Team:UCL Academy/Project

Cyanobuster

Project Introduction

We aim to reduce or even eradicate the harmful effects of algal blooms. To achieve this goal, we plan to engineer a genetically modified organism (GMO) that degrades Microcystin: a toxin produced by cyanobacteria [1].

Cyanobacteria are prokaryotic and due to their diverse genus, they are able to survive in many different aquatic environments from marine to estuarine water [2]. The toxin cyanobacteria produce, microcystin, is not only toxic to the immediate environment, but also to humans, domestic animals, and livestock [3]. Algal blooms are found mainly in regions with high concentrations of nitrates and phosphates in the body of water. High nitrates and phosphates result in increased algal growth, and hence increased toxin concentrations, such as microcystin, in the water.

What functions does our GMO require? Our GMO needs to break down microcystin, remain buoyant at the level of algae, survive in harsh conditions and contain a kill switch for selective removal. We worked towards a four-module system, which we have described in detail below.

Breakdown of Microcystin

Microcystin is resistant to many common bacterial proteases due to its cyclical structure [4]. We selected the mlrA enzyme, which uses hydrolytic cleaving to linearize the structure of microcystin, and thus render microcystin more susceptible to degradation [5].

When the GMO detects microcystin, the mlrA gene is expressed in response to the detection mechanism. The mlrA gene expression triggers the production of the mlrA enzyme, which degrades microcystin.

In order to extract this gene we used 3 different organisms: Escherichia coli, Bacillus flexus, and Oceanibulbus indoliflex. By using more than one organism, we aim to expand the Registry of Standard Biological Parts.</p>

We plan to express the mlrA gene after 2 different promoters.

Promoter 1: Lead sensitive promoter, part BBa_I721001. Research shows that high levels of lead are commonly found in algal blooms [6], and so this promoter will induce the mlrA gene to be expressed in presence of high levels of lead.

Promoter 2: UV sensitive promoter, part BBa_I765001. When algal blooms occur, they occupy the surface of the water and block incoming sunlight. This reduces the amount of UV radiation in the body of water. We used a more complicated mechanism of the UV promoter that induces a repressor gene - part BBa_C0040 - when there are high levels of UV, which corresponds to little to no algal growth. The mlrA gene is inserted after a repressor-regulated promoter - part BBa_R0040 - which will continue inducing the mlrA until the repressor binds and prevents induction. This works as a reverse mechanism: if there is high UV, the repressor is expressed, which inhibits mlrA production.

Buoyancy

Cyanobacteria grow on the surface of bodies of water. Therefore our GMO needs to float at the level with the highest concentration of toxins. We aim to engineer this process by introducing a buoyancy module. The production of gas permeable vesicles within the cell raises the partial pressure of the cell and causes the GMO to become buoyant.

We planned to take the GVP gene cluster from the organism Bacillus Megaterium, based on the previous work of the 2012 UCL iGEM team. It contains 14 putative gens: gvp-A,-P,-Q,-B,-R,-N,-F,-G,-L,-S,-K,-J,-T and –U. The last 11 genes, in a 5.7-kb gene cluster, are the minimum genes required for gas vesicle synthesis and function in E. coli.

Resistance to Harsh conditions

In order to make our GMO resistant to microcystin, we inserted the IrrE gene found in Deinococcus radiodurans. The 2012 UCL iGEM team used the IrrE gene in their composite biobrick BBa_K729005. We placed the IrrE gene (BBa_K729001) after the same promoters we used in our degradation gene; the lead sensitive promoter, the UV sensitive promoter, and the repressor regulated promoter. We constructed the same circuit as for the degradation gene, however with the IrrE gene in place of the mlrA gene.

Kill switch

In the case that Microcystin are able to enter our GMO, the metabolism of the cell would change and cause the bacterium to become unstable. In water, organisms have a high tendency to accept foreign DNA, and the effects of other organisms accepting our bacterium’s DNA may not be positive. In order to prevent this from occurring, we planned to implement a kill switch in our GMO. The kill switch we planned on using was used by Paris’ Bettencourt team, based on the wild type Colicin E2 operon, which has a system for degrading our GMO’s DNA. This would have been induced by genes/proteins that are activated by stress conditions, in this instance the entry of microcystin into our bacterium.

Bibliography

[1] Carmichael W.W. Health effects of toxin-producing cyanobacteria: the CyanoHABs. Hum. Ecolog. Risk Assess. 2001;7:1393–1407.

[2] Dawson R.M. The toxicology of microcystins. Toxicon. 1998;36:953–962. [PubMed]

[3] Stewart I and Falconer IR (2008) "Cyanobacteria and cyanobacterial toxins" Pages 271–296 in Oceans and human health: risks and remedies from the seas, Eds: Walsh PJ, Smith SL and Fleming LE. Academic Press, ISBN 0-12-372584-4.

[4] Harada K.I. Chemistry and detection for microcystins. In: Watanabe M.F., Harada K., Carmichael W.W., Fujiki H., editors. Toxic Microcystis. CRC Press; Boca Raton, MI USA: 1996. pp. 103–148.

[5] Bourne D.G., Jones G.J., Blakeley R.L., Jones A., Negri A.P., Riddles P. Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin-LR. Appl. Environ. Microbiol. 1996;62:4086–4094. + Saito T., Okano K., Park H.D., Itayama T., Inamori Y., Neilan B.A., Burns B.P., Sugiura N. Detection and sequencing of the microcystin LR-degrading gene, mlrA, from new bacteria isolated from Japanese lakes. FEMS Microbiol. Lett. 2003;229:271–276.

[6] García-Hernández J1, García-Rico L, Jara-Marini ME, Barraza-Guardado R, Hudson Weaver A. Concentrations of heavy metals in sediment and organisms during a harmful algal bloom (HAB) at Kun Kaak Bay, Sonora, Mexico.

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