There are several methods to clone the yak-1 into the expression vector. What wi

Question

There are several methods to clone the yak-1 into the expression vector. What will you use? Please list the steps and the require enzymes. Cody attempts to manufacture an antigen for vaccine development. The DNA sequence corresponding to the coding region of yak-1 contains 1000 base pairs, and has the following structure (ATG and TAA correspond to the sites of the start and stop codons for translation, respectively): Sall BamHI SGTCGAC3 5-ATGcttaggcat... (976 intervening base pairs omitted)... tggcagcccTAA-3 CCT Restriction enzyme sites for BamHl and Sall are provided below CAGCTO 3CCTAGG5 A map of the E. coli vector containing yak-1gene is provided below. Ori denotes the origin of replication, amp denotes the ampicillin resistance gene. Hindill, BamHl, Sall, and Ndel designate restriction enzyme sites. There are no other restriction enzyme sites found on this vector This vector has high copy number for maintaining the gene, but it does not have a promoter and terminator for transcription and subsequent translation (A). It needs to subclone the yak-1 gene into an expression vector that contains the required gene structure for transcription and translation (B) Multiple Kanamycin resistant genePromoter sCloning sites Containing BamHI and Sall sites Maintaining plasmid Expressiorn vector Sall sin a-1-1000 lotal (including yak-1)-3000 There are several methods to clone the yak-1 into the expression vector. What will you use? Please list the steps and the require enzymes.

Explanation / Answer

Answer: as we know that vectors are dilivering system of any target gene. vectors are generally two types 1. cloning vector 2. expression vector. The ideal vector must contain 1) Origin of replication 2) multiple cloning sequences, 3) selectable marker sequences

As in the cloning vector our target is just replicate our desired gene by the process of transcription only.

while in Expression vector as the name suggest there should be some extra things so that along with the transcription the process of translation also takes place to produce protein of our interest. so here in the expression vector we have some additional requirements as 1) Origin of replication 2) multiple cloning site 3)selectable marker site 4) strong promoter site that is induciable promoter ,5) Translation initiation sequences as start and stop codons, 6)the element for expression ( Shine Dalgerno sequence in prokeryotes and Kozak consensus sequences in eukaryotes, 6) Purification tag that is generally 6x histidin complex and 7) fusion protein insert which is the enzyme composition as Glutathione S transferase or maltose binding protein or green florescent protein GFP as a reporter gene.

so for expression vector all the above described things will be required and if we are liking to clone the yak-1 in the expression vector all these are needed .

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