Minimal Cells

Question 1

Describe the bottom up approach

Answer 1

The creation of new synthetic or chemically derived life from basic building blocks that were never alive

Question 2

What is the technical phrase for a cell free system?

Answer 2

‘in vitro’ transcription and translation

Question 3

What is the advantage of the ‘bottom up’ approach

Answer 3

No interferences from other pathways

Question 4

What is the disadvantage of the ‘bottom up’ approach?

Answer 4

Not self sustaining, expensive

Question 5

What are the ‘building blocks’ that are used in the ‘bottom up’ approach?

Answer 5

DNA, enzymes, ribosomes, t-RNAs and the metabolic pool (NTPs, amino acids)

Question 6

What does the ‘bottom up’ approach create?

Answer 6

A lipid vesicle

Question 7

What is the advantage of using a whole cell?

Answer 7

It is designed to survive and reproduce, so you don’t need to keep adding catalyst, the catalyst is always being renewed. When the cell is being renewed your design is also being renewed.

Question 8

What is the ‘top down approach’?

Answer 8

New functinoalities are added into an existing system and can involve adding in a gene or sets of genes and/or deleting genes from an existing organism.

Question 9

Are all the metabolic pathways present in a cell necessary?

Answer 9

It depends. It may be that not all of them are required at the same time.

Question 10

What is the minimal genome?

Answer 10

A minimum set of genes that are required to sustain a self sustaining, independently replicating organism

Question 11

What a genome?

Answer 11

A set of all the genes that are available in an organism

Question 12

What is a minimal cell?

Answer 12

Cells containing a minimal genome which can be used as a chassis organism for the synthetic biology parts, devices or systems

Question 13

What are the advantages of using minimal cells?

Answer 13

1) Efficiency - optimise a biofactory (lower the metabolic and genetic burden on the chassis) - limit the number of uncontrolled interactions within the chassis - reduce the cost of building a synthetic organism
2) Safety - ensure better safety (organism unable to survive in non-laboratory conditions) - ensure full control of the abilities an organism is endowed with
3) Increased fundamental understanding - understand the function of essential and dispensable genes

Question 14

Why is a minimal cell unable to survive in non-laboratory conditions?

Answer 14

It will require a very defined st of conditions in order to be able to sustain itself and grow

Question 15

What are the advantages of using Mycoplasma as a starting point for minimal cells?

Answer 15

1. Smallest known genome among independently living organisms
2. UGA (TGA) encodes tryptophan and not a stop codon; codon bias implies that cloning steps can be carried out in E.coli without interference (we can engineer DNA in E.coli without changing its function)
3. Lack of a cell wall implies that genome transplantation could be done by methods already established for mammalian cells i.e. PEG mediated
4. Fast growing i.e. doubling time is ~60 minutes
5. Non pathogenic to humans

Question 16

What is a cytoplasm?

Answer 16

A gel-like matrix that is available in a normal cell

Question 17

Why do we need a cytoplasm?

Answer 17

In order to express the DNA

Question 18

Can we synthesise cytoplasm?

Answer 18

No

Question 19

How was genome transplantation tested?

Answer 19

The whole genome was taken from one species of mycoplasma (A), an antibiotic resistance and beta galactosidase (a colour indicator) into another species of mycoplasma (B). This was then subject to a stressful environment which meant that only the genome that contained the antibiotic resistance survived. This means that mycoplasma (B) had been turned into mycoplasma (A) completely due to the stressful environment.

Question 20

What does beta galactosidase do?

Answer 20

It can convert chloroform in the cytoplasma from being colourless to blue so it can be used as a marker.

Question 21

What is tetracycline resistance?

Answer 21

An antibiotic resistance gene

Question 22

What are cells containing both sets of genomes called?

Answer 22

Recombinants

Question 23

How does inserting an extra genome put stress on the cell?

Answer 23

By increasing metabolic burden

Question 24

What happens when you expose a recombinant to the high concentrations o the antibiotic it is resistant to?

Answer 24

The stress on the organism becomes too large and it will neglect the unnecessary pathway for survival - the genome not containing the antibiotic resistance. This genome will not be passed onto the future generations of cells.

Question 25

What is required for mycoplasma genome maintenance?

Answer 25

To be able to work on the mycoplasma within yeast, as we have the tools for this.

Question 26

How is the mycoplasma genome inserted into yeast?

Answer 26

Adding a yeast vector into the bacterial genome, isolating it and using methylation if necessary.

Question 27

How do we assemble an entire genome?

Answer 27

If you synthesise the genes you require with overlapping fragments, and ensure the yeast takes up all the fragments, then the yeast will assemble the DNA for you.

Question 28

How do you synthesize gene fragments?

Answer 28

Input the sequence you require into a DNA synthesizer

Question 29

How was mycoplasma mycoides syn 1.0 made?

Answer 29

Using a three tiered assembly approach. Over 1000 DNA fragments were synthesized with overhangs. Ten of these were simultaneously assembled in yeast to form just over 100 fragments with overhangs. This was repeated to yield 11 fragments with overhangs. This was repeated a for a third time to assemble the whole mycoplama mycoides genome in yeast. (Plus four watermarks and the yeast vector sequence).
This was then transplanted into mycoplasma capricolum as proven before.

Question 30

Why was the original method of reducing mycoplasma mycoides to a minimal genome unsuccessful?

Answer 30

Outgrowing the selected unwanted genes turned out to be non-viable and produce poor growth after a few rounds.

Question 31

How does transposon mutagenesis work?

Answer 31

It allows you to go in and delete individual genes

Question 32

Why did the mycoplasma mycoides with only 240 genes not work?

Answer 32

There were genes that without which some sort of growth defect would occur (either minimal or severe) missing. This reduced metabolic burden. This was missed the first time as functional redundancy of proteins was not taken into account.

Question 33

How was mycoplasma mycoides 3.0 achieved (the most minimal cell ever)?

Answer 33

They took 2.0 and removed the colour indicator and antibiotic resistance that was inserted in the first iteration.