DESIGN

Overview: How to Fix Compounds on Cellulose

Background: Cellulose and Industry

Thanks to its various interesting properties such as high purity or biocompatibility, bacterial cellulose has already been applied to a wide range of fields. The biomedical industry is one that uses the most the bacterial cellulose¹. It has been developed as an artificial skin for the covering of wounds as it has a high mechanical strength, even when wet, and it doesn’t irritate the user’s skin². Companies have been using it in surgeries and dental implants. It has also been used as a part of artificial blood vessels, as bacterial cellulose has a lower risk of blood clots than the current materials.

But bacterial cellulose has also been used in other industries such as the paper and the food industries. It is used as a food matrix to make nata de coco, a dietary fibre from the South-East Asia¹. Also, bio-based materials made of bacterial cellulose in the food packaging industry are under development as it has proven its efficiency with its water resistance and the fact that it is biodegradable.

Problematic: The Necessity of Improving the Actual Process

Its interesting and unique properties may give bacterial cellulose a bright future in the industries, unfortunately, there are still limitations that explain why it is not already the case. For example, bacterial cellulose lacks antibacterial properties or stress bearing capability³. Today, to overcome these problems, cellulose is often used as a matrix or composite in order to welcome various particles inside.

How can Synthetic Biology Help?

A generic square placeholder image with rounded corners in a figure. — **Figure 1:** Overview of ou experimental procedure

Our Project: Creating Endless Possibilities with Cellulose

To exploit the potential of bacterial cellulose and to increase its added value to make it more competitive against plant cellulose, we designed a three headed linker protein named Cerberus. The protein will allow us to bind an infinite range of molecules to cellulose, thanks to its three fixating protein structures representing the three heads of the system.

The platform binds to cellulose through its protein domain of the type 3 Carbohydrate Binding Module (CBM3) family. The first linker consists of a streptavidin head, which enables us to bind biotin and biotinylated compounds by using the strong affinity these two molecules have for each other. The second linker of our three heads system is composed of 4-azido-L-phenylalanine (AzF), an unnatural amino acid, used to covalently bind alkyne-decorated molecules by click chemistry.

Cellulose Binding

The Carbohydrate Binding Module

- Why does anybody would like to bind protein on cellulose?[4]

The Binding Affinity and How to Use It

*insert clever descrition here*

Experimental Plan: Sirius

Our first protein, Sirius, was constructed by fusing CBM3a to mRFP1, which is an engineered version of DsRED but with a monomeric characteristic. This part was cloned into pET28 containing a pT7Lac promoter and a C-terminus His-tag. Once the cloning was validated by sequencing, we chose to transform it into E. coli BL21 because of its high protein production yield. Once produced, Sirius was purified by Immobilized Metal Affinity Chromatography (IMAC) on cobalt resin column. The binding affinity to cellulose was proved by using regenerated AviCell and measuring the fluorescence intensity, but also by mixing Sirius in presence of bacterial cellulose produced by Gluconacetobacter hansenii followed by several washes.

The Avidin Protein Family: The Strongest Non-Covalent Bond

Domain Diversity

During our experiments, we used two types of streptavidin: the wild-type, which is tetrameric, and the mSA2, which is monomeric and the result of a fusion of streptavidin and rhizavidin. Both have their pros and cons, that is why we chose to work with both. Due to its capacity to bind four molecules of biotin per tetramer, the wild-type has an unmatched affinity for biotin⁶. However, the monomeric version mSA2 has a shorter nucleic sequence and it is less prone to have non-specific interactions. Of all the existing non-tetrameric streptavidin, mSA has the highest biotin affinity (K_D = 2.8 nM)⁶, which increases its ability to detect biotinylated compounds. Additionally, mSA2 is easier to produce as our experiments have shown us, which explains why we did not conduct all our proofs of concept with the tetrameric type.

Biotinylation Diversity and Usages

Of all the interacting pairs known and used today, we chose the biotin-streptavidin because of its appealing characteristics that can be used in a whole range of applications⁷. First, this interaction is one of the strongest non-covalent bond found in Nature with a dissociation constant of 10^-13 M, which is 10³-10⁶ greater than any interaction between a ligand and its specific antibodies. The high affinity ensures that once formed the complex will not be changed by pH variations or washes. Secondly, this strong affinity ensures that the binding is highly specific and will only specifically target certain molecules. When attached to macromolecules, thanks to its small size, biotin does not alter their biological activity or other properties. Finally, what makes streptavidin a great choice for our fusion protein is that it is very stable and its ability to bind to biotin is not easily destroyed as it can survive hard conditions.

Also, the fact that biotinylated compounds such as fluorophores, enzymes and proteins are commercially available encouraged us to use this system in our project. All these aspects also explain why the use of the streptavidin-biotin interaction was developed in the field of biotechnology. It opens a broad range of applications, especially on immunological and nucleic acid hybridization assays.

Experimental Plan: Orthos

Orthos is our second protein used to validate our project. It is the result of the fusion of binding molecular platform, CBM3a, and the streptavidin linker, mSA2, and this part ends with an Amber stop codon, preventing the His-tag from the pET28 environment to be translated. After validating it by sequencing, we produced it in E. coli BL21 and purified it by using the cellulose affinity proven with Sirius. To study its affinity for biotinylated proteins, we needed to in vivo biotinylate proteins. To do that, we fused mTagBFP with an AviTag, and cloned it in pETDuet1. The AviTag will be recognised by a small protein called BirA and the molecule will be biotinylated by it. Once the system was transformed into E. coli BL21 and induced with IPTG, we used the cellular lysate to prove the affinity of Orthos for biotinylated compounds by measuring the fluorescence.

UnNatural Amino Acid

Expanding the Genetic Code with the Orthogonal Machinery of Incorporation

In many cases, proteins are built within organisms by combining the 20 common amino acids. To genetically encode more amino acids, referred to as unnatural, opens the possibilities of controlling the chemical functions of proteins and thus expanding their chemical space. For example, proteins can be expressed with novel side chains⁸. The strategy is to use the cellular machinery to introduce these unnatural amino acids (UnAAs). Today, more than 50 UnAAs have already been incorporated in different microorganisms. This is done by using orthogonal amino acyl tRNA synthetase (aaRS)/tRNA pairs, and an unassigned or reassigned codon. The 20 common amino acids are encoded by 61 triplet codons, which leaves the three stop codons (TAG, amber; TAA, ochre; and TGA, opal) to serve to specify the UnAA. For the 4-azido-L-phenylalanine, the amber stop codon was used⁹.

4-Azido-L-Phenylalanine: The Click Chemistry

At the beginning of the 21^st century, the emergence of the reaction called “click chemistry” revolutionized bioconjugate chemistry. Thanks to this, coupling molecular fragments to synthesize conjugates has become easier. Following that, biorthogonal reactions were developed. The most widely used is the copper-catalyzed azide-alkyne cycloaddition reaction, shortened as CuAAC¹⁰. Catalysed by Cu(I) ions, this reaction involves the azide group of our UnAA and an alkyne group. The main problem of this method is the cytotoxicity of copper on cells limits its use in assays that take place in the cellular environment. To circumvent this problem, the strain-promoted azide−alkyne cycloaddition reaction, shortened as SPAAC, was introduced in 2004 and have been more and more used nowadays. This reaction presents the advantage of not needing a catalyst, so it not toxic for the cells, and it can be implemented in physiologic pH condition (around 7.5).

Experimental Plan: Cerberus

Finally, the third protein we produced was Cerberus. The construction is similar to Orthos’ but to incorporate the UnAA, we had to hack into the genetic code. To do so, we brought to E. coli an orthogonal aaRS/tRNA pair. The amber stop codon present in our construction is now recognised by the tRNA and it becomes an incorporation site for the 4-azido-L-phenylalanine (AzF). This strategy was thought for specific purification because the His-tag will only be attached to AzF-containing proteins. Once validated, the part was co-transformed into E. coli BL21 with pEVOL-AzF, a plasmid containing tRNA and aaRS from Methylococcus ianaschii. We purified the production by using IMAC purification on cobalt column.

To validate the activity of the UnAA, we used fluorescein grafted with DBCO by using SPAAC. This compound clicked with Cerberus’s head and after several washes we measured the difference of fluorescence between cellulose samples containing or not our Cerberus protein with the fluorophore.

Cerberus: The Versatility of a Simple System

Primary Usage: The Tricephalic Protein

Cerberus was first thought for binding different types of compounds at the same time thanks to its two very unlike linkers. The streptavidin linker can bind biotin and biotinylated compounds while the UnAA-containing linker will click molecules that bear an alkyne function. The main advantage of such a system is that we can bring not one but two varied and distinct functions. This way, the possibilities of functionalizing cellulose and bringing new innovative materials are expanded.

Secondary Usage, or How to Recycle?

Imagine that someone wants to double the amount of a molecule bound onto a surface. We have thought about using a biological “adapter”, DBCO-Biotin. This compound can be purchased, and it allows us to turn one linker into another or to form dimers and more of Cerberus. Components with alkyl functions can now be grafted on the streptavidin linker while the streptavidin head of the second Cerberus can bond on the AzF linker.