biotechnology and genetic engineering pbio 450/550 eukaryotic gene organization restriction enzymes...
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Biotechnology and Genetic EngineeringPBIO 450/550
•Eukaryotic gene organization•Restriction enzymes•Cloning vectors
Eukaryotic gene organization
enhancerssilencers
Eukaryotic gene organization & RNA processing
Basic Transcriptional Mechanism and mRNA Splicing Animations
• MCB Chapter 4-Basic Transcriptional Mechanism animation• http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=
00010&n=04000&i=04010.01&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=0
• MCB Chapter 12-mRNA splicing animation• http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=
00010&n=12000&i=12010.02&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=1211
Prokaryotic vs. eukaryotic gene organization
Alternative splicing of eukaryotic 1° RNA transcripts
Eukaryotic gene expression
MCB Chapter 4-Life Cycle of mRNA
• http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=04000&i=04010.02&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|&ns=0
Recombinant DNA cloning procedure
Recombinant DNA cloning procedure
• See MCB Chapter 9 – Plasmid Cloning• http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?
v=category&s=00010&n=09000&i=09010.05&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|&ns=437
Restriction enzymes & DNA methylation
Recognition sequences of some REs
Enzyme Recognition site Type of cut end
EcoRI G ↓ A-A-T-T-C 5’ P extension
BamHI G ↓ G-A-T-C-C 5’ P extension
PstI C-T-G-C-A ↓ G 3’ P extension
Sau3A1 ↓ G-A-T-C 5’ P extension
PvuII C-A-G ↓ C-T-G Blunt end
HpaI G-T-T ↓ A-A-C Blunt end
HaeIII G-G ↓ C-C Blunt end
NotI G ↓ C-G-G-C-C-G-C
5’ P extension
Mapping of restriction enzyme sites
Vector system Host cell Insert capacity (kb)
Plasmid E. coli 0.1-10
Bacteriophage E. coli 10-20
Cosmid E. coli 35-45
Bacteriophage P1 E. coli 80-100
BAC (bacterial artificial chromosome)
E. coli 50-300
P1 bacteriophage-derived AC
E. coli 100-300
YAC Yeast 100-2,000
Human AC Cultured human cells
>2,000
Cloning vectors and their insert capacities
Plasmid cloning vectors
Three important features1. Cloning site2. Ori-an origin of replication3. A selectable marker (ampr)
pBR322
The plasmid pBR322 is one of the most commonly used E.coli cloning vectors. pBR322 is 4361 bp in length and contains: (1) the replicon rep responsible for the replication of plasmid (source – plasmid pMB1); (2) rop gene coding for the Rop protein, which promotes conversion of the unstable RNA I – RNA II complex to a stable complex and serves to decrease copy number (source – plasmid pMB1); (3) bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – transposon Tn3); (4) tet gene, encoding
tetracycline resistance protein (source – plasmid pSC101).
ori
pUC18/19pUC18 and pUC19 vectors are small, high copy number, E.coli plasmids, 2686 bp in length. They are identical except that they contain multiple cloning sites (MCS) arranged in opposite orientations. pUC18/19 plasmids contain: (1) the pMB1 replicon rep responsible for the replication of plasmid (source – plasmid pBR322). The high copy number of pUC plasmids is a result of the lack of the rop gene and a single point mutation in rep of pMB1; (2) bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – plasmid pBR322); (3) region of E.coli operon lac containing CAP protein binding site, promoter Plac, lac repressor binding site and 5’-terminal part of the lacZ gene encoding the N-terminal fragment of beta-galactosidase (source – M13mp18/19). This fragment, whose synthesis can be induced by IPTG, is capable of intra-allelic (alfa) complementation with a defective form of beta-galactosidase encoded by host (mutation lacZDM15). In the presence of IPTG, bacteria synthesize both fragments of the enzyme and form blue colonies on media with X-Gal. Insertion of DNA into the MCS located within the lacZ gene (codons 6-7 of lacZ are replaced by MCS) inactivates the N-terminal fragment of beta-galactosidase and abolishes alfa-complementation. Bacteria carrying recombinant plasmids therefore give rise to white colonies.
pGEM-3Z
Cloning foreign DNA into a plasmid vector
Alkaline phosphatase-removes 5’ phosphate (P) groups of DNA molecules; BAP is more stable but less active than CIP
T4 DNA ligase –joins 5’ phosphate (P) groups of DNA molecules to 3’ hydroxyl (OH) groups of DNA
Some antibiotics commonly used as selective agents
Antibiotic Description
Ampicillin (Amp) Inhibits bacterial cell wall synthesis; inactivated by -lactamase, which cleaves the -lactam ring of amp
Hygromycin B (HygB)
Kanamycin (Kan) Binds to 30S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase
Neomycin (Neo) Binds to 30S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase
Streptomycin (Str)
Tetracycline (Tet) Binds to 30S ribosomal subunit and inhibits protein synthesis; tetr gene encodes a protein which prevents transport of tet into the cell