MCB 150 The Molecular and Cellular Basis of Life Plasmids and Genetic Engineering I Today s Learning Catalytics Session ID is: 20000966 1 Announcements: Check gradebook for discrepancies by Wednesday at 5 PM Deadline to sign up for Conflict Final Exam is Wed. at 5 PM - You ll be contacted with details by Noon Thursday Submit HCLA projects by Wednesday at 5 PM Final Exam is Monday, May 8 at 7:00 PM in Foellinger Office Hours prior to the final: - Tuesday 12:00 2:00, Wednesday 3:00 5:00 (Regular locations) - Friday 11:15 1:15 (124 Burrill Hall) - Monday 11:15 1:15 (124 Burrill Hall) 2
Mobile Genetic Elements (viruses and plasmids) Plasmids: Extrachromosomal molecules of circular, double-stranded DNA Carry from a few to a few dozen genes Found in many prokaryotes, some yeast, plants, protozoans - Nonessential in wild-type conditions 3 Plasmids usually confer some useful property to host: Can carry genes that enable bacteria to live in inhospitable environments - Soils contaminated with toxic metals (mercury, lead) - Industrial chemicals, petroleum by-products (oil slicks) - Pesticides & herbicides - Competition with other bacteria (bacteriocins) Can carry resistance factors that destroy/modify antibiotics - More detail on this in a minute Can carry genes for transferring plasmid via conjugation 4
Plasmids: Have their own origin of replication - Replication is not synchronized with replication of host chromosome, but will not happen in absence of host machinery Can be present in multiple copies in cell - Copy number varies - Naturally-occurring plasmids: copy number is 1 or 2 to 10 - Engineered plasmids can be up to 500 copies per cell - Invaluable tools for molecular biology 5 Plasmids: Can be moved between or among species of bacteria Plasmid ori 6
Major consequence of trading of plasmids among bacteria: Antibiotic resistant bacteria (some resistant to multiple antibiotics) are becoming a huge medical problem Penicillin first antibiotic discovered (A. Fleming) Discovered by accident in 1928 (fungal contaminant) Initially effective against virtually all Staph & Strep Today, 90% of S. aureus strains are resistant 7 Resistance to a traditional antibiotic appears to only be a matter of time: 8
Plasmids also have beneficial uses in genetic engineering Modified plasmids (and modified phage) can be used as transport molecules to move DNA between cells These modified carriers (plasmids & phage) are called vectors We can use endonucleases to open the circular DNA, insert a new fragment of double-stranded DNA, and then use ligase to close the circle back up The nucleases we use are found naturally in bacteria, and are called restriction endonucleases or restriction enzymes 9 Restriction enzymes: Found in most bacteria Absent in eukaryotes Made by the bacteria to degrade foreign DNA - Given names based on organism which produces it - Examples: EcoRI from E. coli, SmaI from S. marcescens Cut double-stranded DNA in very specific sites - called Recognition Sequences or Recognition Sites - usually 4 or 6 base pairs, but can be 5, 8, 12, etc. - if even one bp is changed, enzyme will not cut that site 10
Question: if restriction enzymes recognize specific sequences in double-stranded DNA, and bacteria have genomes of double-stranded DNA, why don t the restriction enzymes degrade host DNA? Could be the bacteria does not have that sequence Could be the bacteria modifies its own DNA to mark it DNA within recognition sites is methylated in host Methylated Not cut -GAATTC- * -CTTAAG- * -GAATTC- -CTTAAG- Unmethylated -G AATTC- -CTTAA G- 11 What s notable about restriction sequences? If the 5-3 sequence of the top strand is: 5 GAATTC 3 then what s the 5-3 sequence of the bottom strand? 5 GAATTC 3 A double-stranded sequence that reads the same in the 5-3 direction on both strands is called a palindrome - Type II restriction enzymes are the most useful for genetic engineering, and most Type II sequences are palindromes 5 GGATCC 3 5 CTGCAG 3 3 CCTAGG 5 3 GACGTC 5 12
When restriction enzymes cut double-stranded DNA, two types of ends are possible: Sticky (or staggered): Blunt: GAATTC CTTAAG GATATC CTATAG Eco RI Eco RV G AATTC CTTAA G GAT ATC CTA TAG single-stranded DNA double-stranded DNA 13 Ok, so what does this have to do with genetic engineering? Sticky ends can be brought together and religated If you cut one DNA molecule with BamHI, and another molecule with BamHI, all of those fragments will have the same single stranded overhanging sequence This means that one overhang will be complementary to another overhang, and can hybridize That temporary complementary base pairing can be made permanent by sealing it with DNA ligase DNA fragments from different organisms brought together is called Recombinant DNA (= genetic engineering) 14
BamHI GATCC G G CCTAG GATCC G G CCTAG DNA ligase BamHI GGATCC CCTAGG BamHI G CCTAG AATTC G EcoRI DNA ligase AATTC G**** G CCTAG 15 What about blunt cutters? No base pairing is involved, so any two blunt fragments can be brought together GATATC CTATAG CCCGGG GGGCCC EcoRV GAT CTA GGG CCC SmaI GATGGG CTACCC DNA Ligase 16
Recognition Sequences occur randomly throughout the genome If you know roughly how often they cut, and you know roughly how many base pairs there are in a given DNA molecule, you can estimate the size and number of fragments that will result from complete digestion with a given enzyme So, you can use restriction enzymes to cut large DNA molecules into smaller, manageable sized fragments And, maybe, you can isolate a gene or some other region of interest in one of those pieces But... molecular biology experiments are inefficient, so we need a lot of DNA to start with 17 MCB 150 The Molecular and Cellular Basis of Life Next: Flipped Class Period Continuing Genetic Engineering and Recombinant DNA Technology