1. Class #11: iGEM and The Synthetic Cell
Plans Today:
iGEM v.6.0
The first exam …
Special Interest Short Reports (5%)
Transition in class format
Topics
Synthetic cells,
Safe cells,
Metagenomics
Glycome in synthetic biology.
October 10, 2012
Class #11: iGEM and The Synthetic Cell

2. Plans Today:
iGEM v.6.0
The first exam …
Special Interest Short Reports (5%)
Transition in class format
Topics
Synthetic cells,
Safe cells,
Metagenomics
Glycome in synthetic biology.
Oct 17 – Dali Lama visit – no class
Oct 24 – Kathy Williams, PhD, J.D. Patent Law
Oct 31 – Halloween and the remarkable coincidence of the Mid-term creative project! Part 1

3. Midterm Project Rules and Regulations.
(This carefully crafted plan supersedes the plan in the syllabus.) 
Rule #1 Be Creative!
Rule #2 Be Brief.
Rule #3 (the fine print): Students will present a creative project making use of the materials covered in class, and beyond. The emphasis is creativity, and the intent is stimulation. Hopefully this project will evolve continuously from discussions in class and between class members and may form the basis for your final project. The project format has two parts: On Oct 31 - each student will get 60 seconds to present an overview of their project. Feedback from the audience is encouraged to help each student maximize the impact of their project.  Being as this is Halloween day, costumes/props/enhancements are encouraged to facilitate this communication (for extra credit of course)! On November 5th, a one page written description of the project is due, hopefully incorporating any comments/improvements resulting from the oral presentation. Both parts of the presentation are evaluated and constitute 10% of the final grade for the course. 

5. The Future of Synthetic Biology
Key considerations:
Levels of expression needs to be regulated precisely
Host pathways need to be known to minimize deleterious interactions
Part characterization is essential
Appropriate Chassis is key –
So is safety…..

6. Synthetic cells?

12. Why create a minimal cell?
Why create a synthetic cell?
Why create a minimal cell?

13. Why M. genitalium?

14. Mycoplasma
Genus of bacteria containing the smallest known free-living bacterium (M. genitalium)
Parasitic bacteria of primates, genital and respiratory tracts
Small genomes ( kb)
M. genitalium: ,970bp
M. capricolum: 1,155,500bp
M. mycoides: 1,077,947bp
Nanoarchaeum equitans is a species of tiny microbe, discovered in 2002 in a hydrothermal vent; its genome is only 490,885 nucleotides long; but has an obligate host

15. Whole genome sequencing could be accomplished by shot-gun (random) sequencing approaches

16. Gene Map of M. genitalium

17. Gene
ontology

18. Metabolic pathways and substrate transport mechanisms encoded by M
Metabolic pathways and substrate transport mechanisms encoded by M. genitalium.
Metabolic pathways and substrate transport mechanisms encoded by M. genitalium. Metabolic products are colored red, and mycoplasma proteins are black. White letters on black boxes mark nonessential functions or proteins based on our current gene disruption study. Question marks denote enzymes or transporters not identified that would be necessary to complete pathways, and those missing enzyme and transporter names are colored green. Transporters are colored according to their substrates: yellow, cations; green, anions and amino acids; orange, carbohydrates; purple, multidrug and metabolic end product efflux. The arrows indicate the predicted direction of substrate transport. The ABC type transporters are drawn as follows: rectangle, substrate-binding protein; diamonds, membrane-spanning permeases; circles, ATP-binding subunits.
Glass J I et al. PNAS 2006;103:
©2006 by National Academy of Sciences

19. Metabolic pathways and substrate transport mechanisms encoded by M
Metabolic pathways and substrate transport mechanisms encoded by M. genitalium.
Metabolic pathways and substrate transport mechanisms encoded by M. genitalium. Metabolic products are colored red, and mycoplasma proteins are black. White letters on black boxes mark nonessential functions or proteins based on our current gene disruption study. Question marks denote enzymes or transporters not identified that would be necessary to complete pathways, and those missing enzyme and transporter names are colored green. Transporters are colored according to their substrates: yellow, cations; green, anions and amino acids; orange, carbohydrates; purple, multidrug and metabolic end product efflux. The arrows indicate the predicted direction of substrate transport. The ABC type transporters are drawn as follows: rectangle, substrate-binding protein; diamonds, membrane-spanning permeases; circles, ATP-binding subunits.
Glass J I et al. PNAS 2006;103:
©2006 by National Academy of Sciences

20. Metabolic pathways and substrate transport mechanisms encoded by M
Metabolic pathways and substrate transport mechanisms encoded by M. genitalium.
Metabolic pathways and substrate transport mechanisms encoded by M. genitalium. Metabolic products are colored red, and mycoplasma proteins are black. White letters on black boxes mark nonessential functions or proteins based on our current gene disruption study. Question marks denote enzymes or transporters not identified that would be necessary to complete pathways, and those missing enzyme and transporter names are colored green. Transporters are colored according to their substrates: yellow, cations; green, anions and amino acids; orange, carbohydrates; purple, multidrug and metabolic end product efflux. The arrows indicate the predicted direction of substrate transport. The ABC type transporters are drawn as follows: rectangle, substrate-binding protein; diamonds, membrane-spanning permeases; circles, ATP-binding subunits.
Glass J I et al. PNAS 2006;103:
©2006 by National Academy of Sciences

21. Legend to molecular anatomy
(previous slide)
Metabolic pathways and substrate transport mechanisms encoded by M. genitalium. Metabolic products are colored red, and mycoplasma proteins are black. White letters on black boxes mark nonessential functions or proteins based on our current gene disruption study. Question marks denote enzymes or transporters not identified that would be necessary to complete pathways, and those missing enzyme and transporter names are colored green. Transporters are colored according to their substrates: yellow, cations; green, anions and amino acids; orange, carbohydrates; purple, multidrug and metabolic end product efflux. The arrows indicate the predicted direction of substrate transport. The ABC type transporters are drawn as follows: rectangle, substrate-binding protein; diamonds, membrane-spanning permeases; circles, ATP-binding subunits.
Glass J I et al. PNAS 2006;103:

22. Biology of The Mycoplasmas
The Mycoplasmas evolved through a process of degenerate evolution from Gram-positive bacteria. They have lost many genes such as those involved with cell wall synthesis and various biosynthetic pathways, as they adapted to a more parasitic lifestyle. This allows them to have some of the smallest genomes of any bacterial organism.
Their UGA codon is used as another codon for tryptophan instead of a stop codon found in other organisms.[1]
M. genitalium lack any genes for amino acid biosynthesis and contain few genes for nucleic acid, vitamins, and fatty acid biosynthesis. They must acquire these components from their host or through an artificial medium. This makes them difficult to study in the lab under in vitro conditions, due to their strict growth requirements.
They also lack genes for oxidative metabolism (Kreb cycle), gluconeogenesis, catalase and peroxidase, or other toxic oxygen protective enzymes. They also appear to possess few regulatory proteins, such as two-component systems and no identifiable transcription factors.
They do however possess the genes necessary for DNA replication, transcription, and translation, but even these contain a minimal set of rRNA and tRNA genes.[10] They also contain genes for glycolysis, phospholipids metabolism, and for converting vitamins to cofactors. They have less DNA repair genes compared to E. coli and H. influenzae. It is assumed however that the genes detected such as uracil DNA glycosylase, exinuclease ABC genes, and recA must be essential for proper DNA repair in all organisms.[9]
While M. genitalium have been able to reduce its genome during the course of its evolution as a parasite, it must maintain genes necessary for this mode of life. A significant portion of its genome is devoted to the transport of nutrients from its host such as glucose and fructose, as well as genes for attachment organelles, adhesins, and antigenic variation to evade the host immune system. In fact it’s estimated that 5% of its total DNA is devoted to repeat fragments used for antigenic recombination of adhesin proteins.[1]
Due to its minimal set of genes needed for protein synthesis and DNA replication, protein synthesis and cell replication occur much slower compared to E. coli. M. genitalium grow much more slowly with a generation time of about 24 hours.[1]

23. two bacterial genomes: Haemophilus influenzae and
Comparison of
two bacterial genomes:
Haemophilus influenzae
and
Mycoplasma genitalium
H. influenzae was the first free-living organism to have its entire genome sequenced: 1,830,140 base pairs of DNA in a single circular chromosome that contains 1740 protein-coding genes, 58 tRNA genes, and 18 other RNA genes. The sequencing method used was whole genome shotgun (1995). Discovered in 1892, mistakenly thought to be causative agent of influenza until 1933, when the influenza virus was discovered.
E.coli? Genome, 4,639,221 bp; genes 4,288 genes

25. Linear representation of the synthesized circular genome
Genetic Watermark: So that the assembled genome would be recognizable as synthetic, four of the ordered DNA sequences contained strings of bases that, in code, spell out an address, the names of many of the people involved in the project, and a few famous quotations.

27. DNA shuttling

28. The Flow of stepwise construction

29. Genome assembly

30. Stage 1 – Cassettes 1,078 one-kb cassettes
Cassettes and a vector were recombined in yeast and transferred to E. coli.
Plasmid DNA was then isolated from individual E. coli clones and digested to screen for cells containing a vector with an assembled 10-kb insert.

31. Stage 2 – 10kb Intermediates
109 ten-kb sections
Too large for E.coli, so DNA was extracted from yeast to assemble 100kb sections
Analyzed by multiplex PCR

32. Stage 3 – 100kb intermediates
11 100kb sections
Isolated in circular plasmid form
Removed linear yeast chromosomes by pooling samples in solidifying agarose
Multiplex PCR and restriction to verify full assembly

33. Obstacles
Bacterial genomes grown in yeast are unmethylated and thus not protected from the single restriction system of the recipient cell
Methylate the donor DNA with purified methylases or crude M. mycoides or M. capricolum extracts
Disrupt the recipient cell’s restriction system
Success was thwarted for many weeks by a single base pair deletion in the essential gene dnaA.
One wrong base out of over one million in an essential gene rendered the genome inactive!

34. Successful Transplants!
Synthetic
Wild-type

35. Notable results
Sequence matched the intended design with the exception of:
The 19 known polymorphisms
8 new single nucleotide polymorphisms
E. coli transposon insertion
85-bp duplication
No sequences from M. capricolum (recipient)
Proteomic analysis shows almost the exact protein pattern as WT M. mycoides

36. Implications Synthesis of a custom organism is possible
Our sequencing technology is accurate enough to produce an entire genome
DNA synthesis costs “will follow what has happened with DNA sequencing and continue to exponentially decrease”
Synthetic biology is now in the public eye

37. Critiques Patents may stifle synthetic biology in this field
Currently, far less efficient than gene insertion to achieve similar goals

38. How many of the genes are essential for viability?

39. How to create a minimal Genome
Venter’s group estimated that M. genitalium might be able to survive on only 265 to 350 genes (Science, 10 December 1999, p. 2165). But testing one gene at a time could not
tell the researchers exactly what would constitute such a minimal genome. It’s possible,
for example, that any one of five genes can do some vital task. Tested one at a time, all
five may seem unnecessary, but if all of them are removed, the microbe dies. “We didn’t have a way of doing cumulative knockouts to get down to just having a chromosome with just 300 genes in it,” Venter explains. “So we don’t know which are the right 300 genes, or even if 300 is the right number. That study drove my thinking that we needed a synthetic genome.”
The biggest genome synthesized to date is that of the 7500-nucleotide poliovirus (Science, 9 August 2002, p. 1016). “It’s not trivial to do these syntheses,”

41. Transposon insertions in M. genitaium.

42. Transposon insertions have been identified in 140
different genes in M. genitalium, so that the minimal (essential) genome may be 470 – 140 ~ 350 genes
What is essential?
What is redundancy?
Conditions for testing?
Compensatory gene activity?