Epidermal staphylococcal tryptophanyl-tRNA synthetase and its use in screening drugs
A technology of Staphylococcus epidermidis and tryptophan, which can be used in DNA/RNA fragmentation, recombinant DNA technology, determination/inspection of microorganisms, etc., and can solve problems such as high antibiotic resistance and chronic infection
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example 1
[0017] Example 1 Three-dimensional modeling of tryptophanyl-tRNA synthetase protein of Staphylococcus epidermidis
[0018] Using PMODELING software, the three-dimensional structure of tryptophanyl-tRNA synthetase protein was simulated on a computer workstation, and the tRNA trp The two regions of action are the 234-238 region (ENKPG) and the 108-122 region (QFKDKAQKRADGVPAG). In addition, two regions that constitute the Rossmann fold and can bind ATP in space have been identified, namely the 15-18 region (TIGN) and the 193-197 region (KMSKS), see figure 1 .
example 2
[0019] Example 2 Cloning and identification of tryptophanyl-tRNA synthetase gene and expression and purification of protein
[0020] (1) Cloning of tryptophanyl-tRNA synthetase gene and construction of prokaryotic expression plasmid TrpRS-PKK223-3
[0021] The target gene was amplified by PCR, and then digested with restriction endonucleases (SmaI and PstI) and T 4 DNA ligase ligation reaction, clone tryptophanyl-tRNA synthetase gene into pkk223-3 vector, transform JM109 bacteria, select ampicillin-resistant colonies, a small amount of amplification to extract plasmid DNA, confirmed positive by double enzyme digestion clone.
[0022] The method for obtaining tryptophanyl-tRNA synthetase gene is as follows:
[0023] Design a SmaI restriction site at the 5′ end of the sense strand primer, and design a PstI restriction site at the 5′ end of the antisense strand primer:
[0024] sense:
[0025] 5′tcc ccc ggg atg gaa act tta ttc tca gga att ca 3′
[0026] anti-sense:
[00...
example 3
[0041] Example 3 Staphylococcus epidermidis tRNA trp Cloning, identification and in vitro transcription of genes
[0042] tRNA trp The gene is used for in vitro transcription after digestion, so the tRNA must be trp Modifications to the genetic sequence. tRNA trp The method of gene acquisition is as follows: Design primers to amplify the target fragment tRNA by PCR method trpGene, when designing primers, a T7 promoter sequence was designed at the 5' end, and a MvaI restriction site was designed at the 3' end (the CCA end can be obtained after enzyme digestion), and the length of the obtained PCR product was 94bp.
[0043] sense:
[0044] 5′ taa tac gac tca cta ta a ggg gca tag ttc aac ggt ag 3′
[0045] Antisense:
[0046] 5′gcc tgg cag ggg cag tag ga 3′
[0047] The underlined sequence is the T7 promoter, and the italicized sequence is the restriction site of Mva I. After obtaining the gene by PCR method, add dATP at both ends of it, and then pass T 4 DNA ligase c...
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