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what is often the easiest way to introduce new genes into a bacterial cell and have them stably maintained in the strain put them on a plasmid TERM 2 () are replicating extrachromosomal DNA elements DEFINITION 2 plasmids TERM 3 "replicating" indicates that ALL plasmids must have their own () DEFINITION 3 origin of replication TERM 4 (classical plasmid types) () can be transferred from one species/strain to another via conjugation DEFINITION 4 F (fertility) plasmid TERM 5 (classical plasmid types) () provid resistance to some toxic substance (antibiotic, heavy metal) ex - ColE DEFINITION 5 R (resistance) plasmid
(classical plasmid types) () encodes production of an antibiotic that inhibits competitors in the environment Col (Colicinogenic) plasmid TERM 7 (true/false) some plasmids can fall into multiple categories DEFINITION 7 true TERM 8 plasmids mostly encode () under certain growth conditions but become () under other conditions DEFINITION 8 not essential or very important things, very important TERM 9 the ability to () functions on a plasmid allows a culture to pick these functions up rapidly = adaptibility DEFINITION 9 mobilize TERM 10 (plasmid types) DNA molecule that will carry a foreign DNA sequence from one place to another AND allow it to replicate DEFINITION 10 vector
requirements of expression vector antibiotic resistance, origin of replication, promoter, cloned gene TERM 17 (expression vector) often the proteins can be produced with additional small () fused to them making them easy to purify DEFINITION 17 domains (ex: his tags are a series of 6-8 consecutive His codons. the his residues int he protein product cause the protein to bind specifically to metals such as zinc, the protein can be purified by metal-affinity chromotography) TERM 18 (true/false) his tags can be on the beginning or the end of sequence DEFINITION 18 true TERM 19 () vectors - contains origins of replication that function in E. coli AND in another species DEFINITION 19 shuttle vector TERM 20 steps of shuttle vector DEFINITION 20 take plasmid from another species and clone into E. coli, alter plasmid in E. coli, and reintroduce back into original species
ways of plasmid transfer (4 ways) conjugation (transfer of DNA via cell-cell contact) transformation (introduction of naked DNA into a cell) electroporation, transduction (rare) TERM 22 (plasmid transfer) selection for plasmid maintenance by a strain is commonly done by selecting () DEFINITION 22 antibiotic resistance TERM 23 (true/false) an infinite variety of plasmid replication mechanisms DEFINITION 23 true, many plasmids in many different species TERM 24 plasmids must generally utilize some host factors such as DEFINITION 24 DNA pol I or III TERM 25 plasmids generally utilize some plasmid encoded factors like DEFINITION 25 an origin specificity protein that plays the role of DnaA
(looped rolling circle replication) the 3' OH end serves as a primer for leading strand rolling circle replication TERM 32 (looped rolling circle replication) when the replication fork gets al the way around circle and passes origin again, the rep protein.. DEFINITION 32 cuts at the same origin position as before, ligates the 5' phosphate it is bound to to the 3' OH that is created TERM 33 (looped rolling circle replication) leading strand uses () as primer, lagging strand uses () as primer, okazaki fragments DEFINITION 33 DNA, RNA TERM 34 linear plasmids are rare and found in these species DEFINITION 34 Borrelia (lyme disease), some streptomyces (soil bacteria that produce a variety of antibiotics) TERM 35 in some linear plasmids and bacteriophage, a () binds the end of the plasmid and serves as the primer DEFINITION 35 protein (found on every 5' end of the linear plasmid
in () the apparent linear plasmids are not actually linear, actually single stranded circles with complementary bases on the two sides Borrelia (two strands are continuous at the ends so replication areound the end results in no loss of DNA) TERM 37 circular single stranded plasmid replicates using DEFINITION 37 RNA polymerase TERM 38 the plasmid origin of replication functions in determining three plasmid properties DEFINITION 38 copy number, incompatibily, host range TERM 39 (copy number) () = low copy number (1- copies per cell) DEFINITION 39 stringent TERM 40 (copy number) () = high copy number (10-
DEFINITION 40 relaxed
what happens if we put a partial mutation in the repE gene so that the protein doesnt interact with itself as well A. do repress transcription of the repE gene so normal levels of repE protein B. dont repress replication as well so get higher copy number C. do activate replication as well, so WILL get higher copy number TERM 47 example of high copy numer DEFINITION 47 ColE TERM 48 (high copy number plasmid) ColE1 orgin is DEFINITION 48 oriV TERM 49 (high copy number plasmid) near the origin is a region that is transcribed by RNA polymerase to produce DEFINITION 49 RNAII (small) TERM 50 (high copy number plasmid) RNAII serves as DEFINITION 50 the primer for plasmid DNA replication (RNAII base pairs with part of oriV and DNA pol I uses it as a primer.
(high copy number plasmid) in the same region of the plasmid there is transcription in the opposite direction to produce RNAI TERM 52 (high copy number plasmid) because RNAI and RNAII are transcribed from the same region but from opposite template strands... DEFINITION 52 these two RNAs are complementary and can base pair with one another. TERM 53 the base pairing of RNAI and RNAII.. DEFINITION 53 prevents RNAII from functioning as a primer for replication TERM 54 () catalyzes formation of the RNAI-RNAII duplex and thus helps () replication DEFINITION 54 ROP (also encoded on the plasmid), inhibit TERM 55 (high copy number plasmid) the copy number is determined by DEFINITION 55 the relative amount of RNAI in the cell available to inhibit replication
incompatibility is often a function of the two plasmids having the same repressor of replication TERM 62 (plasmid incompatibility) classification is done by DEFINITION 62 transform/conjugate plasmid B into different groups of plasmid A cells. select for plasmid B, screen for plasmid A TERM 63 (true/false) two IncP plasmids can be maintained in the same strain DEFINITION 63 false, need to be in different "Inc" groups ex one IncP plasmid and one IncQ plasmid can be maintained in the same strain TERM 64 some plasmids can replicate and be maintained in a wide variety of species DEFINITION 64 broad host range ex RK2, pUB TERM 65 some plasmids can replicate and be maintained in a limited number of species DEFINITION 65 narrow host range ex ColE
Host range is usually determined by the ability of the plasmid-encoded proteins and plasmid origin or replication to interact and function with the particular bacterial species DNA replication proteins TERM 67 (plasmid host range) () often carry more of their own replication functions DEFINITION 67 broad host range plasmids (some cases, everything except DNA polymerase, most commonly DNA pol I and rolling circle replication) TERM 68 to be stably maintained in a strain, a plasmid must not only be replicated, it must also be DEFINITION 68 partitioned to the two daughter cells during cell division TERM 69 high copy number plasmids generally are () partitioned DEFINITION 69 randomly TERM 70 (high copy number plasmids) even if the cell only gets one copy... DEFINITION 70 it can rapidly replicate to increase the copy number up again
(true/false) par systems have to involve membrane association false TERM 77 (plasmid partioning) () protein of some plasmids just forms a long filament near the center of the cell DEFINITION 77 ParM TERM 78 it is possible to get plasmid incompatibility due to two plasmids having similar DEFINITION 78 par functions (results in each daughter cell getting only one of the two incompatible plasmids) TERM 79 (plasmid curing) () in an intercalating agent that gets in between the DNA bases DEFINITION 79 Acridine TERM 80 when acridine is added to a culture of a plasmid-containing strain... DEFINITION 80 it inhibits plasmid replication but not chromosome replication (too much acridine, chromosomal replication is blocked, too little acridine and plasmid replication continues, reason unknown)
(plasmid curing) the number of cell divisions after addition of acridine to cure strain of plasmids depends on the plasmid copy number
