5. A cloning vector is cut with the restriction endonuclease Sma 1, whose restri
ID: 256025 • Letter: 5
Question
5. A cloning vector is cut with the restriction endonuclease Sma 1, whose restriction site is and treated with alkaline phosphatase to prevent the relegation of the plasmid. The synthetic, double-stranded DNA fragment (foreign DNA) with sequence 5' -GGACTTACTACCCAAGTA-OH 3' 3' OH-CCTGAATGATGGGTTCAT- 5 is inserted into the Smal site of the vector, whose location with respect to the ?-galactosidase gene in the cloning vector is shown below. (it is a blunt-end ligation!). [partial sequence shown here] H3N-Met-Thr-Met - lle -Thr -Asn -Ser -Arg -Gly-> functional ? galactosidase gene Starts here. -ATG/ACC/ATG/ATT/ACG/AAT/ TCC/ cGG/GGA Cut her Upon transformation of the ligation mixture into E. coli and in the presence of the indicator X-gal, 52% of the plaques on the plate are blue and 48% are clear. Give the most likely explanation for this result. (7 points) (Blue colonies are not due to relegation of the plasmid!!) SHOW ALL YOUR WORK! (including ligation, transcription and translation of the ligated products) HINT: Make sure to identify the coding/non-coding strands PLEASE BE NEAT!Explanation / Answer
One of the known screening techniques called blue white screen and it can allows recombinant bacteria rapid and convenient detection in the vector based the molecular cloning. Most of the inserts can disrupt the b galactisidose expression (By shifting the reading frame and inserting stop codons) and thus removing the enzyme activity required to cleave X gal and produce blue colour. Thus cells transformed with vector containing recombinant DNA will produce white colonies whereas cells transformed with non recombinant plasmids grow in blue colour. Thus 52% of plaques are blue and therefore these cells have no recombinant plasmids while 48% plaques have been transformed with plasmids containing inserts.
Work Explanation:
Coding region 5' with ligation ATG/ACC/ATG/ATT/ACG/AAT/TGG/ACT/TAC/TAC/CCA/AGT/AGA-OH 3'
Non-coding:
3' TAC/TGG/TAC/TAA/TGC/TTA/ACC/TGA/ATG/ATG/GGT/TCA/TCT- 5'
TRANSCRIPTION:
THE CODING STRAND IS COPIES INTO RNA
5' AUG/ACC/AUG/AUU/ACG/AAU/UCC/ACU/UAC/UAC/CCA/AGU/AGA 3'
TRANSLATION:
CONVERSION OF RNA CODINGS INTO PROTEIN MADE UP OF AMINO ACIDS
Met/thr/Met/Ile/Thr/Asn/Ser/Thr/Tyr/Tyr/Pro/Ser/Arg
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