It explains different methods used to synthesize microbiol genomes.

1. Synthesis of Microbial
genomes
Seminar by
Sugandh Chauhan

2. Introduction
• The discipline involving synthesis of genomes is described as synthetic
genomics, which can be traced back to a report in 1970, when Har
Gobind Khorana and his coworkers reported the synthesis of 77 base
pairs long gene for non-coding yeast alanyl tRNA. This was followed by
successful synthesis of a number of much longer protein-coding genes.

3. Steps for synthesis of genome

5. TECHNIQUES USED FOR SYNTHESIS OF GENOMES
1. Polymerase Cycling Assembly (PCA)
• Polymerase cycling assembly (PCA) is used for the synthesis of
oligonucleotides (oligos), approximately 40 to 60 nucleotides long, which
constitute both strands of the genome to be synthesized. These oligos are
designed such that a single oligo from one strand contains a length of
approximately 20 nucleotides at each end that is complementary to
sequences of two different oligos on the opposite strand, thereby creating
regions of overlap.
• PCA was actually used to generate the first synthetic genome (the genome
of Phi X 174 virus).

6. Different steps involved in polymerase cycling
assembly (PCA)

7. 2. Gibson Assembly
• The Gibson assembly method, designed by Daniel Gibson. The approach
requires a set of double-stranded DNA cassettes that constitute the entire
genome being synthesized. These cassettes contain regions of homology
to other cassettes for the purposes of recombination.
• In contrast to PCA, which is cyclic in nature, Gibson Assembly is a single-
step, isothermal reaction with larger sequence-length capacity. It is used
for genomes larger than 6 kb.
• Gibson assembly is often used in conjunction with transformation
associated recombination to synthesize genomes several hundred
kilobases in size.

8. Different steps involved in Gibson assembly

9. Synthesis of Mycoplasma genitalium genome
• Mycoplasma genitalium is a bacterium with the smallest genome of any
independently replicating cell that has been grown in pure culture.
• In 2008, the Craig Venter Institute (CVI) reported the chemical synthesis of
the genome of Mycoplasma genitalium (583kb).
• Synthetic genome named as M. genitalium JCVI-1.0.

10. Methodology
• The native 580,076-bp M. genitalium genome sequence was partitioned
into 101 cassettes of approximately 5 to 7 kb in length that were
individually synthesized, verified by sequencing, and then joined together
in stages.
• Most cassettes overlapped their adjacent neighbors by 80 bp; however,
some segments overlapped by as much as 360 bp. Cassette 101
overlapped cassette 1, thus completing the circle
• To identify the genome as synthetic,“watermarks” were inserted at
intergenic sites known to tolerate transposon insertions.

11. Cont.
• Overlapping “cassettes” of 5 to 7 kilobases (kb), assembled from
chemically synthesized oligonucleotides, were joined by in vitro
recombination to produce intermediate assemblies of approximately 24 kb,
72 kb and 144 kb which were all cloned as bacterial artificial chromosomes
in Escherichia coli.
• Most of these intermediate clones were sequenced, and clones of all four
1/4 genomes with the correct sequence were identified.
• The complete synthetic genome was assembled by transformation-
associated recombination cloning in the yeast Saccharomyces cerevisiae.

12. Assembly of a synthetic M. mycoides genome in
yeast
• In the first step, 1080-bp cassettes
(orange arrows), produced from
overlapping synthetic
oligonucleotides, were recombined
in sets of 10 to produce 109 ~10-
kb assemblies (blue arrows).
These were then recombined in
sets of 10 to produce 11 ~100-kb
assemblies (green arrows). In the
final stage of assembly, these 11
fragments were recombined into
the complete genome (red circle).

13. Design and synthesis of a minimal bacterial genome
• Whole-genome design and complete chemical synthesis were used to
minimize the synthetic genome of Mycoplasma mycoides JCVI-syn1.0
(1079 kb, 901 genes)
• Design-build-test (DBT) cycle provide a way to build a new genome as a
centromeric plasmid in yeast and to test it for viability and other phenotypic
traits after transplantation into an M. capricolum recipient cell.
• Starting from syn1.0, a reduced genome was designed by removing non-
essential genes by transposon insertions. Genes that could be disrupted
by transposon insertions without affecting cell viability were considered to
be nonessential.

14. Cont.
• The reduced genome was divided into eight overlapping segments that
could be independently synthesized and tested.
• Each of eight reduced segments was tested in the context of a seven-
eighths syn1.0 genome.
• At each cycle, gene essentiality was reevaluated by transposon
mutagenesis. Four cycles of design, synthesis, and testing, (DBT) with
retention of quasi-essential genes, produced JCVI-syn3.0 (531 kbp, 473
genes)
• JCVI-syn3.0 has a doubling time of ~180 min, produces colonies that are
morphologically similar to those of JCVI-syn1.0 and appears to be
polymorphic in appearance.

15. DBT cycle for bacterial genomes

16. Comparison of JCVI-syn1.0 with JCVI-syn3.0
• The red bars inside the
outer circle indicate
regions that are retained
in JCVI-syn3.0.

17. Strategy for whole-genome synthesis
• Overlapping oligonucleotides (oligos)
were designed, chemically synthesized,
and assembled into 1.4-kbp fragments
(red).
• After error correction and PCR
amplification, five fragments were
assembled into 7-kbp cassettes (blue).
• Cassettes were sequence verified and
then assembled in yeast to generate one-
eighth molecules (green).
• The eight molecules were amplified by
RCA and then assembled in yeast to
generate the complete genome (orange).

18. Chemical Synthesis of C. ethensis-2.0
• In 2019, the chemical synthesis of Caulobacter ethensis-2.0 (C. eth-2.0), a
bacterial minimized genome composed of the most fundamental functions
of a bacterial cell was reported.
• C. eth-1.0 consists of 1,761 DNA parts, including 676 protein-coding, 54
noncoding, and 1,015 intergenic sequences. To select for assembly and
stable maintenance in S. cerevisiae, auxotrophic marker genes (TRP1,
HIS3, MET14, LEU2, ADE2) and a set of 10 autonomous replicating
sequences (ARSs) were seeded across the genome design.

19. Cont.
• A four-tier DNA assembly strategy starting from 3- to 4-kb assembly blocks
was designed to build the complete C. eth-2.0 chromosome in yeast
• Assembly of 236 DNA blocks into 37 chromosome segments (19–22 kb in
size) and further into 16 megasegments (38–65 kb in size) using yeast
transformation.
• To select for the complete chromosome assembly, a click marker strategy
was used by introducing five auxotrophic yeast genes (TRP1, HIS3,
MET14, LEU2, and ADE2) split between adjacent megasegments.
• Transformation of the 16 megasegments into yeast spheroplasts yielded
two clones, one of which restored prototrophy for all six auxotrophic click
markers, indicating complete assembly of C. eth-2.0

20. Assembly of C. eth-2.0 in S. cerevisiae
• Circular 785,701-bp C. eth-2.0
chromosome with six auxotrophic
selection markers (red), 11 ARSs
(black), and the restriction sites for
PmeI and PacI (blue); 236 DNA
blocks (green boxes) were
assembled into 37 genome
segments (blue boxes) and 16
megasegments (orange boxes)
and further assembled into the
complete C. eth-2.0 genome
(outermost gray track).

21. Synthesis of Escherichia coli with a recoded genome
• In 2019, chemical synthesis of Escherichia coli was successfully achieved.
In case of E. coli, a variant four-megabase long synthetic genome was
synthesized.
• The synthesized genome, corrections were made at seven positions—to
replace every known occurrence of two sense codons and a stop codon in
the genome. Thus, 18,214 codons were recoded to create an organism
with a 61-codon genome.
• The synthetic genome is refactored and recoded for the genome-wide
removal of two sense codons and a stop codon, which creates a synthetic
E. coli that uses 61 codons for protein synthesis.

22. Design of a recoded genome
A genome was designed in which the serine codons TCG and TCA, and the
stop codon TAG, in open reading frames (ORFs) of MDS42 E. coli are
systematically replaced by their synonyms AGC, AGT and TAA, respectively.
Synthesis of recoded sections
• The genome was disconnected into eight sections, each of approximately
0.5 Mb in length, which were labelled A to H, then disconnected each
section into 4 or 5 fragments.
• This yielded 37 fragments that were between 91 kb and 136 kb in length.
Assembling bacterial artificial chromosomes (BACs) for REXER that contained
each fragment, using homologous recombination in S. cerevisiae.

23. • Numerous single-step REXERs with individual fragments in parallel with
GENESIS (Genome stepwise interchange synthesis) completely recoded
the targeted genome.
• Replicon excision enhanced recombination (REXER)—an approach for
replacing more than 100 kb of the E. coli genome with synthetic DNA in a
single step.
Assembly of a recoded genome
• Conjugation-based strategy was used to assemble the recoded sections
into a single genome. This strategy assembles the recoded genome in a
clockwise manner, by conjugating recoded ‘donor’ sections that contain
the origin of transfer (oriT), into adjacent recoded ‘recipient’ sections that
have been extended to provide homology to the donor.

24. Assembly using Conjugation strategy
• Synthetic genomic sections (pink)
from multiple individual partially
recoded genomes were assembled
into a single fully recoded genome
using conjugative assembly.
• The donor (d) and recipient (r)
strains contain unique recoded
genomic sections labelled in pink;
recoded overlapping homology
regions (3 kb to 400 kb in size)
were used to recombine the
strains, and are shown in dark
pink.

25. Synthesis of a 57- codon E. coli genome (rE.coli-57)
• Synthesis and assembly of a 3.97-megabase, 57-codon Escherichia
coli genome in which all 62,214 instances of seven codons were
replaced with synonymous alternatives across all protein-coding
genes was reported.
• The recoded genome was divided into 87 segments of ~50 kb. Codons
AGA, AGG, AGC, AGU, UUA, UUG, and UAG were computationally
replaced by synonymous alternatives (center).
• Other codons (e.g., UGC) remain unchanged. Color-coded histograms
represent the abundance of the seven forbidden codons in each
segment.

26. 57 codon E.coli genome