Cloning into M13 vector
.docx
School
University of the West Indies at Mona**We aren't endorsed by this school
Course
BIOTECHNOL 3113
Subject
Biology
Date
Dec 16, 2024
Pages
3
Uploaded by MateOxide23178
1.Cloning the Desired Gene into M13 Vector:oThe gene of interest is inserted into the single-stranded M13 bacteriophage vector.oThe M13 vector produces single-stranded DNA (ssDNA), which serves as the starting template for mutagenesis.2.Transformation into E. colidut/ung Strain:oThe M13 vector is transformed into an E. colistrain defective in:dut: dUTPase, an enzyme that reduces dUTP levels.ung: Uracil N-glycosylase, an enzyme that removes uracil from DNA.oIn this strain, dUTP accumulates and is misincorporated into DNA, replacing ~1% of thymine (T) with uracil (U). This creates single-stranded DNA templates containing U.3.Addition of Mutagenic Oligonucleotide:oA synthetic mutagenic oligonucleotide containing the desired mutation is added in excess.oThis oligonucleotide anneals to the ssDNA, forming a short double-stranded region where the mutation is introduced.4.Second-Strand Synthesis and Ligation:oDNA polymerase synthesizes the second strand using the oligonucleotide as a primer and the uracil-containing ssDNA as the template.oThe resulting double-stranded DNA (dsDNA) consists of:The original uracil-containing strand (template).A newly synthesized mutant strand.oT4 DNA ligase seals nicks in the mutant strand.5.Transformation into Wild-Type E. coli:oThe dsDNA is introduced into a wild-type E. colistrain with functional ung.oThe ungenzyme recognizes and removes uracil from the uracil-containing strand (original template), creating abasic (AP) sites.oAP sites are unstable and are rapidly degraded by the bacterial DNA repair machinery.6.Selective Retention of Mutant DNA:oThe original strand with uracil is degraded.oOnly the mutant strand (lacking uracil) remains intact.
oThis ensures that phage particles produced by the bacteria primarily contain the mutant DNA.7.Enrichment of Mutant Plaques:oThe phage particles containing the mutant strand are used to infect E. coli, producing plaques.oSince the uracil-containing wild-type DNA is selectively degraded, most of theplaques will carry the desired mutation, significantly enriching for mutants.Key Points:The dut/ung E. colistrain allows incorporation of uracil into the original DNA strand, creating a selective marker.The wild-type E. coliwith functional ungensures degradation of uracil-containing wild-type DNA.The process selectively enriches for the mutant DNA by taking advantage of the differential stability of the two strands in the presence of ung.This strategy greatly increases the proportion of mutant plaques, simplifying downstream screening for successful mutations.4oYou said:the mutant strand doesn't have uracil? ChatGPT said:ChatGPTCorrect, the mutant strand does not have uracil. Here's why:Nature of the Mutant Strand:The mutant strand is synthesized in vitroduring the second-strand synthesis step using the mutagenic oligonucleotide as a primer and standard dNTPs (including dTTP,not dUTP).Since the synthesis occurs under controlled conditions and with standard dNTPs, no uracil is incorporated into the mutant strand.As a result, the mutant strand is uracil-freeand structurally stable.Nature of the Original Template Strand:The original M13 single-stranded DNA (produced in the dut/ungstrain) contains uracil instead of some thymine bases due to the absence of functional dUTPase and Uracil N-glycosylase in the dut/ungstrain.
This strand is prone to recognition and degradation in the wild-type E. colistrain, which has a functional UNGenzyme.Selective Degradation of the Original Strand:When the double-stranded DNA (mutant strand + uracil-containing template) is introduced into the wild-type E. coli:1.The UNGenzyme recognizes uracil in the template strand and removes it, creating abasic (AP) sites.2.The AP sites make the template strand unstable and lead to its degradation.3.The mutant strand, which lacks uracil, remains intact and serves as the template for replication.Result:Only the mutant strandis replicated and packaged into phage particles.This enrichment step ensures that the majority of plaques produced carry the desired mutation.This mechanism is what makes the mutant strand selectively enriched while eliminating the original, uracil-containing wild-type strand.