Lecture 3. Larger Scale Synthetic Biology - Genomes, Organisms, Tissues Flashcards
What is the majority of the human genome taken up by?
Non-coding RNAs (coding regulatory and structural RNAs)
What is the size of the “minimal cell”?
~ 400 genes (0.5 Mbp)
Volume ≈ 0.1 μm³
What is the “minimal cell”?
How many genes you need to have the attributes of life
What are the ultimate goals of the “minimal cell”?
Define function of every gene in a cell (standard genetic methods inadequate)
Define a (hypothetical) minimal genome – how small can you go?
Develop methods for exploring whole-genome design and construction
What were the three categories of genes that made up the Syn1.0 genome?
i-gene (quasi-essential). In the full genome context, some of these were categorised as minimal (in) or extreme (ie)
e-gene (essential; very few, or no, transposon insertions were viable)
n-gene (non-essential)
How was research into a hypothetical minimal genome (HMG) conducted?
Use transposons to insert themselves at various positions int he genome to analyse the phenotypes of the strains after transposon insertions and examined phenotypes
How was Syn2.0 designed?
Eight blocks, done separately and then brought together
Removed a further 42 genes from Syn1.0
How many genes does Syn3.0 have and what 4 major functional groups can they be assigned under?
473 genes
195 genes (41%) involved in gene expression
84 genes (18%) involved in cell membrane synthesis and maintenance
81 genes (17%) involved in metabolism
36 genes (7%) involved in replication and maintenance of the genome
79 genes (17%) have no unequivocally defined function
What are some features of Syn3.0 cells?
Smaller, grow more slowly, and polymorphic
Tends to aggregate
When is Syn3.0 a ‘working approximation’ to a viable minimal genome?
Under the highly permissive conditions defined by a rich growth medium
Only viable under a certain precise circuit of living conditions
What will Syn3.0 facilitate?
The creation of the Syn3.0 genome will facilitate progress towards cell creation from scratch
This will help facilitate complete computational cellular design at some point in the future
This does not (yet) constitute the creation of a life-form as such
However, (semi-) automated de novo cell manufacture will become feasible
How can microtissue assays be assembled?
Using DPAC (DNA-programmed Assembly of Cells)
What happens during microtissue array formation?
Amino-modified spots of DNA on aldehyde-coated glass; in Step 1, lipid-modified complementary oligos mediate binding of (epithelial) cells; in Step 2, another layer of cells, carrying oligos complementary to those in Step 1, is added, and so on to build hemispherical microtissues.
Reductive amination reaction binds them to glass slides
How can the assembly of cylindrical microtissues with defined patterns of spatial heterogeneity be illustrated?
Using mammalian epithelial cells labelled with two different fluorescent tags
The patterns are dictated by the DNA template patterns on the glass slide and a poly(dimethylsiloxane) PDMS flow cell was placed above this to introduce reagents and cell suspensions
All patterns dictated by specific initial oligos
What will the methods for fabricating three-dimensional multi-cellular structures (microtissues) be used for in the future?
Fabrication of organoids in the future, and will likely find multiple uses in basic research as well as applications
