1. Minimal and Compact
Design and synthesis of a minimal bacterial genome.
Clyde A. Hutchison III et al. Science 351(2016)
Joshua Gefen
2 May 2016

2. J. Craig Venter Institute
Research Timeline
Beginning
Mycoplasma genitalium genome sequenced and minimal genome project begins.
PhiX synthesized
Publication of the synthesized version PhiX174
Bacterial Genome Transplantation
JCVI synthetic genomics team makes breakthrough in transforming one bacterial
species into another through genome transplantation.
First Synthetic Bacterial Genome and First Self-Replicating Synthetic Cell
First Minimal Cell Published
1995-99
2004
2007
2008-10
2016

3. Research Rationale
Purpose:
To ascertain the core sets of conserved genetic functions and to
distinguish them from non-essential genes, building a powerful tool
for molecular and genomic research.
Problem:
More than one
gene product can
perform a
particular
essential function
and an individual
gene product can
have multiple
functions.
Method:
Systematic deletions (knockouts) of genes from the
synthetic genome of M. mycoides (JCVI-syn1.0) with
DBT repetitions.
Hypothetical Result:
To produce a minimal cell that is simpler than any natural one.

4. Work Flow
• HMG: Hypothetical Minimal
Genome
• RGD: Reduced Genome
Design
• DBT: Design, Build, Test

5. Building the synthetic genome

6. Creating the eight segments
Using TREC deletion to generate scarless deletion and specific Not1 sites

8. Using RMCE to construct a hybrid of 1/8 RGD segment plus 7/8 syn1.0
segments

9. HMG designing (deletion) rules
1. In the non-essential genes, most of the coding
region, including start and stop codons, was
deleted.
2. An intergenic region was also deleted when a
cluster of more than one consecutive gene was
deleted.
3. Intergenic regions flanking a deleted gene or a
consecutive cluster of deleted genes were
retained.
4. If part of the gene to be deleted overlapped a
retained gene, that part of the former was also
retained.

10. 6. The assumption is that when two genes were
divergently transcribed the intergenic region
between them contains promoters for both of
them.
7. When deletion resulted in converging transcripts,
a b-directional terminator was inserted (if it
wasn’t already present before).
5. If part of the gene to be deleted contained a
ribosome binding site or a promoter for a retained
gene, that part of the former was also retained.
HMG designing (deletion) rules

11. Failure (“partial success”)
Only one segment, HMG segment 2, on a syn1.0 background produced
viable colonies!!!! Time for a different strategy.

12. Identification of gene clusters through Tn5 mutagenesis

13. Results
→ P0 “hits” – insertion events
→ P4 “hits” – insertion events

14. Results
Long brown arrows → the eight segments
Blue arrows → genes that were retained
Yellow arrows → genes that were deleted
Green arrows → genes that were added back
Pink arrows → genes that were deleted between syn2.0 and syn3.0
DBT

15. • RGD1.0 wasn’t viable but designed segments 2, 6, 7, 8,
with preliminary segments (syn1.0) 1, 3, 4, 5, produced a
viable cell – RGD2678.
Doubling time 105mins compared with 60mins for syn1.0.
• Functions can be provided by more than one e- and i-
genes. Thus, one gene knockout will give us a viable cell
but when both genes are deleted, the function disappears
and the cell stops being viable. At least one of the genes
need to be added back.
REDUNDANT GENES ARE COMMON IN BACTERIAL
GENOME AND THIS LETHAL COMBINATION OF
MUTATIONS IS CALLED SYNTHETIC LETHAL PAIR.
Results

16. Results
Syn3.0 contains 149 genes
with unknown biological
functions.

17. Results

18. Result analysis
Comparison of syn1.0 and syn3.0 growth features.
• Syn1.0 has a bigger
colony size
(although very
similar
characteristics).
• Syn3.0 has a slower
growth rate (3hrs
doubling time).
• Syn3.0 cells formed
matted sediments
and segmented
filament structures.

19. Conclusions
• A minimal cell is usually defined as a cell in which all genes
are essential. This definition is incomplete and must be made
to suit the ecological and survival requirements of the model.
• There is a high probability that more genes could be removed
while retaining viability but it seems pretty sure that the
growth rate would be heavily compromised.
• The largest group of genes retained in Syn3.0 is involved in
gene expression.
• One of the biggest clusters to be removed are genes dealing
with cytoskeletal structures and anchors and also lipoprotein
production.
• JCVI-syn3.0 — Minimal Cell

20. More results and future research

21. • Assessment of plasticity of gene content in
terms of sequence and functionality
• Recoding an rRNA gene replacement
• Codon usage manipulation
More results and future research

22. THANK YOU!