Emzo de low Santos

Question 1

What is top down engineering?

Answer 1

modifying existing DNA

Question 2

What techniques can be carried out to do top down engineering?

Answer 2

MAGE
Zinc Finger nucleases and TALENS
CRISPR-Cas9

Question 3

What does MAGE stand for?

Answer 3

multiplexed
automated
genome
engineering

Question 4

What is a limitation of MAGE?

Answer 4

It only works in fast replicating organisms like E coli and other model organisms so cannot be done to the human or plant genome

Question 5

What is the concept of MAGE?

Answer 5

Taking advantage of replicating DNA being vulnerable

Trying to introduce new oligonucleotides to the lagging strand of DNA

Question 6

Whats the best technique for top down engineering?

Answer 6

Crispr Cas9 = customisable and generalisable
MAGE is not generalisable
TALENs are not customisable

Question 7

What is genome engineering?

Answer 7

Extensive/large scale changes

Intentional, for a specific purpose

Question 8

What is the principle of 2. Zinc Finger nucleases and TALENs?

Answer 8

Engineer proteins to recognise specific sites and cut DNA/make DSBs.
You alter the DNA binding domain to alter specificity.
Protein engineering is difficult so it is not very customisable.
It is generalisable because most organisms have DSB repair mechanisms to take advantage of

Question 9

What does in silico mean?

Answer 9

on a computer

Question 10

Name three examples of synthetic organisms

Answer 10

1. T7.1 was a bacteriophage in 2005
2. JCVI-syn 3.0 was a bacterium in 2013
3. Saccharomyces was a yeast (ongoing)

Question 11

What was the aim with T7.1?

Answer 11

An early attempt at refactoring a well studied virus with essential and non-essential genes already identified.
They wanted to edit the genome to make it easier to engineer without changing its functionality

Question 12

What did they do to T7.1?

Answer 12

• They separated overlapping genes (RBS of one gene can be found in another for example)
• Added restriction sites between genes to remove to test essential-ness or to replace genes
• Divide genome into 6 parts – alpha and beta fragments
• Used lots of types of restriction enzymes – only type 2 used these days
• They did not mutate any genes

Question 13

What was the aim with JCVI-syn 3.0?

Answer 13

Tried to make a minimal genome

Question 14

What did they do to JCVI-syn 3.0?

Answer 14

• Use transposons (which randomly insert sequence into genomes)
• Use next gen sequencing techniques – transposons identify non-essential and essential – when inserted in essential, cell is no viable
• Identified quasi-essential genes (which when removed caused slow growth) – usually at start or end which shows protein only kinda important

Question 15

What was the aim with Sc2.0?

Answer 15

To refactor and redesign saccharomyces cerevisiae

Question 16

What was learned with Sc2.0?

Answer 16

Some genes were essential in some contexts – two that work together were quasi essential but when one is removed the other becomes essential.
Still don’t know the function of 17% gens required for viabiliy – BLAST to homolgous genes with unknown function or no homolog

Question 17

Describe a tryptophan biosensor circuit.

Answer 17

In the absence of tryptophan, genes are expressed to make tryptophan and in the presence of tryptophan, tryptophan binds to trpR which represses/prevents the transcription of the tryptophan operon.

Question 18

Why T7.1?

Answer 18

It infects ecoli
extensively studied but contained unknown elements
60 genes - we knew which were non essential and essential
Failed to predict behaviour so wanted to make it predictable
Wanted to see how parts fit together

Question 19

What was done completely denovo?

Answer 19

Printed mycoplasma genome.
Put in ecoli
Made it minimal - divided into 8 and removed non-essential genes but didn’t work
then Reduced Genome