Genetic Engineering Timothy G. Standish, Ph. D.

Genetic Engineering Genetic engineering involves taking fragments of DNA and manipulating them using enzymes and in other ways to make new genetic constructs The “recombinant” DNA made during genetic engineering can be inserted into organisms to change their genetic make-up In the transformation experiment you have been doing, you have inserted a recombinant piece of DNA called the pBLU plasmid into bacteria. On that pBLU plasmid is the lacZ gene and a gene for antibiotic resistance, both of which the bacteria lacked before you put them into it

Vectors If a fragment of DNA is ligated into an appropriate vector, it can be inserted into cells which will then make many copies of it Vectors are typically plasmids or viruses that have been engineered to both accept DNA insertions and reproduce inside cells Cloning is the process of inserting DNA encoding a gene of interest into a vector, then establishing it as a stable part of a cell line.

pUC 18 A Typical Plasmid 2,686 bp Ampr Gene Lac Z Gene Origin Multiple Cloning Site aagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattc HindIII SphI PstI SalI XbaI BamHI XmaI KpnI SstI EcoRI AccI SmaI BanII HincII BspMI Lac Z Gene Origin of Replication

pUC 18 Sequence tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaaagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc

R. E.s and DNA Ligase Can be used to make recombinant DNA EcoRI GAATTC CTTAAG G CTTAA AATTC 1 Digestion 2 Annealing of sticky ends 3 Ligation Ligase G CTTAA AATTC G G CTTAA AATTC 4 Recombinant DNA

Cloning Into pUC18 R. E. R. E. Digestion Digestion Matching sticky LacZ Ampr R. E. Digestion R. E. Digestion Matching sticky ends anneal Host Cell Addition of ligase joins nicks and makes a single recombinant plasmind Transformation of cells with the recombinant plasmid

So How Do You Know If You Cloned Something? IPTG - Induces expression of lacZ X-Gal - A lactose analog which turns blue when split by b-galactosidase Ampicillin - Kills all bacteria that lack the plasmid

X-Gal 5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside OH HOCH2 HO Galactose OH O HOCH2 HO Glucose Lactose O-b-D-galactopyranosyl-(1->4)-b-D-glucopyranose

X-Gal 5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside b-Galactosidease Lac Z gene product O OH HOCH2 HO Galactose H2O N H Br Cl X-Gal (Colorless)

X-Gal 5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside b-Galactosidease OH O HOCH2 HO Galactose N H Br Cl HO Blue

So How Do You Know If You Cloned Something? Blue colonies - Express b-galactosidase which metabolizes colorless X-gal to blue and turn blue thus lacZ is not disrupted and there is no foreign DNA cloned Cloned fragments disrupt lacZ thus make no b-galactosidase and colonies remain white IPTG - Induces expression of lacZ X-Gal - A lactose analog which turns blue when split by b-galactosidase Ampicillin - Kills all bacteria that lack the plasmid

Libraries If all the DNA from an organism is digested with a restriction enzyme and cloned into a plasmid, many different recombinant plasmids will be made, each with a different fragment of DNA cloned into it Once inserted into host cells or viruses, this collection of many different recombinant plasmids is called a “library” When the whole genome of an organism is used as the starting point for cloning, it is called a “shotgun clone” A library constructed using shotgun cloning may contain hundreds of thousands of different recombinant plasmids Screening is the process of sifting through the library to find the clone of interest

A Library The clone of interest

Library Screening Libraries tend to have a lot of clones, only one of which has the sequence of interest Screening a library is the process of eliminating those clones that do not contain the sequence of interest and locating the clone that does There two major techniques are used for screening: Hybridization screening - In which DNA from a library is bound to a membrane, then the membrane is exposed to a probe that should base pair (hybridize) to the sequence of interest Expression vectors may be used so that if the gene for a protein is cloned, the protein is made. To do this, you must be able to detect the protein

cDNA Libraries Because of the large size of libraries and the tedium of screening, anything that can be done to limit library size is a good thing Protein coding regions of most eukaryotic genomes make up only a small percentage of the total DNA (3% in humans) Most cells only express a small subset of an organism’s genes By using reverse transcriptase, a cDNA copies of the mRNA being produced in a group of cells can be made Cloning cDNA to make a library produces a much smaller library enriched with the part of an organism’s genome that is of most interest

cDNA Library Construction AAAAAAAAAAA3’ 5’ mRNA AAAAAAAAAAA3’ 5’ Reverse transcription TTTTTTTTTTTT5’ AAAAAAAAAAA3’ 5’ mRNA cDNA hybrid Rev. Trans. TTTTTTTTTTTT5’ 5’ TTTTTTTTTTTT5’ 5’ cDNA after RNase treatment RN ase A TTTTTTTTTTTT5’ 5’ Double-stranded cDNA after DNA polymerase AAAAAAAAAAA3’ 5’ DNA Pol Insert into vector

The End

An Expression Vector I II pPROTet.E II Myc tag EK site MCS pPROTet.E is a commercially available plasmid sold by Clontech It is specifically designed to allow efficient control of expression AatII XbaI I II T1 Cmr pPROTet.E II ColE1 t0 SacI