Difference between revisions of "Part:BBa K2570002"

Latest revision as of 03:13, 8 December 2018

TyrB

Phenyllactic acid（PLA）is widely found in kimchi, honey and other foods. It is a new type of natural antibacterial substance and preservative, which can inhibit a series of gram-negative, gram-positive bacteria and fungi. There are two isomers of phenyllactic acid, and D-phenyllactic acid (D-PLA) has higher antibacterial activity. In addition, D-PLA has obvious improvement in protection of the cardiovascular system and has been widely used in the food and pharmaceutical industries. In order to enhance the production of D-PLA, we expressed D-lactate dehydrogenase BBa_K2570012, phenylalanine aminotransferase BBa_K2570002 and rocG BBa_K2570013 which is used to introduce the cofactor circulatory system and optimize the transformation system conditions to express D-PLA effectively with the phenylalanine as a substrate.

Fig.1 Schematic diagram and equation of D-PLA production by introducing a self-sufficient system.

This sequence encodes for Phenylalanine aminotransferase Tyrb that is one of the two key enzymes in the anabolic pathway of phenyllactic acid biosynthesis. Phenylalanine aminotransferase (PheATs), also known as aromatic transaminase (AroATs) or tyrosine aminotransferase (TyrATs), is the key enzyme responsible for formation of the compound pyridoxa-l-5'-phosphate (PLP), and is also essential for the biosynthesis of phenylalanine and tyrosinase. We used phenylalanine transaminase from Escherichia coli 21B to deaminate the substrate phenylalanine combined with aminotransferase to produce phenylpyruvic acid that is the raw material of the target product D-PLA.

Our project uses phenylalanine aminotransferase (PheATs) to catalyze the substrate phenylalanine to produce more intermediate metabolite phenylpyruvic acid, which can be transformed to D-PLA by D-lactate dehydrogenase.

Fig.2 PCR amplification for Tyrb.

The picture above is an electrophoresis of the product of Tyrb amplification. We amplified PCR by Tyrb and purified the product. Finally, we used agarose gel electrophoresis to verify whether the recovered product was purified.

Fig.3 PCR verification of Bacterial colony.

The agarose gel electrophoresis map of colony PCR products is shown above. We connected recombinant plasmid vectors of pRB1s-Dldh and Tyrb by Gibson system, and later transferred them into E. coli. After that, we took the recombinant E. coli (BWpRB1s-Dldh-Tyrb ) to make PCR, and the products obtained were verified by gel electrophoresis.

Fig.4 The SDS-PAGE results of the recombinant strain.

The picture above is a result of the SDS-page of the Tyrb protein expressed by the engineered bacteria. We induced culture of engineered bacteria（BW/pRB1s-Dldh-Tyrb）, and then performed SDS-page to verify its protein expression.

Sequence and Features