A standardized, high-precision, and general-purpose method for building functional modules
A functional module and high-precision technology, applied in the field of synthetic biology, can solve problems that do not involve the assembly of multiple modules
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Embodiment 1
[0043] Example 1 Construction of pLD-Blunt plasmid vector, pLD-Blunt plasmid vector is shown by SEQ ID NO.28.
[0044] In the present invention, pLD-Blunt is the only cloning vector artificially constructed for placing genetic elements, and its construction process is as follows:
[0045](1) Select the pEASY-Blunt kit of full gold, take out 1 μL of pEASY-Blunt reagent, add 4 μL of water, react at 25°C for 5 minutes, transform Escherichia coli, pre-coat IPTG and X-gal on the plate, and use blue-white screening to obtain blue colonies.
[0046] (2) Insert the blue colony into LB-Amp liquid medium, culture overnight at 37°C, and extract the pEASY-Blunt empty plasmid vector.
[0047] (3) Use the primer pLD-Blunt1-F shown in SEQ ID NO.19 and the primer pLD-Blunt1-R shown in SEQ ID NO.20 as the upstream primer and downstream primer, and use the pEASY-Blunt empty plasmid vector as a template, The BsaI point mutation was introduced into the Amp resistance gene, and the main part of ...
Embodiment 2
[0052] Example 2 Construction of pRS425K plasmid vector, the sequence of pRS425K is shown in SEQ ID NO.29.
[0053] In the present invention, pRS425K is an artificially constructed cloning vector for placing the module tool sequence, and its construction process is as follows:
[0054] (1) Select the pEASY-Blunt kit of full gold, take out 1 μL of pEASY-Blunt reagent, add 4 μL of water, react at 25°C for 5 minutes, transform Escherichia coli, pre-coat IPTG and X-gal on the plate, and use blue-white screening to obtain blue colonies.
[0055] (2) Insert the blue colony into LB-Amp liquid medium, culture overnight at 37°C, and extract the plasmid, which is the pEASY-Blunt empty plasmid vector. At the same time, the Escherichia coli strain containing pRS425 was inoculated to extract the pRS425 empty plasmid vector.
[0056] (3) Using the primer pRS425K1-F shown in SEQ ID NO.23 and the primer pRS425K1-R shown in SEQ ID NO.24 as the upstream primer and downstream primer, and using...
Embodiment 3
[0061] Example 3 Constructing the functional module of the green precursor deoxychromoviridans for the synthesis of violacein
[0062] Violacein is a bacteriostatic compound derived from Chromobacterium violaceum. Violacein is synthesized by cells using tryptophan as a substrate through five enzyme reactions: VioA, VioB, VioE, VioD, and VioC. After only the first three steps, the cells synthesize a green product called deoxychromoviridans, which makes yeast colonies appear green.
[0063] Therefore, in this example, three strength functional modules were designed for the genes of the three enzymes VioA, VioB, and VioE. On this basis, different functional modules were used to co-transform yeast, and different modules were combined to obtain combined functional modules. Enables Saccharomyces cerevisiae to produce deoxychromoviridans, making colonies green.
[0064] In this embodiment, VioA is represented by SEQ ID NO.1, VioB is represented by SEQ ID NO.2, and VioE is represent...
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