Francesca Ceroni

Question 1

Define Promoter

Answer 1

Docks for RNA polymerase

-10 region & -35 region determine how easy it is for polymerse to bind

Question 2

Difference between constitutive & inducible promoter

Answer 2

• Constitutive promoters are always on

- Can be turned on or off by activator or repressor

Question 3

Define Gene

Answer 3

Sequence of DNA that encodes for a protein

Question 4

Define Plasmid

Answer 4

DNA sequence/construct that is repsonsible for carrying/propogating the inserted gene

Question 5

What is the MCS

Answer 5

the Multiple Cloning Site where genes are often inserted into DNA plasmids

Question 6

why are antibiotic resitance genes inserted whilst engineering cells

Answer 6

• Ensures only the cells with the inserted genes survive

- Need to make it beneficial for cell to have the inserted gene

Question 7

Define Cell Burden

Answer 7

• It is caused by competition for shared intracellular resources in the cell as teh inserted construct takes away resources (e.g. ribosomes) from the rest of the cell
• Cell burden is the resultant reduction in cell growth rate and capacity as a result of its increased output of the desired protein
• Cause of poor predictability of the behaviour of synthetic constructs

Question 8

What has the highest impact on burden

Answer 8

• Rate of translation as this requires more resources than transcription. Therefore the RBS has a greater impact than the promoter.

Question 9

Why do cells with high burden reduce their output to zero over time

Answer 9

• The cells struggle with resources, resulting in limited cell growth
• Some cells will mutate to inhibit the production of the protein encoded by the inserted gene to reduce the cell burden
• These cells will have a higher growth rate, giving them a higher population which will dominate over time, thereby reducing output

Question 10

Why is functional modularity not always possible

Answer 10

• Context dependencies affect the behaviour of DNA constructs
  e. g. LacI binds to a Trans sequence more effectively than a Cis sequence such that even when IPTG is added it still effectively represses the production of the trans sequence

Question 11

Define repressor

Answer 11

Protein that binds to operator site and prevents or slows RNA polymerase binding thereby decreasing transcription rate

Question 12

Methods of counteracting burden

Answer 12

Select components of synthetic construct that recruit less cellular resources

• weak RBS
• weak promoter
• deoptimised coding sequence

Question 13

Define DNA construct

Answer 13

Synthetic DNA fragment that is composed of biological parts and performs a specific function into the cells

Question 14

Example of context dependency

Answer 14

• Construct engineered into cells may behave differently to what is expected
• Cellular burden is an example