Part:BBa_K4765118
ribozyme connected: MysABCDH
Contents
Introduction
Mycosporine-like amino acids (MAAs), act as a sunscreen for the biofilm. This composite part contains five enyzmes in the MAA biosynthesis pathway. All the enzymes are constructed into ribozyme-assisted polycistronic co-expression system：pRAP.
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| Figure 1. The biosynthetic pathway of shinorine, porphyra-334, palythine-Ser, and palythine-Thr |
Usage and Biology
The biosynthetic route of MAA initiates with the generation of 4-deoxygadusol (4-DG) from sedoheptulose 7-phosphate, an intermediate within the pentose phosphate pathway. This process is catalyzed by two enzymes: a dimethyl 4-degadusol synthase (DDGS; MysA) and an Omethyltrans-ferase (O-MT; MysB). Subsequently, 4-DG undergoes a transformation into mycosporine-glycine(MG) through an ATP-grasp enzyme MysC, which introduces an amino acid moiety, primarily L-Gly. MAA analogues such as shinorine or porphyra-334 are further derived from MG by the D-Ala-D-Ala ligase-like enzyme MysD. In the final step, the biosynthesis is completed with a nonheme iron-(II)- and 2oxoglutarate-dependent (Fe/2OG) oxygenase MysH, leading to the production of palythines.
Characterization
Agarose gel electrophoresis
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| Figure 2. Agarose gel electrophoresis of PCR products, amplified from bacterial colonies/cultures.
From right lane(4) to left lane(1) indicate the successful construction of MysD, MysDH, MysDHB, and MysDHBA. |
Successful Protein Expression
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| Figure 3. SDS-PAGE electrophoresis of ribozyme connected: MysABCDH
We successfully constructed MysDHB and MysDHA into the pET28a plasmid and transformed it into E. coli BL21 DE3. Lane 1 to 2 represents the IPTG uninduced and induced version of MysDHB.Lane 1 to 2 represents the IPTG uninduced and induced version of MysDHA. As indicated by the red arrow, we successfully expressed MysDHBA in this part.
Anti-UV Survival AssayWe employed the Colony-Forming Unit (CFU) assay. After plasmid transformation and plating, we shielded one/half of the agar plate from UV light using a black paperboard, while the other one/half was exposed to UV irradiation (6W power) with wavelengths of 254 nm and 365 nm for 10 seconds.
Our experiments demonstrated that introducing three or four of the five enzymes from the MAA biosynthetic pathway into E. coli did not yield a significant enhancement in the bacterium's UV resistance. We postulate that this lack of effect may arise from two factors: Firstly, the synthetic pathway may not play a pivotal role amidst the numerous routes involved in MAA synthesis. Secondly, to augment MAA levels within E. coli through protein expression in the pathway, additional substrates like ATP and amino acids would likely be required in the reaction.
Assembly Compatibility:
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