Standardized cells with completely predictable and controllable behavior is a key focus of synthetic biology. Recent advances such as the implantation of a synthesized genome into a DNA-free bacterial shell at the J. Craig Venter Institute marked the dawn of these whole-genome scale projects. A first step towards predictable synthetic minimal cells is made through further reduction of the cellular complexity by minimizing the bacterial genome. This has lead to a short list of essential genes for cells to still be considered living entities. Although these minimal cells increase our fundamental understanding of key cellular processes, there are other important criteria to consider for synthetic cells to become biotechnologically relevant. In this colloquium I will address the current state of research concerning biotechnological useful cells that can be used as a platform for operating precisely designed and independent biological systems. Top-down methods for the design of these organisms will be reviewed, highlighting remarkable experiments.

1. The White-box Model Usefulpredictable and controllablecells Pieter van Boheemen 17-12-2010 1

2. The cell 2

3. 3

4. Predictable Definedcellularprocesses Defined medium Controllable Pure culture Geneticmodifications / stability Safe Useful Biotechnological relevant Wishlist 4

6. antisense RNA

7. gene knock outZhanget al, 2010, Protein & Cell 5

8. Somegenesneverinactivated Toxicintermediatesor incomplete proteincomplexes Effect onneighboringgenes Culture conditions Growthrate Inactivationcritisism 6

9. 0.58 MB genome, 518 genes 68% annotated 100 genesindividuallydispensable No definedgrowth medium (FCS) No cellwall M. genitalium 7 Frantz, Al­bay and Bott, UNC/Chapel Hill

10. E. coli4.64 MB genome, 4312 genes B. subitilis4.21 MB genome, 4114 genes Robustecells Easy to transform Rapidgrowthon minimal (defined) media Genome reductions E. coli, 1.38 MB (30%) (Hashimotoet al, 2005) B. subtilis, 1.4 MB (33%) (Tanakaet al, 2010) E. coli & B. subtilis 8

11. DNA synthesis RNA synthesis RNA modification Ribosomeassembly PTMs Proteinsynthesis 115 proteins & 37 RNA Bottom-upapproach Forster & Church, 2006, Mol SystBiol. 9

12. Purifiedsubsystems Integration Self-replication Encapsulation 10 In vitrominimal cellproject (MCP) Jewett & Forster, 2010, Curr. Opin. Biotech.

13. L'Évolutioncréatrice: l’élan vital, « force créant de façon imprévisible des formes toujours plus complexes » Philosophy 11 Henri-LouisBergson (Nobel prize 1927)

14. Oudenaarden & Knight (MIT) Endy & Smolke (Stanford) Carr, Wang, Forster & Church (Harvard) Kitney & Freemont (Imperial College) Elfick, Tyers & French (University of Edinburgh) Gibson, Lartigue & Venter (J. Craig Venter Institute) SyntheticBiologists 12

15. 13

16. 14 Gibson et al, 2010, Science

17. 10 KB assemblies: 10-100% succes rate 100 KB assemblies: 25% successrate 1 MB assemblies: 2% successrate Oneerror free genome per 10^5 assemblyreactions Single-base pair deletion in dnaAcaused lethal frameshift 200 personyears of work and $ 40 million Onlyproof of principle 15 Gibson et al, 2010, Science

18. Robustreproduction Robustcellenvelope Simplecontrol Decreasedgenetic drift Predictablemetabolicinteractions No competingpathways Towardsusefulcells 16

19. Reducingcomplexity X X X X X X 17 X Trinhet al, 2008, Appl. and Environ. Microbio.

20. Inactivategenes E. coliK12 contains 45 ISes ctxvp60 Capsidprotein VP60 of RHD virus Cholera toxin CTX 18 InsertionSequences Umenhofferet al, 2010, Microbio. CellFactories

21. IS problems 19 Umenhofferet al, 2010, Microbio. CellFactories

22. 20 Umenhofferet al, 2010, Microbio. CellFactories

23. 50 oligos at a time 90mer Cycle time of 2hrs >30% efficiency Multiplex-automatedgenomic engineering (MAGE) 21 Wang et al, 2010, Nature

24. 22 Wang

25. 23 Lycopene: 20 genes up, 4 down Wang et al, 2010, Nature

26. Minimal cells Continue deletion of genes Removemetabolism (orsymbionts) In vitro minimal cell project (MCP) Forster & Church paper There is no ‘simple’ organism Conclusions & Future 24

28. 27

29. Gibson Gels 28 Gibson et al

30. Lartigue 2009 Cleaned-up or uncleaned yeast agarose plugs were washed two times for approximately 30 minutes each in 200 mMTris-HCl pH 7.5; 50 mM EDTA and equilibrated two times for approximately 30 minutes each in methylation buffer (100 mMTris-HCl pH 7.5; 10 mM EDTA; 3 µM DTT; 200 µM S-adenosylmethionine) with gentle agitation. Following equilibration, each yeast plug was cut into 4-5 pieces and added to 100 µl of 1X methylation buffer plus Lartigue Supporting Online Material 8 methyltransferases and incubated 16 hours at 37 ºC. For 100 µl of reaction, we used either approximately 125 µg of wild type M. mycoides cellular extracts, M. capricolum RE(-) (clone 10 15P) cellular extracts or or 2.5 µl of each purified M. mycoides LC specific methyltransferases. The methylation reactions with the M. mycoides extracts were also supplemented with 40 units of dam methyltransferase (NEB). Following methylation, each yeast plug was incubated for 4 hours at 50 °C in 1ml of Proteinase K Reaction Buffer supplemented with 40µl of Proteinase K. The plugs were then washed 4 times for 45 minutes each with 1 ml of 1X TE buffer and 2 times for 30 minutes each in 0.1X TE buffer with gentle agitation at room temperature. After removing the final wash buffer, the plugs were melted as above. Methylases 29 Lartigueet al

31. Base per buck 30 Carr & Church, 2009, Nature Biotechnology

32. 31 Church

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34. 33 Keasling

35. 34 Keasling