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1. '''Van den Ent, F., Amos, L.A., and Lowe, J.''' (2001). Prokaryotic origin of the actin cytoskeleton. Nature 413, 39-44.
*1. '''Van den Ent, F., Amos, L.A., and Lowe, J.''' (2001). Prokaryotic origin of the actin cytoskeleton. Nature 413, 39-44.
2. '''Salje J., van den Ent, F., De Boer P., Lowe J.''' (2011). Direct Membrane binding by bacterial actin MreB. Mol Cell 43(3-6):478-487
*2. '''Salje J., van den Ent, F., De Boer P., Lowe J.''' (2011). Direct Membrane binding by bacterial actin MreB. Mol Cell 43(3-6):478-487
3. '''Esue O., Wirtz D., Tseng Y.''' (2006) GTPase activity, Structure, and mechanical properties of filaments assembled from bacterial cytoskeleton protein MreB. Journal of bacteriology 968-976
*3. '''Esue O., Wirtz D., Tseng Y.''' (2006) GTPase activity, Structure, and mechanical properties of filaments assembled from bacterial cytoskeleton protein MreB. Journal of bacteriology 968-976
4. '''J. Bean G., J. Amann K.''' (2008) Polymerization properties of the T.Maritima actin, MreB: roles of temperature, nucleotides and ions. Biochemistry January 15;47(2)826-835
*4. '''J. Bean G., J. Amann K.''' (2008) Polymerization properties of the T.Maritima actin, MreB: roles of temperature, nucleotides and ions. Biochemistry January 15;47(2)826-835
5. '''Van Teeffelen S., Wang S., Furchtgott L., Huang K., Wingreen N., Shaevitz J., Gitai Z.''' (2011) The bacterial actin MreB rotates, and rotation depends on cell-wall assembly  
*5. '''Van Teeffelen S., Wang S., Furchtgott L., Huang K., Wingreen N., Shaevitz J., Gitai Z.''' (2011) The bacterial actin MreB rotates, and rotation depends on cell-wall assembly  
6. '''Oda T., Lwasa M., Aihara T., Maeda Y., Narita A.''' (2009) The nature of the globular-to fibrous-actin transition. Nature 457;22 January
*6. '''Oda T., Lwasa M., Aihara T., Maeda Y., Narita A.''' (2009) The nature of the globular-to fibrous-actin transition. Nature 457;22 January
7. '''Carballido-Lopez R., Errington J.''' (2003) A dynamic bacterial cytoskeleton. Cell biology Vol.13 No.11 November
*7. '''Carballido-Lopez R., Errington J.''' (2003) A dynamic bacterial cytoskeleton. Cell biology Vol.13 No.11 November
8. '''Wang S., Arellano H., A.Combs P., W. Shaevitz J.''' (2009) Actin-like cytoskeleton filaments contribute to cell mechanics in bacteria.  
*8. '''Wang S., Arellano H., A.Combs P., W. Shaevitz J.''' (2009) Actin-like cytoskeleton filaments contribute to cell mechanics in bacteria.  
9. '''Van den Ent F., M Johnson C., Persons L., de Boer P., Lowe J.''' (2010) Bacterial actin MreB assembles in complex with cell shape protein RodZ. EMBO Journal 29, 1081-1090.
*9. '''Van den Ent F., M Johnson C., Persons L., de Boer P., Lowe J.''' (2010) Bacterial actin MreB assembles in complex with cell shape protein RodZ. EMBO Journal 29, 1081-1090.
10. '''J Osborn M., Rothfield L.''' (2007) Cell shape determionation in Escherichia coli. Current Opinion in Microbiology 10:606-610
*10. '''J Osborn M., Rothfield L.''' (2007) Cell shape determionation in Escherichia coli. Current Opinion in Microbiology 10:606-610
11. '''Peter L. Graumann''' (2007) Cytoskeletal elements in Bacteria.Annu. Rev. Microbiol 61:589-618
*11. '''Peter L. Graumann''' (2007) Cytoskeletal elements in Bacteria.Annu. Rev. Microbiol 61:589-618
12. Huebner A., Sharma S., Srisa-Art M., Hollfelder F., B.Edel J., J.deMello A.(2008) Microdroplets: A sea of application? The Royal Society of Chemistry 1244-1254
*12. Huebner A., Sharma S., Srisa-Art M., Hollfelder F., B.Edel J., J.deMello A.(2008) Microdroplets: A sea of application? The Royal Society of Chemistry 1244-1254
13. '''J Doktycz M., L Simpson Michael''' (2007) Nano-enabled synthetic biology. Molecular Systems Biology 3:125
*13. '''J Doktycz M., L Simpson Michael''' (2007) Nano-enabled synthetic biology. Molecular Systems Biology 3:125
14. '''Gulati S., Rouilly V., Niu X., Chappell J., I. Kitney R., B. Edel B., S. Freemont P., J. deMello A''' (2009) Opportunities for microfluidic technologies insynthetic biology. J. R. Soc. Interface 6, S493-S506
*14. '''Gulati S., Rouilly V., Niu X., Chappell J., I. Kitney R., B. Edel B., S. Freemont P., J. deMello A''' (2009) Opportunities for microfluidic technologies insynthetic biology. J. R. Soc. Interface 6, S493-S506
15. '''Gitai Z.''' (2005) The new bacterial cell biology: moving parts and subcellular architecture. Cell, Vol. 120, 577-586  
*15. '''Gitai Z.''' (2005) The new bacterial cell biology: moving parts and subcellular architecture. Cell, Vol. 120, 577-586  
16. '''Mattei P., Neves D., Dessen A.''' (2010) Bridging cell wall biosynthesis and bacterial morphogenesis. Current Opinion in Structural Biology. 20:749-755.
*16. '''Mattei P., Neves D., Dessen A.''' (2010) Bridging cell wall biosynthesis and bacterial morphogenesis. Current Opinion in Structural Biology. 20:749-755.
17. '''A. Mayer J., J. Amann K.''' (2009) Assembly properties of the Bacillus subtilis actin, MreB. Cell Motility and the Cytoskeleton 66: 109-118.  
*17. '''A. Mayer J., J. Amann K.''' (2009) Assembly properties of the Bacillus subtilis actin, MreB. Cell Motility and the Cytoskeleton 66: 109-118.  
18. '''Bendezu F., Hale C., Bernhardt T., de Boer AJ.''' (2009) RodZ(YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in E. coli. The EMBO Journal 28, 193-204.
*18. '''Bendezu F., Hale C., Bernhardt T., de Boer AJ.''' (2009) RodZ(YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in E. coli. The EMBO Journal 28, 193-204.
19. '''Gerdes K.''' (2009) RodZ, a new player in bacterial cell morphogenesis.The EMBO journal 28, 171-172.
*19. '''Gerdes K.''' (2009) RodZ, a new player in bacterial cell morphogenesis.The EMBO journal 28, 171-172.
20. '''Alyahya S., Alexander R., Costa T., O. Henriques A., Emonet T., Jacobs-Wagner C.''' (2009) RodZ, a component of the bacterial core morphogenic apparatus. PNAS January 27 vol.106 no.4 1239-1244.
*20. '''Alyahya S., Alexander R., Costa T., O. Henriques A., Emonet T., Jacobs-Wagner C.''' (2009) RodZ, a component of the bacterial core morphogenic apparatus. PNAS January 27 vol.106 no.4 1239-1244.


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Revision as of 09:50, 31 October 2011


Home            Project            Results            Video            Team            Acknowledgements

Cell-free synthesis of actin-like cytoskeleton filaments

Introduction and Background

Since the successful chemical total synthesis of entire bacteria genome by J. C. Venter, the wave of synthesizing genome and creating artificial life has been set off worldwide. The recognition of life phenomenon for human has already transformed from “read” to write. Nevertheless, even the chemical synthesis of bacteria genome of J. C. Venter has to rely on the intracellular environment of living cells, and the artificial synthesis of cellular microenvironment (cell membrane and cytoplasm) is greatly fall behind the paces of total chemical synthesis of genome.

Fig 1.

Synthetic biology is an emerging field of science that offers the prospect of the design and construction of new biological pathways, systems and even cell entities that do not exist in nature. Our project aims at using a “bottom-up’ approach of assembling components to create artificial cell surrogates, or protocells, which enable biochemical pathways to be reconstructed, to perform complex processing steps.

Filamentous cytoskeleton protein

The very first beginning of create artificial cell surrogates or protocells is to construct a basic cytoskeleton, which can support the bacterial cell shape and serve as membrane environment to become an interface between a predominantly aqueous stable internal (segmented) and external (continuous) environment. Based on these principles, we set our target on microfilament protein MreB, an actin homolog in E. coli encoded by MreB gene, which is essential for cell-shape determination. The actin-like properties of MreB render itself to form large fibrous spirals under the cell membrane of rod-shaped cells (E. coli). In prokaryotes, MreB is rigidly linked to the cell wall, increasing the mechanical stiffness of the overall system, and served as bacterial cytoskeleton contributes to the mechanical integrity of a cell in much the same way as it does in eukaryotes.

Fig 2.

CFS (cell-free system)

The construct of protocells need the expression of filamentous cytoskeleton protein in vitro. That’s why we involve Cell-Free System (CFS) in our project. Cell-Free System is a mixture of cytoplasmic and/or nuclear components from cells and used for in vitro protein synthesis or transcription or DNA replication or other purposes. Following the Central Dogma in terms of two essential processes – the transcription of DNA into messenger RNA (mRNA) and the translation of mRNA into polypeptides, the CFS can serve as a compatible chassis for the various genes and proteins. Not only does CFS house the molecular machinery necessary for transcription and translation, they are also optimized for these two processes.

Fig 3.

Coupled transcription-translation systems usually combine a bacteriophage RNA polymerase and promoter with eukaryotic or prokaryotic extracts rich in ribosomes, transfer RNAs and aminoacyl tRNA transferase enzymes. Buffers are also added to maintain the appropriate magnesium and salt concentrations required for efficient translation. Protease inhibitors can be added to minimize degradation of synthesized proteins. In addition, an ATP regenerating system involving either creatine phosphate and creatine kinase or phosphoenol pyruvate and pyruvate kinase is used to power and prolong the lifespan of the expression machinery. Simply by adding the DNA template to the cell extract and feeding solution, the CFS would be able to express the encoded genetic circuit. The pure system has recently been developed as a reconstituted CFS for synthesizing proteins using recombinant elements. This purely synthetic expression system enables even better quality control over the reaction conditions. The CFS reveals a short and discrete lifespan because of the limited energy system even in the presence of an ATP regenerating system and no sustained metabolism.

Fig 4.

Different compartmentalization strategies have been explored to prolong the expression lifespan of the CFS. In our experiments, batch mode CFS and vesicle-encapsulated CFS are conducted. More details will be showed in the Microfluidcs Part later. Batch mode CFS: Transcription-translation reaction is carried out in bulk solution. Expression is usually limited by nutrient (nucleotides and amino acids) and energy supplies.

Fig 5.

Vesicle-encapsulated CFS: The reaction is separated from the feeding solution by a phospholipid bilayer. Expression is maintained for a longer time period than batch-mode CFS because of exchange of materials between the reaction and the feeding solution across the membrane. More reliable exchange of materials is established by inserting a non-specific pore protein with a suitable channel size into the phospholipid bilayer.

Fig 6.

CFS and Microfluidcs

To mimic the natural cell more faithfully, cell-free expression systems have been combined and encapsulated into double emulsion droplets, which is produced by microfluidcs technology. Water in oil in water (W/O/W) droplets are created by using a hybrid Lab-chip devices comprising a hydrophobic network (supporting a continuous oil phase), interfaced with a hydrophilic network (supporting an aqueous phase).

Fig 7.

These microdroplets are of comparable scales to natural cells and could serve as a platform where biochemical reactions can take place, for example the cell-free expression of water-soluble protein Green Fluorescent Protein (GFP). Using mineral oil as the oil phase would provide a robust and stable membrane, and the hydrophobic interactions generated within this membrane environment produce driving forces which account for the distribution of different intracellular proteins, and mimic the natural cell membrane to provide a non-polar environment which separates the extracellular aqueous environment from the cell’s internal aqueous environment.

Fig 8.

The size of the oil droplet was dependent on the oil and external water flow rate, as well as the storing temperature. In the following figure, the mean diameter of the inner compartment was 37.4μm while that of the outer membrane (whole drop) was 41.0μm, indicating the average thickness of 3.6μm. Emulsions stored at 3℃remained stable for two months with no coalescence or shrinkage observed.

Fig 9:The distribution of the inner (black) and outer (grey) diameter of the emulsion with relative coefficient of variations (c.v.).

B

C

D

  • 1. Van den Ent, F., Amos, L.A., and Lowe, J. (2001). Prokaryotic origin of the actin cytoskeleton. Nature 413, 39-44.
  • 2. Salje J., van den Ent, F., De Boer P., Lowe J. (2011). Direct Membrane binding by bacterial actin MreB. Mol Cell 43(3-6):478-487
  • 3. Esue O., Wirtz D., Tseng Y. (2006) GTPase activity, Structure, and mechanical properties of filaments assembled from bacterial cytoskeleton protein MreB. Journal of bacteriology 968-976
  • 4. J. Bean G., J. Amann K. (2008) Polymerization properties of the T.Maritima actin, MreB: roles of temperature, nucleotides and ions. Biochemistry January 15;47(2)826-835
  • 5. Van Teeffelen S., Wang S., Furchtgott L., Huang K., Wingreen N., Shaevitz J., Gitai Z. (2011) The bacterial actin MreB rotates, and rotation depends on cell-wall assembly
  • 6. Oda T., Lwasa M., Aihara T., Maeda Y., Narita A. (2009) The nature of the globular-to fibrous-actin transition. Nature 457;22 January
  • 7. Carballido-Lopez R., Errington J. (2003) A dynamic bacterial cytoskeleton. Cell biology Vol.13 No.11 November
  • 8. Wang S., Arellano H., A.Combs P., W. Shaevitz J. (2009) Actin-like cytoskeleton filaments contribute to cell mechanics in bacteria.
  • 9. Van den Ent F., M Johnson C., Persons L., de Boer P., Lowe J. (2010) Bacterial actin MreB assembles in complex with cell shape protein RodZ. EMBO Journal 29, 1081-1090.
  • 10. J Osborn M., Rothfield L. (2007) Cell shape determionation in Escherichia coli. Current Opinion in Microbiology 10:606-610
  • 11. Peter L. Graumann (2007) Cytoskeletal elements in Bacteria.Annu. Rev. Microbiol 61:589-618
  • 12. Huebner A., Sharma S., Srisa-Art M., Hollfelder F., B.Edel J., J.deMello A.(2008) Microdroplets: A sea of application? The Royal Society of Chemistry 1244-1254
  • 13. J Doktycz M., L Simpson Michael (2007) Nano-enabled synthetic biology. Molecular Systems Biology 3:125
  • 14. Gulati S., Rouilly V., Niu X., Chappell J., I. Kitney R., B. Edel B., S. Freemont P., J. deMello A (2009) Opportunities for microfluidic technologies insynthetic biology. J. R. Soc. Interface 6, S493-S506
  • 15. Gitai Z. (2005) The new bacterial cell biology: moving parts and subcellular architecture. Cell, Vol. 120, 577-586
  • 16. Mattei P., Neves D., Dessen A. (2010) Bridging cell wall biosynthesis and bacterial morphogenesis. Current Opinion in Structural Biology. 20:749-755.
  • 17. A. Mayer J., J. Amann K. (2009) Assembly properties of the Bacillus subtilis actin, MreB. Cell Motility and the Cytoskeleton 66: 109-118.
  • 18. Bendezu F., Hale C., Bernhardt T., de Boer AJ. (2009) RodZ(YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in E. coli. The EMBO Journal 28, 193-204.
  • 19. Gerdes K. (2009) RodZ, a new player in bacterial cell morphogenesis.The EMBO journal 28, 171-172.
  • 20. Alyahya S., Alexander R., Costa T., O. Henriques A., Emonet T., Jacobs-Wagner C. (2009) RodZ, a component of the bacterial core morphogenic apparatus. PNAS January 27 vol.106 no.4 1239-1244.

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