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Research on the Cloning Bop Gene and Its Promoter from the Genome DNA of the H.salinarium S9, Constructing BR Mutants and Studying Their Photochromic Properties

Author: SongJingJiao
Tutor: CaoJunWei
School: Wuhan University
Course: Microbiological techniques
Keywords: Bacteriorhodopsin bop gene Mutant M412 intermediate state Proton pump
CLC: Q936
Type: Master's thesis
Year: 2005
Downloads: 65
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Abstract


In this study, by recombinant PCR amplified from the salt marsh salt bacilli (Halobactrium salinarium) S9 genome encoding bacterial rhodopsin (bacteriorhodopsin, BR) protein the bop genes and promoter part . The sequencing results showed that in the NCBI database bop gene and promoter sequences are identical , and its expression product molecular weight and a molecular weight of the wild-type BR protein , and exhibit at 568nm at the characteristic absorption peak of the BR protein . Using gene -directed mutagenesis to construct three mutants that single mutations bopG649C , the three mutations the the bop T395C/A400T/G415C < / sub> and four mutations in the bop G649C/T395C/A400T/G415C < / sub>. The mutant gene as well as the wild-type gene are respectively connected to the the expression vector pXLNovR , L33 of ( BOP - < / SUP > ) in the expression host strain . Mutations in the gene expression of BR mutants were BR E204Q , BR I119T/T121S/A126T , BR ( E204Q/I119T/T121S/A126T. after the UV-visible spectra , a single mutation BR E204Q and have a relatively broad absorption peak between 552 and 570nm , the triple mutant BR I119T/T121S/A126T 555nm and 572 nm has a relatively broad absorption peaks, between the four mutant BR E204Q/I119T/T121S/A126T has an absorption peak at 410nm and 552 nm through its M 412 flash kinetic studies show that a single mutation body BR E204Q < / sub> , the lifetime of the M state to 7.10ms, three mutants BR I119T/T121S/A126T < / sub > M state lifetime of 8.23ms, the four mutant BR E204Q/I119T/T121S/A126T M state lifetimes 19.85ms, compared with the wild -type the BR protein M state lifetimes (6.23ms) has been extended , and four mutants performance most significantly , an increase of 218.6% in the M state lifetime than the wild-type BR protein . proton pump function mutants change detection of mutations proton pump function , including single mutation body BR E204Q and three mutants

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CLC: > Biological Sciences > Microbiology > Microbial biochemistry
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