Coding

Part:BBa_K190028:Design

Designed by: Jolanda Witteveen   Group: iGEM09_Groningen   (2009-08-26)

GlpF


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 283
  • 1000
    COMPATIBLE WITH RFC[1000]

Modelling

The import of As(III) via GlpF is modelled as a simple import reaction with [http://2009.igem.org/Team:Groningen/Glossary#MichaelisMenten Michaelis-Menten kinetics], in part because this makes it easy to specify, but also because we only have very high level data. The following allows a comparison with the data acquired from figure 1B from [http://2009.igem.org/Team:Groningen/Literature#Meng2004 Meng 2004].

Initial values
As(III)ex = µM
                (10µM · 1mL / 1.092mL)
Volumes
Vtotal = mL
Vcells = mL
                (0.1ml · 80mg/mL / 1100mg/mL)
E. coli has a density of approximately 1100mg/mL, see our gas vesicle page for more information.
 i 
Kinetic Constants
v5 = µmol/(s·L)
K5 = µM
Loading graph...

To determine the constants v5 and K5 we performed the following steps:

  1. Read the wild-type line in figure 1B of [http://2009.igem.org/Team:Groningen/Literature#Meng2004 Meng 2004] by pasting it in a drawing program and aligning/scaling the axes and then manually determining the coordinates of each data point.
  2. Converted to units of concentration using the data in Meng 2004 and [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi the CCDB] (assuming that the cells are resting/non-growing), see our [http://spreadsheets.google.com/pub?key=t4gilzCbEaCFAvpEVWUE_zQ Google Docs spreadsheet]. Here we disregarded the fact that the measurements were made by taking out 0.1mL samples, as this does not change the concentrations. Specifically (note that uptake is in nmol/mg):
    • uptaketotal (nmol) = uptake · 8mg · 0.3
      The ratio between dry and wet weight is 0.3 (see the [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB]).
       i 
    • As(III)ex (µM=nmol/mL) = (10nmol/mL · 1mL - uptaketotal) / (1.1-0.0073)mL
      The experiment started with 1mL of a 10µM=10nmol/mL solution of As(III). After adding the cells the total volume of the solution was 1.1mL, and 0.0073mL is an estimate of the total volume of cells in the solution, see below.
       i 
  3. Fit the Michaelis-Menten equation to find the constants v5 and K5 in Mathematica (see [http://igemgroningen.googlecode.com/svn/trunk/buoyant/Models/Meng2004%20Figure%201B.nb the Mathematica notebook in SVN]) using the method from [http://2009.igem.org/Team:Groningen/Literature#Goudar1999 Goudar 1999] (a least squares fit of a closed-form solution of the differential equation).

As it turns out the data from Meng2004 can be fit almost equally well by first-order behaviour, meaning that v5 is only likely to be a minimum for the maximum import rate and only the ratio between v5 and K5 is reliable.

Design Notes

This part has been obtained from the genome of E.coli 356 in two steps with PCR. First the whole gene was obtained from the genome by using PCR and in the second step an EcoR1 restiction site was removed.

Source

This part comes from genomic DNA of the Escherichia coli 536.

References

Some useful papers about GlpF:

  • [http://2009.igem.org/Team:Groningen/Literature#Meng2004 Meng 2004] (As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli)
  • [http://2009.igem.org/Team:Groningen/Literature#Rosen2009 Rosen 2009] (Transport pathways for arsenic and selenium: A minireview)
  • [http://2009.igem.org/Team:Groningen/Literature#Porquet,A,etal2007 Porquet, A, et al.2007] (structural similarity between As(OH)3 and glycerol)
  • [http://2009.igem.orgTeam:Groningen/Literature#Fu,DX,etal2000 Fu, DX, et al.2000] (Structure of the GlpF channel)
  • [http://2009.igem.orgTeam:Groningen/Literature#Heller,KB,etal1980 Heller, KB, et al.1980] (Glycerol transport properties of GlpF)