This year, BNU-China is truly fascinated by BIOMINERALIZATION, by which biological organisms produce hierarchically structured minerals with marvellous functions. We are aiming at modifying the genome of Chaetomium globosum to display E.coli K12 50S ribosomal protein L2 onto its surface. Meanwhile, with the help of Mcfp3 which acts as a ﬂocculating protein, cells could automatically cement into solid granules as an inactive and stable form.
● Chaetomium globosum is a widely used fungi with developed mycelia, large spreading area and strong tolerance.
● E.coli K12 50S ribosomal protein L2 has been demonstrated to have higher ability in binding silica. The protein is encoded by rplB, which was ampliﬁed by polymerase chain reaction.
● MCFP3 is a filopodia protein secreted from the mussel Mytilus californianus, it could slowly consolidate the binding of cells to particles.
Biology is closely linked with the real life--every basic research in lab may be a universal finding. After transfer those parts into Chaetomium globosum, we are aiming at cementing sand in the real desert. For which we created a word termed “desandilization”.
Bacterial species degradation is a common problem in laboratory and bioindustrial production. Usually, the strain degradation means the ability of the bacteria to produce target products is reduced or completely disappeared. When some engineering bacterium lose the expression vectors of product, they have a smaller pressure to survive, with strong competitiveness, but keeping the pathway, the engineering bacterium will be behind the competition. As a result, the macroscopic display is degradation.
It is mainly produced for the following reasons:
(1) Gene mutation. Bacterium often degrade because of mutations of gene that control products in the genome or by the loss of plasmids, resulting in inability or inefficiency to produce the target products.
(2) The continuous passages of the strain cause degradation. Mutations always occurs in the cell cycle. The more times of passages happen, the more mutations appear. At the same time, a small amount of degraded bacterium multiply quickly.
(3) The culture medium and preservation environment also affect the degradation of the strain.
Currently, the commonly methods to prevent degradations generally include :
(1) to minimize the passages;
(2) purification of strains;
(3) improve the culture medium and living environment;
(4) use good saving methods.
All of these methods have defects and need to be explored blindly for a long time. What’s more, none of them can give full play to the potential of the bacterium .
With the knowledge of the synthetic biology, we design a system that bacteria can screen out the degraded bacterium themselves, and the non-degraded strains will also get some compensations. Ideally, by using this system, the strain's life cycle would be greatly increased and the cost of the products would be greatly reduced.
In order to verify the feasibility of the designed anti-degradation circuit, we use the strain producing salicylic acid as the experimental strain.
There are mainly the following reasons:
(1) Some laboratories have already got the metabolic pathway in e. coli to produce salicylic acid ;
(2) E. coli contains regulatory proteins that specifically bind with salicylic acid and the special promoter.
(3) salicylic acid has broad utilization in many fields.
The Kolbe-Schmitt method is an industrial synthesis method for salicylic acid production. Salicylic acid was obtained from phenol by three-step reaction. Although it is the main method of producing salicylic acid in industry at present, the cost of this method is expensive and the production is very low.
By constructing the expression plasmid of ICS and IPL in E.coli to produce the key enzyme, the strain has the ability to produce salicylic acid. However, the ability to produce salicylic acid is gradually lost due to the degradation of the strain during the passages. In order to find the degraded bacterium and promote the growth of normal strains, we will introduce a plasmid containing the emrR protein gene and the glucose dehydrogenase GENE sequence.
EmrR protein is a regulator, it can combine with specific promoter, thus inhibiting the promoter downstream gene’s expression. But when cells can synthesize salicylic acid, emrR proteins will combine with salicylic acid , as a result, its conformation changes, loss of the ability to combine with the promoter, then the downstream gene expresses .
When the gene can promote the growth of normal bacteria, the bacteria that can continue to synthesize salicylic acid are more competitive. Through reading the papers , we chose to overexpress glucose dehydrogenase to promote the growth of the normal bacterial.
This project through the establishment of the positive regulation to give high-producing bacterial more survival advantages. When the bacterial produce salicylic acid ,it can express glucose dehydrogenase, then the overexpression of glucose dehydrogenase can promote the growth of the bacterial. Thus, they defeat the degraded bacterial in the competition.
Through this selection and positive regulation, this system can effectively ensure that the non-degraded strains in the reaction vessel have an advantage, and it is even possible to screen out the strains with high production efficiency.
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