Design and construction methods of standardized biological components and their applications
A component and biological technology, applied in the direction of biochemical equipment and methods, microorganism-based methods, and other methods for inserting foreign genetic materials, can solve the problem of huge costs, can not be directly applied to eukaryotic microorganisms such as yeast, and is difficult to construct and optimize Exogenous metabolic pathways and other issues
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Embodiment 1
[0126] Example 1. Construction of HCkan-P, HCkan-O and HCkan-T
[0127] 1. Use pSMART HCKan as template, SEQ ID No.1 and SEQ ID No.2 as primers, and use ultra-fidelity DNA polymerase Q5 for PCR amplification (PCR program: 94℃ 3min; 94℃ 30s, 55℃ 30s, 72℃40s, 30 cycles; 72℃7min; 4℃+∞), the PCR amplification product is obtained, and the PCR amplification product is digested with BsaI to obtain the vector large fragment 1. The sequence of the vector large fragment 1 is as SEQ ID No.3 shown.
[0128] Replace the primers with SEQ ID No. 4 and SEQ ID No. 5. The remaining experimental steps are the same as the above experimental steps to obtain the PCR amplified product. BsaI digests the PCR amplified product to obtain the vector large fragment 2, and the vector large fragment The sequence of 2 is shown in SEQ ID No.6.
[0129] Replace the primers with SEQ ID No.7 and SEQ ID No.8. The rest of the experimental steps are the same as the above experimental steps to obtain the PCR amplificatio...
Embodiment 2
[0137] Example 2. Construction of POT1-11
[0138] 1. Removal of BsaI and BsmBI sites on yeast vector pRS416
[0139] Using pRS416 as template, F1 and R1 as primers, PCR amplification was carried out using ultra-fidelity DNA polymerase Q5 enzyme (PCR program: 94℃ 3min; 94℃ 30s, 55℃ 30s, 72℃ 30s, 30 cycles; 72 ℃7min; 4℃+∞), the PCR amplification product of 2426bp was obtained, and the PCR amplification product was digested by BsaI to obtain gene fragment A.
[0140] Replace the primers with F2 and R2, and the remaining experimental steps are the same as the above experimental steps to obtain a 1484bp PCR amplified product. BsaI digests the PCR amplified product to obtain gene fragment B.
[0141] Replace the primers with F3 and R3, and the remaining experimental steps are the same as the above experimental steps to obtain a 1054 bp PCR amplified product. BsaI digests the PCR amplified product to obtain gene fragment C.
[0142] The gene fragment A, the gene fragment B, and the gene frag...
Embodiment 3
[0161] Example 3. In vitro rapid assembly of β-carotene exogenous synthesis pathway
[0162] 1. The construction of each plasmid HCKan-P-TDH3, HCKan-P-ADH1, HCKan-P-TEF2 containing promoter
[0163] (1) Acquisition of TDH3 promoter, ADH1 promoter and TEF2 promoter
[0164] Extract the genomic DNA of yeast BY4741, use it as a template, use F5 and R5 as primers, and use ultra-fidelity DNA polymerase Q5 for PCR amplification (PCR program: 94℃ 3min; 94℃ 30s, 55℃ 30s, 72℃ 40s, 30 cycles; 72°C 7min; 4°C+∞) to obtain a 687bp PCR amplification product a1, which contains the TDH3 promoter, and the sequence of the TDH3 promoter is shown in SEQ ID No. 42.
[0165] F5: 5’-agcgtgggtc tcgggcttca ttatcaatac tgccatttca aagaatacg-3’;
[0166] R5: 5’-gtgctgggtc tcacatcttt gtttgtttat gtgtgtttat tcgaaact-3’.
[0167] Replace the primers with F6 and R6, and the remaining experimental steps are the same as the above experimental steps to obtain a 519 bp PCR amplification product b1, which contains the ADH1 p...
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