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Lec 11 (synthetic genomes) Flashcards

1
Q

what does ‘PCA’ stand for

A

polymerase cycling(chain) assembly

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2
Q

What does ‘TAR cloning’ stand for

A

transformation association recombination

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3
Q

synthetic genomics uses artificial DNA and _____ by…

_____ is what is put into a cell

A

uses artificial DNA and activates it by putting it into a cell. The DNA will act as computer software to build the hardware of the cell.

synthetic DNA is put into a cell.

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4
Q

the field of synthetic genomics is an emerging field since ___ and is thought to be interdisciplinary with biologists, ___, ___ etc.

A

2000s

biologists
chemists
physicists
engineers
computer scientists
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5
Q

What is the overall aim of synthetic genomics

A

to understand life and to figure out the minimum amount of DNA that is required for life.

we can also re-design

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6
Q

What is the overall aim of synthetic genomics

A

to understand life and to figure out the minimum amount of DNA that is required for life.

we can also re-design organisms with the aim of creating new molecules

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7
Q

Synthetic genomics involves the ______of DNA with _______ techniques to design it

A

Synthetic genomics involves the chemical synthesis of DNA with computational techniques to design it

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8
Q

In terms of manipulating genomes, what occurred first and when?

A

1972

covalently join duplex DNA molecules

2 ssDNA were combined together to form a double-stranded duplex

molecule size=77nt
encoded for a yeast alanine transfer RNA

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9
Q

DNA ligases were identified in ____. What benefit did this provide for genome manipulation?

A

1972

the size of forming a new synthetic gene increased from 77nt in 1972 to 207bp in 1979

the fragment length became longer

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10
Q

What occurred in 1979

A

the synthesis of a 207bp gene of tyrosine suppressor tRNA

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11
Q

1st to develop and create a synthetic DNA gene, + won nobel prize for discovering the genetic code

A

khorana

1968

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12
Q

When were the E.coli and Yeast synthetic genomes created? And how long were they?

A

2018 the E.coli synthetic genome was made (4 mill bp)

2020 (yeast synthetic genome ) = 1 billion bp

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13
Q

In terms of synthetic genomes, what occurred in 2008 and 2020

A

2008 = E.coli synthetic genome was made = 4 mill bp

2020 = yeast synthetic genome = 1 bill bp

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14
Q

The first protein coding gene that was synthetic was made on 1977. What was it and how long was it ?

A

It was a hormone peptide 14 aa

Somatostain gene

Itakura

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15
Q

When was the full length complimentary DNA sequence produced? And what was this for

A

2002

For a virus (infectious polio virus)

7558bp

by Cello et al

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16
Q

What occurred in 2003

A

(greek pi) X174 bacteriophage synthesis

53886bp

Venter et al

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17
Q

When was the polyketide gene cluster produced

A

2004

32kb

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18
Q

polyketide

A

many genes

secondary metabolite

19
Q

What significant event happened after the polyketide gene cluster was produced?

A

In 2007, Craig Venter and colleagues showed that they could transplant natural chromosomes from 1 microbial species to another

Microplasma capricolum into microplasma mycoides

20
Q

In 2008 ___ occured

A

Mycoplasma genitalium genome synthesis

Gibson

21
Q

2010

A

Take genome and implant into a bacterial cell

Creation of a bacterial cell controlled by a chemically synthetized genome

22
Q

When was the first synthetic organism created

A

2010

23
Q

Why has gene synthesis become easier

A

Due to automated synthesis

An oligosynthesiser is required to make the DNA

-became cheaper

24
Q

Why was piX174 Bacteriophage selected as one of the synthetic DNA molecules made (LA)

A

1) well characterised since teh 1950s (we know the structure, icosahedral head, tail, fibers)

2) was the first molecule to be purified to homogeny
- its sequence was the first to be sequenced by Sanger in 1978

3) has a small genome size (5386nt) that codes for 11 genes

4) is infectious, infects bacteria such as E.Coli
but is not a risk to humans, animals, and plants

works well with the safety risk factors and ethical considerations building a new genome from scratch.

25
Q

What are the basic steps of creating a piX174bacteriphage from synthetic oliginucleotides

A

3

1) design the oligonucleotides (what DNA seq.s will be-using comp software)
2) make and purify the oligonucl
3) assemble oligonucl

26
Q

Describe the first basic step of creating a synthetic genome using the piX174bacteriophage

A

The genome has been sequenced. The sequence is on a FASTA file on a computer. Use geneious

1)Design oligonucleotides around 42 nt in length

(the oligos will be from start to finish 5386. Over 5kb a sequence.But will be split into overlapping fragments of 42nt

2)
use oligo synthesiser to make the oligos
(the oligos that are synthesised are single stranded)

3)Purify the oligos by using gel electrophoresis. Loading 2 oligos (a top and a bottom) that are complimentary to eachother so they will form a ds-seq that is 42nt

4)
Add ezy T4 polynucleotide kinase = adds a phosphate group to the 5’ end (Since the oligos don’t have a 5’ phosphate group at the 5’end)

5) join oligos together to make ds-DNA

27
Q

What occurs after you’ve used oligo synthesiser to create ss-oligos?

A

Purify the oligos by using gel electrophoresis. Loading 2 oligos (a top and a bottom) that are complimentary to eachother so they will form a ds-seq that is 42nt

28
Q

what do the following represent:

Arrow

red dot

A

arrow= single strand of DNA that is 42nt

red dot = phosphate group that has been added

Because we need to add the oligos together. If there is no phosphate group, we can’t form out phsphodiester bond

29
Q

What is a minimal organism?

A

a living, dividing cell where every gene in the genome is indispensable for variability in a defined chemical environment

30
Q

What is the minimal genome concept

A

what are the minimal no. of genes required to sustain life?

One way to do that is to use bacteria as a model

31
Q

How would you find the minimal no. of genes required for life?

A

find minimal set of genomes = need a genome that is sequenced

-known seq =>
predict the translation

  • use BLAST to do alignments
  • can get rid of the candidates that aren’t applicable
32
Q

What were the minimal gene set for cellular life

A

made in 1996 using the complete protein list from 2 bacterial genomes sequenced.

33
Q

What were the 2 bacterial genomes sequenced for use in 1996

A

Haemophilius influenzae (gram -)

mycoplasma genitalium (gram +)

34
Q

Why were those particular bacteria used in 1996?

A

They were the first 2 species that were available in 1996

they differed evolutionary (gram - and +)

if we have 2 extremes (- &+) we can figure out what genes are required for them)

35
Q

What is the taxonomic classification of Mycoplasma

A

class: mollicutes
genus: mycoplasma

36
Q

briefly describe mycoplasma

A

5

1) wall-less, obligate pathogenic parasites
2) evolved by massive genome reduction
3) smallest known genome of any free-living organism capable of growing in pure culture
4) lack genomic redundancy
5) unique for their altered genetic code (UGA encodes for tryptophan instead of stop codon)

37
Q

massive genome reduction is __

A

shrinking of the genome size

38
Q

what is genomic redundancy

A

one gene that has many functions

39
Q

A transposon can be thought of as

A

a seq that can jump from 1 gene to another

40
Q

describe global transposon mutagenesis

long answer

A

5

1) a biological process
2) allows genes to be transfered to a host organism’s c/some (jumping gene)
3) interrupt and modify the function of an extant gene on the c/some and cause mutation
4) used to determine experimentally the genes not essential for lab growth of M.genitalium
5) clone/transform/DNA-PCR to track genes for lacking insertion E.g Plasmid with Tn4001

41
Q

What is the transposon used was

A

Tn4001

is contained within a plasmid

42
Q

Tn in Tn4001 stands for

A

Transposon

43
Q

Take the plasmid that contains the transposon gene and tranforming it into m. genitalium

A

if there is a non-essential gene= the jumping gene will insert into the non-essential gene

44
Q

What happens if there is a non-essential gene

A

if there is a non-essential gene= the jumping gene will insert into the non-essential gene

. (12 decks)

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