Bio-circuitry

Question 1

Linking environment-responsive promoters to engineered gene circuits is what type of bio-circuitry?

Answer 1

Transcriptional.

Question 2

What domains are utilised in translational bio-circuitry?

Answer 2

RNA aptamer domains.

Question 3

What type of bio-circuitry uses protein receptors to trigger signal cascades?

Answer 3

Post-translational.

Question 4

Operons are a key element of which type of bio-circuit?

Answer 4

Transcriptional.

Question 5

Describe broadly the mechanism of the arsenic operon.

Answer 5

The arsenic repressor disassociates from the operon if it is bound by arsenite, activating the detoxifying pathway.

Question 6

Why might we create a constantly-active ‘decoupled’ arsenic repressor operon?

Answer 6

If it is all part of the same operon, there will be constant ‘leaky’ basal activity and thus constant reporter expression.

Question 7

A two-input biosensor that produces an inducer for a second biosensor is called a…?

Answer 7

Three-input biosensor.

Question 8

Name three advantages of cell-free biosensors.

Answer 8

1. Low cost.
2. Non-GMO
3. Highly stable.
4. Faster response than a whole-cell biosensor.

Question 9

A gate that is on with no input is?

Answer 9

A ‘not’ gate.

Question 10

A gate that is off if either/both inputs are present is?

Answer 10

A ‘nor’ gate.

Question 11

When combining gates, repressors can’t bind other promoters. This idea is called:

Answer 11

Orthogonality.

Question 12

An RNA molecule with a complex secondary structure that can regulate translation is known as:

Answer 12

A riboswitch.

Question 13

In which region of RNA are riboswitches found?

Answer 13

5’ untranslated region (UTR).

Question 14

Binding to the _____ causes conformational changes to the riboswitch.

Answer 14

Aptamer region.

Question 15

Name two advantages of a riboswitch as a biosensor.

Answer 15

1. Transcription step is skipped.
2. Sensor + regulator + output, all in one.
3. High specificity.

Question 16

What is a trans-activating riboswitch?

Answer 16

A riboswitch activated by another RNA molecule.

Question 17

What is the example application of a trans-activating ‘toehold switch’?

Answer 17

Viral RNA (e.g. Ebola) could trigger the switch.

Question 18

Creating an aptamer region for DNT involved trial-and-error with the addition of a bunch of random bases. True or false?

Answer 18

True.

Question 19

Why would we want to detect DNT? Give two reasons.

Answer 19

Industrial pollutant + precursor to TNT

Question 20

What two categories of variant can we dismiss when screening for DNT-inducable riboswitches?

Answer 20

1. Variants that fluoresce WITHOUT any DNT (the riboswitch hasn’t folded properly)
2. Variants that don’t fluoresce WITH DNT (aptamer isn’t optimised for DNT binding)

Question 21

Does DNT riboswitch produce GFP? Explain.

Answer 21

It doesn’t produce GFP - the switch activates a protease which unlocks GFP from another protein and allows it to fluoresce.

Question 22

Why create a DNT riboswitch that produces the protease to unlock GFP rather than just produce GFP?

Answer 22

Producing each GFP in response to DNT would be time-consuming - instead, having the GFP constantly present but dormant allows the DNT-induced protease to rapidly activate fluorescence.

Question 23

What is the central advantage of protein switches over genetic circuits?

Answer 23

No transcription/translation events = instantaneous responses.

Question 24

What are the three main types of post-translational events?

Answer 24

1. Conformational changes.
2. Protein interactions.
3. Post-translation modifications (PTMs).

Question 25

Name three possible choices of sensor for post-translational bio-circuits.

Answer 25

1. Ligand binding domains.
2. Metal binding motifs.
3. Protein binding domains.
4. Cleavage sites.

Question 26

How does adding calmodulin to GDH work as a biosensor?

Answer 26

Calmodulin will bind to any present calcium, inducing a conformational change in the entire chimeric protein and causing a measurable increase in electron activity.