BCHM 4116 1st Edition Lecture 7Outline of Last Lecture I. Basic manipulation of DNAII. Restriction Endonucleases and hydrolysis of nucleic acidsIII. Type II Restriction Endonucleases a. Related Issues IV. Ligation of Restriction Fragments Outline of Current Lecture V. Vectors and Molecular CloningVI. Alpha-complementationVII. Strategies to increase number of clonesCurrent LectureVectors and Molecular CloningClone: two organisms that have identical genetic material Essentially asexuall reproduction: bacteria can do itSexual reproduction: exchange genetic material, genes from mom and dadThese 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.Sheep CloneTook a somatic cell, and an egg. Remove the nuclei from egg, and fuse the donor cell with the egg. This equals a new cell with the genetic information of the donor cell. Put the egg into a host, and the embryo will have its entire genetic material from the donor cell. Cloning is done in-vivo, inside an organism. Requirements for molecular cloning:1. Cloning vector and methods to insert DNA into a vector2. Methods to introduce vector in a cell (Transformation)3. Methods to produce copies of recombinant molecules (replication)4. Methods to enrich for and identify transformants (selection)5. Methods to detect inserts (screening)Transformation1. Chemical treatment to produce competent cells—use of calcium chloride to prepare cell walls2. Electroporation—using an electrical impulse to introduce transient pores in membrane3. Phase transfection—rely on the ability of the recombinant phage to infect host cellsThe essential components of plasmid vector1. Origin of replication: allows the replication of the plasmid in a bacterial cell2. Selectable marker: allows the selection of bacterial cells that contain the plasmid3. Cloning site: often engineered to have multiple restriction sites, inserting DNA of interestAlpha-ComplementationUsed to determine if the insert has been placed in the vector. Insertional inactivation of lacZ geneA vector contains the alpha fragment of the lacZ gene which encodes beta-galactosidaseThe lacZ is a portion of beta-galactosidase gene, and the remainder of the gene is in the bacterial genome The cloning site is present within the lacZ coding sequenceBeta-galactosidase cleaves lactose or other beta-galactosides such as X-gal X-gal is 5-bromo-4-chloro-3-indolyl-beta-D-galactoside a chromogenic substrate for the enzyme - gives a blue color when cleaved When no DNA fragment is inserted into the cloning site (in lacZ' gene), both portions of the beta-galactosidase enzyme are produced (one from vector, one from bacterial genome). They form a hybrid and complement to yield active enzyme, capable of cleaving X-gal to give blue color (bacterial colony appears blue) When a DNA fragment is inserted in the cloning site of lacZ', the gene is interrupted. As a result,no alpha fragment can be synthesized and therefore no complementation occurs. Without active enzyme, no cleavage of X-gal substrate results and no blue color is produced (the bacterial colony remains colorless)Without insert With insertIntact lacZ Interrupted lacZFunctional alpha Defective alphaBacteria with omega Bacteria with omgaFunctional galactosidase Defective galactosidaseBlue on x-gal plate White on x-gal plateStrategies to increase number of clones that contain insertsPhosphatase treatment: prevents the two ends from ligating together before the insert is in. It cleaves the 5’ phosphate Before putting in insert, treat the vector with phosphatase, to remove 5’ phosphate. Then add insert, and perform ligation reactionInsert/vector ratio: have lots of inserts for a certain amount of vector to increase changesDouble cuts: Double cuts help to increase % of recombinant clones. Double cuts also help in directional cloningDNA to be cloned is inserted into a vector. The vector with the insert in then placed in the host cell via transformation. Transformation can be done using chemical treatment (CaCl2), electroporation, or infection. Things that could happen1) The host cell could uptake a vector that doesn’t have an insert. 2) the host cell could fail to uptake anything (vector or vector+insert)3) the vector could uptake a vector with the insert (Ideal situation)The desired bacterial cells can be separated from the failed ones by adding an antibiotic resistance gene in the insert. Strategies to find the right clone that contains insert of your interest: A) ensure that the cells contain a clone--selectable markers B) ensure that the cells contain a clone w/ an insert--lacZ inactivation (color) C) find the clone that contains insert of your interest: screening w/ a specific probe or antibody. nly ones that will survive will be the bacteria with the