Application of ddb1 protein
A protein and protein function technology, applied in the field of gene therapy, can solve problems that do not involve genetic diseases
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Embodiment 1
[0026] Construction of embodiment 1 eco1-1 mutant strain
[0027] 1. Construct the expression vector of acetyltransferase Eco1 in Saccharomyces cerevisiae: pRS316-Eco1, pRS313-Eco1
[0028] Note: pRS316 is a yeast expression vector with URA as a nutritional selection marker,
[0029] pRS313 is a yeast expression vector with HIS as a nutritional selection marker
[0030] (1) Using the genomic DNA of Saccharomyces cerevisiae BY4741 as a template, use Pfu polymerase PCR to amplify the Eco1 target fragment:
[0031] Eco1-F: 5'-CGCGGATCCCAATTTGATCTTTTTATAAA-3';
[0032] Eco1-R: 5'-CCGGAATTCTCATATGTATACCGGCAATA-3' then use
[0033] Multifunctional DNA Purification Kit to recover PCR products.
[0034] (2) Digest Eco1 target fragment and vector pRS316 with BamHI and EcoRI. Reaction system: 5 μL of reaction buffer, 1.5 μL of BamHI and EcoRI, 2 μg of DNA, added water to 50 μL, digested at 37°C for 2 hours.
[0035] (3) Electrophoresis, using a gel recovery kit to recover the dige...
Embodiment 2
[0075] Example 2 Ubiquitin ligase Rtt101 -Mms1 Identification of the subunit Rtt101 anaplerotic acetyltransferase-deficient mutant eco1-1
[0076] 1. Ubiquitin ligase Rtt101 -Mms1 Construction of Related Vectors pGADT7-Eco1 and pGADT7-Mms1 Complementing Acetyltransferase Deficient Mutant eco1-1
[0077] pGADT7 is a yeast expression vector with LEU as a nutritional selection marker
[0078] (1) Genomic DNA of Saccharomycescerevisiae BY4741 was used as a template, and target fragments of Eco1 and Mms1 were amplified by PCR with Pfu polymerase.
[0079] The primer sequences used are as follows:
[0080] Eco1-2-F: 5'-CCGGAATTCATGAAAGCTAGGAAATCGCA-3'
[0081] Eco1-2-R: 5'-CGCGGATCCTCATATGTATACCGGCAATA-3'
[0082] Mms1-F: 5'-TCCCCCCGGGTATGCTAGGTTTGCGAACTCAT-3'
[0083] Mms1-R: 5'-CCGCTCGAGGGGAATTATACTGTGTTCTTGC-3'
[0084] The PCR product was then recovered with a multifunctional DNA purification kit.
[0085] (2) Digest the target fragment ECO1 and the vector pGADT7 with Ba...
Embodiment 3
[0115] Example 3 Overexpression of Mms1 (equivalent to human Ddb1 protein) in yeast can inhibit the growth defect caused by the deletion of Eco1 gene
[0116] The strains obtained in Example 2 were used for gradient sensitivity experiments. which is:
[0117] BY4741 eco1Δ::NAT pRS313-eco1-1 pGADT7;
[0118] BY4741 eco1Δ::NAT pRS313-eco1-1 pGADT7-ECO1;
[0119] BY4741 eco1Δ::NAT pRS313-eco1-1 pGADT7-MMS1
[0120] The specific operation is as follows:
[0121] 1) Cultivate the above strains overnight, count the cells on the day of the experiment, and adjust the cell concentration of all experimental strains to 2×10 6 cells / mL.
[0122] 2) Take 250 microliters of cells of each experimental strain, drop them into the first column of the 96-well plate, and add 200 microliters of sterilized ultrapure water dropwise to the second to fifth columns of the 96-well plate with a multi-channel micropipette.
[0123] 3) Set up five dilutions from the first column to the sixth column o...
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