Difference between revisions of "Team:USP UNIFESP-Brazil/Project"

Spider silk is an amazing bionanomaterial due to its exceptional features. It exhibits high tensile strength, high fracture resistance, high elasticity and it has biocompatibility and low degradability. Some studies show silk can be compared to a synthetic polymer called Kevlar, that is made of aramid and is used to make body armor (Lewis). Silks are polymers composed of proteins. These proteins and their genes present specific repetitive sequences that are associated with silk properties, making it possible to handle it in order to obtain the most suitable material for each application. Moreover, some studies suggest that the terminal domains of silk proteins play important roles in the polymerization process. In this way, it can be possible to add N- and C- terminal domains in several interesting proteins or motifs and spun it with native proteins to obtain a customized silk.

The size of genes, their repetitive sequences and high content of Cytosine and Guanine establish a challenge to many expression systems. However Chlamydomonas reinhardtii has naturally high CG content on its genome and it could be able to deal with this problem, it can also perform post translational modifications and it can secrete complex proteins, easing the purification process. Furthermore, it exhibits low cost production, rapid growth, scalability and stable transgenic line generation, all desired characteristics to an expression system (Wijffels, Kruse, and Hellingwerf; Rosenberg et al.). It is important to highlight the low cost of microalgae cultivation, while production of antibodies in mammalian cells presents an average cost about US$150.00/per gram of raw materials and plants US$0.05/per gram (Dove), Maylfield has estimated that microalgae can reach US$ 0.002/liter (Rosenberg et al.; Mayfield, Franklin, and Lerner). Chlamydomonas reinhardtii also is Generally Recognized As Safe (GRAS), that is, it has low risk of being contaminated by virus, prions and bacterial endotoxins (Mayfield et al.). Easy purification and low risk of contamination are very significant issues when we are talking about making products to biomedical applications.

Figure 1. Cassette construction to be inserted in C. renhardtii nuclear genome. Promoter hsp70A/rbcs2: fusion of the promoters hsp70A and rbcs2 (Eichler-Stahlberg et al.; Schroda, Blöcker, and Beck). Sh-Ble: resistance gene to Zeomycin. 2A: self-cleavage peptide from Foot and Mouth Disease Virus (FMDV) (Rasala et al.). SP: secretion signal peptide of the gene Ars1. GOI: gene of interest. His: histidine tag. RbcS2 3’ UTR: terminal sequence of the gene RbcS2 (Fuhrmann et al.). Introns were added as the figure shows (Eichler-Stahlberg et al.; Lumbreras et al.).

Our project was inspired by the UCLA’s projects presented at iGEM in 2014 and 2015 about synthetic silks. Like we said above, silks are very interesting material but one of the main problems is dealing with its high content of Cytosine and Guanine. Most of the expression systems are not able to deal with that, however we have known Chlamydomonas reinhardtii has naturally high CG content which makes it interesting as an expression chassis. One of UCLA’s projects was called Silk Functionalization: Developing the Next Generation of Performance Fibers. The team has created a genetic construct using the Green Fluorescent Protein (GFP) inserted between N- and C- terminal domains of Bombyx mori’s silk to express proteins able to bind to native silk proteins. When this customized GFPs and native proteins were mixed and spun they were able to recognize each other by the terminal domains, making a fluorescent silk.

From this idea we thought: “Silk is a very interesting material and could be expressed in Chlamydomonas reinhardtii, a model organism that has many advantages and that remains underexplored. What could be put in place of a GFP to express in microalgae? What biomolecule could take advantage of this amazing silk feature? It has to work while immobilized... Why not enzymes? There are many studies about enzymes immobilization, enzymes are very appropriate and useful in many applications around the world!”. But we knew it could not to be any enzyme linked to silk, we knew it need to address technical, social and economic issues. So we read that spider silk has been studied to many biomedical applications, like the treatment of burn victims. Then we studied about burns in order to find an interesting enzyme that could solve some problem and we learned that one of the most important problems affecting burn victims is sepsis. In this way, we sought for antimicrobial enzymes and we found the endolysins. Endolysins are very interesting enzymes because they are able to combat multiresistant bacterias and they are specific, avoiding problems like the antibiotic resistance.

So we decided to make a project about spider silk production in microalgae, creating possibilities to future works with silk proteins from this amazing organism model, like the association between them and endolysins by UCLA’s approach in order to make an antimicrobial silk to be applied on burn victims to combat sepsis.

Eichler-Stahlberg, Alke et al. “Strategies to Facilitate Transgene Expression in Chlamydomonas Reinhardtii.” Planta 229.4 (2009): 873–883. Print.

Fuhrmann, Markus et al. “A Synthetic Gene Coding for the Green Fluorescent Protein (GFP) Is a Versatile Reporter in Chlamydomonas Reinhardtii.” The Plant journal: for cell and molecular biology 19.3 (1999): 353–361. Print.

Lewis, Randolph V. “Spider Silk: Ancient Ideas for New Biomaterials.” Chemical reviews 106.9 (2006): 3762–3774. Print.

Lumbreras, Victoria et al. “Efficient Foreign Gene Expression in Chlamydomonas Reinhardtii Mediated by an Endogenous Intron.” The Plant journal: for cell and molecular biology 14.4 (1998): 441–447. Print.

Mayfield, S. P., S. E. Franklin, and R. A. Lerner. “Expression and Assembly of a Fully Active Antibody in Algae.” Proceedings of the National Academy of Sciences 100.2 (2003): 438–442. Print.

Mayfield, Stephen P. et al. “Chlamydomonas Reinhardtii Chloroplasts as Protein Factories.” Current opinion in biotechnology 18.2 (2007): 126–133. Print.

Rasala, Beth A. et al. “Robust Expression and Secretion of Xylanase1 in Chlamydomonas Reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide.” PloS one 7.8 (2012): e43349. Print.

Rosenberg, Julian N. et al. “A Green Light for Engineered Algae: Redirecting Metabolism to Fuel a Biotechnology Revolution.” Current opinion in biotechnology 19.5 (2008): 430–436. Print.

Schroda, M., D. Blöcker, and C. F. Beck. “The HSP70A Promoter as a Tool for the Improved Expression of Transgenes in Chlamydomonas.” The Plant journal: for cell and molecular biology 21.2 (2000): 121–131. Print.

Wijffels, René H., Olaf Kruse, and Klaas J. Hellingwerf. “Potential of Industrial Biotechnology with Cyanobacteria and Eukaryotic Microalgae.” Current opinion in biotechnology 24.3 (2013): 405–413. Print.

In our project, we propose to explore the modular characteristic of spider silk proteins, by using it as an immobilization support to other proteins. We were inspired by UCLA’s iGEM Team’s project in 2014 and 2015, where they presented the idea of using silk fibers to integrate other functional proteins to the silk’s structure. We tried to expand on this concept by expressing proteins with antimicrobial activity, enzybiotics (Fig.1). By combining these proteins and their properties, we tried to tackle a major problem with wound dressings for burn victims.

We tried to express the recombinant proteins, spider silk proteins and enzybiotics in the microalgae Chlamydomonas reinhardtii strains by nuclear transformation. Each recombinant strain would express a different protein, which would contain the N- and C-terminal polymerization domains from native spider silk proteins. These domains are essential to the polymerization step and, subsequently, for production of a material very similar to silk. Having been able to build our design, the antimicrobial activity and mechanical properties of the product would be evaluated, as well as the system productivity, shedding some light on spider silk-based immobilization support effectiveness, even for other biotechnological applications, such as the one idealized here. However, there are other possible applications with economic and academic interest.

Check the full description of the "Proof of concept" part of our projet here!

Microalgae present various desirable characteristics in an expression system: fast growth, fast making of stable transgenic lineages, scalability and low production cost, for example ​(Wijffels 2013, Rosenberg 2008)​. Unlike bacterial expression systems, microalgae are capable of producing and secreting complex proteins with post-transcriptional modifications. Mammalian cells also wouldn’t be an optimal expression system when considering production costs. Molecules such as monoclonal antibodies (mAbs) are mainly produced in these cells and their average production cost in this system is estimated to be $ 150.00 per gram of raw materials (Dove 2002), but the estimated value for algae reaches US $ 0.002 per liter, making them potential competitors (Mayfield et al. 2003). Another problem with spider silk expression is the G-C rich content of its sequences, often clogging the heterologous expression of this kind of protein in non-GC-rich systems (Yang et al. 2016). But Chlamydomonas reinhardtii presents a GC-rich genome, which may play an important role in spider silk protein expression.

Immobilization techniques are applied to a wide range of treatments and processes, from medical applications to biotransformations in industrial plants. This stabilization is normally achieved by protein binding to a scaffold (Liese and Hilterhaus 2013). Recent studies explored spider silks as a possible immobilization support (Blüm et al. 2013, Monier 2013). Spider silk is known mainly for its tensile strength and fracture resistance, but also exhibits elasticity, adhesion, biocompatibility and low degradation. Its strength can be compared to Kevlar synthetic polymer, which is composed of aramid and is used in for manufacturing body armor (Lewis 2006). Furthermore, medical applications are possible due to its biocompatibility and biodegradability, as coating for implants and transplanted organs, drug delivery and scaffolding for cell lines (Lewis 2006, Hardy et al. 2008, Kluge et al. 2008).

Check the full description of the "Notebook" part of our projet here!

In the development process of synthetic biology projects not everything can be understood easily. Sometimes, in order to save money, reactants and lab time it is necessary to appeal to other realm of tool sets. This is when modeling comes in, with mathematics as it's hammer and every project as it's nail. Jokes apart, modeling is a useful tool to understand possible limitations in complex systems that could lead to project failure, or even give some insights into underlying processes that would be otherwise hidden. During the initial development of Algaranha several issues that could be tackled within a mathematical framework were found, but one in particular caught our attention: the great difficulty to achieve large polymer chains in the insert. While catching up with the bibliography we found that most of silk chains that could be generated had at average 10-20mers, and the record length was a bit less than 100mer. Several possible reasons were described, such as the large quantity of Cytosine and Guanine in the gene composition, rendering it difficult to synthesize; the impossibility to insert large genes into the plasmid, and the impossibility to insert this large plasmid inside its vector organism.<\p>

As an approach to overcome these difficulties we decided to build our plasmid via repeated addition of smaller sub units in hope of achieving a larger chain. The system consists in a plasmid with a specific sticky end in both sides that pairs only with a specific coding sub unit, which we will call them A and C. These sub units have a specific sticky end as well, which binds only with an intermediate sub unit B. This B sub unit can bind either with the end terminals A and C or with another B sub unit. So we then have the possibility of constructing large chains by adding B units to the plasmid. The system described above can be understood as a set of coupled reactions, represented in the scheme bellow. As we can see, there are a few ramifications the reactions could take, where theoretically it could go on to infinite chain size. This scheme can be put on series of coupled reactions as we can see below:

In the first step we have the reactions for A and C binding in the plasmid (Pl):

At the second step we have the B sub unit binding to an Pl + A or a Pl + C:

And we can keep adding n B's to the chain until it finally ads a C and closes the loop:

These reactions can be further understood by a set of rate equations given by:

But since the number o equations and constants become untreatable it is necessary to take a few simplifications so we can continue. First of all we will consider only the first two steps, where the largest chain possible will be PlAB₂C, so we can keep track of what is going on. This way there are only a few equations left to be integrated. Since we don't have any of the rate constants we will assume three scenarios: homogeneous consumption constants (k₁ = k₂ = ... = k_n) and homogeneous dissociation constants (γ₁ = γ₂ = ... = γ_n); progressively decreasing consumption constants (k₁ > k₂ > ... > k_n) and homogeneous dissociation constants (γ₁ = γ₂ = ... = γ_n); homogeneous consumption constants (k₁ = k₂ = ... = k_n) and progressively increasing dissociation constants (γ₁ < γ₂ < ... < γ_n). This way it is possible to have some insights on what processes may be ruling our system.

Now it is possible to integrate these equations numerically and find the equilibrium conditions for each sub product of the reactions. Our interest is to find the ratios between [PlABC] and [PlAB₂C] when it is in equilibrium, i.e.:

Unfortunately the equations demonstrated to be considerably unstable, probably due to its coupling and the presence of the reactants A,B and C in almost all of the equations. But it is possible to have some insights on the behavior of theses equations given the scenarios described above. In the first scenario (homogeneous constants) it is expected that the final concentration of PlABC and PlAB₂C should be equal or at least approximately equal. The final concentrations of A,B,C and Pl should be dependent on the ratio of the dissociation constant γ and the consumption rate k, if γ = 0 all of the reactants should be consumed and all of the final products would be in the end reactions. But if γ ≠ 0 the amount of reactants would be also different from zero. In the second scenario (progressively decreasing k) it is expected that [PlABC] > [PlAB₂C], since its reaction ratio would be larger and given that k>> γ the amounts of the end sub units C and A would be close to 0, since it would be harder for them to break than to form a closed plasmid. Finally, in the third scenario (progressively increasing γ) the behavior should be similar, given that the larger molecules of PlAB_n and B_nCPl would be rather unstable, dissociating before a C or A sub unit could bind and close the loop.