Interpretation Questions 2: Restriction Enzymes and Subcloning 1. Restriction en

Question

Interpretation Questions 2: Restriction Enzymes and Subcloning 1. Restriction enzymes digest DNA at a specific recognition site, leaving a 5 overhang. 3 overhang or a blunt end. For each enzyme below, does it leave a 5 overhang 3overhang ora blunt end? 5 " A-CTAG T 3. T GATC A..5 5... G CTAGC..3 .CGATCG.. 5 5 CAG·CTG 3. Spel Pull denotes the cut sites 2. In the lab you are given a pUC cloning vector that contains the open reading frame (ORF) for gene X encoding protein X. The schematic diagram of gene X shows all of the restriction sites within the gene and in the flanking pUC vector sequences (vector is shown as dashed lines and the sequence of the sites are given on the next page). ...TCTAGA Start A GATCTStop codon Psti You wish to subclone this gene into the expression vector pETSAC. The MCS region in pETSAC is shown in more detail below MCS promoter rbs After subcloning gene X into pETSAC your plan is to transform the appropriate bacterial expression strain with this new construct to express the entire protein X You are provided with the following restriction enzymes (that have the recognition sites shown below) to cut gene X out of the pUC cloning vector for subsequent insertion into pETSAC. ..TCTAG A...3 3".. A GATC"T..5 3.. C TTAA'G... 5 3"...T GGCC A. .5 Xhal Agel 5..G CATG C...3 C'GTAC G... Sphl 5".. A GATC T...3 T CTAG A.. 3".. GGTAC C...5 Nool

Explanation / Answer

ANSWER TO QUESTION 1.

SpeI: 5' overhang

PvuII: blunt end

BmtI: 3' overhang

ANSWER TO QUESTION 2.

a) The 3' overhangs generated in the vector by SphI digestion will be complementary in sequence to the overhangs flanking the geneX created by NcoI (5'overhang) and SphI (3' overhangs), but both the overhangs flanking the insert (geneX) will be positioned in the 5'-3' strand and none in the 3'-5' strand of geneX; therefore the excised gene won't be able to ligate at its either ends on the digested vector, as only the SphI complementary overhangs will base pair properly, while the 3' overhangs of NcoI wont be able to base pair and ligate

b) Although BglII and EcoRI sites flank the gene X at 5' and 3' end respectively in pUC, in the pETSAC vector the EcoRI and BglII sites are in the 5' and 3' respectively, i.e. in reverse order compared to the pUC cloning vector. Therefore the gene X will be inserted, but in reverse orientation. Therefore the gene wont be aligned with the promoter and rbs, that is not in the frame required for transcription and subsequent expression (translation) of gene X. The start and stop codons will be placed in the 3' and 5' ends respectively after insertioninto pETSAC.

c) The ClaI and SphI sites flank the gene X in the pUC cloning vector, but in pETSAC the ClaI is placed within the promoter region. So even if the gene is positioned in the correct orientation, there will be no transcription initiated as the promoter will be disrupted upon gene insertion.

d)   The restriction sites of AgeI and XmaI differ, but the 5' overhang sequences in both cases are identical and are complementary.The 5' overhang of CCGG created by the AgeI upstream of gene X in its 5'-3'strand can base pair with the GGCC 5' overhang created by XmaI within the pETSAC vector in its 3'-5' strand. On the other hand, the blunt end created by AfeI can ligate with that created by AluI in the vector, as no complementarity or base pairing is required here for the latter case. So the gene X will be inserted in the correct direction, downstream to promoter and rbs and in frame for the gene to be encoded into its corresponding protein.

e) The Xba1 site is within the gene X, therefore using this enzyme will cleave the gene at this restriction site. Although the gene will be inserted in the correct orientation in the pETSAC vector following ligation at BglII and XbaI overhangs created in the vector, the nucleotide sequence downstream to reconstituted XbaI site after ligation will not pertain to gene X, but will correpond to the MCS sequence following XbaI site in the pETSAC vector. So the protein X when encoded will contain a wrong amino acid sequence (different from the original) at its 3' end or it may encounter a stop codon within the MCS to give rise to a truncated protein. The gene will read correctly till the XbaI site, but will encounter the abovementioned two possibilities following the XbaI site.

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