Hansenula polymorpha genetic operation strategy and application thereof
A Hansenula polymorpha, a part of the technology, applied in the field of Hansenula polymorpha genetic manipulation strategy, can solve the problems of cell lethal effect, high false positive rate, low screening efficiency, etc., to achieve a wide range of applications and overcome low efficiency. Effect
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Embodiment 1
[0062] Example 1. Expression and biological function analysis of Escherichia coli toxic protein mazF
[0063] 1. Construction of recombinant plasmid pMOXZ-mazF
[0064] (1) According to the mazF sequence of Escherichia coli JM109 toxic protein gene (GenBank Access No.U00096.2), the following primers were designed and synthesized:
[0065] Primer 1: 5'-CGG AAGCTT ATGGTAAGCCGATACGTAC-3';
[0066] (The underlined part is the recognition site of HindⅢ enzyme digestion)
[0067] Primer 2: 5'-CCC TCTAGA AGTAACACTACCCCAATCAGT-3'.
[0068] (The underlined part is the recognition site of XbaⅠ digestion)
[0069] (2) Extract the genomic DNA of Escherichia coli JM109, use the genomic DNA as a template, and perform PCR amplification with primers 1 and 2.
[0070] PCR reaction system: genomic DNA 50ng, primer 1 final concentration 0.3μmol / L, primer 2 final concentration 0.3μmol / L, KOD-Plus-Neo DNA polymerase 1μL, 10×KOD buffer 5μL, 2mM dNTPs 5μL, 25mM Mg 2+ 2 μL, make up the syst...
Embodiment 2
[0093] Example 2, application of mazF expression cassette and zeoR expression cassette in gene marker-free knockout
[0094] 1. Construction of recombinant plasmid pEBCMMZC
[0095] (1) According to the CYC1TT sequence on the vector pMOXZα-A, design the following primers:
[0096] Primer 3: 5'-ATCCAATTGTGACACGTCCGAC-3';
[0097] Primer 4: 5'-CCTTTTACGGTTCCTGGCC-3'.
[0098] (2) Using pMOXZα-A as a template, PCR was performed with primers 3 and 4 to amplify the CYC1TT fragment.
[0099] PCR reaction system: pMOXZα-A50ng, primer 3 final concentration 0.3μmol / L, primer 4 final concentration 0.3μmol / L, KOD-Plus-Neo DNA polymerase 1μL, 10×KOD buffer 5μL, 2mM dNTPs 5μL, 25mM Mg 2+ 2 μL, make up the system to 50 μL with deionized water, and mix well.
[0100] PCR reaction conditions: 94°C for 5 minutes, 1 cycle; 94°C for 30 seconds, 56°C for 30 seconds, 68°C for 20 seconds, 30 cycles; 68°C for 10 minutes, 1 cycle.
[0101] (3) After the PCR product was detected by agarose gel e...
Embodiment 3
[0192] Example 3, application of mazF expression cassette and zeoR expression cassette in gene traceless knockout
[0193] 1. Amplification of PEP45'UP and mazF-zeoR
[0194] (1) According to the PEP4 gene sequence and the mazF-zeoR sequence in pMOXZ-mazF, the following primers were designed and synthesized:
[0195] Primer 15: 5'-AAA GAGCTC TCGACGCGGAGAACGATCTC-3';
[0196] (The underlined part is the recognition site of SacⅠ enzyme digestion)
[0197] Primer 16: 5'-GATGAGCATTCAGAGCTGTTGCAAATTAAAGCCTTCGAGC-3';
[0198] Primer 17: 5'-GCTCGAAGGCTTTAATTTGCAACAGCTCTGAATGCTCATC-3';
[0199] Primer 18: 5'-AAA GGTCACC AGCTCGGCCCGCCAGAAAT-3'.
[0200] (The underlined part is the recognition site of BstEⅡ restriction enzyme)
[0201] (2) Using pMOXZ-mazF as a template, use primers 15 and 16 to perform PCR amplification to obtain a 3.5kb sequence of mazF-zeoR, which is as shown in SEQ ID No.6 from the 1st position to the 3449th position from the 5' end indicated by nucleotide...
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