Coay attempts to manufacture an antigen for vaccine development. The DNA sequenc
ID: 147114 • Letter: C
Question
Coay 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 BamH 5 GTCGAC3 5-ATGcttaggcat... (976 intervening base pairs omitted)...tggcagcccTAA-3' Restriction enzyme sites for BamHl and Sall are provided below: 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) HndIII Multiple Cloning sites Containing BamHI and Sall sites EcoRI Kanamycin resistant gene Promoter BseR Maintaining plasmid Sal ( Bantll Expression Sall vector Pre yak-t 1000 b Pacl Awt total (including yak-7).30001 Fet DUC Origin Ndel Swul There are several methods to clone the yak-1 into the expression vector. What will you use? Pease list the steps and the require enzymes. LIVEExplanation / Answer
There are several types of vectors found in the recombinant DNA technology, in which cloning vector and expression vectors are very normally used. So, the cloning vector must have the origin of replication (Ori), multiple cloning sites (MCS) and at least one antibiotic resistance gene for the screening. So, there is no need for promoter region and termination sequence because, with all three specialities, promotor region and ending sites are necessary for expression of the gene of interest. So for cloning the gene of interest in expression, we use restriction enzyme cloning technique.
Steps for clone the yak-1 gene into the expression vector:
1. Primer designing for the yek-1 gene:
Both forward and reverse primer designed for the sequence in which 5'-3' is the forward primer and 3'-5' is the reverse primer. we can design these primer manually or by the help of some online sites. now we only want to clone the DNA, there is no need to express it so we can add BamHI to the forward primer's 5' end with some overhangs and SaII at the 5' end of the reverse primer.
2. PCR of the yek-1 gene with both primers
3. Restriction digestion of PCR product and the expression vector (BamH1 and SaII)
4. Ligation of the digested product of the vector and insert DNA:
now both vector and insert DNA have sticky ends and ready for ligation process.
5. Transformation of the ligated product into the host E.coli strain:
here mainly use those strain which mainly increase the copy number, not in the expression. for example DH-5alpha/DH-10beta of E.coli strains. transformation can be done by the chemical process or electroporation process.
6. After the transformation of the vector which have the insert, then incubate the transformed cells into the specific medium.
7. Now from here screening or direct DNA isolation can take place.
8. Restriction digestion of isolated DNA.
9. DNA purification by agarose gel extraction method.