Difference between revisions of "Part:BBa I750016:Design"

Revision as of 09:26, 18 October 2009

Gas Vesicle polycistonic gene

Design Notes

Site directed mutagenesis was performed in four rounds to remove 3 PstI sites and one EcoRI site from gvpL in the sequence. Primers with biobrick prefix and suffix were then used in a PCR reaction to amplify the part. Finally BBa_J61035 was used to provide the plasmid backbone which introduced a RBS at the front of the part.

The part was sequenced and found to have 10 x 40bp repeat inserted into GvpL related to the mutagenic primer for the gvpL-g696a mutagenic primer. Surprisingly this does not appear to inhibit the operation of the system in bouyancy tests.

There are also a number of SNP in comparison to the referance sequence, presumably due to the error rate with pfuultra.

Repeat Removal

ToDo

Modelling

The gas vesicles are shaped roughly like a cylinder with a cone at each end, whose cross-section we model as (based mostly on [http://2009.igem.org/Team:Groningen/Literature#Walsby1994| 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 [http://2009.igem.org/Team:Groningen/Literature#Walsby1994| 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 [http://2009.igem.org/Team:Groningen/Literature#Baldwin1995| Baldwin 1995], [http://2009.igem.org/Team:Groningen/Literature#Bylund1991| Bylund 1991] and [http://2009.igem.org/Team:Groningen/Literature#Poole1977| 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)

Source

This 5.7Kbp part contains 11 open reading frames coding for gas vesicle genes (gvpB,R,N,F,G,L,S,K,J,T and U) from Bacillus megaterium VT1660 AF053765. The original DNA (sequence= AF053765) was provided by Maura Cannon in pBluescriptIIKS plasmid (pNL29 in "Gas Vesicle genes identified in Bacillus megaterium and Functional Expression in Escherichia coli", Ning Li and Maura C. Cannon, Jounal of bacteriology, May 1998 p2450-2458). Site dirrected mutagenesis was performed to produce four silent changes, removing restriction sites.

References

pNL29 in "Gas Vesicle genes identified in Bacillus megaterium and Functional Expression in Escherichia coli", Ning Li and Maura C. Cannon, Jounal of bacteriology, May 1998, p2450-2458