Team:Groningen/Project/Vesicle

From 2009.igem.org

Our goal in this project is to make cells bouyant in the presence of certain concentrations of metals like copper, zinc and arsenic. Metal induced gas vesicle production can provide our cells with this bouyancy. Gas vesicles are bacterial organelles consisting entirely of proteins that envelop a gas filled space. We made, and send to the registry, parts in which the metal sensitive promoters for copper, zinc and arsenic were cloned in front of the GVP (Gas Vesicle Protein) gene cluster. For further characterization of the GVP gene cluster inducible and constitutive promoters were also cloned in front of this cluster. Buoyancy tests showed that our constructs were able to increase cell buoyancy and electron micrographs showed the presence of gas vesicles. A model was made to predict what volume fraction of a cell would have to be gas vesicle for this cell to have a density equal to that of water.

Background

Gas vesicles are organelles made entirely out of proteins that envelop a gas filled space. Because only gas can penetrate into the gas vesicles the total density of the cell is lowered. This lower cell density leads in turn to a buoyancy phenotype. Outside of the laboratory this buoyancy is used by microorganisms to vertically position themselves in the water column or simply to reduce their sinking rates. Organisms can regulate buoyancy by reducing gas vesicle production or by accumulation of denser compounds like carbohydrates. For certain cyanobacteria this regulation depends on light intensities.

For a number of organisms it has been shown that all proteins important for the expression of gas vesicle lie in a single gene cluster. The GVP gene cluster used in this project was cloned from Bacillus megaterium into E. coli (Li & Cannon 1998). This gene cluster now containing 11 genes was turned into a biobrick by Melbourne 2007 (link to part). Figure 1 shows the gene cluster as it was send in by Melbourne in 2007.

For more info see: Walsby 1994.

Figure 1: Gas vesicle gene cluster ()

Goal

The goal of our project is to engineer an organism that can remove heavy metals from water. To facilitate easy separation the cells that have taken up metals should float so they can be removed from the water. The introduction of GVP gene cluster provides the cell with the required buoyancy.

To make the buoyancy level of the cell responsive to the metal concentration inside the cell, metal sensitive promoters would have to be cloned in front of the GVP gene cluster. In this project these metal sensitive promoters were responsive to zinc, copper and arsenic. We also wanted to make a construct with constitutive and inducible promoters in front of the GVP cluster to be able to characterize this biobrick.

Finally we wanted to improve the Melbourne 2007 biobrick () by removing a repeat that was accidentally introduced during the removal of forbidden restriction sites.

Cloning strategy

figure 2: The floating device will be built up of an inducible promoter which can be induced by a certain intracellular concentration of metal-ions, and a gas vesicle cluster.

To create a floating device, we will use the biobrick gas vesicle cluster (GVP).We will try to improve this biobrick by removing the 11x repeat. The GVP cluster will be expressed under control of a metal sensitive promoter.

Alternative cloning strategy

Another possibility is to use e.g. XbaI and SpeI and also cut a vector with these enzymes, this would lead to ligation of the gvp-cluster in one direction. A possible vector for this strategy on the partsregistry is: [http://partsregistry.org/Part:BBa_J23018 BBa_J23018] (2298 bp).

Bouyancy Tests

Electron Microscopy

Conclusions

Modelling

Buoyancy

The gas vesicles are shaped roughly like a cylinder with a cone at each end, whose cross-section we model as (based mostly on Walsby 1994):

We assume the interior of the wall of the gas vesicle is similarly shaped to the exterior, just slightly smaller (the right-most part of the image above illustrates this situation for the left tip of the gas vesicle). This means the different dimensions are related through the equations below. To determine the total volume, just use them with the given width/diameter (at least for the dimensions given in Walsby 1994). To determine the gas volume, use them with w_gas and d_gas.

w = total width
tw = thickness of wall (1.8-1.95nm)
d = diameter
a = 77 degrees
ρgas = density of gas in vesicle (kg/m^3 = yg/nm^3)
ρwall = density of vesicle wall (kg/m^3)
wwt = tw/sin(a/2)
wt = (1/2)*d/tan(a/2)
wc = w - 2*wt
Vc = (1/4)*pi*d^2*wc
Vt = (1/12)*pi*d^2*wt
V = Vc+2*Vt
M = ρ*V

wgas = w-2*wwt = width of gas space
dgas = d-2*tw = diameter of gas space
V = Vgas + Vwall

Now we can consider the buoyant density of E. coli with gas vesicles. We have chosen to approach this problem using densities and volume ratios. According to Baldwin 1995, Bylund 1991 and Poole 1977, the density of (wild-type) E. coli is 1100 kg/m³ ±3% under wildly varying conditions. This makes our method easier than trying to directly compute the density of a single cell, due to the fact that the volume can differ wildly (both during the life cycle and from strain to strain) and a lack of concrete data on the number of gas vesicles produced (in E. coli). Note that the computations below assume that the gas vesicles simply add to the existing structures.

Vc = volume of a cell without gas vesicles
Vv = volume of gas vesicles in cell
Vcv = volume of a cell with gas vesicles (assumed to be Vc+Vv)
ρc = density of a cell without gas vesicles
ρv = density of gas vesicles
ρm = density of medium

The following has to be true if the cell floats:
 Vc*ρc + Vv*ρv < Vcv*ρm
 (Vcv-Vv)*ρc + Vv*ρv < Vcv*ρm
 ρc + (Vv/Vcv)*(ρv-ρc) < ρm
Assume (ρv - ρc)<0
 Vv/Vcv > (ρm - ρc)/(ρv - ρc)
 Vv/Vcv > 1 - (ρm - ρv)/(ρc - ρv)

Discussion

Missing information

• Used promotor for expression of the gvp-cluster:
  • Inducible (may be used for proof-of-principle)
  • Constitutive (may be used for proof-of-principle)
  • Metal sensitive
• What kind of vector was used by Li and Cannon (1995) or Melbourne (2007)? Is there a negative effect of high copy number?
• What is the maximum amount of pressure gas vesicles can handle? At which depth would this be, how can one put this kind of pressure on a water column?
• What is the density of gas vesicles in cells (normally or in case of over-expression)
• Modelling parameters (to be measured):
  • What is the density of the cell without gas vesicles/metal (largely known, but would be good to check), and how is this affected by letting the cell make gas vesicles and/or metal transporters/accumulators and so on?
  • How many gas vesicles are produced? (As volume percentage?)
  • (How fast are they produced?)

-Cyanobacteria Bowen 1965: gas vacuoles made up of gas vesicles (75 nm in diameter and up to 1.0 ,um in length, single wall layer only 2 nm thick) 0,7MPa gives irreversible loss of buoyancy fenotype, but found in next generation.

you can use a nephelometer, to measurement the light scattering of the gas vesicles (Walsby 1994)

Planning and requirements:

• Modelling:
  • Buoyancy
    • Permeability
    • Number of gasvesicles
    • Where do the gasvesciles end, in hight.
  • Mass of the bacteria (E. coli)
  • How long it takes before it floates
    • How long it take untill it is being expressed
    • How long it will take untill there are enough gasvesicles
    • How does it stay floating
• Lab:
  • Gvp
    • Cluster of biobricks
    • Vector ordered from article
    • First expression with constutatieve promotor, later with metal sensitive promotor
  • Measurements
    • Where are the bacteria, what are bac concentrations on a certain hight (not necessary???)
      • Making pictures on certain hight and compare with picture of known concentration
    • Do we have gas vesicles?
      • Measurement vesicle volume using a nephelometer (Holland 2009,Walsby 1994)
      • Measurement of cell density by centrifuging in density (Percoll) gradients Walsby1994
      • Measurement the volume of gass using a compression tube (Walsby 1979, Walsby 1994)