BCHM 4116 1st Edition Lecture 7 Outline of Last Lecture I Basic manipulation of DNA II Restriction Endonucleases and hydrolysis of nucleic acids III Type II Restriction Endonucleases a Related Issues IV Ligation of Restriction Fragments Outline of Current Lecture V Vectors and Molecular Cloning VI Alpha complementation VII Strategies to increase number of clones Current Lecture Vectors and Molecular Cloning Clone two organisms that have identical genetic material Essentially asexuall reproduction bacteria can do it Sexual reproduction exchange genetic material genes from mom and dad These 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 Clone Took 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 vector 2 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 Transformation 1 Chemical treatment to produce competent cells use of calcium chloride to prepare cell walls 2 Electroporation using an electrical impulse to introduce transient pores in membrane 3 Phase transfection rely on the ability of the recombinant phage to infect host cells The essential components of plasmid vector 1 Origin of replication allows the replication of the plasmid in a bacterial cell 2 Selectable marker allows the selection of bacterial cells that contain the plasmid 3 Cloning site often engineered to have multiple restriction sites inserting DNA of interest Alpha Complementation Used to determine if the insert has been placed in the vector Insertional inactivation of lacZ gene A vector contains the alpha fragment of the lacZ gene which encodes beta galactosidase The 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 sequence Beta galactosidase cleaves lactose or other beta galactosides such as X gal X gal is 5 bromo 4chloro 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 insert Intact lacZ Interrupted lacZ Functional alpha Defective alpha Bacteria with omega Bacteria with omga Functional galactosidase Defective galactosidase Blue on x gal plate White on x gal plate Strategies to increase number of clones that contain inserts Phosphatase 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 reaction Insert vector ratio have lots of inserts for a certain amount of vector to increase changes Double cuts Double cuts help to increase of recombinant clones Double cuts also help in directional cloning DNA 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 happen 1 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 vector insert