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Differential Transcriptome Sequencing And Critical Gene Identification On Escherichia Coli With Soluble Over-Expression

Posted on:2018-04-29Degree:MasterType:Thesis
Country:ChinaCandidate:Q Y HongFull Text:PDF
GTID:2370330518983165Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
With the rapid development of biotechnology,the need for rapid access to recombinant proteins is increasing,and how to obtain efficient protein expression systems is becoming increasingly critical.Protein expression system is the core of genetic engineering technology,usually refers to the use of model organisms,such as bacterial cells,yeast cells,animal cells,plant cells as expression of the host expression of foreign biological system.Escherichia coli expression system with Escherichia coli as the host is widely used because of its rapid cell proliferation,high yield,low price,high expression capacity,strong anti-pollution ability and clear genetic background.At the same time,the expression system of E.coli is the earliest Studied and the most mature protein expression system.Nevertheless,the system will still be affected by various factors in practical application,so that the expression efficiency is difficult to reach the highest level.The expression efficiency of Escherichia coli expression system is affected by a variety of factors such as temperature,medium,pH,oxygen content,the characteristics of the foreign gene itself(such as foreign gene A + T composition,exogenous gene codon selection,(Such as the selection of the vector,the choice of host bacteria),the interaction between the foreign gene and the system(such as the regulation of the expression gene,the copy number and the stability of the host bacteria to the plasmid,etc.)and so on.Studies have shown that the optimum growth temperature of Escherichia coli was at 37?.At this temperature,the cell metabolism was strong and the expression rate of exogenous protein was faster,but it was very easy to form inclusion body.And the cell autolysis intensifies,the stability of the plasmid is reduced,the yield and quality of the target protein are affected.At present,the use of low temperature in the production stage can prevent excessive folding rate to produce the wrong protein intermediates that leads to the formation of insoluble inclusion body structure,and at lower temperatures,acetic acid and other inhibition of exogenous protein expression byproducts will also be reduced.However,too low temperatures can lead to a decrease in nutrient intake and growth rates.Therefore,how to make the E.coli system in high-speed growth with efficiently expressing foreign protein,more in-depth study is need for the temperature control mechanismIn the expression of exogenous protein hpv16L1(human papillomavirus type 16 L1),we found that hpv16L1 protein could form soluble expression at 24?,but the expression level was low.At 37?,the expression of hpv16L1 protein was low,but it was almost in the form of inclusion body.Therefore,this study attempts to use transcriptome sequencing(RNA-Seq)method to analyze the difference of transcripts of engineering bacteria C2566 induced by different genes at different temperatures,to find the genes that were significantly differentially expressed and to analyze their related biological functions.The factors influencing the protein soluble expression and its mechanism were studied from the level of transcriptional regulation by investigating the impact of temperature on the transcriptome of Escherichia coli expression system.The results of this study show that the expression level of the transcriptome of Escherichia coli expression system is more abundant at 24?,and most of the high expression genes are related to protein translation and ribosome assembly.Others include glycine metabolism and hydrogen ion transmembrane transport.When the temperature increased from 24? to 37?,genes included the motor-related pathways and sulfur-containing amino acid-related metabolic pathways were significantly down-regulated,and the latter differences were probably the key to the low-temperature induction of E.coli expression system.In this paper,the effect of temperature on the soluble expression of exogenous protein of Escherichia coli was studied from the perspective of transcriptome regulation,which provided a new research ideas and methods for further transformation and promotion of Escherichia coli expression system.
Keywords/Search Tags:RNA-Seq, Soluble expression, C2566
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