Team:LMSU/Description

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# 🥉 Description

## Why Arthrospira platensis?
![https://static.igem.org/mediawiki/2021/thumb/9/96/T--LMSU--Arthr.png/800px-T--LMSU--Arthr.png]
Microalgae are a group of ubiquitous organisms. They have a huge potential as bioresources for various industries, thanks to their unique qualities. Due to the ubiquity of microalgae, they are forced to adapt to different conditions, so their ability to synthesize various substances is incredibly developed - almost all algae are able to **photosynthesize**, and some can **fix nitrogen gas** and convert it into forms assimilable for plants. Therefore, many compounds can be found in microalgae: enzymes, pigments, lipids, sugars, vitamins, sterols, phytohormones, compounds with antimicrobial activity, etc.

One of most important currently used microalgae is *Arthrospira* (*Spirulina*). Dried *Spirulina*, used in dietary supplements, contains **about 60% protein**, the composition of which is **rich in all vital amino acids**. At the same time, the cell wall of *Spirulina* quite fragile, so the contents of the cells are easily digestible can be consumed in pure form.(Sataloff, 2002)

*Spirulina* contains a large variety of **aliphatic acids**, which are used **in medicine** for certain diseases (arthritis, thrombosis, atherosclerosis). After the separation of these oils from microalgae, remaining algae biomass can be used as fertilizers.
The content of these substances allows the use of microalgae for the production and isolation of substances separately, and not only for the use of microalgae in its pure form. (Sataloff, 2002) 

In consequence of variety of organic substances, *Spirulina* is used not only in the **agricultural industry**, but also **in cosmetics**: they are used as
thickeners, antioxidants and water-binding agents, and micro- and macroelements, vitamins and antioxidants contained in large quantities improve the condition of the skin. Now these microalgae are used in the manufacture of creams, toothpastes, lotions and antibacterial creams.

In addition to their biochemical properties, *Spirulina* distinguishes itself with the fact that its biomass can be doubled in a fairly short period of time (in approximately 6 hours). Moreover, these microalgae need only lighting, a nutrient medium and a constant temperature for growth, which is quite easily provided in bioreactors.

## How ASCEND was born
The idea of our project was born in the beginning of 2021 after a long and vivid brainstorming period. We all were fascinated to contribute space exploration and upgrade the already existing solutions of on-board food supply, such as [ESA MELiSSA project](https://www.melissafoundation.org). 

We came to a conclusion, that the most profitable, easy in handling and facing most of the requirements for food source (**safety**, stability, **usability**, **reliability**, **nutritiousness**, palatability, variety, and **resource efficiency**) in case of space is cyanobacteria *Arthrospira platensis*. This microalgae was approved for nutritional use by FDA and GRAS already in 2002. In other words, it is safe for consumption and, what is more, possesses sufficient nutritiousness, according to the literature data. We suppose, that this algae will be able not only to supply space crew with nutrients, but also produce oxygen as byproduct of photosynthesis and also recycle urea, maintaining the fresh water supply on-board. 

However, even such elegant solution has flaws. *Arthrospira platensis* is **tasteless**, which would be a significant disadvantage of such space diet. We decided to introduce *Spirulina* with “flavouring genes” and make astronauts’ diet more appealing and variable. As a proof of concept we chose vanillin biosynthesis, as our cyanobacterium genome already contains primary genes of this metabolic pathway. We are planning to bring this solution to life by embedding new biosynthesis cascades into spirulina metabolism, and creating a new vanillin synthesis platform using two related enzymes that convert ferulic acid (precursor of vanillin) into vanillin: feruloyl-CoA-synthetase (FCS gene) and enoyl-CoA-hydratase (ECH gene).

But what if we optimise the system and separate acquiring of biomass and production of flavouring genes in time? — we asked ourselves. This way our optogenetic approach was rooted. 

## Our focus in 2021
Transformation of *Arthrospira platensis* appears very challenging, according to the literature data and our discussions with algologists and microbiologist. There are few cases of successful *Arthrospira platensis* transformations, however, in all cases genetic constructions are inserted into genome with homologous recombination. Therefore, the whole or at least partial genome sequencing is required. According to NCBI database there is no data of any strain of the whole *Arthrospira platensis* genome sequence. As we could not afford whole genome sequencing of *Arthrospira platensis* IPPAS B-256 this year either, we were unable to implement insertion into genome. Despite that, we have made some attempts to transform IPPAS B-256 according to naturally activated competence without homologous recombination, although, these attempts were unsuccessful. Thus, we decided to postpone optimisation of transformation of *Arthrospira platensis* IPPAS B-256 until we get the whole genome sequence (hopefully, very soon!). 

For the aforementioned reasons we decided to focus on construction of optogenetic system, as the regulating approach to control of biomass accumulation and target genes biosynthesis, this year. 

## Our optogenetic system

![https://static.igem.org/mediawiki/2021/1/15/T--LMSU--Optgen.jpeg](Main players of our optogenetic system)
Properties of light-dependent promoters and proteins which are sensitive to wavelength shift underly our optogenetic solution. 
BphP1(Part:BBa_K3032016), Q-PAS1+Gal4 and BcLOV — are the main components of our optogenetic system. BphP1, bacterial phytochrome P1, is a bacterial protein which has photosensitive domain and α-spiral. Under the 760nm radiation this protein remains in a monomer form and releases its α-spiral. 


![https://static.igem.org/mediawiki/2021/2/20/T--LMSU--BphP1.jpeg](BphP1 forms)
![https://static.igem.org/mediawiki/2021/b/bd/T--LMSU--Team_LMSU_GifOptogene1.gif](BphP1 binding to QPAS1+GAL4)
After this, monomer of BphP1 can interact with Q-PAS1+Gal4, releasing promoters and letting the polymerase begin expression. Q-PAS1+Gal4 is a fusion protein which in dimer form interacts with DNA, blocking promotors and not letting the polimerase occupity its site. HTH-domains of Gal4 bind to the DNA (CCG and CGG triplets). 


![https://static.igem.org/mediawiki/2021/4/40/T--LMSU--Team_LMSU_GifOptogene2.gif](HTH-domains in Gal4 binding sites)
When the wavelentgth is switched to FR (far-red), α-spiral of released BphP1 monomer interacts with α-spiral of Q-PAS1, what causes the break down of the dimer and releases one of the Q-PAS1+Gal4 monomers. Another Q-PAS1+Gal4 monomer dissociates from the DNA by itself. Eventually, polymerase is free to occupy the promoter and start expression. When the light is switched to R (red) (640nm) BphP1 α-spiral folds and interaction between BphP1 and Q-PAS1+Gal4 weakens. This causes dimerisation of Q-PAS1+Gal4. Dimerised Q-PAS1+Gal4 can specifically bind to the DNA CCG and CGG triplets domain, stopping expression. 

However, FR light can not be circumvented absolutely as leaks take place. This is the most common radiation in the Universe. Therefore, expression of the target gene would not be stopped completely (Redchuk, 2017). To prevent this, we fused BphP1 with the BcLOV protein which is also sensitive to the wavelength shift. BcLOV reacts to the blue - 450nm light. In these conditions, BcLOV releases its amphiphilic α-spiral, which is able to adhere to the cell membrane. So, when the 450nm light is active, fused protein of BphP1 - would not have any other way than clinging to the lipid cell membrane. Ultimately, this removes the minor quantities of active BphP1 and stops the expression of target gene completely. 

**States of our optogenetic system**
![https://static.igem.org/mediawiki/2021/d/d2/T--LMSU--FRl.jpeg]
![https://static.igem.org/mediawiki/2021/5/59/T--LMSU--Rl.jpeg]
![https://static.igem.org/mediawiki/2021/d/db/T--LMSU--BFRl.jpeg]

Our system is flexible and reliable. To test it we used YFP as the gene of interest and examined the level of expression by measuring intensity of YFP fluorescence under certain promoters. However, any other protein can be used in this circuit. For the next step we are going to insert genes that can provide vanillin (FCS and ECH), chemical of vanilla taste.