Minimal and CompactDesign and synthesis of a minimal bacterial genome.

Clyde A. Hutchison III et al. Science 351(2016)

Joshua Gefen2 May 2016

J. Craig Venter Institute Research Timeline

Beginning Mycoplasma genitalium genome sequenced and minimal genome project begins.

PhiX synthesizedPublication of the synthesized version PhiX174

Bacterial Genome TransplantationJCVI 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

Research RationalePurpose: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.

Work Flow

• HMG: Hypothetical Minimal Genome

• RGD: Reduced Genome Design

• DBT: Design, Build, Test

Building the synthetic genome

Creating the eight segmentsUsing TREC deletion to generate scarless deletion and specific Not1 sites

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

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.

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

Failure (“partial success”)

Only one segment, HMG segment 2, on a syn1.0 background produced viable colonies!!!! Time for a different strategy.

Identification of gene clusters through Tn5 mutagenesis

Results

→ P0 “hits” – insertion events→ P4 “hits” – insertion events

Results

Long brown arrows → the eight segmentsBlue arrows → genes that were retainedYellow arrows → genes that were deletedGreen arrows → genes that were added backPink arrows → genes that were deleted between syn2.0 and syn3.0 DBT

• 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

Results

Syn3.0 contains 149 genes with unknown biological functions.

Results

Result analysisComparison 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.

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

More results and future research

• 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

THANK YOU!