Homework: Design an useful synthetic minimal cell.

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1) Pick a function.

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1A) What would your synthetic cell do? What is the entrance and what is the exit?

 

The synthetic cell will produce lectin components (coagulant natural) in regulation with photosensors, when the environment is in the dark (turbidty medium).

Input: low light, liquid medium in turbidity

Output: Lectin (coagulant)

 

Sources:

Abd Wahid, M. A., M. J. Megat Mohd Noor, M. Goto, N. Sugiura, N. Othman, Z. Zakaria, T. Ahmad Mohammed, A. Jusoh, and H. Hara. 2017. 'Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment', Biosci Biotechnol Biochem, 81: 1642-49.

Ferreira, R. S., T. H. Napoleao, A. F. Santos, R. A. Sa, M. G. Carneiro-da-Cunha, M. M. Morais, R. A. Silva-Lucca, M. L. Oliva, L. C. Coelho, and P. M. Paiva. 2011. 'Coagulant and antibacterial activities of the water-soluble seed lectin from Moringa oleifera', Lett Appl Microbiol, 53: 186-92.

Moura, M. C., T. H. Napoleao, M. C. Coriolano, P. M. Paiva, R. C. Figueiredo, and L. C. Coelho. 2015. 'Water-soluble Moringa oleifera lectin interferes with growth, survival and cell permeability of corrosive and pathogenic bacteria', J Appl Microbiol, 119: 666-76.

Coelho, J. S., N. D. Santos, T. H. Napoleao, F. S. Gomes, R. S. Ferreira, R. B. Zingali, L. C. Coelho, S. P. Leite, D. M. Navarro, and P. M. Paiva. 2009. 'Effect of Moringa oleifera lectin on development and mortality of Aedes aegypti larvae', Chemosphere, 77: 934-8.

1B) Could this function be performed only without Tx / Tl cells, without encapsulation?

Previous studies have developed systems for the production of lectins in yeast, which are produced in bioreactors. However, the application of autonomous systems that contain a specific alternative to decontaminate liquid media

The lectins can have activity on components, therefore they require encapsultation and the whole system can be protected.

 

1C) Could this function be done by natural genetically modified cells?

 

Yes, but in bacteria it has shown a deleterious effect in E. coli with the production of lectins. However in yeasts the production of lectins turned out to be more optimal. In these cases, the production of lectins occurs in bioreactors. For this proposal it is required that the system can reach autonomy.

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1D) Describe the desired result of your synthetic cell operation.

 

The synthetic cell produces lectins when the liquid medium contains turbidity and alkaline pH.

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**Proteins that are naturally light responsive can be found in organisms involved in photosynthesis or phototropism. The particular protein domains responsible for the photoactivity have been used to create synthetic systems that are triggered by light. Ellington and co-workers applied the light-responsive protein Cph1 to the construction of a synthetic edge detection program in E. coli. This edge detector functions by distinguishing between inputs of either light or dark (NOT light), and generating a diffusible signal in response to “NOT light”. The diffusion of the signal from cells that sense dark to neighboring cells that sense light creates the edge image by producing an output of black pigment as a result of β-galactosidase expression (Gardner and Deiters 2012).

The core of the light sensor is composed of the membrane proteins PCB and Cph8. PCB is a chromophore (phycocyabilin) originating from cyanobacteria Synechocystis. Cph8 is a hybrid protein between the red light response domain of Cph1 (a phytochrome-like protein from Synechocystis ) and the intracellular domain of the histidin kinase EnvZ (an osmolarity sensor protein) from E. coli. The synthesis of PCB requires the expression of both Ho1 (heme oxygenase gene) and PcyA (biliverdin reductase gene).

-Dark system:

 Cph8 autophosphorylates its EnvZ intracellular domain while consuming one molecule of ATP. The phosphoryl group will be subsequently transfered to the transcription factor OmpR, which then, will upregulate genes expressed from the POmpC promoter.

-Bright system: 

PCB prevents the Cph8 autophosphorylation. OmpR will not be activated and the genes not be expressed.

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**A form of gene regulation common in the microbial world, enabling organisms to adapt to differing environments, is control of gene expression by ambient pH. Such regulation tailors the syntheses of molecules operating outside the protection of the organism's internal pH homeostatic system, such as permeases, secreted enzymes, and exported metabolites, to the pH of the growth environment. The zinc finger-containing transcriptional regulator PacC mediates such pH regulation. In response to a signal transduced by the products of the six pal genes at alkaline ambient pH, which facilitates the expression of genes expressed at alkaline ambient pH and prevents the expression of genes expressed under acidic growth conditions. (Espeso and Arst 2000)

 

a. Espeso, E. A., and H. N. Arst, Jr. 2000. 'On the mechanism by which alkaline pH prevents expression of an acid-expressed gene', Mol Cell Biol, 20: 3355-63.

b. Gardner, L., and A. Deiters. 2012. 'Light-controlled synthetic gene circuits', Curr Opin Chem Biol, 16: 292-9.

c. http://2015.igem.org/Team:Toulouse/Description/Regulation

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2) Design all components that would need to be part of your synthetic cell.

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2A) What would be the membrane made of?

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The membranes can be liposomes, so they can contain the information and avoid the agglutination of components. These semipermeable characteristics will allow the release of lectins, through external signaling sequences.

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2B) What would you encapsulate inside? Enzymes, small molecules.

 

Plasmid (contains gene lectin, sequence regulator to light and and regulater expression pH that included PacC and Cphl), water and Mix containing all TXTL reagents (E. coli cell extract, amino acids, and energy buffer)

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http://www.arborbiosci.com/wp-content/uploads/2018/02/myTXTL-Manual-v01.pdf

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2C) Which organism your tx/tl system will come from? is bacterial OK, or do you need mammalian system for some reason? (hint: for example, if you want to use small molecule modulated promotors, like Tet-ON, you need mammalian system.

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Bacterial, the Escherichia coli extract is good.

 

2D) How will your synthetic cell communicate with the environment? (hints: are substrates permeable? or do you need to express membrane channel?

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It does not require permeable substrates, it is necessary to express components in the membrane channel. For the case of pH detection, we need to express the pall and palH in the membrane, for the regulation of expression by light, the Cph1, Envz and PCB

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3) Experimental details

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Scheme (bonus)

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3B) How will you measure the function of your system?

The lectin output of the cells can be measured by ELISA or effect floculantors.

It will be carried out under the conditions of alkaline pH and water turbidity.