Difference between revisions of "Team:Toulouse-INSA-UPS/Demonstrate"

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                         We successfully set up a workflow allowing the synthesis of Cerberus,  a proteic platform that is composed of a cellulose-binding molecular module (CBM3a) fused at its <i>N</i>-terminal part to a biotinylated molecule-binding head (monomeric streptavidin) and at its <i>C</i>-terminal part to an unnatural amino acid (UnAA) 4-azido-L-phenylalanine moiety. Combining molecular modeling, synthetic biology and chemistry, we proved that Cerberus is a valuable and versatile platform allowing to functionalize cellulose with organic and inorganic compounds.
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                         We successfully set up a workflow describing the whole process from the synthesis of Cerberus to the production of functionalized cellulose <i>in vivo</i>. By combining molecular modeling, synthetic biology and chemistry, we proved that Cerberus is a valuable and versatile platform that should pave the way to a large range of applications.
 
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                        We can also plan to associate carbon nanotubes and graphene to AzF head in the aim to change cellulose fibre rigidity and create conductive cellulose, respectively. In order to do that, we successfully activated these compounds by diazotization reaction. The click chemistry assay and validation protocol need improvement to obtain our goal.  
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                      More functionalization are underway such as the association of carbon nanotubes or graphene to modify cellulose fiber rigidity and or to create conductive cellulose, respectively. The unique properties of bacterial cellulose such as its natural purity may give it a bright future in the industries, especially with the new possibilities offered by Cerberus. Combined with <i>in vivo</i> production in bioreactor, this should ensure a profitable and sustainable business. This is why we engaged in an entrepreneurship approach and based our Human Practices on the acceptance of biotechnologies.
 
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                        Bacterial cellulose has unique properties such as its natural purity may give it a bright future in the industries, that is why we decided to produce functionalized bacterial cellulose <i>in vivo</i> in reactor. As a first step towards the production of functionalized bacterial cellulose in a reactor, we demonstrate that CBM3a module of Cerberus fused to Red fluorescent protein is able to make bacterial cellulose fluorescent. Moreover, our first production of Orthos in <i>Pichia pastoris</i> are very conclusive, but we have to optimize the production.
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We truly feel that Cerberus platform is a very convenient and efficient solution to facilitate molecular work for iGEM projects and laboratory work in general. In fact, we can combine the use of two binding sites to purify simply molecules of interest (cellulose/streptavidin or cellulose/AzF or streptavidin/AzF). For example, we can imagine purify cells via biotinylated membrane proteins. An addition of Cerberus protein can permit the interaction between the monomeric streptavidin of Cerberus and the biotin of protein. Then, two possibilities are open to us to purify using either cellulose or paramagnetic beads. After attraction, we can release molecule of interest by using the TEV protease cleavage.
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                        Finally, we can also imagine to change our CBM3a to another proteic domain to associate functionalizing proteins to new materials as plastic to facilitate its degradation for example.
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Revision as of 20:02, 16 October 2018

DEMONSTRATE

Cerberus was a huge challenge on many levels. From the modelling to the in vitro validations, from functionalizing cellulose to laying the ground for in vivo production, we went a lot farther than we had dreamed about. Both functionalities of the Cerberus platform and its applicability have been demonstrated. This page summarizes these achievements.

Validation of the Three Binding Heads

Cerberus is composed of three different domains: CBM3a for cellulose binding, mSA2 for biotin affinity and AzF to click molecules with an alkyne moiety. They all have been demonstrated sequentially.

The CBM3a head and cellulose binding

The cellulose binding property was based on the CBM3a cellulose-binding domain. To demonstrate its functionality, we fused it to the mRFP1 fluorescent protein. Our results clearly proved its efficiency to bind cellulose (Figure 1). This was the first step from which we built the Cerberus platform.

Fluorescence Retained
Figure 1: Picture of cellulose pull-down assay with mRFP1 alone (left tube) or with the Sirius construction (right tube)

The mSA2 domain and biotin binding

To demonstrate the streptavidin head functionality, we combined it to the already demonstrated CBM3a domain. We then validated this construction with biotinylated fluorophores from both in vivo (Figure 2) or in vitro origins. This also demonstrated the capacity of the construction to add new properties to cellulose.

Fluorescence Retained
Figure 2: Functionalization of cellulose with Orthos bound to biotinylated BFP. Fluorescence remaining on cellulose fraction was measured after several washes (*Mann Whitney test p-value 0.01)

The AzF head and click chemistry

To assess the functionality of the AzF head, we used the previous construction with CBM3a and streptavidin and added the UnAA to the second linker (i.e, the whole Cerberus platform). We confirmed the activity of this integration using DBCO-Fluorescein as showed in Figure 3, but also DBCO-paramagnetic beads. This completed the validation of our system.

Fluorescence Retained
Figure 3: Functionalization of cellulose with FITC conjugated to Cerberus. Fluorescence remaining in cellulose fraction was measured after several washes (quadruplicate test). (*Mann Whitney test p-value 0.03)

Adding new properties to cellulose

Color

It was not initially planned to add colors to cellulose. However, mRFP1 has the property to provide a red hue without excitation. This was easily observable on bacterial cellulose after incubation with CBM3a-mRFP1 (Figure 1). This could be of special interest since a lot of proteic dyes are available (see iGEM Imperial 2016). The current dyes used in the textile industry are often toxic to people or the environment. Cerberus association with proteic dyes could thus be a safer alternative solution.

Fluorescence Retained
Figure 1: Picture of functionalized cellulose with CBM3a-mRFP1. Left: control mRFP1 alone; Right: CBM3a-mRFP1

Fluorescence

The first sought-after property of mRFP1 is for sure fluorescence. We successfuly produced fluorescent cellulose by combining cellulose, our platform and fluorescent dyes in several ways : by adding biotinylated fluorescent compounds, by clicking fluorescent molecules with an alkyne function or even by testing the CBM3a-mRFP1 construction on bacterial cellulose (Figure 2). This proves that physico-chemical properties of protein could be transfered to cellulose and allow to imagine many other developments (luminescence for instance).

Fluorescence Retained
Figure 2: Picture of functionalized cellulose under UV ligth with CBM3a-mRFP1). Left: control mRFP1 alone; Right: CBM3a-mRFP1

Antibiotic

We also conjugated an antimicrobial peptide, scygonadin, to the streptavidin. The first results seemed to indicate this could work (Figure 3) but it was not as a definitive result as the other functionalization assays we have performed. This will require further testings since such a functionalization arouse very important expectations from the public (see Human Practices).

Fluorescence Retained
Figure 3: Halo of inhibition of Orthos alone ( C), scygonadin alone (S) and Orthos+scygonadin (C+S) samples

Paramagnetism

Last but not least, we validated the grafting of paramagnetic beads to cellulose (Figure 4). So, our Cerberus platform has allowed us to create paramagnetic cellulose. In our opinion, this is the functionalization that should permit the largest panel of applications, especially in combination with other functions as Cerberus allows. Just imagine all the application in purification of compounds or cells!

Figure 4: Video of the Cerberus-functionalized paramagnetic cellulose: on the left, control with paramagnetic beads alone; on the right, Cerberus-paramagnetic beads.

Perspectives and conclusion

We successfully set up a workflow describing the whole process from the synthesis of Cerberus to the production of functionalized cellulose in vivo. By combining molecular modeling, synthetic biology and chemistry, we proved that Cerberus is a valuable and versatile platform that should pave the way to a large range of applications.

More functionalization are underway such as the association of carbon nanotubes or graphene to modify cellulose fiber rigidity and or to create conductive cellulose, respectively. The unique properties of bacterial cellulose such as its natural purity may give it a bright future in the industries, especially with the new possibilities offered by Cerberus. Combined with in vivo production in bioreactor, this should ensure a profitable and sustainable business. This is why we engaged in an entrepreneurship approach and based our Human Practices on the acceptance of biotechnologies.

A workflow to produce functionalized cellulose

This project was not only about functionalizing cellulose but ambitioned to define the whole process from component purification to production in vivo (Figure 1).

Fluorescence Retained
Figure 1: Workflow of our Cerberus project experimental validation process.

Indeed, we have defined and validated the basis to model our platform. This allows testing in silico any further modifications that could be of interest for Cerberus. Next, we demonstrated the efficiency of the Cerberus production in E. coli. This validated our choices for the strain, vector and culture conditions (IPTG, AzF, arabinose…). Then, we also successfully purified Cerberus and shown this through SDS-PAGE and Western Blot. As previously described, production of biotinylated compounds was validated as well as cellulose functionalization using Cerberus. We went even further and started the production of cellulose in vivo with Gluconacetobacter hansenii. The next step would have been to demonstrate the possibility to co-culture this strain and a strain synthesizing Cerberus and the function. Then, the final stage will be to start the production of cellulose in vivo. Even if we did not have enough time to each this final step, it was thought from the beginning of the project, as it was for this whole workflow.

No dogs were harmed over the course of this iGEM project.

INSA Toulouse UPS LISBP TWB Defi Adisseo Foundation ESF Catalyses


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