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Reinforcement Converter Functionalization Reference

Design

Reinforcement

BC Production

Method selection:

From the visit to Jingzhou Conservation and Restoration Center, we learned that BC(bacteria cellulose) can be applied to the restoration of silk relics, but which method should we choose, the ex-situ or in-situ?

• Ex-situ means spraying the BC on the silk directly, the advantage of this method is its speediness and does not involve in living organisms so that the uncontrolled factors is decreased.


• In-situ means spraying the bacteria that can secret BC (such as G.xylinus ) on the silk. Since the BC fiber can be secreted along the silk fibroin, so this kind of reinforcement will not change the texture of silk.

To validate these two methods, we did a preliminary experiment.

For simulating the situation of the silk to be reinforced, we treated the silk by NaOH for hours and got the aging silk. We smashed BC into a homogenate by beating crusher, and spayed it on the aged silk, this was the ex-situ group; And we sprayed G.xylinus with medium onto the ageing silk, this was the in-situ group. Both of them were inoculated in 37℃ for 7 days, and then we observed them under inverted microscopy.

We can apparently see that the BC produced in-situ can grow along the fibers of silk, while the ex-situ method will cause lumps of BC cover on the silk, which may influence the texture and fabric handle of the silk.

Thus, we chose the way of in-situ to reinforce silk relics in our project!

Chassis selection:

We've chose the in-situ reinforcement, but which kind of bacteria should we use? We may had two choices, the G.xylinus or the E.coli. We compared them by literature research and visiting the Jingzhou Cultural Conservation and Restoration Center.

• Growth Condition:


• --G.xylinus are aerobic bacteria, so that we have to ensure that the survival conditions are oxygenated. It may cause trouble when applied to real condition;


• --G.xylinus prefer acidic conditions. Although the isoelectric point of silk fibroin is near 5.5, we still concerned about its negative effect on the cultural relics, as the production of BC may cause continuous decrease in pH. What's more, we hope to use BC to restore other materials of cultural relics, a neutral pH would be better.



• Growth cycle:


• --G.xylinus grow slowly, usually it needs three days’ inoculation in seed medium, and then be migrated into fermentation medium for half a day. To realize a good reinforcement effect, it needs at least 7 days. This is the core reason why the Jingzhou Conservation and Restoration Center rarely use it for restoration.



• Engineered potential:


• --G.xylinus is not a model strain, it may be difficult to do further engineering. This constrains the development potential of our project.

After comparing the G.xylinus and E.coli, we decided to choose E.coli as the chassis of our project.

Pathway construction:

Cellulose biosynthesis is a process by which glucose is polymerized by a β1 → 4 linkage, and bunches of β1 → 4- glucan chains are simultaneously bundled into fibers called microfibrils, in which all chains are oriented in the same direction and are crystallized into the specific crystallographic form of cellulose.

This complex process is facilitated by cellulose synthase, which exists as a heterosubunit protein complex in the cell membrane. Imperial_2014 introduced four genes, gxcesA, gxcesB, gxcesC, and gxcesD (formerly bcsA, bcsB, bcsC, and bcsD) into E.coli to achieve the production of BC.

But in the latest study, in the case of bacteria, the gene (gx)CesA, (gx)CesB and diguanyl cyclase (DGC) represent the minimum subunit requirements for cellulose synthesis.

GxCesA protein is a GT-2 glycosyltransferase containing transmembrane helices and the catalytic subunit of cellulose synthase. Thus CesA and CesB, coexpressed with DGC which can synthesize C-di-GMP, can activate cellulose-synthesizing activity in E.coli.

To achieve the expression and secretion of BC in E.coli, we recombined these 3 genes into 2 plasmids and co-transduced into the strain of BL21(DE3).

Irregular reinforcement

In order to achieve the homogenized repair, we designed to control the secretion of BC at specific sites and the duration of the secretion. We applied light systems here to realize this control.

--First of all, we scan the silk surface, and get the shapes of weakness, then give a corresponding light signal to the silk to induce the engineered bacteria to produce the BC. And by the luminousness detection, we can know when the reinforcement is finished and stop it.

We built a light aging model to find out the relationship between wavelength and the light aging damage. According to it. We designed to apply several types of light system, for not bring damage to the relics.

• Light system 1.0--Blue light



• To test our idea in advance, we did a collaboration withShanghaiTech_China They applied a blue light system pDawn to their project, and we got pDawn and used it to control the bacteria to produce target proteins in specific areas.






• Considering the application objects in our project are those extremely delicate and precious silk relics, we concerned that the blue light (470nm or so) may cause damage to them. And we established a model to confirm it. So we improved our light system to use light with lower energy.
  
  
• Light system 2.0--Red light



• We found a red-light system designed by iGEM13_NCTU_Formosa. The excitation light’s wavelength is 600nm or so.
  
• This picture shows the details of the system
  
• --the part of the photoreceptor that responds to light, phycocyanobilin(PCB), is produced by introducing two genes (ho1 and pcyA) that can convert heme into PCB. It can help the formation of cph1,a phytochrome. And Cph1 fused with a membrane protein called ‘EnvZ-ompR’ to form the cph8-the complete red-light sensor.
  
• --In this circuit, under the exposure to red light, Cph1 is deactivated, inhibiting the autophosphorylation of ompR, thus turning off gene expression. In the dark, the phosphorylated OmpR will turn on PompC to turn on the following pathway. In order to realize the positive control via the red light, a NOT gate was added here.
  


• Soon we found that the promoter pOmpC is related to osmosis, we concerned that the system may have osmotic noise. Thus we used mathematics model here in order to access the impact of this osmotic noise. And we got verified that by adding a NOT gate after the promoter pOmpC the osmosis may not cause severe leakage of the red light system..
  
• But, it may cause the problem of low efficiency. Thus, we also improved this promoter by adding a riboswitch here to realize a tighter control of this promoter via another regulator, theophylline.
  




• Light system 3.0--Near Infrared Light



• Besides red light system, we also found another light system, near infrared light system. Near infrared light (760nm or so)has longer wavelength and lower energy than red light. But because of the time limited, we didn’t do any measurement on this system, but we hope to achieve this in the future.
  

Converter

Primary Linkage

Bacteria cellulose is a high molecular weight polysaccharide of β1 → 4-Dglucan. Cellulose Binding Domain (CBD) can bind to cellulose, it creates more possibilities for functionalization.

We learned from Imperial_2014 that double the CBD can increase the combination strength and from 18-Toulouse that CBM3a is a kind of strong CBD monomer. Inspired by these 2 teams, we got the idea to double the CBM3a monomer to form the dCBM3a, hoping to get a stronger combination.

Secondary Linkage

We’ve got the primary linkage by a cellulose binding domain, to realize the whole assembling, we still need another linkage, a biotin-avidin linkage.

• We fused streptavidin (SA) with the N terminal of cellulose binding domain, to get a streptomycinated dCBM3a.





• We added a tag with a length of 15 amino acids, the Avi-Tag on the N/C terminal of our functional peptides, the AOP and AMP. With the existing of an enzyme, the BirA, which can synthesis biotin in vivo, the biotin can link to the Avi-Tag, so that biotinylate the target peptides.


• Thus, the secondary linkage, the biotin-avidin linkage is completed. We’d like to use this complex linkage to stable the functional peptides on the reinforced materials, and another reason we are doing this to realize a platform here and provided much more potential of the functions. We called this platform the ‘Converter’.

Functionalization

During our experiments, we found that the silks was easy to be damaged and contaminated by molds. To confirmation of this conjecture further, we paid a visit to museums and read a lot of literature. We found that aging and molds taint were really big problems on silk conservation. So we got the idea to assemble AOPs and AMPs onto the reinforcement materials to gain lasting antioxidant and antimicrobial abilities.

AOP

The aging of silk usually means the oxidation of silk fibroin. Anti-Oxidation Peptides (AOPs) are groups of oligopeptides that can clear free radicals, such as superoxide anions (O·) or hydroxyl radicals (HO·)

We designed a kind of AOP that produced from Pinctada fucata, called DSAOP, which has good anti oxidation ability and is safety for application on food and cosmetic. This DSAOP was designed to be produced in E.coli so it has been optimized for expressing and has high stability and solubility. And the mechanism of anti-oxidant ability is by amino acid residues producing H+, thus the fusion with Avi-Tag may not cause influence on its function.

AMP

The molds taint on silk usually caused by the family of Aspergillus and Penicillium. To inhibit their growth, we found antimicrobial peptides that are especially against for filamentous fungi.

• PgD5 is a typical plant defensin found in Picea glauca. It shows a significant inhibitory ability to filamentous fungi by interrupting the structure of membrane and switch the permeability. Experiments to test its ability have been done on several kinds of fungi including B. cinereal, Rhizoctonia solani, F. oxysporum.



• As the PgD5 is a short peptides contains only 33 amino acids, inclusion bodies form easily in the expression process, we added a GST tag at N-terminal, and used Prescission Protease to cleave the GST tag in purification.

And We will add Avi-Tag a tag with 15 amino acids on them. The Avi-Tag can linked with biotin catalyzed by a enzyme called BirA. And the interaction between CBD with streptavidin to the functional peptides can form complete converters.

Reference

1. Tomoya Imai et al. Functional Reconstitution of Cellulose Synthase in Escherichia coli. Biomac.
   
2. Mark R. Bleackley et al. Nicotiana alata Defensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant. Antimicrob Agents Chemother. 2016 Oct; 60(10): 6302–6312.
   
3. Pere Picart et al. Identification of defensin-encoding genes of Picea glauca: characterization of PgD5, a conserved spruce defensin with strong antifungal activity. BMC Plant Biol. 2012; 12: 180.
   
4. Yanyan Wu et al.Preparation and Antioxidant Activities In Vitro of a Designed Antioxidant Peptide from Pinctada fucata by Recombinant Escherichia coli. J. Microbiol. Biotechnol. (2018), 28(1), 1–11.
   
5. Nicholas T. Ong et al.Engineering an E. coli Near-Infrared Light Sensor. Acs synthetic Biology.
   
6. Wenjing Cui et al. Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch.Microb Cell Fact. 2016; 15: 199.
   
7. Ashraf S S, Benson R E, Payne E S, et al. A novel multi-affinity tag system to produce high levels of soluble and biotinylated proteins in Escherichia coli [J]. Protein Expression and Purification, 2004, 33(2):0-245.
   
8. Chapman-Smith A, Cronan J E. In vivo enzymatic protein biotinylation [J]. Biomolecular Engineering, 2000, 16(1-4):119-125.