Team:Nanjing-China/Contribution

We made some contributions to the expression and purification of spidroin gene, that is, we found that the expression level and purifying productivity were low when using PET29a vector.

We attached spidroin gene sequence to PET29a vector and obtain the recombinant plasmid PET29a-sp, which was transformed to the competent cell BL21 (DE3) and induced by 1M IPTG at 25℃ overnight for later protein purification. The protein purification result shows that spidroin produced by recombinant plasmid PET29a-sp has weaker interaction with Ni column, causing poor purifying results.

In the study from GreatBay SZ, spidroin gene sequence was usually attached to PET29a vector. And in the spidroin expressed by recombinant plasmid PET28a-sp, there were two 6-residue His tags in both C terminal and N terminal, which could interact with Ni column and help the purification of spidroin. However, the spidroin expressing by recombinant plasmid PET29a-sp had one 6-residue His tag in C terminal. This difference resulted in the poor purifying results of spidroin by using Ni column.

2、New documentation to ppk1 gene

We found that PolyP productivity would reach the highest after inoculation for 14 hours and 100ml of synthetic wastewater in a 200ml Erlenmeyer flask has the highest output and the highest conversion efficiency.

PolyP formation is closely associated with ppk1 gene in CPP. This means the cultivation time and volume will affect P consuming a lot. After comparing different cultivation conditions, we found that PolyP productivity will reach the highest after inoculation for 14 hours, that is approximately 20mg/L. Besides, 100ml of synthetic wastewater in a 200ml Erlenmeyer flask has the highest output and the highest conversion efficiency. Without bacterial contamination and normal bacterial viability，it can almost convert all inorganic phosphates into polyphosphates.

Improvement of plasmid

1. The improvement of +36GFP plasmid.

GFP green fluorescent protein has a positive charge of 36 charges after improvement, which is convenient for binding with negatively charged proteins through electrostatic force. Because green fluorescent protein itself has green fluorescence, it is easier to be in cells and tissues. Medium positioning determines the distribution of the negatively charged protein bound to it, and to a certain extent can indicate the electrical and structural changes of the bound protein. Positively charged green fluorescent protein provides a way for negatively charged molecules to change their electrical properties and to bind to negatively charged molecules and label negatively charged proteins.

2. The construction of +36GFP and spidroin fusion protein particles.

Through the fusion of spidroin and positively charged green fluorescent protein, the whole protein is positively charged, and at the same time, it combines the advantages of spidroin and green fluorescent protein, so that the position of this protein has green fluorescence, which is separated in phase. Moreover, it can be clearly seen when the cells are positioned, making it convenient for the observation of experimental results. At the same time, good properties of spidroin is maintained. After combining with other organic and inorganic substances, it can be made into spider silk, which has good toughness and strength. In subsequent experiments, we combined the spidroin green fluorescent protein with polyphosphate, which has better thermal stability, PH stability and better mechanical resistance. The positively charged spidroin can easily and tightly combine with negatively charged molecules, which is convenient for studying the physical and chemical properties of different organic-inorganic hybrid materials.

3. Improvement of spidroin.

We changed the negatively charged amino acids in the non-helical and folded loop structure of the spidroin to lysine (the most positive amino acid) to make the spidroin positively charged without affecting the main structure of the protein, in order to combine with negatively charged polyphosphate through electrostatic force. In future experiments, the properties of spidroins can also be improved through the combination of positively charged spidroins and negatively charged inorganic molecules.

Application prospects of hybrid materials obtained from literature reading

1. Organic-inorganic hybrid materials

Organic-inorganic hybrid materials (HOI) are micro-composite materials composed of organic and inorganic compounds, and the scale of their hybrid mixture is between 10-10m and 10-8m. Under normal circumstances, inorganic compounds help to improve the dielectric properties and magnetic properties of HOI, so that hybrid materials have excellent electrical conductivity, thermal and chemical stability, and mechanical properties, and thus have a wider range of applications. The organic material part of HOI is usually used as a support to provide high structural elasticity and low density for hybrid materials. In addition, organic components also make a greater contribution to the photoconductivity, luminous efficiency, and ability of the hybrid material to gain and lose electrons. In view of the characteristics of organic-inorganic hybrid materials, it is widely used in protection, catalysis, energy-related and biological-related fields. In the field of protection, HOI can be used as an anti-corrosion coating for the protection of metal surfaces. It can also be used as a fireproof material for the flame retardant and heat insulation of general objects, buildings and professional fire protection appliances. It can also be used as a wear-resistant coating to protect the surface of easily worn objects. In the field of catalysis, HOI can use the structural properties of its central metal atom to bind to specific organic ligands to mimic the structure of an enzyme, so as to have a catalytic function similar to that of an enzyme. In the energy-related field, because HOI can have the characteristics of low density, high electronic activity (high energy density) and high stability, it is usually used in batteries, supercapacitors and photoelectric cells. In biological related fields, HOI is used in emerging drug delivery systems. This system can fulfill two functions: drug loading and selective drug release. Due to the properties of both organic and inorganic substances, HOI can experience structural changes due to changes in external pH, temperature, light and other factors to achieve the function of drug loading and fixed-point release. In addition, in the field of biology, HOI can also be used as a biosensor to monitor biological agents and sense certain biological reactions. A typical biosensor includes an inorganic part responsible for detecting target biomolecular events and an organic part responsible for selective interaction with the biological environment.

2. Organic-inorganic hybrid polymer

Organic-inorganic hybrid polymer is a hybrid material formed by inorganic substances and organic polymers through chemical bonds, ionic bonds or other weak bonds. Among these materials, organic polymers are usually used as the "main chain" to provide "connection" sites for inorganic substances, thereby forming stable hybrid materials. This type of material has unique thermal conductivity, electrical conductivity and strong mechanical strength.

Application of organic-inorganic hybrid polymer materials in the field of fire protection: Pratibha Sharma and Leena Nebhani have developed a hybrid thermosetting polymer of bio-based benzoxazine monomers and inorganic siloxane (Si-O-Si) hybrids, which has good thermal and dielectric properties; Li et al. developed an organic-inorganic hybrid polyphosphazene modified manganese hypophosphite material, which combines PET and PZS-MnH, and uses the flame retardant properties of PZS-MnH, Make the hybrid material have excellent fire resistance. Xu et al. improved the polypropylene (PP) material, combining metal oxide and PP to form a hybrid material, which significantly improved the flame retardancy of PP.