Biomi 2900- Wk 13

Front 1

Transformation

Back 1

uptake of naked DNA into the bacterial chromosome
1. Naked DNA is recognized by transformasomes
2. transformasomes degrade one of the strands, while the other strand enters the cell
3. Rec A recognizes the single strand and searches for homology on the recipient's chromosome and inserts DNA
4. DNA ligase seals the inserted fragment into the DNA

Front 2

Transduction

Back 2

virus mediated transfer of DNA; mistakes in assembly and packaging of viral DNA are the basis for transduction

Front 3

life cycle of virus

Back 3

1. attachment
2. penetration
3. synthesis of nucleic acids and proteins
4. assembly and packaging
5. release

Front 4

generalized transduction

Back 4

when a transducing particle is released from the host cell via lytic cycle and taken up by another bacteria
1. transduced DNA is integrated via homologous recombinatio
2. transducing particle does not replicate

Front 5

specialized transduction

Back 5

specific genes are transferred into the cell
1. temperate phage integrates DNA into host cell (lysogeny)
2. some of the host DNA is excised with it and replicated = lysogenized cell
3. lysogenized cell can divide or undergo induction an enter lytic cycle

Front 6

conjugation

Back 6

transfer of genetic material between bacterial cells via cell-cell contact
1. link between sex pilus
2. conjugative element is a plasmid

Front 7

plasmid

Back 7

1. circular, double stranded DNA molecule
2. not essential for cell survival
3. can carry antibiotic resistant and virulent genes
4. often multiple copies

Front 8

F plasmid

Back 8

1. fertility plasmid
2. allows for bacteria to produce a sex pilus
3. conjugative plasmid using tra gene
4. F+ = donors, F- = recipiens

Front 9

conjugation process

Back 9

1. F+ cell has sex pilus attach to F- cell and bring close together
2. F plasmid is nicked at oriT on one strand
3. nicked strand unwinds and is transferred into the F-
4. synthesis of complementary strand occurs (rolling circle replication)
5. cells separate and have 2 F+ cells

Front 10

Recombinant DNA technology

Back 10

inserting a gene of one organisms into another, so that the host propagates0 the products the gene encodes for

Front 11

restriction endonucleases

Back 11

1. integral to recombinant DNA technology
2. cut double-stranded or single-stranded DNA at specific restriction sites
3. dimers
4. naturally present in bacteria

Front 12

defense mechanisms of restriction endonucleases

Back 12

1. foreign DNA is cut by RE before it inserts itself into teh host genome
2. bacteria prevent its own DNA from being cut by methylase
3. there is a corresponding methylase for each RE

Front 13

RE in crafting recombinant DNA

Back 13

1. both plasmid and foreign DNA are cut using the same RE (EcoRI)
2. makes sticky ends
3. plasmid and foreign DNA combine
4. after they anneal, DNA ligase seals the chain
5. recombinant plasmid put into host

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sticky ends

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generated by RE are complementary to each other and bind readily

Front 15

verify DNA has been implanted into a plasmid

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1. study gel electrophoresis
2. inserted plasmids are larger and will be closer to the negative side

Front 16

to amplify large amounts of DNA

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1. use polymerase chain reaction (PCR)
2. series of heating and cooling steps
3. makes about 10^9

Front 17

maximization of target gene transltation

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1. fuse a strong promoter to the gene that encodes the desired product
2. T7 system

Front 18

visualization of promoter regulation/efficacy

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1. fuse the promoter to a gene whose product is easy to detect/measure
2. GFP

Front 19

protein purification

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selective binding

Front 20

DNA sequencing using fluorescent ddNTPs

Back 20

fluorescent ddNTPs indicate at what base chain elongation is halted