John McCarthy

Question 1

How are models created and optimised?

Answer 1

A cycle of:
Design
Modelling 
Construction
Testing

Question 2

What is the purpose of negative feedback circuits?

Answer 2

To stabilise systems/prevent oscillations/maintain a certain level of transcription, whereby the concentration of the repressor controls the level of suppression.

Question 3

Give two examples of naturally occurring negative feedback circuits

Answer 3

1. Physiological response to increase in blood sugar in humans
2. Lac operon in bacteria

Question 4

Describe the feedback mechanism in the Lac Operon

Answer 4

When there is plenty of glucose, a repressor binds to the lac operon to suppress transcription of genes that enable bacteria to use lactose.

When there is low glucose, there is high lactose so the repressor molecule does not bind and the lac operon is expressed

Question 5

What is the purpose of naturally occuring positive feedback circuits?

Answer 5

To generate transient, surge-like behaviour leading to excitability, oscillations, bistability…

Question 6

Give two examples of positive feedback circuits

Answer 6

1. phoP-phoQ

2. Sporulation

Question 7

Describe the feedback mechanism in PhoP-PhoQ

Answer 7

Found in E.coli and Salmonella.
It is a two- component virulence system that allows pathogen survival in the host.
Low [Mg2+] causes autophosphorylation of PhoQ (sensor kinase) which activates PhoP.
PhoP regulates responses and activates more PhoP and other genes, such as ones for resistance to cationic antimicrobials

Question 8

What is an AND Gate?

Answer 8

when an output is made if both inputs are ACTIVE

Question 9

What is a NAND Gate?

Answer 9

an output is made if both inputs are INACTIVE

Question 10

Give two examples of AND Gates

Answer 10

1. E.coli: one input activates gene with nonsense stop codon and the other input suppresses the stop codon to create an output
2. Yeast: the Y2H system is use - one gene promotes AD, one promotes BD and the combination of AD and BD causes gene expression

Question 11

What are the three challenges associated with designing and constructing synthetic circuitry?

Answer 11

1. Circuitry components have variable properties
2. Circuits can interact with hosts, i.e it is not orthogonal enough
3. There is noisy gene expression/variability between cells

Question 12

How can we overcome variable properties of circuit components?

Answer 12

Characterise the properties of component quantitatively so that we can improve their performance and put them into advanced computational algorithms to facilitate design and construction

Question 13

What is cell-cell heterogeneity?

Answer 13

When cells differ in terms of processes going on, protein content etc

Question 14

How can we optimise components?

Answer 14

By matching the outputs and inputs of linked components

e.g. so the concentration of a TF matches the sensitivity range of a promoter

Question 15

What changes can you make to optimise output?

Answer 15

• vertical scaling: to amplify response level or dampen – this is when k’ and k are scaled equally
• vertical shifting
• vertical extension = affects responsiveness but same basal activity
• leakage: can affect dynamic range
• horizontal scaling
• steepness: changing ‘n’ in the hill equation aka cooperativity (increasing ‘n’ makes the curve steeper)

Question 16

Why do we want to make curves steeper in synthetic biology?

Answer 16

Biology only has analogue (graded) responsiveness rather than digital
Steepening the curve makes it more like digital

Question 17

How would you tune output characteristics?

Answer 17

Change the genetic context i.e. where an operon or transcription factor is on the chromosome
Change the position of regulatory elements i.e. moving operator sites relative to the TATA box, which will tune steepness and leakage

Question 18

What causes noise?

Answer 18

random encounters between molecules which are strong enough to generate rate fluctuations

Question 19

What technique is used to measure noise?

Answer 19

smFISH (single molecular fluorescent in situ hybridisation)

Question 20

How is transcriptional noise measured?

Answer 20

smFISH - specially designed probes with fluorescent tags bind to specific mRNA. fluorescence microscopy is then used to find where the mRNA is distributed.
Compare the intensity near and far from the transcription site (new vs mature mRNA)

Question 21

What is a downfall of increasing promoter strengths?

Answer 21

It increases noise

Question 22

How can you automate synthetic biology?

Answer 22

Use robotics instead of manual pipetting
Use Cello to automate genetic circuit design
Verilog

Question 23

What is a NOT gate?

Answer 23

often called an inverter

when the input is true the output is false

Question 24

What is a NOR gate?

Answer 24

when all inputs are false, the output is true

Question 25

How would you design circuitry in E. coli?

Answer 25

1. Design logic architecture using Verilog 2. Put it in a truth table 3. Generate the circuit diagram 4. Assign regulatable promoters to fit the diagram requirements 5. Design genetic circuit DNA sequence 6. Insert synthetic DNA into two plasmids; circuit plasmid and output plasmid (circuit plasmid contains all of the regulatory components; output plasmid contains the final output promoter and ‘actuator’ e.g. encoding a fluorescent protein)

Question 26

What are the three genome minimisation projects we learned about?

Answer 26

1. Mycoplasma Syn3.0 2. Sc2.0 3. HeLa?

Question 27

What did they do in Mycoplasma?

Answer 27

Used transposons to remove genes. Classified genes into essential, quasi-essential and non-essential. The final product had 473 genes but it grew slower and was polymorphic (a colony of different cells).

Question 28

What did we learn from the mycoplasma project?

Answer 28

Knocking out all non-essential genes would not make a viable cell. There was a 'trade off' between transport proteins and biosynthetic pathway proteins. More transport proteins were needed to import nutrients when it couldn't make its own. We still don't know the function of genes which were classed as essential.

Question 29

What was their aim with the synthetic yeast genome?

Answer 29

Tried to streamline it but maintain characteristics like adaptability Assembled synthetic chromosomes with different genes, and the new chromosomes slowly replaced the old ones

Question 30

What did they do for the synthetic yeast project?

Answer 30

- Removed 'non-essential genes and introns, reducing genome to 8% smaller than mycoplasma Syn3.0 - Changed all stop codons from amber to ochre so amber codons can be assigned to an amino acid - Added loxPsym sites to facilitate recombination - SCRaMbLE genome to learn about genome organisation - tRNA genes were removed and put on a dedicated chromosome - added PCR tags to differentiate between WT and synthetic genes - added restriction enzyme sites.

Question 31

What technique is used to construct tissues?

Answer 31

DPAC - DNA Programmed Assembly of Cells

Question 32

How would you assemble a microtissue array?

Answer 32

Bind epithelial cells to an aldehyde-coated glass surface using lipid-modified complementary oligos Add another layer of cells carrying oligos complementary to those in step 1 Add more players to build hemispherical microtissues Liquid ECM gel containing DNAase is added, releasing microtissues from the template. Gel is lifted off with cells trapped inside and additional ECM is laid down, giving a fully embedded 3D culture.