CH391L/S13/Mechanosensing

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'''2008 Tokyo Tech'''
'''2008 Tokyo Tech'''
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This team focused on creating a strain of E. Coli which could be used in a biologically-based touch screen device ([http://2008.igem.org/Team:Tokyo_Tech "Coli Touch"]). Their system applied the pressure-sensitive tet promoter to the expression of GFP. They created a device containing a thin film of cells with this construct, which resulted in the surface selectively fluorescing only in areas which had pressure applied. Since this promoter requires very high pressures (30 MPA according to their page), they also worked on using directed evolution to engineering a promoter which can respond at a lower pressure. In addition, they designed a genetic toggle switch would allow a user to "erase" any writing on the device by activating a heat-sensitive construct which represses GFP production. Beyond these experimental aspects of the project, they also developed a mathematical model which related the pressure input to fluorescence level in order to understand the limitations they needed to develop for an effective write/erase cycle using their "Coli touch" systems.
This team focused on creating a strain of E. Coli which could be used in a biologically-based touch screen device ([http://2008.igem.org/Team:Tokyo_Tech "Coli Touch"]). Their system applied the pressure-sensitive tet promoter to the expression of GFP. They created a device containing a thin film of cells with this construct, which resulted in the surface selectively fluorescing only in areas which had pressure applied. Since this promoter requires very high pressures (30 MPA according to their page), they also worked on using directed evolution to engineering a promoter which can respond at a lower pressure. In addition, they designed a genetic toggle switch would allow a user to "erase" any writing on the device by activating a heat-sensitive construct which represses GFP production. Beyond these experimental aspects of the project, they also developed a mathematical model which related the pressure input to fluorescence level in order to understand the limitations they needed to develop for an effective write/erase cycle using their "Coli touch" systems.

Revision as of 02:52, 16 April 2013

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Contents

Introduction

Mechanosensing refers to the ability of an organism to respond to changes in mechanical force on them or their environment. The mechanical stress can be in a variety of forms:

  • Hydrostatic pressure, as in the case of deep ocean environments
  • Fluid shear stress, as in the case of blood flowing through veins
  • Direct force, as in the case of body weight on a bone
  • Osmotic pressure, resulting from a difference in solute concentrations across a semi-permeable membrane

There are several levels of regulation which may respond to changes in mechanical stress. Channel proteins which respond to pressure act quickly to change the environment or metabolic state of the cell. Transcriptional control is also employed by a wide variety of organisms to adapt the cellular machinery to changing mechanical stresses.

Effects of High Hydrostatic Pressure

High hydrostatic pressure (HHP) can cause a host of problems (reviewed in [1]). Complexes of biomolecules are forced to dissociate, resulting in inhibition of protein synthesis and other essential cellular processes. Very high pressures can cause unfolding of proteins or nucleic acids by interfering with hydrogen bonding and other non-covalent interactions. The equilibria of reactions accompanied by volume change are shifted, resulting in altered metabolic fluxes. Membrane fluidity is changed by close-packing of lipid tails in the bilayer. Together, these effects require many adaptations for organisms to react and survive.

Due to the deleterious effects on cellular machinery, some pasteurization processes for food employ extremely high pressures [2]. High pressures can kill pathogens effectively similar to heat pasteurization, but pressure doesn't damage the integrity and flavor of food products as much as high temperatures.

Mechanosensing Channels

Many organisms have mechanosensitive channels which are involved in changing metabolism more quickly than transcriptional changes allow. Changes in the shape of the membrane cause conformational changes that open the cahnnel, allowing solvent or solutes to flow across the membrane.

Non-Specific Channels

One example of well-studied mechanosensitive channel protein is MscS (summarized in [3]). This protein forms a non-specific ion channel across the cell membrane in E. Coli which opens in response to osmotic changes in the environment. When the osmotic pressure across the membrane changes, the change in lipid bilayer structure causes MscS to open and allow ions and water to move across the membrane, which prevents cell lysis from osmotic pressure. Several other classes of mechanosensitive channels have been identified, which are generally classified by their size and the magnitude of pressure which elicits a response.

Specific Channels

Transcriptional Responses to Pressure

In some cases, changes in mechanical stress result in differential gene expression driven by mechanosensitive promoters.

In Prokaryotes

In microbes responding to high pressure environments, genes that have increased expression might include cold- and heat-shock and other stress response proteins[4], barostable polymerases[5], or membrane proteins[6, 7]. Down-regulated genes might include nutrient transporters[8, 9]. In other cases, porin proteins which provide ion diffusion pathways are opened in response to osmotic stress across the membrane.

The first high pressure induced gene in bacteria was found in 1989[6] in a deep-ocean bacterium, Photobacterium profundum strain SS9. The gene encodes for OmpH, a large transmembrane protein which is involved in nutrient uptake. Later work found that the operon also contained two outer membrane proteins, OmpL[9] (induced at lower pressures ~1atm) and OmpI[7] (induced at much higher pressures ~400atm). Interestingly, all of these gene products encode for channel proteins, a subclass of which are the other main mechanosensing proteins. It has also been found that the lac promoter can be induced by extremely high pressures (~30 MPa) [10].

In addition to genes which are induced in response to pressure, many genes are repressed by HHP. The MalB operon is one example of a regulon repressed by high pressure [8]. Since there is no evidence of a repressor which acts only under high pressure, it is more likely that the transcription factors which induce genes constitutively are inactivated by the effects of pressure on protein folding.


In Eukaryotes

iGEM Connection

There is a brief mention of an idea regarding mechanosensing as an earthquake sensor in the notebook section for the 2012 Baskent University team, but it seems that they chose to pursue a different idea related to quorum sensing.

2010 MIT

In one of their projects, this team focused on engineering pressure sensitive promoters in mammalian cells. They were interested in using pressure as a way to control formation of biomaterials, with a goal of being able to engineer tissues and organs. Specifically, they created a touch pad which would form bone after a pressure change input. They first characterized fluid shear stress responsive promoters and used these to create a genetic toggle switch sensitive to pressure changes. Their touch pad contained human endothelial kidney (HEK) cells with production Bone Morphogenic Protein 2 (BMP2) under control of the pressure sensitive toggle switch, along with undifferentiated stem cells. When pressure is applied, BMP2 production turns on and causes the production of bone tissue via the differentiated stem cells.


2008 Tokyo Tech

This team focused on creating a strain of E. Coli which could be used in a biologically-based touch screen device ("Coli Touch"). Their system applied the pressure-sensitive tet promoter to the expression of GFP. They created a device containing a thin film of cells with this construct, which resulted in the surface selectively fluorescing only in areas which had pressure applied. Since this promoter requires very high pressures (30 MPA according to their page), they also worked on using directed evolution to engineering a promoter which can respond at a lower pressure. In addition, they designed a genetic toggle switch would allow a user to "erase" any writing on the device by activating a heat-sensitive construct which represses GFP production. Beyond these experimental aspects of the project, they also developed a mathematical model which related the pressure input to fluorescence level in order to understand the limitations they needed to develop for an effective write/erase cycle using their "Coli touch" systems.

Future Directions

It's worth noting that extremely high pressure is gaining popularity as a way to pasteurize foods without heat treatment[1]. The high pressure takes less of a toll on the quality of the food products than heat, but can be just as effective at killing microorganisms. It will be interesting to see if baro-resistant organisms arise from this trend.

References

Error fetching PMID 2479840:
Error fetching PMID 8759872:
Error fetching PMID 22404681:
Error fetching PMID 8244922:
Error fetching PMID 11111034:
Error fetching PMID 20198911:
Error fetching PMID 22290643:
Error fetching PMID 8598266:
Error fetching PMID 21210167:
Error fetching PMID 21035541:
Error fetching PMID 7958783:
Error fetching PMID 20616037:
Error fetching PMID 10202137:
  1. Error fetching PMID 22290643: [biotechapplications]
    Review of biotechnological applications of high hydrostatic pressure

  2. Error fetching PMID 20198911: [biotechreview]
    Review of practical applications of extremophiles

  3. Error fetching PMID 22404681: [MscSpaper]
    Review about MscS function theories

  4. Error fetching PMID 21210167: [stressprots]
    Review of stress-induced proteins in marine extremophiles

  5. Error fetching PMID 11111034: [RNApolviolacea]
    Study of high pressure adapted RNA polymerase from S. Violacea

  6. Error fetching PMID 2479840: [OmpHdiscovery]
    First discovery of bacterial pressure-regulated gene, channel protein OmpH

  7. Error fetching PMID 8244922: [OmpIgene]
    OmpL, OmpH, and OmpI identified as pressure regulated by mutational studies

  8. Error fetching PMID 8598266: [MalBinterval]
    High Hydrostatic Pressure interrupts expression of MalB operon

  9. Error fetching PMID 8759872: [OmpLgene]
    Identification of low pressure induced channel protein

  10. Error fetching PMID 7958783: [HHPlac]
    lac promoter is induced by very high pressures

  11. Error fetching PMID 21035541: [copingHHP]
    Review about high hydrostatic pressure environments

  12. Error fetching PMID 20616037: [YbdG]
    Characterization of YbdG as MscM protein

  13. Error fetching PMID 10202137: [MscKS]
    Genes in E Coli predicted to code for MscS and MscK

All Medline abstracts: PubMed HubMed
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