14. Synthetic biology 3

Question 1

What is Transformation?

Answer 1

where DNA is introduced into bacteria, replicated and then expressed

Question 2

What are the 2 methods of artificial transformation?

Answer 2

chemical transformation and electroporation

Question 3

What is chemical transformation?

Answer 3

heat shocks improve DNA uptake (calcium ions)

Question 4

What is electroporation?

Answer 4

exogenous DNA

Question 5

What is the first step of standard transformation protocol?

Answer 5

1. freeze competent cells and then store them at -80°C

Question 6

What can happen if you freeze the cells multiple times?

Answer 6

thaw cycles can reduce competent cell viability

Question 7

What is the second step of the standard transformation protocol?

Answer 7

2. Add plasmid DNA to competent cells and mix by flicking the tube

Question 8

What type of cells do we use for transformation and why?

Answer 8

DH5 alpha cells because they are efficiently transformed by many plasmids

Question 9

What is the third step of the standard transformation protocol?

Answer 9

3. plate the mixture

Question 10

What is the fourth step of the standard transformation protocol? (Heat shock step)

Answer 10

4. Heat competent cell mixture in a 42°C water bath for 45 secs then place the tube on ice for 2 mins to allow the plasmid to enter the cell

Question 11

What is the fifth step of the standard transformation protocol? (outgrowth step)

Answer 11

5. place in a shaking incubator to allow cells to grow in rich medium SOC containing glucose

Question 12

Describe the protocol for the outgrowth step

Answer 12

1. add SOC to the tube containing transformed bacterial cells
2. place tube in shaking incubator at 37°C for 45 mins
3. centrifuge to increase number of colonies for 5 mins
4. plate cells and place in incubator for overnight growth (30-37°C)
5. inspect plate for visible colony growth

Question 13

What could make this transformation process less efficient?

Answer 13

• due to reagents or size of contruct e.g. larger than 10 kb- use electropotent cells and electroporator instead of heat shock

Question 14

What does the plasmid/ DNA include in cloning?

Answer 14

• recombinant plasmid
• plasmid insert
• marker gene

Question 15

Describe the steps of selection

Answer 15

• transfer bacteria onto a Petri dish filled with agar for the bacteria to colonise
• use the antibiotic which allows the plasmid to grow
• use an L-spreader as the plating device to evenly distribute culture around the plate
• leave the agar plate for 12 hours to give the antibiotic a chance to work

Question 16

What are the result scenarios?

Answer 16

results worked - transformation hasn’t worked/ wrong antibiotic used - antibiotic selection not working - issue with digestion of ligation of plasmid

Question 17

What equipment do you need for the spread plate technique?

Answer 17

• bunsen burner
• agar plate containing solid growth medium
• bacterial culture
• ethanol (used to sterilise L-spreader

Question 18

What are the steps of the collection of bacteria?

Answer 18

1. loosen lid of bacterial culture
2. collect bacteria- sterilise neck and collect using a pipette
3. close sample tube (sterilise again beforehand)

Question 19

What are the steps for spreading plate?

Answer 19

1. dip spreader in ethanol and allow excess ethanol to drip off
2. pass spreader through Bunsen burner flame and cool before using
3. spread the bacteria
4. sterilise spreader
5. incubate (loop orientation to prevent condensation/ water dripping)