Lab 4

Question 1

Direct cloning (cloning for function)

Advantages & Disadvantes

Answer 1

Advantages

• Most direct method to identify gene
• If multiple genes encode the same phenotype (e.g., isozymes), a mutagenesis approach will unlikely identify appropriate mutants (masking effect)

Disadvantages

• requires a compatible, heterologous expression system
  
  • a host to take DNA from one organism and express it in another organism that does not have the same phenotype from the 1st organism
• needs to ensure the mutant organism has the capabilities to express the cloned genes
  
  • i.e. lipase gene from S. marcescens, it’s product is secreted extracellularly
  • mutated cell must have the appropriate mechanism to provide secretion of gene product from cell as well
• needs to be a single gene that functions autonomously or genes that are physically linked in a small area
  
  • genes spread across the genome, must be reengineered closer, prior to cloning
• Appropriate/compatible plasmid vector cloning system
  
  • able to replicate in new host
  • Appropriate antibiotic selection marker

Question 2

Direct cloning (cloning for function)

steps

Answer 2

• Isolate genomic DNA
• Construct genomic DNA library
• Transfer library to non‐lipase producing host strain (E. coli)
• Screen E. coli containing clones for lipase activity
• Characterize clones

Question 3

Cosmid cloning vector

Answer 3

• large plasmid cloning
• uses phage to transfer large fragments of DNA
• we use cosmid vector plasmid pLAFR3
  
  • 21.6 kb in size
  • Tetracycline resistance (Tcr)
  • Multicloning sites
    
    • use of different restriction enzymes: RI‐Sm‐B‐Sal‐P‐H
  • Cos sites
  • transferable by conjugal mating
  • Broad host range
    
    • RP4 origin of replication allows for this
  • Low copy number per cell (1‐10)
    
    • similar to bacterial chromosome

Question 4

Genomic Library Construction

Cos sites and phage packaging requirements

Answer 4

• Cos (cohesive end) sites of lambda phage
  
  • approx. 400 bp
  • circularizes the genome once it infects a cell
• Phage enzymes
  
  • recognize cos sites for packaging DNA into phage heads
• Packaging size limitations
  
  • standard lambda genome 40‐45 kb
  • Phage head can accommodate
    
    • 37-50 kb
    • 70‐110% of standard lambda genome
• pLAFR3
  
  • 21.6 kb in size
  • can package a plasmid vector containing around 15‐30 kb
• How many clones in a library
  
  • Average genome size of a cell: 5000 kb (5 Mb)
  • Average clone size: 25 kb
  • thus need 200 clones of 25 kb to represent entire library
• You typically want to have a 5X coverage
  
  • in this example it would be with library of 1000 clones
  • 1X coverage is often not enough

Question 5

Genomic Library Construction

Process

Answer 5

• Ingredients required
  
  • heads
  • tails
  • enzymes
• Add DNA to be packaged
• Cos sites recognize DNA and package it into the head
• Phage assembles w/tail
• Now can be used to infect
• Infection site is a lamb product
  
  • codes for a porin in E. coli induceable by maltose
  • growing E. coli in maltose enhances infectivity
• Mutants can be selected by looking for tetracyclin resistance

Question 6

Characterizing clones

Answer 6

• create a partial digest w/restriction enzymes
• The longer the recognition sequence, the less it will cut the DNA
  
  • the shorter it is, the more it can occur
  • the longer it is, the less it can occur
• Frequency of cutting is based mostly on length of recognition sequence (1/4ⁿ)
  
  • 4 bp recog. Seq = 1/4⁴ = 1/256
  • 6 bp recog. Seq. = 1/4⁶ = 1/4096
  • 8 bp recog. Seq. = 1/4⁸ = 1/65,536
• tend to be palindromic