32. Plasmids, cloning and protein expression

Question 1

what si enzyme biotechnology?**

Answer 1

–

Question 2

what are the main types of vectors?

Answer 2

• plasmids
  – easiest to work with
• bacteriophages / other viruses
  – more conveniantly stored for long periods
• cosmids
  – cloning large pieces of DNA
• artificial chromosomes
  – include: bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs)
  – cloning large pieces of DNA

Question 3

what are the common characteristics of vectors?

Answer 3

• typically small well-characterised molecules of DNA
• contain at least one replication origin
  – can be replicated within appropriate host, even when the contain foreign DNA
• code for a phenotypic trait
  – can be used to detect their presence

Question 4

what are examples of recombinant DNA coning vectors?**

Answer 4

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Question 5

what are plasmids?**

Answer 5

• small circulat DNA molecules
  – exist independantly of host chromosome (present in many bacteria)
• DNa duplicated before very cell division
  – during cell division, at least one copy of plasmid DNA segregated to each daughter cell. Assuring continued progagation of plasmid through successive generations of host cell
• present in some yeast and fungi
• naturally occuring plasmids
  – encode genes providing benefit to host cell
  –
  –

Question 6

what is important about plasmids?

Answer 6

• first cloning vectors
  – antibiotic R
  – REs (same occur in antibiotic R gene)
• easy to isolate and purify
  – can be reintroduced into a bacterium by transformation
• plasmids often bear antibiotic resistant genes
  – used to select bacterial hosts

Question 7

what are the feartures of plasmids?

Answer 7

• own replication origins
• autonomously replicate
  – stably inherited as bacterial cells reproduce
• single copy plasmids produce one copy per host cell
  – multi-copy may present concentrations of >40
• have relatively few genes
  – less than 30
• genetic information not essential to host
  – bacteria lacking this usually function normally
• often confer antibiotic resistance on bacteria that contain them
• plasmid cloning vectors contain multiple cloning sites (MCS)
• some protein expression vectors contain portions of lac operon and effectively regulate expression of clones genes in same manner as operon
• others contain reporter gene, based on lac operon

Question 8

what are the specificities of the features of plasmids?

Answer 8

• own replication origins (ORI)
• autonomously replicate
  – stable inherited as bacterial cells themselves reproduce
  – shuttle vectors constructed so can propogate in two different host species. Thus, E.coli / Bacillus shuttle vector can be propogates in either of host systems
• single copy plasmids produce only one copy per host cell
  – multi-copy plasmids may be present at concentrations of >40
  – induction to go from single copy to multiple in short space of time
  – host can be geown to high cell numbers prior to induction of high plasmid copy number
  – prevents negative effect due to metabolic load, allowing extraction of high concentration of plasmids
• have relatively few genes
  – less than 30
  – small
• genetic information not essential to host
  – bacteria lacking this usually function normally
  – either beneficial for host or used in recombinant system
• often confer antibiotic resistance on bacteria that contain them
  – typically have genes encoding eznymes capable of destroying / modifying antibiotics
  – genes coding for resistance to antibiotics (eg. ampicillin, chloramphenicol, kanamycin found in plasmids). Can have as many as 8 resistive properties.
• plasmid cloning vectors contain multiple cloning sites (MCS)
  – useful restriction sites, only cut once in vector improving ease of cloning DNA fragment
  – RE’s don’t cut inside GOI
• some protein expression vectors contain portions of lac operon and effectively regulate expression of clones genes in same manner as operon
• others contain reporter gene, based on lac operon
  – based on lac operon, indicate bacterial cell harbouring a recombinant plasmid with clone insert
  – eg. pGEM series of cloning vectors

Question 9

what are the key features of clonign vectors?

Answer 9

• origin of replication
• multiple cloning sites
  – flanked by sequencing primer binding sites
• selectable marker / antibiotic resistance genes
• reporter gene
  – LacZ
• expression vectors
  – strong inducible promoter upstream of MCS to drive expression of cloned gene for heterologous protein expression

Question 10

what are the advantages of plasmid vectors?

Answer 10

• circular
  – prevents endo/exo-nucleases from digesting DNA
• derived from natural plasmids occuring in bacterial cells
  – easier to manipulate and propogate
• more stable vector to maintain particular clone
• grate variation in size
  – 1-400 kb

Question 11

what are the disadvantage of plasmid vectors?

Answer 11

• small fragments (10-15 kb) of DNA can be inserted
• low transformation efficiency

Question 12

what is the comparison of the expression systems?**

Answer 12

• table:

Question 13

what are E.coli as microbial expression hosts?**

Answer 13

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Question 14

how are E.coli typically expressed as plasmid vectors?**

Answer 14

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Question 15

what is the heterologous protein expression in industry?**

Answer 15

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