Bacterial transformation

Question 1

what is bacterial transformation?

Answer 1

the process by which bacteria take up free DNA from the environment and incorporate it into their chromosome

Question 2

what are prototrophs

Answer 2

bacteria that can synthesise all essential nutrients

Question 3

what are auxotrophs

Answer 3

bacterial that require supplement (e.g. amino acids)

Question 4

what is the process of transformation

Answer 4

• dsDNA fragment binds to pilus on bacterial membrane
• ssDNA is take up (if bacteria is competent)
• ssDNA incorporated into bacterial chromosome via recombination (2 crossovers)
• after replication and division, one of the 2 resulting cells are transformed

Question 5

what does competent mean?

Answer 5

• state that bacteria enter to take up naked DNA from their environment
• induced by the expression and assembly of DNA uptake molecular machinery

Question 6

why do bacteria go through natural transformation

Answer 6

• nutrition: source of nucleotides
• Repair: uptake of homologous DNA allow repair of damage caused by recombination
• diversity: increase genetic diversity by horizontal gene transfer

Question 7

How can artificial transformation be induced

Answer 7

• transient cell envelope permabilisation
• by e.g. chemical transformation and electroporation

Question 8

what is chemical transformation?

Answer 8

• a type of artificial transformation
• CaCl₂ treatment of bacterial cells at 4 degrees celcius => causes changes in membrane permeability, allow entry of plasmid DNA

Question 9

what is electroporation

Answer 9

• a type of artificial transformation
• electric field (10-20kV/vm) changes the permeability properties of bacterial membranes => transiently introduce pores

Question 10

what is considered a transformed bacterial cell?

Answer 10

a cell that has gone through:

• chromosomal integration of the incoming DNA via double crossing over event (not just the internalisation of DNA)

Question 11

what is a cloning vector

Answer 11

a plasmid, serves as a vector for bacterial transformation, self cloning because it has origin of replication (so it is passed on to the population)

Question 12

what is a polylinker

Answer 12

a region on plasmid that contains unique restrictions sites for DNA insertion

Question 13

How to transform bacteria via plasmid?

Answer 13

• cut the DNA of interest out using restriction enzyme
• mix with open plasmid, DNA fragment sticks to restriction site
• use ligase to close plasmid
• transfer plasmid into bacteria

Question 14

how can you identify bacteria that has take up the plasmid with the gene of interest?

Answer 14

• plasmid itself contains antibacterial resistance, which can be used to select for cells that has taken up the plasmid
• blue white screening:
  
  • incorporation of gene of interest interupts lacZ
  • lacZ codes for alpha fragment of betagalactosidase
  • recipient cell chromosome codes for the rest of betagalatosidase
  • cells that has plasmids that has gene of interest do not have functional betagalatosidase => cannot cleave X-gal
  • X is a chromophore, when cleaved, appears blue
  • cells with plasmid that did not take up gene of interest will appear blue

Question 15

how do you map genes with co-transformation?

Answer 15

• extract DNA of donor bacteria, DNA breaks into fragments
• the genes that are closer together have a higher chance of being on the same fragment
• the cotransformation frequency of genes close together is higher than those that are far away
• percentage co-transfomed cell = co-transformation frequency