BCHM 4116 1st Edition Lecture 8Outline of Last Lecture I. Vectors and Molecular CloningII. Alpha-complementationIII. Strategies to increase number of clonesOutline of Current Lecture IV. Gibson AssemblyV. Gateway Cloning (Recombination-based cloning)VI. DNA library ConstructionVII. Genomic DNA library construction: Phage vector, human DNAVIII. Making a cDNA libraryCurrent LectureGibson Assembly There are three parts to this: DNA preparation, Gibson assembly, and transformationDNA PreparationI. Require a linear vector II. Requires DNA inserts with overlapping ends Gibson Assembly I. An exonuclease chews back 5’ ends to create single stranded 3’ overhangsThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.II. DNA polymerase fills in gaps within each annealed fragmentIII. DNA ligase seals nicks in the assembled DNAAdvantages of Gibson Assembly include being able to do dozens of molecules at a time. TransformationI. This is when the completed vector is mixed with bacteria that can then uptake the vector and start transcription/translationOverview of Gibson AssemblyGateway Cloning (Recombination-based cloning) Step A is used to get the gene of interest into a vector. The gene is inserted into the donor vector, and the final product is called the entry clone. The donor vector has a selectable marker, that gives it resistance to a certain antibiotic. When grown on a plate with this antibiotic, only the bacteria that have the insert of choice and the selectable marker donor vector will survive. This entry clone however cannot express the genes as proteins because it doesn’t have all the promoter sites it needs.Step B takes the product on step A, and mixes it with a destination vector that has a different selectable marker. Through recombination, the gene of choice is transferred to the destination vector. This destination vector is called the expression clone as the finished product. This expression clone has the promotors to express proteins for bacteria or mammalian expression systems. DNA Library ConstructionA DNA library is a set of individual units (or clones) that represents the total sequences from a particular source. Libraries can originate from:I. The entire genome of an organism (a genomic library)II. The total expressed sequences (mRNA) in a sample (a cDNA library)Whatever the source, the DNA inserts are introduced into a vector for propagation in E. coli or other hostsGenomic libraryContains DNA sequences from the total genome of an organism, but expressed sequences and non-expressed (untranscribed) sequences.DNA is identical for essentially all cells so the source of DNA is unimportant—all genes present in all cellsContains intergenic regions and signals for proper eukaryotic gene expression, i.e, promoters, enhancers, terminators, introns. Genomic DNA library construction: Phage vector, human DNA1. Phage bacteria is cut with Bam H1 into two pieces: left and right arm. 2. The internal fragment is discarded. 3. Human genomic DNA is cut up into 20 kb fragment using Sau 3A (4 base cutter). 4. Sau 3A forms overhangs that are compatible with Bam H15. The human genomic DNA fragments are then inserted between the left and right arms of the phage DNAMaking a cDNA library1. Add a solution of total RNA in a column with salt The column has a cellulose matrix that has oligo(dT) chains attached2. The column is washed with salt solution to remove any non mRNA material such as rRNAand tRNA. Eukaryotic mRNA has a poly(A) tail that hybridizes with oligo (dT) chains on the cellulose, whereas rRNA and tRNA pass right through the column.3. The mRNA (bound to column with oligo(dT) is eluted by adding water. Low salt concentration destabilizes DNA, allowing the collection and evaluation of the mRNA eluted from the column. A visual of the process:How is cDNA made?mRNA isolated from the column is mixed with oligo(dT) primers which anneals to the poly A tail of the mRNA. Then reverse transcriptase is added along with substrates (dATP, dTTP, dGTP, dCTP) This synthesizes the first strand of cDNA from the mRNA template. Then RNase H is addedto the mix. RNase H is a endonuclease which recognizes the mRNA cDNA duplex, and cuts the mRNA leaving behind large gaps in the sequence. DNA polymerase then fills in the gaps, which are then ligated together by ligase. This is then the cDNA duplex. Overhanging ends are added at the end to aid in cloning. Here is a