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=='''Object'''==
=='''Objective'''==
*Establishing a stable bionic membrane system, an cell-free microenvironment enabling E. coli actin-like cytoskeleton?filaments synthesis.
*Establishing a stable bionic membrane system, an cell-free microenvironment enabling E. coli actin-like cytoskeleton?filaments synthesis.
*Exploring E. coli actin-like cytoskeleton?filaments location and aggregation in vitro.
*Exploring E. coli actin-like cytoskeleton?filaments location and aggregation in vitro.

Revision as of 06:48, 2 November 2011


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Cell-free synthesis of actin-like cytoskeleton filaments

Objective

  • Establishing a stable bionic membrane system, an cell-free microenvironment enabling E. coli actin-like cytoskeleton?filaments synthesis.
  • Exploring E. coli actin-like cytoskeleton?filaments location and aggregation in vitro.

Introduction

  • Recently, Synthetic biology rewriting efforts have usually followed a bottom-up strategy of constructing deliberately simplified systems to build an understanding of cellular regulatory processes. Since the successful chemical 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, the artificial synthesis of cellular microenvironment (cell membrane and cytoplasm) is greatly fall behind the paces of total chemical synthesis of genome.
  • An efficient bottom-up approach to the rewriting of synthetic biology is to follow a cell-free strategy to minimize unintended interactions by eliminating cellular support of synthetic gene circuits. A Cell-Free System (CFS) involves the in-vitro expression of genes into proteins and can serve as a compatible chassis for the various parts and devices.
  • Our project aims at using a “bottom-up’ approach of assembling components to create artificial cell surrogates in a cell-free system, which enable biochemical pathways to be reconstructed, to perform complex processing steps. By synthesizing MreB, a filamentous cytoskeleton protein, and combining with microfluidics technology and bionanotechnology, we construct the actin-like cytoskeleton filaments in vitro, which can support the bacterial cell shape and serve as membrane environment.

Filamentous cytoskeleton protein

Fig1. Three dimensional-structure of MreB Protein (Van den Ent, F., Amos, L.A., and Lowe, J. 2001)
  • Based on these principles, we set our target on microfilament protein MreB, an actin homolog in E. coli encoded by MreB gene. MreB are organized into helical filamentous structures that coil around the rod-shaped cell. The extended coiled structures consist of two interwined helices and are located on the undersuface of the cytoplasmic membrane and frequently extend along the entire length of the cell. The MreB involves in the synthesis of cell wall with other related protein including MreC, MreD, PBP, RodZ, etc. The shape of the cell wall determines the shape of the cell ultimately. Two properties are very important for MreB to have this function. One is that MreB can self-assembles into long filamentous structure in the presence ATP without any other auxiliary proteins in vitro; Another is membrane binding property, the N-terminal region that forms amphipathic helix is essential for MreB to bind membrane of the cell.

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 and optimized chassis for the various genes and proteins.
Fig 2. Comparison of whole-cell and cell-free synthetic biology (Michael L Simpson, 2006)
  • In a cell-free system, 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 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.
  • Despite the lack of experience and characterization, CFS shows huge advantages on the expression, control and purification special exogenous proteins which interrupt the normal metabolism of host cell. Here CFS is particularly suited for constructing artificial cell surrogates.

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 3.Double emulsion generation: a microfluidic system made of two glass chips with different wetability characteristics.
  • 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.

CFS and SWNTs (Single-Walled Carbon Nanotubes)

  • Single-walled carbon nanotubes (SWNTs) have become an area of wide ranging research activity due to their exceptional chemical and physical properties. In our project, to take advantage of the remarkable tubular framework structure of SWNTs in various applications, the SWNTs need to be derivatized with organic acyl peroxides of dicarboxylic acids, such as those containing terminal carboxylic acid groups to provide a high binding affinity and selectivity with proteins through formation of either hydrogen or covalent bonds.
Fig 4.Chemical Routes for Preparation of SWNT-Derivatives
  • After the successful sideway carboxylic acid functionalization, we immersed these SWNTs into nickel nitrate solution for the electrostatic interaction between nickel ions and carboxylic acid groups. The binding of nickel ions to SWNTs is indispensible for the absorption of MreB to SWNTs in vitro.
Fig 5.Nickel ions binding with sideway functinalized SWNTs

Hypothesis

  • 1. The similarity of the oil phase in our double emulsion generated by microfluidics technology with natural cell membrane implies that the original actin-like cytoskeleton filaments synthesis could be achieved in this artificial microenvironment, for the N-terminal region that forms amphipathic helix is essential for MreB to bind both the cell membrane and the artificial oil phase.
  • 2. Considering the special hydrophobic interactions and electrostatic interaction between nanomaterials and proteins, SWNTs after surface treatment might specifically organize the distribution of actin-like cytoskeleton filaments. As MreB can self-assembles into long filamentous structure in the presence ATP without any other auxiliary proteins in vitro, we assume that MreB would aggregate and spirally wind to SWNTs.

Design and Characterization

DNA expression cassette

Fig 6.Gene expression cassette of N-terminal His-tagged MreB-RFP fusion protein
  • For the overall expression and characterization of MreB, we designed and built a DNA cassette as shown in Fig 6. The original gene expression was obtained from a BioBrick (BBa_I719015) from the Registry of Standard Biological Parts in pSB1A2. This plasmid was reconstructed by introducing BamHI and XhoI sites between the RBS and terminator. Then MreB-RFP fragment with GS linker betweem was inserted, resulting in the vector pSB1A2_BX (MreB-RFP), which contains the target gene expression cassette T7 promoter-RBS-MreB-RFP-terminator. For the further control experiment and the interaction of the fusion protein to SWNT, we also construct plasmid pMreB-NT containing the N-terminal His6-tagged fusion protein gene MreB-RFP with GS linker.
Fig 7.The simulation diagram of MreB-RFP fusion protein

Characterization of MreB-RFP synthesis in vitro

Cell-free expression in W/O/W microdroplets

  • After the expression of gene cassette, we proceeded with the combination of cell-free system with the microfluidics technology to see whether the synthesis of MreB in vitro could continue in microdroplets and assign to the oil phase. Two different DNA templates were used, fusion protein MreB-RFP to study the protein’s phase separation at the membrane, and the water soluble RFP as a control.

Aggregation with SWNTs

  • The fusion protein MreB-RFP would be affiliated to SWNTs after surface treatment and its aggregation patterns is mainly determined by the nickel ions distribution on the sidewall and the polymerization state of itself.

Protocol

Gene expression Cassette

  • Plasmid construction: BioBrick ref BBa_I719015 was obtained from the Registry of Standard Biological Parts, which contains gene expression cassette of T7 promoter-RBS-mGFP-terminator in pSB1A2. This plasmid was reconstructed as pB1A2_BX (A) by introducing BamHI and XhoI sites between the RBS and terminator instead of mGFP gene. MreB gene was PCR-amplified from the genomic DNA isolated from E.coli strain KO11 using the TIANamp bacteria DNA kit (Tiangen, China). The reporter gene RFP was amplified from BioBrick BBa_E1010, using RFP_forward and RFP_reverse primers. The fusion protein gene MreB-RFP was assembled by overlap extension PCR, with GS linker inserted between the two genes. Then the MreB-RFP fragment was inserted into pSB1A2_BX with BamHI and XhoI, resulting in the vector pSB1A2_BX (MreB-RFP) (B), which contains the target gene expression cassette T7 promoter-RBS-MreB-RFP-terminator.
Fig 12. Plasmid constructed for DNA expression cassette in vitro. pSB1A2_BX (MreB-RFP) contains the target gene of MreB-RFP fusion protein between RBS and terminator.
  • The fusion protein gene MreB-RFP with GS linker and the RFP gene alone were PCR-amplified from plasmid pSB1A2_BX (MreB-RFP) using Platinum® Taq DNA Polymerase High Fidelity (Invitrogen, UK) and primers MreB_NT_for and MreB_NT_rev or RFP_NT_for and MreB_NT_rev. The general protocol for the polymerase was followed with an additional final 30 min extension step at 68 C. After gel extraction (Qiagen, UK) the PCR products were used in pEXP5-NT/TOPO® Cloning reactions (Invitrogen, UK) to obtain the plasmid pMreB-NT containing the N-terminal His6 tagged fusion protein gene MreB-RFP with GS linker and the control plasmid pRFP-NT containing the RFP gene with an N-terminal His6 tag.

Cell-free Expression

  • By using S30 T7 High-Yield Protein Expression System (Promega), we express our target protein MreB-RFP in a cell-free system. We set up the following reations on ice in a DNase- and RNase-free 1.5.ml microcentrifuge tube:
Chart 1. Formula and reaction options of S30 T7 High-Yield Protein Expression System
  • Mix thoroughly by pipetting several times or vortexing gently, then centrifuge in a microcentrifuge for 5 seconds to force the reaction mixture to the bottom of the tube. Then quickly bring reaction to 37℃ and incubate the reactions with vigorous shaking for 1 hour. Stop the reaction by placing the tubes in an ice-water bath for 5 minutes. At last, Analyze the results of reaction by SDS-PAGE.

Microfluidics Technology

  • The size of the oil droplet was dependent on the oil and external water flow rate, as well as the storing temperature. In our project, 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.

SWNTs (Single-walled carbon nanotubes)

  • The reactions of single-walled carbon nanotubes (SWNTs) with succinic or glutaric acid acyl peroxides in o-dichlorobenzene at 80-90 °C resulted in the addition of 2-carboxyethyl or 3-carboxypropyl groups, respectively, to the sidewalls of the SWNTs. These SWNTs were then immersed into nickel nitrate solution for the further interaction between nickel ions and carboxylic acid groups.

Supporting Material

Fig 8.Dual-channel confocal microscopy of W/O/W double emulsions made in an aqueous solution: (a) The inner aqueous (green) and (b) interfacial hydrophobic phases (red) consisted of 100 nM FITC (fluorescein isothiocyanate) in deionised water and mineral oil (Sigma) with 2% (w/w) Span 80 and 2 nM DiI (dialkylcarbocyanine iodide), respectively.
Fig 9.LCSM (Laser confocal Scanning Microscopy) of fusion protein MreB-RFP. Red fluorescence can be observed all over the cell membrane, indicating that fusion protein was anchoring on the cell membrane and the intracellular synthsis was successful.
Fig 10. SDS-PAGE of Fusion protein MreB-RFP in vivo: 15% separation gel, 5% stacking gel. Size of fusion protein is 62.8 kD. Samples in the four lanes are: 1. Marker; 2. total protein of BL21(DE3) with pSB1A2_BX; 3. total protein of BL21(DE3) with pSB1A2_BX (fusion protein); 4. soluble protein of BL21(DE3) with pSB1A2_BX (fusion protein), respectively.
Fig 11. Comparison between fluorescence curves of E. Coli with the target plasmid pEXP5-NT (6AA) with the gene of MreB-RFP fusion protein and control plasmid pSB1A2_BX: the cell growth rate measured by OD is corresponding, whereas the tremendously higher fluorescence of target group illustrating the successful expression of target plasmid.


References

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  • 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
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