Animations Slide Show Review View tion and might be unsafe. Cick for more detail
ID: 3508804 • Letter: A
Question
Animations Slide Show Review View tion and might be unsafe. Cick for more details. Enable Eating Homework: this can be combined with homework from next class. Search for 1 paper that has used gene knockout technique to study E. coli ene function. Outline the method, gene function and conclusion. (Do not copy and paste) . I wish you to obtain a E, coli knockout strain, arsC KO, please search for the available resource and list the 1) website 2) Catalog number 3) the selective marker they used. . Design step-wise approach to knockout a gene in E.coli. Choose any gene that you are interested (not a lethal gene though). Include the gene sequence, regions that you wish to remove, vectors for cloning and primers for PCR that you are using and stepwise protocol. Describe how you verify the deletion.Explanation / Answer
1.CONSTRUCTION OF ESCHERICHIA COLI K-12 in-FRAME,SINGLE GENE KNOCKOUT MUTANTS:THE KEIO COLLECTION:
A set of precisely defined single gene deletion of all non essential genes in ESCHERICHIA COLI K-12.4288 genes were targeted and mutants were obtained for 3985.In this study two independent mutanst ere sved for every deleted gene.The mutants wew KEIO COLLECTION.This was taken not only for systematic analyses of unknown gene function and gene regulatory network but also to test mutational effects in common strain background;303 genes wew disrupted which had 37 of unknown function
3.The knockout protocol for E.coli employs 3 components:
Step1 :It includes construction of these 3 components.
STEP2:selection of the targets of Cas9 target sites and construction of pCRISPR-SacB-gDNA PLsmid
STEP3:mutgenic donor DNA must be designed and PCR screening must be done
STEP4:bacterial strains and culture conditions
STEP5:GENE KNOCKOUT USING CRISPR/Cas9