Building DNA and genomes

Question 1

What are the two types of enzyme needed for traditional DNA cloning?

Answer 1

Restriction enzymes and DNA ligase.

Question 2

What enzyme integrates the digested gene into the plasmid DNA?

Answer 2

DNA ligase.

Question 3

Name three disadvantages of traditional DNA cloning methods.

Answer 3

1. Time-consuming
2. Only joins two parts at a time
3. Restriction sites leave a ‘scar’
4. Requires compatible restriction sites in the DNA

Question 4

What is the name of the example of modular DNA building blocks?

Answer 4

BioBrick.

Question 5

What is a limitation of modular DNA building like BioBrick?

Answer 5

They leave a bunch of unwanted regions (i.e. BioBrick ‘prefix/suffix’ DNA)

Question 6

What is the rapid method of DNA assembly we look at?

Answer 6

Gibson assembly.

Question 7

Name three advantages of Gibson over traditional assembly.

Answer 7

1. Leaves no scars.
2. Up to 15 fragments at the same time.
3. Rapid (~15 mins)

Question 8

Name the three enzymes involved in Gibson assembly.

Answer 8

1. Exonucleases
2. DNA polymerase
3. DNA ligase

Question 9

What is the role of exonucleases in Gibson assembly?

Answer 9

Creating single-strand overhangs (at the 5’ end) of around 20-40bp.

Question 10

What are the two sources of DNA for cloning?

Answer 10

1. Genomic DNA
2. Reverse-transcribed cDNA

Question 11

What is the difference in terms of content between genomic DNA and cDNA?

Answer 11

cDNA is derived from mRNA transcripts, thus contains no introns.

Question 12

What is the type of chemistry involved in oligonucleotide synthesis?

Answer 12

Phosphoramidite chemistry.

Question 13

We can add a number of overlapping oligonucleotides together and combine them with DNA polymerase. This new DNA can then be amplified. What is the name of this process?

Answer 13

Polymerase cycling assembly.

Question 14

What are two weaknesses of polymerase cycling assembly?

Answer 14

1. High G-C content leads to issues.
2. Oligonucleotide synthesis is error-prone (even commercial options)

Question 15

Why can codon optimisation for some gene transformations necessary?

Answer 15

Some organisms are biassed towards using certain codons - thus, the same sequence may be less efficient in a non-native host.

Question 16

What are two reasons for using DNA as a digital storage medium?

Answer 16

1. Dry DNA can survive stably for hundreds of millennia.
2. Highly efficient (700tb = 1g of DNA)

Question 17

When was the first synthetic genome produced? What type of organism was it?

Answer 17

A bacteriophage genome was synthesised in 2003.

Question 18

What is the very first step of genome synthesis?

Answer 18

Having the entire natural genome correctly sequenced.

Question 19

What are three applications of a synthetic bacteriophage genome?

Answer 19

1. Study function (e.g. create new variants)
2. Create attenuated viruses for vaccines.
3. Resurrect viruses for biocontrol of invasive pests.

Question 20

What was the first bacterial genome to be synthesised? Why this one?

Answer 20

Mycoplasma genitalium, as it has the smallest known genome.

Question 21

For biosecurity and intellectual property reasons, what is added to synthetic genomes?

Answer 21

DNA ‘watermarks’.

Question 22

What failed with the first attempt at synthesising a bacterial genome?

Answer 22

BACs were unstable at large sizes - yeast artificial chromosomes and a yeast cell host had to be used instead.

Question 23

What was the natural method used for assembly in the second attempt at synthesising a bacterial genome?

Answer 23

Homologous recombination.

Question 24

Did the second attempt at synthesising a bacterial genome utilise Mycoplasma species again?

Answer 24

Yes.

Question 25

What can show researchers which genes are critical for cell survival?

Answer 25

Mutagenesis of the genome.

Question 26

Do minimal bacterial cells evolve at the same rate as normal cells?

Answer 26

Yes.

Question 27

Approximately how much of a bacterial genome was removed by researchers to create a minimal viable cell?

Answer 27

50%.

Question 28

Were researchers able to synthesise a yeast genome?

Answer 28

Still underway.

Question 29

What is the big hurdle when jumping between synthesising bacterial and yeast genomes?

Answer 29

Yeast has multiple (16) chromosomes.

Question 30

What type of codon is a good candidate for reprogramming (i.e. assigning a non-canonical amino acid to it)?

Answer 30

Stop codons (UAG, UAA, UGA)

Question 31

What would be the three main steps of assigning a new amino acid to a stop codon?

Answer 31

1. Knock out the gene for the stop codon release factor. 2. Add a tRNA synthetase for the new amino acid. 3. Remove all naturally-occurring appearances of the reassigned stop codon.

Question 32

The first synthesised bacterial genome was ~1,000,000bp. How big is the candidate yeast genome?

Answer 32

14,000,000bp - 14x bigger.

Question 33

Approximately how many DNA fragments were needed in the first and second attempts at bacterial genome synthesis?

Answer 33

~100 and ~1,000.

Question 34

Did assembly occur in a bacterial cell or a yeast cell for the first successful synthesis of a bacterial genome?

Answer 34

Assembly occurred via homologous recombination in a yeast host cell.