The Role Of Imitated Nacre Recombinant Proteins And Gene Sequences In Controlling Calci?m Carbonate Mineralization

Biominerals in nature have many amazing properties such as sophistication,hybridation,hierarchical organizations and excellent mechanical performance,which always attract lots of life scientists,chemists and materials scientists.They are trying to find out the basic components and principles of synthesis of biominerals and direct the bio-process inspired fabrication of other materials.As we know,nacre has unique hierarchical assembly structure and extraordinary mechanical properties,which is a kind of natural organic-inorganic composite materials.It has been a research hotspot to many scientists.Nacre is composed of the inorganic phase and organic phase.About 95% is inorganic phase and the rest is consisted of three parts.Firstly it's insoluble chitin and secondly it's acid-rich soluble protein and thirdly it's protein which is similar to the silk protein.The organic macromolecules in the crystallization process of calcium carbonate is important to the synthesis of nacre,so researching the principles of biomineralization is a key link to understand the bio-process inspired synthesis.In this thesis,we use molecular biology to synthesis recombinant proteins which are similar to the composition of nacre organic macromolecules.The recombinant protein is composed of three domains: chitin binding domain,calcium binding region,silk protein repeat segments.Because the latter two parts can exchange the order when combined with chitin domain,we design two different recombinant proteins so as to figure out the different order may cause what kinds of biomineralization.We respectively named these two proteins as CCaS(chitin domain-calcium region-silk protein)and CSCa(chitin domain-silk protein-calcium region).Then we use PCR amplificate techniques and digestion,ligation methods to make the designed gene inserted into the plasmid pTWIN1.After transformation the protein is expresses in the E.coli.Through purification operation and gradient dialysis,the protein can refold.Finally we get the concentration of recombinant proteins by the quantitative experiment.The chitin resin and two different proteins are respectively mixed and vibrated at low temperature for above 12 hours.Join the chitin into the mineralization solution and let it stays at experiment designed temperature and time.We study the influence of these two proteins working to calcium carbonate at different pH and protein concentration and time.Finally we find that the vaterite appears when the recombinant proteins join into the solution,and the CSCa protein can make the vaterite phase appears earlier than the CCaS protein.Analyse the different results about calcium carbonate mineralization and we find that there are many difference between the proteins sequences.At the mean time,we understand that they have different repeat times of silk protein and the kind and number of the amino acid.So are the secondary structures.All these factors can have different effects on the minerals.