Microbiology (1997): 779–787. Journal.

ZENG Jin, ZHAO DaYong, JI YongBan & WU QingLong . “Comparison of heavy metal accumulation by a bloom-forming cyanobacterium, Microcystis aeruginosa .” Chinese Science Bulletin (2012): 3970-3797. Article.

Fengchang Wu, Fuhong Sun, Shan Wu, Yuanbo Yan, Baoshan Xing. “Removal of antimony (III) from aqueous solution by freshwater cyanobacteria Microcystis biomass.” Chemical Engineer Journal (2011): 172-179. Journal.

Beversdorf, Lucas J et al. “The role of nitrogen fixation in cyanobacterial bloom toxicity in a temperate, eutrophic lake.” PloS one vol. 8,2 (2013): e56103. doi:10.1371/journal.pone.0056103