1. Genetic Engineering
Timothy G. Standish, Ph. D.

2. 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

3. 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.

4. 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

5. pUC 18 Sequence
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaaagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc

6. 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

7. 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

8. 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

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

10. 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)

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

12. 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

13. 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

14. A Library
The clone of interest

15. 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

16. 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

17. 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

18. The
End

19. 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