| The study on unknown gene function or specific gene both requires a properate vector to clone into,which makes the selection of vector even more crucial.The development and extensive use of T-A clone technology has overcome the disadvantage occurred in ligation of cohesive or blunt ends of sequences by the unique cohesive-ends-linkage principle.Common commercial T vector requires IPTG as inducer and selecting positive clone by blue-white selection.However,there are several disadvantage of this method:1.High false positive rate occurred.2.Deficient expression of taget gene.3.Insertion of target gene is unidirectional,reverse insertion can't promote expression of target gene,and determination of direction makes the selection and construction of expression vectors more difficult.In consequence,our research focusing on development and applications of a novel multi-functional T vector:pHsh-T,whichprovides molecular cloning,high-level gene expression,library construction and target gene activity screening for gene engineering.pHsh-Cm was sequenced and the Bfu ? site at 1874 bp was removed.Design a constitutive promotor Hkg pro based on the conserved sequence of E.coli ?70 recognizing sequence,reverse PCR was used to introduce the Hkg pro and relevant RBS into pHsh-Cm,based on that,a terminator Hkg term which is not depending on p factor was designed and introduced into the vecotor.two back-to-back Bfu ? sites were introduced between Hkg pro site and Hsh pro,the vector was designated as pre-pHsh-T,at last the vector was digested by Bfu ?,a positive band was formed when electrophresed on agar gel,which indicated the pHsh-T was constructed correctely.16S rDNAs from 3 unidentified cholesterol oxidase produced strains which were isolated by previous work were cloned into pHsh-T,sequencing and blasting against NCBI database demonstrated those three strains were Geobacillus.RSP gene from Thermoanaerobacter ethanolicus JW200 and Endo-1,4-?-xylanase gene from Thermotoga neapolitana DSM 4359 were cloned into pHsh-T,and positive transformants were selected from plates randomly for sequencing,all seleceted transformants were correct and can express targed gene efficiently.RSP gene was cloned into commercial pET-28a vector,designated as pET-RSP.A transformation rate analysis was performed by electrotransforming pHsh-RSP and pET-RSP into same host and compare the transformation rate of each in the same procedure.The result shows,pHsh-RSP gets higher transformation efficiency.This indicates that pHsh-T is beneficial to clone free lowers the cost of industry enzyme and medicinal protein production,and the and high-level expression of gene,inducer bi-directional expression elements guarantee forword or reverse insertion of target gene expressing,which can be broadly used in library construction and target gene product active screening.A novel method of genetic library and screening was developed base on the characterastics of pHsh-T.A genome from T.ethanolicus JW200 were digested by Sau3A I partially,the products with appropriate lenth were recovered from agar gel,and an additional A base was added by Taq DNA polymerase after filling the sticky ends of the fragments.The theoretical transformants are 6900 while the actual transformants of the experiment are 9000,which is 1.3 times as many as theoretical value.pHsh-T,combined with a previously constructed vector pTrc99A-ap,was used to develop a method for screening interaction between DNA and proteins,the study of this method is still underway. |
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